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Sample records for drug resistance assay

  1. Fluorometric assay for phenotypic differentiation of drug-resistant HIV mutants

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    Zhu, Qinchang; Yu, Zhiqiang; Kabashima, Tsutomu; Yin, Sheng; Dragusha, Shpend; El-Mahdy, Ahmed F. M.; Ejupi, Valon; Shibata, Takayuki; Kai, Masaaki

    2015-01-01

    Convenient drug-resistance testing of viral mutants is indispensable to effective treatment of viral infection. We developed a novel fluorometric assay for phenotypic differentiation of drug-resistant mutants of human immunodeficiency virus-I protease (HIV-PR) which uses enzymatic and peptide-specific fluorescence (FL) reactions and high-performance liquid chromatography (HPLC) of three HIV-PR substrates. This assay protocol enables use of non-purified enzyme sources and multiple substrates for the enzymatic reaction. In this study, susceptibility of HIV mutations to drugs was evaluated by selective formation of three FL products after the enzymatic HIV-PR reaction. This proof-of-concept study indicates that the present HPLC-FL method could be an alternative to current phenotypic assays for the evaluation of HIV drug resistance. PMID:25988960

  2. Rapid detection of drug resistance and mutational patterns of extensively drug-resistant strains by a novel GenoType® MTBDRsl assay

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    A K Singh

    2013-01-01

    Full Text Available Background: The emergence of extensively drug-resistant tuberculosis (XDR-TB is a major concern in the India. The burden of XDR-TB is increasing due to inadequate monitoring, lack of proper diagnosis, and treatment. The GenoType ® Mycobacterium tuberculosis drug resistance second line (MTBDRsl assay is a novel line probe assay used for the rapid detection of mutational patterns conferring resistance to XDR-TB. Aim: The aim of this study was to study the rapid detection of drug resistance and mutational patterns of the XDR-TB by a novel GenoType ® MTBDRsl assay. Materials and Methods: We evaluated 98 multidrug-resistant (MDR M. tuberculosis isolates for second line drugs susceptibility testing by 1% proportion method (BacT/ALERT 3D system and GenoType ® MTBDRsl assay for rapid detection of conferring drug resistance to XDR-TB. Results: A total of seven (17.4% were identified as XDR-TB by using standard phenotypic method. The concordance between phenotypic and GenoType ® MTBDRsl assay was 91.7-100% for different antibiotics. The sensitivity and specificity of the MTBDRsl assay were 100% and 100% for aminoglycosides; 100% and 100% for fluoroquinolones; 91.7% and 100% for ethambutol. The most frequent mutations and patterns were gyrA MUT1 (A90V in seven (41.2% and gyrA + WT1-3 + MUT1 in four (23.5%; rrs MUT1 (A1401G in 11 (64.7%, and rrs WT1-2 + MUT1 in eight (47.1%; and embB MUT1B (M306V in 11 (64.7% strains. Conclusions: These data suggest that the GenoType ® MTBDRsl assay is rapid, novel test for detection of resistance to second line anti-tubercular drugs. This assay provides additional information about the frequency and mutational patterns responsible for XDR-TB resistance.

  3. Fluorometric assay for phenotypic differentiation of drug-resistant HIV mutants

    OpenAIRE

    Zhu, Qinchang; Yu, Zhiqiang; Kabashima, Tsutomu; Yin, Sheng; Dragusha, Shpend; El-Mahdy, Ahmed F. M.; Ejupi, Valon; Shibata, Takayuki; Kai, Masaaki

    2015-01-01

    Convenient drug-resistance testing of viral mutants is indispensable to effective treatment of viral infection. We developed a novel fluorometric assay for phenotypic differentiation of drug-resistant mutants of human immunodeficiency virus-I protease (HIV-PR) which uses enzymatic and peptide-specific fluorescence (FL) reactions and high-performance liquid chromatography (HPLC) of three HIV-PR substrates. This assay protocol enables use of non-purified enzyme sources and multiple substrates f...

  4. Dynamic optical tweezers based assay for monitoring early drug resistance

    International Nuclear Information System (INIS)

    Wu, Xiaojing; Zhu, Siwei; Feng, Jie; Zhang, Yuquan; Min, Changjun; Yuan, X-C

    2013-01-01

    In this letter, a dynamic optical tweezers based assay is proposed and investigated for monitoring early drug resistance with Pemetrexed-resistant non-small cell lung cancer (NSCLC) cell lines. The validity and stability of the method are verified experimentally in terms of the physical parameters of the optical tweezers system. The results demonstrate that the proposed technique is more convenient and faster than traditional techniques when the capability of detecting small variations of the response of cells to a drug is maintained. (letter)

  5. Evaluation of the Roche prototype 454 HIV-1 ultradeep sequencing drug resistance assay in a routine diagnostic laboratory.

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    Garcia-Diaz, A; Guerrero-Ramos, A; McCormick, A L; Macartney, M; Conibear, T; Johnson, M A; Haque, T; Webster, D P

    2013-10-01

    Studies have shown that low-frequency resistance mutations can influence treatment outcome. However, the lack of a standardized high-throughput assay has precluded their detection in clinical settings. To evaluate the performance of the Roche prototype 454 UDS HIV-1 drug resistance assay (UDS assay) in a routine diagnostic laboratory. 50 plasma samples, previously characterized by population sequencing and that had shown ≥1 resistance associated mutation (RAM), were retrospectively tested by the UDS assay, including 18 B and 32 non-B subtypes; viral loads between 114-1,806,407 cp/ml; drug-naive (n=27) and drug-experienced (n=23) individuals. The UDS assay was successful for 37/50 (74%) samples. It detected all RAMs found by population sequencing at frequencies above 20%. In addition, 39 low-frequency RAMs were exclusively detected by the UDS assay at frequencies below 20% in both drug-naïve (19/26, 73%) and drug-experienced (9/18, 50%) individuals. UDS results would lead to changes from susceptible to resistant to efavirenz (EFV) in one drug-naive individual with suboptimal response to an EFV-containing regimen and from susceptible to resistance to lamivudine (3TC) in one drug naïve subject who subsequently failed a 3TC-containing regimen and in a treatment experienced subject who had failed a 3TC-containing regimen. The UDS assay performed well across a wide range of subtypes and viral loads; it showed perfect agreement with population sequencing for all RAMs analyzed. In addition, the UDS assay detected additional mutations at frequencies below 20% which correlate with patients' treatment history and had in some cases important prognostic implications. Copyright © 2013 Elsevier B.V. All rights reserved.

  6. Development of a TaqMan Allelic Discrimination Assay for detection of Single Nucleotides Polymorphisms associated with anti-malarial drug resistance

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    Kamau Edwin

    2012-01-01

    Full Text Available Abstract Background Anti-malarial drug resistance poses a threat to current global efforts towards control and elimination of malaria. Several methods are used in monitoring anti-malarial drug resistance. Molecular markers such as single nucleotide polymorphism (SNP for example are increasingly being used to identify genetic mutations related to anti-malarial drug resistance. Several methods are currently being used in analysis of SNP associated with anti-malarial drug resistance and although each one of these methods has unique strengths and shortcoming, there is still need to improve and/or develop new methods that will close the gap found in the current methods. Methods TaqMan Allelic Discrimination assays for detection of SNPs associated with anti-malarial drug resistance were designed for analysis on Applied Biosystems PCR platform. These assays were designed by submitting SNP sequences associated with anti-malarial drug resistance to Applied Biosystems website. Eleven SNPs associated with resistance to anti-malarial drugs were selected and tested. The performance of each SNP assay was tested by creating plasmid DNAs carrying codons of interests and analysing them for analysis. To test the sensitivity and specificity of each SNP assay, 12 clinical samples were sequenced at codons of interest and used in the analysis. Plasmid DNAs were used to establish the Limit of Detection (LoD for each assay. Results Data from genetic profiles of the Plasmodium falciparum laboratory strains and sequence data from 12 clinical samples was used as the reference method with which the performance of the SNP assays were compared to. The sensitivity and specificity of each SNP assay was establish at 100%. LoD for each assay was established at 2 GE, equivalent to less than 1 parasite/μL. SNP assays performed well in detecting mixed infection and analysis of clinical samples. Conclusion TaqMan Allelic Discrimination assay provides a good alternative tool in

  7. Use of GenoType® MTBDRplus assay to assess drug resistance and mutation patterns of multidrug-resistant tuberculosis isolates in northern India

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    A K Maurya

    2013-01-01

    Full Text Available Purpose: The emergence and spread of multidrug-resistant tuberculosis (MDR-TB is a major public health problem. The diagnosis of MDR-TB is of paramount importance in establishing appropriate clinical management and infection control measures. The aim of this study was to evaluate drug resistance and mutational patterns in clinical isolates MDR-TB by GenoType® MTBDRplus assay. Material and Methods: A total of 350 non-repeated sputum specimens were collected from highly suspected drug-resistant pulmonary tuberculosis (PTB cases; which were processed by microscopy, culture, differentiation and first line drug susceptibility testing (DST using BacT/ALERT 3D system. Results: Among a total of 125 mycobacterium tuberculosis complex (MTBC strains, readable results were obtained from 120 (96% strains by GenoType® MTBDRplus assay. Only 45 MDR-TB isolates were analysed for the performance, frequency and mutational patterns by GenoType® MTBDRplus assay. The sensitivity of the GenoType® MDRTBplus assay for detecting individual resistance to rifampicin (RIF, isoniazid (INH and multidrug resistance was found to be 95.8%, 96.3% and 97.7%, respectively. Mutation in codon S531L of the rpoB gene and codon S315T1 of katG genes were dominated in MDR-TB strains, respectively (P < 0.05. Conclusions: The GenoType® MTBDRplus assay is highly sensitive with short turnaround times and a rapid test for the detection of the most common mutations conferring resistance in MDR-TB strains that can readily be included in a routine laboratory workflow.

  8. Simple PCR assays improve the sensitivity of HIV-1 subtype B drug resistance testing and allow linking of resistance mutations.

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    Jeffrey A Johnson

    Full Text Available BACKGROUND: The success of antiretroviral therapy is known to be compromised by drug-resistant HIV-1 at frequencies detectable by conventional bulk sequencing. Currently, there is a need to assess the clinical consequences of low-frequency drug resistant variants occurring below the detection limit of conventional genotyping. Sensitive detection of drug-resistant subpopulations, however, requires simple and practical methods for routine testing. METHODOLOGY: We developed highly-sensitive and simple real-time PCR assays for nine key drug resistance mutations and show that these tests overcome substantial sequence heterogeneity in HIV-1 clinical specimens. We specifically used early wildtype virus samples from the pre-antiretroviral drug era to measure background reactivity and were able to define highly-specific screening cut-offs that are up to 67-fold more sensitive than conventional genotyping. We also demonstrate that sequencing the mutation-specific PCR products provided a direct and novel strategy to further detect and link associated resistance mutations, allowing easy identification of multi-drug-resistant variants. Resistance mutation associations revealed in mutation-specific amplicon sequences were verified by clonal sequencing. SIGNIFICANCE: Combined, sensitive real-time PCR testing and mutation-specific amplicon sequencing provides a powerful and simple approach that allows for improved detection and evaluation of HIV-1 drug resistance mutations.

  9. Evaluation of GenoType® MTBDRplus assay for rapid detection of drug susceptibility testing of multi-drug resistance tuberculosis in Northern India

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    Anand Kumar Maurya

    2013-01-01

    Full Text Available Background: The problem of multi-drug resistance tuberculosis (MDR-TB is growing in several hotspots throughout the world. Rapid and accurate diagnosis of MDR-TB is crucial to facilitate early treatment and to reduce its spread in the community. The aim of the present study was to evaluate the new, novel GenoType® MTBDRplus assay for rapid detection of drug susceptibility testing (DST of MDR-TB cases in Northern India. Materials and Methods: A total of 550 specimens were collected from highly suspected drug resistant from pulmonary and extra-pulmonary TB cases. All the specimens were processed by Ziehl- Neelsen staining, culture, differentiation by the GenoType® CM assay, first line DST using BacT/ALERT 3D system and GenoType® MTBDRplus assay. The concordance of the GenoType® MTBDRplus assay was calculated in comparison with conventional DST results. Results: Overall the sensitivity for detection of rifampicin, isoniazid and MDR-TB resistance by GenoType® MTBDRplus assay was 98.0%, 98.4% and 98.2% respectively. Out of 55 MDR-TB strains, 45 (81.8%, 52 (94.5% and 17 (30.9% strains showed mutation in rpoB, katG and inhA genes respectively (P < 0.05. The most prominent mutations in rpoB, katG and inhA genes were; 37 (67.3% in S531L, 52 (94.5% in S315T1 and 11 (20% in C15T regions respectively (P < 0.05. Conclusions: Our study demonstrated a high concordance between the GenoType® MTBDRplus assay resistance patterns and those were observed by conventional DST with good sensitivity, specificity with short turnaround times and to control new cases of MDR-TB in countries with a high prevalence of MDR-TB.

  10. The value of microscopic-observation drug susceptibility assay in the diagnosis of tuberculosis and detection of multidrug resistance.

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    Sertel Şelale, Denİz; Uzun, Meltem

    2018-01-01

    Inexpensive, rapid, and reliable tests for detecting the presence and drug susceptibility of Mycobacterium tuberculosis complex (MTBC) are urgently needed to control the transmission of tuberculosis. In this study, we aimed to assess the accuracy and speed of the microscopic-observation drug susceptibility (MODS) assay in the identification of MTBC and detection of multidrug resistance. Sputum samples from patients suspected to have tuberculosis were simultaneously tested with MODS and conventional culture [Löwenstein-Jensen (LJ) culture, BACTEC MGIT™ 960 (MGIT) system], and drug susceptibility testing (MGIT system) methods. A total of 331 sputum samples were analyzed. Sensitivity and specificity of MODS assay for detection of MTBC strains were 96% and 98.8%, respectively. MODS assay detected multidrug resistant MTBC isolates with 92.3% sensitivity and 96.6% specificity. Median time to culture positivity was similar for MGIT (8 days) and MODS culture (8 days), but was significantly longer with LJ culture (20 days) (p tuberculosis and detection of multidrug resistance. © 2017 APMIS. Published by John Wiley & Sons Ltd.

  11. Laboratory determination of chemotherapeutic drug resistance in tumor cells from patients with leukemia, using a fluorometric microculture cytotoxicity assay (FMCA).

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    Larsson, R; Kristensen, J; Sandberg, C; Nygren, P

    1992-01-21

    An automated fluorometric microculture cytotoxicity assay (FMCA) based on the measurement of fluorescence generated from cellular hydrolysis of fluorescein diacetate (FDA) to fluorescein was employed for chemotherapeutic-drug-sensitivity testing of tumor-cell suspensions from patients with leukemia. Fluorescence was linearly related to cell number, and reproducible measurements of drug sensitivity could be performed using fresh or cryopreserved leukemia cells. A marked heterogeneity with respect to chemotherapeutic drug sensitivity was observed for a panel of cytotoxic drugs tested in 43 samples from 35 patients with treated or untreated acute and chronic leukemia. For samples obtained from patients with chronic lymphocytic and acute myelocytic leukemia, sensitivity profiles for standard drugs corresponded to known clinical activity and the assay detected primary and acquired drug resistance. Individual in vitro/in vivo correlations indicated high specificity with respect to the identification of drug resistance. The results suggest that the FMCA may be a simple and rapid method for in vivo-representative determinations of chemotherapeutic drug resistance in tumor cells obtained from patients with leukemia.

  12. Evaluation of the microscopic observational drug susceptibility assay for rapid and efficient diagnosis of multi-drug resistant tuberculosis

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    R P Lazarus

    2012-01-01

    Full Text Available Purpose: Tuberculosis (TB is endemic in India and the burden of multi-drug-resistant tuberculosis (MDR-TB is high. Early detection of MDR-TB is of primary importance in controlling the spread of TB. The microscopic observational drug susceptibility (MODS assay has been described as a cost-effective and rapid method by which mycobacterial culture and the drug susceptibility test (DST can be done at the same time. Materials and Methods: A total of 302 consecutive sputum samples that were received in an accredited mycobacteriology laboratory for conventional culture and DST were evaluated by the MODS assay. Results: In comparison with conventional culture on Lowenstein Jensen (LJ media, the MODS assay showed a sensitivity of 94.12% and a specificity of 89.39% and its concordance with the DST by the proportion method on LJ media to isoniazid and rifampicin was 90.8% and 91.5%, respectively. The turnaround time for results by MODS was 9 days compared to 21 days by culture on LJ media and an additional 42 days for DST by the 1% proportion method. The cost of performing a single MODS assay was Rs. 250/-, compared to Rs. 950/- for culture and 1st line DST on LJ. Conclusion: MODS was found to be a sensitive and rapid alternative method for performing culture and DST to identify MDR-TB in resource poor settings.

  13. Resistance patterns and trends of extensively drug-resistant tuberculosis: 5-year experience

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    Amresh Kumar Singh

    2013-12-01

    Full Text Available Objective:Extensively drug-resistant tuberculosis (XDR-TB strains were emerged when multidrug-resistant TB (MDR- TB was inadequately treated. Inadequate treatment of MDR-TB cases may result in additional resistance especially non-XDR-TB and then XDR-TB. The aim of this study was to know the prevalence, resistance patterns and trends of the XDR-TB strains among the MDR-TB at a tertiary care hospital in Lucknow, India Methods: A total of 430 Mycobacterium isolates were underwent NAP test and TB MPT64 Ag test for the identification of Mycobacterium tuberculosis complex (MTBC. Drug-susceptibility test (DST was performed over MTBC for the first line drugs by 1% proportion method (Bactec and for the second-line drugs by 1% proportion method (Lowenstein- Jensen media. The XDR-TB status was further confirmed by line probe assay (GenoType® MTBDRsl assay. Results: Among the 430 isolates of mycobacterium, 365 (84.9% were MTBC and 139 (38.1% were MDR-TB respectively. Further 97 MDR-TB from “highly suspected drug resistant-TB (DR-TB” cases among MDR-TB were tested with second line drugs in which 15 (15.5% XDR-TB and 82 (84.5% were non-XDR-TB. Regarding XDR-TB status, using the 1% proportion method a 100% agreement was seen with the GenoType® MTBDRsl assay. Resistance patterns of XDR-TB were as; 10/15 (66.7% as isoniazid + rifampicin + ciprofloxacin + amikacin resistance and 5/15 (33.3% as isoniazid + rifampicin + ciprofloxacin + amikacin + kanamycin resistance. Conclusion:The prevalence of XDR-TB was 15.5% among MDR-TB. Hence laboratory testing of “highly suspected drug resistant-TB” isolates should be done for both first and second line drugs simultaneously especially in developing countries.J Microbiol Infect Dis 2013;3(4: 169-175

  14. Characterization of the Drug Resistance Profiles of Patients Infected with CRF07_BC Using Phenotypic Assay and Ultra-Deep Pyrosequencing.

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    Szu-Wei Huang

    Full Text Available The usefulness of ultra-deep pyrosequencing (UDPS for the diagnosis of HIV-1 drug resistance (DR remains to be determined. Previously, we reported an explosive outbreak of HIV-1 circulating recombinant form (CRF 07_BC among injection drug users (IDUs in Taiwan in 2004. The goal of this study was to characterize the DR of CRF07_BC strains using different assays including UDPS. Seven CRF07_BC isolates including 4 from early epidemic (collected in 2004-2005 and 3 from late epidemic (collected in 2008 were obtained from treatment-naïve patient's peripheral blood mononuclear cells. Viral RNA was extracted directly from patient's plasma or from cultural supernatant and the pol sequences were determined using RT-PCR sequencing or UDPS. For comparison, phenotypic drug susceptibility assay using MAGIC-5 cells (in-house phenotypic assay and Antivirogram were performed. In-house phenotypic assay showed that all the early epidemic and none of the late epidemic CRF07_BC isolates were resistant to most protease inhibitors (PIs (4.4-47.3 fold. Neither genotypic assay nor Antivirogram detected any DR mutations. UDPS showed that early epidemic isolates contained 0.01-0.08% of PI DR major mutations. Furthermore, the combinations of major and accessory PI DR mutations significantly correlated with the phenotypic DR. The in-house phenotypic assay is superior to other conventional phenotypic assays in the detection of DR variants with a frequency as low as 0.01%.

  15. Estimating Fitness by Competition Assays between Drug Susceptible and Resistant Mycobacterium tuberculosis of Predominant Lineages in Mumbai, India

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    Bhatter, Purva; Chatterjee, Anirvan; D'souza, Desiree; Tolani, Monica; Mistry, Nerges

    2012-01-01

    Background Multi Drug Resistant Tuberculosis (MDR TB) is a threat to global tuberculosis control. A significant fitness cost has been associated with DR strains from specific lineages. Evaluation of the influence of the competing drug susceptible strains on fitness of drug resistant strains may have an important bearing on understanding the spread of MDR TB. The aim of this study was to evaluate the fitness of MDR TB strains, from a TB endemic region of western India: Mumbai, belonging to 3 predominant lineages namely CAS, Beijing and MANU in the presence of drug susceptible strains from the same lineages. Methodology Drug susceptible strains from a single lineage were mixed with drug resistant strain, bearing particular non synonymous mutation (rpoB D516V; inhA, A16G; katG, S315T1/T2) from the same or different lineages. Fitness of M.tuberculosis (M.tb) strains was evaluated using the difference in growth rates obtained by using the CFU assay system. Conclusion/Significance While MANU were most fit amongst the drug susceptible strains of the 3 lineages, only Beijing MDR strains were found to grow in the presence of any of the competing drug susceptible strains. A disproportionate increase in Beijing MDR could be an alarm for an impending epidemic in this locale. In addition to particular non synonymous substitutions, the competing strains in an environment may impact the fitness of circulating drug resistant strains. PMID:22479407

  16. A histidine-rich protein 2-based malaria drug sensitivity assay for field use.

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    Noedl, Harald; Attlmayr, Bernhard; Wernsdorfer, Walther H; Kollaritsch, Herwig; Miller, Robert S

    2004-12-01

    With the spread of antimalarial drug resistance, simple and reliable tools for the assessment of antimalarial drug resistance, particularly in endemic regions and under field conditions, have become more important than ever before. We therefore developed a histidine-rich protein 2 (HRP2)-based drug sensitivity assay for testing of fresh isolates of Plasmodium falciparum in the field. In contrast to the HRP2 laboratory assay, the field assay uses a procedure that further simplifies the handling and culturing of malaria parasites by omitting centrifugation, washing, the use of serum, and dilution with uninfected red blood cells. A total of 40 fresh Plasmodium falciparum isolates were successfully tested for their susceptibility to dihydroartemisinin, mefloquine, quinine, and chloroquine (50% inhibitory concentration [IC50] = 3.43, 61.89, 326.75, and 185.31 nM, respectively). Results very closely matched those obtained with a modified World Health Organization schizont maturation assay (R2 = 0.96, P < 0.001; mean log difference at IC50 = 0.054).

  17. Pharmacological modification of multi-drug resistance (MDR) in vitro detected by a novel fluorometric microculture cytotoxicity assay. Reversal of resistance and selective cytotoxic actions of cyclosporin A and verapamil on MDR leukemia T-cells.

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    Larsson, R; Nygren, P

    1990-07-15

    A novel fluorometric microculture cytotoxicity assay (FMCA), based on measurements of fluorescein diacetate (FDA) hydrolysis and DNA staining by Hoechst 33342, was used for drug sensitivity testing and detection of resistance reversal in acute lymphoblastic leukemia (ALL) cell lines. The 72-hr assay was found to be sensitive, reproducible and linearly related to the number of viable cells within a broad range of cell concentrations. At clinically achievable drug concentrations, the calcium channel blocker Verapamil (ver) and the immunosuppressant Cyclosporin A (csA) were found to partly reverse acquired Vincristine (vcr) resistance in multi-drug resistant (MDR) T-ALL L100 cells with little or no effect on the drug-sensitive parental L0 cell line. By combining the fluorometric indices, we found that low concentrations of csA were growth-inhibitory, whereas higher concentrations (greater than 10 micrograms/ml) were progressively cytotoxic for drug-sensitive L0 cells. In MDR L100 cells, on the other hand, csA produced significant cell kill even at low drug concentrations. Ver had no effects on sensitive L0 cells but showed considerable cytotoxic action towards MDR L100 cells. There was no apparent relationship between drug reversal of vcr resistance and the cytotoxic actions of the drug per se since the calcium channel blocker diltiazem (dil) significantly potentiated the actions of vcr on MDR L100 cells without being more toxic to these cells (compared to vcr-sensitive L0 cells).

  18. A novel high throughput assay for anthelmintic drug screening and resistance diagnosis by real-time monitoring of parasite motility.

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    Michael J Smout

    Full Text Available BACKGROUND: Helminth parasites cause untold morbidity and mortality to billions of people and livestock. Anthelmintic drugs are available but resistance is a problem in livestock parasites, and is a looming threat for human helminths. Testing the efficacy of available anthelmintic drugs and development of new drugs is hindered by the lack of objective high-throughput screening methods. Currently, drug effect is assessed by observing motility or development of parasites using laborious, subjective, low-throughput methods. METHODOLOGY/PRINCIPAL FINDINGS: Here we describe a novel application for a real-time cell monitoring device (xCELLigence that can simply and objectively assess anthelmintic effects by measuring parasite motility in real time in a fully automated high-throughput fashion. We quantitatively assessed motility and determined real time IC(50 values of different anthelmintic drugs against several developmental stages of major helminth pathogens of humans and livestock, including larval Haemonchus contortus and Strongyloides ratti, and adult hookworms and blood flukes. The assay enabled quantification of the onset of egg hatching in real time, and the impact of drugs on hatch rate, as well as discriminating between the effects of drugs on motility of drug-susceptible and -resistant isolates of H. contortus. CONCLUSIONS/SIGNIFICANCE: Our findings indicate that this technique will be suitable for discovery and development of new anthelmintic drugs as well as for detection of phenotypic resistance to existing drugs for the majority of helminths and other pathogens where motility is a measure of pathogen viability. The method is also amenable to use for other purposes where motility is assessed, such as gene silencing or antibody-mediated killing.

  19. In-vitro antimycobacterial drug susceptibility testing of non-tubercular mycobacteria by tetrazolium microplate assay.

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    Sankar, Manimuthu Mani; Gopinath, Krishnamoorthy; Singla, Roopak; Singh, Sarman

    2008-07-11

    Non-tubercular mycobacteria (NTM) has not been given due attention till the recent epidemic of HIV. This has led to increasing interest of health care workers in their biology, epidemiology and drug resistance. However, timely detection and drug susceptibility profiling of NTM isolates are always difficult in resource poor settings like India. Furthermore, no standardized methodology or guidelines are available to reproduce the results with clinical concordance. To find an alternative and rapid method for performing the drug susceptibility assay in a resource limited settings like India, we intended to evaluate the utility of Tetrazolium microplate assay (TEMA) in comparison with proportion method for reporting the drug resistance in clinical isolates of NTM. A total of fifty-five NTM isolates were tested for susceptibility against Streptomycin, Rifampicin, Ethambutol, Ciprofloxacin, Ofloxacin, Azithromycin, and Clarithromycin by TEMA and the results were compared with agar proportion method (APM). Of the 55 isolates, 23 (41.8%) were sensitive to all the drugs and the remaining 32 (58.2%) were resistant to at least one drug. TEMA had very good concordance with APM except with minor discrepancies. Susceptibility results were obtained in the median of 5 to 9 days by TEMA. The NTM isolates were highly sensitive against Ofloxacin (98.18% sensitive) and Ciprofloxacin (90.09% sensitive). M. mucogenicum was sensitive only to Clarithromycin and resistant to all the other drugs tested. The concordance between TEMA and APM ranged between 96.4 - 100%. Tetrazolium Microplate Assay is a rapid and highly reproducible method. However, it must be performed only in tertiary level Mycobacteriology laboratories with proper bio-safety conditions.

  20. Exosomes from adriamycin-resistant breast cancer cells transmit drug resistance partly by delivering miR-222.

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    Yu, Dan-Dan; Wu, Ying; Zhang, Xiao-Hui; Lv, Meng-Meng; Chen, Wei-Xian; Chen, Xiu; Yang, Su-Jin; Shen, Hongyu; Zhong, Shan-Liang; Tang, Jin-Hai; Zhao, Jian-Hua

    2016-03-01

    Breast cancer (BCa) is one of the major deadly cancers in women. However, treatment of BCa is still hindered by the acquired-drug resistance. It is increasingly reported that exosomes take part in the development, metastasis, and drug resistance of BCa. However, the specific role of exosomes in drug resistance of BCa is poorly understood. In this study, we investigate whether exosomes transmit drug resistance through delivering miR-222. We established an adriamycin-resistant variant of Michigan Cancer Foundation-7 (MCF-7) breast cancer cell line (MCF-7/Adr) from a drug-sensitive variant (MCF-7/S). Exosomes were isolated from cell supernatant by ultracentrifugation. Cell viability was assessed by MTT assay and apoptosis assay. Individual miR-222 molecules in BCa cells were detected by fluorescence in situ hybridization (FISH). Then, FISH was combined with locked nucleic acid probes and enzyme-labeled fluorescence (LNA-ELF-FISH). Individual miR-222 could be detected as bright photostable fluorescent spots and then the quantity of miR-222 per cell could be counted. Stained exosomes were taken in by the receipt cells. MCF-7/S acquired drug resistance after co-culture with exosomes from MCF-7/Adr (A/exo) but did not after co-culture with exosomes from MCF-7/S (S/exo). The quantity of miR-222 in A/exo-treated MCF-7/S was significantly greater than in S/exo-treated MCF-7/S. MCF-7/S transfected with miR-222 mimics acquired adriamycin resistance while MCF-7/S transfected with miR-222 inhibitors lost resistance. In conclusion, exosomes are effective in transmitting drug resistance and the delivery of miR-222 via exosomes may be a mechanism.

  1. Rapid detection of multidrug-resistant Mycobacterium tuberculosis using the malachite green decolourisation assay

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    Coban, Ahmet Yilmaz; Uzun, Meltem

    2013-01-01

    Early detection of drug resistance in Mycobacterium tuberculosis isolates allows for earlier and more effective treatment of patients. The aim of this study was to investigate the performance of the malachite green decolourisation assay (MGDA) in detecting isoniazid (INH) and rifampicin (RIF) resistance in M. tuberculosis clinical isolates. Fifty M. tuberculosis isolates, including 19 multidrug-resistant, eight INH-resistant and 23 INH and RIF-susceptible samples, were tested. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and agreement of the assay for INH were 92.5%, 91.3%, 92.5%, 91.3% and 92%, respectively. Similarly, the sensitivity, specificity, PPV, NPV and agreement of the assay for RIF were 94.7%, 100%, 100%, 96.8% and 98%, respectively. There was a major discrepancy in the tests of two isolates, as they were sensitive to INH by the MGDA test, but resistant by the reference method. There was a minor discrepancy in the tests of two additional isolates, as they were sensitive to INH by the reference method, but resistant by the MGDA test. The drug susceptibility test results were obtained within eight-nine days. In conclusion, the MGDA test is a reliable and accurate method for the rapid detection of INH and RIF resistance compared with the reference method and the MGDA test additionally requires less time to obtain results. PMID:24402143

  2. Rapid detection of multidrug-resistant Mycobacterium tuberculosis using the malachite green decolourisation assay

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    Ahmet Yilmaz Coban

    2013-12-01

    Full Text Available Early detection of drug resistance in Mycobacterium tuberculosis isolates allows for earlier and more effective treatment of patients. The aim of this study was to investigate the performance of the malachite green decolourisation assay (MGDA in detecting isoniazid (INH and rifampicin (RIF resistance in M. tuberculosis clinical isolates. Fifty M. tuberculosis isolates, including 19 multidrug-resistant, eight INH-resistant and 23 INH and RIF-susceptible samples, were tested. The sensitivity, specificity, positive predictive value (PPV, negative predictive value (NPV and agreement of the assay for INH were 92.5%, 91.3%, 92.5%, 91.3% and 92%, respectively. Similarly, the sensitivity, specificity, PPV, NPV and agreement of the assay for RIF were 94.7%, 100%, 100%, 96.8% and 98%, respectively. There was a major discrepancy in the tests of two isolates, as they were sensitive to INH by the MGDA test, but resistant by the reference method. There was a minor discrepancy in the tests of two additional isolates, as they were sensitive to INH by the reference method, but resistant by the MGDA test. The drug susceptibility test results were obtained within eight-nine days. In conclusion, the MGDA test is a reliable and accurate method for the rapid detection of INH and RIF resistance compared with the reference method and the MGDA test additionally requires less time to obtain results.

  3. Molecular approaches for detection of the multi-drug resistant tuberculosis (MDR-TB in Bangladesh.

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    Tafsina Haque Aurin

    Full Text Available The principal obstacles in the treatment of tuberculosis (TB are delayed and inaccurate diagnosis which often leads to the onset of the drug resistant TB cases. To avail the appropriate treatment of the patients and to hinder the transmission of drug-resistant TB, accurate and rapid detection of resistant isolates is critical. Present study was designed to demonstrate the efficacy of molecular techniques inclusive of line probe assay (LPA and GeneXpert MTB/RIF methods for the detection of multi-drug resistant (MDR TB. Sputum samples from 300 different categories of treated and new TB cases were tested for the detection of possible mutation in the resistance specific genes (rpoB, inhA and katG through Genotype MTBDRplus assay or LPA and GeneXpert MTB/RIF tests. Culture based conventional drug susceptibility test (DST was also carried out to measure the efficacy of the molecular methods employed. Among 300 samples, 191 (63.7% and 193 (64.3% cases were found to be resistant against rifampicin in LPA and GeneXpert methods, respectively; while 189 (63% cases of rifampicin resistance were detected by conventional DST methods. On the other hand, 196 (65.3% and 191 (63.7% isolates showed isoniazid resistance as detected by LPA and conventional drug susceptibility test (DST, respectively. Among the drug resistant isolates (collectively 198 in LPA and 193 in conventional DST, 189 (95.6% and 187 (96.9% were considered to be MDR as examined by LPA and conventional DST, respectively. Category-II and -IV patients encountered higher frequency of drug resistance compared to those from category-I and new cases. Considering the higher sensitivity, specificity and accuracy along with the required time to results significantly shorter, our study supports the adoption of LPA and GeneXpert assay as efficient tools in detecting drug resistant TB in Bangladesh.

  4. RAS - Screens & Assays - Drug Discovery

    Science.gov (United States)

    The RAS Drug Discovery group aims to develop assays that will reveal aspects of RAS biology upon which cancer cells depend. Successful assay formats are made available for high-throughput screening programs to yield potentially effective drug compounds.

  5. Neratinib resistance and cross-resistance to other HER2-targeted drugs due to increased activity of metabolism enzyme cytochrome P4503A4.

    Science.gov (United States)

    Breslin, Susan; Lowry, Michelle C; O'Driscoll, Lorraine

    2017-02-28

    Neratinib is in Phase 3 clinical trials but, unfortunately, the development of resistance is inevitable. Here, we investigated the effects of acquired neratinib resistance on cellular phenotype and the potential mechanism of this resistance. Neratinib-resistant variants of HER2-positive breast cancer cells were developed and their cross-resistance investigated using cytotoxicity assays. Similarly, sensitivity of trastuzumab-resistant and lapatinib-resistant cells to neratinib was assessed. Cellular phenotype changes were evaluated using migration, invasion and anoikis assays. Immunoblotting for HER family members and drug efflux pumps, as well as enzyme activity assays were performed. Neratinib resistance conferred cross-resistance to trastuzumab, lapatinib and afatinib. Furthermore, the efficacy of neratinib was reduced in trastuzumab- and lapatinib-resistant cells. Neratinib-resistant cells were more aggressive than their drug-sensitive counterparts, with increased CYP3A4 activity identified as a novel mechanism of neratinib resistance. The potential of increased CYP3A4 activity as a biomarker and/or target to add value to neratinib warrants investigation.

  6. In vitro determination of cytotoxic drug response in ovarian carcinoma using the fluorometric microculture cytotoxicity assay (FMCA).

    Science.gov (United States)

    Csóka, K; Tholander, B; Gerdin, E; de la Torre, M; Larsson, R; Nygren, P

    1997-09-17

    The fluorometric microculture cytotoxicity assay (FMCA), a short-term in vitro assay based on the concept of total tumor cell kill, was used for testing the cytotoxic drug sensitivity of tumor cells from patients with ovarian carcinoma. A total of 125 fresh specimens was obtained, 98 (78%) of which were analyzed successfully. Data from 45 patients were available for clinical correlations. The FMCA appeared to yield clinically relevant cytotoxic drug sensitivity data for ovarian carcinoma as indicated by a comparison with tumor samples obtained from patients with non-Hodgkin's lymphoma or kidney carcinoma. Considering the most active single agent in vitro actually given in vivo, and using the median drug activity among all ovarian carcinoma samples as a cut-off, the sensitivity of the assay and its specificity were 75 and 52%, respectively. Cross-resistance in vitro was frequently observed between standard drugs but not between standard drugs and Taxol. Ten percent of the specimens showed an extreme resistance for at least 4 of 6 of the drugs investigated.

  7. The microculture-kinetic (MiCK) assay: the role of a drug-induced apoptosis assay in drug development and clinical care.

    Science.gov (United States)

    Bosserman, Linda; Prendergast, Franklyn; Herbst, Roy; Fleisher, Martin; Salom, Emery; Strickland, Steven; Raptis, Anastasios; Hallquist, Allan; Perree, Mathieu; Rajurkar, Swapnil; Karimi, Misagh; Rogers, Karl; Davidson, Dirk; Willis, Carl; Penalver, Manuel; Homesley, Howard; Burrell, Matthew; Garrett, Audrey; Rutledge, James; Chernick, Michael; Presant, Cary A

    2012-08-15

    A drug-induced apoptosis assay, termed the microculture-kinetic (MiCK) assay, has been developed. Blinded clinical trials have shown higher response rates and longer survival in groups of patients with acute myelocytic leukemia and epithelial ovarian cancer who have been treated with drugs that show high apoptosis in the MiCK assay. Unblinded clinical trials in multiple tumor types have shown that the assay will be used frequently by clinicians to determine treatment, and when used, results in higher response rates, longer times to relapse, and longer survivals. Model economic analyses suggest possible cost savings in clinical use based on increased generic drug use and single-agent substitution for combination therapies. Two initial studies with drugs in development are promising. The assay may help reduce costs and speed time to drug approval. Correlative studies with molecular biomarkers are planned. This assay may have a role both in personalized clinical therapy and in more efficient drug development. ©2012 AACR.

  8. Evaluation of the larval migration inhibition assay for detecting macrocyclic lactone resistance in Dirofilaria immitis.

    Science.gov (United States)

    Evans, Christopher C; Moorhead, Andrew R; Storey, Bobby E; Blagburn, Byron L; Wolstenholme, Adrian J; Kaplan, Ray M

    2017-11-15

    Anthelmintics of the macrocyclic lactone (ML) drug class are widely used as preventives against the canine heartworm (Dirofilaria immitis). Over the past several years, however, reports of ML lack of efficacy (LOE) have emerged, in which dogs develop mature heartworm infection despite the administration of monthly prophylactics. More recently, isolates from LOE cases have been used to infect laboratory dogs and the resistant phenotype has been confirmed by the establishment of adult worms in the face of ML treatment at normally preventive dosages. Testing for and monitoring resistance in D. immitis requires a validated biological or molecular diagnostic assay. In this study, we assessed a larval migration inhibition assay (LMIA) that we previously optimized for use with D. immitis third-stage larvae (L 3 ). We used this assay to measure the in vitro ML susceptibilities of a known-susceptible laboratory strain of D. immitis and three highly suspected ML-resistant isolates originating from three separate LOE cases; progeny from two of these isolates have been confirmed ML-resistant by treatment of an infected dog in a controlled setting. A nonlinear regression model was fit to the dose-response data, from which IC 50 values were calculated. The D. immitis LMIA yielded consistent and reproducible dose-response data; however, no statistically significant differences in drug susceptibility were observed between control and LOE parasites. Additionally, the drug concentrations needed to paralyze the L 3 were much higher than those third- and fourth-stage larvae would experience in vivo. IC 50 values ranged from 1.57 to 5.56μM (p≥0.19). These data could suggest that ML resistance in this parasite is not mediated through a reduced susceptibility of L 3 to the paralytic effects of ML drugs, and therefore motility-based assays are likely not appropriate for measuring the effects of MLs against D. immitis in this target stage. Published by Elsevier B.V.

  9. [A novel chemo-resistant gene MSX2 discovered by establishment of two pancreatic cancer drug resistant cell lines JF305/CDDP and PANC-1/GEM].

    Science.gov (United States)

    Yuan, W; Sui, C G; Ma, X; Ma, J

    2018-05-23

    Objective: To explore new multidrug resistant genes of pancreatic cancer by establishment and characterization of chemo-resistant cell lines. Methods: The cisplatin-resistant cell line JF305/CDDP and the gemcitabine-resistant cell line PANC-1/GEM were induced by high-dose intermittent treatment. CCK-8 assay was used to detect the 50% inhibiting concentration (IC(50)), drug resistance index (R), cross-resistance, and growth difference of different cells. The changes of cell cycle and migration ability of drug-resistant cells were determined by flow cytometry and transwell assay, respectively. And then real-time fluorescence quantitative PCR was used to detect the expression of multidrug resistance-related genes. Results: The drug resistance indexes of JF305/CDDP and PANC-1/GEM were 15.3 and 27.31, respectively, and there was cross-resistance. Compared with the parental cells, the proliferation rate of JF305/CDDP was decreased by 40% on the fourth day ( P PANC-1 cells upregulated MRP2 level ( P PANC-1/GEM, were successfully established. MSX2 might be a new drug resistance related gene in pancreatic cancer cells by up-regulation of MRP2 expression.

  10. PCR (Polymerase Chain Reaction) Assay On Antibiotics Resistant Clinical Isolates Of Mycobacterium tuberculosis

    International Nuclear Information System (INIS)

    R, Maria Lina; S, Dadang; Suhadi, F.

    2000-01-01

    To detect to DNA of 9 drug-resistant isolates of m. tuberculosis such as isoniazid, streptomycin, isoniazid + streptomycin and isoniazid + rifampisin- resistant isolates, the DNA amplification by using PCR assay was carried out after lysing the bacterial cells. Two primer pairs for amplification used were Pt8 and Pt9 and Pt3 and Pt6. The amplified DNA taeget of 8 drug-resistant isolates and 1 drug-resistant isolate by means Pt8 8 Pt9 primer, gave the positive and negative result, respectively. Presence of amplified DNA target fragmens/bands on agarose gel, showed the positive result and vice verse. PCR process by using Pt3 and Pt6 primer revealed the positive results on 2 drug-resistant islates, whereas there was no amplified DNA bands from the other 7 isolates. DNA amplification by using either Pt8 and Pt9 or Pt3 and Pt6 primers occurred on H sub.37Rv strain DNA. Size of the amplified DNA products with Pt8 and Pt9 and Pt3 and Pt6 primers were 541 bp and 188 bp, respectively

  11. Simplified Paper Format for Detecting HIV Drug Resistance in Clinical Specimens by Oligonucleotide Ligation

    Science.gov (United States)

    Panpradist, Nuttada; Beck, Ingrid A.; Chung, Michael H.; Kiarie, James N.; Frenkel, Lisa M.; Lutz, Barry R.

    2016-01-01

    Human immunodeficiency virus (HIV) is a chronic infection that can be managed by antiretroviral treatment (ART). However, periods of suboptimal viral suppression during lifelong ART can select for HIV drug resistant (DR) variants. Transmission of drug resistant virus can lessen or abrogate ART efficacy. Therefore, testing of individuals for drug resistance prior to initiation of treatment is recommended to ensure effective ART. Sensitive and inexpensive HIV genotyping methods are needed in low-resource settings where most HIV infections occur. The oligonucleotide ligation assay (OLA) is a sensitive point mutation assay for detection of drug resistance mutations in HIV pol. The current OLA involves four main steps from sample to analysis: (1) lysis and/or nucleic acid extraction, (2) amplification of HIV RNA or DNA, (3) ligation of oligonucleotide probes designed to detect single nucleotide mutations that confer HIV drug resistance, and (4) analysis via oligonucleotide surface capture, denaturation, and detection (CDD). The relative complexity of these steps has limited its adoption in resource-limited laboratories. Here we describe a simplification of the 2.5-hour plate-format CDD to a 45-minute paper-format CDD that eliminates the need for a plate reader. Analysis of mutations at four HIV-1 DR codons (K103N, Y181C, M184V, and G190A) in 26 blood specimens showed a strong correlation of the ratios of mutant signal to total signal between the paper CDD and the plate CDD. The assay described makes the OLA easier to perform in low resource laboratories. PMID:26751207

  12. Drug resistance detection and mutation patterns of multidrug resistant tuberculosis strains from children in Delhi

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    Jyoti Arora

    2017-06-01

    Full Text Available A total of 312 sputum samples from pediatric patients presumptive of multidrug resistant tuberculosis were tested for the detection of drug resistance using the GenoTypeMTBDRplus assay. A total of 193 (61.8% patients were smear positive and 119 (38.1% were smear negative by Ziehl–Neelsen staining. Line probe assay (LPA was performed for 208 samples/cultures (193 smear positive samples and 15 cultures from smear negative samples. Valid results were obtained from 198 tests. Of these, 125/198 (63.1% were sensitive to both rifampicin (RIF and isoniazid (INH. 73/198 (36.9% were resistant to at least INH/RIF, out of which 49 (24.7% were resistant to both INH and RIF (multidrug resistant. Children with tuberculosis are often infected by someone close to them, so strengthening of contact tracing in the program may help in early diagnosis to identify additional cases within the household. There is a need to evaluate newer diagnostic assays which have a high sensitivity in the case of smear negative samples, additional samples other than sputum among young children not able to expectorate, and also to fill the gap between estimated and reported cases under the program.

  13. Evaluation of genotype MTBDRplus assay for rapid detection of isoniazid and rifampicin resistance in Mycobacterium tuberculosis clinical isolates from Pakistan

    Directory of Open Access Journals (Sweden)

    Hasnain Javed

    2016-01-01

    Conclusions: As evidenced in this study, the major concern with the GenoType MTBDRplus assay were false negative results. In comparison to conventional drug susceptibility testing, the assay was unable to detect 30 (30/100; 30% strains resistant to INH and 23 (23/100; 23% strains resistant to RMP. The GenoType MTBDRplus failed to identify 38 MDR (38/100; 38% strains. Resistance in those strains probably originate from mutations in other codons and/or genes than those covered by the test. For detecting INH and RMP resistance in TB cases, especially in high TB incidence countries, such as Pakistan, molecular approaches should still be a complement rather than areplacement to conventional drug susceptibility testing.

  14. An FDA-Drug Library Screen for Compounds with Bioactivities against Meticillin-Resistant Staphylococcus aureus (MRSA

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    Qiu Ying Lau

    2015-10-01

    Full Text Available The lack of new antibacterial drugs entering the market and their misuse have resulted in the emergence of drug-resistant bacteria, posing a major health crisis worldwide. In particular, meticillin-resistant Staphylococcus aureus (MRSA, a pathogen responsible for numerous human infections, has become endemic in hospitals worldwide. Drug repurposing, the finding of new therapeutic indications for approved drugs, is deemed a plausible solution to accelerate drug discovery and development in this area. Towards this end, we screened 1163 drugs approved by the Food and Drug Administration (FDA for bioactivities against MRSA in a 10 μM single-point assay. After excluding known antibiotics and antiseptics, six compounds were identified and their MICs were determined against a panel of clinical MRSA strains. A toxicity assay using human keratinocytes was also conducted to gauge their potential for repurposing as topical agents for treating MRSA skin infections.

  15. Phenotypic and genotypic analysis of anti-tuberculosis drug resistance in Mycobacterium tuberculosis isolates in Myanmar.

    Science.gov (United States)

    Aung, Wah Wah; Ei, Phyu Win; Nyunt, Wint Wint; Swe, Thyn Lei; Lwin, Thandar; Htwe, Mi Mi; Kim, Kyung Jun; Lee, Jong Seok; Kim, Chang Ki; Cho, Sang Nae; Song, Sun Dae; Chang, Chulhun L

    2015-09-01

    Tuberculosis (TB) is one of the most serious health problems in Myanmar. Because TB drug resistance is associated with genetic mutation(s) relevant to responses to each drug, genotypic methods for detecting these mutations have been proposed to overcome the limitations of classic phenotypic drug susceptibility testing (DST). We explored the current estimates of drug-resistant TB and evaluated the usefulness of genotypic DST in Myanmar. We determined the drug susceptibility of Mycobacterium tuberculosis isolated from sputum smear-positive patients with newly diagnosed pulmonary TB at two main TB centers in Myanmar during 2013 by using conventional phenotypic DST and the GenoType MTBDRplus assay (Hain Lifescience, Germany). Discrepant results were confirmed by sequencing the genes relevant to each type of resistance (rpoB for rifampicin; katG and inhA for isoniazid). Of 191 isolates, phenotypic DST showed that 27.7% (n=53) were resistant to at least one first-line drug and 20.9% (n=40) were resistant to two or more, including 18.3% (n=35) multidrug-resistant TB (MDR-TB) strains. Monoresistant strains accounted for 6.8% (n=13) of the samples. Genotypic assay of 189 isolates showed 17.5% (n=33) MDR-TB and 5.3% (n=10) isoniazid-monoresistant strains. Genotypic susceptibility results were 99.5% (n=188) concordant and agreed almost perfectly with phenotypic DST (kappa=0.99; 95% confidence interval 0.96-1.01). The results highlight the burden of TB drug resistance and prove the usefulness of the genotypic DST in Myanmar.

  16. In vitro drug sensitivity testing of tumor cells from patients with non-Hodgkin's lymphoma using the fluorometric microculture cytotoxicity assay.

    Science.gov (United States)

    Nygren, P; Hagberg, H; Glimelius, B; Sundström, C; Kristensen, J; Christiansen, I; Larsson, R

    1994-01-01

    Tumor cell drug sensitivity is an important determinant of chemotherapy response. Its measurement in vitro would aid in therapy individualization and new drug development. The fluorometric microculture cytotoxicity assay (FMCA), based on production by viable cells of fluorescent fluorescein after 3 days of culture, was used for cytotoxic drug sensitivity testing of 73 samples of tumor cells from patients with non-Hodgkin's lymphoma (NHL). The technical success rate was 92%, and FMCA data showed good correlation to the Disc assay. NHL samples were considerably more drug sensitive than were samples from in vivo resistant tumors. There was no obvious difference in drug sensitivity for high- vs. low-grade or untreated vs. previously treated low-grade NHL. For 26 patients, clinical outcome was correlated to in vitro response giving a sensitivity and specificity of 93 and 48%, respectively. Cross-resistance between standard drugs was frequent in vitro. Resistance modulators potentiated the effect of vincristine and doxorubicin in 10-29% of the samples, most frequently from previously treated patients. The FMCA seems to report clinically relevant drug sensitivity data for NHL, and thus it could serve as a tool for optimization of chemotherapy in the future.

  17. A locked nucleic acid (LNA-based real-time PCR assay for the rapid detection of multiple bacterial antibiotic resistance genes directly from positive blood culture.

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    Lingxiang Zhu

    Full Text Available Bacterial strains resistant to various antibiotic drugs are frequently encountered in clinical infections, and the rapid identification of drug-resistant strains is highly essential for clinical treatment. We developed a locked nucleic acid (LNA-based quantitative real-time PCR (LNA-qPCR method for the rapid detection of 13 antibiotic resistance genes and successfully used it to distinguish drug-resistant bacterial strains from positive blood culture samples. A sequence-specific primer-probe set was designed, and the specificity of the assays was assessed using 27 ATCC bacterial strains and 77 negative blood culture samples. No cross-reaction was identified among bacterial strains and in negative samples, indicating 100% specificity. The sensitivity of the assays was determined by spiking each bacterial strain into negative blood samples, and the detection limit was 1-10 colony forming units (CFU per reaction. The LNA-qPCR assays were first applied to 72 clinical bacterial isolates for the identification of known drug resistance genes, and the results were verified by the direct sequencing of PCR products. Finally, the LNA-qPCR assays were used for the detection in 47 positive blood culture samples, 19 of which (40.4% were positive for antibiotic resistance genes, showing 91.5% consistency with phenotypic susceptibility results. In conclusion, LNA-qPCR is a reliable method for the rapid detection of bacterial antibiotic resistance genes and can be used as a supplement to phenotypic susceptibility testing for the early detection of antimicrobial resistance to allow the selection of appropriate antimicrobial treatment and to prevent the spread of resistant isolates.

  18. Antimicrobial (Drug) Resistance

    Science.gov (United States)

    ... with facebook share with twitter share with linkedin Antimicrobial (Drug) Resistance Go to Information for Researchers ► Credit: ... and infectious diseases. Why Is the Study of Antimicrobial (Drug) Resistance a Priority for NIAID? Over time, ...

  19. Differentiation of drug and non-drug Cannabis using a single nucleotide polymorphism (SNP) assay.

    Science.gov (United States)

    Rotherham, D; Harbison, S A

    2011-04-15

    Cannabis sativa is both an illegal drug and a legitimate crop. The differentiation of illegal drug Cannabis from non-drug forms of Cannabis is relevant in the context of the growth of fibre and seed oil varieties of Cannabis for commercial purposes. This differentiation is currently determined based on the levels of tetrahydrocannabinol (THC) in adult plants. DNA based methods have the potential to assay Cannabis material unsuitable for analysis using conventional means including seeds, pollen and severely degraded material. The purpose of this research was to develop a single nucleotide polymorphism (SNP) assay for the differentiation of "drug" and "non-drug"Cannabis plants. An assay was developed based on four polymorphisms within a 399 bp fragment of the tetrahydrocannabinolic acid (THCA) synthase gene, utilising the snapshot multiplex kit. This SNP assay was tested on 94 Cannabis plants, which included 10 blind samples, and was able to differentiate between "drug" and "non-drug"Cannabis in all cases, while also differentiating between Cannabis and other species. Non-drug plants were found to be homozygous at the four sites assayed while drug Cannabis plants were either homozygous or heterozygous. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

  20. Multidrug-resistant tuberculosis: Rapid molecular detection with MTBDRplus® assay in clinical samples

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    Rita Macedo

    2009-05-01

    Full Text Available Nowadays, the greatest concern of tuberculosis control programmes is the appearance of multidrug-resistant tuberculosis and extensively drug-resistant tuberculosis. Rapid determination of drug resistance in clinical samples, with Mycobacterium tuberculosis complex (MTC, is the prerequisite for initiating effective chemotherapy, ensuring successful treatment of the patient and preventing further spread of drugresistant isolates.The aim of our study was to determine the sensitivity of the new MTBDRplus® assay in comparison to culture, identification and classic DST, directly from smear-positive clinical specimens.A total of 68 smear-positive sputum specimens were processed by both the classical mycobacteriological methods and the molecular assay, MTBDRplus®.MTBDRplus® assay allowed an accurate identification of MTC species by detection of the specific band in all samples, from which we also isolated and identified MTC strains by culture methods. In the samples from which we isolated susceptible strains (63.2%, wild type patterns were found using MTBDRplus® assay. The samples from which we isolated resistant strains (36.8% showed specific mutations associated with the correspondent resistant phenotype.Our study indicated that this assay allows rapid detection of resistance, always in agreement with classic methods. Resumo: Uma das principais problematicas no controlo da tuberculose e o aparecimento de casos de tuberculose multirresistente (TB-MR e tuberculose extensivamente resistente (TB-XDR. A deteccao precoce da resistencia a farmacos, directamente a partir de amostras respiratorias, e essencial para que se assegure o tratamento atempado, adequado e eficaz da tuberculose, bem como para prevenir a disseminacao destes casos de especial gravidade.O nosso objectivo foi avaliar a sensibilidade e comparar os resultados obtidos com um metodo de genetica molecular disponivel comercialmente – MTBDRplus® – e o isolamento

  1. Drug-resistant tuberculosis in Sindh

    International Nuclear Information System (INIS)

    Almani, S.A.; Memon, N.M.; Qureshi, A.F.

    2002-01-01

    Objective: To assess the prevalence of primary and secondary drug resistance amongst the clinical isolates of M.tuberculosis, to identify risk factors and how to overcome this problem. Design: A case series of 50 indoor patients with sputum smear-positive pulmonary tuberculosis. Place and duration of Study: Department of Medicine, Liaquat University of Medical and Health Sciences Jamshoro, Sindh, (Pakistan) from January 1999 to December 2000. Patients and methods: Four first line anti-tuberculous drugs rifampicine, ethambutol and streptomycin were tested for sensitivity pattern. Results: Twelve (26.66%) were sensitive to all four drugs, 12(26.66%) were resistant to one drug, 14 (31.11%) were resistant to two drugs, 2 (4.44%) were resistant to three drugs, and 5(11.11%) were resistant to all four drugs. Resistance to isoniazid was the most common in 27 cases (60%) with primary resistance in 6(13.33%) and secondary resistance in 21(46.66%), followed by resistance to streptomycin in 17 cases (37.77%) with primary resistance in 5(11.11%) and secondary resistance in 12 (26.66%). Resistance to ethambutol in 10 cases (22.22%) and rifampicine in 11 (24.44%) and all cases were secondary. Similarly multi-drugs resistance (MRD) TB was found in 11(24.44%) isolates. Conclusion: This study showed high prevalence of drug resistance among clinical isolates of M. tuberculosis. Their is a need to establish centers at number of places with adequate facilities for susceptibility testing so that the resistant pattern could be ascertained and treatment regimens tailored accordingly. (author)

  2. Riboflavin-mediated photosensitization of Vinca alkaloids distorts drug sensitivity assays.

    Science.gov (United States)

    Granzow, C; Kopun, M; Kröber, T

    1995-11-01

    Poor reproducibility of cytotoxicity tests with Vinca alkaloids has frequently been reported. A commonly presumed light sensitivity of the drugs could not be confirmed. However, we found that they are photosensitized by riboflavin (vitamin B2), an obligatory component of cell culture media. Light of wavelengths below 500 nm triggered rapid photoreactions of riboflavin with vinblastine, vincristine, and vindesine in aqueous solutions. The photoreactions altered the absorption spectra of these alkaloids and yielded degradation products that could be separated by TLC. In cell cultures, both immediate and persisting, riboflavin-mediated photoreactivity could be distinguished. They preclude reliable determinations of sensitivity and resistance to Vinca alkaloids, as exemplified on chemosensitive and multidrug-resistant mouse ascites cells. In experiments involving photosensitization, the 50% inhibitory concentration values of sensitive and resistant cells were overlapping and fluctuated in the ranges from 3 to 30 nM and 15 to 360 nM vinblastine, respectively. Corresponding values from series of experiments protected from photosensitization were 1.02 +/- 0.22 nM and 18.5 +/- 3.42 nM. Hence, riboflavin-mediated photoreactions must be fully prevented in assays of cellular drug sensitivity. Procedures for eliminating immediate as well as persisting photoreactivity were established.

  3. Withanolide D Exhibits Similar Cytostatic Effect in Drug-Resistant and Drug-Sensitive Multiple Myeloma Cells

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    Mark E. Issa

    2017-09-01

    Full Text Available In spite of recent therapeutic advances, multiple myeloma (MM remains a malignancy with very low curability. This has been partly attributed to the existence of a drug-resistant subpopulation known as cancer stem cells (CSCs. MM-CSCs are equipped with the necessary tools that render them highly resistant to virtually all conventional therapies. In this study, the growth inhibitory effects of withanolide D (WND, a steroidal lactone isolated from Withania somnifera, on drug-sensitive tumoral plasma cells and drug-resistant MM cells have been investigated. In MTT/XTT assays, WND exhibited similar cytostatic effects between drug-resistant and drug-sensitive cell lines in the nM range. WND also induced cell death and apoptosis in MM-CSCs and RPMI 8226 cells, as examined by the calcein/ethidium homodimer and annexin V/propidium iodide stainings, respectively. To determine whether P-glycoprotein (P-gp efflux affected the cytostatic activity of WND, P-gp was inhibited with verapamil and results indicated that the WND cytostatic effect in MM-CSCs was independent of P-gp efflux. Furthermore, WND did not increase the accumulation of the fluorescent P-gp substrate rhodamine 123 in MM-CSCs, suggesting that WND may not inhibit P-gp at the tested relevant doses. Therefore, the WND-induced cytostatic effect may be independent of P-gp efflux. These findings warrant further investigation of WND in MM-CSC animal models.

  4. Whole animal automated platform for drug discovery against multi-drug resistant Staphylococcus aureus.

    Directory of Open Access Journals (Sweden)

    Rajmohan Rajamuthiah

    Full Text Available Staphylococcus aureus, the leading cause of hospital-acquired infections in the United States, is also pathogenic to the model nematode Caenorhabditis elegans. The C. elegans-S. aureus infection model was previously carried out on solid agar plates where the bacteriovorous C. elegans feeds on a lawn of S. aureus. However, agar-based assays are not amenable to large scale screens for antibacterial compounds. We have developed a high throughput liquid screening assay that uses robotic instrumentation to dispense a precise amount of methicillin resistant S. aureus (MRSA and worms in 384-well assay plates, followed by automated microscopy and image analysis. In validation of the liquid assay, an MRSA cell wall defective mutant, MW2ΔtarO, which is attenuated for killing in the agar-based assay, was found to be less virulent in the liquid assay. This robust assay with a Z'-factor consistently greater than 0.5 was utilized to screen the Biomol 4 compound library consisting of 640 small molecules with well characterized bioactivities. As proof of principle, 27 of the 30 clinically used antibiotics present in the library conferred increased C. elegans survival and were identified as hits in the screen. Surprisingly, the antihelminthic drug closantel was also identified as a hit in the screen. In further studies, we confirmed the anti-staphylococcal activity of closantel against vancomycin-resistant S. aureus isolates and other Gram-positive bacteria. The liquid C. elegans-S. aureus assay described here allows screening for anti-staphylococcal compounds that are not toxic to the host.

  5. Whole animal automated platform for drug discovery against multi-drug resistant Staphylococcus aureus.

    Science.gov (United States)

    Rajamuthiah, Rajmohan; Fuchs, Beth Burgwyn; Jayamani, Elamparithi; Kim, Younghoon; Larkins-Ford, Jonah; Conery, Annie; Ausubel, Frederick M; Mylonakis, Eleftherios

    2014-01-01

    Staphylococcus aureus, the leading cause of hospital-acquired infections in the United States, is also pathogenic to the model nematode Caenorhabditis elegans. The C. elegans-S. aureus infection model was previously carried out on solid agar plates where the bacteriovorous C. elegans feeds on a lawn of S. aureus. However, agar-based assays are not amenable to large scale screens for antibacterial compounds. We have developed a high throughput liquid screening assay that uses robotic instrumentation to dispense a precise amount of methicillin resistant S. aureus (MRSA) and worms in 384-well assay plates, followed by automated microscopy and image analysis. In validation of the liquid assay, an MRSA cell wall defective mutant, MW2ΔtarO, which is attenuated for killing in the agar-based assay, was found to be less virulent in the liquid assay. This robust assay with a Z'-factor consistently greater than 0.5 was utilized to screen the Biomol 4 compound library consisting of 640 small molecules with well characterized bioactivities. As proof of principle, 27 of the 30 clinically used antibiotics present in the library conferred increased C. elegans survival and were identified as hits in the screen. Surprisingly, the antihelminthic drug closantel was also identified as a hit in the screen. In further studies, we confirmed the anti-staphylococcal activity of closantel against vancomycin-resistant S. aureus isolates and other Gram-positive bacteria. The liquid C. elegans-S. aureus assay described here allows screening for anti-staphylococcal compounds that are not toxic to the host.

  6. Drug resistance in Mexico: results from the National Survey on Drug-Resistant Tuberculosis.

    Science.gov (United States)

    Bojorquez-Chapela, I; Bäcker, C E; Orejel, I; López, A; Díaz-Quiñonez, A; Hernández-Serrato, M I; Balandrano, S; Romero, M; Téllez-Rojo Solís, M M; Castellanos, M; Alpuche, C; Hernández-Ávila, M; López-Gatell, H

    2013-04-01

    To present estimations obtained from a population-level survey conducted in Mexico of prevalence rates of mono-, poly- and multidrug-resistant strains among newly diagnosed cases of pulmonary tuberculosis (TB), as well as the main factors associated with multidrug resistance (combined resistance to isoniazid and rifampicin). Study data came from the National Survey on TB Drug Resistance (ENTB-2008), a nationally representative survey conducted during 2008-2009 in nine states with a stratified cluster sampling design. Samples were obtained for all newly diagnosed cases of pulmonary TB in selected sites. Drug susceptibility testing (DST) was performed for anti-tuberculosis drugs. DST results were obtained for 75% of the cases. Of these, 82.2% (95%CI 79.5-84.7) were susceptible to all drugs. The prevalence of multidrug-resistant TB (MDR-TB) was estimated at 2.8% (95%CI 1.9-4.0). MDR-TB was associated with previous treatment (OR 3.3, 95%CI 1.1-9.4). The prevalence of drug resistance is relatively low in Mexico. ENTB-2008 can be used as a baseline for future follow-up of drug resistance.

  7. Development and customization of a color-coded microbeads-based assay for drug resistance in HIV-1 reverse transcriptase.

    Science.gov (United States)

    Gu, Lijun; Kawana-Tachikawa, Ai; Shiino, Teiichiro; Nakamura, Hitomi; Koga, Michiko; Kikuchi, Tadashi; Adachi, Eisuke; Koibuchi, Tomohiko; Ishida, Takaomi; Gao, George F; Matsushita, Masaki; Sugiura, Wataru; Iwamoto, Aikichi; Hosoya, Noriaki

    2014-01-01

    Drug resistance (DR) of HIV-1 can be examined genotypically or phenotypically. Although sequencing is the gold standard of the genotypic resistance testing (GRT), high-throughput GRT targeted to the codons responsible for DR may be more appropriate for epidemiological studies and public health research. We used a Japanese database to design and synthesize sequence-specific oligonucleotide probes (SSOP) for the detection of wild-type sequences and 6 DR mutations in the clade B HIV-1 reverse transcriptase region. We coupled SSOP to microbeads of the Luminex 100 xMAP system and developed a GRT based on the polymerase chain reaction (PCR)-SSOP-Luminex method. Sixteen oligoprobes for discriminating DR mutations from wild-type sequences at 6 loci were designed and synthesized, and their sensitivity and specificity were confirmed using isogenic plasmids. The PCR-SSOP-Luminex DR assay was then compared to direct sequencing using 74 plasma specimens from treatment-naïve patients or those on failing treatment. In the majority of specimens, the results of the PCR-SSOP-Luminex DR assay were concordant with sequencing results: 62/74 (83.8%) for M41, 43/74 (58.1%) for K65, 70/74 (94.6%) for K70, 55/73 (75.3%) for K103, 63/73 (86.3%) for M184 and 68/73 (93.2%) for T215. There were a number of specimens without any positive signals, especially for K65. The nucleotide position of A2723G, A2747G and C2750T were frequent polymorphisms for the wild-type amino acids K65, K66 and D67, respectively, and 14 specimens had the D67N mutation encoded by G2748A. We synthesized 14 additional oligoprobes for K65, and the sensitivity for K65 loci improved from 43/74 (58.1%) to 68/74 (91.9%). We developed a rapid high-throughput assay for clade B HIV-1 DR mutations, which could be customized by synthesizing oligoprobes suitable for the circulating viruses. The assay could be a useful tool especially for public health research in both resource-rich and resource-limited settings.

  8. Analytical and clinical performance characteristics of the Abbott RealTime MTB RIF/INH Resistance, an assay for the detection of rifampicin and isoniazid resistant Mycobacterium tuberculosis in pulmonary specimens.

    Science.gov (United States)

    Kostera, Joshua; Leckie, Gregor; Tang, Ning; Lampinen, John; Szostak, Magdalena; Abravaya, Klara; Wang, Hong

    2016-12-01

    Clinical management of drug-resistant tuberculosis patients continues to present significant challenges to global health. To tackle these challenges, the Abbott RealTime MTB RIF/INH Resistance assay was developed to accelerate the diagnosis of rifampicin and/or isoniazid resistant tuberculosis to within a day. This article summarizes the performance of the Abbott RealTime MTB RIF/INH Resistance assay; including reliability, analytical sensitivity, and clinical sensitivity/specificity as compared to Cepheid GeneXpert MTB/RIF version 1.0 and Hain MTBDRplus version 2.0. The limit of detection (LOD) of the Abbott RealTime MTB RIF/INH Resistance assay was determined to be 32 colony forming units/milliliter (cfu/mL) using the Mycobacterium tuberculosis (MTB) strain H37Rv cell line. For rifampicin resistance detection, the Abbott RealTime MTB RIF/INH Resistance assay demonstrated statistically equivalent clinical sensitivity and specificity as compared to Cepheid GeneXpert MTB/RIF. For isoniazid resistance detection, the assay demonstrated statistically equivalent clinical sensitivity and specificity as compared to Hain MTBDRplus. The performance data presented herein demonstrate that the Abbott RealTime MTB RIF/INH Resistance assay is a sensitive, robust, and reliable test for realtime simultaneous detection of first line anti-tuberculosis antibiotics rifampicin and isoniazid in patient specimens. Copyright © 2016 The Author. Published by Elsevier Ltd.. All rights reserved.

  9. Real-time polymerase chain reaction assay for the rapid detection and characterization of chloroquine-resistant Plasmodium falciparum malaria in returned travelers.

    Science.gov (United States)

    Farcas, Gabriella A; Soeller, Rainer; Zhong, Kathleen; Zahirieh, Alireza; Kain, Kevin C

    2006-03-01

    Imported drug-resistant malaria is a growing problem in industrialized countries. Rapid and accurate diagnosis is essential to prevent malaria-associated mortality in returned travelers. However, outside of a limited number of specialized centers, the microscopic diagnosis of malaria is slow, unreliable, and provides little information about drug resistance. Molecular diagnostics have the potential to overcome these limitations. We developed and evaluated a rapid, real-time polymerase chain reaction (PCR) assay to detect Plasmodium falciparum malaria and chloroquine (CQ)-resistance determinants in returned travelers who are febrile. A real-time PCR assay based on detection of the K76T mutation in PfCRT (K76T) of P. falciparum was developed on a LightCycler platform (Roche). The performance characteristics of the real-time assay were compared with those of the nested PCR-restriction fragment-length polymorphism (RFLP) and the sequence analyses of samples obtained from 200 febrile returned travelers, who included 125 infected with P. falciparum (48 of whom were infected CQ-susceptible [K76] and 77 of whom were CQ-resistant [T76] P. falciparum), 22 infected with Plasmodium vivax, 10 infected with Plasmodium ovale, 3 infected with Plasmodium malariae malaria, and 40 infected with other febrile syndromes. All patient samples were coded, and all analyses were performed blindly. The real-time PCR assay detected multiple pfcrt haplotypes associated with CQ resistance in geographically diverse malaria isolates acquired by travelers. Compared with nested-PCR RFLP (the reference standard), the real-time assay was 100% sensitive and 96.2% specific for detection of the P. falciparum K76T mutation. This assay is rapid, sensitive, and specific for the detection and characterization of CQ-resistant P. falciparum malaria in returned travelers. This assay is automated, standardized, and suitable for routine use in clinical diagnostic laboratories.

  10. Whole genome sequencing-based characterization of extensively drug resistant (XDR) strains of Mycobacterium tuberculosis from Pakistan

    KAUST Repository

    Hasan, Zahra; Ali, Asho; McNerney, Ruth; Mallard, Kim; Hill-Cawthorne, Grant A.; Coll, Francesc; Nair, Mridul; Pain, Arnab; Clark, Taane G.; Hasan, Rumina

    2015-01-01

    Objectives: The global increase in drug resistance in Mycobacterium tuberculosis (MTB) strains increases the focus on improved molecular diagnostics for MTB. Extensively drug-resistant (XDR) - TB is caused by MTB strains resistant to rifampicin, isoniazid, fluoroquinolone and aminoglycoside antibiotics. Resistance to anti-tuberculous drugs has been associated with single nucleotide polymorphisms (SNPs), in particular MTB genes. However, there is regional variation between MTB lineages and the SNPs associated with resistance. Therefore, there is a need to identify common resistance conferring SNPs so that effective molecular-based diagnostic tests for MTB can be developed. This study investigated used whole genome sequencing (WGS) to characterize 37 XDR MTB isolates from Pakistan and investigated SNPs related to drug resistance. Methods: XDR-TB strains were selected. DNA was extracted from MTB strains, and samples underwent WGS with 76-base-paired end fragment sizes using Illumina paired end HiSeq2000 technology. Raw sequence data were mapped uniquely to H37Rv reference genome. The mappings allowed SNPs and small indels to be called using SAMtools/BCFtools. Results: This study found that in all XDR strains, rifampicin resistance was attributable to SNPs in the rpoB RDR region. Isoniazid resistance-associated mutations were primarily related to katG codon 315 followed by inhA S94A. Fluoroquinolone resistance was attributable to gyrA 91-94 codons in most strains, while one did not have SNPs in either gyrA or gyrB. Aminoglycoside resistance was mostly associated with SNPs in rrs, except in 6 strains. Ethambutol resistant strains had embB codon 306 mutations, but many strains did not have this present. The SNPs were compared with those present in commercial assays such as LiPA Hain MDRTBsl, and the sensitivity of the assays for these strains was evaluated. Conclusions: If common drug resistance associated with SNPs evaluated the concordance between phenotypic and

  11. Whole genome sequencing-based characterization of extensively drug resistant (XDR) strains of Mycobacterium tuberculosis from Pakistan

    KAUST Repository

    Hasan, Zahra

    2015-03-01

    Objectives: The global increase in drug resistance in Mycobacterium tuberculosis (MTB) strains increases the focus on improved molecular diagnostics for MTB. Extensively drug-resistant (XDR) - TB is caused by MTB strains resistant to rifampicin, isoniazid, fluoroquinolone and aminoglycoside antibiotics. Resistance to anti-tuberculous drugs has been associated with single nucleotide polymorphisms (SNPs), in particular MTB genes. However, there is regional variation between MTB lineages and the SNPs associated with resistance. Therefore, there is a need to identify common resistance conferring SNPs so that effective molecular-based diagnostic tests for MTB can be developed. This study investigated used whole genome sequencing (WGS) to characterize 37 XDR MTB isolates from Pakistan and investigated SNPs related to drug resistance. Methods: XDR-TB strains were selected. DNA was extracted from MTB strains, and samples underwent WGS with 76-base-paired end fragment sizes using Illumina paired end HiSeq2000 technology. Raw sequence data were mapped uniquely to H37Rv reference genome. The mappings allowed SNPs and small indels to be called using SAMtools/BCFtools. Results: This study found that in all XDR strains, rifampicin resistance was attributable to SNPs in the rpoB RDR region. Isoniazid resistance-associated mutations were primarily related to katG codon 315 followed by inhA S94A. Fluoroquinolone resistance was attributable to gyrA 91-94 codons in most strains, while one did not have SNPs in either gyrA or gyrB. Aminoglycoside resistance was mostly associated with SNPs in rrs, except in 6 strains. Ethambutol resistant strains had embB codon 306 mutations, but many strains did not have this present. The SNPs were compared with those present in commercial assays such as LiPA Hain MDRTBsl, and the sensitivity of the assays for these strains was evaluated. Conclusions: If common drug resistance associated with SNPs evaluated the concordance between phenotypic and

  12. A real-time PCR antibiogram for drug-resistant sepsis.

    Directory of Open Access Journals (Sweden)

    John R Waldeisen

    Full Text Available Current molecular diagnostic techniques for susceptibility testing of septicemia rely on genotyping for the presence of known resistance cassettes. This technique is intrinsically vulnerable due to the inability to detect newly emergent resistance genes. Traditional phenotypic susceptibility testing has always been a superior method to assay for resistance; however, relying on the multi-day growth period to determine which antimicrobial to administer jeopardizes patient survival. These factors have resulted in the widespread and deleterious use of broad-spectrum antimicrobials. The real-time PCR antibiogram, described herein, combines universal phenotypic susceptibility testing with the rapid diagnostic capabilities of PCR. We have developed a procedure that determines susceptibility by monitoring pathogenic load with the highly conserved 16S rRNA gene in blood samples exposed to different antimicrobial drugs. The optimized protocol removes heme and human background DNA from blood, which allows standard real-time PCR detection systems to be employed with high sensitivity (<100 CFU/mL. Three strains of E. coli, two of which were antimicrobial resistant, were spiked into whole blood and exposed to three different antibiotics. After real-time PCR-based determination of pathogenic load, a ΔC(t<3.0 between untreated and treated samples was found to indicate antimicrobial resistance (P<0.01. Minimum inhibitory concentration was determined for susceptible bacteria and pan-bacterial detection was demonstrated with 3 gram-negative and 2 gram-positive bacteria. Species identification was performed via analysis of the hypervariable amplicons. In summary, we have developed a universal diagnostic phenotyping technique that assays for the susceptibility of drug-resistant septicemia with the speed of PCR. The real-time PCR antibiogram achieves detection, susceptibility testing, minimum inhibitory concentration determination, and identification in less than 24

  13. Preventing drug resistance in severe influenza

    Science.gov (United States)

    Dobrovolny, Hana; Deecke, Lucas

    2015-03-01

    Severe, long-lasting influenza infections are often caused by new strains of influenza. The long duration of these infections leads to an increased opportunity for the emergence of drug resistant mutants. This is particularly problematic for new strains of influenza since there is often no vaccine, so drug treatment is the first line of defense. One strategy for trying to minimize drug resistance is to apply periodic treatment. During treatment the wild-type virus decreases, but resistant virus might increase; when there is no treatment, wild-type virus will hopefully out-compete the resistant virus, driving down the number of resistant virus. We combine a mathematical model of severe influenza with a model of drug resistance to study emergence of drug resistance during a long-lasting infection. We apply periodic treatment with two types of antivirals: neuraminidase inhibitors, which block release of virions; and adamantanes, which block replication of virions. We compare the efficacy of the two drugs in reducing emergence of drug resistant mutants and examine the effect of treatment frequency on the emergence of drug resistant mutants.

  14. Role of drug transporters and drug accumulation in the temporal acquisition of drug resistance

    International Nuclear Information System (INIS)

    Hembruff, Stacey L; Laberge, Monique L; Villeneuve, David J; Guo, Baoqing; Veitch, Zachary; Cecchetto, Melanie; Parissenti, Amadeo M

    2008-01-01

    Anthracyclines and taxanes are commonly used in the treatment of breast cancer. However, tumor resistance to these drugs often develops, possibly due to overexpression of drug transporters. It remains unclear whether drug resistance in vitro occurs at clinically relevant doses of chemotherapy drugs and whether both the onset and magnitude of drug resistance can be temporally and causally correlated with the enhanced expression and activity of specific drug transporters. To address these issues, MCF-7 cells were selected for survival in increasing concentrations of doxorubicin (MCF-7 DOX-2 ), epirubicin (MCF-7 EPI ), paclitaxel (MCF-7 TAX-2 ), or docetaxel (MCF-7 TXT ). During selection cells were assessed for drug sensitivity, drug uptake, and the expression of various drug transporters. In all cases, resistance was only achieved when selection reached a specific threshold dose, which was well within the clinical range. A reduction in drug uptake was temporally correlated with the acquisition of drug resistance for all cell lines, but further increases in drug resistance at doses above threshold were unrelated to changes in cellular drug uptake. Elevated expression of one or more drug transporters was seen at or above the threshold dose, but the identity, number, and temporal pattern of drug transporter induction varied with the drug used as selection agent. The pan drug transporter inhibitor cyclosporin A was able to partially or completely restore drug accumulation in the drug-resistant cell lines, but had only partial to no effect on drug sensitivity. The inability of cyclosporin A to restore drug sensitivity suggests the presence of additional mechanisms of drug resistance. This study indicates that drug resistance is achieved in breast tumour cells only upon exposure to concentrations of drug at or above a specific selection dose. While changes in drug accumulation and the expression of drug transporters does occur at the threshold dose, the magnitude of

  15. The role of the time-kill kinetics assay as part of a preclinical modeling framework for assessing the activity of anti-tuberculosis drugs.

    Science.gov (United States)

    Bax, Hannelore I; Bakker-Woudenberg, Irma A J M; de Vogel, Corné P; van der Meijden, Aart; Verbon, Annelies; de Steenwinkel, Jurriaan E M

    2017-07-01

    Novel treatment strategies for tuberculosis are urgently needed. Many different preclinical models assessing anti-tuberculosis drug activity are available, but it is yet unclear which combination of models is most predictive of clinical treatment efficacy. The aim of this study was to determine the role of our in vitro time kill-kinetics assay as an asset to a predictive preclinical modeling framework assessing anti-tuberculosis drug activity. The concentration- and time-dependent mycobacterial killing capacities of six anti-tuberculosis drugs were determined during exposure as single drugs or in dual, triple and quadruple combinations towards a Mycobacterium tuberculosis Beijing genotype strain and drug resistance was assessed. Streptomycin, rifampicin and isoniazid were most active against fast-growing M. tuberculosis. Isoniazid with rifampicin or high dose ethambutol were the only synergistic drug combinations. The addition of rifampicin or streptomycin to isoniazid prevented isoniazid resistance. In vitro ranking showed agreement with early bactericidal activity in tuberculosis patients for some but not all anti-tuberculosis drugs. The time-kill kinetics assay provides important information on the mycobacterial killing dynamics of anti-tuberculosis drugs during the early phase of drug exposure. As such, this assay is a valuable component of the preclinical modeling framework. Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. Emerging Technologies and Generic Assays for the Detection of Anti-Drug Antibodies

    Directory of Open Access Journals (Sweden)

    Michael A. Partridge

    2016-01-01

    Full Text Available Anti-drug antibodies induced by biologic therapeutics often impact drug pharmacokinetics, pharmacodynamics response, clinical efficacy, and patient safety. It is critical to assess the immunogenicity risk of potential biotherapeutics in producing neutralizing and nonneutralizing anti-drug antibodies, especially in clinical phases of drug development. Different assay methodologies have been used to detect all anti-drug antibodies, including ELISA, radioimmunoassay, surface plasmon resonance, and electrochemiluminescence-based technologies. The most commonly used method is a bridging assay, performed in an ELISA or on the Meso Scale Discovery platform. In this report, we aim to review the emerging new assay technologies that can complement or address challenges associated with the bridging assay format in screening and confirmation of ADAs. We also summarize generic anti-drug antibody assays that do not require drug-specific reagents for nonclinical studies. These generic assays significantly reduce assay development efforts and, therefore, shorten the assay readiness timeline.

  17. Drug-resistant spinal tuberculosis

    Directory of Open Access Journals (Sweden)

    Anil K Jain

    2018-01-01

    Full Text Available Drug-resistant spinal tuberculosis (TB is an emerging health problem in both developing and developed countries. In this review article, we aim to define management protocols for suspicion, diagnosis, and treatment of such patients. Spinal TB is a deep-seated paucibacillary lesion, and the demonstration of acid-fast bacilli on Ziehl-Neelsen staining is possible only in 10%–30% of cases. Drug resistance is suspected in patients showing the failure of clinicoradiological improvement or appearance of a fresh lesion of osteoarticular TB while on anti tubercular therapy (ATT for a minimum period of 5 months. The conventional culture of Mycobacterium tuberculosis remains the gold standard for both bacteriological diagnosis and drug sensitivity testing (DST; however, the high turn around time of 2–6 weeks for detection with added 3 weeks for DST is a major limitation. To overcome this problem, rapid culture methods and molecular methods have been introduced. From a public health perspective, reducing the period between diagnosis and treatment initiation has direct benefits for both the patient and the community. For all patients of drug-resistant spinal TB, a complete Drug-O-Gram should be prepared which includes details of all drugs, their doses, and duration. Patients with confirmed multidrug-resistant TB strains should receive a regimen with at least five effective drugs, including pyrazinamide and one injectable. Patients with resistance to additional antitubercular drugs should receive individualized ATT as per their DST results.

  18. Nucleic acid amplification of HIV-1 integrase sequence subtypes CRF01_AE and B for development of HIV anti-integrase drug resistance genotyping assay

    Science.gov (United States)

    Adlar, F. R.; Bela, B.

    2017-08-01

    To anticipate the potential use of anti-integrase drugs in Indonesia for treatment of HIV-1 infection, the development of a drug resistance genotyping assay for anti-integrase is crucial in identifying the genetic drug resistance profile of Indonesian HIV-1 strains. This experiment aimed to amplify a target region in the integrase gene of Indonesian HIV-1 subtypes CRF01_AE and B that contain genetic mutations known to confer resistance to anti-integrase drug. Eleven archived plasma samples from individuals living with HIV-1 were obtained from the Virology and Cancer Pathobiology Research Center for Health Service (VCPRC FKUI-RSCM) laboratory. One of the plasma samples contained HIV-1 subtype B, and the remaining plasma samples contained subtype CRF01_AE. The target regions for all samples were amplified through RT-PCR, with an annealing temperature of 55 °C, using the primer pair AE_POL 4086F and AE_POL 5232R that were designed by VCPRC FKUI-RSCM. The results of this experiment show that 18.2% (2/11) of the samples were successfully amplified using the one-step RT-PCR. While the primer pair was effective in amplifying the target region in the integrase gene sequence for subtype B (100%; 1/1), it had a low efficacy (10%, 1/10) for subtype CRF01_AE. In conclusion, the primer pair can be used to amplify the target region in Indonesian HIV-1 strain subtypes CRF01_AE and B. However, optimization of the PCR condition and an increased number of samples would help to determine an accurate representation of the efficacy of the primer pair.

  19. Performance of PCR-reverse blot hybridization assay for detection of rifampicin-resistant Mycobacterium leprae.

    Science.gov (United States)

    Wang, Hye-young; Kim, Hyunjung; Kim, Yeun; Bang, Hyeeun; Kim, Jong-Pill; Hwang, Joo Hwan; Cho, Sang-Nae; Kim, Tae Ue; Lee, Hyeyoung

    2015-10-01

    Drug resistance in Mycobacterium leprae is a significant problem in countries where leprosy is endemic. A sensitive, specific, and high-throughput reverse blot hybridization assay (REBA) for the detection of genotypic resistance to rifampicin (RIF) was designed and evaluated. It has been shown that resistance to RIF in M. leprae involves mutations in the rpoB gene encoding the -subunit of the RNA polymerase. The PCR-REBA simultaneously detects both 6 wild-type regions and 5 different mutations (507 AGC, 513 GTG, 516 TAT, 531 ATG, and 531 TTC) including the most prevalent mutations at positions 507 and 531. Thirty-one clinical isolates provided by Korea Institute of Hansen-s Disease were analyzed by PCR-REBA with RIF resistance of rpoB gene. As a result, missense mutations at codons 507 AGC and 531 ATG with 2-nucleotide substitutions were found in one sample, and a missense mutation at codon 516 TAT and ΔWT6 (deletion of 530-534) was found in another sample. These cases were confirmed by DNA sequence analysis. This rapid, simple, and highly sensitive assay provides a practical alternative to sequencing for genotypic evaluation of RIF resistance in M. leprae.

  20. Performance of an in-house human immunodeficiency virus type 1 genotyping system for assessment of drug resistance in Cuba.

    Science.gov (United States)

    Alemán, Yoan; Vinken, Lore; Kourí, Vivian; Pérez, Lissette; Álvarez, Alina; Abrahantes, Yeissel; Fonseca, Carlos; Pérez, Jorge; Correa, Consuelo; Soto, Yudira; Schrooten, Yoeri; Vandamme, Anne-Mieke; Van Laethem, Kristel

    2015-01-01

    As commercial human immunodeficiency virus type 1 drug resistance assays are expensive, they are not commonly used in resource-limited settings. Hence, a more affordable in-house procedure was set up taking into account the specific epidemiological and economic circumstances of Cuba. The performance characteristics of the in-house assay were evaluated using clinical samples with various subtypes and resistance patterns. The lower limit of amplification was determined on dilutions series of 20 clinical isolates and ranged from 84 to 529 RNA copies/mL. For the assessment of trueness, 14 clinical samples were analyzed and the ViroSeq HIV-1 Genotyping System v2.0 was used as the reference standard. The mean nucleotide sequence identity between the two assays was 98.7% ± 1.0. Additionally, 99.0% of the amino acids at drug resistance positions were identical. The sensitivity and specificity in detecting drug resistance mutations was respectively 94.1% and 99.5%. Only few discordances in drug resistance interpretation patterns were observed. The repeatability and reproducibility were evaluated using 10 clinical samples with 3 replicates per sample. The in-house test was very precise as nucleotide sequence identity among paired nucleotide sequences ranged from 98.7% to 99.9%. The acceptance criteria were met by the in-house test for all performance characteristics, demonstrating a high degree of accuracy. Subsequently, the applicability in routine clinical practice was evaluated on 380 plasma samples. The amplification success rate was 91% and good quality consensus sequences encoding the entire protease and the first 335 codons in reverse transcriptase could be obtained for 99% of the successful amplicons. The reagent cost per sample using the in-house procedure was around € 80 per genotyping attempt. Overall, the in-house assay provided good results, was feasible with equipment and reagents available in Cuba and was half as expensive as commercial assays.

  1. Nonradioactive heteroduplex tracking assay for the detection of minority-variant chloroquine-resistant Plasmodium falciparum in Madagascar

    Science.gov (United States)

    Juliano, Jonathan J; Randrianarivelojosia, Milijaona; Ramarosandratana, Benjamin; Ariey, Frédéric; Mwapasa, Victor; Meshnick, Steven R

    2009-01-01

    Background Strains of Plasmodium falciparum genetically resistant to chloroquine (CQ) due to the presence of pfcrt 76T appear to have been recently introduced to the island of Madagascar. The prevalence of such resistant genotypes is reported to be low (chloroquine resistant parasites were described in Malawian patients using an isotopic heteroduplex tracking assay (HTA), which can detect pfcrt 76T-bearing P. falciparum minority variants in individual patients that were undetectable by conventional PCR. However, as this assay required a radiolabeled probe, it could not be used in many resource-limited settings. Methods This study describes a digoxigenin (DIG)-labeled chemiluminescent heteroduplex tracking assay (DIG-HTA) to detect pfcrt 76T-bearing minority variant P. falciparum. This assay was compared to restriction fragment length polymorphism (RFLP) analysis and to the isotopic HTA for detection of genetically CQ-resistant parasites in clinical samples. Results Thirty one clinical P. falciparum isolates (15 primary isolates and 16 recurrent isolates) from 17 Malagasy children treated with CQ for uncomplicated malaria were genotyped for the pfcrt K76T mutation. Two (11.7%) of 17 patients harboured genetically CQ-resistant P. falciparum strains after therapy as detected by HTA. RFLP analysis failed to detect any pfcrt K76T-bearing isolates. Conclusion These findings indicate that genetically CQ-resistant P. falciparum are more common than previously thought in Madagascar even though the fitness of the minority variant pfcrt 76T parasites remains unclear. In addition, HTAs for malaria drug resistance alleles are promising tools for the surveillance of anti-malarial resistance. The use of a non-radioactive label allows for the use of HTAs in malaria endemic countries. PMID:19291288

  2. Drug Target Interference in Immunogenicity Assays: Recommendations and Mitigation Strategies.

    Science.gov (United States)

    Zhong, Zhandong Don; Clements-Egan, Adrienne; Gorovits, Boris; Maia, Mauricio; Sumner, Giane; Theobald, Valerie; Wu, Yuling; Rajadhyaksha, Manoj

    2017-11-01

    Sensitive and specific methodology is required for the detection and characterization of anti-drug antibodies (ADAs). High-quality ADA data enables the evaluation of potential impact of ADAs on the drug pharmacokinetic profile, patient safety, and efficacious response to the drug. Immunogenicity assessments are typically initiated at early stages in preclinical studies and continue throughout the drug development program. One of the potential bioanalytical challenges encountered with ADA testing is the need to identify and mitigate the interference mediated by the presence of soluble drug target. A drug target, when present at sufficiently high circulating concentrations, can potentially interfere with the performance of ADA and neutralizing antibody (NAb) assays, leading to either false-positive or, in some cases, false-negative ADA and NAb assay results. This publication describes various mechanisms of assay interference by soluble drug target, as well as strategies to recognize and mitigate such target interference. Pertinent examples are presented to illustrate the impact of target interference on ADA and NAb assays as well as several mitigation strategies, including the use of anti-target antibodies, soluble versions of the receptors, target-binding proteins, lectins, and solid-phase removal of targets. Furthermore, recommendations for detection and mitigation of such interference in different formats of ADA and NAb assays are provided.

  3. Study on drug resistance of mycobacterium tuberculosis in patients with pulmonary tuberculosis by drug resistance gene detecting

    International Nuclear Information System (INIS)

    Wang Wei; Li Hongmin; Wu Xueqiong; Wang Ansheng; Ye Yixiu; Wang Zhongyuan; Liu Jinwei; Chen Hongbing; Lin Minggui; Wang Jinhe; Li Sumei; Jiang Ping; Feng Bai; Chen Dongjing

    2004-01-01

    To investigate drug resistance of mycobacterium tuberculosis in different age group, compare detecting effect of two methods and evaluate their the clinical application value, all of the strains of mycobacterium tuberculosis were tested for resistance to RFP, INH SM PZA and EMB by the absolute concentration method on Lowenstein-Jensen medium and the mutation of the rpoB, katG, rpsL, pncA and embB resistance genes in M. tuberculosis was tested by PCR-SSCP. In youth, middle and old age group, the rate of acquired drug resistance was 89.2%, 85.3% and 67.6% respectively, the gene mutation rate was 76.2%, 81.3% and 63.2% respectively. The rate of acquired drug resistance and multiple drug resistance in youth group was much higher than those in other groups. The gene mutation was correlated with drug resistance level of mycobacterium tuberculosis. The gene mutation rate was higher in strains isolated from high concentration resistance than those in strains isolated from low concentration resistance. The more irregular treatment was longer, the rate of drug resistance was higher. Acquired drug resistance varies in different age group. It suggested that surveillance of drug resistence in different age group should be taken seriously, especially in youth group. PCR - SSCP is a sensitive and specific method for rapid detecting rpoB, katG, rpsL, pncA and embB genes mutations of MTB. (authors)

  4. Activity of siderophores against drug-resistant Gram-positive and Gram-negative bacteria

    Directory of Open Access Journals (Sweden)

    Gokarn K

    2018-01-01

    Full Text Available Karuna Gokarn,1,2 Ramprasad B Pal1 1Department of Microbiology, Sir Hurkisondas Nurrotumdas Medical Research Society, 2Caius Research Laboratory, St Xavier’s College, Mumbai, India Abstract: Infections by drug-resistant bacteria are life-threatening. As iron is a vital element for the growth of bacteria, iron-chelating agents (siderophores can be used to arrest their multiplication. Exogenous siderophores – exochelin-MS and deferoxamine-B – were evaluated for their inhibitory activity against methicillin-resistant Staphylococcus aureus and metallo-β-lactamase producers – Pseudomonas aeruginosa and Acinetobacter baumannii – by disc diffusion, micro-broth dilution, and turbidimetric growth assays. The drug-resistant isolates were inhibited by the synergistic activity of siderophores and antibiotics. Minimum inhibitory concentration of exochelin-MS+ampicillin for different isolates was between 0.05 and 0.5 mg/mL. Minimum inhibitory concentration of deferoxamine-B+ampicillin was 1.0 mg/mL and greater. Iron-chelation therapy could provide a complementary approach to overcome drug resistance in pathogenic bacteria. Keywords: iron-chelation, xenosiderophores, exochelin MS, deferoxamine B

  5. Development and validation of a luminescence-based, medium-throughput assay for drug screening in Schistosoma mansoni.

    Directory of Open Access Journals (Sweden)

    Cristiana Lalli

    2015-01-01

    Full Text Available Schistosomiasis, one of the world's greatest neglected tropical diseases, is responsible for over 280,000 human deaths per annum. Praziquantel, developed in the 1970s, has high efficacy, excellent tolerability, few and transient side effects, simple administration procedures and competitive cost and it is currently the only recommended drug for treatment of human schistosomiasis. The use of a single drug to treat a population of over 200 million infected people appears particularly alarming when considering the threat of drug resistance. Quantitative, objective and validated methods for the screening of compound collections are needed for the discovery of novel anti-schistosomal drugs.The present work describes the development and validation of a luminescence-based, medium-throughput assay for the detection of schistosomula viability through quantitation of ATP, a good indicator of metabolically active cells in culture. This validated method is demonstrated to be fast, highly reliable, sensitive and automation-friendly. The optimized assay was used for the screening of a small compound library on S. mansoni schistosomula, showing that the proposed method is suitable for a medium-throughput semi-automated screening. Interestingly, the pilot screening identified hits previously reported to have some anti-parasitic activity, further supporting the validity of this assay for anthelminthic drug discovery.The developed and validated schistosomula viability luminescence-based assay was shown to be successful and suitable for the identification of novel compounds potentially exploitable in future schistosomiasis therapies.

  6. The culturable soil antibiotic resistome: a community of multi-drug resistant bacteria.

    Science.gov (United States)

    Walsh, Fiona; Duffy, Brion

    2013-01-01

    Understanding the soil bacterial resistome is essential to understanding the evolution and development of antibiotic resistance, and its spread between species and biomes. We have identified and characterized multi-drug resistance (MDR) mechanisms in the culturable soil antibiotic resistome and linked the resistance profiles to bacterial species. We isolated 412 antibiotic resistant bacteria from agricultural, urban and pristine soils. All isolates were multi-drug resistant, of which greater than 80% were resistant to 16-23 antibiotics, comprising almost all classes of antibiotic. The mobile resistance genes investigated, (ESBL, bla NDM-1, and plasmid mediated quinolone resistance (PMQR) resistance genes) were not responsible for the respective resistance phenotypes nor were they present in the extracted soil DNA. Efflux was demonstrated to play an important role in MDR and many resistance phenotypes. Clinically relevant Burkholderia species are intrinsically resistant to ciprofloxacin but the soil Burkholderia species were not intrinsically resistant to ciprofloxacin. Using a phenotypic enzyme assay we identified the antibiotic specific inactivation of trimethoprim in 21 bacteria from different soils. The results of this study identified the importance of the efflux mechanism in the soil resistome and variations between the intrinsic resistance profiles of clinical and soil bacteria of the same family.

  7. The culturable soil antibiotic resistome: a community of multi-drug resistant bacteria.

    Directory of Open Access Journals (Sweden)

    Fiona Walsh

    Full Text Available Understanding the soil bacterial resistome is essential to understanding the evolution and development of antibiotic resistance, and its spread between species and biomes. We have identified and characterized multi-drug resistance (MDR mechanisms in the culturable soil antibiotic resistome and linked the resistance profiles to bacterial species. We isolated 412 antibiotic resistant bacteria from agricultural, urban and pristine soils. All isolates were multi-drug resistant, of which greater than 80% were resistant to 16-23 antibiotics, comprising almost all classes of antibiotic. The mobile resistance genes investigated, (ESBL, bla NDM-1, and plasmid mediated quinolone resistance (PMQR resistance genes were not responsible for the respective resistance phenotypes nor were they present in the extracted soil DNA. Efflux was demonstrated to play an important role in MDR and many resistance phenotypes. Clinically relevant Burkholderia species are intrinsically resistant to ciprofloxacin but the soil Burkholderia species were not intrinsically resistant to ciprofloxacin. Using a phenotypic enzyme assay we identified the antibiotic specific inactivation of trimethoprim in 21 bacteria from different soils. The results of this study identified the importance of the efflux mechanism in the soil resistome and variations between the intrinsic resistance profiles of clinical and soil bacteria of the same family.

  8. Extensively Drug-Resistant Tuberculosis: Principles of Resistance, Diagnosis, and Management.

    Science.gov (United States)

    Wilson, John W; Tsukayama, Dean T

    2016-04-01

    Extensively drug-resistant (XDR) tuberculosis (TB) is an unfortunate by-product of mankind's medical and pharmaceutical ingenuity during the past 60 years. Although new drug developments have enabled TB to be more readily curable, inappropriate TB management has led to the emergence of drug-resistant disease. Extensively drug-resistant TB describes Mycobacterium tuberculosis that is collectively resistant to isoniazid, rifampin, a fluoroquinolone, and an injectable agent. It proliferates when established case management and infection control procedures are not followed. Optimized treatment outcomes necessitate time-sensitive diagnoses, along with expanded combinations and prolonged durations of antimicrobial drug therapy. The challenges to public health institutions are immense and most noteworthy in underresourced communities and in patients coinfected with human immunodeficiency virus. A comprehensive and multidisciplinary case management approach is required to optimize outcomes. We review the principles of TB drug resistance and the risk factors, diagnosis, and managerial approaches for extensively drug-resistant TB. Treatment outcomes, cost, and unresolved medical issues are also discussed. Copyright © 2016 Mayo Foundation for Medical Education and Research. Published by Elsevier Inc. All rights reserved.

  9. Identifying clinically relevant drug resistance genes in drug-induced resistant cancer cell lines and post-chemotherapy tissues.

    Science.gov (United States)

    Tong, Mengsha; Zheng, Weicheng; Lu, Xingrong; Ao, Lu; Li, Xiangyu; Guan, Qingzhou; Cai, Hao; Li, Mengyao; Yan, Haidan; Guo, You; Chi, Pan; Guo, Zheng

    2015-12-01

    Until recently, few molecular signatures of drug resistance identified in drug-induced resistant cancer cell models can be translated into clinical practice. Here, we defined differentially expressed genes (DEGs) between pre-chemotherapy colorectal cancer (CRC) tissue samples of non-responders and responders for 5-fluorouracil and oxaliplatin-based therapy as clinically relevant drug resistance genes (CRG5-FU/L-OHP). Taking CRG5-FU/L-OHP as reference, we evaluated the clinical relevance of several types of genes derived from HCT116 CRC cells with resistance to 5-fluorouracil and oxaliplatin, respectively. The results revealed that DEGs between parental and resistant cells, when both were treated with the corresponding drug for a certain time, were significantly consistent with the CRG5-FU/L-OHP as well as the DEGs between the post-chemotherapy CRC specimens of responders and non-responders. This study suggests a novel strategy to extract clinically relevant drug resistance genes from both drug-induced resistant cell models and post-chemotherapy cancer tissue specimens.

  10. Performance of the Abbott RealTime MTB RIF/INH resistance assay when used to test Mycobacterium tuberculosis specimens from Bangladesh

    Directory of Open Access Journals (Sweden)

    Kostera J

    2018-05-01

    Full Text Available Joshua Kostera, Gregor Leckie, Klara Abravaya, Hong Wang Abbott Molecular, Abbott Laboratories, Des Plaines, IL, USA Introduction: The Abbott RealTime MTB RIF/INH Resistance Assay (RT MTB RIF/INH is an assay for the detection of rifampicin (RIF- and/or isoniazid (INH-resistant Mycobacterium tuberculosis (MTB. The assay can be used to test sputum, bronchial alveolar lavage, and N-Acetyl-L-Cysteine (NALC/NaOH pellets prepared from these samples. The assay can be used in direct testing mode, or in reflex mode following a MTB positive result produced by its companion assay, Abbott RT MTB. Methods: In this study, the direct testing mode was used to test paired sputum and NALC/NaOH pellets prepared from sputum collected from Bangladesh TB patients. One hundred and thirty two paired samples were tested. Results: The RT MTB RIF/INH inhibition rate was 0%. One hundred and twenty-two paired samples had results above the assay limit of detection and were analyzed by comparing with results from phenotypic drug sensitivity testing, GeneXpert MTB/RIF (Xpert, and MTBDR plus (Hain. RT MTB RIF/INH results were in good agreement with those of GeneXpert and Hain. Conclusion: The ability of this assay to detect RIF and INH resistance may contribute to the global control of multidrug resistant tuberculosis. Keywords: tuberculosis, rifampicin, isoniazid, resistance

  11. Drug-permeability and transporter assays in Caco-2 and MDCK cell lines.

    Science.gov (United States)

    Volpe, Donna A

    2011-12-01

    The human colon adenocarcinoma Caco-2 and Madin-Darby canine kidney epithelial cell lines provide in vitro tools to assess a drug's permeability and transporter interactions during discovery and development. The cells, when cultured on semiporous filters, form confluent monolayers that model the intestinal epithelial barrier for permeability, transporter and drug-interaction assays. The applications of these assays in pharmaceutical research include qualitative prediction and ranking of absorption, determining mechanism(s) of permeability, formulation effects on drug permeability, and the potential for transporter-mediated drug-drug interactions. This review focuses on recent examples of Caco-2 and Madin-Darby canine kidney cells assays for drug permeability including transfected and knock-down cells, miniaturization and automation, and assay combinations to better understand and predict intestinal drug absorption.

  12. Antibacterial activity of combined medicinal plants extract against multiple drug resistant strains

    Directory of Open Access Journals (Sweden)

    Rafiqul Islam

    2015-06-01

    Full Text Available Objective: To find out the combined antibacterial efficacy of Aegle marmelos, Aphanamixis polystachya, Cuscuta reflexa and Aesclynomene indica against bacterial pathogens. Methods: Antibacterial potency of combined plant extracts has been tested against Bacillus subtilis IFO 3026, Sarcina lutea IFO 3232, Xanthomonas campestris IAM 1671, Escherichia coli IFO 3007, Klebsiella pneumoniae ATTC 10031, Proteus vulgaris MTCC 321 and Pseudomonas denitrificans KACC 32026 by disc diffusion assay. Commercially available standard antibiotic discs were also used to find out antibiotic resistance pattern of test organisms. Results: Among the test organisms, Escherichia coli, Proteus vulgaris, Klebsiella pneumoniae and Proteus denitrificans showed resistance against multiple commercially available antibiotics. On the other hand, these multiple drug resistant organisms showed susceptibility against combined plant extracts. Conclusions: These combined plants extracts showed synergistic antibacterial activity and could lead to new antibacterial drug designing.

  13. Drug-resistance patterns of Mycobacterium tuberculosis strains and associated risk factors among multi drug-resistant tuberculosis suspected patients from Ethiopia.

    Science.gov (United States)

    Mesfin, Eyob Abera; Beyene, Dereje; Tesfaye, Abreham; Admasu, Addisu; Addise, Desalegn; Amare, Miskir; Dagne, Biniyam; Yaregal, Zelalem; Tesfaye, Ephrem; Tessema, Belay

    2018-01-01

    Multidrug drug-resistant tuberculosis (MDR-TB) is a major health problem and seriously threatens TB control and prevention efforts globally. Ethiopia is among the 30th highest TB burden countries for MDR-TB with 14% prevalence among previously treated cases. The focus of this study was on determining drug resistance patterns of Mycobacterium tuberculosis among MDR-TB suspected cases and associated risk factors. A cross-sectional study was conducted in Addis Ababa from June 2015 to December 2016. Sputum samples and socio-demographic data were collected from 358 MDR-TB suspected cases. Samples were analyzed using Ziehl-Neelsen technique, GeneXpert MTB/RIF assay, and culture using Lowenstein-Jensen and Mycobacterial growth indicator tube. Data were analyzed using SPSS version 23. A total of 226 the study participants were culture positive for Mycobacterium tuberculosis, among them, 133 (58.8%) participants were males. Moreover, 162 (71.7%) had been previously treated for tuberculosis, while 128 (56.6%) were TB/HIV co-infected. A majority [122 (54%)] of the isolates were resistant to any first-line anti-TB drugs. Among the resistant isolates, 110 (48.7%) were determined to be resistant to isoniazid, 94 (41.6%) to streptomycin, 89 (39.4%) to rifampicin, 72 (31.9%) to ethambutol, and 70 (30.9%) to pyrazinamide. The prevalence of MDR-TB was 89 (39.4%), of which 52/89 (58.4%) isolates were resistance to all five first-line drugs. Risk factors such as TB/HIV co-infection (AOR = 5.59, p = 0.00), cigarette smoking (AOR = 3.52, p = 0.045), alcohol drinking (AOR = 5.14, p = 0.001) hospital admission (AOR = 3.49, p = 0.005) and visiting (AOR = 3.34, p = 0.044) were significantly associated with MDR-TB. The prevalence of MDR-TB in the study population was of a significantly high level among previously treated patients and age group of 25-34. TB/HIV coinfection, smoking of cigarette, alcohol drinking, hospital admission and health facility visiting were identified as risk factors

  14. Performance of an in-house human immunodeficiency virus type 1 genotyping system for assessment of drug resistance in Cuba.

    Directory of Open Access Journals (Sweden)

    Yoan Alemán

    Full Text Available As commercial human immunodeficiency virus type 1 drug resistance assays are expensive, they are not commonly used in resource-limited settings. Hence, a more affordable in-house procedure was set up taking into account the specific epidemiological and economic circumstances of Cuba. The performance characteristics of the in-house assay were evaluated using clinical samples with various subtypes and resistance patterns. The lower limit of amplification was determined on dilutions series of 20 clinical isolates and ranged from 84 to 529 RNA copies/mL. For the assessment of trueness, 14 clinical samples were analyzed and the ViroSeq HIV-1 Genotyping System v2.0 was used as the reference standard. The mean nucleotide sequence identity between the two assays was 98.7% ± 1.0. Additionally, 99.0% of the amino acids at drug resistance positions were identical. The sensitivity and specificity in detecting drug resistance mutations was respectively 94.1% and 99.5%. Only few discordances in drug resistance interpretation patterns were observed. The repeatability and reproducibility were evaluated using 10 clinical samples with 3 replicates per sample. The in-house test was very precise as nucleotide sequence identity among paired nucleotide sequences ranged from 98.7% to 99.9%. The acceptance criteria were met by the in-house test for all performance characteristics, demonstrating a high degree of accuracy. Subsequently, the applicability in routine clinical practice was evaluated on 380 plasma samples. The amplification success rate was 91% and good quality consensus sequences encoding the entire protease and the first 335 codons in reverse transcriptase could be obtained for 99% of the successful amplicons. The reagent cost per sample using the in-house procedure was around € 80 per genotyping attempt. Overall, the in-house assay provided good results, was feasible with equipment and reagents available in Cuba and was half as expensive as commercial

  15. Automation of cell-based drug absorption assays in 96-well format using permeable support systems.

    Science.gov (United States)

    Larson, Brad; Banks, Peter; Sherman, Hilary; Rothenberg, Mark

    2012-06-01

    Cell-based drug absorption assays, such as Caco-2 and MDCK-MDR1, are an essential component of lead compound ADME/Tox testing. The permeability and transport data they provide can determine whether a compound continues in the drug discovery process. Current methods typically incorporate 24-well microplates and are performed manually. Yet the need to generate absorption data earlier in the drug discovery process, on an increasing number of compounds, is driving the use of higher density plates. A simple, more efficient process that incorporates 96-well permeable supports and proper instrumentation in an automated process provides more reproducible data compared to manual methods. Here we demonstrate the ability to perform drug permeability and transport assays using Caco-2 or MDCKII-MDR1 cells. The assay procedure was automated in a 96-well format, including cell seeding, media and buffer exchanges, compound dispense, and sample removal using simple robotic instrumentation. Cell monolayer integrity was confirmed via transepithelial electrical resistance and Lucifer yellow measurements. Proper cell function was validated by analyzing apical-to-basolateral and basolateral-to-apical movement of rhodamine 123, a known P-glycoprotein substrate. Apparent permeability and efflux data demonstrate how the automated procedure provides a less variable method than manual processing, and delivers a more accurate assessment of a compound's absorption characteristics.

  16. Molecular detection of multi drug resistant tuberculosis (mdr-tb) in mdr-tb patients' attendant in north western pakistan

    International Nuclear Information System (INIS)

    Shah, T.; Hayat, A.; Shah, Z.; Hayat, A.; Khan, S.B.

    2017-01-01

    Objective: To determine the drugs susceptibility pattern of mycobacterium tuberculosis (M.TB) in multi-drug resistant tuberculosis (MDR-TB) patients' attendants in North Western, Pakistan. Study Design: Cross sectional study. Place and Duration of Study: This study was conducted at Peshawar Tuberculosis Research Laboratory (PTRL), Provincial TB Control Program Hayatabad Medical Complex Peshawar, (KP) from August 2013 to March 2014. Material and Methods: A cross sectional study in which four hundred and eighty sputum samples from MDR-TB patients' attendants were processed for the detection of M.TB through Ziehl-Neelsen staining, Lowenstein-Jensen, BACTEC MGIT-960 culture and line probe assay. Results: Out of 480 samples, 06 (2.1%) were found positive for M.TB through Ziehl-Neelsen staining while 10 (2.8%) were positive through LJ and BACTEC MGIT-960 culture. The 10 positive samples were further subjected to drugs susceptibility testing and line probes assay test to find out rifampicin, isoniazid, streptomycin and ethambutol resistant and it was found that 6 M.TB isolates were resistant while 4 were sensitive to rifampicin and isoniazid. Among the 6 resistant M.TB strains, 4 showed mutation in rpoB gene at 531, 516 and 526 codons. Conclusion: Majority of MDR-TB patients' attendants had drug-resistant tuberculosis and the rate of drug susceptible TB was low. (author)

  17. Radiosensitivity of drug-resistant human tumour xenografts

    International Nuclear Information System (INIS)

    Mattern, J.; Bak, M. Jr.; Volm, M.; Hoever, K.H.

    1989-01-01

    The radiosensitivity of three drug-resistant sublines of a human epidermoid lung carcinoma growing as xenografts in nude mice was investigated. Drug resistance to vincristine, actinomycin D and cisplatin was developed in vivo by repeated drug treatment. It was found that all three drug-resistant tumour lines were not cross-resistant to irradiation. (orig.) [de

  18. Development of a real-time PCR melt curve assay for simultaneous detection of virulent and antibiotic resistant Salmonella.

    Science.gov (United States)

    Singh, Prashant; Mustapha, Azlin

    2014-12-01

    Multiple drug resistance in Salmonella is an emerging problem in the area of food safety. Depending on the virulence and antibiotic resistance characteristics of the Salmonella strain, infections of varying severity could result. In this study, a multiplex melt curve real-time PCR assay for the detection of virulent and antibiotic resistance strains of Salmonella was developed with two primer sets. The first set targets the virulence gene, invasin (invA), and tetracycline (tetG), streptomycin (aadA2) and sulphonamide (sulI) antibiotic resistance genes, and the second set amplifies ampicillin (blaPSE,blaTEM) and chloramphenicol (floR) resistance genes. The multiplex assay was evaluated using 41 Salmonella strains and was further tested on eight different artificially inoculated food samples. The fluorescent DNA intercalating dye, SYTO9, generated high resolution melt curve peaks and, hence, was used for the development of the assay. This multiplex assay worked efficiently over a DNA concentration range of 20 ng-200 fg and showed a sensitivity of 290 CFU/mL with serially diluted broth cultures. The detection limit for un-enriched artificially inoculated food samples was 10(4) CFU/g, but an enrichment period of 6 h allowed for detection of 10 CFU/g of cells in the samples. Copyright © 2014 Elsevier Ltd. All rights reserved.

  19. P-glycoprotein inhibition of drug resistant cell lines by nanoparticles.

    Science.gov (United States)

    Singh, Manu Smriti; Lamprecht, Alf

    2016-01-01

    Several pharmaceutical excipients are known for their ability to interact with cell membrane lipids and reverse the phenomenon of multidrug resistance (MDR) in cancer. Interestingly, many excipients act as stabilizers and are key ingredients in a variety of nano-formulations. In this study, representatives of ionic and non-ionic excipients were used as surface active agents in nanoparticle (NP) formulations to utilize their MDR reversing potential. In-vitro assays were performed to elucidate particle-cell interaction and accumulation of P-glycoprotein (P-gp) substrates-rhodamine-123 and calcein AM, in highly drug resistant glioma cell lines. Chemosensitization achieved using NPs and their equivalent dose of free excipients was assessed with the co-administered anti-cancer drug doxorubicin. Among the excipients used, non-ionic surfactant, Cremophor® EL, and cationic surfactant, cetyltrimethylammonuium bromide (CTAB), demonstrated highest P-gp modulatory activity in both free solution form (up to 7-fold lower IC50) and as a formulation (up to 4.7-fold lower IC50) as compared to doxorubicin treatment alone. Solutol® HS15 and Tween® 80 exhibited considerable chemosensitization as free solution but not when incorporated into a formulation. Sodium dodecyl sulphate (SDS)-based nanocarriers resulted in slightly improved cytotoxicity. Overall, the results highlight and envisage the usage of excipient in nano-formulations in a bid to improve chemosensitization of drug resistant cancer cells towards anti-cancer drugs.

  20. Synergy against extensively drug-resistant Acinetobacter baumannii in vitro by two old antibiotics: colistin and chloramphenicol.

    Science.gov (United States)

    Wei, Wen-Juan; Yang, Hai-Fei

    2017-03-01

    Combination antimicrobial therapy is an important option in the fight against Gram-negative 'superbugs'. This study systematically investigated the synergistic effect of colistin (CST) and chloramphenicol (CHL) in combination against extensively drug-resistant Acinetobacter baumannii (XDR-AB). The microtitre plate chequerboard assay was used to test synergy against 50 XDR-AB clinical strains. Then, three XDR-AB clinical isolates and the type strain A. baumannii ATCC 19606 were chosen for further synergy studies using time-kill assay, mutant prevention concentration (MPC) assay and real-time population analysis profile (PAP) assay. In the chequerboard assays, synergistic or additive effects [defined as a fractional inhibitory concentration index (FICI) of ≤0.5 and 0.5 synergy testing, the results of time-kill assays indicated that CST monotherapy produced rapid bacterial killing followed by rapid re-growth, with the emergence of CST resistance; CHL monotherapy was largely ineffective. The combination CST/CHL, however, showed a synergistic effect and enhanced bacterial killing in the four tested strains. It also significantly delayed re-growth and suppressed the emergence of CST resistance. In the MPC assay, a decrease in MPCs for CST was observed in the two CST-susceptible strains. PAP assay showed that both CST-resistant strains were heteroresistant. Copyright © 2017 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.

  1. Prediction of resistance development against drug combinations by collateral responses to component drugs

    DEFF Research Database (Denmark)

    Munck, Christian; Gumpert, Heidi; Nilsson Wallin, Annika

    2014-01-01

    the genomes of all evolved E. coli lineages, we identified the mutational events that drive the differences in drug resistance levels and found that the degree of resistance development against drug combinations can be understood in terms of collateral sensitivity and resistance that occurred during...... adaptation to the component drugs. Then, using engineered E. coli strains, we confirmed that drug resistance mutations that imposed collateral sensitivity were suppressed in a drug pair growth environment. These results provide a framework for rationally selecting drug combinations that limit resistance......Resistance arises quickly during chemotherapeutic selection and is particularly problematic during long-term treatment regimens such as those for tuberculosis, HIV infections, or cancer. Although drug combination therapy reduces the evolution of drug resistance, drug pairs vary in their ability...

  2. Characterization and drug resistance patterns of Ewing's sarcoma family tumor cell lines.

    Directory of Open Access Journals (Sweden)

    William A May

    Full Text Available Despite intensive treatment with chemotherapy, radiotherapy and surgery, over 70% of patients with metastatic Ewing's Sarcoma Family of Tumors (EFT will die of their disease. We hypothesize that properly characterized laboratory models reflecting the drug resistance of clinical tumors will facilitate the application of new therapeutic agents to EFT. To determine resistance patterns, we studied newly established EFT cell lines derived from different points in therapy: two established at diagnosis (CHLA-9, CHLA-32, two after chemotherapy and progressive disease (CHLA-10, CHLA-25, and two at relapse after myeloablative therapy and autologous bone marrow transplantation (post-ABMT (CHLA-258, COG-E-352. The new lines were compared to widely studied EFT lines TC-71, TC-32, SK-N-MC, and A-673. These lines were extensively characterized with regard to identity (short tandem repeat (STR analysis, p53, p16/14 status, and EWS/ETS breakpoint and target gene expression profile. The DIMSCAN cytotoxicity assay was used to assess in vitro drug sensitivity to standard chemotherapy agents. No association was found between drug resistance and the expression of EWS/ETS regulated genes in the EFT cell lines. No consistent association was observed between drug sensitivity and p53 functionality or between drug sensitivity and p16/14 functionality across the cell lines. Exposure to chemotherapy prior to cell line initiation correlated with drug resistance of EFT cell lines in 5/8 tested agents at clinically achievable concentrations (CAC or the lower tested concentration (LTC: (cyclophosphamide (as 4-HC and doxorubicin at CAC, etoposide, irinotecan (as SN-38 and melphalan at LTC; P<0.1 for one agent, and P<0.05 for four agents. This panel of well-characterized drug-sensitive and drug-resistant cell lines will facilitate in vitro preclinical testing of new agents for EFT.

  3. Rapid molecular diagnostics for multi-drug resistant tuberculosis in India.

    Science.gov (United States)

    Ramachandran, Rajeswari; Muniyandi, M

    2018-03-01

    Rapid molecular diagnostic methods help in the detection of TB and Rifampicin resistance. These methods detect TB early, are accurate and play a crucial role in reducing the burden of drug resistant tuberculosis. Areas covered: This review analyses rapid molecular diagnostic tools used in the diagnosis of MDR-TB in India, such as the Line Probe Assay and GeneXpert. We have discussed the burden of MDR-TB and the impact of recent diagnostic tools on case detection and treatment outcomes. This review also discusses the costs involved in establishing these new techniques in India. Expert commentary: Molecular methods have considerable advantages for the programmatic management of drug resistant TB. These include speed, standardization of testing, potentially high throughput and reduced laboratory biosafety requirements. There is a desperate need for India to adopt modern, rapid, molecular tools with point-of-care tests being currently evaluated. New molecular diagnostic tests appear to be cost effective and also help in detecting missing cases. There is enough evidence to support the scaling up of these new tools in India.

  4. Affordable HIV drug-resistance testing for monitoring of antiretroviral therapy in sub-Saharan Africa.

    Science.gov (United States)

    Inzaule, Seth C; Ondoa, Pascale; Peter, Trevor; Mugyenyi, Peter N; Stevens, Wendy S; de Wit, Tobias F Rinke; Hamers, Raph L

    2016-11-01

    Increased provision of antiretroviral therapy in sub-Saharan Africa has led to a growing number of patients with therapy failure and acquired drug-resistant HIV, driving the demand for more costly further lines of antiretroviral therapy. In conjunction with accelerated access to viral load monitoring, feasible and affordable technologies to detect drug-resistant HIV could help maximise the durability and rational use of available drug regimens. Potential low-cost technologies include in-house Sanger and next-generation sequencing in centralised laboratories, and point mutation assays and genotype-free systems that predict response to antiretroviral therapy at point-of-care. Strengthening of centralised high-throughput laboratories, including efficient systems for sample referral and results delivery, will increase economies-of-scale while reducing costs. Access barriers can be mitigated by standardisation of in-house assays into commercial kits, use of polyvalent instruments, and adopting price-reducing strategies. A stepwise rollout approach should improve feasibility, prioritising WHO-recommended population-based surveillance and management of complex patient categories, such as patients failing protease inhibitor-based antiretroviral therapy. Implementation research, adaptations of existing WHO guidance, and political commitment, will be key to support the appropriate investments and policy changes. In this Personal View, we discuss the potential role of HIV drug resistance testing for population-based surveillance and individual patient management in sub-Saharan Africa. We review the strengths and challenges of promising low-cost technologies and how they can be implemented. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. Plasmodium falciparum drug resistance in Angola.

    Science.gov (United States)

    Fançony, Cláudia; Brito, Miguel; Gil, Jose Pedro

    2016-02-09

    Facing chloroquine drug resistance, Angola promptly adopted artemisinin-based combination therapy as the first-line to treat malaria. Currently, the country aims to consolidate malaria control, while preparing for the elimination of the disease, along with others African countries in the region. However, the remarkable capacity of Plasmodium to develop drug resistance represents an alarming threat for those achievements. Herein, the available, but relatively scarce and dispersed, information on malaria drug resistance in Angola, is reviewed and discussed. The review aims to inform but also to encourage future research studies that monitor and update the information on anti-malarial drug efficacy and prevalence of molecular markers of drug resistance, key fields in the context and objectives of elimination.

  6. [Change in drug resistance of Staphylococcus aureus].

    Science.gov (United States)

    Lin, Yan; Liu, Yan; Luo, Yan-Ping; Liu, Chang-Ting

    2013-11-01

    To analyze the change in drug resistance of Staphylococcus aureus (SAU) in the PLA general hospital from January 2008 to December 2012, and to provide solid evidence to support the rational use of antibiotics for clinical applications. The SAU strains isolated from clinical samples in the hospital were collected and subjected to the Kirby-Bauer disk diffusion test. The results were assessed based on the 2002 American National Committee for Clinical Laboratory Standards (NCCLS) guidelines. SAU strains were mainly isolated from sputum, urine, blood and wound excreta and distributed in penology, neurology wards, orthopedics and surgery ICU wards. Except for glycopeptide drugs, methicillin-resistant Staphylococcus aureus (MRSA) had a higher drug resistance rate than those of the other drugs and had significantly more resistance than methicillin-sensitive Staphylococcus aureus (MSSA) (P resistance, we discovered a gradual increase in drug resistance to fourteen test drugs during the last five years. Drug resistance rate of SAU stayed at a higher level over the last five years; moreover, the detection ratio of MRSA keeps rising year by year. It is crucial for physicians to use antibiotics rationally and monitor the change in drug resistance in a dynamic way.

  7. Drug Resistance

    Science.gov (United States)

    ... Drug-resistance testing is also recommended for all pregnant women with HIV before starting HIV medicines and also in some pregnant women already taking HIV medicines. Pregnant women will work with their health ...

  8. Prediction of individual patient response to chemotherapy by the fluorometric microculture cytotoxicity assay (FMCA) using drug specific cut-off limits and a Bayesian model.

    Science.gov (United States)

    Larsson, R; Nygren, P

    1993-01-01

    The semiautomated fluorometric microculture cytotoxicity assay (FMCA) based on the measurement of fluorescence generated from cellular hydrolysis of fluorescein diacetate (FDA) to fluorescein in microtiter plates, has been used for determination of cytotoxic drug resistance of tumor cells from patients with hematological and solid tumors. In the present study we describe a calibration procedure based on statistically derived cut-off limits and assay-predicted response probabilities using Bayes' theorem. Test results at a specified drug concentration were divided into three categories: low, intermediate or extreme drug resistance (LDR, IDR and EDR, respectively) using the median and median +1 standard deviation as the cut-off limits. When correlated with clinical outcome, LDR samples showed a higher response rate than expected, IDR a lower and EDR samples no response at all. The sensitivity and specificity of the test, using the median as cut-off limit, were 0.92 and 0.69 respectively. By fitting these test characteristics to a statistical model based on Bayes' theorem it is possible to calculate response probabilities for each individual patient taking into consideration not only the test characteristics and the particular assay result, but also the clinical and patient specific characteristics influencing the pre-test probability of response. EDR predicts clinical drug resistance with high specificity and is also observed in tumor types with high response rate.

  9. Circumvention of inherent or acquired cytotoxic drug resistance in vitro using combinations of modulating agents.

    Science.gov (United States)

    Cadagan, David; Merry, Stephen

    2013-10-01

    Modulating agents are used to circumvent drug resistance in the clinical setting. However achievable serum concentrations are often lower than those which are optimal in vitro. Combination of modulating agents with non-overlapping toxicities may overcome this obstacle. We have investigated combinations of three modulating agents (quinine, verapamil, and cinnarizine) to circumvent inherent or acquired resistance to the cytotoxic drugs doxorubicin, vincristine and paclitaxel. Dose-response curves to cytotoxic drugs in the presence/absence of modulating agents were determined using colony formation and cell proliferation assays. Doxorubicin accumulation into cell monolayers was measured by fluorescence spectrophotometry. Greater (1.9-fold) sensitisation to particular cytotoxic drugs was observed for certain combinations of modulating agents compared to individual effects. The most effective combination was quinine-plus-verapamil with the cytotoxic drug doxorubicin. This increase in sensitivity was associated with increased doxorubicin accumulation. Such enhanced activity was, however, not observed for all combinations of modulating agents or for all studied cytotoxic drugs. The findings of the present study suggest certain combinations of modulating agents to have a clinical role in circumventing drug resistance. Particular combinations of modulating agents must be carefully chosen to suit particular cytotoxic drug treatments.

  10. Mechanisms of drug resistance in cancer cells

    International Nuclear Information System (INIS)

    Iqbal, M.P.

    2003-01-01

    Development of drug resist chemotherapy. For the past several years, investigators have been striving hard to unravel mechanisms of drug resistance in cancer cells. Using different experimental models of cancer, some of the major mechanisms of drug resistance identified in mammalian cells include: (a) Altered transport of the drug (decreased influx of the drug; increased efflux of the drug (role of P-glycoprotein; role of polyglutamation; role of multiple drug resistance associated protein)), (b) Increase in total amount of target enzyme/protein (gene amplification), (c) alteration in the target enzyme/protein (low affinity enzyme), (d) Elevation of cellular glutathione, (e) Inhibition of drug-induced apoptosis (mutation in p53 tumor suppressor gene; increased expression of bcl-xl gene). (author)

  11. Drug resistance in leishmaniasis: current drug-delivery systems and future perspectives.

    Science.gov (United States)

    Yasinzai, Masoom; Khan, Momin; Nadhman, Akhtar; Shahnaz, Gul

    2013-10-01

    Leishmaniasis is a complex of diseases with numerous clinical manifestations for instance harshness from skin lesions to severe disfigurement and chronic systemic infection in the liver and spleen. So far, the most classical leishmaniasis therapy, despite its documented toxicities, remains pentavalent antimonial compounds. The arvailable therapeutic modalities for leishmaniasis are overwhelmed with resistance to leishmaniasis therapy. Mechanisms of classical drug resistance are often related with the lower drug uptake, increased efflux, the faster drug metabolism, drug target modifications and over-expression of drug transporters. The high prevalence of leishmaniasis and the appearance of resistance to classical drugs reveal the demand to develop and explore novel, less toxic, low cost and more promising therapeutic modalities. The review describes the mechanisms of classical drug resistance and potential drug targets in Leishmania infection. Moreover, current drug-delivery systems and future perspectives towards Leishmaniasis treatment are also covered.

  12. Clinical Management of HIV Drug Resistance

    Science.gov (United States)

    Cortez, Karoll J.; Maldarelli, Frank

    2011-01-01

    Combination antiretroviral therapy for HIV-1 infection has resulted in profound reductions in viremia and is associated with marked improvements in morbidity and mortality. Therapy is not curative, however, and prolonged therapy is complicated by drug toxicity and the emergence of drug resistance. Management of clinical drug resistance requires in depth evaluation, and includes extensive history, physical examination and laboratory studies. Appropriate use of resistance testing provides valuable information useful in constructing regimens for treatment-experienced individuals with viremia during therapy. This review outlines the emergence of drug resistance in vivo, and describes clinical evaluation and therapeutic options of the individual with rebound viremia during therapy. PMID:21994737

  13. Detection of systemic hypersensitivity to drugs using standard guinea pig assays.

    Science.gov (United States)

    Weaver, James L; Staten, David; Swann, Joslyn; Armstrong, George; Bates, Melissa; Hastings, Kenneth L

    2003-12-01

    The most commonly used assays designed to detect either skin or systemic immune-based hypersensitivity reactions are those using guinea pigs (GP). We obtained data from various FDA records to evaluate the correlation between GP assay results and reported post-marketing systemic hypersensitivity reactions. We examined the new drug application (NDA) reviews of approved drugs for the results of GP assays. Post-marketing human data were extracted from the FDA adverse event reporting system (AERS). Drug usage data were obtained from a commercial database maintained by IMS Health Inc. We found 83 (21%) of 396 drugs approved between 1978 and 1998 had reported GP test results. Among these 83 drugs, 14 (17%) were found to have positive results in at least one GP assay. Simple reporting index (RI) values for systemic hypersensitivity reactions were calculated from AERS data and usage to produce the index of adverse event reports per million shipping units of drug. A variety of definitions of positive human response were examined. A statistically significant association was seen for rash between post-marketing and clinical trials adverse event reports. No statistically significant associations between human data and GP test results were observed. These data suggest that standard GP assays have limited ability to predict human systemic hypersensitivity potential for pharmaceuticals.

  14. Determination of antipsychotic drug in human serum by radioreceptor assay

    International Nuclear Information System (INIS)

    Wu Jinchang; Jiang Yimin

    1989-01-01

    Serum antipsychotic drug in 50 psychosis cases were measured by radioreceptor assay (RRA) and the values were compared in parallel with that by radioimmunoassay (RIA). The results showed that the RRA values were lower than the RIA values, but both assays gave significant correlation between the serum drug level and antipsychotic dose

  15. Surveillance of transmitted antiretroviral drug resistance among HIV-1 infected women attending antenatal clinics in Chitungwiza, Zimbabwe.

    Directory of Open Access Journals (Sweden)

    Mqondisi Tshabalala

    Full Text Available The rapid scale-up of highly active antiretroviral therapy (HAART and use of single dose Nevirapine (SD NVP for prevention of mother-to-child transmission (pMTCT have raised fears about the emergence of resistance to the first line antiretroviral drug regimens. A cross-sectional study was conducted to determine the prevalence of primary drug resistance (PDR in a cohort of young (<25 yrs HAART-naïve HIV pregnant women attending antenatal clinics in Chitungwiza, Zimbabwe. Whole blood was collected in EDTA for CD4 counts, viral load, serological estimation of duration of infection using the BED Calypte assay and genotyping for drug resistance. Four hundred and seventy-one women, mean age 21 years; SD: 2.1 were enrolled into the study between 2006 and 2007. Their median CD4 count was 371cells/µL; IQR: 255-511 cells/µL. Two hundred and thirty-six samples were genotyped for drug resistance. Based on the BED assay, 27% were recently infected (RI whilst 73% had long-term infection (LTI. Median CD4 count was higher (p<0.05 in RI than in women with LTI. Only 2 women had drug resistance mutations; protease I85V and reverse transcriptase Y181C. Prevalence of PDR in Chitungwiza, 4 years after commencement of the national ART program remained below WHO threshold limit (5%. Frequency of recent infection BED testing is consistent with high HIV acquisition during pregnancy. With the scale-up of long-term ART programs, maintenance of proper prescribing practices, continuous monitoring of patients and reinforcement of adherence may prevent the acquisition and transmission of PDR.

  16. Fluorescence lifetime assays: current advances and applications in drug discovery.

    Science.gov (United States)

    Pritz, Stephan; Doering, Klaus; Woelcke, Julian; Hassiepen, Ulrich

    2011-06-01

    Fluorescence lifetime assays complement the portfolio of established assay formats available in drug discovery, particularly with the recent advances in microplate readers and the commercial availability of novel fluorescent labels. Fluorescence lifetime assists in lowering complexity of compound screening assays, affording a modular, toolbox-like approach to assay development and yielding robust homogeneous assays. To date, materials and procedures have been reported for biochemical assays on proteases, as well as on protein kinases and phosphatases. This article gives an overview of two assay families, distinguished by the origin of the fluorescence signal modulation. The pharmaceutical industry demands techniques with a robust, integrated compound profiling process and short turnaround times. Fluorescence lifetime assays have already helped the drug discovery field, in this sense, by enhancing productivity during the hit-to-lead and lead optimization phases. Future work will focus on covering other biochemical molecular modifications by investigating the detailed photo-physical mechanisms underlying the fluorescence signal.

  17. Lysosomes as mediators of drug resistance in cancer.

    Science.gov (United States)

    Zhitomirsky, Benny; Assaraf, Yehuda G

    2016-01-01

    Drug resistance remains a leading cause of chemotherapeutic treatment failure and cancer-related mortality. While some mechanisms of anticancer drug resistance have been well characterized, multiple mechanisms remain elusive. In this respect, passive ion trapping-based lysosomal sequestration of multiple hydrophobic weak-base chemotherapeutic agents was found to reduce the accessibility of these drugs to their target sites, resulting in a markedly reduced cytotoxic effect and drug resistance. Recently we have demonstrated that lysosomal sequestration of hydrophobic weak base drugs triggers TFEB-mediated lysosomal biogenesis resulting in an enlarged lysosomal compartment, capable of enhanced drug sequestration. This study further showed that cancer cells with an increased number of drug-accumulating lysosomes are more resistant to lysosome-sequestered drugs, suggesting a model of drug-induced lysosome-mediated chemoresistance. In addition to passive drug sequestration of hydrophobic weak base chemotherapeutics, other mechanisms of lysosome-mediated drug resistance have also been reported; these include active lysosomal drug sequestration mediated by ATP-driven transporters from the ABC superfamily, and a role for lysosomal copper transporters in cancer resistance to platinum-based chemotherapeutics. Furthermore, lysosomal exocytosis was suggested as a mechanism to facilitate the clearance of chemotherapeutics which highly accumulated in lysosomes, thus providing an additional line of resistance, supplementing the organelle entrapment of chemotherapeutics away from their target sites. Along with these mechanisms of lysosome-mediated drug resistance, several approaches were recently developed for the overcoming of drug resistance or exploiting lysosomal drug sequestration, including lysosomal photodestruction and drug-induced lysosomal membrane permeabilization. In this review we explore the current literature addressing the role of lysosomes in mediating cancer drug

  18. Systematic comparison of drug-tolerant assays for anti-drug antibodies in a cohort of adalimumab-treated rheumatoid arthritis patients

    NARCIS (Netherlands)

    Bloem, Karien; van Leeuwen, Astrid; Verbeek, Gerrit; Nurmohamed, Michael T.; Wolbink, Gerrit Jan; van der Kleij, Desiree; Rispens, Theo

    2015-01-01

    Drug interference complicates assessment of immunogenicity of biologicals and results in an underestimation of anti-drug antibody (ADA) formation. Drug-tolerant assays have the potential to overcome such limitations. However, to which extent drug-tolerant assays provide an unbiased picture of the

  19. Simple strategy to assess linezolid exposure in patients with multi-drug-resistant and extensively-drug-resistant tuberculosis

    NARCIS (Netherlands)

    Kamp, Jasper; Bolhuis, Mathieu S.; Tiberi, Simon; Akkerman, Onno W.; Centis, Rosella; de lange, Wiel C.; Kosterink, Jos G.; van der Werf, Tjip S.; Migliori, Giovanni B.; Alffenaar, Jan-Willem C.

    Linezolid is used increasingly for the treatment of multi-drug-resistant (MDR) and extensively-drug-resistant (XDR) tuberculosis (TB). However, linezolid can cause severe adverse events, such as peripheral and optical neuropathy or thrombocytopenia related to higher drug exposure. This study aimed

  20. Identifying rapidly parasiticidal anti-malarial drugs using a simple and reliable in vitro parasite viability fast assay.

    Science.gov (United States)

    Linares, María; Viera, Sara; Crespo, Benigno; Franco, Virginia; Gómez-Lorenzo, María G; Jiménez-Díaz, María Belén; Angulo-Barturen, Íñigo; Sanz, Laura María; Gamo, Francisco-Javier

    2015-11-05

    The emergence of Plasmodium falciparum resistance to artemisinins threatens to undermine the effectiveness of artemisinin-based combination anti-malarial therapy. Developing suitable drugs to replace artemisinins requires the identification of new compounds that display rapid parasite killing kinetics. However, no current methods fully meet the requirements to screen large compound libraries for candidates with such properties. This study describes the development and validation of an in vitro parasite viability fast assay for identifying rapidly parasiticidal anti-malarial drugs. Parasite killing kinetics were determined by first culturing unlabelled erythrocytes with P. falciparum in the presence of anti-malarial drugs for 24 or 48 h. After removing the drug, samples were added to erythrocytes pre-labelled with intracellular dye to allow their subsequent identification. The ability of viable parasites to re-establish infection in labelled erythrocytes could then be detected by two-colour flow cytometry after tagging of parasite DNA. Thus, double-stained erythrocytes (with the pre-labelled intracellular dye and the parasite DNA dye) result only after establishment of new infections by surviving parasites. The capacity of the test anti-malarial drugs to eliminate viable parasites within 24 or 48 h could, therefore, be determined. The parasite viability fast assay could be completed within 48 h following drug treatment and distinguished between rapidly parasiticidal anti-malarial drugs versus those acting more slowly. The assay was validated against ten standard anti-malarial agents with known properties and results correlated well with established methods. An abbreviated assay, suitable for adaption to medium-high throughput screening, was validated and applied against a set of 20 compounds retrieved from the publically available Medicines for Malaria Venture 'Malaria Box'. The quantification of new infections to determine parasite viability offers important

  1. Development of in-vitro radiometric assay for the rapid assessment of chloroquine resistant plasmodium vivax

    International Nuclear Information System (INIS)

    Myint Oo; Myo Khin; Nwe Nwe Oo

    1997-01-01

    Previously, resistance of malaria parasite to chloroquine has been restricted only to Plasmodium falciparum. Recently, there have been many reports of chloroquine-resistant Plasmodium vivax. One of the mechanisms of chloroquine resistance is the decreased uptake of chloroquine or rapid efflux of the drug from the food vacuole of the parasite. In this study, we have measured the rapid efflux of IH-chloroquine in fifty blood samples from patients with P Vivax infection. All 50 patients were hospitalised for 28 days for the standard treatment with chloroquine. It was found that seven patients who did not respond to the standard regimen of chloroquine have parasites with rapid effluxes of IH-chloroquine. Since rapid effluxes of IH-chloroquine in the resistant parasites showed strong correlation with in vivo 28 days clinical trial, this assay could be used as rapid assessment of chloroquine resistance in patients with P vivax infection

  2. Development of in-vitro radiometric assay for the rapid assessment of chloroquine resistant plasmodium vivax

    Energy Technology Data Exchange (ETDEWEB)

    Oo, Myint; Khin, Myo; Oo, Nwe Nwe [Department of Medical Research, Yangon (Myanmar)

    1997-12-01

    Previously, resistance of malaria parasite to chloroquine has been restricted only to Plasmodium falciparum. Recently, there have been many reports of chloroquine-resistant Plasmodium vivax. One of the mechanisms of chloroquine resistance is the decreased uptake of chloroquine or rapid efflux of the drug from the food vacuole of the parasite. In this study, we have measured the rapid efflux of IH-chloroquine in fifty blood samples from patients with P Vivax infection. All 50 patients were hospitalised for 28 days for the standard treatment with chloroquine. It was found that seven patients who did not respond to the standard regimen of chloroquine have parasites with rapid effluxes of IH-chloroquine. Since rapid effluxes of IH-chloroquine in the resistant parasites showed strong correlation with in vivo 28 days clinical trial, this assay could be used as rapid assessment of chloroquine resistance in patients with P vivax infection.

  3. Drug resistance

    NARCIS (Netherlands)

    Gorter, J.A.; Potschka, H.; Noebels, J.L.; Avoli, M.; Rogawski, M.A.; Olsen, R.W.; Delgado-Escueta, A.V.

    2012-01-01

    Drug resistance remains to be one of the major challenges in epilepsy therapy. Identification of factors that contribute to therapeutic failure is crucial for future development of novel therapeutic strategies for difficult-to-treat epilepsies. Several clinical studies have shown that high seizure

  4. Antimicrobial resistance determinant microarray for analysis of multi-drug resistant isolates

    Science.gov (United States)

    Taitt, Chris Rowe; Leski, Tomasz; Stenger, David; Vora, Gary J.; House, Brent; Nicklasson, Matilda; Pimentel, Guillermo; Zurawski, Daniel V.; Kirkup, Benjamin C.; Craft, David; Waterman, Paige E.; Lesho, Emil P.; Bangurae, Umaru; Ansumana, Rashid

    2012-06-01

    The prevalence of multidrug-resistant infections in personnel wounded in Iraq and Afghanistan has made it challenging for physicians to choose effective therapeutics in a timely fashion. To address the challenge of identifying the potential for drug resistance, we have developed the Antimicrobial Resistance Determinant Microarray (ARDM) to provide DNAbased analysis for over 250 resistance genes covering 12 classes of antibiotics. Over 70 drug-resistant bacteria from different geographic regions have been analyzed on ARDM, with significant differences in patterns of resistance identified: genes for resistance to sulfonamides, trimethoprim, chloramphenicol, rifampin, and macrolide-lincosamidesulfonamide drugs were more frequently identified in isolates from sources in Iraq/Afghanistan. Of particular concern was the presence of genes responsible for resistance to many of the last-resort antibiotics used to treat war traumaassociated infections.

  5. Digital Drug Dosing: Dosing in Drug Assays by Light-Defined Volumes of Hydrogels with Embedded Drug-Loaded Nanoparticles

    DEFF Research Database (Denmark)

    Faralli, Adele; Melander, Fredrik; Larsen, Esben Kjær Unmack

    2014-01-01

    Polyethylene glycol (PEG)-based hydrogels are widely used for biomedical applications, including matrices for controlled drug release. We present a method for defining drug dosing in screening assays by light-activated cross-linking of PEG-diacrylate hydrogels with embedded drug-loaded liposome...

  6. Drug-resistant gram-negative uropathogens: A review.

    Science.gov (United States)

    Khoshnood, Saeed; Heidary, Mohsen; Mirnejad, Reza; Bahramian, Aghil; Sedighi, Mansour; Mirzaei, Habibollah

    2017-10-01

    Urinary tract infection(UTI) caused by Gram-negative bacteria is the second most common infectious presentation in community medical practice. Approximately 150 million people are diagnosed with UTI each year worldwide. Drug resistance in Gram-negative uropathogens is a major global concern which can lead to poor clinical outcomes including treatment failure, development of bacteremia, requirement for intravenous therapy, hospitalization, and extended length of hospital stay. The mechanisms of drug resistance in these bacteria are important due to they are often not identified by routine susceptibility tests and have an exceptional potential for outbreaks. Treatment of UTIs depends on the access to effective drugs, which is now threatened by antibiotic resistant Gram-negative uropathogens. Although several effective antibiotics with activity against highly resistant Gram-negatives are available, there is not a unique antibiotic with activity against the high variety of resistance. Therefore, antimicrobial susceptibility tests, correlation between clinicians and laboratories, development of more rapid diagnostic methods, and continuous monitoring of drug resistance are urgent priorities. In this review, we will discuss about the current global status of drug-resistant Gram-negative uropathogens and their mechanisms of drug resistance to provide new insights into their treatment options. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  7. Adenosine triphosphate-based chemotherapy response assay (ATP-CRA)-guided platinum-based 2-drug chemotherapy for unresectable nonsmall-cell lung cancer.

    Science.gov (United States)

    Moon, Yong Wha; Choi, Sung Ho; Kim, Yong Tai; Sohn, Joo Hyuk; Chang, Joon; Kim, Se Kyu; Park, Moo Suk; Chung, Kyung Young; Lee, Hyoun Ju; Kim, Joo-Hang

    2007-05-01

    The study investigated correlations between adenosine triphosphate / chemotherapy response assay (ATP-CRA) and clinical outcomes after ATP-CRA-guided platinum-based chemotherapy for unresectable nonsmall-cell lung cancer (NSCLC). The authors performed an in vitro chemosensitivity test, ATP-CRA, to evaluate the chemosensitivities of anticancer drugs such as cisplatin, carboplatin, paclitaxel, docetaxel, gemcitabine, and vinorelbine for chemonaive, unresectable NSCLC. The cell death rate was determined by measuring the intracellular ATP levels of drug-exposed cells compared with untreated controls. A sensitive drug was defined as a drug producing 30% or more reduction in ATP compared with untreated controls. Assay-guided platinum-based 2-drug chemotherapy was given to patients with pathologically confirmed NSCLC. Thirty-four patients were enrolled. Thirty tumor specimens were obtained by bronchoscopic biopsies and 4 obtained surgically. The median age was 61 years and 27 patients had an Eastern Cooperative Oncology Group (ECOG) performance status of 0-1. The response rate was 43.8%. At a median follow-up period of 16.9 months, the median progression-free and overall survivals were 3.6 and 11.2 months, respectively. Patients were dichotomized into the platinum-sensitive (S; 20 patients) and resistant (R; 14 patients) groups. The positive/negative predictive values were 61.1% and 78.6% with a predictive accuracy of 68.8%. Although without significant differences in pretreatment parameters, the S-group showed better clinical response (P=.036), longer progression-free survival (P=.060), and longer overall survival (P=.025). Despite using bronchoscopic biopsied specimens, ATP-CRA and clinical outcomes correlated well after assay-guided platinum-based 2-drug chemotherapy for unresectable NSCLC. There was a favorable response and survival in the platinum-sensitive vs resistant groups. Copyright (c) 2007 American Cancer Society

  8. Gene expression analysis of two extensively drug-resistant tuberculosis isolates show that two-component response systems enhance drug resistance.

    Science.gov (United States)

    Yu, Guohua; Cui, Zhenling; Sun, Xian; Peng, Jinfu; Jiang, Jun; Wu, Wei; Huang, Wenhua; Chu, Kaili; Zhang, Lu; Ge, Baoxue; Li, Yao

    2015-05-01

    Global analysis of expression profiles using DNA microarrays was performed between a reference strain H37Rv and two clinical extensively drug-resistant isolates in response to three anti-tuberculosis drug exposures (isoniazid, capreomycin, and rifampicin). A deep analysis was then conducted using a combination of genome sequences of the resistant isolates, resistance information, and related public microarray data. Certain known resistance-associated gene sets were significantly overrepresented in upregulated genes in the resistant isolates relative to that observed in H37Rv, which suggested a link between resistance and expression levels of particular genes. In addition, isoniazid and capreomycin response genes, but not rifampicin, either obtained from published works or our data, were highly consistent with the differentially expressed genes of resistant isolates compared to those of H37Rv, indicating a strong association between drug resistance of the isolates and genes differentially regulated by isoniazid and capreomycin exposures. Based on these results, 92 genes of the studied isolates were identified as candidate resistance genes, 10 of which are known resistance-related genes. Regulatory network analysis of candidate resistance genes using published networks and literature mining showed that three two-component regulatory systems and regulator CRP play significant roles in the resistance of the isolates by mediating the production of essential envelope components. Finally, drug sensitivity testing indicated strong correlations between expression levels of these regulatory genes and sensitivity to multiple anti-tuberculosis drugs in Mycobacterium tuberculosis. These findings may provide novel insights into the mechanism underlying the emergence and development of drug resistance in resistant tuberculosis isolates and useful clues for further studies on this issue. Copyright © 2015 Elsevier Ltd. All rights reserved.

  9. Drug resistance pattern of mycobacterial isolates in HIV and non-HIV population in South India

    Directory of Open Access Journals (Sweden)

    Umamaheshwari Shivaswamy

    2016-01-01

    Full Text Available Background: Emergence of drug resistance has complicated the treatment of tuberculosis (TB. WHO reports India to be one among 27 “high burden” multidrug-resistant (MDR TB countries. Objective: To diagnose TB and detect drug resistance of mycobacterial isolates in acid-fast bacilli (AFB smear negative HIV reactive patients (Group A and compare them with HIV seropositive AFB smear positive (Group B and HIV-seronegative AFB positive cases (Group C. Materials and Methods: Clinical specimens collected in all groups were processed as per the standard protocol except blood, which was processed by lysis centrifugation technique. They were then inoculated with Lowenstein-Jensen media and the isolates obtained were subjected to drug susceptibility test (DST by proportion method and genotype MTBDR plus assay. Results: In Group A, 162 patients were included. Of the 443 clinical samples collected, 76 mycobacterial strains were obtained from 67 (41% patients. Of these, 50 (65.8% were sensitive to all drugs and 26 (34.2% resistant to one or more anti-tubercular drugs. Antibiogram of Group A when compared with Group B and C showed that the MDR rate 6.6%, 6.7% and 8% respectively did not differ much; but resistance to at least single drug was (26 [34.2%], 3 [10%], and 8 [16%], respectively. Conclusion: Our study suggests that HIV has no influence on the anti-tubercular resistance pattern, but increased MDR rate along with HIV in high TB burden setting stresses the need for early diagnosis and DST in providing proper regimens and improve prognosis.

  10. Whole Genome Sequencing Based Characterization of Extensively Drug-Resistant Mycobacterium tuberculosis Isolates from Pakistan

    KAUST Repository

    Ali, Asho; Hasan, Zahra; McNerney, Ruth; Mallard, Kim; Hill-Cawthorne, Grant A.; Coll, Francesc; Nair, Mridul; Pain, Arnab; Clark, Taane G.; Hasan, Rumina

    2015-01-01

    Improved molecular diagnostic methods for detection drug resistance in Mycobacterium tuberculosis (MTB) strains are required. Resistance to first- and second- line anti-tuberculous drugs has been associated with single nucleotide polymorphisms (SNPs) in particular genes. However, these SNPs can vary between MTB lineages therefore local data is required to describe different strain populations. We used whole genome sequencing (WGS) to characterize 37 extensively drug-resistant (XDR) MTB isolates from Pakistan and investigated 40 genes associated with drug resistance. Rifampicin resistance was attributable to SNPs in the rpoB hot-spot region. Isoniazid resistance was most commonly associated with the katG codon 315 (92%) mutation followed by inhA S94A (8%) however, one strain did not have SNPs in katG, inhA or oxyR-ahpC. All strains were pyrazimamide resistant but only 43% had pncA SNPs. Ethambutol resistant strains predominantly had embB codon 306 (62%) mutations, but additional SNPs at embB codons 406, 378 and 328 were also present. Fluoroquinolone resistance was associated with gyrA 91-94 codons in 81% of strains; four strains had only gyr B mutations, while others did not have SNPs in either gyrA or gyrB. Streptomycin resistant strains had mutations in ribosomal RNA genes; rpsL codon 43 (42%); rrs 500 region (16%), and gidB (34%) while six strains did not have mutations in any of these genes. Amikacin/kanamycin/capreomycin resistance was associated with SNPs in rrs at nt1401 (78%) and nt1484 (3%), except in seven (19%) strains. We estimate that if only the common hot-spot region targets of current commercial assays were used, the concordance between phenotypic and genotypic testing for these XDR strains would vary between rifampicin (100%), isoniazid (92%), flouroquinolones (81%), aminoglycoside (78%) and ethambutol (62%); while pncA sequencing would provide genotypic resistance in less than half the isolates. This work highlights the importance of expanded

  11. Whole Genome Sequencing Based Characterization of Extensively Drug-Resistant Mycobacterium tuberculosis Isolates from Pakistan

    KAUST Repository

    Ali, Asho

    2015-02-26

    Improved molecular diagnostic methods for detection drug resistance in Mycobacterium tuberculosis (MTB) strains are required. Resistance to first- and second- line anti-tuberculous drugs has been associated with single nucleotide polymorphisms (SNPs) in particular genes. However, these SNPs can vary between MTB lineages therefore local data is required to describe different strain populations. We used whole genome sequencing (WGS) to characterize 37 extensively drug-resistant (XDR) MTB isolates from Pakistan and investigated 40 genes associated with drug resistance. Rifampicin resistance was attributable to SNPs in the rpoB hot-spot region. Isoniazid resistance was most commonly associated with the katG codon 315 (92%) mutation followed by inhA S94A (8%) however, one strain did not have SNPs in katG, inhA or oxyR-ahpC. All strains were pyrazimamide resistant but only 43% had pncA SNPs. Ethambutol resistant strains predominantly had embB codon 306 (62%) mutations, but additional SNPs at embB codons 406, 378 and 328 were also present. Fluoroquinolone resistance was associated with gyrA 91-94 codons in 81% of strains; four strains had only gyr B mutations, while others did not have SNPs in either gyrA or gyrB. Streptomycin resistant strains had mutations in ribosomal RNA genes; rpsL codon 43 (42%); rrs 500 region (16%), and gidB (34%) while six strains did not have mutations in any of these genes. Amikacin/kanamycin/capreomycin resistance was associated with SNPs in rrs at nt1401 (78%) and nt1484 (3%), except in seven (19%) strains. We estimate that if only the common hot-spot region targets of current commercial assays were used, the concordance between phenotypic and genotypic testing for these XDR strains would vary between rifampicin (100%), isoniazid (92%), flouroquinolones (81%), aminoglycoside (78%) and ethambutol (62%); while pncA sequencing would provide genotypic resistance in less than half the isolates. This work highlights the importance of expanded

  12. MRJP1-containing glycoproteins isolated from honey, a novel antibacterial drug candidate with broad spectrum activity against multi-drug resistant clinical isolates

    Directory of Open Access Journals (Sweden)

    Katrina eBrudzynski

    2015-07-01

    Full Text Available The emergence of extended- spectrum β-lactamase (ESBL is the underlying cause of growing antibiotic resistance among Gram-negative bacteria to β-lactam antibiotics. We recently reported the discovery of honey glycoproteins (glps that exhibited a rapid, concentration-dependent antibacterial activity against both Gram-positive Bacillus subtilis and Gram-negative Escherichia coli that resembled action of cell wall-active β-lactam drugs. Glps showed sequence identity with the Major Royal Jelly Protein 1 (MRJP1 precursor that harbors three antimicrobial peptides: Jelleins 1, 2 and 4. Here, we used semi-quantitative radial diffusion assay and broth microdilution assay to evaluate susceptibility of a number of multi-drug resistant (MDR clinical isolates to the MRJP1-contaning honey glycoproteins. The MDR bacterial strains comprised 3 MRSA, 4 Pseudomonas aeruginosa, 2 Klebsiella pneumoniae, 2 VRE and 5 Extended-spectrum beta-lactamase (ESBL identified as 1 Proteus mirabilis, 3 Escherichia coli and 1 Escherichia coli NDM. Their resistance to different classes of antibiotics was confirmed using automated system Vitek 2. MDR isolates differred in their susceptibility to glps with MIC90 values ranging from 4.8μg/ml against B. subtilis to 14.4μg/ml against ESBL K. pneumoniae, Klebsiella spp ESBL and E. coli and up to 33μg/ml against highly resistant strains of P. aeruginosa. Glps isolated from different honeys showed a similar ability to overcome bacterial resistance to β-lactams suggesting that (a their mode of action is distinct from other classes of β-lactams and that (b the common glps structure was the lead structure responsible for the activity. The results of the current study together with our previous evidence of a rapid bactericidal activity of glps demonstrate that glps possess suitable characteristics to be considered a novel antibacterial drug candidate.

  13. Kinetically Controlled Drug Resistance

    DEFF Research Database (Denmark)

    Sun, Xin E.; Hansen, Bjarne Gram; Hedstrom, Lizbeth

    2011-01-01

    The filamentous fungus Penicillium brevicompactum produces the immunosuppressive drug mycophenolic acid (MPA), which is a potent inhibitor of eukaryotic IMP dehydrogenases (IMPDHs). IMPDH catalyzes the conversion of IMP to XMP via a covalent enzyme intermediate, E-XMP*; MPA inhibits by trapping E...... of resistance is not apparent. Here, we show that, unlike MPA-sensitive IMPDHs, formation of E-XMP* is rate-limiting for both PbIMPDH-A and PbIMPDH-B. Therefore, MPA resistance derives from the failure to accumulate the drug-sensitive intermediate....

  14. Overview of drug-resistant tuberculosis worldwide

    Directory of Open Access Journals (Sweden)

    Ali A Velayati

    2016-01-01

    Full Text Available Even in the 21st century, we are losing the battle against eradication of tuberculosis (TB. In 2015, 9.6 million people were estimated to have fallen ill with TB, of which 1.5 million people died. This is the real situation despite the well-structured treatment programs and availability of effective treatment options since the 1950s. The high mortality rate has been associated with other risk factors, such as the HIV epidemic, underlying diseases, and decline of socioeconomic standards. Furthermore, the problem of drug resistance that was recognized in the early days of the chemotherapeutic era raises serious concerns. Although resistance to a single agent is the most common type, resistance to multiple agents is less frequent but of greater concern. The World Health Organization estimated approximately 5% of all new TB cases involved multidrug-resistant (MDR-TB. The estimation for MDR-TB is 3.3% for new cases, and 20.5% for previously treated cases. Failure to identify and appropriately treat MDR-TB patients has led to more dangerous forms of resistant TB. Based on World Health Organization reports, 5% of global TB cases are now considered to be extensively drug resistant (XDR, defined as MDR with additional resistance to both fluoroquinolones and at least one second-line injectable drug. XDR-TB had been reported by 105 countries by 2015. An estimated 9.7% of people with MDR-TB have XDR-TB. More recently, another dangerous form of TB bacillus was identified, which was named totally drug resistant (TDR-TB or extremely drug resistant TB. These strains were resistant to all first- and second-line anti-TB drugs. Collectively, it is accepted that 2% of MDR-TB strains turn to be TDR-TB. This number, however, may not reflect the real situation, as many laboratories in endemic TB countries do not have proper facilities and updated protocols to detect the XDR or TDR-TB strains. Nevertheless, existing data emphasize the need for additional control

  15. The molecular basis of drug resistance against hepatitis C virus NS3/4A protease inhibitors.

    Directory of Open Access Journals (Sweden)

    Keith P Romano

    Full Text Available Hepatitis C virus (HCV infects over 170 million people worldwide and is the leading cause of chronic liver diseases, including cirrhosis, liver failure, and liver cancer. Available antiviral therapies cause severe side effects and are effective only for a subset of patients, though treatment outcomes have recently been improved by the combination therapy now including boceprevir and telaprevir, which inhibit the viral NS3/4A protease. Despite extensive efforts to develop more potent next-generation protease inhibitors, however, the long-term efficacy of this drug class is challenged by the rapid emergence of resistance. Single-site mutations at protease residues R155, A156 and D168 confer resistance to nearly all inhibitors in clinical development. Thus, developing the next-generation of drugs that retain activity against a broader spectrum of resistant viral variants requires a comprehensive understanding of the molecular basis of drug resistance. In this study, 16 high-resolution crystal structures of four representative protease inhibitors--telaprevir, danoprevir, vaniprevir and MK-5172--in complex with the wild-type protease and three major drug-resistant variants R155K, A156T and D168A, reveal unique molecular underpinnings of resistance to each drug. The drugs exhibit differential susceptibilities to these protease variants in both enzymatic and antiviral assays. Telaprevir, danoprevir and vaniprevir interact directly with sites that confer resistance upon mutation, while MK-5172 interacts in a unique conformation with the catalytic triad. This novel mode of MK-5172 binding explains its retained potency against two multi-drug-resistant variants, R155K and D168A. These findings define the molecular basis of HCV N3/4A protease inhibitor resistance and provide potential strategies for designing robust therapies against this rapidly evolving virus.

  16. Effect of radiation decontamination on drug-resistant bacteria

    International Nuclear Information System (INIS)

    Ito, Hitoshi

    2006-01-01

    More than 80% of food poisoning bacteria such as Salmonella are reported as antibiotic-resistant to at least one type antibiotic, and more than 50% as resistant to two or more. For the decontamination of food poisoning bacteria in foods, radiation resistibility on drug-resistant bacteria were investigated compared with drug-sensitive bacteria. Possibility on induction of drug-resistant mutation by radiation treatment was also investigated. For these studies, type strains of Escherichia coli S2, Salmonella enteritidis YK-2 and Staphylococcus aureus H12 were used to induce drug-resistant strains with penicillin G. From the study of radiation sensitivity on the drug-resistant strain induced from E. coli S2, D 10 value was obtained to be 0.20 kGy compared with 0.25 kGy at parent strain. On S. enteritidis YK-2, D 10 value was obtained to be 0.14 kGy at drug-resistant strain compared with 0.16 kGy at parent strain. D 10 value was also obtained to be 0.15 kGy at drug-resistant strain compared with 0.21 kGy at parent strain of St. aureus H12. Many isolates of E. coli 157:H7 or other type of E. coli from meats such as beef were resistant to penicillin G, and looked to be no relationship on radiation resistivities between drug-resistant strains and sensitive strains. On the study of radiation sensitivity on E. coli S2 at plate agars containing antibiotics, higher survival fractions were obtained at higher doses compared with normal plate agar. The reason of higher survival fractions at higher doses on plate agar containing antibiotics should be recovery of high rate of injured cells by the relay of cell division, and drug-resistant strains by mutation are hardly induced by irradiation. (author)

  17. Spectrofluorometric determination of intracellular levels of reactive oxygen species in drug-sensitive and drug-resistant cancer cells using the 2',7'-dichlorofluorescein diacetate assay

    Energy Technology Data Exchange (ETDEWEB)

    Loetchutinat, Chatchanok [Laboratory of Physical Chemistry, Molecular and Cellular Biology, Faculty of Science, Burapha University, Bangsaen, Chonburi 20131 (Thailand); Kothan, Suchart [Laboratory of Physical Chemistry, Molecular and Cellular Biology, Faculty of Science, Burapha University, Bangsaen, Chonburi 20131 (Thailand); Dechsupa, Samarn [Laboratory of Physical Chemistry, Molecular and Cellular Biology, Faculty of Science, Burapha University, Bangsaen, Chonburi 20131 (Thailand); Meesungnoen, Jintana [Laboratory of Physical Chemistry, Molecular and Cellular Biology, Faculty of Science, Burapha University, Bangsaen, Chonburi 20131 (Thailand); Departement de medecine nucleaire et de radiobiologie, Faculte de medecine, Groupe en sciences des radiations, Universite de Sherbrooke, Sherbrooke, Quebec, J1H 5N4 (Canada); Jay-Gerin, Jean-Paul [Departement de medecine nucleaire et de radiobiologie, Faculte de medecine, Groupe en sciences des radiations, Universite de Sherbrooke, Sherbrooke, Quebec, J1H 5N4 (Canada); Mankhetkorn, Samlee [Laboratory of Physical Chemistry, Molecular and Cellular Biology, Faculty of Science, Burapha University, Bangsaen, Chonburi 20131 (Thailand)]. E-mail: samlee@bucc4.buu.ac.th

    2005-02-01

    This article examines a non-invasive spectrofluorometric method using the 2',7'-dichlorofluorescein diacetate (DCHF-DA) assay for quantifying the intracellular reactive oxygen species (ROS{sub i}) produced in four cultured cancer cell lines: drug-sensitive (K562) and drug-resistant (K562/adr) human erythromyelogenous leukemia cell lines, and drug-sensitive (GLC4) and drug-resistant (GLC4/adr) human small cell lung carcinoma cell lines. The oxidation of the probe to the fluorescent dichlorofluorescein (DCF) was continuously monitored by following the DCF fluorescence intensity as a function of time using a standard spectrofluorometer in the presence of an extracellular DCF fluorescence quencher (Co{sup 2+}). By fitting the spectrofluorometric data to a kinetic model based on the following two reactions: (i) deacetylation of DCHF-DA to the oxidant-sensitive compound 2',7'-dichlorofluorescein (DCHF) by cellular esterase enzymes (pseudo-first-order rate constant: k{sub e}) and (ii) oxidation of DCHF by ROS{sub i} (second-order rate constant: k{sub 2}), the parameters intervening in DCF formation, k{sub e} and the product of k{sub 2} by the ROS{sub i} concentration, were quantitatively determined for the different cell lines studied. The results revealed that the intracellular esterase content or activity is similar in K562, K562/adr, and GLC4 cells, but 5-fold higher in GLC4/adr cells. The product k{sub 2}[ROS{sub i}] was found to be similar in the four cell lines considered, with a mean value of (5.3+/-0.9)x10{sup -7}cell{sup -1}s{sup -1}. Assuming that H{sub 2}O{sub 2} (in combination with peroxidases) is the primary responsible species for DCHF oxidation in intact cells, and using the rate constant value k2=790+/-62M-1s-1 established in our laboratory for the reaction of DCHF with H{sub 2}O{sub 2} in the presence of horseradish peroxidase, the mean value of the intracellular levels of ROS{sub i} in those cells was estimated to be 0.67+/-0.16n

  18. The Effect of Primary Cancer Cell Culture Models on the Results of Drug Chemosensitivity Assays: The Application of Perfusion Microbioreactor System as Cell Culture Vessel

    Science.gov (United States)

    Chen, Yi-Dao; Huang, Shiang-Fu; Wang, Hung-Ming

    2015-01-01

    To precisely and faithfully perform cell-based drug chemosensitivity assays, a well-defined and biologically relevant culture condition is required. For the former, a perfusion microbioreactor system capable of providing a stable culture condition was adopted. For the latter, however, little is known about the impact of culture models on the physiology and chemosensitivity assay results of primary oral cavity cancer cells. To address the issues, experiments were performed. Results showed that minor environmental pH change could significantly affect the metabolic activity of cells, demonstrating the importance of stable culture condition for such assays. Moreover, the culture models could also significantly influence the metabolic activity and proliferation of cells. Furthermore, the choice of culture models might lead to different outcomes of chemosensitivity assays. Compared with the similar test based on tumor-level assays, the spheroid model could overestimate the drug resistance of cells to cisplatin, whereas the 2D and 3D culture models might overestimate the chemosensitivity of cells to such anticancer drug. In this study, the 3D culture models with same cell density as that in tumor samples showed comparable chemosensitivity assay results as the tumor-level assays. Overall, this study has provided some fundamental information for establishing a precise and faithful drug chemosensitivity assay. PMID:25654105

  19. Competitive release of drug resistance following drug treatment of mixed Plasmodium chabaudi infections.

    Science.gov (United States)

    de Roode, Jacobus C; Culleton, Richard; Bell, Andrew S; Read, Andrew F

    2004-09-14

    Malaria infections are often genetically diverse, potentially leading to competition between co-infecting strains. Such competition is of key importance in the spread of drug resistance. The effects of drug treatment on within-host competition were studied using the rodent malaria Plasmodium chabaudi. Mice were infected simultaneously with a drug-resistant and a drug-sensitive clone and were then either drug-treated or left untreated. Transmission was assessed by feeding mice to Anopheles stephensi mosquitoes. In the absence of drugs, the sensitive clone competitively suppressed the resistant clone; this resulted in lower asexual parasite densities and also reduced transmission to the mosquito vector. Drug treatment, however, allowed the resistant clone to fill the ecological space emptied by the removal of the sensitive clone, allowing it to transmit as well as it would have done in the absence of competition. These results show that under drug pressure, resistant strains can have two advantages: (1) they survive better than sensitive strains and (2) they can exploit the opportunities presented by the removal of their competitors. When mixed infections are common, such effects could increase the spread of drug resistance.

  20. High-Throughput Cytochrome P450 Cocktail Inhibition Assay for Assessing Drug-Drug and Drug-Botanical Interactions.

    Science.gov (United States)

    Li, Guannan; Huang, Ke; Nikolic, Dejan; van Breemen, Richard B

    2015-11-01

    Detection of drug-drug interactions is essential during the early stages of drug discovery and development, and the understanding of drug-botanical interactions is important for the safe use of botanical dietary supplements. Among the different forms of drug interactions that are known, inhibition of cytochrome P450 (P450) enzymes is the most common cause of drug-drug or drug-botanical interactions. Therefore, a rapid and comprehensive mass spectrometry-based in vitro high-throughput P450 cocktail inhibition assay was developed that uses 10 substrates simultaneously against nine CYP isoforms. Including probe substrates for CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and two probes targeting different binding sites of CYP3A4/5, this cocktail simultaneously assesses at least as many P450 enzymes as previous assays while remaining among the fastest due to short incubation times and rapid analysis using ultrahigh pressure liquid chromatography-tandem mass spectrometry. The method was validated using known inhibitors of each P450 enzyme and then shown to be useful not only for single-compound testing but also for the evaluation of potential drug-botanical interactions using the botanical dietary supplement licorice (Glycyrrhiza glabra) as an example. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

  1. Distribution of red blood cell antigens in drug-resistant and drug ...

    African Journals Online (AJOL)

    sofo

    Frequency distribution of ABO, Rh-Hr, MN, Kell blood group system antigens were studied in 277 TB patients (151-drug-sensitive and 126 drug-resistant) of pulmonary tuberculosis to know whether there was any association between them, and also between drug resistance and sensitiveness. They were compared with 485 ...

  2. FLIPR assays of intracellular calcium in GPCR drug discovery

    DEFF Research Database (Denmark)

    Hansen, Kasper Bø; Bräuner-Osborne, Hans

    2009-01-01

    Fluorescent dyes sensitive to changes in intracellular calcium have become increasingly popular in G protein-coupled receptor (GPCR) drug discovery for several reasons. First of all, the assays using the dyes are easy to perform and are of low cost compared to other assays. Second, most non...

  3. Synergy of drug combinations in treating multidrug-resistant Pseudomonas aeruginosa.

    Science.gov (United States)

    Rizvi, Meher; Ahmad, Junaid; Khan, Fatima; Shukla, Indu; Malik, Abida; Sami, Hiba

    2015-01-01

    With the emergence of metallo-betalactamases (MBL) in Pseudomonas aeruginosa (P. aeruginosa), the value of carbapenem, the drug of last resort, is being severely compromised. Curtailing the use of carbapenems becomes paramount if resistance is to be reined in. To study the role of synergy between combinations of drugs as an alternative treatment choice for P. aeruginosa. Synergy was studied between combinations of levofloxacin with piperacillin-tazobactam and levofloxacin with cefoperazone-sulbactam by time-kill and chequerboard techniques. P. aeruginosa were tested for antibiotic susceptibility by the disc diffusion assay (260 isolates) and E-test (60 isolates). Synergy testing by chequerboard and time-kill assays was performed with combinations of piperacillin-tazobactam with levofloxacin (11 isolates) and cefoperazone-sulbactam with levofloxacin (10 isolates). Nearly all isolates were susceptible to piperacillin-tazobactam (96.1 per cent), followed by piperacillin (78.5 per cent). Seventy-one isolates (27.3 per cent) were found to be multidrug resistant and 19.6 per cent were ESBL producers. MIC50 of amikacin was 32μg/ml and MIC90 was 64μg/ml. MIC50 and MIC90 of cefoperazone-sulbactam was 32μg/ml and 64μg/ml, and for levofloxacin it was 10μg/ml and 240μg/ml, respectively. Piperacillin-tazobactam had MIC50 and MIC90 of 5μg/ml and 10μg/ml, respectively. Synergy was noted in 72.7 per cent isolates for levofloxacin and piperacillin-tazobactam combination, the remaining 27.3 per cent isolates showed addition by both chequerboard and time-kill assay. For levofloxacin and cefoperazone-sulbactam, only 30 per cent isolates had synergy, 40 per cent showed addition, 20 per cent indifference, and 10 per cent were antagonistic by the chequerboard method. The combination of levofloxacin and piperacillin-tazobactam is a good choice for treatment of such strains.

  4. Direct detection of Mycobacterium tuberculosis and drug resistance in respiratory specimen using Abbott Realtime MTB detection and RIF/INH resistance assay.

    Science.gov (United States)

    Tam, Kingsley King-Gee; Leung, Kenneth Siu-Sing; To, Sabrina Wai-Chi; Siu, Gilman Kit-Hang; Lau, Terrence Chi-Kong; Shek, Victor Chi-Man; Tse, Cindy Wing-Sze; Wong, Samson Sai-Yin; Ho, Pak-Leung; Yam, Wing-Cheong

    2017-10-01

    Abbott RealTime MTB (Abbott-RT) in conjunction with Abbott RealTime MTB RIF/INH Resistance (Abbott-RIF/INH) is a new, high-throughput automated nucleic acid amplification platform (Abbott-MDR) for detection of Mycobacterium tuberculosis complex (MTBC) and the genotypic markers for rifampicin (RIF) and isoniazid (INH) resistance directly from respiratory specimens. This prospective study evaluated the diagnostic performance of this new platform for MTBC and multidrug-resistant tuberculosis (MDR-TB) using 610 sputum specimens in a tuberculosis high-burden setting. Using conventional culture results and clinical background as reference standards, Abbott-RT exhibited an overall sensitivity and specificity of 95.2% and 99.8%, respectively. Genotypic RIF/INH resistance of 178 "MTB detected" specimens was subsequently analyzed by Abbott-RIF/INH. Compared to phenotypic drug susceptibility test results, Abbott-RIF/INH detected resistance genotypic markers in 84.6% MDR-TB, 80% mono-RIF-resistant and 66.7% mono-INH-resistant specimens. Two of the RIF-resistant specimens carried a novel single, nonsense mutation at rpoB Q513 and in silico simulation demonstrated that the truncated RpoB protein failed to bind with other subunits for transcription. Overall, Abbott-MDR platform provided high throughput and reliable diagnosis of MDR-TB within a TB high-burden region. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Molecular Characterization of the Resistance of Mycobacterium ...

    African Journals Online (AJOL)

    Purpose: To characterize the resistance of Mycobacterium tuberculosis to second line drugs using a line probe assay. Methods: Multi-drug resistant strains of Mycobacterium tuberculosis isolated between December 2008 and December 2009 were tested for resistance to fluoroquinolones and second-line injectable drugs ...

  6. Systematic drug screening reveals specific vulnerabilities and co-resistance patterns in endocrine-resistant breast cancer.

    Science.gov (United States)

    Kangaspeska, Sara; Hultsch, Susanne; Jaiswal, Alok; Edgren, Henrik; Mpindi, John-Patrick; Eldfors, Samuli; Brück, Oscar; Aittokallio, Tero; Kallioniemi, Olli

    2016-07-04

    The estrogen receptor (ER) inhibitor tamoxifen reduces breast cancer mortality by 31 % and has served as the standard treatment for ER-positive breast cancers for decades. However, 50 % of advanced ER-positive cancers display de novo resistance to tamoxifen, and acquired resistance evolves in 40 % of patients who initially respond. Mechanisms underlying resistance development remain poorly understood and new therapeutic opportunities are urgently needed. Here, we report the generation and characterization of seven tamoxifen-resistant breast cancer cell lines from four parental strains. Using high throughput drug sensitivity and resistance testing (DSRT) with 279 approved and investigational oncology drugs, exome-sequencing and network analysis, we for the first time, systematically determine the drug response profiles specific to tamoxifen resistance. We discovered emerging vulnerabilities towards specific drugs, such as ERK1/2-, proteasome- and BCL-family inhibitors as the cells became tamoxifen-resistant. Co-resistance to other drugs such as the survivin inhibitor YM155 and the chemotherapeutic agent paclitaxel also occurred. This study indicates that multiple molecular mechanisms dictate endocrine resistance, resulting in unexpected vulnerabilities to initially ineffective drugs, as well as in emerging co-resistances. Thus, combatting drug-resistant tumors will require patient-tailored strategies in order to identify new drug vulnerabilities, and to understand the associated co-resistance patterns.

  7. Radiometric studies on the oxidation of (I-14C) fatty acids by drug-susceptible and drug-resistant mycobacteria

    International Nuclear Information System (INIS)

    Camargo, E.E.; Kopajtic, T.M.; Hopkins, G.K.; Cannon, N.P.; Wagner Junior, H.N.

    1987-01-01

    A radiometric assay system has been used to study oxidation patterns of (l - 14 C) fatty acids by drug-susceptible and drug-resistant organisms of the genus Mycobacterium (M. tuberculosis - H 37 Rv and Erdman, M. bovis, M. avium, M. intracellulare, M.Kansasii and M. chelonei). The organisms were inoculated in sterile reaction vials containing liquid 7H9 medium, 10% ADC enrichment and 1.0 uli of one of the (l- 14 C) fatty acids (butyric, hexanoic, octanoic, decanoic, lauric, myristic, palmitic, stearic, oleic, linoleic, linolenic). Vials were incubated at 37 0 C and the 14 CO 2 envolved was measured daily for 3 days with a Bactec R-301 instrument. (M.A.C.) [pt

  8. A biotin-drug extraction and acid dissociation (BEAD) procedure to eliminate matrix and drug interference in a protein complex anti-drug antibody (ADA) isotype specific assay.

    Science.gov (United States)

    Niu, Hongmei; Klem, Thomas; Yang, Jinsong; Qiu, Yongchang; Pan, Luying

    2017-07-01

    Monitoring anti-drug antibody (ADA) responses in patients receiving protein therapeutics treatment is an important safety assessment for regulatory agencies, drug manufacturers, clinicians and patients. Recombinant human IGF-1/IGFBP-3 (rhIGF-1/rhIGFBP-3) is a 1:1 formulation of naturally occurring protein complex. The individual IGF-1 and IGFBP-3 proteins have multiple binding partners in serum matrix with high binding affinity to each other, which presents challenges in ADA assay development. We have developed a biotin-drug extraction with acid dissociation (BEAD) procedure followed by an electrochemiluminescence (ECL) direct assay to overcome matrix and drug interference. The method utilizes two step acid dissociation and excess biotin-drug to extract total ADA, which are further captured by soluble biotin-drug and detected in an ECL semi-homogeneous direct assay format. The pre-treatment method effectively eliminates interference by serum matrix and free drug, and enhances assay sensitivity. The assays passed acceptance criteria for all validation parameters, and have been used for clinical sample Ab testing. This method principle exemplifies a new approach for anti-isotype ADA assays, and could be an effective strategy for neutralizing antibody (NAb), pharmacokinetic (PK) and biomarker analysis in need of overcoming interference factors. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Development of an opioid self-administration assay to study drug seeking in zebrafish.

    Science.gov (United States)

    Bossé, Gabriel D; Peterson, Randall T

    2017-09-29

    The zebrafish (Danio rerio) has become an excellent tool to study mental health disorders, due to its physiological and genetic similarity to humans, ease of genetic manipulation, and feasibility of small molecule screening. Zebrafish have been shown to exhibit characteristics of addiction to drugs of abuse in non-contingent assays, including conditioned place preference, but contingent assays have been limited to a single assay for alcohol consumption. Using inexpensive electronic, mechanical, and optical components, we developed an automated opioid self-administration assay for zebrafish, enabling us to measure drug seeking and gain insight into the underlying biological pathways. Zebrafish trained in the assay for five days exhibited robust self-administration, which was dependent on the function of the μ-opioid receptor. In addition, a progressive ratio protocol was used to test conditioned animals for motivation. Furthermore, conditioned fish continued to seek the drug despite an adverse consequence and showed signs of stress and anxiety upon withdrawal of the drug. Finally, we validated our assay by confirming that self-administration in zebrafish is dependent on several of the same molecular pathways as in other animal models. Given the ease and throughput of this assay, it will enable identification of important biological pathways regulating drug seeking and could lead to the development of new therapeutic molecules to treat addiction. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Candidate genes for cross-resistance against DNA-damaging drugs

    DEFF Research Database (Denmark)

    Wittig, Rainer; Nessling, Michelle; Will, Rainer D

    2002-01-01

    Drug resistance of tumor cells leads to major drawbacks in the treatment of cancer. To identify candidate genes for drug resistance, we compared the expression patterns of the drug-sensitive human malignant melanoma cell line MeWo and three derived sublines with acquired resistance to the DNA...... as several apoptosis-related genes, in particular STK17A and CRYAB. As MPP1 and CRYAB are also among the 14 genes differentially expressed in all three of the drug-resistant sublines, they represent the strongest candidates for resistance against DNA-damaging drugs....

  11. Emergence of Extensively Drug Resistant Tuberculosis

    Centers for Disease Control (CDC) Podcasts

    Extensively drug-resistant tuberculosis (XDR TB) outbreaks have been reported in South Africa, and strains have been identified on 6 continents. Dr. Peter Cegielski, team leader for drug-resistant TB with the Division of Tuberculosis Elimination at CDC, comments on a multinational team's report on this emerging global public health threat.

  12. Measuring enzymatic HIV-1 susceptibility to two reverse transcriptase inhibitors as a rapid and simple approach to HIV-1 drug-resistance testing.

    Directory of Open Access Journals (Sweden)

    Dieter Hoffmann

    Full Text Available Simple and cost-effective approaches for HIV drug-resistance testing are highly desirable for managing increasingly expanding HIV-1 infected populations who initiate antiretroviral therapy (ART, particularly in resource-limited settings. Non-nucleoside reverse trancriptase inhibitor (NNRTI-based regimens with an NRTI backbone containing lamivudine (3TC or emtricitabine (FTC are preferred first ART regimens. Failure with these drug combinations typically involves the selection of NNRTI- and/or 3TC/FTC-resistant viruses. Therefore, the availability of simple assays to measure both types of drug resistance is critical. We have developed a high throughput screening test for assessing enzymatic resistance of the HIV-1 RT in plasma to 3TC/FTC and NNRTIs. The test uses the sensitive "Amp-RT" assay with a newly-developed real-time PCR format to screen biochemically for drug resistance in single reactions containing either 3TC-triphosphate (3TC-TP or nevirapine (NVP. Assay cut-offs were defined based on testing a large panel of subtype B and non-subtype B clinical samples with known genotypic profiles. Enzymatic 3TC resistance correlated well with the presence of M184I/V, and reduced NVP susceptibility was strongly associated with the presence of K103N, Y181C/I, Y188L, and G190A/Q. The sensitivity and specificity for detecting resistance were 97.0% and 96.0% in samples with M184V, and 97.4% and 96.2% for samples with NNRTI mutations, respectively. We further demonstrate the utility of an HIV capture method in plasma by using magnetic beads coated with CD44 antibody that eliminates the need for ultracentifugation. Thus our results support the use of this simple approach for distinguishing WT from NNRTI- or 3TC/FTC-resistant viruses in clinical samples. This enzymatic testing is subtype-independent and can assist in the clinical management of diverse populations particularly in resource-limited settings.

  13. Early antiretroviral therapy and potent second-line drugs could decrease HIV incidence of drug resistance.

    Science.gov (United States)

    Shen, Mingwang; Xiao, Yanni; Rong, Libin; Meyers, Lauren Ancel; Bellan, Steven E

    2017-06-28

    Early initiation of antiretroviral therapy (ART) reduces the risk of drug-sensitive HIV transmission but may increase the transmission of drug-resistant HIV. We used a mathematical model to estimate the long-term population-level benefits of ART and determine the scenarios under which earlier ART (treatment at 1 year post-infection, on average) could decrease simultaneously both total and drug-resistant HIV incidence (new infections). We constructed an infection-age-structured mathematical model that tracked the transmission rates over the course of infection and modelled the patients' life expectancy as a function of ART initiation timing. We fitted this model to the annual AIDS incidence and death data directly, and to resistance data and demographic data indirectly among men who have sex with men (MSM) in San Francisco. Using counterfactual scenarios, we assessed the impact on total and drug-resistant HIV incidence of ART initiation timing, frequency of acquired drug resistance, and second-line drug effectiveness (defined as the combination of resistance monitoring, biomedical drug efficacy and adherence). Earlier ART initiation could decrease the number of both total and drug-resistant HIV incidence when second-line drug effectiveness is sufficiently high (greater than 80%), but increase the proportion of new infections that are drug resistant. Thus, resistance may paradoxically appear to be increasing while actually decreasing. © 2017 The Author(s).

  14. Repurposing and Revival of the Drugs: A New Approach to Combat the Drug Resistant Tuberculosis

    Directory of Open Access Journals (Sweden)

    Divakar Sharma

    2017-12-01

    Full Text Available Emergence of drug resistant tuberculosis like multi drug resistant tuberculosis (MDR-TB, extensively drug-resistant tuberculosis (XDR-TB and totally drug resistant tuberculosis (TDR-TB has created a new challenge to fight against these bad bugs of Mycobacterium tuberculosis. Repurposing and revival of the drugs are the new trends/options to combat these worsen situations of tuberculosis in the antibiotics resistance era or in the situation of global emergency. Bactericidal and synergistic effect of repurposed/revived drugs along with the latest drugs bedaquiline and delamanid used in the treatment of MDR-TB, XDR-TB, and TDR-TB might be the choice for future promising combinatorial chemotherapy against these bad bugs.

  15. Detection of Anti-Hepatitis B Virus Drug Resistance Mutations Based on Multicolor Melting Curve Analysis.

    Science.gov (United States)

    Mou, Yi; Athar, Muhammad Ammar; Wu, Yuzhen; Xu, Ye; Wu, Jianhua; Xu, Zhenxing; Hayder, Zulfiqar; Khan, Saeed; Idrees, Muhammad; Nasir, Muhammad Israr; Liao, Yiqun; Li, Qingge

    2016-11-01

    Detection of anti-hepatitis B virus (HBV) drug resistance mutations is critical for therapeutic decisions for chronic hepatitis B virus infection. We describe a real-time PCR-based assay using multicolor melting curve analysis (MMCA) that could accurately detect 24 HBV nucleotide mutations at 10 amino acid positions in the reverse transcriptase region of the HBV polymerase gene. The two-reaction assay had a limit of detection of 5 copies per reaction and could detect a minor mutant population (5% of the total population) with the reverse transcriptase M204V amino acid mutation in the presence of the major wild-type population when the overall concentration was 10 4 copies/μl. The assay could be finished within 3 h, and the cost of materials for each sample was less than $10. Clinical validation studies using three groups of samples from both nucleos(t)ide analog-treated and -untreated patients showed that the results for 99.3% (840/846) of the samples and 99.9% (8,454/8,460) of the amino acids were concordant with those of Sanger sequencing of the PCR amplicon from the HBV reverse transcriptase region (PCR Sanger sequencing). HBV DNA in six samples with mixed infections consisting of minor mutant subpopulations was undetected by the PCR Sanger sequencing method but was detected by MMCA, and the results were confirmed by coamplification at a lower denaturation temperature-PCR Sanger sequencing. Among the treated patients, 48.6% (103/212) harbored viruses that displayed lamivudine monoresistance, adefovir monoresistance, entecavir resistance, or lamivudine and adefovir resistance. Among the untreated patients, the Chinese group had more mutation-containing samples than did the Pakistani group (3.3% versus 0.56%). Because of its accuracy, rapidness, wide-range coverage, and cost-effectiveness, the real-time PCR assay could be a robust tool for the detection if anti-HBV drug resistance mutations in resource-limited countries. Copyright © 2016, American Society for

  16. Drug-resistant tuberculosis: emerging treatment options

    Directory of Open Access Journals (Sweden)

    Adhvaryu MR

    2011-12-01

    Full Text Available Meghna Adhvaryu1, Bhasker Vakharia21Department of Biotechnology, SRK Institute of Computer Education and Applied Sciences, 2R&D, Bhuma Research in Ayurvedic and Herbal Medicine, Surat, Gujarat, IndiaAbstract: Multidrug-resistant tuberculosis has emerged worldwide, with an increasing incidence due to failure of implementation of apparently effective first-line antituberculous therapy as well as primary infection with drug-resistant strains. Failure of current therapy is attributed to a long duration of treatment leading to nonadherence and irregular therapy, lack of patient education about the disease, poverty, irregular supply by care providers, drug–drug interactions in patients coinfected with human immunodeficiency virus (HIV, inadequate regulations causing market overlap and irresponsible drug usage in the private sector, and lack of research, with no addition of new drugs in the last four decades. Present standards of care for the treatment of drug-susceptible tuberculosis, multidrug-resistant tuberculosis, tuberculosis-HIV coinfection, and latent tuberculosis infection are all unsatisfactory. Since 2000, the World Health Organization (WHO has focused on drug development for tuberculosis, as well as research in all relevant aspects to discover new regimens by 2015 and to eliminate tuberculosis as a public health concern by 2050. As a result, some 20 promising compounds from 14 groups of drugs have been discovered. Twelve candidates from eight classes are currently being evaluated in clinical trials. Ongoing research should prioritize identification of novel targets and newer application of existing drugs, discovery of multitargeted drugs from natural compounds, strengthening host factors by immunopotentiation with herbal immunomodulators, as well as protective vaccines before and after exposure, consideration of surgical measures when indicated, development of tools for rapid diagnosis, early identification of resistant strains, and

  17. Mechanisms of first-line antimicrobial resistance in multi-drug and extensively drug resistant strains of Mycobacterium tuberculosis in KwaZulu-Natal, South Africa

    Directory of Open Access Journals (Sweden)

    Navisha Dookie

    2016-10-01

    Full Text Available Abstract Background In South Africa, drug resistant tuberculosis is a major public health crisis in the face of the colossal HIV pandemic. Methods In an attempt to understand the distribution of drug resistance in our setting, we analysed the rpoB, katG, inhA, pncA and embB genes associated with resistance to key drugs used in the treatment of tuberculosis in clinical isolates of Mycobacterium tuberculosis in the KwaZulu-Natal province. Results Classical mutations were detected in the katG, inhA and embB genes associated with resistance to isoniazid and ethambutol. Diverse mutations were recorded in the multidrug resistant (MDR and extensively drug resistant (XDR isolates for the rpoB and pncA gene associated with resistance to rifampicin and pyrazinamide. Conclusions M.tuberculosis strains circulating in our setting display a combination of previously observed mutations, each mediating resistance to a different drug. The MDR and XDR TB isolates analysed in this study displayed classical mutations linked to INH and EMB resistance, whilst diverse mutations were linked to RIF and PZA resistance. The similarity of the XDR strains confirms reports of the clonality of the XDR epidemic. The successful dissemination of the drug resistant strains in the province underscores the need for rapid diagnostics to effectively diagnose drug resistance and guide treatment.

  18. In vitro testing of chemotherapeutic drug combinations in acute myelocytic leukaemia using the fluorometric microculture cytotoxicity assay (FMCA).

    Science.gov (United States)

    Larsson, R; Fridborg, H; Kristensen, J; Sundström, C; Nygren, P

    1993-05-01

    The fluorometric microculture cytotoxicity assay (FMCA) was employed for analysing the effect of different chemotherapeutic drug combinations and their single constituents in 44 cases of acute myelocytic leukaemia (AML). A large heterogeneity with respect to cell kill was observed for all combinations tested, the interactions ranging from antagonistic to synergistic in terms of the multiplicative concept for drug interactions. However, an 'additive' model provided a significantly better fit of the data compared to the effect of the most active single agent of the combination (Dmax) for several common antileukaemic drug combinations. When the two interaction models were related to treatment outcome 38% of the non-responders showed preference for the additive model whereas the corresponding figure for responders was 80%. Overall, in 248 of 290 (85%) tests performed with drug combinations, there was an agreement between the effect of the combination and that of the most active single component. Direct comparison of Dmax and the combination for correlation with clinical outcome demonstrated only minor differences in the ability to predict drug resistance. The results show that FMCA appear to report drug interactions in samples from patients with AML in accordance with clinical experience. Furthermore, testing single agents as a substitute for drug combinations may be adequate for detection of clinical drug resistance to combination therapy in AML.

  19. Sample preparation composite and replicate strategy for assay of solid oral drug products.

    Science.gov (United States)

    Harrington, Brent; Nickerson, Beverly; Guo, Michele Xuemei; Barber, Marc; Giamalva, David; Lee, Carlos; Scrivens, Garry

    2014-12-16

    In pharmaceutical analysis, the results of drug product assay testing are used to make decisions regarding the quality, efficacy, and stability of the drug product. In order to make sound risk-based decisions concerning drug product potency, an understanding of the uncertainty of the reportable assay value is required. Utilizing the most restrictive criteria in current regulatory documentation, a maximum variability attributed to method repeatability is defined for a drug product potency assay. A sampling strategy that reduces the repeatability component of the assay variability below this predefined maximum is demonstrated. The sampling strategy consists of determining the number of dosage units (k) to be prepared in a composite sample of which there may be a number of equivalent replicate (r) sample preparations. The variability, as measured by the standard error (SE), of a potency assay consists of several sources such as sample preparation and dosage unit variability. A sampling scheme that increases the number of sample preparations (r) and/or number of dosage units (k) per sample preparation will reduce the assay variability and thus decrease the uncertainty around decisions made concerning the potency of the drug product. A maximum allowable repeatability component of the standard error (SE) for the potency assay is derived using material in current regulatory documents. A table of solutions for the number of dosage units per sample preparation (r) and number of replicate sample preparations (k) is presented for any ratio of sample preparation and dosage unit variability.

  20. Multidrug resistant to extensively drug resistant tuberculosis: What is ...

    Indian Academy of Sciences (India)

    Prakash

    The modern, ... World Health Organization is based on a four-drug regimen ... Better management and control of tuberculosis specially drug resistant TB by experienced and qualified .... a comprehensive approach including the major DOTS.

  1. Systematic drug screening reveals specific vulnerabilities and co-resistance patterns in endocrine-resistant breast cancer

    International Nuclear Information System (INIS)

    Kangaspeska, Sara; Hultsch, Susanne; Jaiswal, Alok; Edgren, Henrik; Mpindi, John-Patrick; Eldfors, Samuli; Brück, Oscar; Aittokallio, Tero; Kallioniemi, Olli

    2016-01-01

    The estrogen receptor (ER) inhibitor tamoxifen reduces breast cancer mortality by 31 % and has served as the standard treatment for ER-positive breast cancers for decades. However, 50 % of advanced ER-positive cancers display de novo resistance to tamoxifen, and acquired resistance evolves in 40 % of patients who initially respond. Mechanisms underlying resistance development remain poorly understood and new therapeutic opportunities are urgently needed. Here, we report the generation and characterization of seven tamoxifen-resistant breast cancer cell lines from four parental strains. Using high throughput drug sensitivity and resistance testing (DSRT) with 279 approved and investigational oncology drugs, exome-sequencing and network analysis, we for the first time, systematically determine the drug response profiles specific to tamoxifen resistance. We discovered emerging vulnerabilities towards specific drugs, such as ERK1/2-, proteasome- and BCL-family inhibitors as the cells became tamoxifen-resistant. Co-resistance to other drugs such as the survivin inhibitor YM155 and the chemotherapeutic agent paclitaxel also occurred. This study indicates that multiple molecular mechanisms dictate endocrine resistance, resulting in unexpected vulnerabilities to initially ineffective drugs, as well as in emerging co-resistances. Thus, combatting drug-resistant tumors will require patient-tailored strategies in order to identify new drug vulnerabilities, and to understand the associated co-resistance patterns. The online version of this article (doi:10.1186/s12885-016-2452-5) contains supplementary material, which is available to authorized users

  2. Computational Studies of Drug Resistance

    DEFF Research Database (Denmark)

    da Silva Martins, João Miguel

    Drug resistance has been an increasing problem in patient treatment and drug development. Starting in the last century and becoming a major worry in the medical and scienti c communities in the early part of the current millennium, major research must be performed to address the issues of viral...... is of the utmost importance in developing better and less resistance-inducing drugs. A drug's in uence can be characterized in many diff erent ways, however, and the approaches I take in this work re ect those same different in uences. This is what I try to achieve in this work, through seemingly unrelated...... approaches that come together in the study of drug's and their in uence on proteins and vice-versa. In part I, I aim to understand through combined theoretical ensemble analysis and free energy calculations the e ects mutations have over the binding anity and function of the M2 proton channel. This research...

  3. Drug resistance patterns in pulmonary tuberculosis

    International Nuclear Information System (INIS)

    Khoharo, H.K.; Shaikh, I.A.

    2011-01-01

    Objective: To determine the resistance patterns of mycobacterium tuberculosis (MTB) isolates among category I and II patients of pulmonary tuberculosis. Methods: This cross sectional study was conducted at the Department of Medicine, Liaquat University of Medical and Health Sciences Jamshoro, from November 2008 to September 2009. Patients were divided into category I and II. The sputa were collected, stained with Ziehl-Nielsen (Z-N) staining and ultimately inoculated on Lowenstein-Jensen (L-J) media for six weeks. Out of 890 pulmonary tuberculosis (PTB) patients, the growth was obtained in 285 cases. The Drug sensitivity testing (DST) for Isoniazid (INH), Rifampicin (RIF), Ethambutol (EMB) Pyrazinamide (PZA) and Streptomycin (SM) were performed. The data was analyzed on SPSS 10.0. A p-value of <0.05 was taken as significant. Result: Out of 285 cases, 176 (61.75%) were male and 109 (38.24%) female. The mean age was 37 +- 19.90 years. The DST showed drug sensitive and drug resistant isolates in 80 (28.05%) and 205 (71.92%) cases respectively (p=0.001). The drug resistant tuberculosis (DR-TB) rates for individual drugs; INH, RIF, EMB, PZA and SM were 51,22%, 15.4%, 13.33%, 9%12, and 3.85% respectively (p=0.03). The MDR-TB isolates were detected in 120 (42.10%) cases, including 5 (5.88%) in category I and 115 (57.50%) in category II patients (p=0.0001). Conclusion: Drug resistant and multidrug resistant tuberculosis was observed mainly in category II patients. However, primary MDR was also observed in category I patients and reflects dissemination of MDR cases within the community. (author)

  4. Rationale and uses of a public HIV drug-resistance database.

    Science.gov (United States)

    Shafer, Robert W

    2006-09-15

    Knowledge regarding the drug resistance of human immunodeficiency virus (HIV) is critical for surveillance of drug resistance, development of antiretroviral drugs, and management of infections with drug-resistant viruses. Such knowledge is derived from studies that correlate genetic variation in the targets of therapy with the antiretroviral treatments received by persons from whom the variant was obtained (genotype-treatment), with drug-susceptibility data on genetic variants (genotype-phenotype), and with virological and clinical response to a new treatment regimen (genotype-outcome). An HIV drug-resistance database is required to represent, store, and analyze the diverse forms of data underlying our knowledge of drug resistance and to make these data available to the broad community of researchers studying drug resistance in HIV and clinicians using HIV drug-resistance tests. Such genotype-treatment, genotype-phenotype, and genotype-outcome correlations are contained in the Stanford HIV RT and Protease Sequence Database and have specific usefulness.

  5. HIV Genetic Diversity and Drug Resistance

    Science.gov (United States)

    Santos, André F.; Soares, Marcelo A.

    2010-01-01

    Most of the current knowledge on antiretroviral (ARV) drug development and resistance is based on the study of subtype B of HIV-1, which only accounts for 10% of the worldwide HIV infections. Cumulative evidence has emerged that different HIV types, groups and subtypes harbor distinct biological properties, including the response and susceptibility to ARV. Recent laboratory and clinical data highlighting such disparities are summarized in this review. Variations in drug susceptibility, in the emergence and selection of specific drug resistance mutations, in viral replicative capacity and in the dynamics of resistance acquisition under ARV selective pressure are discussed. Clinical responses to ARV therapy and associated confounding factors are also analyzed in the context of infections by distinct HIV genetic variants. PMID:21994646

  6. A population-based study of first and second-line drug-resistant tuberculosis in a high-burden area of the Mexico/United States border

    Directory of Open Access Journals (Sweden)

    Pola Becerril-Montes

    2013-04-01

    Full Text Available The resistance of 139 Mycobacterium tuberculosis (MTB isolates from the city of Monterrey, Northeast Mexico, to first and second-line anti-TB drugs was analysed. A total of 73 isolates were susceptible and 66 were resistant to anti-TB drugs. Monoresistance to streptomycin, isoniazid (INH and ethambutol was observed in 29 cases. Resistance to INH was found in 52 cases and in 29 cases INH resistance was combined with resistance to two or three drugs. A total of 24 isolates were multidrug-resistant (MDR resistant to at least INH and rifampicin and 11 MDR cases were resistant to five drugs. The proportion of MDR-TB among new TB cases in our target population was 0.72% (1/139 cases. The proportion of MDR-TB among previously treated cases was 25.18% (35/139 cases. The 13 polyresistant and 24 MDR isolates were assayed against the following seven second-line drugs: amikacin (AMK, kanamycin (KAN, capreomycin (CAP, clofazimine (CLF, ethionamide (ETH, ofloxacin (OFL and cycloserine (CLS. Resistance to CLF, OFL or CLS was not observed. Resistance was detected to ETH (10.80% and to AMK (2.70%, KAN (2.70% and CAP (2.70%. One isolate of MDR with primary resistance was also resistant to three second-line drugs. Monterrey has a high prevalence of MDR-TB among previously treated cases and extensively drug-resistant-MTB strains may soon appear.

  7. Spread of anti-malarial drug resistance: Mathematical model with implications for ACT drug policies

    Directory of Open Access Journals (Sweden)

    Dondorp Arjen M

    2008-11-01

    Full Text Available Abstract Background Most malaria-endemic countries are implementing a change in anti-malarial drug policy to artemisinin-based combination therapy (ACT. The impact of different drug choices and implementation strategies is uncertain. Data from many epidemiological studies in different levels of malaria endemicity and in areas with the highest prevalence of drug resistance like borders of Thailand are certainly valuable. Formulating an appropriate dynamic data-driven model is a powerful predictive tool for exploring the impact of these strategies quantitatively. Methods A comprehensive model was constructed incorporating important epidemiological and biological factors of human, mosquito, parasite and treatment. The iterative process of developing the model, identifying data needed, and parameterization has been taken to strongly link the model to the empirical evidence. The model provides quantitative measures of outcomes, such as malaria prevalence/incidence and treatment failure, and illustrates the spread of resistance in low and high transmission settings. The model was used to evaluate different anti-malarial policy options focusing on ACT deployment. Results The model predicts robustly that in low transmission settings drug resistance spreads faster than in high transmission settings, and treatment failure is the main force driving the spread of drug resistance. In low transmission settings, ACT slows the spread of drug resistance to a partner drug, especially at high coverage rates. This effect decreases exponentially with increasing delay in deploying the ACT and decreasing rates of coverage. In the high transmission settings, however, drug resistance is driven by the proportion of the human population with a residual drug level, which gives resistant parasites some survival advantage. The spread of drug resistance could be slowed down by controlling presumptive drug use and avoiding the use of combination therapies containing drugs with

  8. Antimicrobial Drugs in Fighting against Antimicrobial Resistance

    OpenAIRE

    Cheng, Guyue; Dai, Menghong; Ahmed, Saeed; Hao, Haihong; Wang, Xu; Yuan, Zonghui

    2016-01-01

    The outbreak of antimicrobial resistance, together with the lack of newly developed antimicrobial drugs, represents an alarming signal for both human and animal healthcare worldwide. Selection of rational dosage regimens for traditional antimicrobial drugs based on pharmacokinetic/pharmacodynamic principles as well as development of novel antimicrobials targeting new bacterial targets or resistance mechanisms are key approaches in tackling AMR. In addition to the cellular level resistance (i....

  9. Geographic and Temporal Trends in the Molecular Epidemiology and Genetic Mechanisms of Transmitted HIV-1 Drug Resistance: An Individual-Patient- and Sequence-Level Meta-Analysis

    NARCIS (Netherlands)

    S.Y. Rhee (Soo Yoon); J.L. Blanco (Jose Luis); M.R. Jordan (Michael); J. Taylor (Jonathan); P. Lemey (Philippe); V. Varghese (Vici); R.L. Hamers (Raph); S. Bertagnolio (Silvia); M. De Wit (Meike); A.F. Aghokeng (Avelin); J. Albert (Jan); R. Avi (Radko); S. Avila-Rios (Santiago); P.O. Bessong (Pascal O.); J.I. Brooks (James I.); C.A.B. Boucher (Charles); Z.L. Brumme (Zabrina L.); M.P. Busch (Michael P.); H. Bussmann (Hermann); M.L. Chaix (Marie Laure); B.S. Chin (Bum Sik); T.T. D’Aquin (Toni T.); C. de Gascun (Cillian); A. Derache (Anne); D. Descamps (Diane); A.K. Deshpande (Alaka K.); C.F. Djoko (Cyrille F.); S.H. Eshleman (Susan H.); H. Fleury (Hervé); P. Frange (Pierre); S. Fujisaki (Seiichiro); P. Harrigan (Pr); J. Hattori (Junko); A. Holguin (Africa); G.M. Hunt (Gillian M.); H. Ichimura (Hiroshi); P. Kaleebu (Pontiano); D. Katzenstein (David); S. Kiertiburanakul (Sasisopin); J.H. Kim (Jerome H.); S.S. Kim (Sung Soon); Y. Li (Yanpeng); I. Lutsar (Irja); L. Morris (L.); N. Ndembi (Nicaise); K.P. NG (Kee Peng); R.S. Paranjape (Ramesh S.); M.C. Peeters (Marian); M. Poljak (Mario); M.A. Price (Matt A.); M.L. Ragonnet-Cronin (Manon L.); G. Reyes-Terán (Gustavo); M. Rolland (Morgane); S. Sirivichayakul (Sunee); D.M. Smith (Davey M.); M.A. Soares (Marcelo A.); V. Soriano (Virtudes); D. Ssemwanga (Deogratius); M. Stanojevic (Maja); M.A. Stefani (Mariane A.); W. Sugiura (Wataru); S. Sungkanuparph (Somnuek); A. Tanuri (Amilcar); K.K. Tee (Kok Keng); H.-H.M. Truong (Hong-Ha M.); D.A.M.C. van de Vijver (David); N. Vidal (Nicole); C. Yang (Chunfu); R. Yang (Rongge); G. Yebra (Gonzalo); J.P.A. Ioannidis (John); A.M. Vandamme (Anne Mieke); R.W. Shafer (Robert)

    2015-01-01

    textabstractRegional and subtype-specific mutational patterns of HIV-1 transmitted drug resistance (TDR) are essential for informing first-line antiretroviral (ARV) therapy guidelines and designing diagnostic assays for use in regions where standard genotypic resistance testing is not affordable. We

  10. ZK DrugResist 2.0: A TextMiner to extract semantic relations of drug resistance from PubMed.

    Science.gov (United States)

    Khalid, Zoya; Sezerman, Osman Ugur

    2017-05-01

    Extracting useful knowledge from an unstructured textual data is a challenging task for biologists, since biomedical literature is growing exponentially on a daily basis. Building an automated method for such tasks is gaining much attention of researchers. ZK DrugResist is an online tool that automatically extracts mutations and expression changes associated with drug resistance from PubMed. In this study we have extended our tool to include semantic relations extracted from biomedical text covering drug resistance and established a server including both of these features. Our system was tested for three relations, Resistance (R), Intermediate (I) and Susceptible (S) by applying hybrid feature set. From the last few decades the focus has changed to hybrid approaches as it provides better results. In our case this approach combines rule-based methods with machine learning techniques. The results showed 97.67% accuracy with 96% precision, recall and F-measure. The results have outperformed the previously existing relation extraction systems thus can facilitate computational analysis of drug resistance against complex diseases and further can be implemented on other areas of biomedicine. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Cytotoxic drug sensitivity testing of tumor cells from patients with ovarian carcinoma using the fluorometric microculture cytotoxicity assay (FMCA).

    Science.gov (United States)

    Csoka, K; Larsson, R; Tholander, B; Gerdin, E; de la Torre, M; Nygren, P

    1994-08-01

    The automated fluorometric microculture cytotoxicity assay (FMCA) is based on the measurement of fluorescence generated from cellular hydrolysis of fluorescein diacetate (FDA) to fluorescein by viable cells after a 72-hr culture period in microtiter plates. The FMCA was adopted for chemosensitivity testing of tumor cells from patients with ovarian carcinoma. Thirty-seven samples of solid tumors and malignant effusions were obtained from 35 patients at diagnosis or relapse. Tumor cells from solid samples and effusions were prepared by enzymatic digestion and centrifugation, respectively, followed by Percoll or Ficoll purification. The fluorescence was proportional to the number of cells/well and considerably higher in tumor cells than in contaminating normal cells. The effect of up to 19 cytotoxic drugs was successfully assessed in 70% of the samples and there was a good correlation between drug sensitivity data reported by the FMCA and the DiSC assay performed in parallel. The overall drug sensitivity pattern in vitro corresponded well to the clinical experience. The effect of cisplatin varied considerably between patients and resistance was found also in cases not previously exposed to cytotoxic drugs. The FMCA is a rapid and simple method that seems to report clinically relevant cytotoxic drug sensitivity data in ovarian carcinomas. In the future, this method may contribute to optimizing chemotherapy by assisting in individualized drug selection and new drug development.

  12. Efflux Pump-mediated Drug Resistance in Burkholderia

    Directory of Open Access Journals (Sweden)

    Nicole L Podnecky

    2015-04-01

    Full Text Available Several members of the genus Burkholderia are prominent pathogens. Infections caused by these bacteria are difficult to treat because of significant antibiotic resistance. Virtually all Burkholderia species are also resistant to polymyxin, prohibiting use of drugs like colistin that are available for treatment of infections caused by most other drug resistant Gram-negative bacteria. Despite clinical significance and antibiotic resistance of Burkholderia species, characterization of efflux pumps lags behind other non-enteric Gram-negative pathogens such as Acinetobacter baumannii and Pseudomonas aeruginosa. Although efflux pumps have been described in several Burkholderia species, they have been best studied in B. cenocepacia and B. pseudomallei. As in other non-enteric Gram-negatives, efflux pumps of the resistance nodulation cell division (RND family are the clinically most significant efflux systems in these two species. Several efflux pumps were described in B. cenocepacia, which when expressed confer resistance to clinically significant antibiotics, including aminoglycosides, chloramphenicol, fluoroquinolones, and tetracyclines. Three RND pumps have been characterized in B. pseudomallei, two of which confer either intrinsic or acquired resistance to aminoglycosides, macrolides, chloramphenicol, fluoroquinolones, tetracyclines, trimethoprim, and in some instances trimethoprim+sulfamethoxazole. Several strains of the host-adapted B. mallei, a clone of B. pseudomallei, lack AmrAB-OprA and are therefore aminoglycoside and macrolide susceptible. B. thailandensis is closely related to B. pseudomallei, but non-pathogenic to humans. Its pump repertoire and ensuing drug resistance profile parallels that of B. pseudomallei. An efflux pump in B. vietnamiensis plays a significant role in acquired aminoglycoside resistance. Summarily, efflux pumps are significant players in Burkholderia drug resistance.

  13. Drug Resistance of Mycobacterium tuberculosis Complex among ...

    African Journals Online (AJOL)

    BACKGROUND: In Burkina Faso, there is no recent data about the level of drug resistance in Mycobacterium tuberculosis strains among newly diagnosed tuberculosis cases. OBJECTIVE: To provide an update of the primary drug resistance of mycobacterium tuberculosis among patients in Burkina faso. METHODS: ...

  14. Enhanced transmission of drug-resistant parasites to mosquitoes following drug treatment in rodent malaria.

    Directory of Open Access Journals (Sweden)

    Andrew S Bell

    Full Text Available The evolution of drug resistant Plasmodium parasites is a major challenge to effective malaria control. In theory, competitive interactions between sensitive parasites and resistant parasites within infections are a major determinant of the rate at which parasite evolution undermines drug efficacy. Competitive suppression of resistant parasites in untreated hosts slows the spread of resistance; competitive release following treatment enhances it. Here we report that for the murine model Plasmodium chabaudi, co-infection with drug-sensitive parasites can prevent the transmission of initially rare resistant parasites to mosquitoes. Removal of drug-sensitive parasites following chemotherapy enabled resistant parasites to transmit to mosquitoes as successfully as sensitive parasites in the absence of treatment. We also show that the genetic composition of gametocyte populations in host venous blood accurately reflects the genetic composition of gametocytes taken up by mosquitoes. Our data demonstrate that, at least for this mouse model, aggressive chemotherapy leads to very effective transmission of highly resistant parasites that are present in an infection, the very parasites which undermine the long term efficacy of front-line drugs.

  15. Antimicrobial (Drug) Resistance Prevention

    Science.gov (United States)

    ... June 6, 2018 HIV Vaccine Elicits Antibodies in Animals that Neutralize Dozens of HIV Strains , June 4, 2018 ... Antimicrobial (Drug) Resistance > Understanding share with facebook share with twitter share ...

  16. Disinfectant-susceptibility of multi-drug-resistant Mycobacterium tuberculosis isolated in Japan

    Directory of Open Access Journals (Sweden)

    Noriko Shinoda

    2016-02-01

    Full Text Available Abstract Background Multi-drug-resistant Mycobacterium tuberculosis has been an important problem in public health around the world. However, limited information about disinfectant-susceptibility of multi-drug-resistant strain of M. tuberculosis was available. Findings We studied susceptibility of several Japanese isolates of multi-drug-resistant M. tuberculosis against disinfectants, which are commonly used in clinical and research laboratories. We selected a laboratory reference strain (H37Rv and eight Japanese isolates, containing five drug-susceptible strains and three multi-drug-resistant strains, and determined profiles of susceptibility against eight disinfectants. The M. tuberculosis strains were distinguished into two groups by the susceptibility profile. There was no relationship between multi-drug-resistance and disinfectant-susceptibility in the M. tuberculosis strains. Cresol soap and oxydol were effective against all strains we tested, regardless of drug resistance. Conclusions Disinfectant-resistance is independent from multi-drug-resistance in M. tuberculosis. Cresol soap and oxydol were effective against all strains we tested, regardless of drug resistance.

  17. Functional miRNAs in breast cancer drug resistance

    Directory of Open Access Journals (Sweden)

    Hu WZ

    2018-03-01

    Full Text Available Weizi Hu,1–3,* Chunli Tan,1–3,* Yunjie He,4 Guangqin Zhang,2 Yong Xu,3,5 Jinhai Tang1 1Department of General Surgery, The First Affiliated Hospital of Nanjing Medical University, 2School of Basic Medicine and Clinical Pharmacy, China Pharmaceutical University, 3Nanjing Medical University Affiliated Cancer Hospital, 4The First Clinical School of Nanjing Medical University, 5Jiangsu Key Lab of Cancer Biomarkers, Prevention and Treatment, Nanjing Medical University, Nanjing, People’s Republic of China *These authors contributed equally to this work Abstract: Owing to improved early surveillance and advanced therapy strategies, the current death rate due to breast cancer has decreased; nevertheless, drug resistance and relapse remain obstacles on the path to successful systematic treatment. Multiple mechanisms responsible for drug resistance have been elucidated, and miRNAs seem to play a major part in almost every aspect of cancer progression, including tumorigenesis, metastasis, and drug resistance. In recent years, exosomes have emerged as novel modes of intercellular signaling vehicles, initiating cell–cell communication through their fusion with target cell membranes, delivering functional molecules including miRNAs and proteins. This review particularly focuses on enumerating functional miRNAs involved in breast cancer drug resistance as well as their targets and related mechanisms. Subsequently, we discuss the prospects and challenges of miRNA function in drug resistance and highlight valuable approaches for the investigation of the role of exosomal miRNAs in breast cancer progression and drug resistance. Keywords: microRNA, exosome, breast cancer, drug resistance

  18. Geographic and temporal trends in the molecular epidemiology and genetic mechanisms of transmitted HIV-1 drug resistance: an individual-patient- and sequence-level meta-analysis

    NARCIS (Netherlands)

    Rhee, Soo-Yon; Blanco, Jose Luis; Jordan, Michael R.; Taylor, Jonathan; Lemey, Philippe; Varghese, Vici; Hamers, Raph L.; Bertagnolio, Silvia; Rinke de Wit, Tobias F.; Aghokeng, Avelin F.; Albert, Jan; Avi, Radko; Avila-Rios, Santiago; Bessong, Pascal O.; Brooks, James I.; Boucher, Charles A. B.; Brumme, Zabrina L.; Busch, Michael P.; Bussmann, Hermann; Chaix, Marie-Laure; Chin, Bum Sik; D'Aquin, Toni T.; de Gascun, Cillian F.; Derache, Anne; Descamps, Diane; Deshpande, Alaka K.; Djoko, Cyrille F.; Eshleman, Susan H.; Fleury, Herve; Frange, Pierre; Fujisaki, Seiichiro; Harrigan, P. Richard; Hattori, Junko; Holguin, Africa; Hunt, Gillian M.; Ichimura, Hiroshi; Kaleebu, Pontiano; Katzenstein, David; Kiertiburanakul, Sasisopin; Kim, Jerome H.; Kim, Sung Soon; Li, Yanpeng; Lutsar, Irja; Morris, Lynn; Ndembi, Nicaise; Ng, Kee Peng; Paranjape, Ramesh S.; Peeters, Martine; Poljak, Mario; Price, Matt A.; Ragonnet-Cronin, Manon L.; Reyes-Terán, Gustavo; Rolland, Morgane; Sirivichayakul, Sunee; Smith, Davey M.; Soares, Marcelo A.; Soriano, Vincent V.; Ssemwanga, Deogratius; Stanojevic, Maja; Stefani, Mariane A.; Sugiura, Wataru; Sungkanuparph, Somnuek; Tanuri, Amilcar; tee, Kok Keng; Truong, Hong-Ha M.; van de Vijver, David A. M. C.; Vidal, Nicole; Yang, Chunfu; Yang, Rongge; Yebra, Gonzalo; Ioannidis, John P. A.; Vandamme, Anne-Mieke; Shafer, Robert W.

    2015-01-01

    Regional and subtype-specific mutational patterns of HIV-1 transmitted drug resistance (TDR) are essential for informing first-line antiretroviral (ARV) therapy guidelines and designing diagnostic assays for use in regions where standard genotypic resistance testing is not affordable. We sought to

  19. Epigenetic modulation of the biophysical properties of drug-resistant cell lipids to restore drug transport and endocytic functions.

    Science.gov (United States)

    Vijayaraghavalu, Sivakumar; Peetla, Chiranjeevi; Lu, Shan; Labhasetwar, Vinod

    2012-09-04

    In our recent studies exploring the biophysical characteristics of resistant cell lipids, and the role they play in drug transport, we demonstrated the difference of drug-resistant breast cancer cells from drug-sensitive cells in lipid composition and biophysical properties, suggesting that cancer cells acquire a drug-resistant phenotype through the alteration of lipid synthesis to inhibit intracellular drug transport to protect from cytotoxic effect. In cancer cells, epigenetic changes (e.g., DNA hypermethylation) are essential to maintain this drug-resistant phenotype. Thus, altered lipid synthesis may be linked to epigenetic mechanisms of drug resistance. We hypothesize that reversing DNA hypermethylation in resistant cells with an epigenetic drug could alter lipid synthesis, changing the cell membrane's biophysical properties to facilitate drug delivery to overcome drug resistance. Herein we show that treating drug-resistant breast cancer cells (MCF-7/ADR) with the epigenetic drug 5-aza-2'-deoxycytidine (decitabine) significantly alters cell lipid composition and biophysical properties, causing the resistant cells to acquire biophysical characteristics similar to those of sensitive cell (MCF-7) lipids. Following decitabine treatment, resistant cells demonstrated increased sphingomyelinase activity, resulting in a decreased sphingomyelin level that influenced lipid domain structures, increased membrane fluidity, and reduced P-glycoprotein expression. Changes in the biophysical characteristics of resistant cell lipids facilitated doxorubicin transport and restored endocytic function for drug delivery with a lipid-encapsulated form of doxorubicin, enhancing the drug efficacy. In conclusion, we have established a new mechanism for efficacy of an epigenetic drug, mediated through changes in lipid composition and biophysical properties, in reversing cancer drug resistance.

  20. Emergence of Extensively Drug Resistant Tuberculosis

    Centers for Disease Control (CDC) Podcasts

    2007-03-01

    Extensively drug-resistant tuberculosis (XDR TB) outbreaks have been reported in South Africa, and strains have been identified on 6 continents. Dr. Peter Cegielski, team leader for drug-resistant TB with the Division of Tuberculosis Elimination at CDC, comments on a multinational team's report on this emerging global public health threat.  Created: 3/1/2007 by Emerging Infectious Diseases.   Date Released: 3/26/2007.

  1. Multi drug resistance tuberculosis: pattern seen in last 13 years

    International Nuclear Information System (INIS)

    Iqbal, R.; Shabbir, I.; Munir, K.; Tabassum, M.N.; Khan, S.U.; Khan, M.Z.U.

    2011-01-01

    Background: Drug resistance in tuberculosis is a serious problem throughout the world especially, after the emergence of multi drug resistant TB strains. Objectives: To estimate drug resistance in TB patients and compare it with previous studies to see the changing trends. Materials and Methods: The PMRC Research Centre receives sputum samples from all the leading hospitals of Lahore. This retrospective analysis was done from 1996 to 2008 on the multi drug resistant TB strains that were seen during these years. Five first lines anti tuberculosis drugs were tested on Lowenstein Jensen medium using standard proportion method. Results: A total of 2661 confirmed isolates of Mycobacterium tuberculosis were seen over the past 13 years. Of the total, 2182 were pulmonary and 479 were extra pulmonary specimens. The patients comprised of those with and without history of previous treatment. These specimens were subjected to drug susceptibility testing. Almost half of the patient had some resistance; multiple drug resistance was seen in 12.3% and 23.0% cases without and with history of previous treatment respectively. Overall resistance to rifampicin was 26.4%, isoniazid 24.1% streptomycin 21.6% ethambutol 13.4% and pyrazinamide 28.4% respectively. Statistically significant difference was seen between primary and acquired resistance. When compared with the reports from previous studies from the same area, there was a trend of gradual increase of drug resistance. Conclusions Resistance to anti tuberculosis drugs is high. Policy message. TB Control Program should start 'DOTS Plus' schemes for which drug susceptibility testing facilities should be available for correctly managing the patients. (author)

  2. Multi drug resistance tuberculosis: pattern seen in last 13 years

    Energy Technology Data Exchange (ETDEWEB)

    Iqbal, R; Shabbir, I; Munir, K [King Edward Medical University Hospital, Lahore (Pakistan). Dept. of Research Centre; Tabassum, M N; Khan, S U; Khan, M Z.U. [King Edward Medical University Hospital, Lahore (Pakistan). Dept. of Chest Medicine

    2011-01-15

    Background: Drug resistance in tuberculosis is a serious problem throughout the world especially, after the emergence of multi drug resistant TB strains. Objectives: To estimate drug resistance in TB patients and compare it with previous studies to see the changing trends. Materials and Methods: The PMRC Research Centre receives sputum samples from all the leading hospitals of Lahore. This retrospective analysis was done from 1996 to 2008 on the multi drug resistant TB strains that were seen during these years. Five first lines anti tuberculosis drugs were tested on Lowenstein Jensen medium using standard proportion method. Results: A total of 2661 confirmed isolates of Mycobacterium tuberculosis were seen over the past 13 years. Of the total, 2182 were pulmonary and 479 were extra pulmonary specimens. The patients comprised of those with and without history of previous treatment. These specimens were subjected to drug susceptibility testing. Almost half of the patient had some resistance; multiple drug resistance was seen in 12.3% and 23.0% cases without and with history of previous treatment respectively. Overall resistance to rifampicin was 26.4%, isoniazid 24.1% streptomycin 21.6% ethambutol 13.4% and pyrazinamide 28.4% respectively. Statistically significant difference was seen between primary and acquired resistance. When compared with the reports from previous studies from the same area, there was a trend of gradual increase of drug resistance. Conclusions Resistance to anti tuberculosis drugs is high. Policy message. TB Control Program should start 'DOTS Plus' schemes for which drug susceptibility testing facilities should be available for correctly managing the patients. (author)

  3. Sample preparation composite and replicate strategy case studies for assay of solid oral drug products.

    Science.gov (United States)

    Nickerson, Beverly; Harrington, Brent; Li, Fasheng; Guo, Michele Xuemei

    2017-11-30

    Drug product assay is one of several tests required for new drug products to ensure the quality of the product at release and throughout the life cycle of the product. Drug product assay testing is typically performed by preparing a composite sample of multiple dosage units to obtain an assay value representative of the batch. In some cases replicate composite samples may be prepared and the reportable assay value is the average value of all the replicates. In previously published work by Harrington et al. (2014) [5], a sample preparation composite and replicate strategy for assay was developed to provide a systematic approach which accounts for variability due to the analytical method and dosage form with a standard error of the potency assay criteria based on compendia and regulatory requirements. In this work, this sample preparation composite and replicate strategy for assay is applied to several case studies to demonstrate the utility of this approach and its application at various stages of pharmaceutical drug product development. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Development in Assay Methods for in Vitro Antimalarial Drug Efficacy Testing: A Systematic Review

    Directory of Open Access Journals (Sweden)

    Shweta Sinha

    2017-10-01

    Full Text Available The emergence and spread of drug resistance are the major challenges in malaria eradication mission. Besides various strategies laid down by World Health Organization, such as vector management, source reduction, early case detection, prompt treatment, and development of new diagnostics and vaccines, nevertheless the need for new and efficacious drugs against malaria has become a critical priority on the global malaria research agenda. At several screening stages, millions of compounds are screened (1,000–2,000,000 compounds per screening campaign, before pre-clinical trials to select optimum lead. Carrying out in vitro screening of antimalarials is very difficult as different assay methods are subject to numerous sources of variability across different laboratories around the globe. Despite this, in vitro screening is an essential part of antimalarial drug development as it enables to resource various confounding factors such as host immune response and drug–drug interaction. Therefore, in this article, we try to illustrate the basic necessity behind in vitro study and how new methods are developed and subsequently adopted for high-throughput antimalarial drug screening and its application in achieving the next level of in vitro screening based on the current approaches (such as stem cells.

  5. Cytotoxicity of anthraquinones from the roots of Pentas schimperi towards multi-factorial drug-resistant cancer cells.

    Science.gov (United States)

    Kuete, Victor; Donfack, Arno R Nanfack; Mbaveng, Armelle T; Zeino, Maen; Tane, Pierre; Efferth, Thomas

    2015-08-01

    Multidrug resistance in cancer represents a major problem in chemotherapy. The present study was designed to assess the cytotoxicity of anthraquinones from Pentas schimperi, namely damnacanthal (1), damnacanthol (2), 3-hydroxy-2-hydroxymethyl anthraquinone (3) and schimperiquinone B (4) against nine drug-sensitive and multidrug resistant (MDR) cancer cell lines. The resazurin reduction assay was used to evaluate the cytotoxicity of the above compounds, whilst caspase-Glo assay was used to detect the activation of caspases enzymes by compounds 1 and 2. Cell cycle, mitochondrial membrane potential (MMP) and levels of reactive oxygen species were all analyzed via flow cytometry. Anthraquinones 1 and 2 displayed cytotoxic effects with IC50 values below 81 μM on all the nine tested cancer cell lines whilst 3 and 4 displayed selective activities. The recorded IC50 values for compounds 1 and 2 ranged from 3.12 μM and 12.18 μM (towards leukemia CCRF-CEM cells) and from 30.32 μM and 80.11 μM (towards gliobastoma U87MG.ΔEGFR cells) respectively, and from 0.20 μM (against CCRF-CEM cells) to 195.12 μM (against CEM/ADR5000 cells) for doxorubicin. Compounds 1 and 2 induced apoptosis in CCRF-CEM leukemia cells, mediated by the disruption of the MMP and increase in ROS production. Anthraquinones from Pentas schimperi and mostly 1 and 2 are potential cytotoxic natural products that deserve more investigations to develop novel antineoplastic drugs against multifactorial drug resistant cancers.

  6. A spheroid-based 3-D culture model for pancreatic cancer drug testing, using the acid phosphatase assay

    International Nuclear Information System (INIS)

    Wen, Z.; Liao, Q.; Hu, Y.; You, L.; Zhou, L.; Zhao, Y.

    2013-01-01

    Current therapy for pancreatic cancer is multimodal, involving surgery and chemotherapy. However, development of pancreatic cancer therapies requires a thorough evaluation of drug efficacy in vitro before animal testing and subsequent clinical trials. Compared to two-dimensional culture of cell monolayer, three-dimensional (3-D) models more closely mimic native tissues, since the tumor microenvironment established in 3-D models often plays a significant role in cancer progression and cellular responses to the drugs. Accumulating evidence has highlighted the benefits of 3-D in vitro models of various cancers. In the present study, we have developed a spheroid-based, 3-D culture of pancreatic cancer cell lines MIAPaCa-2 and PANC-1 for pancreatic drug testing, using the acid phosphatase assay. Drug efficacy testing showed that spheroids had much higher drug resistance than monolayers. This model, which is characteristically reproducible and easy and offers rapid handling, is the preferred choice for filling the gap between monolayer cell cultures and in vivo models in the process of drug development and testing for pancreatic cancer

  7. A spheroid-based 3-D culture model for pancreatic cancer drug testing, using the acid phosphatase assay

    Directory of Open Access Journals (Sweden)

    Z. Wen

    2013-08-01

    Full Text Available Current therapy for pancreatic cancer is multimodal, involving surgery and chemotherapy. However, development of pancreatic cancer therapies requires a thorough evaluation of drug efficacy in vitro before animal testing and subsequent clinical trials. Compared to two-dimensional culture of cell monolayer, three-dimensional (3-D models more closely mimic native tissues, since the tumor microenvironment established in 3-D models often plays a significant role in cancer progression and cellular responses to the drugs. Accumulating evidence has highlighted the benefits of 3-D in vitro models of various cancers. In the present study, we have developed a spheroid-based, 3-D culture of pancreatic cancer cell lines MIAPaCa-2 and PANC-1 for pancreatic drug testing, using the acid phosphatase assay. Drug efficacy testing showed that spheroids had much higher drug resistance than monolayers. This model, which is characteristically reproducible and easy and offers rapid handling, is the preferred choice for filling the gap between monolayer cell cultures and in vivo models in the process of drug development and testing for pancreatic cancer.

  8. A Simple Assay to Screen Antimicrobial Compounds Potentiating the Activity of Current Antibiotics

    Directory of Open Access Journals (Sweden)

    Junaid Iqbal

    2013-01-01

    Full Text Available Antibiotic resistance continues to pose a significant problem in the management of bacterial infections, despite advances in antimicrobial chemotherapy and supportive care. Here, we suggest a simple, inexpensive, and easy-to-perform assay to screen antimicrobial compounds from natural products or synthetic chemical libraries for their potential to work in tandem with the available antibiotics against multiple drug-resistant bacteria. The aqueous extract of Juglans regia tree bark was tested against representative multiple drug-resistant bacteria in the aforementioned assay to determine whether it potentiates the activity of selected antibiotics. The aqueous extract of J. regia bark was added to Mueller-Hinton agar, followed by a lawn of multiple drug-resistant bacteria, Salmonella typhi or enteropathogenic E. coli. Next, filter paper discs impregnated with different classes of antibiotics were placed on the agar surface. Bacteria incubated with extract or antibiotics alone were used as controls. The results showed a significant increase (>30% in the zone of inhibition around the aztreonam, cefuroxime, and ampicillin discs compared with bacteria incubated with the antibiotics/extract alone. In conclusion, our assay is able to detect either synergistic or additive action of J. regia extract against multiple drug-resistant bacteria when tested with a range of antibiotics.

  9. Repurposing High-Throughput Image Assays Enables Biological Activity Prediction for Drug Discovery.

    Science.gov (United States)

    Simm, Jaak; Klambauer, Günter; Arany, Adam; Steijaert, Marvin; Wegner, Jörg Kurt; Gustin, Emmanuel; Chupakhin, Vladimir; Chong, Yolanda T; Vialard, Jorge; Buijnsters, Peter; Velter, Ingrid; Vapirev, Alexander; Singh, Shantanu; Carpenter, Anne E; Wuyts, Roel; Hochreiter, Sepp; Moreau, Yves; Ceulemans, Hugo

    2018-05-17

    In both academia and the pharmaceutical industry, large-scale assays for drug discovery are expensive and often impractical, particularly for the increasingly important physiologically relevant model systems that require primary cells, organoids, whole organisms, or expensive or rare reagents. We hypothesized that data from a single high-throughput imaging assay can be repurposed to predict the biological activity of compounds in other assays, even those targeting alternate pathways or biological processes. Indeed, quantitative information extracted from a three-channel microscopy-based screen for glucocorticoid receptor translocation was able to predict assay-specific biological activity in two ongoing drug discovery projects. In these projects, repurposing increased hit rates by 50- to 250-fold over that of the initial project assays while increasing the chemical structure diversity of the hits. Our results suggest that data from high-content screens are a rich source of information that can be used to predict and replace customized biological assays. Copyright © 2018 Elsevier Ltd. All rights reserved.

  10. Drug resistance in the sexually transmitted protozoan Trichomonas vaginalis

    Institute of Scientific and Technical Information of China (English)

    REBECCA L DUNNE; LINDA A DUNN; PETER UPCROFT; PETER J O'DONOGHUE; JACQUELINE A UPCROFT

    2003-01-01

    Trichomoniasis is the most common, sexually transmitted infection. It is caused by the flagellated protozoan parasite Trichomonas vaginalis. Symptoms include vaginitis and infections have been associated with preterm delivery, low birth weight and increased infant mortality, as well as predisposing to HIV/AIDS and cervical cancer. Trichomoniasis has the highest prevalence and incidence of any sexually transmitted infection. The 5-nitroimidazole drugs, of which metronidazole is the most prescribed, are the only approved,effective drugs to treat trichomoniasis. Resistance against metronidazole is frequently reported and crossresistance among the family of 5-nitroimidazole drugs is common, leaving no alternative for treatment, with some cases remaining unresolved. The mechanism of metronidazole resistance in T. vaginalis from treatment failures is not well understood, unlike resistance which is developed in the laboratory under increasing metronidazole pressure. In the latter situation, hydrogenosomal function which is involved in activation of the prodrug, metronidazole, is down-regulated. Reversion to sensitivity is incomplete after removal of drug pressure in the highly resistant parasites while clinically resistant strains, so far analysed, maintain their resistance levels in the absence of drug pressure. Although anaerobic resistance has been regarded as a laboratory induced phenomenon, it clearly has been demonstrated in clinical isolates. Pursuit of both approaches will allow dissection of the underlying mechanisms. Many alternative drugs and treatments have been tested in vivo in cases of refractory trichomoniasis, as well as in vitro with some successes including the broad spectrum anti-parasitic drug nitazoxanide. Drug resistance incidence in T. vaginalis appears to be on the increase and improved surveillance of treatment failures is urged.

  11. Cytotoxicity of the indole alkaloid reserpine from Rauwolfia serpentina against drug-resistant tumor cells.

    Science.gov (United States)

    Abdelfatah, Sara A A; Efferth, Thomas

    2015-02-15

    The antihypertensive reserpine is an indole alkaloid from Rauwolfia serpentina and exerts also profound activity against cancer cells in vitro and in vivo. The present investigation was undertaken to investigate possible modes of action to explain its activity toward drug-resistant tumor cells. Sensitive and drug-resistant tumor cell lines overexpressing P-glycoprotein (ABCB1/MDR1), breast cancer resistance protein (ABCG2/BCRP), mutation-activated epidermal growth factor receptor (EGFR), wild-type and p53-knockout cells as well as the NCI panel of cell lines from different tumor origin were analyzed. Reserpine's cytotoxicity was investigated by resazurin and sulforhodamine assays, flow cytometry, and COMPARE and hierarchical cluster analyses of transcriptome-wide microarray-based RNA expressions. P-glycoprotein- or BCRP overexpressing tumor cells did not reveal cross-resistance to reserpine. EGFR-overexpressing cells were collateral sensitive and p53- Knockout cells cross-resistant to this drug compared to their wild-type parental cell lines. Reserpine increased the uptake of doxorubicin in P-glycoprotein-overexpressing cells, indicating that reserpine inhibited the efflux function of P-glycoprotein. Using molecular docking, we found that reserpine bound with even higher binding energy to P-glycoprotein and EGFR than the control drugs verapamil (P-glycoprotein inhibitor) and erlotinib (EGFR inhibitor). COMPARE and cluster analyses of microarray data showed that the mRNA expression of a panel of genes predicted the sensitivity or resistance of the NCI tumor cell line panel with statistical significance. The genes belonged to diverse pathways and biological functions, e.g. cell survival and apoptosis, EGFR activation, regulation of angiogenesis, cell mobility, cell adhesion, immunological functions, mTOR signaling, and Wnt signaling. The lack of cross-resistance to most resistance mechanisms and the collateral sensitivity in EGFR-transfectants compared to wild

  12. Combined antiretroviral and anti- tuberculosis drug resistance ...

    African Journals Online (AJOL)

    these epidemics, many challenges remain.[3] Antiretroviral and anti-TB drug resistance pose considerable threats to the control of these epidemics.[4,5]. The breakdown in HIV/TB control within prisons is another emerging threat.[6,7] We describe one of the first reports of combined antiretroviral and anti-TB drug resistance ...

  13. The Culture Repopulation Ability (CRA) Assay and Incubation in Low Oxygen to Test Antileukemic Drugs on Imatinib-Resistant CML Stem-Like Cells.

    Science.gov (United States)

    Cheloni, Giulia; Tanturli, Michele

    2016-01-01

    Chronic myeloid leukemia (CML) is a stem cell-driven disorder caused by the BCR/Abl oncoprotein, a constitutively active tyrosine kinase (TK). Chronic-phase CML patients are treated with impressive efficacy with TK inhibitors (TKi) such as imatinib mesylate (IM). However, rather than definitively curing CML, TKi induces a state of minimal residual disease, due to the persistence of leukemia stem cells (LSC) which are insensitive to this class of drugs. LSC persistence may be due to different reasons, including the suppression of BCR/Abl oncoprotein. It has been shown that this suppression follows incubation in low oxygen under appropriate culture conditions and incubation times.Here we describe the culture repopulation ability (CRA) assay, a non-clonogenic assay capable - together with incubation in low oxygen - to reveal in vitro stem cells endowed with marrow repopulation ability (MRA) in vivo. The CRA assay can be used, before moving to animal tests, as a simple and reliable method for the prescreening of drugs potentially active on CML and other leukemias with respect to their activity on the more immature leukemia cell subsets.

  14. Mechanisms of Candida biofilm drug resistance

    Science.gov (United States)

    Taff, Heather T; Mitchell, Kaitlin F; Edward, Jessica A; Andes, David R

    2013-01-01

    Candida commonly adheres to implanted medical devices, growing as a resilient biofilm capable of withstanding extraordinarily high antifungal concentrations. As currently available antifungals have minimal activity against biofilms, new drugs to treat these recalcitrant infections are urgently needed. Recent investigations have begun to shed light on the mechanisms behind the profound resistance associated with the biofilm mode of growth. This resistance appears to be multifactorial, involving both mechanisms similar to conventional, planktonic antifungal resistance, such as increased efflux pump activity, as well as mechanisms specific to the biofilm lifestyle. A unique biofilm property is the production of an extracellular matrix. Two components of this material, β-glucan and extracellular DNA, promote biofilm resistance to multiple antifungals. Biofilm formation also engages several stress response pathways that impair the activity of azole drugs. Resistance within a biofilm is often heterogeneous, with the development of a subpopulation of resistant persister cells. In this article we review the molecular mechanisms underlying Candida biofilm antifungal resistance and their relative contributions during various growth phases. PMID:24059922

  15. Streptococcus pneumoniae Drugs Resistance in Acute Rhinosinusitis

    Directory of Open Access Journals (Sweden)

    Chong Jie Hao

    2016-03-01

    Full Text Available Background: Acute rhinosinusitis that usually caused by Streptococcus pneumoniae becomes the reason why patients seek for medical care. Drugs resistance in Streptococcus pneumoniae is increasing worldwide. This study was conducted to determine drugs resistance of Streptococcus pneumonia from acute rhinosinusitis in Dr. Hasan Sadikin General Hospital. Methods: A descriptive laboratory study was conducted in June–October 2014 at the Laboratory of Microbiology Faculty of Medicine Universitas Padjadjaran. The sample was taken using nasopharyngeal swabbing from 100 acute rhinosinusitis patients in Dr. Hasan Sadikin General Hospital and planted on tryptic soy agar containing 5% sheep blood and 5 μg/ml of gentamicin sulphate and then incubated in 5% CO2 incubator at 37°C for 24 hours. The identification of Streptococcus pneumonia was performed by optochin test. The susceptibility test against Streptococcus pneumoniae was done using disk diffusion method.The antibiotic disks were trimethoprim-sulfamethoxazole, oxacillin, levofloxacin, azithromycin, and doxycycline. Results: Out of 100 samples, 8 of them were tested positive for Streptococcus pneumoniae. Three of Streptococcus pneumoniae isolates died with unknown reason after it were stored at -80 .The drugs resistance test showed the resistance of Streptococcus pneumonia to oxacillin, azithromycin and trimethoprim were 6, whereas levofloxacin and doxycycline are 4. Conclusions: Streptococcus pneumonia drugs resistance in acute rhinosinusitis shows the resistance of Streptococcus pneumoniae to oxacillin, azithromycin and trimethoprim are 6, whereas the resistance to levofloxacin and doxycycline are 4.

  16. Cell-patterned glass spray for direct drug assay using mass spectrometry

    International Nuclear Information System (INIS)

    Wu, Jing; Wang, Shiqi; Chen, Qiushui; Jiang, Hao; Liang, Shuping; Lin, Jin-Ming

    2015-01-01

    In this work, the establishment of a glass spray mass spectrometry (GS-MS) platform for direct cell-based drug assay was described. Cell co-culture, drug-induced cell apoptosis, proliferation analysis and intracellular drug absorption measurement were performed simultaneously on this specifically designed platform. Two groups of co-cultured cells (NIH-3T3/HepG2 and HepG2/MCF-7) were cultivated and they showed high viability within 3 days. The biocompatibility of the platform facilitated the subsequent bioassays, in which, cyclophosphamide (CPA) and genistein were used as the model drugs. The distinctions of cell apoptosis and proliferation between the mono-cultured and co-cultured cells were clearly observed and well explained by in situ GS-MS measurements. A satisfactory linearity of the calibration curve between the relative MS intensity and CPA concentrations was obtained using stable isotope labeling method (y = 0.16545 + 0.0985x, R"2 = 0.9937). The variations in the quantity of absorbed drug were detected and the results were consistent with the concentration-dependence of cell apoptosis. All the results demonstrated that direct cell-based drug assay could be performed on the stable isotope labeling assisted GS-MS platform in a facile and quantitative manner. - Highlights: • A versatile glass spray mass spectrometry (GS-MS) platform for direct cell-based drug assay was developed in this paper. • It has characteristics of the atmospheric pressure ionization method. • It is multifunctional for cell co-culture, bioassays, qualitative and quantitative intracellular drug absorption measurement. • GS-MS has the potential to increase the use of mass spectrometry in biological analysis.

  17. A Structural View on Medicinal Chemistry Strategies against Drug Resistance.

    Science.gov (United States)

    Agnello, Stefano; Brand, Michael; Chellat, Mathieu F; Gazzola, Silvia; Riedl, Rainer

    2018-05-30

    The natural phenomenon of drug resistance represents a generic impairment that hampers the benefits of drugs in all major clinical indications. Antibacterials and antifungals are affected as well as compounds for the treatment of cancer, viral infections or parasitic diseases. Despite the very diverse set of biological targets and organisms involved in the development of drug resistance, underlying molecular processes have been identified to understand the emergence of resistance and to overcome this detrimental mechanism. Detailed structural information of the root causes for drug resistance is nowadays frequently available to design next generation drugs anticipated to suffer less from resistance. This knowledge-based approach is a prerequisite in the fight against the inevitable occurrence of drug resistance to secure the achievements of medicinal chemistry in the future. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Changing prevalence and resistance patterns in children with drug-resistant tuberculosis in Mumbai.

    Science.gov (United States)

    Shah, Ira; Shah, Forum

    2017-05-01

    The prevalence of drug-resistant (DR) tuberculosis (TB) in children is increasing. Although, in India, multi-drug-resistant (MDR) TB rates have been relatively stable, the number of children with pre-extensively drug-resistant and extensively drug-resistant (XDR) TB is increasing. To determine whether the prevalence of DR TB in children in Mumbai is changing and to study the evolving patterns of resistance. A retrospective study was undertaken in 1311 paediatric patients referred between April 2007 and March 2013 to the Paediatric TB clinic at B. J. Wadia Hospital for Children, Mumbai. Children were defined as having DR TB on the basis of drug susceptibility testing (DST) of Mycobacterium tuberculosis grown on culture of body fluids (in the case of extra pulmonary TB) or from gastric lavage/bronchi-alveolar lavage/sputum in patients with pulmonary TB or from DST of the contacts. The prevalence of DR TB was calculated and the type of DR was evaluated yearly and in the pre-2010 and post-2010 eras. The overall prevalence of DR TB was 86 (6.6%) with an increase from 23 (5.6%) patients pre-2010 to 63 (7%) post-2010 (P = 0.40). Nine (10.4%) patients were diagnosed on the basis of contact with a parent with DR TB. Overall fluoroquinolone resistance increased from 9 (39.1%) pre-2010 to 59 (93.7%) post-2010 (P = 0.0001): moxifloxacin resistance increased from 2 (8.7%) to 29 (46%) (P = 0.0018) and ofloxacin resistance increased from 7 (30.4%) to 30 (47.6%) (P = 0.14). Ethionamide resistance also increased from 6 (26.1%) to 31 (49.2%) (P = 0.04), aminoglycoside resistance was one (4.3%) pre-2010 and 12 (19%) post-2010 (P = 0.17) and resistance remained virtually the same for both amikacin [0 pre-2010 and 6 (9.5%) after 2010] and kanamycin [one (4.3%) pre- and 6 (9.5%) post-2010]. Of the first-line drugs, resistance remained the same for isoniazid [23 (100%) to 61 (96.8%)], rifampicin [22 (95.7%) to 51 (80.9%),P = 0.17], pyrazinamide [15 (65.2%) to

  19. Fitness of Leishmania donovani parasites resistant to drug combinations.

    Directory of Open Access Journals (Sweden)

    Raquel García-Hernández

    2015-04-01

    Full Text Available Drug resistance represents one of the main problems for the use of chemotherapy to treat leishmaniasis. Additionally, it could provide some advantages to Leishmania parasites, such as a higher capacity to survive in stress conditions. In this work, in mixed populations of Leishmania donovani parasites, we have analyzed whether experimentally resistant lines to one or two combined anti-leishmanial drugs better support the stress conditions than a susceptible line expressing luciferase (Luc line. In the absence of stress, none of the Leishmania lines showed growth advantage relative to the other when mixed at a 1:1 parasite ratio. However, when promastigotes from resistant lines and the Luc line were mixed and exposed to different stresses, we observed that the resistant lines are more tolerant of different stress conditions: nutrient starvation and heat shock-pH stress. Further to this, we observed that intracellular amastigotes from resistant lines present a higher capacity to survive inside the macrophages than those of the control line. These results suggest that resistant parasites acquire an overall fitness increase and that resistance to drug combinations presents significant differences in their fitness capacity versus single-drug resistant parasites, particularly in intracellular amastigotes. These results contribute to the assessment of the possible impact of drug resistance on leishmaniasis control programs.

  20. Nanoparticles: Alternatives Against Drug-Resistant Pathogenic Microbes

    Directory of Open Access Journals (Sweden)

    Gudepalya Renukaiah Rudramurthy

    2016-06-01

    Full Text Available Antimicrobial substances may be synthetic, semisynthetic, or of natural origin (i.e., from plants and animals. Antimicrobials are considered “miracle drugs” and can determine if an infected patient/animal recovers or dies. However, the misuse of antimicrobials has led to the development of multi-drug-resistant bacteria, which is one of the greatest challenges for healthcare practitioners and is a significant global threat. The major concern with the development of antimicrobial resistance is the spread of resistant organisms. The replacement of conventional antimicrobials by new technology to counteract antimicrobial resistance is ongoing. Nanotechnology-driven innovations provide hope for patients and practitioners in overcoming the problem of drug resistance. Nanomaterials have tremendous potential in both the medical and veterinary fields. Several nanostructures comprising metallic particles have been developed to counteract microbial pathogens. The effectiveness of nanoparticles (NPs depends on the interaction between the microorganism and the NPs. The development of effective nanomaterials requires in-depth knowledge of the physicochemical properties of NPs and the biological aspects of microorganisms. However, the risks associated with using NPs in healthcare need to be addressed. The present review highlights the antimicrobial effects of various nanomaterials and their potential advantages, drawbacks, or side effects. In addition, this comprehensive information may be useful in the discovery of broad-spectrum antimicrobial drugs for use against multi-drug-resistant microbial pathogens in the near future.

  1. Validation of a modified fluorimetric assay for the screening of trichomonacidal drugs

    Directory of Open Access Journals (Sweden)

    Alexandra Ibáñez Escribano

    2012-08-01

    Full Text Available A fluorimetric microassay that uses a redox dye to determine the viability of the flagellate Trichomonas vaginalis has been optimised to provide a more sensitive method to evaluate potential trichomonacidal compounds. Resazurin has been used in recent years to test drugs against different parasites, including trichomonadid protozoa; however, the reproducibility of these resazurin-based methods in our laboratory has been limited because the flagellate culture medium spontaneously reduces the resazurin. The objective of this work was to refine the fluorimetric microassay method previously developed by other research groups to reduce the fluorescence background generated by the media and increase the sensitivity of the screening assay. The experimental conditions, time of incubation, resazurin concentration and media used in the microtitre plates were adjusted. Different drug sensitivity studies against T. vaginalis were developed using the 5-nitroimidazole reference drugs, new 5-nitroindazolinones and 5-nitroindazole synthetic derivatives. Haemocytometer count results were compared with the resazurin assay using a 10% solution of 3 mM resazurin dissolved in phosphate buffered saline with glucose (1 mg/mL. The fluorimetric assay and the haemocytometer counts resulted in similar percentages of trichomonacidal activity in all the experiments, demonstrating that the fluorimetric microtitre assay has the necessary accuracy for high-throughput screening of new drugs against T. vaginalis.

  2. Aggressive chemotherapy and the selection of drug resistant pathogens.

    Directory of Open Access Journals (Sweden)

    Silvie Huijben

    2013-09-01

    Full Text Available Drug resistant pathogens are one of the key public health challenges of the 21st century. There is a widespread belief that resistance is best managed by using drugs to rapidly eliminate target pathogens from patients so as to minimize the probability that pathogens acquire resistance de novo. Yet strong drug pressure imposes intense selection in favor of resistance through alleviation of competition with wild-type populations. Aggressive chemotherapy thus generates opposing evolutionary forces which together determine the rate of drug resistance emergence. Identifying treatment regimens which best retard resistance evolution while maximizing health gains and minimizing disease transmission requires empirical analysis of resistance evolution in vivo in conjunction with measures of clinical outcomes and infectiousness. Using rodent malaria in laboratory mice, we found that less aggressive chemotherapeutic regimens substantially reduced the probability of onward transmission of resistance (by >150-fold, without compromising health outcomes. Our experiments suggest that there may be cases where resistance evolution can be managed more effectively with treatment regimens other than those which reduce pathogen burdens as fast as possible.

  3. Multiplex TaqMan® detection of pathogenic and multi-drug resistant Salmonella.

    Science.gov (United States)

    Singh, Prashant; Mustapha, Azlin

    2013-09-02

    Overuse of antibiotics in the medical and animal industries is one of the major causes for the development of multi-drug-resistant (MDR) food pathogens that are often difficult to treat. In the past few years, higher incidences of outbreaks caused by MDR Salmonella have been increasingly documented. The objective of this study was to develop a rapid multiplex real-time polymerase chain reaction (PCR) assay for simultaneous detection of pathogenic and MDR Salmonella spp. A multiplex TaqMan®real-time PCR was designed by targeting the invasin virulence gene (invA), and four commonly found antibiotic resistance genes, viz. ampicillin, chloramphenicol, streptomycin and tetracycline. To avoid false negative results and to increase the reliability of the assay, an internal amplification control (IAC) was added which was detected using a locked nucleic acid (LNA) probe. In serially diluted (5 ng-50 fg) DNA samples, the assay was able to detect 100 genomic equivalents of Salmonella, while in a multiplex format, the sensitivity was 1000 genomic equivalents. The assay performed equally well on artificially contaminated samples of beef trim, ground beef of different fat contents (73:27, 80:20, 85:15 and 93:7), chicken rinse, ground chicken, ground turkey, egg, spinach and tomato. While the detection limit for un-enriched inoculated food samples was 10(4) CFU/g, this was improved to 10 CFU/g after a 12-h enrichment in buffered peptone water, with 100% reproducibility. The multiplex real-time assay developed in this study can be used as a valuable tool to detect MDR virulent Salmonella, thus enhancing the safety of food. © 2013.

  4. Risk Factors for Acquisition of Drug Resistance during Multidrug-Resistant Tuberculosis Treatment, Arkhangelsk Oblast, Russia, 2005–2010

    Science.gov (United States)

    Ershova, Julia; Vlasova, Natalia; Nikishova, Elena; Tarasova, Irina; Eliseev, Platon; Maryandyshev, Andrey O.; Shemyakin, Igor G.; Kurbatova, Ekaterina; Cegielski, J. Peter

    2015-01-01

    Acquired resistance to antituberculosis drugs decreases effective treatment options and the likelihood of treatment success. We identified risk factors for acquisition of drug resistance during treatment for multidrug-resistant tuberculosis (MDR TB) and evaluated the effect on treatment outcomes. Data were collected prospectively from adults from Arkhangelsk Oblast, Russia, who had pulmonary MDR TB during 2005–2008. Acquisition of resistance to capreomycin and of extensively drug-resistant TB were more likely among patients who received 3 effective drugs (9.4% vs. 0% and 8.6% vs. 0.8%, respectively). Poor outcomes were more likely among patients with acquired capreomycin resistance (100% vs. 25.9%), acquired ofloxacin resistance (83.6% vs. 22.7%), or acquired extensive drug resistance (100% vs. 24.4%). To prevent acquired drug resistance and poor outcomes, baseline susceptibility to first- and second-line drugs should be determined quickly, and treatment should be adjusted to contain >3 effective drugs. PMID:25988954

  5. Mathematical modeling and computational prediction of cancer drug resistance.

    Science.gov (United States)

    Sun, Xiaoqiang; Hu, Bin

    2017-06-23

    Diverse forms of resistance to anticancer drugs can lead to the failure of chemotherapy. Drug resistance is one of the most intractable issues for successfully treating cancer in current clinical practice. Effective clinical approaches that could counter drug resistance by restoring the sensitivity of tumors to the targeted agents are urgently needed. As numerous experimental results on resistance mechanisms have been obtained and a mass of high-throughput data has been accumulated, mathematical modeling and computational predictions using systematic and quantitative approaches have become increasingly important, as they can potentially provide deeper insights into resistance mechanisms, generate novel hypotheses or suggest promising treatment strategies for future testing. In this review, we first briefly summarize the current progress of experimentally revealed resistance mechanisms of targeted therapy, including genetic mechanisms, epigenetic mechanisms, posttranslational mechanisms, cellular mechanisms, microenvironmental mechanisms and pharmacokinetic mechanisms. Subsequently, we list several currently available databases and Web-based tools related to drug sensitivity and resistance. Then, we focus primarily on introducing some state-of-the-art computational methods used in drug resistance studies, including mechanism-based mathematical modeling approaches (e.g. molecular dynamics simulation, kinetic model of molecular networks, ordinary differential equation model of cellular dynamics, stochastic model, partial differential equation model, agent-based model, pharmacokinetic-pharmacodynamic model, etc.) and data-driven prediction methods (e.g. omics data-based conventional screening approach for node biomarkers, static network approach for edge biomarkers and module biomarkers, dynamic network approach for dynamic network biomarkers and dynamic module network biomarkers, etc.). Finally, we discuss several further questions and future directions for the use of

  6. HIV resistance testing and detected drug resistance in Europe

    DEFF Research Database (Denmark)

    Schultze, Anna; Phillips, Andrew N; Paredes, Roger

    2015-01-01

    to Southern Europe. CONCLUSIONS: Despite a concurrent decline in virological failure and testing, drug resistance was commonly detected. This suggests a selective approach to resistance testing. The regional differences identified indicate that policy aiming to minimize the emergence of resistance......OBJECTIVES: To describe regional differences and trends in resistance testing among individuals experiencing virological failure and the prevalence of detected resistance among those individuals who had a genotypic resistance test done following virological failure. DESIGN: Multinational cohort...... study. METHODS: Individuals in EuroSIDA with virological failure (>1 RNA measurement >500 on ART after >6 months on ART) after 1997 were included. Adjusted odds ratios (aORs) for resistance testing following virological failure and aORs for the detection of resistance among those who had a test were...

  7. Molecular chess? Hallmarks of anti-cancer drug resistance.

    Science.gov (United States)

    Cree, Ian A; Charlton, Peter

    2017-01-05

    The development of resistance is a problem shared by both classical chemotherapy and targeted therapy. Patients may respond well at first, but relapse is inevitable for many cancer patients, despite many improvements in drugs and their use over the last 40 years. Resistance to anti-cancer drugs can be acquired by several mechanisms within neoplastic cells, defined as (1) alteration of drug targets, (2) expression of drug pumps, (3) expression of detoxification mechanisms, (4) reduced susceptibility to apoptosis, (5) increased ability to repair DNA damage, and (6) altered proliferation. It is clear, however, that changes in stroma and tumour microenvironment, and local immunity can also contribute to the development of resistance. Cancer cells can and do use several of these mechanisms at one time, and there is considerable heterogeneity between tumours, necessitating an individualised approach to cancer treatment. As tumours are heterogeneous, positive selection of a drug-resistant population could help drive resistance, although acquired resistance cannot simply be viewed as overgrowth of a resistant cancer cell population. The development of such resistance mechanisms can be predicted from pre-existing genomic and proteomic profiles, and there are increasingly sophisticated methods to measure and then tackle these mechanisms in patients. The oncologist is now required to be at least one step ahead of the cancer, a process that can be likened to 'molecular chess'. Thus, as well as an increasing role for predictive biomarkers to clinically stratify patients, it is becoming clear that personalised strategies are required to obtain best results.

  8. Malaria medicines to address drug resistance and support malaria elimination efforts.

    Science.gov (United States)

    Achan, Jane; Mwesigwa, Julia; Edwin, Chinagozi Precious; D'alessandro, Umberto

    2018-01-01

    Antimalarial drugs are essential weapons to fight malaria and have been used effectively since the 17 th century. However, P.falciparum resistance has been reported to almost all available antimalarial drugs, including artemisinin derivatives, raising concerns that this could jeopardize malaria elimination. Areas covered: In this article, we present a historical perspective of antimalarial drug resistance, review current evidence of resistance to available antimalarial drugs and discuss possible mitigating strategies to address this challenge. Expert commentary: The historical approach to drug resistance has been to change the national treatment policy to an alternative treatment. However, alternatives to artemisinin-based combination treatment are currently extremely limited. Innovative approaches utilizing available schizonticidal drugs such as triple combination therapies or multiple first line treatments could delay the emergence and spread of drug resistance. Transmission blocking drugs like primaquine may play a key role if given to a substantial proportion of malaria infected persons. Deploying antimalarial medicines in mass drug administration or mass screening and treatment campaigns could also contribute to containment efforts by eliminating resistant parasites in some settings. Ultimately, response to drug resistance should also include further investment in the development of new antimalarial drugs.

  9. Adaptation and evolution of drug-resistant Mycobacterium tuberculosis

    NARCIS (Netherlands)

    Bergval, I.L.

    2013-01-01

    Many studies have been conducted on drug resistance and the evolution of Mycobacterium tuberculosis. Notwithstanding, many molecular mechanisms facilitating the emergence, adaptation and spread of drug-resistant tuberculosis have yet to be discovered. This thesis reports studies of the adaptive

  10. Drug resistance following irradiation of RIF-1 tumors: Influence of the interval between irradiation and drug treatment

    International Nuclear Information System (INIS)

    Hopwood, L.E.; Davies, B.M.; Moulder, J.E.

    1990-01-01

    RIF-1 tumors contain a small number of cells (1 to 100 per 10(6) cells) that are resistant to 5-fluorouracil, methotrexate, or adriamycin. The frequency of drug-resistant cells among individual untreated tumors is highly variable. Radiation, delivered in vivo at doses of 3 to 12 Gy, increases the frequency of methotrexate- and 5-fluorouracil-resistant cells, but not the frequency of adriamycin-resistant cells. The magnitude of induction of 5-fluorouracil and methotrexate resistance shows a complex dependence on the radiation dose and on the interval between irradiation and assessment of drug resistance. For a dose of 3 Gy, induced 5-fluorouracil and methotrexate resistance is seen only after an interval of 5 to 7 days, whereas for a dose of 12 Gy, high levels of induced resistance are observed 1 to 3 days after irradiation. The maximum absolute risk for induction of resistance is 4 per 10(4) cells per Gy for methotrexate, and 3 per 10(6) cells per Gy for 5-fluorouracil. These results indicate that tumor hypoxia may play a role in the increased levels of drug resistance seen after irradiation, and that both genetic and environmental factors may influence radiation-induction of drug resistance. These studies provide essential data for models of the development of tumor drug resistance, and imply that some of the drug resistance seen when chemotherapy follows radiotherapy may be caused by radiation-induced drug resistance

  11. [Tuberculosis and drug-resistance tuberculosis in prisoners. Colombia, 2010-2012].

    Science.gov (United States)

    Gómez, Ingrid T; Llerena, Claudia R; Zabaleta, Angie P

    2015-01-01

    To characterize tuberculosis drug-resistance using anti-tuberculosis drug-sensitivity tests in Colombian prisoners. Descriptive-retrospective analyses were performed on cases of tuberculosis in prisoners. Samples were evaluated by the National Reference Laboratory. Conditions like gender, TB/VIH co-infection and drug-resistance were evaluated. Anti-tuberculosis drug-sensitivity tests were carried out on 72 prisoners. Results showed a distribution of 90.7 % of cases in males and 9.3 % of cases in females. 12 % of cases were TB/VIH co-infections, 94 % of the cases had not received any anti-tuberculosis treatment before, six isolates were drug-resistant corresponding to 8.8 % of total cases, and two cases were multi drug-resistant representing 1.3 % of the cases. Of the drug-resistant cases, 83.3 % were TB/VIH co-infected. Previously treated cases corresponded to 5.6 % of the total cases analyzed. One case with TB/VIH co-infection and rifampicin resistance was observed, representing 1.3 % of the total cases. The government must create a clear policy for prisoners in Colombia, because a high rate of disease in prisoners was observed. In addition, the results showed an association between drug-resistance and TB/VIH co-infection. Overcrowding and low quality of life in penitentiaries could become an important public health problem.

  12. Evaluation of drug-induced hematotoxicity using novel in vitro monkey CFU-GM and BFU-E colony assays.

    Science.gov (United States)

    Goto, Koichi; Goto, Mayumi; Ando-Imaoka, Masako; Kai, Kiyonori; Mori, Kazuhiko

    2017-01-01

    In order to evaluate drug-induced hematotoxicity in monkey cells in vitro, colony-forming unit-granulocyte, macrophage (CFU-GM), and burst-forming unit-erythroid (BFU-E) colony assays were established using mononuclear cells in the bone marrow collected from male cynomolgus monkeys. Furthermore, the effects of doxorubicin, chloramphenicol, and linezolid on CFU-GM and BFU-E colony formation were investigated using established monkey CFU-GM and BFU-E colony assays in comparison with those on human CFU-GM and BFU-E colonies acquired from human umbilical cord blood cells. Bone marrow mononuclear cells were collected from the ischial or iliac bone of male cynomolgus monkeys. The cells were subsequently processed by density gradient separation at 1.067, 1.070, or 1.077 g/mL for CFU-GM or 1.077 g/mL for BFU-E, and then cultured in methylcellulose medium for 9 or 13 days, respectively. A sufficient number of CFU-GM colonies were formed from mononuclear cells processed at a density of 1.070 g/mL. Moreover, the number of BFU-E colonies from the cells processed at a density of 1.077 g/mL was sufficient for the colony assay. The number of CFU-GM or BFU-E colonies decreased after treatment with the drugs of interest in a concentration-dependent manner. Compared with human CFU-GM, monkey CFU-GM were more sensitive to chloramphenicol and resistant to doxorubicin, whereas monkey BFU-E were more sensitive to all compounds in comparison to the sensitivity of human BFU-E. In conclusion, monkey CFU-GM and BFU-E colony assays were established and considered useful tools to evaluate the differences in drug-induced hematotoxicity between species.

  13. Pathogenicity of pan-drug-resistant Serratia marcescens harbouring blaNDM-1.

    Science.gov (United States)

    Gruber, Teresa M; Göttig, Stephan; Mark, Laura; Christ, Sara; Kempf, Volkhard A J; Wichelhaus, Thomas A; Hamprecht, Axel

    2015-04-01

    To characterize a pan-drug-resistant Serratia marcescens clinical isolate carrying the New Delhi metallo-β-lactamase (NDM)-1. The presence of β-lactamase genes was examined by PCR and sequencing. Antibiotic susceptibility was determined by antibiotic gradient test. Transformation assays, transconjugation assays, PFGE and PCR-based replicon typing were used for plasmid analysis. Horizontal gene transfer was evaluated by liquid mating using Escherichia coli J53 as a recipient. Pathogenicity of NDM-1 expressing S. marcescens was analysed using the Galleria mellonella infection model. S. marcescens isolate SM1890 was non-susceptible to all tested antibiotics, with minocycline retaining intermediate activity. blaNDM-1 was located on a 140 kb IncA/C-type plasmid which was transferable to E. coli and Klebsiella pneumoniae by conjugation. The LD50 of the NDM-positive, SM1890 isolate was higher than that of other, NDM-1 negative, S. marcescens strains. The presence of a blaNDM-1-harbouring IncA/C plasmid resulted in marked resistance to β-lactam antibiotics, but had no significant effect on virulence of isogenic strains. Because of the intrinsic resistance of S. marcescens to colistin and reduced susceptibility to tigecycline, treatment options for infections by NDM-1-positive isolates are extremely limited in this species. © The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  14. Quantifying the Determinants of Evolutionary Dynamics Leading to Drug Resistance.

    Directory of Open Access Journals (Sweden)

    Guillaume Chevereau

    Full Text Available The emergence of drug resistant pathogens is a serious public health problem. It is a long-standing goal to predict rates of resistance evolution and design optimal treatment strategies accordingly. To this end, it is crucial to reveal the underlying causes of drug-specific differences in the evolutionary dynamics leading to resistance. However, it remains largely unknown why the rates of resistance evolution via spontaneous mutations and the diversity of mutational paths vary substantially between drugs. Here we comprehensively quantify the distribution of fitness effects (DFE of mutations, a key determinant of evolutionary dynamics, in the presence of eight antibiotics representing the main modes of action. Using precise high-throughput fitness measurements for genome-wide Escherichia coli gene deletion strains, we find that the width of the DFE varies dramatically between antibiotics and, contrary to conventional wisdom, for some drugs the DFE width is lower than in the absence of stress. We show that this previously underappreciated divergence in DFE width among antibiotics is largely caused by their distinct drug-specific dose-response characteristics. Unlike the DFE, the magnitude of the changes in tolerated drug concentration resulting from genome-wide mutations is similar for most drugs but exceptionally small for the antibiotic nitrofurantoin, i.e., mutations generally have considerably smaller resistance effects for nitrofurantoin than for other drugs. A population genetics model predicts that resistance evolution for drugs with this property is severely limited and confined to reproducible mutational paths. We tested this prediction in laboratory evolution experiments using the "morbidostat", a device for evolving bacteria in well-controlled drug environments. Nitrofurantoin resistance indeed evolved extremely slowly via reproducible mutations-an almost paradoxical behavior since this drug causes DNA damage and increases the mutation

  15. Rapid identification of drug-type strains in Cannabis sativa using loop-mediated isothermal amplification assay.

    Science.gov (United States)

    Kitamura, Masashi; Aragane, Masako; Nakamura, Kou; Watanabe, Kazuhito; Sasaki, Yohei

    2017-01-01

    In Cannabis sativa L., tetrahydrocannabinol (THC) is the primary psychoactive compound and exists as the carboxylated form, tetrahydrocannabinolic acid (THCA). C. sativa is divided into two strains based on THCA content-THCA-rich (drug-type) strains and THCA-poor (fiber-type) strains. Both strains are prohibited by law in many countries including Japan, whereas the drug-type strains are regulated in Canada and some European countries. As the two strains cannot be discriminated by morphological analysis, a simple method for identifying the drug-type strains is required for quality control in legal cultivation and forensic investigation. We have developed a novel loop-mediated isothermal amplification (LAMP) assay for identifying the drug-type strains of C. sativa. We designed two selective LAMP primer sets for on-site or laboratory use, which target the drug-type THCA synthase gene. The LAMP assay was accomplished within approximately 40 min. The assay showed high specificity for the drug-type strains and its sensitivity was the same as or higher than that of conventional polymerase chain reaction. We also showed the effectiveness of melting curve analysis that was conducted after the LAMP assay. The melting temperature values of the drug-type strains corresponded to those of the cloned drug-type THCA synthase gene, and were clearly different from those of the cloned fiber-type THCA synthase gene. Moreover, the LAMP assay with simple sample preparation could be accomplished within 1 h from sample treatment to identification without the need for special devices or techniques. Our rapid, sensitive, specific, and simple assay is expected to be applicable to laboratory and on-site detection.

  16. Hospital costs of nosocomial multi-drug resistant Pseudomonas aeruginosa acquisition.

    Science.gov (United States)

    Morales, Eva; Cots, Francesc; Sala, Maria; Comas, Mercè; Belvis, Francesc; Riu, Marta; Salvadó, Margarita; Grau, Santiago; Horcajada, Juan P; Montero, Maria Milagro; Castells, Xavier

    2012-05-23

    We aimed to assess the hospital economic costs of nosocomial multi-drug resistant Pseudomonas aeruginosa acquisition. A retrospective study of all hospital admissions between January 1, 2005, and December 31, 2006 was carried out in a 420-bed, urban, tertiary-care teaching hospital in Barcelona (Spain). All patients with a first positive clinical culture for P. aeruginosa more than 48 h after admission were included. Patient and hospitalization characteristics were collected from hospital and microbiology laboratory computerized records. According to antibiotic susceptibility, isolates were classified as non-resistant, resistant and multi-drug resistant. Cost estimation was based on a full-costing cost accounting system and on the criteria of clinical Activity-Based Costing methods. Multivariate analyses were performed using generalized linear models of log-transformed costs. Cost estimations were available for 402 nosocomial incident P. aeruginosa positive cultures. Their distribution by antibiotic susceptibility pattern was 37.1% non-resistant, 29.6% resistant and 33.3% multi-drug resistant. The total mean economic cost per admission of patients with multi-drug resistant P. aeruginosa strains was higher than that for non-resistant strains (15,265 vs. 4,933 Euros). In multivariate analysis, resistant and multi-drug resistant strains were independently predictive of an increased hospital total cost in compared with non-resistant strains (the incremental increase in total hospital cost was more than 1.37-fold and 1.77-fold that for non-resistant strains, respectively). P. aeruginosa multi-drug resistance independently predicted higher hospital costs with a more than 70% increase per admission compared with non-resistant strains. Prevention of the nosocomial emergence and spread of antimicrobial resistant microorganisms is essential to limit the strong economic impact.

  17. High prevalence of multidrug-resistant tuberculosis among patients with rifampicin resistance using GeneXpert Mycobacterium tuberculosis/rifampicin in Ghana

    NARCIS (Netherlands)

    Boakye-Appiah, Justice K; Steinmetz, Alexis R; Pupulampu, Peter; Ofori-Yirenkyi, Stephen; Tetteh, Ishmael; Frimpong, Michael; Oppong, Patrick; Opare-Sem, Ohene; Norman, Betty R; Stienstra, Ymkje; van der Werf, Tjip S; Wansbrough-Jones, Mark; Bonsu, Frank; Obeng-Baah, Joseph; Phillips, Richard O

    2016-01-01

    OBJECTIVE/BACKGROUND: Drug-resistant strains of tuberculosis (TB) represent a major threat to global TB control. In low- and middle-income countries, resource constraints make it difficult to identify and monitor cases of resistance using drug susceptibility testing and culture. Molecular assays

  18. Enhanced Transmission of Drug-Resistant Parasites to Mosquitoes following Drug Treatment in Rodent Malaria

    OpenAIRE

    Bell, Andrew S.; Huijben, Silvie; Paaijmans, Krijn P.; Sim, Derek G.; Chan, Brian H. K.; Nelson, William A.; Read, Andrew F.

    2012-01-01

    The evolution of drug resistant Plasmodium parasites is a major challenge to effective malaria control. In theory, competitive interactions between sensitive parasites and resistant parasites within infections are a major determinant of the rate at which parasite evolution undermines drug efficacy. Competitive suppression of resistant parasites in untreated hosts slows the spread of resistance; competitive release following treatment enhances it. Here we report that for the murine model Plasm...

  19. Rapid diagnosis of drug resistance to fluoroquinolones, amikacin, capreomycin, kanamycin and ethambutol using genotype MTBDRsl assay: a meta-analysis.

    Directory of Open Access Journals (Sweden)

    Yan Feng

    Full Text Available BACKGROUND: There are urgent needs for rapid and accurate drug susceptibility testing of M. tuberculosis. GenoType MTBDRsl is a new molecular kit designed for rapid identification of the resistance to the second-line antituberculosis drugs with a single strip. In recent years, it has been evaluated in many settings, but with varied results. The aim of this meta-analysis was to synthesize the latest data on the diagnostic accuracy of GenoType MTBDRsl in detecting drug resistance to fluoroquinolones, amikacin, capreomycin, kanamycin and ethambutol, in comparison with the phenotypic drug susceptibility test. METHODS: This systematic review followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA guideline. The search terms of "MTBDRsl" and "tuberculosis" were used on PubMed, EMBASE, and Web of Science. QUADAS-2 was used to assess the quality of included studies. Data were analyzed by Meta-Disc 1.4. We calculated the sensitivity, specificity, positive likelihood ratio (PLR, negative likelihood ratio (NLR, diagnostic odds ratio (DOR and corresponding 95% confidence interval (CI for each study. From these calculations, forest plots and summary receiver operating characteristic (SROC curves were produced. RESULTS: Patient selection bias as well as flow and timing bias were observed in most studies. The summarized sensitivity (95% CI was 0.874(0.845-0.899, 0.826(0.777-0.869, 0.820(0.772-0.862, 0.444(0.396-0.492, and 0.679(0.652-0.706 for fluoroquinolones, amikacin, capreomycin, kanamycin, and ethambutol, respectively. The specificity (95% CI was 0.971(0.961-0.980, 0.995(0.987-0.998, 0.973(0.963-0.981, 0.993(0.985-0.997, and 0.799(0.773-0.823, respectively. The AUC (standard error were 0.9754(0.0203, 0.9300(0.0598, 0.9885(0.0038, 0.9689(0.0359, and 0.6846(0.0550, respectively. CONCLUSION: Genotype MTBDRsl showed good accuracy for detecting drug resistance to fluoroquinolones, amikacin and capreomycin, but it may not be an

  20. Developing artemisinin based drug combinations for the treatment of drug resistant falciparum malaria: A review

    Directory of Open Access Journals (Sweden)

    Olliaro P

    2004-01-01

    Full Text Available The emergence and spread of drug resistant malaria represents a considerable challenge to controlling malaria. To date, malaria control has relied heavily on a comparatively small number of chemically related drugs, belonging to either the quinoline or the antifolate groups. Only recently have the artemisinin derivatives been used but mostly in south east Asia. Experience has shown that resistance eventually curtails the life-span of antimalarial drugs. Controlling resistance is key to ensuring that the investment put into developing new antimalarial drugs is not wasted. Current efforts focus on research into new compounds with novel mechanisms of action, and on measures to prevent or delay resistance when drugs are introduced. Drug discovery and development are long, risky and costly ventures. Antimalarial drug development has traditionally been slow but now various private and public institutions are at work to discover and develop new compounds. Today, the antimalarial development pipeline is looking reasonably healthy. Most development relies on the quinoline, antifolate and artemisinin compounds. There is a pressing need to have effective, easy to use, affordable drugs that will last a long time. Drug combinations that have independent modes of action are seen as a way of enhancing efficacy while ensuring mutual protection against resistance. Most research work has focused on the use of artesunate combined with currently used standard drugs, namely, mefloquine, amodiaquine, sulfadoxine/pyrimethamine, and chloroquine. There is clear evidence that combinations improve efficacy without increasing toxicity. However, the absolute cure rates that are achieved by combinations vary widely and depend on the level of resistance of the standard drug. From these studies, further work is underway to produce fixed dose combinations that will be packaged in blister packs. This review will summarise current antimalarial drug developments and outline recent

  1. Influence of multidrug resistance and drug transport proteins on chemotherapy drug metabolism.

    Science.gov (United States)

    Joyce, Helena; McCann, Andrew; Clynes, Martin; Larkin, Annemarie

    2015-05-01

    Chemotherapy involving the use of anticancer drugs remains an important strategy in the overall management of patients with metastatic cancer. Acquisition of multidrug resistance remains a major impediment to successful chemotherapy. Drug transporters in cell membranes and intracellular drug metabolizing enzymes contribute to the resistance phenotype and determine the pharmacokinetics of anticancer drugs in the body. ATP-binding cassette (ABC) transporters mediate the transport of endogenous metabolites and xenobiotics including cytotoxic drugs out of cells. Solute carrier (SLC) transporters mediate the influx of cytotoxic drugs into cells. This review focuses on the substrate interaction of these transporters, on their biology and what role they play together with drug metabolizing enzymes in eliminating therapeutic drugs from cells. The majority of anticancer drugs are substrates for the ABC transporter and SLC transporter families. Together, these proteins have the ability to control the influx and the efflux of structurally unrelated chemotherapeutic drugs, thereby modulating the intracellular drug concentration. These interactions have important clinical implications for chemotherapy because ultimately they determine therapeutic efficacy, disease progression/relapse and the success or failure of patient treatment.

  2. Acetohydroxyacid synthase (AHAS) in vivo assay for screening imidazolinone-resistance in sunflower (Helianthus annuus L.).

    Science.gov (United States)

    Vega, T; Breccia, G; Gil, M; Zorzoli, R; Picardi, L; Nestares, G

    2012-12-01

    The objective of this work was to evaluate the in vivo acetohydroxyacid synthase (AHAS) activity response to imidazolinones and its possible use as a selection method for evaluating AHAS inhibitor resistance. In vivo AHAS assay and the comparison of parameters from dose-response curves have been used as a valid tool for comparing sunflower lines and hybrids differing in imidazolinone resistance. The sunflower resistant genotypes evaluated here were 100-fold and 20-fold more resistant compared with the susceptible line for imazethapyr and imazapyr, respectively. This assay also allowed discrimination of homozygous from heterozygous genotypes for I(mr1) locus that codify for the catalytic subunit of AHAS. The in vivo AHAS assay described in this study was useful for the selection of sunflower genotypes differing in herbicide resistance and could be a useful tool when breeding for imidazolinone resistance in sunflower. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

  3. Molecular Genetics of Drug-resistance in Epilepsies

    Directory of Open Access Journals (Sweden)

    Kurupath Radhakrishnan

    2015-06-01

    Full Text Available Nearly one-third of newly diagnosed patients with epilepsy remain unresponsive to antiepileptic drugs (AEDs, etiopathogenesis of which is poorly understood. The genes encoding the proteins that regulate the pharmacokinetics such as P-glycoprotein [ABCBI], major vault protein [MVP gene] and drug metabolizing enzymes [ABCB1, ABCG2, MVP, CYP2C9, CYP2C19, CYP3A4, CYP3A5, EPHX1, UGT1A1, UGT2B7], and pharmacodynamics such as sodium channels [SCN1A, SCN2A] and GABA receptors [GABRA1, GABRA6, GABRB2, GABRG2] of AEDs are under intense investigation to unravel the mysteries of AED-resistance. However, till today, a consistent and reliable result that could help the clinician either to predict drug resistance or to overcome it has not been forthcoming. The discrepant results may be related to variations in the definition of drug-resistance, heterogeneous patient populations, ethnic variations in the frequency distribution of single nucleotide polymorphisms (SNPs and the selection of SNPs. Understanding of these limitations of existing studies, hopefully, will help in designing better studies. Nearly one-third of newly diagnosed patients with epilepsy remain unresponsive toantiepileptic drugs (AEDs, etiopathogenesis of which is poorly understood. The genesencoding the proteins that regulate the pharmacokinetics such as P-glycoprotein[ABCBI], major vault protein [MVP gene] and drug metabolizing enzymes [ABCB1,ABCG2, MVP, CYP2C9, CYP2C19, CYP3A4, CYP3A5, EPHX1, UGT1A1, UGT2B7],and pharmacodynamics such as sodium channels [SCN1A, SCN2A] and GABAreceptors [GABRA1, GABRA6, GABRB2, GABRG2] of AEDs are under intenseinvestigation to unravel the mysteries of AED-resistance. However, till today, aconsistent and reliable result that could help the clinician either to predict drugresistanceor to overcome it has not been forthcoming. The discrepant results may berelated to variations in the definition of drug-resistance, heterogeneous patientpopulations, ethnic

  4. Complete genome sequence of Acinetobacter baumannii XH386 (ST208, a multi-drug resistant bacteria isolated from pediatric hospital in China

    Directory of Open Access Journals (Sweden)

    Youhong Fang

    2016-03-01

    Full Text Available Acinetobacter baumannii is an important bacterium that emerged as a significant nosocomial pathogen worldwide. The rise of A. baumannii was due to its multi-drug resistance (MDR, while it was difficult to treat multi-drug resistant A. baumannii with antibiotics, especially in pediatric patients for the therapeutic options with antibiotics were quite limited in pediatric patients. A. baumannii ST208 was identified as predominant sequence type of carbapenem resistant A. baumannii in the United States and China. As we knew, there was no complete genome sequence reproted for A. baumannii ST208, although several whole genome shotgun sequences had been reported. Here, we sequenced the 4087-kilobase (kb chromosome and 112-kb plasmid of A. baumannii XH386 (ST208, which was isolated from a pediatric hospital in China. The genome of A. baumannii XH386 contained 3968 protein-coding genes and 94 RNA-only encoding genes. Genomic analysis and Minimum inhibitory concentration assay showed that A. baumannii XH386 was multi-drug resistant strain, which showed resistance to most of antibiotics, except for tigecycline. The data may be accessed via the GenBank accession number CP010779 and CP010780. Keywords: Acinetobacter baumannii, Multi-drug resistance, Paediatric

  5. Rapid susceptibility testing of Mycobacterium tuberculosis by bioluminescence assay of mycobacterial ATP

    International Nuclear Information System (INIS)

    Nilsson, L.E.; Hoffner, S.E.; Ansehn, S.

    1988-01-01

    Mycobacterial growth was monitored by bioluminescence assay of mycobacterial ATP. Cultures of Mycobacterium tuberculosis H37Rv and of 25 clinical isolates of the same species were exposed to serial dilutions of ethambutol, isoniazid, rifampin, and streptomycin. A suppression of ATP, indicating growth inhibition, occurred for susceptible but not resistant strains within 5 to 7 days of incubation. Breakpoint concentrations between susceptibility and resistance were determined by comparing these results with those obtained by reference techniques. Full agreement was found in 99% of the assays with the resistance ratio method on Lowenstein-Jensen medium, and 98% of the assays were in full agreement with the radiometric system (BACTEC). A main advantage of the bioluminescence method is its rapidity, with results available as fast as with the radiometric system but at a lower cost and without the need for radioactive culture medium. The method provides kinetic data concerning drug effects within available in vivo drug concentrations and has great potential for both rapid routine susceptibility testing and research applications in studies of drug effects on mycobacteria

  6. Hospital costs of nosocomial multi-drug resistant Pseudomonas aeruginosa acquisition

    Directory of Open Access Journals (Sweden)

    Morales Eva

    2012-05-01

    Full Text Available Abstract Background We aimed to assess the hospital economic costs of nosocomial multi-drug resistant Pseudomonas aeruginosa acquisition. Methods A retrospective study of all hospital admissions between January 1, 2005, and December 31, 2006 was carried out in a 420-bed, urban, tertiary-care teaching hospital in Barcelona (Spain. All patients with a first positive clinical culture for P. aeruginosa more than 48 h after admission were included. Patient and hospitalization characteristics were collected from hospital and microbiology laboratory computerized records. According to antibiotic susceptibility, isolates were classified as non-resistant, resistant and multi-drug resistant. Cost estimation was based on a full-costing cost accounting system and on the criteria of clinical Activity-Based Costing methods. Multivariate analyses were performed using generalized linear models of log-transformed costs. Results Cost estimations were available for 402 nosocomial incident P. aeruginosa positive cultures. Their distribution by antibiotic susceptibility pattern was 37.1% non-resistant, 29.6% resistant and 33.3% multi-drug resistant. The total mean economic cost per admission of patients with multi-drug resistant P. aeruginosa strains was higher than that for non-resistant strains (15,265 vs. 4,933 Euros. In multivariate analysis, resistant and multi-drug resistant strains were independently predictive of an increased hospital total cost in compared with non-resistant strains (the incremental increase in total hospital cost was more than 1.37-fold and 1.77-fold that for non-resistant strains, respectively. Conclusions P. aeruginosa multi-drug resistance independently predicted higher hospital costs with a more than 70% increase per admission compared with non-resistant strains. Prevention of the nosocomial emergence and spread of antimicrobial resistant microorganisms is essential to limit the strong economic impact.

  7. GEAR: A database of Genomic Elements Associated with drug Resistance

    Science.gov (United States)

    Wang, Yin-Ying; Chen, Wei-Hua; Xiao, Pei-Pei; Xie, Wen-Bin; Luo, Qibin; Bork, Peer; Zhao, Xing-Ming

    2017-01-01

    Drug resistance is becoming a serious problem that leads to the failure of standard treatments, which is generally developed because of genetic mutations of certain molecules. Here, we present GEAR (A database of Genomic Elements Associated with drug Resistance) that aims to provide comprehensive information about genomic elements (including genes, single-nucleotide polymorphisms and microRNAs) that are responsible for drug resistance. Right now, GEAR contains 1631 associations between 201 human drugs and 758 genes, 106 associations between 29 human drugs and 66 miRNAs, and 44 associations between 17 human drugs and 22 SNPs. These relationships are firstly extracted from primary literature with text mining and then manually curated. The drug resistome deposited in GEAR provides insights into the genetic factors underlying drug resistance. In addition, new indications and potential drug combinations can be identified based on the resistome. The GEAR database can be freely accessed through http://gear.comp-sysbio.org. PMID:28294141

  8. Malaria epidemic and drug resistance, Djibouti.

    Science.gov (United States)

    Rogier, Christophe; Pradines, Bruno; Bogreau, H; Koeck, Jean-Louis; Kamil, Mohamed-Ali; Mercereau-Puijalon, Odile

    2005-02-01

    Analysis of Plasmodium falciparum isolates collected before, during, and after a 1999 malaria epidemic in Djibouti shows that, despite a high prevalence of resistance to chloroquine, the epidemic cannot be attributed to a sudden increase in drug resistance of local parasite populations.

  9. Collateral Resistance and Sensitivity Modulate Evolution of High-Level Resistance to Drug Combination Treatment in Staphylococcus aureus

    DEFF Research Database (Denmark)

    de Evgrafov, Mari Cristina Rodriguez; Gumpert, Heidi; Munck, Christian

    2015-01-01

    As drug-resistant pathogens continue to emerge, combination therapy will increasingly be relied upon to treat infections and to help combat further development of multidrug resistance. At present a dichotomy exists between clinical practice, which favors therapeutically synergistic combinations......, to reflect drug concentrations more likely to be encountered during treatment. We performed a series of adaptive evolution experiments using Staphylococcus aureus. Interestingly, no relationship between drug interaction type and resistance evolution was found as resistance increased significantly beyond wild......-type levels. All drug combinations, irrespective of interaction types, effectively limited resistance evolution compared with monotreatment. Cross-resistance and collateral sensitivity were found to be important factors in the extent of resistance evolution toward a combination. Comparative genomic analyses...

  10. Pan Drug-Resistant Environmental Isolate of Acinetobacter baumannii from Croatia.

    Science.gov (United States)

    Goic-Barisic, Ivana; Seruga Music, Martina; Kovacic, Ana; Tonkic, Marija; Hrenovic, Jasna

    2017-06-01

    Acinetobacter baumannii is an emerging nosocomial pathogen with also emerging resistance to different antibiotics. Multidrug and pan drug-resistant clinical isolates were reported worldwide. Here we report the first evidence of pan drug-resistant environmental isolate of A. baumannii. The isolate was recovered from the effluent of secondary treated municipal wastewater of the City of Zagreb, Croatia. The isolate was resistant to penicillins/β-lactamase inhibitors, carbapenems, fluoroquinolones, aminoglycosides, folate pathway inhibitors, and polymyxins, except intermediately susceptible to minocycline and tigecycline. Intrinsic chromosomally located bla OXA-51-like gene and acquired plasmid-located bla OXA-23-like gene were related to clinical isolates. Pan drug-resistant A. baumannii can occur in natural environments outside of the hospital. Secondary treated municipal wastewater represents a potential epidemiological reservoir of pan drug-resistant A. baumannii and carbapenem resistance gene.

  11. Extensively and Pre-Extensively Drug Resistant Tuberculosis in Clinical Isolates of Multi-Drug Resistant Tuberculosis Using Classical Second Line Drugs (Levofloxacin and Amikacin)

    International Nuclear Information System (INIS)

    Mirza, I. A.; Khan, F. A.; Khan, K. A.; Satti, L.; Ghafoor, T.; Fayyaz, M.

    2015-01-01

    Objective:To find out the frequency of Extensively Drug Resistant (XDR) and pre-XDR tuberculosis in clinical isolates of Multi-Drug Resistant (MDR) Tuberculosis (TB) by determining the susceptibilities against Levofloxacin and Amikacin (classical second line antituberculosis drugs). Study Design: A descriptive cross-sectional study. Place and Duration of Study: Microbiology Department, Armed Forces Institute of Pathology (AFIP), Rawalpindi, from September 2011 to August 2013. Methodology: Amikacin (AK) and Levofloxacin (LEVO) were obtained in chemically pure form from Sigma (Taufkirchen, Germany). The breakpoint concentration used for AK was 1.0 micro g/ml and for LEVO 2.0 micro g/ml. Mycobacterial Growth Indicator Tube (MGIT) 960 system was used to carry out drug susceptibility testing as per recommended protocol. Results: A total of 3 MDR-TB isolates (3 percentage) turned out to be XDR-TB based upon simultaneous resistance to injectable second line antituberculosis drug AK and one of the fluoro-quinolones (LEVO). A total of 24 MDR-TB isolates (24 percentage) were found to be pre-XDR based upon resistance to LEVO alone. Treatment status record of patients with XDR and pre-XDRTB isolates revealed that majority of patients had received fluoroquinolones (FQs) during the course of treatment. Conclusion: XDR-TB has started to emerge in MDR-TB isolates in our set up. The worrying sign is the high frequency of pre-XDR tuberculosis. Urgent steps need to be taken to stem the tide of pre-XDR-TB in our population. It is thus recommended to develop facilities to carry out drug susceptibility testing to monitor the status of pre-XDR and XDR-TB in our population. (author)

  12. Importance of the Genetic Diversity within the Mycobacterium tuberculosis Complex for the Development of Novel Antibiotics and Diagnostic Tests of Drug Resistance

    KAUST Repository

    Koser, C. U.; Feuerriegel, S.; Summers, D. K.; Archer, John A.C.; Niemann, S.

    2012-01-01

    Despite being genetically monomorphic, the limited genetic diversity within the Mycobacterium tuberculosis complex (MTBC) has practical consequences for molecular methods for drug susceptibility testing and for the use of current antibiotics and those in clinical trials. It renders some representatives of MTBC intrinsically resistant against one or multiple antibiotics and affects the spectrum and consequences of resistance mutations selected for during treatment. Moreover, neutral or silent changes within genes responsible for drug resistance can cause false-positive results with hybridization-based assays, which have been recently introduced to replace slower phenotypic methods. We discuss the consequences of these findings and propose concrete steps to rigorously assess the genetic diversity of MTBC to support ongoing clinical trials.

  13. Importance of the Genetic Diversity within the Mycobacterium tuberculosis Complex for the Development of Novel Antibiotics and Diagnostic Tests of Drug Resistance

    KAUST Repository

    Koser, C. U.

    2012-09-24

    Despite being genetically monomorphic, the limited genetic diversity within the Mycobacterium tuberculosis complex (MTBC) has practical consequences for molecular methods for drug susceptibility testing and for the use of current antibiotics and those in clinical trials. It renders some representatives of MTBC intrinsically resistant against one or multiple antibiotics and affects the spectrum and consequences of resistance mutations selected for during treatment. Moreover, neutral or silent changes within genes responsible for drug resistance can cause false-positive results with hybridization-based assays, which have been recently introduced to replace slower phenotypic methods. We discuss the consequences of these findings and propose concrete steps to rigorously assess the genetic diversity of MTBC to support ongoing clinical trials.

  14. Resistance of solanum species to phytophthora infestans evaluated in the detached-leaf and whole-plant assays

    International Nuclear Information System (INIS)

    Akhtar, K.P.; Saleem, M.Y.; Asghar, M.

    2012-01-01

    The reaction of 82 tomato genotypes belonging to 8 Solanum and a Lycopersicon species against Phytophthora infestans causing late blight was determined using detached-leaf and whole-plant assays. None of the test genotypes was immune or highly resistant. Of the 82 commercial and wild genotypes only TMS-2 (male-sterile and characterized by indeterminate growth) belonging to Lycopersicon esculentum was resistant with severity index of 2.4 in the detached-leaf assay on 0-5 scale (where 5 was highly susceptible) and percent disease index (%DI) of 23.3% under the whole-plant assay. Among the remaining genotypes, 41 were susceptible and 40 were highly susceptible under the detached-leaf assay, while 18 were susceptible and 63 were highly susceptible under the whole-plant assay. However, there was a significant difference in %DI for genotypes under the whole-plant assay. The response of whole-plants to inoculation with P. infestans in the detached-leaf assay was similar in all cases. The overall screening results indicate that TMS-2 is a good source of resistance and it can be useful for the development of tomato hybrid cultivars resistant to late blight. (author)

  15. Prevalence of drug resistant tuberculosis in Arsi Zone, Ethiopia ...

    African Journals Online (AJOL)

    Background: Wide spread of occurrence of multi-drug resistance tuberculosis is becoming a major challenge to effective tuberculosis control. Thus, it is imperative to monitor the sensitivity of anti-TB drugs regularly. Objective: To determine the prevalence resistance to anti-TB drugs in a well established control program area ...

  16. A high content screening assay to predict human drug-induced liver injury during drug discovery.

    Science.gov (United States)

    Persson, Mikael; Løye, Anni F; Mow, Tomas; Hornberg, Jorrit J

    2013-01-01

    Adverse drug reactions are a major cause for failures of drug development programs, drug withdrawals and use restrictions. Early hazard identification and diligent risk avoidance strategies are therefore essential. For drug-induced liver injury (DILI), this is difficult using conventional safety testing. To reduce the risk for DILI, drug candidates with a high risk need to be identified and deselected. And, to produce drug candidates without that risk associated, risk factors need to be assessed early during drug discovery, such that lead series can be optimized on safety parameters. This requires methods that allow for medium-to-high throughput compound profiling and that generate quantitative results suitable to establish structure-activity-relationships during lead optimization programs. We present the validation of such a method, a novel high content screening assay based on six parameters (nuclei counts, nuclear area, plasma membrane integrity, lysosomal activity, mitochondrial membrane potential (MMP), and mitochondrial area) using ~100 drugs of which the clinical hepatotoxicity profile is known. We find that a 100-fold TI between the lowest toxic concentration and the therapeutic Cmax is optimal to classify compounds as hepatotoxic or non-hepatotoxic, based on the individual parameters. Most parameters have ~50% sensitivity and ~90% specificity. Drugs hitting ≥2 parameters at a concentration below 100-fold their Cmax are typically hepatotoxic, whereas non-hepatotoxic drugs typically hit based on nuclei count, MMP and human Cmax, we identified an area without a single false positive, while maintaining 45% sensitivity. Hierarchical clustering using the multi-parametric dataset roughly separates toxic from non-toxic compounds. We employ the assay in discovery projects to prioritize novel compound series during hit-to-lead, to steer away from a DILI risk during lead optimization, for risk assessment towards candidate selection and to provide guidance of safe

  17. Molecular detection methods of resistance to antituberculosis drugs in Mycobacterium tuberculosis.

    Science.gov (United States)

    Brossier, F; Sougakoff, W

    2017-09-01

    Molecular methods predict drug resistance several weeks before phenotypic methods and enable rapid implementation of appropriate therapeutic treatment. We aimed to detail the most representative molecular tools used in routine practice for the rapid detection of resistance to antituberculosis drugs among Mycobacterium tuberculosis strains. The molecular diagnosis of resistance to antituberculosis drugs in clinical samples or from in vitro cultures is based on the detection of the most common mutations in the genes involved in the development of resistance in M. tuberculosis strains (encoding either protein targets of antibiotics, or antibiotic activating enzymes) by commercial molecular kits or by sequencing. Three hypotheses could explain the discrepancies between the genotypic results and the phenotypic drug susceptibility testing results: a low percentage of resistant mutants precluding the detection by genotypic methods on the primary culture; a low level of resistance not detected by phenotypic testing; and other resistance mechanisms not yet characterized. Molecular methods have varying sensitivity with regards to detecting antituberculosis drug resistance; that is why phenotypic susceptibility testing methods are mandatory for detecting antituberculosis drug-resistant isolates that have not been detected by molecular methods. The questionable ability of existing phenotypic and genotypic drug susceptibility testing to properly classify strains as susceptible or resistant, and at what level of resistance, was raised for several antituberculosis agents. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  18. Emergence of fluoroquinolone resistance among drug resistant tuberculosis patients at a tertiary care facility in Karachi, Pakistan.

    Science.gov (United States)

    Zaidi, Syed Mohammad Asad; Haseeb, Abdul; Habib, Shifa Salman; Malik, Amyn; Khowaja, Saira; SaifUllah, Nausheen; Rizvi, Nadeem

    2017-07-25

    Pakistan is classified as one of the high multi-drug resistant tuberculosis (MDR-TB) burden countries. A poorly regulated private sector, over-prescription of antibiotics and self-medication has led to augmented rates of drug-resistance in the country. Pakistan's first national anti-tuberculosis drug resistance survey identified high prevalence of fluoroquinolone resistance among MDR-TB patients. Further institutional evidence of fluoroquinolone drug-resistance can support re-evaluation of treatment regimens as well as invigorate efforts to control antibiotic resistance in the country. In this study, data for drug-susceptibility testing (DST) was retrospectively analyzed for a total of 133 patients receiving MDR-TB treatment at the Chest Department of Jinnah Postgraduate Medical Center, Karachi, Pakistan. Frequency analyses for resistance patterns was carried out and association of fluoroquinolone (ofloxacin) resistance with demographics and past TB treatment category were assessed. Within first-line drugs, resistance to isoniazid was detected in 97.7% of cases, followed by rifampicin (96.9%), pyrazinamide (86.4%), ethambutol (69.2%) and streptomycin (64.6%). Within second-line drugs, ofloxacin resistance was detected in 34.6% of cases. Resistance to ethionamide and amikacin was 2.3% and 1.6%, respectively. Combined resistance of oflaxacin and isoniazid was detected in 33.9% of cases. Age, gender and past TB treatment category were not significantly associated with resistance to ofloxacin. Fluoroquinolone resistance was observed in an alarmingly high proportion of MDR-TB cases. Our results suggest caution in their use for empirical management of MDR-TB cases and recommended treatment regimens for MDR-TB may require re-evaluation. Greater engagement of private providers and stringent pharmacy regulations are urgently required.

  19. Role of 14-3-3σ in poor prognosis and in radiation and drug resistance of human pancreatic cancers

    International Nuclear Information System (INIS)

    Li, Zhaomin; Dong, Zizheng; Myer, David; Yip-Schneider, Michele; Liu, Jianguo; Cui, Ping; Schmidt, C Max; Zhang, Jian-Ting

    2010-01-01

    Pancreatic cancer is the fourth leading cause of death in the US. Unlike other solid tumors such as testicular cancer which are now curable, more than 90% of pancreatic cancer patients die due to lack of response to therapy. Recently, the level of 14-3-3σ mRNA was found to be increased in pancreatic cancers and this increased expression may contribute to the failure in treatment of pancreatic cancers. In the present study, we tested this hypothesis. Western blot analysis was used to determine 14-3-3σ protein level in fresh frozen tissues and was correlated to clinical outcome. A stable cell line expressing 14-3-3σ was established and the effect of 14-3-3σ over-expression on cellular response to radiation and anticancer drugs were tested using SRB assay and clonogenic assays. Cell cycle distribution and apoptosis analyses were performed using propidium iodide staining and PARP cleavage assays. We found that 14-3-3σ protein level was increased significantly in about 71% (17 of 24) of human pancreatic cancer tissues and that the 14-3-3σ protein level in cancers correlated with lymph node metastasis and poor prognosis. Furthermore, we demonstrated that over-expression of 14-3-3σ in a pancreatic cancer cell line caused resistance to γ-irradiation as well as anticancer drugs by causing resistance to treatment-induced apoptosis and G2/M arrest. The increased level of 14-3-3σ protein likely contributes to the poor clinical outcome of human pancreatic cancers by causing resistance to radiation and anticancer drugs. Thus, 14-3-3σ may serve as a prognosis marker predicting survival of pancreatic cancer patients and guide the clinical treatment of these patients

  20. Antiretroviral drug resistance in HIV-1 therapy-naive patients in Cuba.

    Science.gov (United States)

    Pérez, Lissette; Kourí, Vivian; Alemán, Yoan; Abrahantes, Yeisel; Correa, Consuelo; Aragonés, Carlos; Martínez, Orlando; Pérez, Jorge; Fonseca, Carlos; Campos, Jorge; Álvarez, Delmis; Schrooten, Yoeri; Dekeersmaeker, Nathalie; Imbrechts, Stijn; Beheydt, Gertjan; Vinken, Lore; Soto, Yudira; Álvarez, Alina; Vandamme, Anne-Mieke; Van Laethem, Kristel

    2013-06-01

    In Cuba, antiretroviral therapy rollout started in 2001 and antiretroviral therapy coverage has reached almost 40% since then. The objectives of this study were therefore to analyze subtype distribution, and level and patterns of drug resistance in therapy-naive HIV-1 patients. Four hundred and one plasma samples were collected from HIV-1 therapy-naive patients in 2003 and in 2007-2011. HIV-1 drug resistance genotyping was performed in the pol gene and drug resistance was interpreted according to the WHO surveillance drug-resistance mutations list, version 2009. Potential impact on first-line therapy response was estimated using genotypic drug resistance interpretation systems HIVdb version 6.2.0 and Rega version 8.0.2. Phylogenetic analysis was performed using Neighbor-Joining. The majority of patients were male (84.5%), men who have sex with men (78.1%) and from Havana City (73.6%). Subtype B was the most prevalent subtype (39.3%), followed by CRF20-23-24_BG (19.5%), CRF19_cpx (18.0%) and CRF18_cpx (10.3%). Overall, 29 patients (7.2%) had evidence of drug resistance, with 4.0% (CI 1.6%-4.8%) in 2003 versus 12.5% (CI 7.2%-14.5%) in 2007-2011. A significant increase in drug resistance was observed in recently HIV-1 diagnosed patients, i.e. 14.8% (CI 8.0%-17.0%) in 2007-2011 versus 3.8% (CI 0.9%-4.7%) in 2003 (OR 3.9, CI 1.5-17.0, p=0.02). The majority of drug resistance was restricted to a single drug class (75.8%), with 55.2% patients displaying nucleoside reverse transcriptase inhibitor (NRTI), 10.3% non-NRTI (NNRTI) and 10.3% protease inhibitor (PI) resistance mutations. Respectively, 20.7% and 3.4% patients carried viruses containing drug resistance mutations against NRTI+NNRTI and NRTI+NNRTI+PI. The first cases of resistance towards other drug classes than NRTI were only detected from 2008 onwards. The most frequent resistance mutations were T215Y/rev (44.8%), M41L (31.0%), M184V (17.2%) and K103N (13.8%). The median genotypic susceptibility score for the

  1. Radiation induction of drug resistance in RIF-1 tumors and tumor cells

    International Nuclear Information System (INIS)

    Hopwood, L.E.; Moulder, J.E.

    1989-01-01

    The RIF-1 tumor cell line contains a small number of cells (1-20 per 10(6) cells) that are resistant to various single antineoplastic drugs, including 5-fluorouracil (5FU), methotrexate (MTX), and adriamycin (ADR). For 5FU the frequency of drug resistance is lower for tumor-derived cells than for cells from cell culture; for MTX the reverse is true, and for ADR there is no difference. In vitro irradiation at 5 Gy significantly increased the frequency of drug-resistant cells for 5FU, MTX, and ADR. In vivo irradiation at 3 Gy significantly increased the frequency of drug-resistant cells for 5FU and MTX, but not for ADR. The absolute risk for in vitro induction of MTX, 5FU, and ADR resistance, and for in vivo induction of 5FU resistance, was 1-3 per 10(6) cells per Gy; but the absolute risk for in vivo induction of MTX resistance was 54 per 10(6) cells per Gy. The frequency of drug-resistant cells among individual untreated tumors was highly variable; among individual irradiated tumors the frequency of drug-resistant cells was significantly less variable. These studies provide supporting data for models of the development of tumor drug resistance, and imply that some of the drug resistance seen when chemotherapy follows radiotherapy may be due to radiation-induced drug resistance

  2. Pattern of secondary acquired drug resistance to antituberculosis drug in Mumbai, India--1991-1995.

    Science.gov (United States)

    Chowgule, R V; Deodhar, L

    1998-01-01

    A retrospective observational study was conducted to find out whether secondary acquired drug resistance to isoniazid and ethambutol is high and to rifamycin and pyrazinamide is low, as is commonly believed in India. There were 2033 patients, whose sputum samples (6099) were reviewed from a specimen registry of the microbiology laboratory for the years 1991 to 1995. Of these, 521 (25.6%) patients [335 males and 186 females; age ranged from 11 to 75 years] had sputum positive culture and sensitivity for acid-fast bacilli (AFB). The drug resistance patterns in our study were: isoniazid (H) 15%, rifamycin (R) 66.8%, pyrazinamide (Z) 72.2%, ethambutol (E) 8.4%, streptomycin (S) 53.6%, cycloserine (C) 39.2% kanamycin (K) 25.1% and ethionamide (Eth) 65.3%. The resistance to streptomycin showed a significant fall over a year while there was a rise in resistance to cycloserine and kanamycin which is significant. The rate of secondary acquired resistance of isoniazid and ethambutol was low, and the rate of secondary acquired resistance to rifamycin and pyrazinamide was high, which is contarary to the common belief regarding these drugs in India. This implies that isoniazid is still a valuable drug in the treatment of multidrug resistance in India.

  3. SPIN1, negatively regulated by miR-148/152, enhances Adriamycin resistance via upregulating drug metabolizing enzymes and transporter in breast cancer.

    Science.gov (United States)

    Chen, Xu; Wang, Ya-Wen; Gao, Peng

    2018-05-09

    Spindlin1 (SPIN1), a protein highly expressed in several human cancers, has been correlated with tumorigenesis and development. Alterations of drug metabolizing enzymes and drug transporters are major determinants of chemoresistance in tumor cells. However, whether the metabolizing enzymes and transporters are under the control of SPIN1 in breast cancer chemoresistance has not yet been defined. SPIN1 expression in breast cancer cells and tissues was detected by quantitative real-time PCR (qRT-PCR) and immunohistochemistry. Chemosensitivity assays in vitro and in vivo were performed to determine the effect of SPIN1 on Adriamycin resistance. Downstream effectors of SPIN1 were screened by microarray and confirmed by qRT-PCR and Western blot. Luciferase assay and Western blot were used to identify miRNAs regulating SPIN1. We showed that SPIN1 was significantly elevated in drug-resistant breast cancer cell lines and tissues, compared with the chemosensitive ones. SPIN1 enhanced Adriamycin resistance of breast cancer cells in vitro, and downregulation of SPIN1 by miRNA could decrease Adriamycin resistance in vivo. Mechanistically, drug metabolizing enzymes and transporter CYP2C8, UGT2B4, UGT2B17 and ABCB4 were proven to be downstream effectors of SPIN1. Notably, SPIN1 was identified as a direct target of the miR-148/152 family (miR-148a-3p, miR-148b-3p and miR-152-3p). As expected, miR-148a-3p, miR-148b-3p or miR-152-3p could increase Adriamycin sensitivity in breast cancer cells in vitro. Moreover, high expression of SPIN1 or low expression of the miR-148/152 family predicted poorer survival in breast cancer patients. Our results establish that SPIN1, negatively regulated by the miR-148/152 family, enhances Adriamycin resistance in breast cancer via upregulating the expression of drug metabolizing enzymes and drug transporter.

  4. Extensively Drug-Resistant TB

    Centers for Disease Control (CDC) Podcasts

    2016-12-16

    Dr. Charlotte Kvasnovsky, a surgery resident and Ph.D. candidate in biostatistics, discusses various types of drug resistance in TB patients in South Africa.  Created: 12/16/2016 by National Center for Emerging and Zoonotic Infectious Diseases (NCEZID).   Date Released: 12/16/2016.

  5. Diversity and evolution of drug resistance mechanisms in Mycobacterium tuberculosis

    Directory of Open Access Journals (Sweden)

    Al-Saeedi M

    2017-10-01

    Full Text Available Mashael Al-Saeedi, Sahal Al-Hajoj Department of Infection and Immunity, Mycobacteriology Research Section, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia Abstract: Despite the efficacy of antibiotics to protect humankind against many deadly pathogens, such as Mycobacterium tuberculosis, nothing can prevent the emergence of drug-resistant strains. Several mechanisms facilitate drug resistance in M. tuberculosis including compensatory evolution, epistasis, clonal interference, cell wall integrity, efflux pumps, and target mimicry. In this study, we present recent findings relevant to these mechanisms, which can enable the discovery of new drug targets and subsequent development of novel drugs for treatment of drug-resistant M. tuberculosis. Keywords: Mycobacterium tuberculosis, antibiotic resistance, compensatory evolution, epistasis, efflux pumps, fitness cost

  6. Laboratory-Based Surveillance of Extensively Drug-Resistant Tuberculosis in Eastern China.

    Science.gov (United States)

    Huang, Yu; Wu, Qingqing; Xu, Shuiyang; Zhong, Jieming; Chen, Songhua; Xu, Jinghang; Zhu, Liping; He, Haibo; Wang, Xiaomeng

    2017-03-01

    With 25% of the global burden, China has the highest incidence of drug-resistant tuberculosis (TB) in the world. However, surveillance data on extensively drug-resistant TB (XDR-TB) from China are scant. To estimate the prevalence of XDR-TB in Zhejiang, Eastern China, 30 of 90 TB treatment centers in Zhejiang were recruited. Patients with suspected TB who reported to the clinics for diagnosis were requested to undergo a smear sputum test. Positive sputum samples were tested for drug susceptibility. Data on anti-TB drug resistance from 1999 to 2008 were also collected to assess drug resistance trends. A total of 931 cases were recruited for drug susceptibility testing (DST). Among these, 23.6% (95% confidence interval [CI], 18.8-24.4) were resistant to any of the following drugs: isoniazid, rifampin, streptomycin, and ethambutol. Multidrug resistant (MDR) strains were identified in 5.1% of all cases (95% CI, 3.61-6.49). Among MDR-TB cases, 6.4% were XDR (95% CI, 1.7-18.6) and 8.9% (95% CI, 7.0-10.8) of all cases were resistant to either isoniazid or rifampin (but not both). Among MDR-TB cases, 23.4% (95% CI, 12.8-38.4) were resistant to either fluoroquinolones or a second-line anti-TB injectable drug, but not both. From 1999 to 2014, the percentage of MDR cases decreased significantly, from 8.6% to 5.1% (p = 0.00). The Global Fund to Fight TB program showed signs of success in Eastern China. However, drug-resistant TB, MDR-TB, and XDR-TB still pose a challenge for TB control in Eastern China. High-quality directly observed treatment, short-course, and universal DST for TB cases to determine appropriate treatment regimens are urgently needed to prevent acquired drug resistance.

  7. Incidence of multidrug-resistant, extensively drug-resistant and pan-drug-resistant bacteria in children hospitalized at Dr. Hasan Sadikin general hospital Bandung Indonesia

    Science.gov (United States)

    Adrizain, R.; Suryaningrat, F.; Alam, A.; Setiabudi, D.

    2018-03-01

    Antibiotic resistance has become a global issue, with 700,000 deaths attributable to multidrug-resistance (MDR) occurring each year. Centers for Disease Control and Prevention (CDC) show rapidly increasing rates of infection due to antibiotic-resistant bacteria. The aim of the study isto describe the incidence of MDR, extensively drug-resistant (XDR) and pan drug-resistant (PDR) in Enterococcus spp., Staphylococcus aureus, K. pneumonia, Acinetobacter baumanii, P. aeruginosin, and Enterobacter spp. (ESKAPE) pathogens in children admitted to Dr. Hasan Sadikin Hospital. All pediatric patients having blood culture drawn from January 2015 to December 2016 were retrospectively studied. Data include the number of drawn blood culture, number of positive results, type of bacteria, sensitivity pattern. International standard definitions for acquired resistance by ECDC and CDC was used as definitions for MDR, XDR and PDR bacteria. From January 2015 to December 2016, 299 from 2.542 (11.7%) blood culture was positive, with Staphylococcus aureus, Enterococcus spp., Enterobacteriaceae, Pseudomonas aeruginosa, Acinetobacter spp., respectively 5, 6, 24, 5, 20 with total 60 (20%). The MDR and XDR pathogen found were 47 and 13 patients, respectively.

  8. Detection of First-Line Drug Resistance Mutations and Drug-Protein Interaction Dynamics from Tuberculosis Patients in South India.

    Science.gov (United States)

    Nachappa, Somanna Ajjamada; Neelambike, Sumana M; Amruthavalli, Chokkanna; Ramachandra, Nallur B

    2018-05-01

    Diagnosis of drug-resistant tuberculosis predominantly relies on culture-based drug susceptibility testing, which take weeks to produce a result and a more time-efficient alternative method is multiplex allele-specific PCR (MAS-PCR). Also, understanding the role of mutations in causing resistance helps better drug designing. To evaluate the ability of MAS-PCR in the detection of drug resistance and to understand the mechanism of interaction of drugs with mutant proteins in Mycobacterium tuberculosis. Detection of drug-resistant mutations using MAS-PCR and validation through DNA sequencing. MAS-PCR targeted five loci on three genes, katG 315 and inhA -15 for the drug isoniazid (INH), and rpoB 516, 526, and 531 for rifampicin (RIF). Furthermore, the sequence data were analyzed to study the effect on interaction of the anti-TB drug molecule with the target protein using in silico docking. We identified drug-resistant mutations in 8 out of 114 isolates with 2 of them as multidrug-resistant TB using MAS-PCR. DNA sequencing confirmed only six of these, recording a sensitivity of 85.7% and specificity of 99.3% for MAS-PCR. Molecular docking showed estimated free energy of binding (ΔG) being higher for RIF binding with RpoB S531L mutant. Codon 315 in KatG does not directly interact with INH but blocks the drug access to active site. We propose DNA sequencing-based drug resistance detection for TB, which is more accurate than MAS-PCR. Understanding the action of resistant mutations in disrupting the normal drug-protein interaction aids in designing effective drug alternatives.

  9. Effect of pretreatment HIV-1 drug resistance on immunological, virological, and drug-resistance outcomes of first-line antiretroviral treatment in sub-Saharan Africa: a multicentre cohort study

    NARCIS (Netherlands)

    Hamers, Raph L.; Schuurman, Rob; Sigaloff, Kim C. E.; Wallis, Carole L.; Kityo, Cissy; Siwale, Margaret; Mandaliya, Kishor; Ive, Prudence; Botes, Mariette E.; Wellington, Maureen; Osibogun, Akin; Wit, Ferdinand W.; van Vugt, Michèle; Stevens, Wendy S.; de Wit, Tobias F. Rinke

    2012-01-01

    Background The effect of pretreatment HIV-1 drug resistance on the response to first-line combination antiretroviral therapy (ART) in sub-Saharan Africa has not been assessed. We studied pretreatment drug resistance and virological, immunological, and drug-resistance treatment outcomes in a large

  10. Sulfonylureas and Glinides as New PPARγ Agonists:. Virtual Screening and Biological Assays

    Science.gov (United States)

    Scarsi, Marco; Podvinec, Michael; Roth, Adrian; Hug, Hubert; Kersten, Sander; Albrecht, Hugo; Schwede, Torsten; Meyer, Urs A.; Rücker, Christoph

    2007-12-01

    This work combines the predictive power of computational drug discovery with experimental validation by means of biological assays. In this way, a new mode of action for type 2 diabetes drugs has been unvealed. Most drugs currently employed in the treatment of type 2 diabetes either target the sulfonylurea receptor stimulating insulin release (sulfonylureas, glinides), or target PPARγ improving insulin resistance (thiazolidinediones). Our work shows that sulfonylureas and glinides bind to PPARγ and exhibit PPARγ agonistic activity. This result was predicted in silico by virtual screening and confirmed in vitro by three biological assays. This dual mode of action of sulfonylureas and glinides may open new perspectives for the molecular pharmacology of antidiabetic drugs, since it provides evidence that drugs can be designed which target both the sulfonylurea receptor and PPARγ. Targeting both receptors could in principle allow to increase pancreatic insulin secretion, as well as to improve insulin resistance.

  11. The Association between Mycobacterium Tuberculosis Genotype and Drug Resistance in Peru.

    Directory of Open Access Journals (Sweden)

    Louis Grandjean

    Full Text Available The comparison of Mycobacterium tuberculosis bacterial genotypes with phenotypic, demographic, geospatial and clinical data improves our understanding of how strain lineage influences the development of drug-resistance and the spread of tuberculosis.To investigate the association of Mycobacterium tuberculosis bacterial genotype with drug-resistance. Drug susceptibility testing together with genotyping using both 15-loci MIRU-typing and spoligotyping, was performed on 2,139 culture positive isolates, each from a different patient in Lima, Peru. Demographic, geospatial and socio-economic data were collected using questionnaires, global positioning equipment and the latest national census.The Latin American Mediterranean (LAM clade (OR 2.4, p<0.001 was significantly associated with drug-resistance and alone accounted for more than half of all drug resistance in the region. Previously treated patients, prisoners and genetically clustered cases were also significantly associated with drug-resistance (OR's 2.5, 2.4 and 1.8, p<0.001, p<0.05, p<0.001 respectively.Tuberculosis disease caused by the LAM clade was more likely to be drug resistant independent of important clinical, genetic and socio-economic confounding factors. Explanations for this include; the preferential co-evolution of LAM strains in a Latin American population, a LAM strain bacterial genetic background that favors drug-resistance or the "founder effect" from pre-existing LAM strains disproportionately exposed to drugs.

  12. Antituberculosis drug resistance patterns in adults with tuberculous meningitis

    DEFF Research Database (Denmark)

    Senbayrak, Seniha; Ozkutuk, Nuri; Erdem, Hakan

    2015-01-01

    BACKGROUND: Tuberculous meningitis (TBM) caused by Mycobacterium tuberculosis resistant to antituberculosis drugs is an increasingly common clinical problem. This study aimed to evaluate drug resistance profiles of TBM isolates in adult patients in nine European countries involving 32 centers...

  13. A targeted proteomics approach to the quantitative analysis of ERK/Bcl-2-mediated anti-apoptosis and multi-drug resistance in breast cancer.

    Science.gov (United States)

    Yang, Ting; Xu, Feifei; Sheng, Yuan; Zhang, Wen; Chen, Yun

    2016-10-01

    Apoptosis suppression caused by overexpression of anti-apoptotic proteins is a central factor to the acquisition of multi-drug resistance (MDR) in breast cancer. As a highly conserved anti-apoptotic protein, Bcl-2 can initiate an anti-apoptosis response via an ERK1/2-mediated pathway. However, the details therein are still far from completely understood and a quantitative description of the associated proteins in the biological context may provide more insights into this process. Following our previous attempts in the quantitative analysis of MDR mechanisms, liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based targeted proteomics was continually employed here to describe ERK/Bcl-2-mediated anti-apoptosis. A targeted proteomics assay was developed and validated first for the simultaneous quantification of ERK1/2 and Bcl-2. In particular, ERK isoforms (i.e., ERK1 and ERK2) and their differential phosphorylated forms including isobaric ones were distinguished. Using this assay, differential protein levels and site-specific phosphorylation stoichiometry were observed in parental drug-sensitive MCF-7/WT cancer cells and drug-resistant MCF-7/ADR cancer cells and breast tissue samples from two groups of patients who were either suspected or diagnosed to have drug resistance. In addition, quantitative analysis of the time course of both ERK1/2 and Bcl-2 in doxorubicin (DOX)-treated MCF-7/WT cells confirmed these findings. Overall, we propose that targeted proteomics can be used generally to resolve more complex cellular events.

  14. Multi-drug resistant tuberculosis in Tanzania: Initial description of ...

    African Journals Online (AJOL)

    Background: Drug resistant Tuberculosis is well documented worldwide and is associated with increasing morbidity and mortality complicating Tuberculosis control with increasing costs of managing the disease. Broad. Objective: To describe clinical and laboratory characteristics of multi-drug resistant Tuberculosis ...

  15. Macropis fulvipes Venom component Macropin Exerts its Antibacterial and Anti-Biofilm Properties by Damaging the Plasma Membranes of Drug Resistant Bacteria.

    Science.gov (United States)

    Ko, Su Jin; Kim, Min Kyung; Bang, Jeong Kyu; Seo, Chang Ho; Luchian, Tudor; Park, Yoonkyung

    2017-11-29

    The abuse of antibiotics for disease treatment has led to the emergence of multidrug resistant bacteria. Antimicrobial peptides, found naturally in various organisms, have received increasing interest as alternatives to conventional antibiotics because of their broad spectrum antimicrobial activity and low cytotoxicity. In a previous report, Macropin, isolated from bee venom, exhibited antimicrobial activity against both gram-positive and negative bacteria. In the present study, Macropin was synthesized and its antibacterial and anti-biofilm activities were tested against bacterial strains, including gram-positive and negative bacteria, and drug resistant bacteria. Moreover, Macropin did not exhibit hemolytic activity and cytotoxicity to keratinocytes, whereas Melittin, as a positive control, showed very high toxicity. Circular dichroism assays showed that Macropin has an α-helical structure in membrane mimic environments. Macropin binds to peptidoglycan and lipopolysaccharide and kills the bacteria by disrupting their membranes. Moreover, the fractional inhibitory concentration index indicated that Macropin has additive and partially synergistic effects with conventional antibiotics against drug resistant bacteria. Thus, our study suggested that Macropin has potential for use of an antimicrobial agent for infectious bacteria, including drug resistant bacteria.

  16. Molecular basis of antifungal drug resistance in yeasts

    DEFF Research Database (Denmark)

    Morio, Florent; Jensen, Rasmus Hare; Le Pape, Patrice

    2017-01-01

    Besides inherent differences in in vitro susceptibilities, clinically-relevant yeast species may acquire resistance upon exposure to most antifungal drugs used in the clinic. In recent years, major fundamental research studies have been conducted to improve our understanding of the molecular basis...... of antifungal resistance. This topic is of major interest as antifungal resistance in yeast is clearly evolving and is correlated with clinical failure. This minireview is an overview of the most recent findings about key molecular mechanisms evolving in human pathogenic yeasts, particularly Candida spp......., in the context of antifungal drug resistance. Also included are the methods currently available for in vitro antifungal susceptibility testing and for molecular detection of mutations associated with resistance. Finally, the genetic drivers of antifungal resistance are discussed in light of the spectra...

  17. Detection of tumor-specific cytotoxic drug activity in vitro using the fluorometric microculture cytotoxicity assay and primary cultures of tumor cells from patients.

    Science.gov (United States)

    Nygren, P; Fridborg, H; Csoka, K; Sundström, C; de la Torre, M; Kristensen, J; Bergh, J; Hagberg, H; Glimelius, B; Rastad, J

    1994-03-01

    The semi-automated fluorometric microculture cytotoxicity assay (FMCA), based on the measurement of fluorescence generated from cellular hydrolysis of fluorescein diacetate (FDA) by viable cells, was employed for cytotoxic drug sensitivity testing of tumor cells from patients with hematological or solid tumors. In total, 390 samples from 20 diagnoses were tested with up to 12 standard cytotoxic drugs. The technical success rate for different tumor types ranged from 67 to 95%. Fluorescence was linearly related to cell number but variably steep depending on tumor type. Samples from most solid tumors thus showed higher signal-to-noise ratios than hematological samples. A wide spectrum of in vitro drug activity was obtained, with acute leukemias and non-Hodgkin's lymphomas being sensitive to almost all tested drugs, whereas renal and adrenocortical carcinomas were essentially totally resistant. Between these extremes were samples of breast and ovarian carcinomas and sarcomas. When in vitro response was compared with known clinical response patterns, a good correspondence was observed. The results indicate that the FMCA is a rapid and efficient method for in vitro measurement of tumor-specific drug activity both in hematological and in solid tumors. The assay may be suitable for new drug development and direction of phase-2 trials to suitable patients.

  18. Application of a drug-induced apoptosis assay to identify treatment strategies in recurrent or metastatic breast cancer.

    Directory of Open Access Journals (Sweden)

    Linda Bosserman

    Full Text Available A drug-induced apoptosis assay has been developed to determine which chemotherapy drugs or regimens can produce higher cell killing in vitro. This study was done to determine if this assay could be performed in patients with recurrent or metastatic breast cancer patients, to characterize the patterns of drug-induced apoptosis, and to evaluate the clinical utility of the assay. A secondary goal was to correlate assay use with clinical outcomes.In a prospective, non-blinded, multi institutional controlled trial, 30 evaluable patients with recurrent or metastatic breast cancer who were treated with chemotherapy had tumor samples submitted for the MiCK drug-induced apoptosis assay. After receiving results within 72 hours after biopsy, physicians could use the test to determine therapy (users, or elect to not use the test (non-users.The assay was able to characterize drug-induced apoptosis in tumor specimens from breast cancer patients and identified which drugs or combinations gave highest levels of apoptosis. Patterns of drug activity were also analyzed in triple negative breast cancer. Different drugs from a single class of agents often produced significantly different amounts of apoptosis. Physician frequently (73% used the assay to help select chemotherapy treatments in patients, Patients whose physicians were users had a higher response (CR+PR rate compared to non-users (38.1% vs 0%, p = 0.04 and a higher disease control (CR+PR+Stable rate (81% vs 25%, p<0.01. Time to relapse was longer in users 7.4 mo compared to non-users 2.2 mo (p<0.01.The MiCK assay can be performed in breast cancer specimens, and results are often used by physicians in breast cancer patients with recurrent or metastatic disease. These results from a good laboratory phase II study can be the basis for a future larger prospective multicenter study to more definitively establish the value of the assay.Clinicaltrials.gov NCT00901264.

  19. Reversion of pH-induced physiological drug resistance: a novel function of copolymeric nanoparticles.

    Directory of Open Access Journals (Sweden)

    Rutian Li

    Full Text Available AIMS: The extracellular pH of cancer cells is lower than the intracellular pH. Weakly basic anticancer drugs will be protonated extracellularly and display a decreased intracellular concentration. In this study, we show that copolymeric nanoparticles (NPs are able to overcome this "pH-induced physiological drug resistance" (PIPDR by delivering drugs to the cancer cells via endocytosis rather than passive diffussion. MATERIALS AND METHODS: As a model nanoparticle, Tetradrine (Tet, Pka 7.80 was incorporated into mPEG-PCL. The effectiveness of free Tet and Tet-NPs were compared at different extracellular pHs (pH values 6.8 and 7.4, respectively by MTT assay, morphological observation and apoptotic analysis in vitro and on a murine model by tumor volume measurement, PET-CT scanning and side effect evaluation in vivo. RESULTS: The cytotoxicity of free Tet decreased prominently (P<0.05 when the extracellular pH decreased from 7.4 to 6.8. Meanwhile, the cytotoxicity of Tet-NPs was not significantly influenced by reduced pH. In vivo experiment also revealed that Tet-NPs reversed PIPDR more effectively than other existing methods and with much less side effects. CONCLUSION: The reversion of PIPDR is a new discovered mechanism of copolymeric NPs. This study emphasized the importance of cancer microenvironmental factors in anticancer drug resistance and revealed the superiority of nanoscale drug carrier from a different aspect.

  20. Tenofovir-based regimens associated with less drug resistance in HIV-1-infected Nigerians failing first-line antiretroviral therapy.

    Science.gov (United States)

    Etiebet, Mary-Ann A; Shepherd, James; Nowak, Rebecca G; Charurat, Man; Chang, Harry; Ajayi, Samuel; Elegba, Olufunmilayo; Ndembi, Nicaise; Abimiku, Alashle; Carr, Jean K; Eyzaguirre, Lindsay M; Blattner, William A

    2013-02-20

    In resource-limited settings, HIV-1 drug resistance testing to guide antiretroviral therapy (ART) selection is unavailable. We retrospectively conducted genotypic analysis on archived samples from Nigerian patients who received targeted viral load testing to confirm treatment failure and report their drug resistance mutation patterns. Stored plasma from 349 adult patients on non-nucleoside reverse transcriptase inhibitor (NNRTI) regimens was assayed for HIV-1 RNA viral load, and samples with more than 1000 copies/ml were sequenced in the pol gene. Analysis for resistance mutations utilized the IAS-US 2011 Drug Resistance Mutation list. One hundred and seventy-five samples were genotyped; the majority of the subtypes were G (42.9%) and CRF02_AG (33.7%). Patients were on ART for a median of 27 months. 90% had the M184V/I mutation, 62% had at least one thymidine analog mutation, and 14% had the K65R mutation. 97% had an NNRTI resistance mutation and 47% had at least two etravirine-associated mutations. In multivariate analysis tenofovir-based regimens were less likely to have at least three nucleoside reverse transcriptase inhibitor (NRTI) mutations after adjusting for subtype, previous ART, CD4, and HIV viral load [P < 0.001, odds ratio (OR) 0.04]. 70% of patients on tenofovir-based regimens had at least two susceptible NRTIs to include in a second-line regimen compared with 40% on zidovudine-based regimens (P = 0.04, OR = 3.4). At recognition of treatment failure, patients on tenofovir-based first-line regimens had fewer NRTI drug-resistant mutations and more active NRTI drugs available for second-line regimens. These findings can inform strategies for ART regimen sequencing to optimize long-term HIV treatment outcomes in low-resource settings.

  1. vPARP Adjusts MVP Expression in Drug-resistant Cell Lines in Conjunction with MDR Proteins.

    Science.gov (United States)

    Wojtowicz, Karolina; Januchowski, Radoslaw; Nowicki, Michal; Zabel, Maciej

    2017-06-01

    The definition of vault (ribonucleoprotein particles) function remains highly complex. Vaults may cooperate with multidrug resistance (MDR) proteins, supporting their role in drug resistance. This topic is the main theme of this publication. The cell viability was determined by an MTT assay. The protein expression was detected by western blot analysis. The proteins were knocked-down using siRNA. No major vault protein (MVP) in the LoVo/Dx and W1PR cell lines after tunicamycin treatment was shown. In W1PR cells with knocked-down MVP, a statistically significant decrease in cell viability was noted. In LoVo/Dx, W1TR and A2780TR cells were vault poly-ADP-ribose polymerase (vPARP) was knockdown, a decrease in cell viability was shown. Also, MVP silencing induced an increase in glycoprotein P (Pgp) expression in LoVo/Dx cells. MVP is important for the drug resistance of cancer cells, but it probably requires the presence of vPARP for full activation. Some correlations between MDR proteins and vaults exist. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  2. Epigenetic Modulation of the Biophysical Properties of Drug-Resistant Cell Lipids to Restore Drug Transport and Endocytic Functions

    OpenAIRE

    Vijayaraghavalu, Sivakumar; Peetla, Chiranjeevi; Lu, Shan; Labhasetwar, Vinod

    2012-01-01

    In our recent studies exploring the biophysical characteristics of resistant cell lipids, and the role they play in drug transport, we demonstrated the difference of drug-resistant breast cancer cells from drug-sensitive cells in lipid composition and biophysical properties, suggesting that cancer cells acquire a drug-resistant phenotype through the alteration of lipid synthesis to inhibit intracellular drug transport to protect from cytotoxic effect. In cancer cells, epigenetic changes (e.g....

  3. Impact of treatment heterogeneity on drug resistance and supply chain costs.

    Science.gov (United States)

    Spiliotopoulou, Eirini; Boni, Maciej F; Yadav, Prashant

    2013-09-01

    The efficacy of scarce drugs for many infectious diseases is threatened by the emergence and spread of resistance. Multiple studies show that available drugs should be used in a socially optimal way to contain drug resistance. This paper studies the tradeoff between risk of drug resistance and operational costs when using multiple drugs for a specific disease. Using a model for disease transmission and resistance spread, we show that treatment with multiple drugs, on a population level, results in better resistance-related health outcomes, but more interestingly, the marginal benefit decreases as the number of drugs used increases. We compare this benefit with the corresponding change in procurement and safety stock holding costs that result from higher drug variety in the supply chain. Using a large-scale simulation based on malaria transmission dynamics, we show that disease prevalence seems to be a less important factor when deciding the optimal width of drug assortment, compared to the duration of one episode of the disease and the price of the drug(s) used. Our analysis shows that under a wide variety of scenarios for disease prevalence and drug cost, it is optimal to simultaneously deploy multiple drugs in the population. If the drug price is high, large volume purchasing discounts are available, and disease prevalence is high, it may be optimal to use only one drug. Our model lends insights to policy makers into the socially optimal size of drug assortment for a given context.

  4. In vitro evaluation of new anticancer drugs, exemplified by vinorelbine, using the fluorometric microculture cytotoxicity assay on human tumor cell lines and patient biopsy cells.

    Science.gov (United States)

    Fridborg, H; Nygren, P; Dhar, S; Csoka, K; Kristensen, J; Larsson, R

    1996-09-01

    The feasibility of combined studies on a cell-line panel and primary cultures of patient tumor cells in the preclinical evaluation of new anticancer drugs was evaluated in a study of the activity and cross-resistance pattern in vitro of the new semi-synthetic vinca alkaloid vinorelbine (Vrb). The activity of Vrb was investigated in ten cell lines representing different resistance mechanisms and in a total of 256 fresh human tumor samples, using the fluorometric microculture cytotoxicity assay (FMCA). Resistance to Vrb in the cell lines was associated with expression of the multidrug resistance-mediating P-glycoprotein and the multidrug resistance-associated protein (MRP) and by a recently described tubulin-associated mechanism, while the cell lines with topoisomerase II- and glutathion-associated resistance did not show decreased sensitivity to the drug. Cross-resistance to vincristine (Vcr) and other tubulin-active agents was high in cell lines as well as in patient cells. As with most commonly used anti-cancer drugs, Vrb was more active in hematological than in solid tumor samples. Among the solid tumors investigated, the highest in vitro response rates were observed in ovarian cancer (27%), sarcoma (25%), non-small cell lung cancer (21%) and bladder cancer (20%), while no response was observed in renal or colorectal cancer. Compared to Vcr, Vrb appeared to be slightly more active in solid tumors and slightly less active in hematological tumors. The results show that although Vrb displays a high degree of cross-resistance to Vcr and other tubulin-active drugs, some difference in the activity spectrum could be detected and that the drug is sensitive to multiple mechanisms of resistance. The results also suggest that leukemias, ovarian cancer, sarcoma and bladder cancer are possible further targets for Vrb. The combination of studies on a cell-line panel and patient tumor cells from a broad spectrum of diagnoses to evaluate a new drug seems feasible and may give

  5. Shigella Antimicrobial Drug Resistance Mechanisms, 2004-2014.

    Science.gov (United States)

    Nüesch-Inderbinen, Magdalena; Heini, Nicole; Zurfluh, Katrin; Althaus, Denise; Hächler, Herbert; Stephan, Roger

    2016-06-01

    To determine antimicrobial drug resistance mechanisms of Shigella spp., we analyzed 344 isolates collected in Switzerland during 2004-2014. Overall, 78.5% of isolates were multidrug resistant; 10.5% were ciprofloxacin resistant; and 2% harbored mph(A), a plasmid-mediated gene that confers reduced susceptibility to azithromycin, a last-resort antimicrobial agent for shigellosis.

  6. Viability, biofilm formation, and MazEF expression in drug-sensitive and drug-resistant Mycobacterium tuberculosis strains circulating in Xinjiang, China.

    Science.gov (United States)

    Zhao, Ji-Li; Liu, Wei; Xie, Wan-Ying; Cao, Xu-Dong; Yuan, Li

    2018-01-01

    Tuberculosis (TB) caused by Mycobacterium tuberculosis (MTB) is one of the most common chronic infectious amphixenotic diseases worldwide. Prevention and control of TB are greatly difficult, due to the increase in drug-resistant TB, particularly multidrug-resistant TB. We speculated that there were some differences between drug-sensitive and drug-resistant MTB strains and that mazEF 3,6,9 toxin-antitoxin systems (TASs) were involved in MTB viability. This study aimed to investigate differences in viability, biofilm formation, and MazEF expression between drug-sensitive and drug-resistant MTB strains circulating in Xinjiang, China, and whether mazEF 3,6,9 TASs contribute to MTB viability under stress conditions. Growth profiles and biofilm-formation abilities of drug-sensitive, drug-resistant MTB strains and the control strain H37Rv were monitored. Using molecular biology experiments, the mRNA expression of the mazF 3, 6, and 9 toxin genes, the mazE 3, 6, and 9 antitoxin genes, and expression of the MazF9 protein were detected in the different MTB strains, H37RvΔ mazEF 3,6,9 mutants from the H37Rv parent strain were generated, and mutant viability was tested. Ex vivo culture analyses demonstrated that drug-resistant MTB strains exhibit higher survival rates than drug-sensitive strains and the control strain H37Rv. However, there was no statistical difference in biofilm-formation ability in the drug-sensitive, drug-resistant, and H37Rv strains. mazE 3,6 mRNA-expression levels were relatively reduced in the drug-sensitive and drug-resistant strains compared to H37Rv. Conversely, mazE 3,9 expression was increased in drug-sensitive strains compared to drug-resistant strains. Furthermore, compared with the H37Rv strain, mazF 3,6 expression was increased in drug-resistant strains, mazF 9 expression was increased in drug-sensitive strains, and mazF 9 exhibited reduced expression in drug-resistant strains compared with drug-sensitive strains. Protein expression of mazF9

  7. Economic implications of resistance to antimalarial drugs.

    Science.gov (United States)

    Phillips, M; Phillips-Howard, P A

    1996-09-01

    The widespread evolution of drug resistance in malarial parasites has seriously hampered efforts to control this debilitating disease. Chloroquine, the mainstay of malaria treatment for many decades, is now proving largely ineffective in many parts of the world, particularly against the most severe form of malaria--falciparum. Alternative drugs have been developed, but they are frequently less safe and are all between 50 and 700% more expensive than chloroquine. Choice of drug clearly has important budgetary implications and national malaria control programmes need to weigh up the costs and benefits in deciding whether to change to more effective but more expensive drugs. The growth in drug resistance also has implications for the choice of diagnostic tool. Clinical diagnosis of malaria is relatively cheap, but less specific than some technological approaches. As more expensive drugs are employed, the cost of wasted treatment on suspected cases who do not in fact have malaria rises and the more worthwhile it becomes to invest in more specific diagnostic techniques. This paper presents an economic framework for analysing the various malaria drug and diagnostic tool options available. It discusses the nature of the key factors that need to be considered when making choices of malaria treatment (including treatment costs, drug resistance, the costs of treatment failure and compliance) and diagnosis (including diagnosis cost and accuracy, and the often overlooked costs associated with delayed treatment), and uses some simple equations to illustrate the impact of these on the relative cost effectiveness of the alternatives being considered. On the basis of some simplifying assumptions and illustrative calculations, it appears that in many countries more effective drugs and more specific and rapid diagnostic approaches will be worth adopting even although they imply additional expense.

  8. Drug-resistance in chronic tuberculosis cases in Southern Nigeria ...

    African Journals Online (AJOL)

    Nigeria has a high burden of tuberculosis but the drug resistant situationwas previously unknown. This report evaluates the firstline drug resistance and associated factors among chronic tuberculosis cases from the tuberculosis control programme in South south and South east zones ofNigeria. Descriptive study of chronic ...

  9. Repurposing salicylanilide anthelmintic drugs to combat drug resistant Staphylococcus aureus.

    Science.gov (United States)

    Rajamuthiah, Rajmohan; Fuchs, Beth Burgwyn; Conery, Annie L; Kim, Wooseong; Jayamani, Elamparithi; Kwon, Bumsup; Ausubel, Frederick M; Mylonakis, Eleftherios

    2015-01-01

    Staphylococcus aureus is a Gram-positive bacterium that has become the leading cause of hospital acquired infections in the US. Repurposing Food and Drug Administration (FDA) approved drugs for antimicrobial therapy involves lower risks and costs compared to de novo development of novel antimicrobial agents. In this study, we examined the antimicrobial properties of two commercially available anthelmintic drugs. The FDA approved drug niclosamide and the veterinary drug oxyclozanide displayed strong in vivo and in vitro activity against methicillin resistant S. aureus (minimum inhibitory concentration (MIC): 0.125 and 0.5 μg/ml respectively; minimum effective concentration: ≤ 0.78 μg/ml for both drugs). The two drugs were also effective against another Gram-positive bacteria Enterococcus faecium (MIC 0.25 and 2 μg/ml respectively), but not against the Gram-negative species Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter aerogenes. The in vitro antimicrobial activity of niclosamide and oxyclozanide were determined against methicillin, vancomycin, linezolid or daptomycin resistant S. aureus clinical isolates, with MICs at 0.0625-0.5 and 0.125-2 μg/ml for niclosamide and oxyclozanide respectively. A time-kill study demonstrated that niclosamide is bacteriostatic, whereas oxyclozanide is bactericidal. Interestingly, oxyclozanide permeabilized the bacterial membrane but neither of the anthelmintic drugs exhibited demonstrable toxicity to sheep erythrocytes. Oxyclozanide was non-toxic to HepG2 human liver carcinoma cells within the range of its in vitro MICs but niclosamide displayed toxicity even at low concentrations. These data show that the salicylanilide anthelmintic drugs niclosamide and oxyclozanide are suitable candidates for mechanism of action studies and further clinical evaluation for treatment of staphylococcal infections.

  10. High prevalence of drug-resistant tuberculosis, Republic of Lithuania, 2002

    DEFF Research Database (Denmark)

    Dewan, P; Sosnovskaja, A; Thomsen, V

    2005-01-01

    BACKGROUND: Nations of the former Soviet Union have the world's highest reported levels of resistance to anti-tuberculosis drugs. We conducted the first national survey of anti-tuberculosis drug resistance in the Republic of Lithuania. METHODS: We tested Mycobacterium tuberculosis isolates from all...... isolates, 475 (41%) were resistant to at least one first-line drug, and 263 (23%) were resistant to at least INH and RMP (MDR); this included 76/818 (9.3%) from new patients and 187/345 (54%) from previously treated patients. Of 52 MDR isolates randomly selected for extended testing at an international...

  11. A modified Plasmodium falciparum growth inhibition assay (GIA) to assess activity of plasma from malaria endemic areas.

    Science.gov (United States)

    Mlambo, Godfree; Kumar, Nirbhay

    2007-02-01

    Plasma samples from patients undergoing treatment in malaria endemic countries often contain anti-malaria drugs, that may overstate effects of specific antibodies in growth inhibition assays (GIA). We describe a modified assay that uses drug resistant P. falciparum parasites (W2) that circumvents the requirement for dialyzing samples that may likely contain drugs such as chloroquine and sulfadoxine/pyrimethamine (SP).

  12. Young Women's Experiences of Resisting Invitations to Use Illicit Drugs

    Science.gov (United States)

    Koehn, Corinne V.; O'Neill, Linda K.

    2011-01-01

    Ten young women were interviewed regarding their experiences of resisting invitations to use illicit drugs. Hermeneutic phenomenology was used to gather and analyze information. One key theme was the motivations that inspired women to refuse drug offers. Young women resisted drug invitations because of their desires to be authentic, protect their…

  13. Status of drug-resistant tuberculosis in China: A systematic review and meta-analysis.

    Science.gov (United States)

    Zhang, Jingya; Gou, Haimei; Hu, Xuejiao; Hu, Xin; Shang, Mengqiao; Zhou, Juan; Zhou, Yi; Ye, Yuanxin; Song, Xingbo; Lu, Xiaojun; Chen, Xuerong; Ying, Binwu; Wang, Lanlan

    2016-06-01

    We conducted a systematic review and meta-analysis on drug-resistant tuberculosis in China to provide useful data for tuberculosis (TB) surveillance and treatment. Several databases, including PubMed, Embase, and the Chinese Biological Medical Database, were systematically searched between January 1, 1999, and August 31, 2015, using strict inclusion and exclusion criteria. The corresponding drug-resistant TB prevalence between the new and previously treated cases was significantly different in almost all of the economic regions. The Eastern coastal region is the most developed economic region with the lowest total drug-resistant TB prevalence (any drug resistance: 28%; 95% confidence interval [CI], 25%-32%; multidrug resistance: 9%; 95% CI, 8%-12%) and the lowest number of new cases (any drug resistance: 21%; 95% CI, 19%-23%; multidrug resistance: 4%; 95% CI, 3%-5%). The Northwest is the least developed area with the lowest drug-resistant TB prevalence for previously treated cases (any drug resistance: 45%; 95% CI, 36%-55%; multidrug resistance: 17%; 95% CI, 11%-26%). The prevalence (multidrug and first-line drug resistance) exhibited a downward trend from 1996-2014. The extensively drug-resistant prevalence in China was 3% (95% CI, 2%-5%) in this review. Overall, the status of drug-resistant tuberculosis in China is notably grim and exhibits regional epidemiologic characteristics. We are in urgent need of several comprehensive and effective control efforts to reverse this situation. Copyright © 2016 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Elsevier Inc. All rights reserved.

  14. A Comparison of Real-Time and Endpoint Cell Viability Assays for Improved Synthetic Lethal Drug Validation.

    Science.gov (United States)

    Single, Andrew; Beetham, Henry; Telford, Bryony J; Guilford, Parry; Chen, Augustine

    2015-12-01

    Cell viability assays fulfill a central role in drug discovery studies. It is therefore important to understand the advantages and disadvantages of the wide variety of available assay methodologies. In this study, we compared the performance of three endpoint assays (resazurin reduction, CellTiter-Glo, and nuclei enumeration) and two real-time systems (IncuCyte and xCELLigence). Of the endpoint approaches, both the resazurin reduction and CellTiter-Glo assays showed higher cell viabilities when compared directly to stained nuclei counts. The IncuCyte and xCELLigence real-time systems were comparable, and both were particularly effective at tracking the effects of drug treatment on cell proliferation at sub-confluent growth. However, the real-time systems failed to evaluate contrasting cell densities between drug-treated and control-treated cells at full growth confluency. Here, we showed that using real-time systems in combination with endpoint assays alleviates the disadvantages posed by each approach alone, providing a more effective means to evaluate drug toxicity in monolayer cell cultures. Such approaches were shown to be effective in elucidating the toxicity of synthetic lethal drugs in an isogenic pair of MCF10A breast cell lines. © 2015 Society for Laboratory Automation and Screening.

  15. CHEMOTHERAPY, WITHIN-HOST ECOLOGY AND THE FITNESS OF DRUG-RESISTANT MALARIA PARASITES

    OpenAIRE

    Huijben, Silvie; Nelson, William A.; Wargo, Andrew R.; Sim, Derek G.; Drew, Damien R.; Read, Andrew F.

    2010-01-01

    A major determinant of the rate at which drug-resistant malaria parasites spread through a population is the ecology of resistant and sensitive parasites sharing the same host. Drug treatment can significantly alter this ecology by removing the drug-sensitive parasites, leading to competitive release of resistant parasites. Here, we test the hypothesis that the spread of resistance can be slowed by reducing drug treatment and hence restricting competitive release. Using the rodent malaria mod...

  16. Tuberculosis drug resistance in the Western Cape | Weyer | South ...

    African Journals Online (AJOL)

    Objectives: Drug resistance is a serious problem in the treatment of tuberculosis and a threat to successful tuberculosis control programmes. Local health workers have expressed concern that the increasing tuberculosis epidemic in the Western Cape is partly attributable to drug resistance. The aim of this study was to ...

  17. Establishing Drug Resistance in Microorganisms by Mass Spectrometry

    Science.gov (United States)

    Demirev, Plamen A.; Hagan, Nathan S.; Antoine, Miquel D.; Lin, Jeffrey S.; Feldman, Andrew B.

    2013-08-01

    A rapid method to determine drug resistance in bacteria based on mass spectrometry is presented. In it, a mass spectrum of an intact microorganism grown in drug-containing stable isotope-labeled media is compared with a mass spectrum of the intact microorganism grown in non-labeled media without the drug present. Drug resistance is determined by predicting characteristic mass shifts of one or more microorganism biomarkers using bioinformatics algorithms. Observing such characteristic mass shifts indicates that the microorganism is viable even in the presence of the drug, thus incorporating the isotopic label into characteristic biomarker molecules. The performance of the method is illustrated on the example of intact E. coli, grown in control (unlabeled) and 13C-labeled media, and analyzed by MALDI TOF MS. Algorithms for data analysis are presented as well.

  18. Initial drug resistance in India

    Indian Academy of Sciences (India)

    First page Back Continue Last page Overview Graphics. Initial drug resistance in India. There is gradual increase in primary MDR all over India : Pondi= Pondicherry 1985; Bangalore =1986; Jaipur = 1991; Jaipur =2000. Overall the MDR is less than 3% (TRC studies).

  19. The validation of an invitro colonic motility assay as a biomarker for gastrointestinal adverse drug reactions

    International Nuclear Information System (INIS)

    Keating, Christopher; Martinez, Vicente; Ewart, Lorna; Gibbons, Stephen; Grundy, Luke; Valentin, Jean-Pierre; Grundy, David

    2010-01-01

    Motility-related gastrointestinal adverse drug reactions (GADRs), such as constipation and diarrhea, are some of the most frequently reported adverse events associated with the clinical development of new chemical entities, and for marketed drugs. However, biomarkers capable of detecting such GADRs are lacking. Here, we describe an in vitro assay developed to detect and quantify changes in intestinal motility as a surrogate biomarker for constipation/diarrhea-type GADRs. In vitro recordings of intraluminal pressure were used to monitor the presence of colonic peristaltic motor complexes (CPMCs) in mouse colonic segments. CPMC frequency, contractile and total mechanical activity were assessed. To validate the assay, two experimental protocols were conducted. Initially, five drugs with known gastrointestinal effects were tested to determine optimal parameters describing excitation and inhibition as markers for disturbances in colonic motility. This was followed by a 'blinded' evaluation of nine drugs associated with or without clinically identified constipation/diarrhea-type GADRs. Concentration-response relationships were determined for these drugs and the effects were compared with their maximal free therapeutic plasma concentration in humans. The assay detected stimulatory and inhibitory responses, likely correlating to the occurrence of diarrhea or constipation. Concentration-related effects were identified and potential mechanisms of action were inferred for several drugs. Based on the results from the fourteen drugs assessed, the sensitivity of the assay was calculated at 90%, with a specificity of 75% and predictive capacity of 86%. These results support the potential use of this assay in screening for motility-related GADRs during early discovery phase, safety pharmacology assessment.

  20. Mesenchymal change and drug resistance in neuroblastoma.

    Science.gov (United States)

    Naiditch, Jessica A; Jie, Chunfa; Lautz, Timothy B; Yu, Songtao; Clark, Sandra; Voronov, Dimitry; Chu, Fei; Madonna, Mary Beth

    2015-01-01

    Metastatic initiation has many phenotypic similarities to epithelial-to-mesenchymal transition, including loss of cell-cell adhesion, increased invasiveness, and increased cell mobility. We have previously demonstrated that drug resistance is associated with a metastatic phenotype in neuroblastoma (NB). The purpose of this project was to determine if the development of doxorubicin resistance is associated with characteristics of mesenchymal change in human NB cells. Total RNA was isolated from wild type (WT) and doxorubicin-resistant (DoxR) human NB cell lines (SK-N-SH and SK-N-BE(2)C) and analyzed using the Illumina Human HT-12 version 4 Expression BeadChip. Differentially expressed genes (DEGs) were identified. Volcano plots and heat maps were generated. Genes of interest with a fold change in expression >1.5 and an adjusted P change via multiple pathways in the transition to a drug-resistant state. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. DNA origami as a carrier for circumvention of drug resistance.

    Science.gov (United States)

    Jiang, Qiao; Song, Chen; Nangreave, Jeanette; Liu, Xiaowei; Lin, Lin; Qiu, Dengli; Wang, Zhen-Gang; Zou, Guozhang; Liang, Xingjie; Yan, Hao; Ding, Baoquan

    2012-08-15

    Although a multitude of promising anti-cancer drugs have been developed over the past 50 years, effective delivery of the drugs to diseased cells remains a challenge. Recently, nanoparticles have been used as drug delivery vehicles due to their high delivery efficiencies and the possibility to circumvent cellular drug resistance. However, the lack of biocompatibility and inability to engineer spatially addressable surfaces for multi-functional activity remains an obstacle to their widespread use. Here we present a novel drug carrier system based on self-assembled, spatially addressable DNA origami nanostructures that confronts these limitations. Doxorubicin, a well-known anti-cancer drug, was non-covalently attached to DNA origami nanostructures through intercalation. A high level of drug loading efficiency was achieved, and the complex exhibited prominent cytotoxicity not only to regular human breast adenocarcinoma cancer cells (MCF 7), but more importantly to doxorubicin-resistant cancer cells, inducing a remarkable reversal of phenotype resistance. With the DNA origami drug delivery vehicles, the cellular internalization of doxorubicin was increased, which contributed to the significant enhancement of cell-killing activity to doxorubicin-resistant MCF 7 cells. Presumably, the activity of doxorubicin-loaded DNA origami inhibits lysosomal acidification, resulting in cellular redistribution of the drug to action sites. Our results suggest that DNA origami has immense potential as an efficient, biocompatible drug carrier and delivery vehicle in the treatment of cancer.

  2. Detection of low frequency multi-drug resistance and novel putative maribavir resistance in immunocompromised paediatric patients with cytomegalovirus

    Directory of Open Access Journals (Sweden)

    Charlotte Jane Houldcroft

    2016-09-01

    Full Text Available Human cytomegalovirus (HCMV is a significant pathogen in immunocompromised individuals, with the potential to cause fatal pneumonitis and colitis, as well as increasing the risk of organ rejection in transplant patients. With the advent of new anti-HCMV drugs there is therefore considerable interest in using virus sequence data to monitor emerging resistance to antiviral drugs in HCMV viraemia and disease, including the identification of putative new mutations. We used target-enrichment to deep sequence HCMV DNA from 11 immunosuppressed paediatric patients receiving single or combination anti-HCMV treatment, serially sampled over 1-27 weeks. Changes in consensus sequence and resistance mutations were analysed for three ORFs targeted by anti-HCMV drugs and the frequencies of drug resistance mutations monitored. Targeted-enriched sequencing of clinical material detected mutations occurring at frequencies of 2%. Seven patients showed no evidence of drug resistance mutations. Four patients developed drug resistance mutations a mean of 16 weeks after starting treatment. In two patients, multiple resistance mutations accumulated at frequencies of 20% or less, including putative maribavir and ganciclovir resistance mutations P522Q (UL54 and C480F (UL97. In one patient, resistance was detected 14 days earlier than by PCR. Phylogenetic analysis suggested recombination or superinfection in one patient. Deep sequencing of HCMV enriched from clinical samples excluded resistance in 7 of eleven subjects and identified resistance mutations earlier than conventional PCR-based resistance testing in 2 patients. Detection of multiple low level resistance mutations was associated with poor outcome.

  3. Ovatodiolide Targets β-Catenin Signaling in Suppressing Tumorigenesis and Overcoming Drug Resistance in Renal Cell Carcinoma

    Directory of Open Access Journals (Sweden)

    Jar-Yi Ho

    2013-01-01

    Full Text Available Dysregulated β-catenin signaling is intricately involved in renal cell carcinoma (RCC carcinogenesis and progression. Determining potential β-catenin signaling inhibitors would be helpful in ameliorating drug resistance in advanced or metastatic RCC. Screening for β-catenin signaling inhibitors involved in silico inquiry of the PubChem Bioactivity database followed by TCF/LEF reporter assay. The biological effects of ovatodiolide were evaluated in 4 RCC cell lines in vitro and 2 RCC cell lines in a mouse xenograft model. The synergistic effects of ovatodiolide and sorafenib or sunitinib were examined in 2 TKI-resistant RCC cell lines. Ovatodiolide, a pure compound of Anisomeles indica, inhibited β-catenin signaling and reduced RCC cell viability, survival, migration/invasion, and in vitro cell or in vivo mouse tumorigenicity. Cytotoxicity was significantly reduced in a normal kidney epithelial cell line with the treatment. Ovatodiolide reduced phosphorylated β-catenin (S552 that inhibited β-catenin nuclear translocation. Moreover, ovatodiolide decreased β-catenin stability and impaired the association of β-catenin and transcription factor 4. Ovatodiolide combined with sorafenib or sunitinib overcame drug resistance in TKI-resistant RCC cells. Ovatodiolide may be a potent β-catenin signaling inhibitor, with synergistic effects with sorafenib or sunitinib, and therefore, a useful candidate for improving RCC therapy.

  4. Update on HIV-1 acquired and transmitted drug resistance in Africa.

    Science.gov (United States)

    Ssemwanga, Deogratius; Lihana, Raphael W; Ugoji, Chinenye; Abimiku, Alash'le; Nkengasong, John; Dakum, Patrick; Ndembi, Nicaise

    2015-01-01

    The last ten years have witnessed a significant scale-up and access to antiretroviral therapy in Africa, which has improved patient quality of life and survival. One major challenge associated with increased access to antiretroviral therapy is the development of antiretroviral resistance due to inconsistent drug supply and/or poor patient adherence. We review the current state of both acquired and transmitted drug resistance in Africa over the past ten years (2001-2011) to identify drug resistance associated with the different drug regimens used on the continent and to help guide affordable strategies for drug resistance surveillance. A total of 161 references (153 articles, six reports and two conference abstracts) were reviewed. Antiretroviral resistance data was available for 40 of 53 African countries. A total of 5,541 adult patients from 99 studies in Africa were included in this analysis. The pooled prevalence of drug resistance mutations in Africa was 10.6%, and Central Africa had the highest prevalence of 54.9%. The highest prevalence of nucleoside reverse transcriptase inhibitor mutations was in the west (55.3%) and central (54.8%) areas; nonnucleoside reverse transcriptase inhibitor mutations were highest in East Africa (57.0%) and protease inhibitors mutations highest in Southern Africa (16.3%). The major nucleoside reverse transcriptase inhibitor mutation in all four African regions was M184V. Major nonnucleoside reverse transcriptase inhibitor as well as protease inhibitor mutations varied by region. The prevalence of drug resistance has remained low in several African countries although the emergence of drug resistance mutations varied across countries. Continued surveillance of antiretroviral therapy resistance remains crucial in gauging the effectiveness of country antiretroviral therapy programs and strategizing on effective and affordable strategies for successful treatment.

  5. HIV-1 evolution, drug resistance, and host genetics: The Indian scenario

    Directory of Open Access Journals (Sweden)

    U Shankarkumar

    2009-03-01

    Full Text Available U Shankarkumar, A Pawar, K GhoshNational Institute of Immunohaematology (ICMR, KEM Hospital, Parel, Mumbai, Maharashtra, IndiaAbstract: A regimen with varied side effects and compliance is of paramount importance to prevent viral drug resistance. Most of the drug-resistance studies, as well as interpretation algorithms, are based on sequence data from HIV-1 subtype B viruses. Increased resistance to antiretroviral drugs leads to poor prognosis by restricting treatment options. Due to suboptimal adherence to antiretroviral therapy there is an emergence of drug-resistant HIV-1 strains. The other factors responsible for this viral evolution are antiretroviral drug types and host genetics, especially major histocompatibility complex (MHC. Both primary and secondary drug resistances occur due to mutations in specific epitopes of viral protein regions which may influence the T cell recognition by immune system through MHC Class I and class II alleles. Mutations in viral epitopes enable the virus to escape the immune system. New drugs under clinical trials are being added but their exorbitant costs limit their access in developing countries. Thus the environmental consequences and, the impact of both viral and host genetic variations on the therapy in persons infected with HIV-1 clade C from India need to be determined.Keywords: HIV-1 C drug resistance, virus adaptation, HARRT, India

  6. Genome-wide mutagenesis and multi-drug resistance in American trypanosomes induced by the front-line drug benznidazole

    KAUST Repository

    Campos, Mônica C.

    2017-10-25

    Chagas disease is caused by the protozoan parasite Trypanosoma cruzi and affects 5–8 million people in Latin America. Although the nitroheterocyclic compound benznidazole has been the front-line drug for several decades, treatment failures are common. Benznidazole is a pro-drug and is bio-activated within the parasite by the mitochondrial nitroreductase TcNTR-1, leading to the generation of reactive metabolites that have trypanocidal activity. To better assess drug action and resistance, we sequenced the genomes of T. cruzi Y strain (35.5 Mb) and three benznidazole-resistant clones derived from a single drug-selected population. This revealed the genome-wide accumulation of mutations in the resistant parasites, in addition to variations in DNA copy-number. We observed mutations in DNA repair genes, linked with increased susceptibility to DNA alkylating and inter-strand cross-linking agents. Stop-codon-generating mutations in TcNTR-1 were associated with cross-resistance to other nitroheterocyclic drugs. Unexpectedly, the clones were also highly resistant to the ergosterol biosynthesis inhibitor posaconazole, a drug proposed for use against T. cruzi infections, in combination with benznidazole. Our findings therefore identify the highly mutagenic activity of benznidazole metabolites in T. cruzi, demonstrate that this can result in multi-drug resistance, and indicate that vigilance will be required if benznidazole is used in combination therapy.

  7. Drug resistance in the mouse cancer clinic

    NARCIS (Netherlands)

    Rottenberg, Sven; Borst, Piet

    2012-01-01

    Drug resistance is one of the most pressing problems in treating cancer patients today. Local and regional disease can usually be adequately treated, but patients eventually die from distant metastases that have become resistant to all available chemotherapy. Although work on cultured tumor cell

  8. Surveillance of drug resistance for tuberculosis control: why and how?

    Science.gov (United States)

    Chaulet, P; Boulahbal, F; Grosset, J

    1995-12-01

    The resistance of Mycobacterium tuberculosis to antibiotics, which reflects the quality of the chemotherapy applied in the community, is one of the elements of epidemiological surveillance used in national tuberculosis programmes. Measurement of drug resistance poses problems for biologists in standardization of laboratory methods and quality control. The definition of rates of acquired and primary drug resistance also necessitates standardization in the methods used to collect information transmitted by clinicians. Finally, the significance of the rates calculated depends on the choice of the patients sample on which sensitivity tests have been performed. National surveys of drug resistance therefore require multidisciplinary participation in order to select the only useful indicators: rates of primary resistance and of acquired resistance. These indicators, gathered in representative groups of patients over a long period, are a measurement of the impact of modern chemotherapy regimens on bacterial ecology.

  9. New-Onset Psychosis in a Multi-Drug Resistant Tuberculosis Patient ...

    African Journals Online (AJOL)

    Drug-resistant tuberculosis poses a serious challenge to global control of TB. These forms of TB do not respond to the standard six-month treatment; it can take two years or more to treat with category IV drugs that are less potent, more toxic and much more expensive. Treatment of multi-drug resistant tuberculosis is still ...

  10. The role of compensatory mutations in the emergence of drug resistance.

    Directory of Open Access Journals (Sweden)

    Andreas Handel

    2006-10-01

    Full Text Available Pathogens that evolve resistance to drugs usually have reduced fitness. However, mutations that largely compensate for this reduction in fitness often arise. We investigate how these compensatory mutations affect population-wide resistance emergence as a function of drug treatment. Using a model of gonorrhea transmission dynamics, we obtain generally applicable, qualitative results that show how compensatory mutations lead to more likely and faster resistance emergence. We further show that resistance emergence depends on the level of drug use in a strongly nonlinear fashion. We also discuss what data need to be obtained to allow future quantitative predictions of resistance emergence.

  11. Predicting changes in cardiac myocyte contractility during early drug discovery with in vitro assays

    International Nuclear Information System (INIS)

    Morton, M.J.; Armstrong, D.; Abi Gerges, N.; Bridgland-Taylor, M.; Pollard, C.E.; Bowes, J.; Valentin, J.-P.

    2014-01-01

    Cardiovascular-related adverse drug effects are a major concern for the pharmaceutical industry. Activity of an investigational drug at the L-type calcium channel could manifest in a number of ways, including changes in cardiac contractility. The aim of this study was to define which of the two assay technologies – radioligand-binding or automated electrophysiology – was most predictive of contractility effects in an in vitro myocyte contractility assay. The activity of reference and proprietary compounds at the L-type calcium channel was measured by radioligand-binding assays, conventional patch-clamp, automated electrophysiology, and by measurement of contractility in canine isolated cardiac myocytes. Activity in the radioligand-binding assay at the L-type Ca channel phenylalkylamine binding site was most predictive of an inotropic effect in the canine cardiac myocyte assay. The sensitivity was 73%, specificity 83% and predictivity 78%. The radioligand-binding assay may be run at a single test concentration and potency estimated. The least predictive assay was automated electrophysiology which showed a significant bias when compared with other assay formats. Given the importance of the L-type calcium channel, not just in cardiac function, but also in other organ systems, a screening strategy emerges whereby single concentration ligand-binding can be performed early in the discovery process with sufficient predictivity, throughput and turnaround time to influence chemical design and address a significant safety-related liability, at relatively low cost. - Highlights: • The L-type calcium channel is a significant safety liability during drug discovery. • Radioligand-binding to the L-type calcium channel can be measured in vitro. • The assay can be run at a single test concentration as part of a screening cascade. • This measurement is highly predictive of changes in cardiac myocyte contractility

  12. Predicting changes in cardiac myocyte contractility during early drug discovery with in vitro assays

    Energy Technology Data Exchange (ETDEWEB)

    Morton, M.J., E-mail: michael.morton@astrazeneca.com [Discovery Sciences, AstraZeneca, Macclesfield, Cheshire SK10 4TG (United Kingdom); Armstrong, D.; Abi Gerges, N. [Drug Safety and Metabolism, AstraZeneca, Macclesfield, Cheshire SK10 4TG (United Kingdom); Bridgland-Taylor, M. [Discovery Sciences, AstraZeneca, Macclesfield, Cheshire SK10 4TG (United Kingdom); Pollard, C.E.; Bowes, J.; Valentin, J.-P. [Drug Safety and Metabolism, AstraZeneca, Macclesfield, Cheshire SK10 4TG (United Kingdom)

    2014-09-01

    Cardiovascular-related adverse drug effects are a major concern for the pharmaceutical industry. Activity of an investigational drug at the L-type calcium channel could manifest in a number of ways, including changes in cardiac contractility. The aim of this study was to define which of the two assay technologies – radioligand-binding or automated electrophysiology – was most predictive of contractility effects in an in vitro myocyte contractility assay. The activity of reference and proprietary compounds at the L-type calcium channel was measured by radioligand-binding assays, conventional patch-clamp, automated electrophysiology, and by measurement of contractility in canine isolated cardiac myocytes. Activity in the radioligand-binding assay at the L-type Ca channel phenylalkylamine binding site was most predictive of an inotropic effect in the canine cardiac myocyte assay. The sensitivity was 73%, specificity 83% and predictivity 78%. The radioligand-binding assay may be run at a single test concentration and potency estimated. The least predictive assay was automated electrophysiology which showed a significant bias when compared with other assay formats. Given the importance of the L-type calcium channel, not just in cardiac function, but also in other organ systems, a screening strategy emerges whereby single concentration ligand-binding can be performed early in the discovery process with sufficient predictivity, throughput and turnaround time to influence chemical design and address a significant safety-related liability, at relatively low cost. - Highlights: • The L-type calcium channel is a significant safety liability during drug discovery. • Radioligand-binding to the L-type calcium channel can be measured in vitro. • The assay can be run at a single test concentration as part of a screening cascade. • This measurement is highly predictive of changes in cardiac myocyte contractility.

  13. Parallel screening of wild-type and drug-resistant targets for anti-resistance neuraminidase inhibitors.

    Directory of Open Access Journals (Sweden)

    Kai-Cheng Hsu

    Full Text Available Infection with influenza virus is a major public health problem, causing serious illness and death each year. Emergence of drug-resistant influenza virus strains limits the effectiveness of drug treatment. Importantly, a dual H275Y/I223R mutation detected in the pandemic influenza A 2009 virus strain results in multidrug resistance to current neuraminidase (NA drugs. Therefore, discovery of new agents for treating multiple drug-resistant (MDR influenza virus infections is important. Here, we propose a parallel screening strategy that simultaneously screens wild-type (WT and MDR NAs, and identifies inhibitors matching the subsite characteristics of both NA-binding sites. These may maintain their potency when drug-resistant mutations arise. Initially, we analyzed the subsite of the dual H275Y/I223R NA mutant. Analysis of the site-moiety maps of NA protein structures show that the mutant subsite has a relatively small volume and is highly polar compared with the WT subsite. Moreover, the mutant subsite has a high preference for forming hydrogen-bonding interactions with polar moieties. These changes may drive multidrug resistance. Using this strategy, we identified a new inhibitor, Remazol Brilliant Blue R (RB19, an anthraquinone dye, which inhibited WT NA and MDR NA with IC(50 values of 3.4 and 4.5 µM, respectively. RB19 comprises a rigid core scaffold and a flexible chain with a large polar moiety. The former interacts with highly conserved residues, decreasing the probability of resistance. The latter forms van der Waals contacts with the WT subsite and yields hydrogen bonds with the mutant subsite by switching the orientation of its flexible side chain. Both scaffolds of RB19 are good starting points for lead optimization. The results reveal a parallel screening strategy for identifying resistance mechanisms and discovering anti-resistance neuraminidase inhibitors. We believe that this strategy may be applied to other diseases with high

  14. Inhibitory effect of Allium sativum and Zingiber officinale extracts on clinically important drug resistant pathogenic bacteria

    Directory of Open Access Journals (Sweden)

    Gull Iram

    2012-04-01

    Full Text Available Abstract Background Herbs and spices are very important and useful as therapeutic agent against many pathological infections. Increasing multidrug resistance of pathogens forces to find alternative compounds for treatment of infectious diseases. Methods In the present study the antimicrobial potency of garlic and ginger has been investigated against eight local clinical bacterial isolates. Three types of extracts of each garlic and ginger including aqueous extract, methanol extract and ethanol extract had been assayed separately against drug resistant Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, Staphylococcus aureus, Klebsiella pneumoniae, Shigella sonnei, Staphylococcusepidermidis and Salmonella typhi. The antibacterial activity was determined by disc diffusion method. Results All tested bacterial strains were most susceptible to the garlic aqueous extract and showed poor susceptibility to the ginger aqueous extract. The (minimum inhibitory concentration MIC of different bacterial species varied from 0.05 mg/ml to 1.0 mg/ml. Conclusion In the light of several socioeconomic factors of Pakistan mainly poverty and poor hygienic condition, present study encourages the use of spices as alternative or supplementary medicine to reduce the burden of high cost, side effects and progressively increasing drug resistance of pathogens.

  15. Drug resistance and genetic diversity of Plasmodium falciparum parasites from Suriname

    NARCIS (Netherlands)

    Peek, Ron; van Gool, Tom; Panchoe, Daynand; Greve, Sophie; Bus, Ellen; Resida, Lesley

    2005-01-01

    Plasmodium falciparum in Suriname was studied for the presence of drug resistance and genetic variation in blood samples of 86 patients with symptomatic malaria. Drug resistance was predicted by determining point mutations in the chloroquine resistance marker of the P. falciparum chloroquine

  16. Cancer stem cells and drug resistance: the potential of nanomedicine

    Science.gov (United States)

    Vinogradov, Serguei; Wei, Xin

    2012-01-01

    Properties of the small group of cancer cells called tumor-initiating or cancer stem cells (CSCs) involved in drug resistance, metastasis and relapse of cancers can significantly affect tumor therapy. Importantly, tumor drug resistance seems to be closely related to many intrinsic or acquired properties of CSCs, such as quiescence, specific morphology, DNA repair ability and overexpression of antiapoptotic proteins, drug efflux transporters and detoxifying enzymes. The specific microenvironment (niche) and hypoxic stability provide additional protection against anticancer therapy for CSCs. Thus, CSC-focused therapy is destined to form the core of any effective anticancer strategy. Nanomedicine has great potential in the development of CSC-targeting drugs, controlled drug delivery and release, and the design of novel gene-specific drugs and diagnostic modalities. This review is focused on tumor drug resistance-related properties of CSCs and describes current nanomedicine approaches, which could form the basis of novel combination therapies for eliminating metastatic and CSCs. PMID:22471722

  17. [Drug resistance reversal of HL-60/ADR cells by simultaneous suppression of XIAP and MRP].

    Science.gov (United States)

    Wang, Xiao-Fang; Wang, Chun; Qin, You-Wen; Yan, Shi-Ke; Gao, Yan-Rong

    2006-12-01

    This study was purposed to explore the mechanisms of drug resistance of HL-60/ADR cells and to compare the reversal drug-resistance effects of antisense oligonucleotides (AS ODN) of XIAP (X-linked inhibitor of apoptosis protein) and AS ODNs of MRP (multidrug resistance-associated protein) by use alone or in combination. Reverse transcription-PCR and Western blot were applied to detect the expression of XIAP, BCL-2, MRP and MDR1 in mRNA and protein levels of HL-60 cells and HL-60/ADR cells, respectively. Fully phosphorothioated AS ODN of XIAP and MRP was delivered into HL-60/ADR cells with Lipofectamine 2000 in the form of liposome-ODN complexes alone or in combination. CCK-8 cell viability assay was used to determine the effect of AS ODN of XIAP and MRP used alone or in combination on the chemotherapy sensitivity of HL-60/ADR cells to daunorubicin (DNR). Reverse transcription-PCR and Western blot were applied to examine the changes of XIAP, MRP in mRNA and protein levels respectively. The results showed that MRP and XIAP were both significantly higher in HL-60/ADR cells than those in HL-60 cells. AS ODN of XIAP and MRP down-regulated the expression of XIAP and MRP in HL-60/ADR cells and increased the sensitivity of HL-60/ADR cells to DNR, respectively. AS ODN of XIAP + MRP did not enhance the inhibition expression of XIAP in HL-60/ADR cells but increased the sensitivity of HL-60/ADR cells to DNR significantly as compared with AS ODN of XIAP (P MRP did not increase the concentration of DNR nor enhanced the inhibition expression of MRP in HL-60/ADR cells but increased the sensitivity of HL-60/ADR cells to DNR significantly (P MRP. It is concluded that both XIAP and MRP may be involved in the drug resistance mechanisms of HL-60/ADR cells. Drug-resistance of HL-60/ADR cells can be reversed significantly when antisense oligonucleotides of XIAP and MRP were used in combination.

  18. Ribonucleotide reductase as a drug target against drug resistance Mycobacterium leprae: A molecular docking study.

    Science.gov (United States)

    Mohanty, Partha Sarathi; Bansal, Avi Kumar; Naaz, Farah; Gupta, Umesh Datta; Dwivedi, Vivek Dhar; Yadava, Umesh

    2018-06-01

    Leprosy is a chronic infection of skin and nerve caused by Mycobacterium leprae. The treatment is based on standard multi drug therapy consisting of dapsone, rifampicin and clofazamine. The use of rifampicin alone or with dapsone led to the emergence of rifampicin-resistant Mycobacterium leprae strains. The emergence of drug-resistant leprosy put a hurdle in the leprosy eradication programme. The present study aimed to predict the molecular model of ribonucleotide reductase (RNR), the enzyme responsible for biosynthesis of nucleotides, to screen new drugs for treatment of drug-resistant leprosy. The study was conducted by retrieving RNR of M. leprae from GenBank. A molecular 3D model of M. leprae was predicted using homology modelling and validated. A total of 325 characters were included in the analysis. The predicted 3D model of RNR showed that the ϕ and φ angles of 251 (96.9%) residues were positioned in the most favoured regions. It was also conferred that 18 α-helices, 6 β turns, 2 γ turns and 48 helix-helix interactions contributed to the predicted 3D structure. Virtual screening of Food and Drug Administration approved drug molecules recovered 1829 drugs of which three molecules, viz., lincomycin, novobiocin and telithromycin, were taken for the docking study. It was observed that the selected drug molecules had a strong affinity towards the modelled protein RNR. This was evident from the binding energy of the drug molecules towards the modelled protein RNR (-6.10, -6.25 and -7.10). Three FDA-approved drugs, viz., lincomycin, novobiocin and telithromycin, could be taken for further clinical studies to find their efficacy against drug resistant leprosy. Copyright © 2018 Elsevier B.V. All rights reserved.

  19. Danshen extract circumvents drug resistance and represses cell growth in human oral cancer cells.

    Science.gov (United States)

    Yang, Cheng-Yu; Hsieh, Cheng-Chih; Lin, Chih-Kung; Lin, Chun-Shu; Peng, Bo; Lin, Gu-Jiun; Sytwu, Huey-Kang; Chang, Wen-Liang; Chen, Yuan-Wu

    2017-12-29

    Danshen is a common traditional Chinese medicine used to treat neoplastic and chronic inflammatory diseases in China. However, the effects of Danshen on human oral cancer cells remain relatively unknown. This study investigated the antiproliferative effects of a Danshen extract on human oral cancer SAS, SCC25, OEC-M1, and KB drug-resistant cell lines and elucidated the possible underlying mechanism. We investigated the anticancer potential of the Danshen extract in human oral cancer cell lines and an in vivo oral cancer xenograft mouse model. The expression of apoptosis-related molecules was evaluated through Western blotting, and the concentration of in vivo apoptotic markers was measured using immunohistochemical staining. The antitumor effects of 5-fluorouracil and the Danshen extract were compared. Cell proliferation assays revealed that the Danshen extract strongly inhibited oral cancer cell proliferation. Cell morphology studies revealed that the Danshen extract inhibited the growth of SAS, SCC25, and OEC-M1 cells by inducing apoptosis. The Flow cytometric analysis indicated that the Danshen extract induced cell cycle G0/G1 arrest. Immunoblotting analysis for the expression of active caspase-3 and X-linked inhibitor of apoptosis protein indicated that Danshen extract-induced apoptosis in human oral cancer SAS cells was mediated through the caspase pathway. Moreover, the Danshen extract significantly inhibited growth in the SAS xenograft mouse model. Furthermore, the Danshen extract circumvented drug resistance in KB drug-resistant oral cancer cells. The study results suggest that the Danshen extract could be a potential anticancer agent in oral cancer treatment.

  20. Alteration of gene expression and DNA methylation in drug-resistant gastric cancer.

    Science.gov (United States)

    Maeda, Osamu; Ando, Takafumi; Ohmiya, Naoki; Ishiguro, Kazuhiro; Watanabe, Osamu; Miyahara, Ryoji; Hibi, Yoko; Nagai, Taku; Yamada, Kiyofumi; Goto, Hidemi

    2014-04-01

    The mechanisms of drug resistance in cancer are not fully elucidated. To study the drug resistance of gastric cancer, we analyzed gene expression and DNA methylation profiles of 5-fluorouracil (5-FU)- and cisplatin (CDDP)-resistant gastric cancer cells and biopsy specimens. Drug-resistant gastric cancer cells were established with culture for >10 months in a medium containing 5-FU or CDDP. Endoscopic biopsy specimens were obtained from gastric cancer patients who underwent chemotherapy with oral fluoropyrimidine S-1 and CDDP. Gene expression and DNA methylation analyses were performed using microarray, and validated using real-time PCR and pyrosequencing, respectively. Out of 17,933 genes, 541 genes commonly increased and 569 genes decreased in both 5-FU- and CDDP-resistant AGS cells. Genes with expression changed by drugs were related to GO term 'extracellular region' and 'p53 signaling pathway' in both 5-FU- and CDDP-treated cells. Expression of 15 genes including KLK13 increased and 12 genes including ETV7 decreased, in both drug-resistant cells and biopsy specimens of two patients after chemotherapy. Out of 10,365 genes evaluated with both expression microarray and methylation microarray, 74 genes were hypermethylated and downregulated, or hypomethylated and upregulated in either 5-FU-resistant or CDDP-resistant cells. Of these genes, expression of 21 genes including FSCN1, CPT1C and NOTCH3, increased from treatment with a demethylating agent. There are alterations of gene expression and DNA methylation in drug-resistant gastric cancer; they may be related to mechanisms of drug resistance and may be useful as biomarkers of gastric cancer drug sensitivity.

  1. [Detection of CRISPR and its relationship to drug resistance in Shigella].

    Science.gov (United States)

    Wang, Linlin; Wang, Yingfang; Duan, Guangcai; Xue, Zerun; Guo, Xiangjiao; Wang, Pengfei; Xi, Yuanlin; Yang, Haiyan

    2015-04-04

    To detect clustered regularly interspaced short palindromic repeats (CRISPR) in Shigella, and to analyze its relationship to drug resistance. Four pairs of primers were used for the detection of convincing CRISPR structures CRISPR-S2 and CRISPR-S4, questionable CRISPR structures CRISPR-S1 and CRISPR-S3 in 60 Shigella strains. All primers were designed using sequences in CRISPR database. CRISPR Finder was used to analyze CRISPR and susceptibilities of Shigella strains were tested by agar diffusion method. Furthermore, we analyzed the relationship between drug resistance and CRISPR-S4. The positive rate of convincing CRISPR structures was 95%. The four CRISPR loci formed 12 spectral patterns (A-L), all of which contained convincing CRISPR structures except type K. We found one new repeat and 12 new spacers. The multi-drug resistance rate was 53. 33% . We found no significant difference between CRISPR-S4 and drug resistant. However, the repeat sequence of CRISPR-S4 in multi- or TE-resistance strains was mainly R4.1 with AC deletions in the 3' end, and the spacer sequences of CRISPR-S4 in multi-drug resistance strains were mainly Sp5.1, Sp6.1 and Sp7. CRISPR was common in Shigella. Variations df repeat sequences and diversities of spacer sequences might be related to drug resistance in Shigella.

  2. A typology and analysis of drug resistance strategies of rural Native Hawaiian youth.

    Science.gov (United States)

    Okamoto, Scott K; Helm, Susana; Giroux, Danielle; Kaliades, Alexis; Kawano, Kaycee Nahe; Kulis, Stephen

    2010-12-01

    This study examines the drug resistance strategies described by Native Hawaiian youth residing in rural communities. Sixty-four youth from 7 middle and intermediate schools on the Island of Hawai'i participated in a series of gender-specific focus groups. Youth responded to 15 drug-related problem situations developed and validated from prior research. A total of 509 responses reflecting primary or secondary drug resistance strategies were identified by the youth, which were qualitatively collapsed into 16 different categories. Primary drug resistance strategies were those that participants listed as a single response, or the first part of a two-part response, while secondary drug resistance strategies were those that were used in tandem with primary drug resistance strategies. Over half of the responses reflecting primary drug resistance strategies fell into three different categories ("refuse," "explain," or "angry refusal"), whereas over half of the responses reflecting secondary drug resistance strategies represented one category ("explain"). Significant gender differences were found in the frequency of using different strategies as well as variations in the frequency of using different strategies based on the type of drug offerer (family versus friends/peers). Implications for prevention practice are discussed.

  3. Apoptosis-related molecular differences for response to tyrosin kinase inhibitors in drug-sensitive and drug-resistant human bladder cancer cells

    Directory of Open Access Journals (Sweden)

    Jixia Li

    2013-01-01

    Full Text Available Context: The epidermal growth factor receptor (EGFR family is reportedly overexpressed in bladder cancer, and tyrosine kinaseinhibitors (TKIs have been suggested as treatment. Gefitinib is a selective inhibitor of the EGFR and lapatinib is a dual inhibitor of both the EGFR and HER2 (human EGFR type 2 receptor. Both compounds compete with the binding of adenosine triphosphate (ATP to the tyrosine kinase domain of the respective receptors to inhibit receptor autophosphorylation causing suppression of signal transduction. Unfortunately, resistance to these inhibitors is a major clinical problem. Aims: To compare the apoptosis signaling pathway(s induced by gefitinib and lapatinib, in UM-UC-5 (drug-sensitive and UM-UC-14 (drug-resistant bladder cancer cells and to identify molecular differences that might be useful predictors of their efficacy. Materials and Methods: Cell proliferation, cell cycle and apoptosis assay were used to detect the effect of TKIs on UM-UC-5 and UM-UC-14 cells. Molecular differences for response to TKIs were examined by protein array. Results: TKIs strongly inhibited cell proliferation and induced cell cycle G1 arrest and apoptosis in UM-UC-5 cells. Most notable apoptosis molecular differences included decreased claspin, trail, and survivin by TKIs in the sensitive cells. In contrast, TKIs had no effect on resistant cells. Conclusions: Claspin, trail, and survivin might be used to determine the sensitivity of bladder cancers to TKIs.

  4. Multi-drug resistance and molecular pattern of erythromycin and ...

    African Journals Online (AJOL)

    The appearance and dissemination of penicillin resistant and macrolide resistant Streptococcus pneumoniae strains has caused increasing concern worldwide. The aim of this study was to survey drug resistance and genetic characteristics of macrolide and penicillin resistance in S. pneumoniae. This is a cross-sectional ...

  5. A Qualitative and Quantitative Assay to Study DNA/Drug Interaction ...

    African Journals Online (AJOL)

    Research Article. A Qualitative and Quantitative Assay to Study. DNA/Drug Interaction Based on Sequence Selective. Inhibition of Restriction Endonucleases. Syed A Hassan1*, Lata Chauhan2, Ritu Barthwal2 and Aparna Dixit3. 1 Faculty of Computing and Information Technology, King Abdul Aziz University, Rabigh-21911 ...

  6. Comet Assay: A Method to Evaluate Genotoxicity of Nano-Drug Delivery System

    Science.gov (United States)

    Vandghanooni, Somayeh; Eskandani, Morteza

    2011-01-01

    Introduction Drug delivery systems could induce cellular toxicity as side effect of nanomaterials. The mechanism of toxicity usually involves DNA damage. The comet assay or single cell gel electrophoresis (SCGE) is a sensitive method for detecting strand damages in the DNA of a cell with applications in genotoxicity testing and molecular epidemiology as well as fundamental research in DNA damage and repair. Methods In the current study, we reviewed recent drug delivery researches related to SCGE. Results We found that one preference for choosing the assay is that comet images may result from apoptosis-mediated nuclear fragmentation. This method has been widely used over the last decade in several different areas. Overall cells, such as cultured cells are embedded in agarose on a microscope slide, lysed with detergent, and treated with high salt. Nucleoids are supercoiled DNA form. When the slide is faced to alkaline electrophoresis any breakages present in the DNA cause the supercoiling to relax locally and loops of DNA extend toward the anode as a ‘‘comet tail’’. Conclusion This article provides a relatively comprehensive review upon potentiality of the comet assay for assessment of DNA damage and accordingly it can be used as an informative platform in genotoxicity studies of drug delivery systems. PMID:23678412

  7. A dual reporter cell assay for identifying serotype and drug susceptibility of herpes simplex virus.

    Science.gov (United States)

    Lu, Wen-Wen; Sun, Jun-Ren; Wu, Szu-Sian; Lin, Wan-Hsuan; Kung, Szu-Hao

    2011-08-15

    A dual reporter cell assay (DRCA) that allows real-time detection of herpes simplex virus (HSV) infection was developed. This was achieved by stable transfection of cells with an expression cassette that contains the dual reporter genes, secreted alkaline phosphatase (SEAP) and enhanced green fluorescent protein (EGFP), under the control of an HSV early gene promoter. Baby hamster kidney (BHK) and Chinese hamster ovary (CHO) cell lines were used as parental cell lines because the former is permissive for both HSV serotypes, HSV-1 and HSV-2, whereas the latter is susceptible to infection only by HSV-2. The DRCA permitted differential detection of HSV-1 and HSV-2 by observation of EGFP-positive cells, as substantiated by screening a total of 35 samples. The BHK-based cell line is sensitive to a viral titer as low as a single plaque-forming unit with a robust assay window as measured by a chemiluminescent assay. Evaluations of the DRCA with representative acyclovir-sensitive and acyclovir-resistant HSV strains demonstrated that their drug susceptibilities were accurately determined by a 48-h format. In summary, this novel DRCA is a useful means for serotyping of HSV in real time as well as a rapid screening method for determining anti-HSV susceptibilities. Copyright © 2011 Elsevier Inc. All rights reserved.

  8. HIV-1 evolution, drug resistance, and host genetics: The Indian scenario

    OpenAIRE

    Shankarkumar, U.; Pawar,Aruna; Ghosh,Kanjaksha

    2009-01-01

    U Shankarkumar, A Pawar, K GhoshNational Institute of Immunohaematology (ICMR), KEM Hospital, Parel, Mumbai, Maharashtra, IndiaAbstract: A regimen with varied side effects and compliance is of paramount importance to prevent viral drug resistance. Most of the drug-resistance studies, as well as interpretation algorithms, are based on sequence data from HIV-1 subtype B viruses. Increased resistance to antiretroviral drugs leads to poor prognosis by restricting treatment optio...

  9. Open innovation for phenotypic drug discovery: The PD2 assay panel.

    Science.gov (United States)

    Lee, Jonathan A; Chu, Shaoyou; Willard, Francis S; Cox, Karen L; Sells Galvin, Rachelle J; Peery, Robert B; Oliver, Sarah E; Oler, Jennifer; Meredith, Tamika D; Heidler, Steven A; Gough, Wendy H; Husain, Saba; Palkowitz, Alan D; Moxham, Christopher M

    2011-07-01

    Phenotypic lead generation strategies seek to identify compounds that modulate complex, physiologically relevant systems, an approach that is complementary to traditional, target-directed strategies. Unlike gene-specific assays, phenotypic assays interrogate multiple molecular targets and signaling pathways in a target "agnostic" fashion, which may reveal novel functions for well-studied proteins and discover new pathways of therapeutic value. Significantly, existing compound libraries may not have sufficient chemical diversity to fully leverage a phenotypic strategy. To address this issue, Eli Lilly and Company launched the Phenotypic Drug Discovery Initiative (PD(2)), a model of open innovation whereby external research groups can submit compounds for testing in a panel of Lilly phenotypic assays. This communication describes the statistical validation, operations, and initial screening results from the first PD(2) assay panel. Analysis of PD(2) submissions indicates that chemical diversity from open source collaborations complements internal sources. Screening results for the first 4691 compounds submitted to PD(2) have confirmed hit rates from 1.6% to 10%, with the majority of active compounds exhibiting acceptable potency and selectivity. Phenotypic lead generation strategies, in conjunction with novel chemical diversity obtained via open-source initiatives such as PD(2), may provide a means to identify compounds that modulate biology by novel mechanisms and expand the innovation potential of drug discovery.

  10. Multi-clonal evolution of multi-drug-resistant/extensively drug-resistant Mycobacterium tuberculosis in a high-prevalence setting of Papua New Guinea for over three decades

    Science.gov (United States)

    Bainomugisa, Arnold; Lavu, Evelyn; Hiashiri, Stenard; Majumdar, Suman; Honjepari, Alice; Moke, Rendi; Dakulala, Paison; Hill-Cawthorne, Grant A.; Pandey, Sushil; Marais, Ben J.; Coulter, Chris; Coin, Lachlan

    2018-01-01

    An outbreak of multi-drug resistant (MDR) tuberculosis (TB) has been reported on Daru Island, Papua New Guinea. Mycobacterium tuberculosis strains driving this outbreak and the temporal accrual of drug resistance mutations have not been described. Whole genome sequencing of 100 of 165 clinical isolates referred from Daru General Hospital to the Supranational reference laboratory, Brisbane, during 2012–2015 revealed that 95 belonged to a single modern Beijing sub-lineage strain. Molecular dating suggested acquisition of streptomycin and isoniazid resistance in the 1960s, with potentially enhanced virulence mediated by an mycP1 mutation. The Beijing sub-lineage strain demonstrated a high degree of co-resistance between isoniazid and ethionamide (80/95; 84.2 %) attributed to an inhA promoter mutation combined with inhA and ndh coding mutations. Multi-drug resistance, observed in 78/95 samples, emerged with the acquisition of a typical rpoB mutation together with a compensatory rpoC mutation in the 1980s. There was independent acquisition of fluoroquinolone and aminoglycoside resistance, and evidence of local transmission of extensively drug resistant (XDR) strains from 2009. These findings underline the importance of whole genome sequencing in informing an effective public health response to MDR/XDR TB. PMID:29310751

  11. Transmitted drug resistance and type of infection in newly diagnosed HIV-1 individuals in Honduras.

    Science.gov (United States)

    Murillo, Wendy; Paz-Bailey, Gabriela; Morales, Sonia; Monterroso, Edgar; Paredes, Mayte; Dobbs, Trudy; Parekh, Bharat S; Albert, Jan; Rivera, Ivette Lorenzana de

    2010-12-01

    Transmitted drug resistance (TDR) reduces the efficacy of antiretroviral treatment and is a public health concern. To gain insight in the epidemiology of TDR in Honduras by evaluating the amount of TDR in a representative sample of newly diagnosed individuals and by determining whether these are recent or established infections. Two hundred treatment-naïve, newly diagnosed HIV-positive individuals representing different population groups (general population, Garifunas ethnic group, female sex workers and men who have sex with men) and different geographic regions were enrolled during April 2004-April 2007. The HIV-1 pol gene was sequenced to identify drug-resistant mutations and TDR was scored as recommended by the WHO. Specimens were classified as recent or established infections using the BED assay. Among 200 samples analyzed from Honduran patients the prevalence of TDR was 7% (95% CI: 3.9-11.5%), 5% for non-nucleoside reverse transcriptase inhibitors (NNRTIs), 3% for nucleoside reverse transcriptase inhibitors (NRTIs) and 0.5% for protease inhibitors (PIs). Testing of these samples with the BED assay revealed that 12% of the specimens were associated with recent infections. TDR was significantly more common in specimens with recent infection (21%) than established infection (5%) (p=0.016). The prevalence of TDR in Honduras was moderate (7%). The percentage of specimens who were recently infected was low (12%), suggesting that late HIV diagnosis is common. The TDR prevalence was higher in recent than in established infections, which may indicate that TDR is increasing over time. The higher prevalence of NNRTI and NRTI mutations as compared to PI mutations is probably due to a broader and longer use of these drugs in Honduras. Copyright © 2010 Elsevier B.V. All rights reserved.

  12. Drug resistance in Mycobacterium leprae from patients with leprosy in China.

    Science.gov (United States)

    Liu, D; Zhang, Q; Sun, Y; Wang, C; Zhang, Y; Fu, X; Chen, M; Zhou, G; Yu, X; Wang, J; Liu, H; Zhang, F

    2015-12-01

    Previous studies of drug resistance have shown that mutations in the drug resistance-determining region (DRDR) in the Folp1, RpoB and GyrA genes of Mycobacterium leprae are responsible for resistance to dapsone, rifampin and ofloxacin, respectively. To investigate the prevalence of mutations in genes associated with drug resistance in M. leprae isolates from patients with leprosy in Shandong Province. The DRDR in the FolP1, RpoB and GyrA genes was analysed by direct sequencing of the PCR product from 85 isolates of M. leprae sampled from patients with leprosy in Shandong, China. Sequencing results were obtained for FolP1, RpoB and GyrA in 67, 57 and 81 of the 85 samples, with mutation rates of 1.5% (1/67), 8.8% 5/57 and 25.9% (21/81). Three multidrug-resistant samples were found among the new cases: one had a mutation in both Folp1 and RpoB, while the other two had a mutation in both RpoB and GyrA. Primary resistance appears to be to either single drugs or combinations of two drugs. The resistance rate to dapsone seems to be low. To our knowledge, this is the first case of multidrug-resistant M. leprae from China. © 2015 British Association of Dermatologists.

  13. High Levels of Transmitted HIV Drug Resistance in a Study in Papua New Guinea.

    Science.gov (United States)

    Lavu, Evelyn; Kave, Ellan; Mosoro, Euodia; Markby, Jessica; Aleksic, Eman; Gare, Janet; Elsum, Imogen A; Nano, Gideon; Kaima, Petronia; Dala, Nick; Gurung, Anup; Bertagnolio, Silvia; Crowe, Suzanne M; Myatt, Mark; Hearps, Anna C; Jordan, Michael R

    2017-01-01

    Papua New Guinea is a Pacific Island nation of 7.3 million people with an estimated HIV prevalence of 0.8%. ART initiation and monitoring are guided by clinical staging and CD4 cell counts, when available. Little is known about levels of transmitted HIV drug resistance in recently infected individuals in Papua New Guinea. Surveillance of transmitted HIV drug resistance in a total of 123 individuals recently infected with HIV and aged less than 30 years was implemented in Port Moresby (n = 62) and Mount Hagen (n = 61) during the period May 2013-April 2014. HIV drug resistance testing was performed using dried blood spots. Transmitted HIV drug resistance was defined by the presence of one or more drug resistance mutations as defined by the World Health Organization surveillance drug resistance mutations list. The prevalence of non-nucleoside reverse transcriptase inhibitor transmitted HIV drug resistance was 16.1% (95% CI 8.8%-27.4%) and 8.2% (95% CI 3.2%-18.2%) in Port Moresby and Mount Hagen, respectively. The prevalence of nucleoside reverse transcriptase inhibitor transmitted HIV drug resistance was 3.2% (95% CI 0.2%-11.7%) and 3.3% (95% CI 0.2%-11.8%) in Port Moresby and Mount Hagen, respectively. No protease inhibitor transmitted HIV drug resistance was observed. The level of non-nucleoside reverse transcriptase inhibitor drug resistance in antiretroviral drug naïve individuals recently infected with HIV in Port Moresby is amongst the highest reported globally. This alarming level of transmitted HIV drug resistance in a young sexually active population threatens to limit the on-going effective use of NNRTIs as a component of first-line ART in Papua New Guinea. To support the choice of nationally recommended first-line antiretroviral therapy, representative surveillance of HIV drug resistance among antiretroviral therapy initiators in Papua New Guinea should be urgently implemented.

  14. Drug-resistant tuberculosis: time for visionary political leadership.

    Science.gov (United States)

    Abubakar, Ibrahim; Zignol, Matteo; Falzon, Dennis; Raviglione, Mario; Ditiu, Lucica; Masham, Susan; Adetifa, Ifedayo; Ford, Nathan; Cox, Helen; Lawn, Stephen D; Marais, Ben J; McHugh, Timothy D; Mwaba, Peter; Bates, Matthew; Lipman, Marc; Zijenah, Lynn; Logan, Simon; McNerney, Ruth; Zumla, Adam; Sarda, Krishna; Nahid, Payam; Hoelscher, Michael; Pletschette, Michel; Memish, Ziad A; Kim, Peter; Hafner, Richard; Cole, Stewart; Migliori, Giovanni Battista; Maeurer, Markus; Schito, Marco; Zumla, Alimuddin

    2013-06-01

    Two decades ago, WHO declared tuberculosis a global emergency, and invested in the highly cost-effective directly observed treatment short-course programme to control the epidemic. At that time, most strains of Mycobacterium tuberculosis were susceptible to first-line tuberculosis drugs, and drug resistance was not a major issue. However, in 2013, tuberculosis remains a major public health concern worldwide, with prevalence of multidrug-resistant (MDR) tuberculosis rising. WHO estimates roughly 630 000 cases of MDR tuberculosis worldwide, with great variation in the frequency of MDR tuberculosis between countries. In the past 8 years, extensively drug-resistant (XDR) tuberculosis has emerged, and has been reported in 84 countries, heralding the possibility of virtually untreatable tuberculosis. Increased population movement, the continuing HIV pandemic, and the rise in MDR tuberculosis pose formidable challenges to the global control of tuberculosis. We provide an overview of the global burden of drug-resistant disease; discuss the social, health service, management, and control issues that fuel and sustain the epidemic; and suggest specific recommendations for important next steps. Visionary political leadership is needed to curb the rise of MDR and XDR tuberculosis worldwide, through sustained funding and the implementation of global and regional action plans. Copyright © 2013 World Health Organization. Published by Elsevier Ltd/Inc/BV. All rights reserved. Published by Elsevier Ltd. All rights reserved.

  15. Hypoxia-induced cytotoxic drug resistance in osteosarcoma is independent of HIF-1Alpha.

    Directory of Open Access Journals (Sweden)

    Jennifer Adamski

    Full Text Available Survival rates from childhood cancer have improved dramatically in the last 40 years, such that over 80% of children are now cured. However in certain subgroups, including metastatic osteosarcoma, survival has remained stubbornly poor, despite dose intensive multi-agent chemotherapy regimens, and new therapeutic approaches are needed. Hypoxia is common in adult solid tumours and is associated with treatment resistance and poorer outcome. Hypoxia induces chemotherapy resistance in paediatric tumours including neuroblastoma, rhabdomyosarcoma and Ewing's sarcoma, in vitro, and this drug resistance is dependent on the oxygen-regulated transcription factor hypoxia inducible factor-1 (HIF-1. In this study the effects of hypoxia on the response of the osteosarcoma cell lines 791T, HOS and U2OS to the clinically relevant cytotoxics cisplatin, doxorubicin and etoposide were evaluated. Significant hypoxia-induced resistance to all three agents was seen in all three cell lines and hypoxia significantly reduced drug-induced apoptosis. Hypoxia also attenuated drug-induced activation of p53 in the p53 wild-type U2OS osteosarcoma cells. Drug resistance was not induced by HIF-1α stabilisation in normoxia by cobalt chloride nor reversed by the suppression of HIF-1α in hypoxia by shRNAi, siRNA, dominant negative HIF or inhibition with the small molecule NSC-134754, strongly suggesting that hypoxia-induced drug resistance in osteosarcoma cells is independent of HIF-1α. Inhibition of the phosphoinositide 3-kinase (PI3K pathway using the inhibitor PI-103 did not reverse hypoxia-induced drug resistance, suggesting the hypoxic activation of Akt in osteosarcoma cells does not play a significant role in hypoxia-induced drug resistance. Targeting hypoxia is an exciting prospect to improve current anti-cancer therapy and combat drug resistance. Significant hypoxia-induced drug resistance in osteosarcoma cells highlights the potential importance of hypoxia as a target

  16. A review of mechanisms of circumvention and modulation of chemotherapeutic drug resistance.

    Science.gov (United States)

    O'Connor, R

    2009-05-01

    Drug resistance is a serious limitation to the effective treatment of a number of common malignancies. Thirty years of laboratory and clinical research have greatly defined the molecular alterations underlying many drug resistance processes in cancer. Based on this knowledge, strategies to overcome the impact of resistance and increase the efficacy of cancer treatment have been translated from laboratory models to clinical trials. This article reviews laboratory and, in particular, clinical attempts at drug resistance circumvention from early forays in the inhibition of cellular efflux pump-mediated drug resistance through to more selective circumvention agent strategies and into inhibition of the other important mechanisms which can allow cancer cells to survive therapy, such as apoptosis resistance. Despite some promising results to date, resistance inhibition strategies have largely failed due to poor understanding of the pharmacology, dynamics and complexity of the resistance phenotype. With the realisation that new molecularly-targeted agents can also be rendered ineffectual by the actions of resistance mechanisms, a major focus is once again emerging on identifying new strategies/pharmaceuticals which can augment the activity of the arsenal of more conventional cytotoxics and newer targeted anti-cancer drugs. Future tactical directions where old and new resistance strategies may merge to overcome this challenge are discussed.

  17. Deciphering mechanisms of drug sensitivity and resistance to Selective Inhibitor of Nuclear Export (SINE) compounds

    International Nuclear Information System (INIS)

    Crochiere, Marsha; Kashyap, Trinayan; Kalid, Ori; Shechter, Sharon; Klebanov, Boris; Senapedis, William; Saint-Martin, Jean-Richard; Landesman, Yosef

    2015-01-01

    Exportin 1 (XPO1) is a well-characterized nuclear export protein whose expression is up-regulated in many types of cancers and functions to transport key tumor suppressor proteins (TSPs) from the nucleus. Karyopharm Therapeutics has developed a series of small-molecule Selective Inhibitor of Nuclear Export (SINE) compounds, which have been shown to block XPO1 function both in vitro and in vivo. The drug candidate, selinexor (KPT-330), is currently in Phase-II/IIb clinical trials for treatment of both hematologic and solid tumors. The present study sought to decipher the mechanisms that render cells either sensitive or resistant to treatment with SINE compounds, represented by KPT-185, an early analogue of KPT-330. Using the human fibrosarcoma HT1080 cell line, resistance to SINE was acquired over a period of 10 months of constant incubation with increasing concentration of KPT-185. Cell viability was assayed by MTT. Immunofluorescence was used to compare nuclear export of TSPs. Fluorescence activated cell sorting (FACS), quantitative polymerase chain reaction (qPCR), and immunoblots were used to measure effects on cell cycle, gene expression, and cell death. RNA from naïve and drug treated parental and resistant cells was analyzed by Affymetrix microarrays. Treatment of HT1080 cells with gradually increasing concentrations of SINE resulted in > 100 fold decrease in sensitivity to SINE cytotoxicity. Resistant cells displayed prolonged cell cycle, reduced nuclear accumulation of TSPs, and similar changes in protein expression compared to parental cells, however the magnitude of the protein expression changes were more significant in parental cells. Microarray analyses comparing parental to resistant cells indicate that a number of key signaling pathways were altered in resistant cells including expression changes in genes involved in adhesion, apoptosis, and inflammation. While the patterns of changes in transcription following drug treatment are similar in parental

  18. Surveillance of extensively drug-resistant tuberculosis in Europe, 2003-2007.

    NARCIS (Netherlands)

    Devaux, I.; Manissero, D.; Fernandez de la Hoz, K.; Kremer, K.; Soolingen, D. van

    2010-01-01

    This paper describes the results of second-line drug (SLD) susceptibility tests among multidrug-resistant tuberculosis (MDR TB) cases reported in 20 European countries aiming to identify extensively drug-resistant tuberculosis (XDR TB) cases. A project on molecular surveillance of MDR TB cases was

  19. Higher Desolvation Energy Reduces Molecular Recognition in Multi-Drug Resistant HIV-1 Protease

    Directory of Open Access Journals (Sweden)

    Ladislau C. Kovari

    2012-05-01

    Full Text Available Designing HIV-1 protease inhibitors that overcome drug-resistance is still a challenging task. In this study, four clinical isolates of multi-drug resistant HIV-1 proteases that exhibit resistance to all the US FDA-approved HIV-1 protease inhibitors and also reduce the substrate recognition ability were examined. A multi-drug resistant HIV-1 protease isolate, MDR 769, was co-crystallized with the p2/NC substrate and the mutated CA/p2 substrate, CA/p2 P1’F. Both substrates display different levels of molecular recognition by the wild-type and multi-drug resistant HIV-1 protease. From the crystal structures, only limited differences can be identified between the wild-type and multi-drug resistant protease. Therefore, a wild-type HIV-1 protease and four multi-drug resistant HIV-1 proteases in complex with the two peptides were modeled based on the crystal structures and examined during a 10 ns-molecular dynamics simulation. The simulation results reveal that the multi-drug resistant HIV-1 proteases require higher desolvation energy to form complexes with the peptides. This result suggests that the desolvation of the HIV-1 protease active site is an important step of protease-ligand complex formation as well as drug resistance. Therefore, desolvation energy could be considered as a parameter in the evaluation of future HIV-1 protease inhibitor candidates.

  20. Bone morphogenetic protein 4 is overexpressed in and promotes migration and invasion of drug-resistant cancer cells.

    Science.gov (United States)

    Zhou, Kairui; Shi, Xiaoli; Huo, Jinling; Liu, Weihua; Yang, Dongxiao; Yang, Tengjiao; Qin, Tiantian; Wang, Cong

    2017-08-01

    Drug resistance and metastasis significantly hinder chemotherapy and worsen prognoses in cancer. Bone morphogenetic protein 4 (BMP4) belongs to the TGF-β superfamily, has broad biological activities in cell proliferation and cartilage differentiation and is also able to induce migration and invasion. Herein, we investigated the role of BMP4 in the regulation of metastasis in paclitaxel-resistant human esophageal carcinoma EC109 cells (EC109/Taxol) and docetaxel-resistant human gastric cancer MGC803 cells (MGC/Doc). In these drug-resistant cell lines, we found the cell motility was enhanced and BMP4 was up-regulated relative to their respective parental cell lines. Consistent with in vitro assays, migration potential and BMP4 expression were increased in EC109/Taxol nude mice. Furthermore, to address whether BMP4 was required to enhance the metastatic in EC109/Taxol cells, the pharmacological inhibitor of BMP signaling dorsomorphin was used; meanwhile, we found that the migration and invasion abilities were inhibited. Moreover, the canonical Smad signaling pathway was investigated. Overall, our studies demonstrated that BMP4 participates in the regulation of invasion and migration by EC109/Taxol cells, and inhibition of BMP4 may be a novel strategy to interfere with metastasis in cancer therapy. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Definition of drug resistance of Mycobacterium tuberculosis to antituberculosis drugs in patients with multidrugresistant tuberculosis and TB with extremely drug resistant depending on the case of the disease

    Directory of Open Access Journals (Sweden)

    Kryzhanovsky D.G.

    2014-11-01

    Full Text Available There was studied the profile of drug resistance to the main (I line and reserve (II line antituberculosis drugs in patients with MDR and XDR tuberculosis, depending of the case of the disease. According to the randomized retrospective research 200 patients with MDR and XDR tuberculosis, who received treatment in the clinic of hospital Municipal institution «Dnipropetrovsk rigional clinical association «Phthisiology» Dnipropetrovsk regional Council» during the period 2010 – 2012 were involved. Data about patients contained the data on a case of the disease and the results of the test of drug sensitivity to MBT. XDR – TB was revealed in 7.5% of patients with MDR tuberculosis. In patients with MDR tuberculosis as compared with patients with XDR tuberculosis «new cases» were diagnosed in 19.5% against 18.5% (p <0.05. In patients with MDR tuberculosis and with XDR tuberculosis resistance to the antituberculosis drug more commonly developed to S - 88.5%, E - 55% and Z - 24%. The presence of MDR-TB and XDR-TB prevails in patients, who underwent previous courses of treatment with anti-TB drugs in case history as compared with patients with «new cases» of treatment. The development of resistance to anti-TB drugs depends on the availability of these drugs in the previous treatment regimens.

  2. A Qualitative and Quantitative Assay to Study DNA/Drug Interaction ...

    African Journals Online (AJOL)

    Purpose: To explore the use of restriction inhibition assay (RIA) to study the binding specificity of some anticancer drugs. Methods: A 448 bp DNA fragment derived from pBCKS+ plasmid (harboring the polylinker region with multiple restriction endonuclease sites) was used as a template for sequence selective inhibition of ...

  3. Re-sensitizing drug-resistant bacteria to antibiotics by designing Antisense Therapeutics

    Science.gov (United States)

    Courtney, Colleen; Chatterjee, Anushree

    2014-03-01

    ``Super-bugs'' or ``multi-drug resistant organisms'' are a serious international health problem, with devastating consequences to patient health care. The Center for Disease Control has identified antibiotic resistance as one of the world's most pressing public health problems as a significant fraction of bacterial infections contracted are drug resistant. Typically, antibiotic resistance is encoded by ``resistance-genes'' which express proteins that carryout the resistance causing functions inside the bacterium. We present a RNA based therapeutic strategy for designing antimicrobials capable of re-sensitizing resistant bacteria to antibiotics by targeting labile regions of messenger RNAs encoding for resistance-causing proteins. We perform in silico RNA secondary structure modeling to identify labile target regions in an mRNA of interest. A synthetic biology approach is then used to administer antisense nucleic acids to our model system of ampicillin resistant Escherichia coli. Our results show a prolonged lag phase and decrease in viability of drug-resistant E. colitreated with antisense molecules. The antisense strategy can be applied to alter expression of other genes in antibiotic resistance pathways or other pathways of interest.

  4. Radiation induction of drug resistance in RIF-1: Correlation of tumor and cell culture results

    International Nuclear Information System (INIS)

    Moulder, J.E.; Hopwood, L.E.; Volk, D.M.; Davies, B.M.

    1991-01-01

    The RIF-1 tumor line contains cells that are resistant to various anti-neoplastic drugs, including 5-fluorouracil (5FU), methotrexate (MTX), adriamycin (ADR), and etoposide (VP16). The frequency of these drug-resistant cells is increased after irradiation. The frequency of drug-resistant cells and the magnitude of radiation-induced drug resistance are different in cell culture than in tumors. The dose-response and expression time relationships for radiation induction of drug resistance observed in RIF-1 tumors are unusual.We hypothesize that at high radiation doses in vivo, we are selecting for cells that are both drug resistant and radiation resistant due to microenvironmental factors, whereas at low radiation doses in vivo and all radiation doses in vitro, we are observing true mutants. These studies indicate that there can be significant differences in drug-resistance frequencies between tumors and their cell lines of origin, and that radiation induction of drug resistance depends significantly on whether the induction is done in tumors or in cell culture. These results imply that theories about the induction of drug resistance that are based on cell culture studies may be inapplicable to the induction of drug resistance in tumors

  5. Decreasing prevalence of multi-drugs resistant Mycobacterium tuberculosis in Nashik City, India

    Directory of Open Access Journals (Sweden)

    Arun P. More

    2013-03-01

    Full Text Available Objective: In India, increasing prevalence of multi-drug resistant tuberculosis (MDR has aggravated the control oftuberculosis problem. In many urban and semi-urban regions of India, no surveillance data of multidrug resistance inMycobacterium tuberculosisis available.Methods: A surveillance study on multidrug resistance was carried out in semi-urban and rural regions in and aroundNashik City of Maharashtra, India. The surveillance study was conducted in this region found that the prevalence ofcombined resistance to first and second-line anti-tuberculosis drugs is remarkably high. The isolates of M. tuberculosiswas identified and subjected to drug susceptibility testing. The patterns of drug susceptibility of isolates of M. tuberculosisduring the periods 2000 and 2004 were compared with drug susceptibility patterns of the organisms during theperiod 2008 to 2011.Results: The 260 isolates identified as M. tuberculosis show mean drug resistance prevalence of 45.6% for more than anytwo drugs and the MDR rate as 37% in the years 2000 to 2004 whereas 305 isolates of the organism show mean drugresistance prevalence of 30.2% and the MDR rate as 25% in the years 2008 to 2011.Conclusion: The researcher found that, though the prevalence of multidrug resistance to the drugs tested is remarkablyhigh, it has come down noticeably during the past seven years due to efforts of State Government and strict implementationof treatment guidelines of WHO by the physicians. J Microbiol Infect Dis 2013; 3(1: 12-17Key words: MDR-TB, XDR-TB, DOTS, drug-resistance prevalence rate.

  6. Multi drug resistant tuberculosis: a challenge in the management of ...

    African Journals Online (AJOL)

    Multi drug resistant tuberculosis (MDR-TB) will not usually respond to short course chemotherapy. Unless the individual infected with this bug is treated appropriately, they can continue spreading resistant strains in the community and further fuel the tuberculosis epidemic. Diagnosis requires drug sensitivity testing and the ...

  7. Engineered reversal of drug resistance in cancer cells--metastases suppressor factors as change agents.

    Science.gov (United States)

    Yadav, Vinod Kumar; Kumar, Akinchan; Mann, Anita; Aggarwal, Suruchi; Kumar, Maneesh; Roy, Sumitabho Deb; Pore, Subrata Kumar; Banerjee, Rajkumar; Mahesh Kumar, Jerald; Thakur, Ram Krishna; Chowdhury, Shantanu

    2014-01-01

    Building molecular correlates of drug resistance in cancer and exploiting them for therapeutic intervention remains a pressing clinical need. To identify factors that impact drug resistance herein we built a model that couples inherent cell-based response toward drugs with transcriptomes of resistant/sensitive cells. To test this model, we focused on a group of genes called metastasis suppressor genes (MSGs) that influence aggressiveness and metastatic potential of cancers. Interestingly, modeling of 84 000 drug response transcriptome combinations predicted multiple MSGs to be associated with resistance of different cell types and drugs. As a case study, on inducing MSG levels in a drug resistant breast cancer line resistance to anticancer drugs caerulomycin, camptothecin and topotecan decreased by more than 50-60%, in both culture conditions and also in tumors generated in mice, in contrast to control un-induced cells. To our knowledge, this is the first demonstration of engineered reversal of drug resistance in cancer cells based on a model that exploits inherent cellular response profiles.

  8. Specific Cell Targeting Therapy Bypasses Drug Resistance Mechanisms in African Trypanosomiasis.

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    Juan D Unciti-Broceta

    2015-06-01

    Full Text Available African trypanosomiasis is a deadly neglected disease caused by the extracellular parasite Trypanosoma brucei. Current therapies are characterized by high drug toxicity and increasing drug resistance mainly associated with loss-of-function mutations in the transporters involved in drug import. The introduction of new antiparasitic drugs into therapeutic use is a slow and expensive process. In contrast, specific targeting of existing drugs could represent a more rapid and cost-effective approach for neglected disease treatment, impacting through reduced systemic toxicity and circumventing resistance acquired through impaired compound uptake. We have generated nanoparticles of chitosan loaded with the trypanocidal drug pentamidine and coated by a single domain nanobody that specifically targets the surface of African trypanosomes. Once loaded into this nanocarrier, pentamidine enters trypanosomes through endocytosis instead of via classical cell surface transporters. The curative dose of pentamidine-loaded nanobody-chitosan nanoparticles was 100-fold lower than pentamidine alone in a murine model of acute African trypanosomiasis. Crucially, this new formulation displayed undiminished in vitro and in vivo activity against a trypanosome cell line resistant to pentamidine as a result of mutations in the surface transporter aquaglyceroporin 2. We conclude that this new drug delivery system increases drug efficacy and has the ability to overcome resistance to some anti-protozoal drugs.

  9. A meta-analysis of Drug resistant Tuberculosis in Sub-Saharan Africa

    African Journals Online (AJOL)

    Background: In Sub-Saharan Africa, the fight against tuberculosis (TB) has encountered a great challenge because of the emergence of drug resistant TB strains and the high prevalence of HIV infection. The aim of this meta-analysis was to determine the association of drug-resistant TB with anti-TB drug treatment history ...

  10. Molecular mechanisms of drug resistance in natural Leishmania populations vary with genetic background.

    Directory of Open Access Journals (Sweden)

    Saskia Decuypere

    Full Text Available The evolution of drug-resistance in pathogens is a major global health threat. Elucidating the molecular basis of pathogen drug-resistance has been the focus of many studies but rarely is it known whether a drug-resistance mechanism identified is universal for the studied pathogen; it has seldom been clarified whether drug-resistance mechanisms vary with the pathogen's genotype. Nevertheless this is of critical importance in gaining an understanding of the complexity of this global threat and in underpinning epidemiological surveillance of pathogen drug resistance in the field. This study aimed to assess the molecular and phenotypic heterogeneity that emerges in natural parasite populations under drug treatment pressure. We studied lines of the protozoan parasite Leishmania (L. donovani with differential susceptibility to antimonial drugs; the lines being derived from clinical isolates belonging to two distinct genetic populations that circulate in the leishmaniasis endemic region of Nepal. Parasite pathways known to be affected by antimonial drugs were characterised on five experimental levels in the lines of the two populations. Characterisation of DNA sequence, gene expression, protein expression and thiol levels revealed a number of molecular features that mark antimonial-resistant parasites in only one of the two populations studied. A final series of in vitro stress phenotyping experiments confirmed this heterogeneity amongst drug-resistant parasites from the two populations. These data provide evidence that the molecular changes associated with antimonial-resistance in natural Leishmania populations depend on the genetic background of the Leishmania population, which has resulted in a divergent set of resistance markers in the Leishmania populations. This heterogeneity of parasite adaptations provides severe challenges for the control of drug resistance in the field and the design of molecular surveillance tools for widespread

  11. Multidrug-resistant and extensively drug-resistant tuberculosis: implications for the HIV epidemic and antiretroviral therapy rollout in South Africa.

    Science.gov (United States)

    Andrews, Jason R; Shah, N Sarita; Gandhi, Neel; Moll, Tony; Friedland, Gerald

    2007-12-01

    Drug-resistant tuberculosis (TB) is emerging as a major clinical and public health challenge in areas of sub-Saharan Africa where there is a high prevalence of human immunodeficiency virus (HIV) infection. TB drug-resistance surveillance in this region has been limited by laboratory capacity and the public health infrastructure; however, with the maturation of the HIV epidemic, the burden of drug-resistant TB is increasing rapidly. The recent discovery of large numbers of cases of multidrug-resistant (MDR) TB and extensively drug-resistant (XDR) TB in South Africa likely represents an unrecognized and evolving epidemic rather than sporadic, localized outbreaks. The combination of a large population of HIV-infected susceptible hosts with poor TB treatment success rates, a lack of airborne infection control, limited drug-resistance testing, and an overburdened MDR-TB treatment program provides ideal conditions for an MDR-TB and XDR-TB epidemic of unparalleled magnitude. In the present article, we review the history of drug-resistant TB in South Africa, describe its interaction with the HIV epidemic and the resultant consequences, and suggest measures necessary for controlling MDR-TB and XDR-TB in this context. A successful response to the emergence of MDR-TB and XDR-TB will necessitate increased resources for and collaboration between TB and HIV programs.

  12. mTOR Signaling Confers Resistance to Targeted Cancer Drugs.

    Science.gov (United States)

    Guri, Yakir; Hall, Michael N

    2016-11-01

    Cancer is a complex disease and a leading cause of death worldwide. Extensive research over decades has led to the development of therapies that target cancer-specific signaling pathways. However, the clinical benefits of such drugs are at best transient due to tumors displaying intrinsic or adaptive resistance. The underlying compensatory pathways that allow cancer cells to circumvent a drug blockade are poorly understood. We review here recent studies suggesting that mammalian TOR (mTOR) signaling is a major compensatory pathway conferring resistance to many cancer drugs. mTOR-mediated resistance can be cell-autonomous or non-cell-autonomous. These findings suggest that mTOR signaling should be monitored routinely in tumors and that an mTOR inhibitor should be considered as a co-therapy. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. Clinical and Drug Resistance Characteristics of New Pediatric Tuberculosis Cases in Northern China.

    Science.gov (United States)

    Wang, Ting; Dong, Fang; Li, Qin-Jing; Yin, Qing-Qin; Song, Wen-Qi; Mokrousov, Igor; Jiao, Wei-Wei; Shen, A-Dong

    2018-05-09

    The aim of this study was to evaluate the clinical features and characteristics of drug resistance in newly diagnosed pediatric tuberculosis (TB) patients in northern China. Mycobacterium tuberculosis isolates were collected from September 2010 to October 2016 at the Beijing Children's Hospital. Patients were divided into two groups (resistant to at least one drug and pan-susceptible) according to drug susceptibility testing (DST) results. A total of 132 new cases, mainly from northern China (87.9%), were included in the study. The median age was 1.9 years (1 month-15 years). Resistance to at least one drug was detected in Mycobacterium tuberculosis isolates from 33 (25%) cases. Eight cases of multidrug-resistant TB (MDR-TB) (6.1%) were detected. The two groups did not differ in clinical presentations (disease site, fever >2 weeks, and cough >2 weeks) or in chest imaging (lesion location, lymphadenitis [mediastinal], and pleural effusion). The rate of Mycobacterium tuberculosis drug resistance in new pediatric TB cases was as high as in the new adult patients surveyed in the national drug resistance survey conducted in 2007. No significant difference was observed in clinical features between patients infected with drug-resistant and drug-susceptible strains. Routine DST is important for prescribing effective antituberculosis treatment regimens.

  14. Nanoparticle-mediated combination chemotherapy and photodynamic therapy overcomes tumor drug resistance.

    Science.gov (United States)

    Khdair, Ayman; Chen, Di; Patil, Yogesh; Ma, Linan; Dou, Q Ping; Shekhar, Malathy P V; Panyam, Jayanth

    2010-01-25

    Tumor drug resistance significantly limits the success of chemotherapy in the clinic. Tumor cells utilize multiple mechanisms to prevent the accumulation of anticancer drugs at their intracellular site of action. In this study, we investigated the anticancer efficacy of doxorubicin in combination with photodynamic therapy using methylene blue in a drug-resistant mouse tumor model. Surfactant-polymer hybrid nanoparticles formulated using an anionic surfactant, Aerosol-OT (AOT), and a naturally occurring polysaccharide polymer, sodium alginate, were used for synchronized delivery of the two drugs. Balb/c mice bearing syngeneic JC tumors (mammary adenocarcinoma) were used as a drug-resistant tumor model. Nanoparticle-mediated combination therapy significantly inhibited tumor growth and improved animal survival. Nanoparticle-mediated combination treatment resulted in enhanced tumor accumulation of both doxorubicin and methylene blue, significant inhibition of tumor cell proliferation, and increased induction of apoptosis. These data suggest that nanoparticle-mediated combination chemotherapy and photodynamic therapy using doxorubicin and methylene blue has significant therapeutic potential against drug-resistant tumors. Copyright 2009 Elsevier B.V. All rights reserved.

  15. Prevalence of resistance to second-line tuberculosis drug among multidrug-resistant tuberculosis patients in Viet Nam, 2011.

    Science.gov (United States)

    Nguyen, Hoa Binh; Nguyen, Nhung Viet; Tran, Huong Thi Giang; Nguyen, Hai Viet; Bui, Quyen Thi Tu

    2016-01-01

    Extensively drug-resistant tuberculosis (XDR-TB) represents an emerging public health problem worldwide. According to the World Health Organization, an estimated 9.7% of multidrug-resistant TB (MDR-TB) cases are defined as XDR-TB globally. The objective of this study was to determine the prevalence of drug resistance to second-line TB drugs among MDR-TB cases detected in the Fourth National Anti-Tuberculosis Drug Resistance Survey in Viet Nam. Eighty clusters of TB cases were selected using a probability-proportion-to-size approach. To identify MDR-TB cases, drug susceptibility testing (DST) was performed for the four major first-line TB drugs. DST of second-line drugs (ofloxacin, amikacin, kanamycin, capreomycin) was performed on isolates from MDR-TB cases to identify pre-XDR and XDR cases. A total of 1629 smear-positive TB cases were eligible for culture and DST. Of those, DST results for first-line drugs were available for 1312 cases, and 91 (6.9%) had MDR-TB. Second-line DST results were available for 84 of these cases. Of those, 15 cases (17.9%) had ofloxacin resistance and 6.0% were resistant to kanamycin and capreomycin. Five MDR-TB cases (6.0%) met the criteria of XDR-TB. This survey provides the first estimates of the proportion of XDR-TB among MDR-TB cases in Viet Nam and provides important information for local policies regarding second-line DST. Local policies and programmes that are geared towards TB prevention, early diagnosis and treatment with effective regimens are of high importance.

  16. Feasibility evaluation of 3 automated cellular drug screening assays on a robotic workstation.

    Science.gov (United States)

    Soikkeli, Anne; Sempio, Cristina; Kaukonen, Ann Marie; Urtti, Arto; Hirvonen, Jouni; Yliperttula, Marjo

    2010-01-01

    This study presents the implementation and optimization of 3 cell-based assays on a TECAN Genesis workstation-the Caspase-Glo 3/7 and sulforhodamine B (SRB) screening assays and the mechanistic Caco-2 permeability protocol-and evaluates their feasibility for automation. During implementation, the dispensing speed to add drug solutions and fixative trichloroacetic acid and the aspiration speed to remove the supernatant immediately after fixation were optimized. Decontamination steps for cleaning the tips and pipetting tubing were also added. The automated Caspase-Glo 3/7 screen was successfully optimized with Caco-2 cells (Z' 0.7, signal-to-base ratio [S/B] 1.7) but not with DU-145 cells. In contrast, the automated SRB screen was successfully optimized with the DU-145 cells (Z' 0.8, S/B 2.4) but not with the Caco-2 cells (Z' -0.8, S/B 1.4). The automated bidirectional Caco-2 permeability experiments separated successfully low- and high-permeability compounds (Z' 0.8, S/B 84.2) and passive drug permeation from efflux-mediated transport (Z' 0.5, S/B 8.6). Of the assays, the homogeneous Caspase-Glo 3/7 assay benefits the most from automation, but also the heterogeneous SRB assay and Caco-2 permeability experiments gain advantages from automation.

  17. HIV Drug Resistance Surveillance in Honduras after a Decade of Widespread Antiretroviral Therapy.

    Science.gov (United States)

    Avila-Ríos, Santiago; García-Morales, Claudia; Tapia-Trejo, Daniela; Meza, Rita I; Nuñez, Sandra M; Parham, Leda; Flores, Norma A; Valladares, Diana; Pineda, Luisa M; Flores, Dixiana; Motiño, Roxana; Umanzor, Víctor; Carbajal, Candy; Murillo, Wendy; Lorenzana, Ivette; Palou, Elsa Y; Reyes-Terán, Gustavo

    2015-01-01

    We assessed HIV drug resistance (DR) in individuals failing ART (acquired DR, ADR) and in ART-naïve individuals (pre-ART DR, PDR) in Honduras, after 10 years of widespread availability of ART. 365 HIV-infected, ART-naïve, and 381 ART-experienced Honduran individuals were enrolled in 5 reference centres in Tegucigalpa, San Pedro Sula, La Ceiba, and Choluteca between April 2013 and April 2015. Plasma HIV protease-RT sequences were obtained. HIVDR was assessed using the WHO HIVDR mutation list and the Stanford algorithm. Recently infected (RI) individuals were identified using a multi-assay algorithm. PDR to any ARV drug was 11.5% (95% CI 8.4-15.2%). NNRTI PDR prevalence (8.2%) was higher than NRTI (2.2%) and PI (1.9%, p500 vs. Honduras remains at the intermediate level, after 10 years of widespread availability of ART. Evidence of ADR influencing the presence of PDR was observed by phylogenetic analyses and ADR/PDR mutation frequency correlations.

  18. Genotypic diversity of multidrug-, quinolone- and extensively drug-resistant Mycobacterium tuberculosis isolates in Thailand.

    Science.gov (United States)

    Disratthakit, Areeya; Meada, Shinji; Prammananan, Therdsak; Thaipisuttikul, Iyarit; Doi, Norio; Chaiprasert, Angkana

    2015-06-01

    Drug-resistant tuberculosis (TB), which includes multidrug-resistant (MDR-TB), quinolone-resistant (QR-TB) and extensively drug-resistant tuberculosis (XDR-TB), is a serious threat to TB control. We aimed to characterize the genotypic diversity of drug-resistant TB clinical isolates collected in Thailand to establish whether the emergence of drug-resistant TB is attributable to transmitted resistance or acquired resistance. We constructed the first molecular phylogeny of MDR-TB (n=95), QR-TB (n=69) and XDR-TB (n=28) in Thailand based on spoligotyping and proposed 24-locus multilocus variable-number of tandem repeat analysis (MLVA). Clustering analysis was performed using the unweighted pair group method with arithmetic mean. Spoligotyping identified the Beijing strain (SIT1) as the most predominant genotype (n=139; 72.4%). The discriminatory power of 0.9235 Hunter-Gaston Discriminatory Index (HGDI) with the 15-locus variable-number tandem repeats of mycobacterial interspersed repetitive units typing was improved to a 0.9574 HGDI with proposed 24-locus MLVA, thereby resulting in the subdivision of a large cluster of Beijing strains (SIT1) into 17 subclusters. We identified the spread of drug-resistant TB clones caused by three different MLVA types in the Beijing strain (SIT1) and a specific clone of XDR-TB caused by a rare genotype, the Manu-ancestor strain (SIT523). Overall, 49.5% of all isolates were clustered. These findings suggest that a remarkable transmission of drug-resistant TB occurred in Thailand. The remaining 50% of drug-resistant TB isolates were unique genotypes, which may have arisen from the individual acquisition of drug resistance. Our results suggest that transmitted and acquired resistance have played an equal role in the emergence of drug-resistant TB. Further characterization of whole genome sequences of clonal strains could help to elucidate the mycobacterial genetic factors relevant for drug resistance, transmissibility and virulence

  19. Drug-Resistant Bacteria: On the Edge of a Crisis | NIH MedlinePlus the Magazine

    Science.gov (United States)

    ... drug-resistant bacteria research program. Why are certain bacteria becoming more resistant to drugs? There is a ... a national, even global crisis of drug-resistant bacteria. Why is that? The more we see this ...

  20. Susceptibility Testing by Polymerase Chain Reaction DNA Quantitation: A Method to Measure Drug Resistance of Human Immunodeficiency Virus Type 1 Isolates

    Science.gov (United States)

    Eron, Joseph J.; Gorczyca, Paul; Kaplan, Joan C.; D'Aquila, Richard T.

    1992-04-01

    Polymerase chain reaction (PCR) DNA quantitation (PDQ) susceptibility testing rapidly and directly measures nucleoside sensitivity of human immunodeficiency virus type 1 (HIV-1) isolates. PCR is used to quantitate the amount of HIV-1 DNA synthesized after in vitro infection of peripheral blood mononuclear cells. The relative amounts of HIV-1 DNA in cell lysates from cultures maintained at different drug concentrations reflect drug inhibition of virus replication. The results of PDQ susceptibility testing of 2- or 3-day cultures are supported by assays measuring HIV-1 p24 antigen production in supernatants of 7- or 10-day cultures. DNA sequence analyses to identify mutations in the reverse transcriptase gene that cause resistance to 3'-azido-3'-deoxythymidine also support the PDQ results. With the PDQ method, both infectivity titration and susceptibility testing can be performed on supernatants from primary cultures of peripheral blood mononuclear cells. PDQ susceptibility testing should facilitate epidemiologic studies of the clinical significance of drug-resistant HIV-1 isolates.

  1. Life cycle synchronization is a viral drug resistance mechanism.

    Directory of Open Access Journals (Sweden)

    Iulia A Neagu

    2018-02-01

    Full Text Available Viral infections are one of the major causes of death worldwide, with HIV infection alone resulting in over 1.2 million casualties per year. Antiviral drugs are now being administered for a variety of viral infections, including HIV, hepatitis B and C, and influenza. These therapies target a specific phase of the virus's life cycle, yet their ultimate success depends on a variety of factors, such as adherence to a prescribed regimen and the emergence of viral drug resistance. The epidemiology and evolution of drug resistance have been extensively characterized, and it is generally assumed that drug resistance arises from mutations that alter the virus's susceptibility to the direct action of the drug. In this paper, we consider the possibility that a virus population can evolve towards synchronizing its life cycle with the pattern of drug therapy. The periodicity of the drug treatment could then allow for a virus strain whose life cycle length is a multiple of the dosing interval to replicate only when the concentration of the drug is lowest. This process, referred to as "drug tolerance by synchronization", could allow the virus population to maximize its overall fitness without having to alter drug binding or complete its life cycle in the drug's presence. We use mathematical models and stochastic simulations to show that life cycle synchronization can indeed be a mechanism of viral drug tolerance. We show that this effect is more likely to occur when the variability in both viral life cycle and drug dose timing are low. More generally, we find that in the presence of periodic drug levels, time-averaged calculations of viral fitness do not accurately predict drug levels needed to eradicate infection, even if there is no synchronization. We derive an analytical expression for viral fitness that is sufficient to explain the drug-pattern-dependent survival of strains with any life cycle length. We discuss the implications of these findings for

  2. Detection of selected antibiotic resistance genes using multiplex PCR assay in mastitis pathogens in the Czech Republic

    Directory of Open Access Journals (Sweden)

    Vladimir Pyatov

    2017-01-01

    Full Text Available The aim of this research was to develop multiplex polymerase chain reaction assays for the detection of aminoglycoside (strA, strB, sulphonamide (sulI, sulII, tetracycline (tetA, tetB, tetK, tetM, tetO, macrolide and lincosamide (msrA, ermA, ermB, ermC, mefA/E genes of resistance in mastitis pathogens (Escherichia coli, Staphylococcus aureus, Streptococcus uberis, Streptococcus agalactiae and Streptococcus dysgalactiae. Applying the established assays, we investigated the distribution of antibiotic resistance genes in the above mentioned species isolated from milk samples in the Czech Republic. Each assay consisted of seven pairs of primers. Six of them amplified fragments of antibiotic resistance genes and one pair a fragment of a species specific gene. Polymerase chain reaction conditions were optimized to amplify seven gene fragments simultaneously in one reaction. In total, 249 isolates were used, among which 111 were positive for E. coli, 52 for S. aureus and 86 for Streptococcus spp. The majority (60.2% of bacteria carried at least one antibiotic resistance gene and 44.6% were multidrug-resistant. The designed multiplex polymerase chain reaction assays may be applied as diagnostic method to replace or complement standard techniques of antibiotic susceptibility testing in the mentioned pathogens.

  3. Implementation of a national anti-tuberculosis drug resistance survey in Tanzania

    NARCIS (Netherlands)

    Chonde, Timothy M.; Doulla, Basra; van Leth, Frank; Mfinanga, Sayoki G. M.; Range, Nyagosya; Lwilla, Fred; Mfaume, Saidi M.; van Deun, Armand; Zignol, Matteo; Cobelens, Frank G.; Egwaga, Saidi M.

    2008-01-01

    BACKGROUND: A drug resistance survey is an essential public health management tool for evaluating and improving the performance of National Tuberculosis control programmes. The current manuscript describes the implementation of the first national drug resistance survey in Tanzania. METHODS:

  4. Occurrence of transmitted HIV-1 drug resistance among Drug-naïve pregnant women in selected HIV-care centres in Ghana.

    Science.gov (United States)

    Martin-Odoom, Alexander; Adiku, Theophilus; Delgado, Elena; Lartey, Margaret; Ampofo, William K

    2017-03-01

    Access to antiretroviral therapy in Ghana has been scaled up across the country over the last decade. This study sought to determine the occurrence of transmitted HIV-1 drug resistance in pregnant HIV-1 positive women yet to initiate antiretroviral therapy at selected HIV Care Centres in Ghana. Plasma specimens from twenty-six (26) HIV seropositive pregnant women who were less than 28weeks pregnant with their first pregnancy and ART naïve were collected from selected HIV care centres in three (3) regions in Ghana. Genotypic testing was done for the reverse transcriptase gene and the sequences generated were analyzed for HIV-1 drug resistance mutations using the Stanford University HIV Drug Resistance Database. Resistance mutations associated with the reverse transcriptase gene were detected in 4 (15.4%) of the participants. At least one major drug resistance mutation in the reverse transcriptase gene was found in 3 (11.5%) of the women. The detection of transmitted HIV-1 drug resistance in this drug-naïve group in two regional HIV care sites is an indication of the need for renewed action in monitoring the emergence of transmitted HIV-1 drug resistance in Ghana. None declared.

  5. Drug-resistant tuberculosis--current dilemmas, unanswered questions, challenges, and priority needs.

    Science.gov (United States)

    Zumla, Alimuddin; Abubakar, Ibrahim; Raviglione, Mario; Hoelscher, Michael; Ditiu, Lucica; McHugh, Timothy D; Squire, S Bertel; Cox, Helen; Ford, Nathan; McNerney, Ruth; Marais, Ben; Grobusch, Martin; Lawn, Stephen D; Migliori, Giovanni-Battista; Mwaba, Peter; O'Grady, Justin; Pletschette, Michel; Ramsay, Andrew; Chakaya, Jeremiah; Schito, Marco; Swaminathan, Soumya; Memish, Ziad; Maeurer, Markus; Atun, Rifat

    2012-05-15

    Tuberculosis was declared a global emergency by the World Health Organization (WHO) in 1993. Following the declaration and the promotion in 1995 of directly observed treatment short course (DOTS), a cost-effective strategy to contain the tuberculosis epidemic, nearly 7 million lives have been saved compared with the pre-DOTS era, high cure rates have been achieved in most countries worldwide, and the global incidence of tuberculosis has been in a slow decline since the early 2000s. However, the emergence and spread of multidrug-resistant (MDR) tuberculosis, extensively drug-resistant (XDR) tuberculosis, and more recently, totally drug-resistant tuberculosis pose a threat to global tuberculosis control. Multidrug-resistant tuberculosis is a man-made problem. Laboratory facilities for drug susceptibility testing are inadequate in most tuberculosis-endemic countries, especially in Africa; thus diagnosis is missed, routine surveillance is not implemented, and the actual numbers of global drug-resistant tuberculosis cases have yet to be estimated. This exposes an ominous situation and reveals an urgent need for commitment by national programs to health system improvement because the response to MDR tuberculosis requires strong health services in general. Multidrug-resistant tuberculosis and XDR tuberculosis greatly complicate patient management within resource-poor national tuberculosis programs, reducing treatment efficacy and increasing the cost of treatment to the extent that it could bankrupt healthcare financing in tuberculosis-endemic areas. Why, despite nearly 20 years of WHO-promoted activity and >12 years of MDR tuberculosis-specific activity, has the country response to the drug-resistant tuberculosis epidemic been so ineffectual? The current dilemmas, unanswered questions, operational issues, challenges, and priority needs for global drug resistance screening and surveillance, improved treatment regimens, and management of outcomes and prevention of DR

  6. Reduced intracellular drug accumulation in drug-resistant leukemia cells is not solely due to MDR-mediated efflux but also to decreased uptake

    Directory of Open Access Journals (Sweden)

    Angela Oliveira Pisco

    2014-10-01

    Full Text Available Expression of ABC family transporter proteins that promote drug efflux from cancer cells is a widely observed mechanism of multi-drug resistance of cancer cells. Cell adaptation in long-term culture of HL60 leukemic cells in the presence of chemotherapy leads to induction and maintenance of the ABC transporters expression, preventing further accumulation of drugs. However, we found that decreased accumulation of drugs and fluorescent dyes was also contributed by a reduced uptake by the resistant cells. Confocal time-lapse microscopy and flow cytometry revealed that fluid-phase endocytosis was diminished in drug-resistant cells compared to drug-sensitive cells. Drug uptake was increased by insulin co-treatment when cells were grown in methylcellulose and monitored under the microscope, but not when cultured in suspension. We propose that multi-drug resistance is not solely achieved by enhanced efflux capacity but also by supressed intake of the drug offering an alternative target to overcome drug resistance or potentiate chemotherapy.

  7. Nanoparticle-mediated combination chemotherapy and photodynamic therapy overcomes tumor drug resistance in vitro.

    Science.gov (United States)

    Khdair, Ayman; Handa, Hitesh; Mao, Guangzhao; Panyam, Jayanth

    2009-02-01

    Drug resistance limits the success of many anticancer drugs. Reduced accumulation of the drug at its intracellular site of action because of overexpression of efflux transporters such as P-glycoprotein (P-gp) is a major mechanism of drug resistance. In this study, we investigated whether photodynamic therapy (PDT) using methylene blue, also a P-gp inhibitor, can be used to enhance doxorubicin-induced cytotoxicity in drug-resistant tumor cells. Aerosol OT (AOT)-alginate nanoparticles were used as a carrier for the simultaneous cellular delivery of doxorubicin and methylene blue. Methylene blue was photoactivated using light of 665 nm wavelength. Induction of apoptosis and necrosis following treatment with combination chemotherapy and PDT was investigated in drug-resistant NCI/ADR-RES cells using flow cytometry and fluorescence microscopy. Effect of encapsulation in nanoparticles on the intracellular accumulation of doxorubicin and methylene blue was investigated qualitatively using fluorescence microscopy and was quantitated using HPLC. Encapsulation in AOT-alginate nanoparticles significantly enhanced the cytotoxicity of combination therapy in resistant tumor cells. Nanoparticle-mediated combination therapy resulted in a significant induction of both apoptosis and necrosis. Improvement in cytotoxicity could be correlated with enhanced intracellular and nuclear delivery of the two drugs. Further, nanoparticle-mediated combination therapy resulted in significantly elevated reactive oxygen species (ROS) production compared to single drug treatment. In conclusion, nanoparticle-mediated combination chemotherapy and PDT using doxorubicin and methylene blue was able to overcome resistance mechanisms and resulted in improved cytotoxicity in drug-resistant tumor cells.

  8. Evaluation of the BACTEC MGIT 960 SL DST Kit and the GenoType MTBDRsl Test for Detecting Extensively Drug-resistant Tuberculosis Cases.

    Science.gov (United States)

    Tekin, Kemal; Albay, Ali; Simsek, Hulya; Sig, Ali Korhan; Guney, Mustafa

    2017-10-01

    The present study aimed to evaluate the performances of the BACTEC MGIT 960 SL DST kit and the GenoType MTBDRsl test for detecting second-line antituberculosis drug resistance in Multidrug-resistant TB (MDR-TB) cases. Forty-six MDR-TB strains were studied. Second-line antituberculosis drug resistances were detected using the BACTEC MGIT 960 SL DST kit and the GenoType MTBDRsl test. The Middlebrook 7H10 agar proportion method was used as the reference test. The sensitivity and specificity values for the BACTEC MGIT 960 SL DST kit were both 100% for amikacin, kanamycin, capreomycin (4 µg/mL), and ofloxacin; 100% and 95.3%, respectively, for capreomycin (10 µg/mL); and 85.7% and 100%, respectively, for moxifloxacin (0.5 µg/mL). The sensitivity and specificity values for the GenoType MTBDRsl test to detect fluoroquinolone and aminoglycoside/cyclic peptide resistance were 88.9% and 100%, respectively, for ofloxacin and 85.7% and 94.9%, respectively, for moxifloxacin (0.5 µg/mL). The accuracy of the GenoType MTBDRsl assay for kanamycin, capreomycin, ofloxacin, and moxifloxacin was lower than that of the BACTEC MGIT 960 SL DST. The BACTEC MGIT 960 SL DST kit and the GenoType MTBDRsl were successful in detecting second-line antituberculosis drug resistance. Preliminary results of the GenoType MTBDRsl are very valuable for early treatment decisions, but we still recommend additional BACTEC MGIT 960 SL DST kit usage in the routine evaluation of drug-resistant tuberculosis.

  9. Evaluation of the BACTEC MGIT 960 SL DST Kit and the GenoType MTBDRsl Test for Detecting Extensively Drug-resistant Tuberculosis Cases

    Science.gov (United States)

    Tekin, Kemal; Albay, Ali; Simsek, Hulya; Sig, Ali Korhan; Guney, Mustafa

    2017-01-01

    Objective: The present study aimed to evaluate the performances of the BACTEC MGIT 960 SL DST kit and the GenoType MTBDRsl test for detecting second-line antituberculosis drug resistance in Multidrug-resistant TB (MDR-TB) cases. Materials and Methods: Forty-six MDR-TB strains were studied. Second-line antituberculosis drug resistances were detected using the BACTEC MGIT 960 SL DST kit and the GenoType MTBDRsl test. The Middlebrook 7H10 agar proportion method was used as the reference test. Results: The sensitivity and specificity values for the BACTEC MGIT 960 SL DST kit were both 100% for amikacin, kanamycin, capreomycin (4 µg/mL), and ofloxacin; 100% and 95.3%, respectively, for capreomycin (10 µg/mL); and 85.7% and 100%, respectively, for moxifloxacin (0.5 µg/mL). The sensitivity and specificity values for the GenoType MTBDRsl test to detect fluoroquinolone and aminoglycoside/cyclic peptide resistance were 88.9% and 100%, respectively, for ofloxacin and 85.7% and 94.9%, respectively, for moxifloxacin (0.5 µg/mL). The accuracy of the GenoType MTBDRsl assay for kanamycin, capreomycin, ofloxacin, and moxifloxacin was lower than that of the BACTEC MGIT 960 SL DST. Conclusion: The BACTEC MGIT 960 SL DST kit and the GenoType MTBDRsl were successful in detecting second-line antituberculosis drug resistance. Preliminary results of the GenoType MTBDRsl are very valuable for early treatment decisions, but we still recommend additional BACTEC MGIT 960 SL DST kit usage in the routine evaluation of drug-resistant tuberculosis. PMID:29123441

  10. Comparison of the pharmacokinetics of two dosage regimens of linezolid in multidrug-resistant and extensively drug-resistant tuberculosis patients.

    NARCIS (Netherlands)

    Alffenaar, J.W.C.; Altena, R. van; Harmelink, I.M.; Filguera, P.; Molenaar, E.; Wessels, A.M.; Soolingen, D. van; Kosterink, J.G.W.; Uges, D.R.A.; Werf, T.S. van der

    2010-01-01

    BACKGROUND AND OBJECTIVES: For the treatment of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB), potent new drugs are urgently needed. Linezolid is a promising drug, but its use is limited by adverse effects with prolonged administration of 600 mg twice daily. In

  11. Comparison of the Pharmacokinetics of Two Dosage Regimens of Linezolid in Multidrug-Resistant and Extensively Drug-Resistant Tuberculosis Patients

    NARCIS (Netherlands)

    Alffenaar, Jan-Willem C.; van Altena, Richard; Harmelink, Ilse M.; Filguera, Patricia; Molenaar, Esther; Wessels, A. Mireille A.; van Soolingen, Dick; Kosterink, Jos G. W.; Uges, Donald R. A.; van der Werf, Tjip S.

    2010-01-01

    Background and Objectives: For the treatment of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB), potent new drugs are urgently needed. Linezolid is a promising drug, but its use is limited by adverse effects with prolonged administration of 600 mg twice daily. In

  12. Population mobility, globalization, and antimicrobial drug resistance.

    Science.gov (United States)

    MacPherson, Douglas W; Gushulak, Brian D; Baine, William B; Bala, Shukal; Gubbins, Paul O; Holtom, Paul; Segarra-Newnham, Marisel

    2009-11-01

    Population mobility is a main factor in globalization of public health threats and risks, specifically distribution of antimicrobial drug-resistant organisms. Drug resistance is a major risk in healthcare settings and is emerging as a problem in community-acquired infections. Traditional health policy approaches have focused on diseases of global public health significance such as tuberculosis, yellow fever, and cholera; however, new diseases and resistant organisms challenge existing approaches. Clinical implications and health policy challenges associated with movement of persons across barriers permeable to products, pathogens, and toxins (e.g., geopolitical borders, patient care environments) are complex. Outcomes are complicated by high numbers of persons who move across disparate and diverse settings of disease threat and risk. Existing policies and processes lack design and capacity to prevent or mitigate adverse health outcomes. We propose an approach to global public health risk management that integrates population factors with effective and timely application of policies and processes.

  13. Modeling HIV-1 drug resistance as episodic directional selection.

    Science.gov (United States)

    Murrell, Ben; de Oliveira, Tulio; Seebregts, Chris; Kosakovsky Pond, Sergei L; Scheffler, Konrad

    2012-01-01

    The evolution of substitutions conferring drug resistance to HIV-1 is both episodic, occurring when patients are on antiretroviral therapy, and strongly directional, with site-specific resistant residues increasing in frequency over time. While methods exist to detect episodic diversifying selection and continuous directional selection, no evolutionary model combining these two properties has been proposed. We present two models of episodic directional selection (MEDS and EDEPS) which allow the a priori specification of lineages expected to have undergone directional selection. The models infer the sites and target residues that were likely subject to directional selection, using either codon or protein sequences. Compared to its null model of episodic diversifying selection, MEDS provides a superior fit to most sites known to be involved in drug resistance, and neither one test for episodic diversifying selection nor another for constant directional selection are able to detect as many true positives as MEDS and EDEPS while maintaining acceptable levels of false positives. This suggests that episodic directional selection is a better description of the process driving the evolution of drug resistance.

  14. Modeling HIV-1 drug resistance as episodic directional selection.

    Directory of Open Access Journals (Sweden)

    Ben Murrell

    Full Text Available The evolution of substitutions conferring drug resistance to HIV-1 is both episodic, occurring when patients are on antiretroviral therapy, and strongly directional, with site-specific resistant residues increasing in frequency over time. While methods exist to detect episodic diversifying selection and continuous directional selection, no evolutionary model combining these two properties has been proposed. We present two models of episodic directional selection (MEDS and EDEPS which allow the a priori specification of lineages expected to have undergone directional selection. The models infer the sites and target residues that were likely subject to directional selection, using either codon or protein sequences. Compared to its null model of episodic diversifying selection, MEDS provides a superior fit to most sites known to be involved in drug resistance, and neither one test for episodic diversifying selection nor another for constant directional selection are able to detect as many true positives as MEDS and EDEPS while maintaining acceptable levels of false positives. This suggests that episodic directional selection is a better description of the process driving the evolution of drug resistance.

  15. Towards an understanding of drug resistance in malaria

    DEFF Research Database (Denmark)

    Lemcke, T; Christensen, I T; Jørgensen, Flemming Steen

    1999-01-01

    and structural differences. Based on this analysis the molecular consequences of point mutations known to be involved in drug resistance were discussed. The significance of the most important point mutation causing resistance, S108N, could be explained by the model, whereas the point mutations associated...... with enhanced resistance, N51I and C59R, seem to have a more indirect effect on inhibitor binding....

  16. Molecular Basis for Drug Resistance in HIV-1 Protease

    Directory of Open Access Journals (Sweden)

    Celia A. Schiffer

    2010-11-01

    Full Text Available HIV-1 protease is one of the major antiviral targets in the treatment of patients infected with HIV-1. The nine FDA approved HIV-1 protease inhibitors were developed with extensive use of structure-based drug design, thus the atomic details of how the inhibitors bind are well characterized. From this structural understanding the molecular basis for drug resistance in HIV-1 protease can be elucidated. Selected mutations in response to therapy and diversity between clades in HIV-1 protease have altered the shape of the active site, potentially altered the dynamics and even altered the sequence of the cleavage sites in the Gag polyprotein. All of these interdependent changes act in synergy to confer drug resistance while simultaneously maintaining the fitness of the virus. New strategies, such as incorporation of the substrate envelope constraint to design robust inhibitors that incorporate details of HIV-1 protease’s function and decrease the probability of drug resistance, are necessary to continue to effectively target this key protein in HIV-1 life cycle.

  17. DRUG RESISTANCE IN HELICOBACTER PYLORI

    Directory of Open Access Journals (Sweden)

    Júlia Silveira VIANNA

    Full Text Available ABSTRACT Background Helicobacter pylori has a worldwide distribution and is associated with the pathogenesis of various diseases of the digestive system. Treatment to eradicate this microorganism involves the use of a combination of antimicrobials, such as amoxicillin, metronidazole, clarithromycin, and levofloxacin, combined with proton pump inhibitors. Although the current therapy is effective, a high rate of treatment failure has been observed, mainly because of the acquisition of point mutations, one of the major resistance mechanisms developed by H. pylori. This phenomenon is related to frequent and/or inappropriate use of antibiotics. Conclusion This review reported an overview of the resistance to the main drugs used in the treatment of H. pylori, confirming the hypothesis that antibacterial resistance is a highly local phenomenon and genetic characteristics of a given population can influence which therapy is the most appropriate.

  18. Characterization of drug resistant Enterobacter species isolated from ...

    African Journals Online (AJOL)

    Enterobacter species are emerging clinical pathogens and they play important roles in the dissemination of drug resistant traits within the food chain due to their intrinsic abilities for resistance to commonly used antibiotics such as cephalosporins. Two Enterobacter cloacae and one Enterobacter hormaechei characterized in ...

  19. The fourth national anti-tuberculosis drug resistance survey in Viet Nam.

    Science.gov (United States)

    Nhung, N V; Hoa, N B; Sy, D N; Hennig, C M; Dean, A S

    2015-06-01

    Viet Nam's Fourth National Anti-Tuberculosis Drug Resistance Survey was conducted in 2011. To determine the prevalence of resistance to the four main first-line anti-tuberculosis drugs in Viet Nam. Eighty clusters were selected using a probability proportion to size approach. Drug susceptibility testing (DST) against the four main first-line anti-tuberculosis drugs was performed. A total of 1629 smear-positive tuberculosis (TB) patients were eligible for culture. Of these, DST results were available for 1312 patients, including 1105 new TB cases, 195 previously treated TB cases and 12 cases with an unknown treatment history. The proportion of cases with resistance to any drug was 32.7% (95%CI 29.1-36.5) among new cases and 54.2% (95%CI 44.3-63.7) among previously treated cases. The proportion of multidrug-resistant TB (MDR-TB) cases was 4.0% (95%CI 2.5-5.4) in new cases and 23.3 (95%CI 16.7-29.9) in previously treated cases. The fourth drug resistance survey in Viet Nam found that the proportion of MDR-TB among new and previously treated cases was not significantly different from that in the 2005 survey. The National TB Programme should prioritise the detection and treatment of MDR-TB to reduce transmission of MDR-TB in the community.

  20. An Aptamer Bio-barCode (ABC) assay using SPR, RNase H, and probes with RNA and gold-nanorods for anti-cancer drug screening.

    Science.gov (United States)

    Loo, Jacky Fong-Chuen; Yang, Chengbin; Tsang, Hing Lun; Lau, Pui Man; Yong, Ken-Tye; Ho, Ho Pui; Kong, Siu Kai

    2017-10-07

    With modifications to an ultra-sensitive bio-barcode (BBC) assay, we have developed a next generation aptamer-based bio-barcode (ABC) assay to detect cytochrome-c (Cyto-c), a cell death marker released from cancer cells, for anti-cancer drug screening. An aptamer is a short single-stranded DNA selected from a synthetic DNA library that is capable of binding to its target with high affinity and specificity based on its unique DNA sequence and 3D structure after folding. Similar to the BBC assay, Cyto-c is captured by a micro-magnetic particle (MMP) coated with capturing antibodies (Ab) and an aptamer specifically against Cyto-c to form sandwich structures ([MMP-Ab]-[Cyto-c]-[Aptamer]). After washing and melting, our aptamers, acting as a DNA bio-barcode, are released from the sandwiches and hybridized with the probes specially designed for RNase H for surface plasmon resonance (SPR) sensing. In an aptamer-probe duplex, RNase H digests the RNA in the probe and releases the intact aptamer for another round of hybridization and digestion. With signal enhancement effects from gold-nanorods (Au-NRs) on probes for SPR sensing, the detection limit was found to be 1 nM for the aptamer and 80 pM for Cyto-c. Without the time-consuming DNA amplification steps by PCR, the detection process of this new ABC assay can be completed within three hours. As a proof-of-concept, phenylarsine oxide was found to be a potent agent to kill liver cancer cells with multi-drug resistance at the nano-molar level. This approach thus provides a fast, sensitive and robust tool for anti-cancer drug screening.

  1. Low-level quinolone-resistance in multi-drug resistant typhoid

    Energy Technology Data Exchange (ETDEWEB)

    Mirza, S H; Khan, M A [Armed Forces Inst. of Pathology, Rawalpindi (Pakistan). Dept. of Microbiolgy

    2008-01-15

    To find out the frequency of low-level quinolone-resistance in Multi-Drug Resistant (MDR) typhoid using nalidixic acid screening disc. Blood was obtained from suspected cases of typhoid fever and cultured in to BacT/ALERT. The positive blood cultures bottles were subcultured. The isolates were identified by colony morphology and biochemical tests using API-20E galleries. Susceptibility testing of isolates was done by modified Kirby-Bauer disc diffusion method on Muellar Hinton Agar. For the isolates, which were resistant to nalidixic acid by disc diffusion method, Minimal Inhibitory Concentrations (MICs) of ciprofloxacin and nalidixic acid were determined by using the E-test strips. Disc diffusion susceptibility tests and MICs were interpreted according to the guidelines provided by National Committee for Control Laboratory Standard (NCCLS). A total of 21(65.5%) out of 32 isolates of Salmonellae were nalidixic acid-resistant by disk diffusion method. All the nalidixic acid-resistant isolates by disc diffusion method were confirmed by MICs for both ciprofloxacin and nalidixic acid. All the nalidixic acid-resistant isolates had a ciprofloxacin MIC of 0.25-1 microg/ml (reduced susceptibility) and nalidixic acid MICs > 32 microg (resistant). Out of all Salmonella isolates, 24 (75%) were found to be MDR, and all were S. typbi. Low-level quinolone-resistance in typhoid was high in this small series. Screening for nalidixic acid resistance with a 30 microg nalidixic acid disk is a reliable and cost-effective method to detect low-level fluoroquinolone resistance, especially in the developing countries. (author)

  2. Low-level quinolone-resistance in multi-drug resistant typhoid

    International Nuclear Information System (INIS)

    Mirza, S.H.; Khan, M.A.

    2008-01-01

    To find out the frequency of low-level quinolone-resistance in Multi-Drug Resistant (MDR) typhoid using nalidixic acid screening disc. Blood was obtained from suspected cases of typhoid fever and cultured in to BacT/ALERT. The positive blood cultures bottles were subcultured. The isolates were identified by colony morphology and biochemical tests using API-20E galleries. Susceptibility testing of isolates was done by modified Kirby-Bauer disc diffusion method on Muellar Hinton Agar. For the isolates, which were resistant to nalidixic acid by disc diffusion method, Minimal Inhibitory Concentrations (MICs) of ciprofloxacin and nalidixic acid were determined by using the E-test strips. Disc diffusion susceptibility tests and MICs were interpreted according to the guidelines provided by National Committee for Control Laboratory Standard (NCCLS). A total of 21(65.5%) out of 32 isolates of Salmonellae were nalidixic acid-resistant by disk diffusion method. All the nalidixic acid-resistant isolates by disc diffusion method were confirmed by MICs for both ciprofloxacin and nalidixic acid. All the nalidixic acid-resistant isolates had a ciprofloxacin MIC of 0.25-1 microg/ml (reduced susceptibility) and nalidixic acid MICs > 32 microg (resistant). Out of all Salmonella isolates, 24 (75%) were found to be MDR, and all were S. typbi. Low-level quinolone-resistance in typhoid was high in this small series. Screening for nalidixic acid resistance with a 30 microg nalidixic acid disk is a reliable and cost-effective method to detect low-level fluoroquinolone resistance, especially in the developing countries. (author)

  3. Drug Targets and Mechanisms of Resistance in the Anaerobic Protozoa

    Science.gov (United States)

    Upcroft, Peter; Upcroft, Jacqueline A.

    2001-01-01

    The anaerobic protozoa Giardia duodenalis, Trichomonas vaginalis, and Entamoeba histolytica infect up to a billion people each year. G. duodenalis and E. histolytica are primarily pathogens of the intestinal tract, although E. histolytica can form abscesses and invade other organs, where it can be fatal if left untreated. T. vaginalis infection is a sexually transmitted infection causing vaginitis and acute inflammatory disease of the genital mucosa. T. vaginalis has also been reported in the urinary tract, fallopian tubes, and pelvis and can cause pneumonia, bronchitis, and oral lesions. Respiratory infections can be acquired perinatally. T. vaginalis infections have been associated with preterm delivery, low birth weight, and increased mortality as well as predisposing to human immunodeficiency virus infection, AIDS, and cervical cancer. All three organisms lack mitochondria and are susceptible to the nitroimidazole metronidazole because of similar low-redox-potential anaerobic metabolic pathways. Resistance to metronidazole and other drugs has been observed clinically and in the laboratory. Laboratory studies have identified the enzyme that activates metronidazole, pyruvate:ferredoxin oxidoreductase, to its nitroso form and distinct mechanisms of decreasing drug susceptibility that are induced in each organism. Although the nitroimidazoles have been the drug family of choice for treating the anaerobic protozoa, G. duodenalis is less susceptible to other antiparasitic drugs, such as furazolidone, albendazole, and quinacrine. Resistance has been demonstrated for each agent, and the mechanism of resistance has been investigated. Metronidazole resistance in T. vaginalis is well documented, and the principal mechanisms have been defined. Bypass metabolism, such as alternative oxidoreductases, have been discovered in both organisms. Aerobic versus anaerobic resistance in T. vaginalis is discussed. Mechanisms of metronidazole resistance in E. histolytica have recently

  4. Genotypic drug resistance and long-term mortality in patients with triple-class antiretroviral drug failure

    DEFF Research Database (Denmark)

    Lohse, Nicolai; Jørgensen, LB; Kronborg, G

    2007-01-01

    OBJECTIVE: To examine the prevalence of drug-resistance-associated mutations in HIV patients with triple-drug class virological failure (TCF) and their association with long-term mortality. DESIGN: Population-based study from the Danish HIV Cohort Study (DHCS). METHODS: We included all patients...... range 2-10), and 81 (61%) patients had mutations conferring resistance towards all three major drug classes. In a regression model adjusted for CD4+ T-cell count, HIV RNA, year of TCF, age, gender and previous inferior antiretroviral therapy, harbouring > or =9 versus ... in the DHCS who experienced TCF between January 1995 and November 2004, and we performed genotypic resistance tests for International AIDS Society (IAS)-USA primary mutations on virus from plasma samples taken around the date of TCF. We computed time to all-cause death from date of TCF. The relative risk...

  5. Assessment of tobramycin RIA for drug monitoring and dosage regimen. Comparison with other assay technics

    International Nuclear Information System (INIS)

    Takahashi, S.; Shinozaki, K.; Tsujino, D.; Ohhara, H.; Tanaka, Y.; Arai, S.; Someya, K.; Sasaki, Y.

    1983-01-01

    Because of wide range of inter-individual difference of pharmacokinetic parameter, importance of monitoring blood concentration of aminoglycoside antibiotics in each patient has been recognized. With the purpose to use for monitoring of serum tobramycin (TOB) levels and for adequate dosage regimen RIA of TOB was evaluated in comparison with other assay technics. Gamma Coat TOB RIA kits (Clinical Assay-Travenol Japan) were used for RIA of TOB. The TOB concentrations in the same samples were also measured by two kinds of enzyme immunoassay (EIA) (EMIT EIA and SLFIA EIA), High Performance Liquid Chromatography (HPLC) and bioassay (BA). RIA of TOB is a useful assay method with high sensitivity and reasonably good precision to be used for drug monitoring and adequate dosage regimen. Modification of the method for rapid assay of a small number of samples will increase the clinical usefulness in individualized drug monitoring

  6. MDM2 Antagonist Nutlin-3a Reverses Mitoxantrone Resistance by Inhibiting Breast Cancer Resistance Protein Mediated Drug Transport

    Science.gov (United States)

    Zhang, Fan; Throm, Stacy L.; Murley, Laura L.; Miller, Laura A.; Zatechka, D. Steven; Guy, R. Kiplin; Kennedy, Rachel; Stewart, Clinton F.

    2011-01-01

    Breast cancer resistance protein (BCRP; ABCG2), a clinical marker for identifying the side population (SP) cancer stem cell subgroup, affects intestinal absorption, brain penetration, hepatobiliary excretion, and multidrug resistance of many anti-cancer drugs. Nutlin-3a is currently under pre-clinical investigation in a variety of solid tumor and leukemia models as a p53 reactivation agent, and has been recently demonstrated to also have p53 independent actions in cancer cells. In the present study, we first report that nutlin-3a can inhibit the efflux function of BCRP. We observed that although the nutlin-3a IC50 did not differ between BCRP over-expressing and vector control cells, nutlin-3a treatment significantly potentiated the cells to treatment with the BCRP substrate mitoxantrone. Combination index calculations suggested synergism between nutlin-3a and mitoxantrone in cell lines over-expressing BCRP. Upon further investigation, it was confirmed that nutlin-3a increased the intracellular accumulation of BCRP substrates such as mitoxantrone and Hoechst 33342 in cells expressing functional BCRP without altering the expression level or localization of BCRP. Interestingly, nutlin-3b, considered virtually “inactive” in disrupting the MDM2/p53 interaction, reversed Hoechst 33342 efflux with the same potency as nutlin-3a. Intracellular accumulation and bi-directional transport studies using MDCKII cells suggested that nutlin-3a is not a substrate of BCRP. Additionally, an ATPase assay using Sf9 insect cell membranes over-expressing wild-type BCRP indicated that nutlin-3a inhibits BCRP ATPase activity in a dose-dependent fashion. In conclusion, our studies demonstrate that nutlin-3a inhibits BCRP efflux function, which consequently reverses BCRP-related drug resistance. PMID:21459080

  7. Drug Resistance to EGFR Inhibitors in Lung Cancer | Office of Cancer Genomics

    Science.gov (United States)

    The discovery of mutations in epidermal growth factor receptor (EGFR) has dramatically changed the treatment of patients with non-small-cell lung cancer (NSCLC), the leading cause of cancer deaths worldwide. EGFR-targeted therapies show considerable promise, but drug resistance has become a substantial issue. We reviewed the literature to provide an overview of the drug resistance to EGFR tyrosine kinase inhibitors (TKIs) in NSCLC. The mechanisms causing primary, acquired and persistent drug resistance to TKIs vary.

  8. Experimental studies on the ecology and evolution of drug-resistant malaria parasites

    OpenAIRE

    Huijben, Silvie

    2010-01-01

    Drug resistance is a serious problem in health care in general, and in malaria treatment in particular, rendering many of our previously considered ‘wonder drugs’ useless. Recently, large sums of money have been allocated for the continuous development of new drugs to replace the failing ones. We seem to be one step behind the evolution of antimalarial resistance; is it possible to get one step ahead? Are interventions which slow down the evolution and spread of drug-resistant ...

  9. Identifying co-targets to fight drug resistance based on a random walk model

    Directory of Open Access Journals (Sweden)

    Chen Liang-Chun

    2012-01-01

    Full Text Available Abstract Background Drug resistance has now posed more severe and emergent threats to human health and infectious disease treatment. However, wet-lab approaches alone to counter drug resistance have so far still achieved limited success due to less knowledge about the underlying mechanisms of drug resistance. Our approach apply a heuristic search algorithm in order to extract active network under drug treatment and use a random walk model to identify potential co-targets for effective antibacterial drugs. Results We use interactome network of Mycobacterium tuberculosis and gene expression data which are treated with two kinds of antibiotic, Isoniazid and Ethionamide as our test data. Our analysis shows that the active drug-treated networks are associated with the trigger of fatty acid metabolism and synthesis and nicotinamide adenine dinucleotide (NADH-related processes and those results are consistent with the recent experimental findings. Efflux pumps processes appear to be the major mechanisms of resistance but SOS response is significantly up-regulation under Isoniazid treatment. We also successfully identify the potential co-targets with literature confirmed evidences which are related to the glycine-rich membrane, adenosine triphosphate energy and cell wall processes. Conclusions With gene expression and interactome data supported, our study points out possible pathways leading to the emergence of drug resistance under drug treatment. We develop a computational workflow for giving new insights to bacterial drug resistance which can be gained by a systematic and global analysis of the bacterial regulation network. Our study also discovers the potential co-targets with good properties in biological and graph theory aspects to overcome the problem of drug resistance.

  10. "A'ole" Drugs! Cultural Practices and Drug Resistance of Rural Hawai'ian Youths

    Science.gov (United States)

    Po'A-Kekuawela, Ka'Ohinani; Okamoto, Scott K.; Nebre, La Risa H.; Helm, Susana; Chin, Coralee I. H.

    2009-01-01

    This qualitative study examined how Native Hawai'ian youths from rural communities utilized cultural practices to promote drug resistance and/or abstinence. Forty-seven students from five different middle schools participated in gender-specific focus groups that focused on the cultural and environmental contexts of drug use for Native Hawai'ian…

  11. Transmission of Drug-Resistant Leprosy in Guinea-Conakry Detected Using Molecular Epidemiological Approaches.

    Science.gov (United States)

    Avanzi, Charlotte; Busso, Philippe; Benjak, Andrej; Loiseau, Chloé; Fomba, Abdoulaye; Doumbia, Glodia; Camara, Idrissa; Lamou, André; Sock, Gouressy; Drame, Tiguidanké; Kodio, Mamadou; Sakho, Fatoumata; Sow, Samba O; Cole, Stewart T; Johnson, Roch Christian

    2016-12-01

    Molecular drug susceptibility testing was performed on skin biopsies from 24 leprosy patients from Guinea-Conakry for the first time. We identified primary drug resistance in 4 cases and a dapsone-resistant cluster caused by the same strain. Primary transmission of drug-resistant Mycobacterium leprae, including a rifampicin-resistant strain, is reported. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

  12. Comparison of Strategies to Overcome Drug Resistance: Learning from Various Kingdoms

    Directory of Open Access Journals (Sweden)

    Hiroshi Ogawara

    2018-06-01

    Full Text Available Drug resistance, especially antibiotic resistance, is a growing threat to human health. To overcome this problem, it is significant to know precisely the mechanisms of drug resistance and/or self-resistance in various kingdoms, from bacteria through plants to animals, once more. This review compares the molecular mechanisms of the resistance against phycotoxins, toxins from marine and terrestrial animals, plants and fungi, and antibiotics. The results reveal that each kingdom possesses the characteristic features. The main mechanisms in each kingdom are transporters/efflux pumps in phycotoxins, mutation and modification of targets and sequestration in marine and terrestrial animal toxins, ABC transporters and sequestration in plant toxins, transporters in fungal toxins, and various or mixed mechanisms in antibiotics. Antibiotic producers in particular make tremendous efforts for avoiding suicide, and are more flexible and adaptable to the changes of environments. With these features in mind, potential alternative strategies to overcome these resistance problems are discussed. This paper will provide clues for solving the issues of drug resistance.

  13. Quadruple-first line drug resistance in Mycobacterium tuberculosis in Vietnam: What can we learn from genes?

    Science.gov (United States)

    Nguyen, Huy Quang; Nguyen, Nhung Viet; Contamin, Lucie; Tran, Thanh Hoa Thi; Vu, Thuong Thi; Nguyen, Hung Van; Nguyen, Ngoc Lan Thi; Nguyen, Son Thai; Dang, Anh Duc; Bañuls, Anne-Laure; Nguyen, Van Anh Thi

    2017-06-01

    In Vietnam, a country with high tuberculosis (137/100.000 population) and multidrug-resistant (MDR)-TB burdens (7.8/100.000 population), little is known about the molecular signatures of drug resistance in general and more particularly of second line drug (SLD) resistance. This study is specifically focused on Mycobacterium tuberculosis isolates resistant to four first-line drugs (FLDs) that make TB much more difficult to treat. The aim is to determine the proportion of SLD resistance in these quadruple drug resistant isolates and the genetic determinants linked to drug resistance to better understand the genetic processes leading to quadruple and extremely drug resistance (XDR). 91 quadruple (rifampicin, isoniazid, ethambutol and streptomycin) FLD resistant and 55 susceptible isolates were included. Spoligotyping and 24-locus MIRU-VNTR techniques were performed and 9 genes and promoters linked to FLD and SLD resistance were sequenced. SLD susceptibility testing was carried out on a subsample of isolates. High proportion of quadruple-FLD resistant isolates was resistant to fluoroquinolones (27%) and second-line injectable drugs (30.2%) by drug susceptibility testing. The sequencing revealed high mutation diversity with prevailing mutations at positions katG315, inhA-15, rpoB531, embB306, rrs1401, rpsL43 and gyrA94. The sensitivity and specificity were high for most drug resistances (>86%), but the sensitivity was lower for injectable drug resistances (resistance. Nevertheless, particular mutation patterns linked to high-level resistance and low fitness costs seem to be favored. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Drug membrane interaction and the importance for drug transport, distribution, accumulation, efficacy and resistance.

    Science.gov (United States)

    Seydel, J K; Coats, E A; Cordes, H P; Wiese, M

    1994-10-01

    Some aspects of drug membrane interaction and its influence on drug transport, accumulation, efficacy and resistance have been discussed. The interactions manifest themselves macroscopically in changes in the physical and thermodynamic properties of "pure membranes" or bilayers. As various amounts of foreign molecules enter the membrane, in particular the main gel to liquid crystalline phase transition can be dramatically changed. This may change permeability, cell-fusion, cell resistance and may also lead to changes in conformation of the embedded receptor proteins. Furthermore, specific interactions with lipids may lead to drug accumulation in membranes and thus to much larger concentrations at the active site than present in the surrounding water phase. The lipid environment may also lead to changes in the preferred conformation of drug molecules. These events are directly related to drug efficacy. The determination of essential molecular criteria for the interaction could be used to design new and more selective therapeutics. This excursion in some aspects of drug membrane interaction underlines the importance of lipids and their interaction with drug molecules for our understanding of drug action, but this is not really a new thought but has been formulated in 1884 by THUDICUM: "Phospholipids are the centre, life and chemical soul of all bioplasm whatsoever, that of plants as well as of animals".

  15. Expression of multidrug resistance proteins in retinoblastoma

    Directory of Open Access Journals (Sweden)

    Swati Shukla

    2017-11-01

    Full Text Available AIM: To elucidate the mechanism of multidrug resistance in retinoblastoma, and to acquire more insights into in vivo drug resistance. METHODS: Three anticancer drug resistant Y79 human RB cells were generated against vincristine, etoposide or carboplatin, which are used for conventional chemotherapy in RB. Primary cultures from enucleated eyes after chemotherapy (PCNC were also prepared. Their chemosensitivity to chemotherapeutic agents (vincristine, etoposide and carboplatin were measured using MTT assay. Western blot analysis was performed to evaluate the expression of p53, Bcl-2 and various multidrug resistant proteins in retinoblastoma cells. RESULTS: Following exposure to chemotherapeutic drugs, PCNC showed less sensitivity to drugs. No significant changes observed in the p53 expression, whereas Bcl-2 expression was found to be increased in the drug resistant cells as well as in PCNC. Increased expression of P-glycoprotein (P-gp was observed in drug resistant Y79 cells; however there was no significant change in the expression of P-gp found between primary cultures of primarily enucleated eyes and PCNC. Multidrug resistance protein 1 (Mrp-1 expression was found to be elevated in the drug resistant Y79 cells as well as in PCNC. No significant change in the expression of lung resistance associated protein (Lrp was observed in the drug resistant Y79 cells as well as in PCNC. CONCLUSION: Our results suggest that multidrug resistant proteins are intrinsically present in retinoblastoma which causes treatment failure in managing retinoblastoma with chemotherapy.

  16. Expression of multidrug resistance proteins in retinoblastoma.

    Science.gov (United States)

    Shukla, Swati; Srivastava, Arpna; Kumar, Sunil; Singh, Usha; Goswami, Sandeep; Chawla, Bhavna; Bajaj, Mandeep Singh; Kashyap, Seema; Kaur, Jasbir

    2017-01-01

    To elucidate the mechanism of multidrug resistance in retinoblastoma, and to acquire more insights into in vivo drug resistance. Three anticancer drug resistant Y79 human RB cells were generated against vincristine, etoposide or carboplatin, which are used for conventional chemotherapy in RB. Primary cultures from enucleated eyes after chemotherapy (PCNC) were also prepared. Their chemosensitivity to chemotherapeutic agents (vincristine, etoposide and carboplatin) were measured using MTT assay. Western blot analysis was performed to evaluate the expression of p53, Bcl-2 and various multidrug resistant proteins in retinoblastoma cells. Following exposure to chemotherapeutic drugs, PCNC showed less sensitivity to drugs. No significant changes observed in the p53 expression, whereas Bcl-2 expression was found to be increased in the drug resistant cells as well as in PCNC. Increased expression of P-glycoprotein (P-gp) was observed in drug resistant Y79 cells; however there was no significant change in the expression of P-gp found between primary cultures of primarily enucleated eyes and PCNC. Multidrug resistance protein 1 (Mrp-1) expression was found to be elevated in the drug resistant Y79 cells as well as in PCNC. No significant change in the expression of lung resistance associated protein (Lrp) was observed in the drug resistant Y79 cells as well as in PCNC. Our results suggest that multidrug resistant proteins are intrinsically present in retinoblastoma which causes treatment failure in managing retinoblastoma with chemotherapy.

  17. Expression of multidrug resistance proteins in retinoblastoma

    Science.gov (United States)

    Shukla, Swati; Srivastava, Arpna; Kumar, Sunil; Singh, Usha; Goswami, Sandeep; Chawla, Bhavna; Bajaj, Mandeep Singh; Kashyap, Seema; Kaur, Jasbir

    2017-01-01

    AIM To elucidate the mechanism of multidrug resistance in retinoblastoma, and to acquire more insights into in vivo drug resistance. METHODS Three anticancer drug resistant Y79 human RB cells were generated against vincristine, etoposide or carboplatin, which are used for conventional chemotherapy in RB. Primary cultures from enucleated eyes after chemotherapy (PCNC) were also prepared. Their chemosensitivity to chemotherapeutic agents (vincristine, etoposide and carboplatin) were measured using MTT assay. Western blot analysis was performed to evaluate the expression of p53, Bcl-2 and various multidrug resistant proteins in retinoblastoma cells. RESULTS Following exposure to chemotherapeutic drugs, PCNC showed less sensitivity to drugs. No significant changes observed in the p53 expression, whereas Bcl-2 expression was found to be increased in the drug resistant cells as well as in PCNC. Increased expression of P-glycoprotein (P-gp) was observed in drug resistant Y79 cells; however there was no significant change in the expression of P-gp found between primary cultures of primarily enucleated eyes and PCNC. Multidrug resistance protein 1 (Mrp-1) expression was found to be elevated in the drug resistant Y79 cells as well as in PCNC. No significant change in the expression of lung resistance associated protein (Lrp) was observed in the drug resistant Y79 cells as well as in PCNC. CONCLUSION Our results suggest that multidrug resistant proteins are intrinsically present in retinoblastoma which causes treatment failure in managing retinoblastoma with chemotherapy. PMID:29181307

  18. Assessing the potential impact of artemisinin and partner drug resistance in sub-Saharan Africa.

    Science.gov (United States)

    Slater, Hannah C; Griffin, Jamie T; Ghani, Azra C; Okell, Lucy C

    2016-01-06

    Artemisinin and partner drug resistant malaria parasites have emerged in Southeast Asia. If resistance were to emerge in Africa it could have a devastating impact on malaria-related morbidity and mortality. This study estimates the potential impact of artemisinin and partner drug resistance on disease burden in Africa if it were to emerge. Using data from Asia and Africa, five possible artemisinin and partner drug resistance scenarios are characterized. An individual-based malaria transmission model is used to estimate the impact of each resistance scenario on clinical incidence and parasite prevalence across Africa. Artemisinin resistance is characterized by slow parasite clearance and partner drug resistance is associated with late clinical failure or late parasitological failure. Scenarios with high levels of recrudescent infections resulted in far greater increases in clinical incidence compared to scenarios with high levels of slow parasite clearance. Across Africa, it is estimated that artemisinin and partner drug resistance at levels similar to those observed in Oddar Meanchey province in Cambodia could result in an additional 78 million cases over a 5 year period, a 7% increase in cases compared to a scenario with no resistance. A scenario with high levels of slow clearance but no recrudescence resulted in an additional 10 million additional cases over the same period. Artemisinin resistance is potentially a more pressing concern than partner drug resistance due to the lack of viable alternatives. However, it is predicted that a failing partner drug will result in greater increases in malaria cases and morbidity than would be observed from artemisinin resistance only.

  19. Antituberculosis drug resistance in the south of Vietnam: prevalence and trends

    NARCIS (Netherlands)

    Huong, Nguyen T.; Lan, Nguyen T. N.; Cobelens, Frank G. J.; Duong, Bui D.; Co, Nguyen V.; Bosman, Maarten C.; Kim, Sang-Jae; van Soolingen, Dick; Borgdorff, Martien W.

    2006-01-01

    BACKGROUND: There is limited evidence that the DOTS (directly observed therapy, short course) strategy for tuberculosis (TB) control can contain the emergence and spread of drug resistance in the absence of second-line treatment. We compared drug-resistance levels between 1996 and 2001 in the south

  20. Interplay between Mutations and Efflux in Drug Resistant Clinical Isolates of Mycobacterium tuberculosis

    Directory of Open Access Journals (Sweden)

    Miguel Viveiros

    2017-04-01

    Full Text Available Numerous studies show efflux as a universal bacterial mechanism contributing to antibiotic resistance and also that the activity of the antibiotics subject to efflux can be enhanced by the combined use of efflux inhibitors. Nevertheless, the contribution of efflux to the overall drug resistance levels of clinical isolates of Mycobacterium tuberculosis is poorly understood and still is ignored by many. Here, we evaluated the contribution of drug efflux plus target-gene mutations to the drug resistance levels in clinical isolates of M. tuberculosis. A panel of 17 M. tuberculosis clinical strains were characterized for drug resistance associated mutations and antibiotic profiles in the presence and absence of efflux inhibitors. The correlation between the effect of the efflux inhibitors and the resistance levels was assessed by quantitative drug susceptibility testing. The bacterial growth/survival vs. growth inhibition was analyzed through the comparison between the time of growth in the presence and absence of an inhibitor. For the same mutation conferring antibiotic resistance, different MICs were observed and the different resistance levels found could be reduced by efflux inhibitors. Although susceptibility was not restored, the results demonstrate the existence of a broad-spectrum synergistic interaction between antibiotics and efflux inhibitors. The existence of efflux activity was confirmed by real-time fluorometry. Moreover, the efflux pump genes mmr, mmpL7, Rv1258c, p55, and efpA were shown to be overexpressed in the presence of antibiotics, demonstrating the contribution of these efflux pumps to the overall resistance phenotype of the M. tuberculosis clinical isolates studied, independently of the genotype of the strains. These results showed that the drug resistance levels of multi- and extensively-drug resistant M. tuberculosis clinical strains are a combination between drug efflux and the presence of target-gene mutations, a reality

  1. Radiation response of drug-resistant variants of a human breast cancer cell line

    International Nuclear Information System (INIS)

    Lehnert, S.; Greene, D.; Batist, G.

    1989-01-01

    The radiation response of drug-resistant variants of the human tumor breast cancer cell line MCF-7 has been investigated. Two sublines, one resistant to adriamycin (ADRR) and the other to melphalan (MLNR), have been selected by exposure to stepwise increasing concentrations of the respective drugs. ADRR cells are 200-fold resistant to adriamycin and cross-resistant to a number of other drugs and are characterized by the presence of elevated levels of selenium-dependent glutathione peroxidase and glutathione-S-transferase. MLNR cells are fourfold resistant to melphalan and cross-resistant to some other drugs. The only mechanism of drug resistance established for MLNR cells to date is an enhancement of DNA excision repair processes. While the spectrum of drug resistance and the underlying mechanisms differ for the two sublines, their response to radiation is qualitatively similar. Radiation survival curves for ADRR and MLNR cells differ from that for wild-type cells in a complex manner with, for the linear-quadratic model, a decrease in the size of alpha and an increase in the size of beta. There is a concomitant decrease in the size of the alpha/beta ratio which is greater for ADRR cells than for MLNR cells. Analysis of results using the multitarget model gave values of D0 of 1.48, 1.43, and 1.67 Gy for MCF-7 cells are not a consequence of cell kinetic differences between these sublines. Results of split-dose experiments indicated that for both drug-resistant sublines the extent of sublethal damage repair reflected the width of the shoulder on the single-dose survival curve. For MCF-7 cells in the stationary phase of growth, the drug-resistant sublines did not show cross-resistance to radiation; however, delayed subculture following irradiation of stationary-phase cultures increased survival to a greater extent for ADRR and MLNR cells than for wild-type cells

  2. Primary drug-resistant tuberculosis in Hanoi, Viet Nam: present status and risk factors.

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    Nguyen Thi Le Hang

    Full Text Available INTRODUCTION: Resistance of Mycobacterium tuberculosis (MTB to anti-tuberculosis (TB drugs presents a serious challenge to TB control worldwide. We investigated the status of drug resistance, including multidrug-resistant (MDR TB, and possible risk factors among newly diagnosed TB patients in Hanoi, the capital of Viet Nam. METHODS: Clinical and epidemiological information was collected from 506 newly diagnosed patients with sputum smear- and culture-positive TB, and 489 (96.6% MTB isolates were subjected to conventional drug susceptibility testing, spoligotyping, and 15-locus variable numbers of tandem repeats typing. Adjusted odds ratios (aORs were calculated to analyze the risk factors for primary drug resistance. RESULTS: Of 489 isolates, 298 (60.9% were sensitive to all drugs tested. Resistance to isoniazid, rifampicin, streptomycin, ethambutol, and MDR accounted for 28.2%, 4.9%, 28.2%, 2.9%, and 4.5%, respectively. Of 24 isolates with rifampicin resistance, 22 (91.7% were MDR and also resistant to streptomycin, except one case. Factors associated with isoniazid resistance included living in old urban areas, presence of the Beijing genotype, and clustered strains [aOR = 2.23, 95% confidence interval (CI 1.15-4.35; 1.91, 1.18-3.10; and 1.69, 1.06-2.69, respectively. The Beijing genotype was also associated with streptomycin resistance (aOR = 2.10, 95% CI 1.29-3.40. Human immunodeficiency virus (HIV coinfection was associated with rifampicin resistance and MDR (aOR = 5.42, 95% CI 2.07-14.14; 6.23, 2.34-16.58, respectively. CONCLUSION: Isoniazid and streptomycin resistance was observed in more than a quarter of TB patients without treatment history in Hanoi. Transmission of isoniazid-resistant TB among younger people should be carefully monitored in urban areas, where Beijing strains and HIV coinfection are prevalent. Choosing an optimal treatment regimen on the basis of the results of drug susceptibility tests and monitoring of treatment

  3. Setting priorities for a research agenda to combat drug-resistant tuberculosis in children.

    Science.gov (United States)

    Velayutham, B; Nair, D; Ramalingam, S; Perez-Velez, C M; Becerra, M C; Swaminathan, S

    2015-12-21

    Numerous knowledge gaps hamper the prevention and treatment of childhood drug-resistant tuberculosis (TB). Identifying research priorities is vital to inform and develop strategies to address this neglected problem. To systematically identify and rank research priorities in childhood drug-resistant TB. Adapting the Child Health and Nutrition Research Initiative (CHNRI) methodology, we compiled 53 research questions in four research areas, then classified the questions into three research types. We invited experts in childhood drug-resistant TB to score these questions through an online survey. A total of 81 respondents participated in the survey. The top-ranked research question was to identify the best combination of existing diagnostic tools for early diagnosis. Highly ranked treatment-related questions centred on the reasons for and interventions to improve treatment outcomes, adverse effects of drugs and optimal treatment duration. The prevalence of drug-resistant TB was the highest-ranked question in the epidemiology area. The development type questions that ranked highest focused on interventions for optimal diagnosis, treatment and modalities for treatment delivery. This is the first effort to identify and rank research priorities for childhood drug-resistant TB. The result is a resource to guide research to improve prevention and treatment of drug-resistant TB in children.

  4. Protease Inhibitors Drug Resistance Mutations in Turkish Patients with Chronic Hepatitis C.

    Science.gov (United States)

    Sargin Altunok, Elif; Sayan, Murat; Akhan, Sila; Aygen, Bilgehan; Yildiz, Orhan; Tekin Koruk, Suda; Mistik, Resit; Demirturk, Nese; Ural, Onur; Kose, Şükran; Aynioglu, Aynur; Korkmaz, Fatime; Ersoz, Gülden; Tuna, Nazan; Ayaz, Celal; Karakecili, Faruk; Keten, Derya; Inan, Dilara; Yazici, Saadet; Koculu, Safiye; Yildirmak, Taner

    2016-09-01

    Drug resistance development is an expected problem during treatment with protease inhibitors (PIs), this is largely due to the fact that Pls are low-genetic barrier drugs. Resistance-associated variants (RAVs) however may also occur naturally, and prior to treatment with Pls, the clinical impact of this basal resistance remains unknown. In Turkey, there is yet to be an investigation into the hepatitis C (HCV) drug associated resistance to oral antivirals. 178 antiviral-naïve patients infected with HCV genotype 1 were selected from 27 clinical centers of various geographical regions in Turkey and included in the current study. The basal NS3 Pls resistance mutations of these patients were analyzed. In 33 (18.5%) of the patients included in the study, at least one mutation pattern that can cause drug resistance was identified. The most frequently detected mutation pattern was T54S while R109K was the second most frequently detected. Following a more general examination of the patients studied, telaprevir (TVR) resistance in 27 patients (15.2%), boceprevir (BOC) resistance in 26 (14.6%) patients, simeprevir (SMV) resistance in 11 (6.2%) patients and faldaprevir resistance in 13 (7.3%) patients were detected. Our investigation also revealed that rebound developed in the presence of a Q80K mutation and amongst two V55A mutations following treatment with TVR, while no response to treatment was detected in a patient with a R55K mutation. We are of the opinion that drug resistance analyses can be beneficial and necessary in revealing which variants are responsible for pre-treatment natural resistance and which mutations are responsible for the viral breakthrough that may develop during the treatment. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  5. Drug Resistance Patterns of Escherichia coli in Ethiopia: A Meta-Analysis.

    Science.gov (United States)

    Tuem, Kald Beshir; Gebre, Abadi Kahsu; Atey, Tesfay Mehari; Bitew, Helen; Yimer, Ebrahim M; Berhe, Derbew Fikadu

    2018-01-01

    Antimicrobial drug resistance is a global threat for treatment of infectious diseases and costs life and money and threatens health delivery system's effectiveness. The resistance of E. coli to frequently utilized antimicrobial drugs is becoming a major challenge in Ethiopia. However, there is no inclusive countrywide study. Therefore, this study intended to assess the prevalence of E. coli resistance and antimicrobial-specific resistance pattern among E. coli clinical isolates in Ethiopia. Articles were retrieved from PubMed, Embase, and grey literature from 2007 to 2017. The main outcome measures were overall E. coli and drug-specific resistance patterns. A random-effects model was used to determine pooled prevalence with 95% confidence interval (CI), using DerSimonian and Laird method. In addition, subgroup analysis was conducted to improve the outcome. The study bias was assessed by Begg's funnel plot. This study was registered in PROSPERO as follows: PROSPERO 2017: CRD42017070106. Of 164 articles retrieved, 35 articles were included. A total of 19,235 study samples participated in the studies and 2,635 E. coli strains were isolated. Overall, E. coli antibacterial resistance was 45.38% (95% confidence interval (CI): 33.50 to 57.27). The resistance pattern ranges from 62.55% in Addis Ababa to 27.51% in Tigray region. The highest resistance of E. coli reported was to ampicillin (83.81%) and amoxicillin (75.79%), whereas only 13.55% of E. coli isolates showed resistance to nitrofurantoin. E. coli antimicrobial resistance remains high with disparities observed among regions. The bacterium was found to be highly resistant to aminopenicillins. The finding implies the need for effective prevention strategies for the E. coli drug resistance and calls for multifaceted approaches with full involvement of all stakeholders.

  6. Drug-resistant post-neurosurgical nosocomial Acinetobacter ...

    African Journals Online (AJOL)

    Drug-resistant post-neurosurgical nosocomial Acinetobacter baumannii meningitis in two Iranian hospitals. ... Vol 11, No 17 (2012) >. Log in or Register to get access to full text downloads. ... Acinetobacter baumannii may cause meningitis and ventriculitis, particularly after head trauma and/or neurosurgery. The rate of ...

  7. Cigarette smoke promotes drug resistance and expansion of cancer stem cell-like side population.

    Directory of Open Access Journals (Sweden)

    Yi An

    Full Text Available It is well known that many patients continue to smoke cigarettes after being diagnosed with cancer. Although smoking cessation has typically been presumed to possess little therapeutic value for cancer, a growing body of evidence suggests that continued smoking is associated with reduced efficacy of treatment and a higher incidence of recurrence. We therefore investigated the effect of cigarette smoke condensate (CSC on drug resistance in the lung cancer and head and neck cancer cell lines A549 and UMSCC-10B, respectively. Our results showed that CSC significantly increased the cellular efflux of doxorubicin and mitoxantrone. This was accompanied by membrane localization and increased expression of the multi-drug transporter ABCG2. The induced efflux of doxorubicin was reversed upon addition of the specific ABCG2 inhibitor Fumitremorgin C, confirming the role of ABCG2. Treatment with CSC increased the concentration of phosphorylated Akt, while addition of the PI3K inhibitor LY294002 blocked doxorubicin extrusion, suggesting that Akt activation is required for CSC-induced drug efflux. In addition, CSC was found to promote resistance to doxorubicin as determined by MTS assays. This CSC-induced doxurbicin-resistance was mitigated by mecamylamine, a nicotinic acetylcholine receptor inhibitor, suggesting that nicotine is at least partially responsible for the effect of CSC. Lastly, CSC increased the size of the side population (SP, which has been linked to a cancer stem cell-like phenotype. In summary, CSC promotes chemoresistance via Akt-mediated regulation of ABCG2 activity, and may also increase the proportion of cancer stem-like cells, contributing to tumor resilience. These findings underscore the importance of smoking cessation following a diagnosis of cancer, and elucidate the mechanisms of continued smoking that may be detrimental to treatment.

  8. Challenges of drug resistance in the management of pancreatic cancer.

    LENUS (Irish Health Repository)

    Sheikh, Rizwan

    2012-02-01

    The current treatment of choice for metastatic pancreatic cancer involves single-agent gemcitabine or a combination of gemcitabine with capecitabine or erlotinib (a tyrosine kinase inhibitor). Only 25–30% of patients respond to this treatment and patients who do respond initially ultimately exhibit disease progression. Median survival for pancreatic cancer patients has reached a plateau due to inherent and acquired resistance to these agents. Key molecular factors implicated in this resistance include: deficiencies in drug uptake, alteration of drug targets, activation of DNA repair pathways, resistance to apoptosis and the contribution of the tumor microenvironment. Moreover, for newer agents including tyrosine kinase inhibitors, overexpression of signaling proteins, mutations in kinase domains, activation of alternative pathways, mutations of genes downstream of the target and\\/or amplification of the target represent key challenges for treatment efficacy. Here we will review the contribution of known mechanisms and markers of resistance to key pancreatic cancer drug treatments.

  9. Label-free recognition of drug resistance via impedimetric screening of breast cancer cells.

    Directory of Open Access Journals (Sweden)

    Bilge Eker

    Full Text Available We present a novel study on label-free recognition and distinction of drug resistant breast cancer cells (MCF-7 DOX from their parental cells (MCF-7 WT via impedimetric measurements. Drug resistant cells exhibited significant differences in their dielectric properties compared to wild-type cells, exerting much higher extracellular resistance (Rextra . Immunostaining revealed that MCF-7 DOX cells gained a much denser F-actin network upon acquiring drug resistance indicating that remodeling of actin cytoskeleton is probably the reason behind higher Rextra , providing stronger cell architecture. Moreover, having exposed both cell types to doxorubicin, we were able to distinguish these two phenotypes based on their substantially different drug response. Interestingly, impedimetric measurements identified a concentration-dependent and reversible increase in cell stiffness in the presence of low non-lethal drug doses. Combined with a profound frequency analysis, these findings enabled distinguishing distinct cellular responses during drug exposure within four concentration ranges without using any labeling. Overall, this study highlights the possibility to differentiate drug resistant phenotypes from their parental cells and to assess their drug response by using microelectrodes, offering direct, real-time and noninvasive measurements of cell dependent parameters under drug exposure, hence providing a promising step for personalized medicine applications such as evaluation of the disease progress and optimization of the drug treatment of a patient during chemotherapy.

  10. Prevalence of genotypic HIV-1 drug resistance in Thailand, 2002

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    Watitpun Chotip

    2003-03-01

    Full Text Available Abstract Background The prices of reverse transcriptase (RT inhibitors in Thailand have been reduced since December 1, 2001. It is expected that reduction in the price of these inhibitors may influence the drug resistance mutation pattern of HIV-1 among infected people. This study reports the frequency of HIV-1 genetic mutation associated with drug resistance in antiretroviral-treated patients from Thailand. Methods Genotypic resistance testing was performed on samples collected in 2002 from 88 HIV-1 infected individuals. Automated DNA sequencing was used to genotype the HIV-1 polymerase gene isolated from patients' plasma. Results Resistance to protease inhibitors, nucleoside and non-nucleoside reverse transcriptase inhibitors were found in 10 (12%, 42 (48% and 19 (21% patients, respectively. The most common drug resistance mutations in the protease gene were at codon 82 (8%, 90 (7% and 54 (6%, whereas resistant mutations at codon 215 (45%, 67 (40%, 41 (38% and 184 (27% were commonly found in the RT gene. This finding indicates that genotypic resistance to nucleoside reverse transcriptase inhibitors was prevalent in 2002. The frequency of resistant mutations corresponding to non-nucleoside reverse transcriptase inhibitors was three times higher-, while resistant mutation corresponding to protease inhibitors was two times lower than those frequencies determined in 2001. Conclusion This study shows that the frequencies of RT inhibitor resistance mutations have been increased after the reduction in the price of RT inhibitors since December 2001. We believe that this was an important factor that influenced the mutation patterns of HIV-1 protease and RT genes in Thailand.

  11. Analysis of metal and biocides resistance genes in drug resistance and susceptible Salmonella enterica from food animals

    Science.gov (United States)

    Background Generally drug resistant bacteria carry antibiotic resistance genes and heavy metal and biocide resistance genes on large conjugative plasmids. The presence of these metal and biocide resistance genes in susceptible bacteria are not assessed comprehensively. Hence, WGS data of susceptib...

  12. Prevalence of transmitted drug resistance and impact of transmitted resistance on treatment success in the German HIV-1 Seroconverter Cohort.

    Directory of Open Access Journals (Sweden)

    Barbara Bartmeyer

    Full Text Available BACKGROUND: The aim of this study is to analyse the prevalence of transmitted drug resistance, TDR, and the impact of TDR on treatment success in the German HIV-1 Seroconverter Cohort. METHODS: Genotypic resistance analysis was performed in treatment-naïve study patients whose sample was available 1,312/1,564 (83.9% October 2008. A genotypic resistance result was obtained for 1,276/1,312 (97.3%. The resistance associated mutations were identified according to the surveillance drug resistance mutations list recommended for drug-naïve patients. Treatment success was determined as viral suppression below 500 copies/ml. RESULTS: Prevalence of TDR was stable at a high level between 1996 and 2007 in the German HIV-1 Seroconverter Cohort (N = 158/1,276; 12.4%; CI(wilson 10.7-14.3; p(for trend = 0.25. NRTI resistance was predominant (7.5% but decreased significantly over time (CI(Wilson: 6.2-9.1, p(for trend = 0.02. NNRTI resistance tended to increase over time (NNRTI: 3.5%; CI(Wilson: 2.6-4.6; p(for trend= 0.07, whereas PI resistance remained stable (PI: 3.0%; CI(Wilson: 2.1-4.0; p(for trend = 0.24. Resistance to all drug classes was frequently caused by singleton resistance mutations (NRTI 55.6%, PI 68.4%, NNRTI 99.1%. The majority of NRTI-resistant strains (79.8% carried resistance-associated mutations selected by the thymidine analogues zidovudine and stavudine. Preferably 2NRTI/1PIr combinations were prescribed as first line regimen in patients with resistant HIV as well as in patients with susceptible strains (susceptible 45.3%; 173/382 vs. resistant 65.5%; 40/61. The majority of patients in both groups were treated successfully within the first year after ART-initiation (susceptible: 89.9%; 62/69; resistant: 7/9; 77.8%. CONCLUSION: Overall prevalence of TDR remained stable at a high level but trends of resistance against drug classes differed over time. The significant decrease of NRTI-resistance in patients newly infected

  13. Managing anthelmintic resistance-Variability in the dose of drug reaching the target worms influences selection for resistance?

    Science.gov (United States)

    Leathwick, Dave M; Luo, Dongwen

    2017-08-30

    The concentration profile of anthelmintic reaching the target worms in the host can vary between animals even when administered doses are tailored to individual liveweight at the manufacturer's recommended rate. Factors contributing to variation in drug concentration include weather, breed of animal, formulation and the route by which drugs are administered. The implications of this variability for the development of anthelmintic resistance was investigated using Monte-Carlo simulation. A model framework was established where 100 animals each received a single drug treatment. The 'dose' of drug allocated to each animal (i.e. the concentration-time profile of drug reaching the target worms) was sampled at random from a distribution of doses with mean m and standard deviation s. For each animal the dose of drug was used in conjunction with pre-determined dose-response relationships, representing single and poly-genetic inheritance, to calculate efficacy against susceptible and resistant genotypes. These data were then used to calculate the overall change in resistance gene frequency for the worm population as a result of the treatment. Values for m and s were varied to reflect differences in both mean dose and the variability in dose, and for each combination of these 100,000 simulations were run. The resistance gene frequency in the population after treatment increased as m decreased and as s increased. This occurred for both single and poly-gene models and for different levels of dominance (survival under treatment) of the heterozygote genotype(s). The results indicate that factors which result in lower and/or more variable concentrations of active reaching the target worms are more likely to select for resistance. The potential of different routes of anthelmintic administration to play a role in the development of anthelmintic resistance is discussed. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Multi drug resistance to cancer chemotherapy: Genes involved and blockers

    International Nuclear Information System (INIS)

    Sayed-Ahmed, Mohamed M.

    2007-01-01

    During the last three decades, important and considerable research efforts had been performed to investigate the mechanism through which cancer cells overcome the cytotoxic effects of a variety of chemotherapeutic drugs. Most of the previously published work has been focused on the resistance of tumor cells to those anticancer drugs of natural source. Multidrug resistance (MDR) is a cellular cross-resistance to a broad spectrum of natural products used in cancer chemotherapy and is believed to be the major cause of the therapeutic failures of the drugs belonging to different naturally obtained or semisynthetic groups including vinca alkaloids, taxans, epipodophyllotoxins and certain antibiotics. This phenomenon results from overexpression of four MDR genes and their corresponding proteins that act as membrane-bound ATP consuming pumps. These proteins mediate the efflux of many structurally and functionally unrelated anticancer drugs of natural source. MDR may be intrinsic or acquired following exposure to chemotherapy. The existence of intrinsically resistant tumor cell clone before and following chemotherapeutic treatment has been associated with a worse final outcome because of increased incidence of distant metasis. In view of irreplaceability of natural product anticancer drugs as effective chemotherapeutic agents, and in view of MDR as a major obstacle to successful chemotherapy, this review is aimed to highlight the genes involved in MDR, classical MDR blockers and gene therapy approaches to overcome MDR. (author)

  15. Perinatal acquisition of drug-resistant HIV-1 infection: mechanisms and long-term outcome

    Directory of Open Access Journals (Sweden)

    Dollfus Catherine

    2009-09-01

    Full Text Available Abstract Background Primary-HIV-1-infection in newborns that occurs under antiretroviral prophylaxis that is a high risk of drug-resistance acquisition. We examine the frequency and the mechanisms of resistance acquisition at the time of infection in newborns. Patients and Methods We studied HIV-1-infected infants born between 01 January 1997 and 31 December 2004 and enrolled in the ANRS-EPF cohort. HIV-1-RNA and HIV-1-DNA samples obtained perinatally from the newborn and mother were subjected to population-based and clonal analyses of drug resistance. If positive, serial samples were obtained from the child for resistance testing. Results Ninety-two HIV-1-infected infants were born during the study period. Samples were obtained from 32 mother-child pairs and from another 28 newborns. Drug resistance was detected in 12 newborns (20%: drug resistance to nucleoside reverse transcriptase inhibitors was seen in 10 cases, non-nucleoside reverse transcriptase inhibitors in two cases, and protease inhibitors in one case. For 9 children, the detection of the same resistance mutations in mothers' samples (6 among 10 available and in newborn lymphocytes (6/8 suggests that the newborn was initially infected by a drug-resistant strain. Resistance variants were either transmitted from mother-to-child or selected during subsequent temporal exposure under suboptimal perinatal prophylaxis. Follow-up studies of the infants showed that the resistance pattern remained stable over time, regardless of antiretroviral therapy, suggesting the early cellular archiving of resistant viruses. The absence of resistance in the mother of the other three children (3/10 and neonatal lymphocytes (2/8 suggests that the newborns were infected by a wild-type strain without long-term persistence of resistance when suboptimal prophylaxis was stopped. Conclusion This study confirms the importance of early resistance genotyping of HIV-1-infected newborns. In most cases (75%, drug

  16. Motility in the L3 stage is a poor phenotype for detecting and measuring resistance to avermectin/milbemycin drugs in gastrointestinal nematodes of livestock

    Directory of Open Access Journals (Sweden)

    Melissa M. George

    2018-04-01

    Full Text Available Motility is a commonly used in vitro phenotype for assessing anthelmintic activity of candidate compounds, and for detecting anthelmintic resistance in nematodes. Third-stage larvae (L3 of parasitic nematodes are commonly used in motility-based assays because L3 are simple to obtain and can remain viable in storage for extended periods. To improve the measurement of motility of microscopic stages of nematodes, our laboratory developed the Worminator, which quantitatively measures motility of parasites. Using the Worminator, we compared the dose-response characteristics of several avermectin/milbemycin (AM compounds using L3 from both AM-susceptible and AM-resistant Cooperia spp. (abamectin, doramectin, eprinomectin, ivermectin, moxidectin and Haemonchus contortus (eprinomectin, ivermectin, moxidectin. Concentrations tested with the Worminator ranged from 0.156 to 40 μM. Differences in EC50 between AM-susceptible and AM-resistant isolates of Cooperia spp. and Haemonchus contortus were small, with resistance ratios ranging from 1.00 to 1.34 for Cooperia spp., 0.99 to 1.65 for Haemonchus contortus. Larval migration inhibition assays were conducted using the same isolates and were equally ineffective for detection of resistance with resistance ratios less than 2.0. These results contrast with those of the Larval Development Assay where we obtained a resistance ratio of 16.48 using the same isolates of Haemonchus contortus. Moreover, even at the highest concentration tested (40 μM, 100% inhibition of motility was never achieved and EC50 for Worminator assays were more than 100× higher than peak plasma levels achieved in vivo following treatment. These data demonstrate that dose-response characteristics for inhibition of motility in L3 of gastrointestinal nematodes of livestock do not significantly differ for AM-susceptible and AM-resistant isolates. These data challenge the suitability of motility as a phenotype for detecting and measuring

  17. Impedimetric toxicity assay in microfluidics using free and liposome-encapsulated anticancer drugs

    DEFF Research Database (Denmark)

    Caviglia, Claudia; Zor, Kinga; Montini, Lucia

    2015-01-01

    In this work, we have developed a microfluidic cytotoxicity assay for a cell culture and detection platform, which enables both fluid handling and electrochemical/optical detection. The cytotoxic effect of anticancer drugs doxorubicin (DOX), oxaliplatin (OX) as well as OX-loaded liposomes, develo...

  18. Characterization of a cadmium resistance Lactococcus lactis subsp. lactis strain by antioxidant assays and proteome profiles methods.

    Science.gov (United States)

    Sheng, Yao; Yang, Xuan; Lian, Yuanyuan; Zhang, Boyang; He, Xiaoyun; Xu, Wentao; Huang, Kunlun

    2016-09-01

    Heavy metal contamination poses a major threat to the environment and human health for their potential toxicity and non-biodegradable properties. At present, some probiotics bacteria are reported to have great potential to eliminate heavy metals from food and water. In this study, resistance properties of a newly isolated Lactococcus lactis subsp. lactis for cadmium were studied by antioxidant assays and proteomics analysis. Antioxidant capacity of this strain was significantly activated under cadmium stress indicated by Fenton reaction, DPPH assay, SOD assay and GSH assay. Intracellular antioxidant enzyme systems, such as superoxide dismutase, glutathione reductase and catalase were suggested to play vital roles in the activated antioxidant capacity. The up-regulated cadA was associated with the activated P-type ATPases that plays an important role in cadmium resistance. Proteomics analysis identified 12 over-expressed proteins under 50mg/L cadmium stress and these proteins are abundant in oxidative stress response and energy metabolism regulation, which were considered as consequences as cadmium resistance of the strain. Thus, the probiotics Lactococcus lactis subsp. lactis may resist cadmium stress through antioxidant approach and enhanced energy metabolism. The food grade lactis strain may be applied in metal decontamination in environment and food/feed. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Genome Analysis of the First Extensively Drug-Resistant (XDR Mycobacterium tuberculosis in Malaysia Provides Insights into the Genetic Basis of Its Biology and Drug Resistance.

    Directory of Open Access Journals (Sweden)

    Chee Sian Kuan

    Full Text Available The outbreak of extensively drug-resistant tuberculosis (XDR-TB has become an increasing problem in many TB-burdened countries. The underlying drug resistance mechanisms, including the genetic variation favored by selective pressure in the resistant population, are partially understood. Recently, the first case of XDR-TB was reported in Malaysia. However, the detailed genotype family and mechanisms of the formation of multiple drugs resistance are unknown. We sequenced the whole genome of the UM 1072388579 strain with a 2-kb insert-size library and combined with that from previously sequenced 500-bp-insert paired-end reads to produce an improved sequence with maximal sequencing coverage across the genome. In silico spoligotyping and phylogenetic analyses demonstrated that UM 1072388579 strain belongs to an ancestral-like, non-Beijing clade of East Asia lineage. This is supported by the presence of a number of lineage-specific markers, including fadD28, embA, nuoD and pks7. Polymorphism analysis showed that the drug-susceptibility profile is correlated with the pattern of resistance mutations. Mutations in drug-efflux pumps and the cell wall biogenesis pathway such as mmpL, pks and fadD genes may play an important role in survival and adaptation of this strain to its surrounding environment. In this work, fifty-seven putative promoter SNPs were identified. Among them, we identified a novel SNP located at -4 T allele of TetR/acrR promoter as an informative marker to recognize strains of East Asian lineage. Our work indicates that the UM 1072388579 harbors both classical and uncommon SNPs that allow it to escape from inhibition by many antibiotics. This study provides a strong foundation to dissect the biology and underlying resistance mechanisms of the first reported XDR M. tuberculosis in Malaysia.

  20. Effect and Safety of Shihogyejitang for Drug Resistant Childhood Epilepsy

    Directory of Open Access Journals (Sweden)

    Jinsoo Lee

    2016-01-01

    Full Text Available Objective. Herbal medicine has been widely used to treat drug resistant epilepsy. Shihogyejitang (SGT has been commonly used to treat epilepsy. We investigated the effect and safety of SGT in children with drug resistant epilepsy. Design. We reviewed medical records of 54 patients with epilepsy, who failed to respond to at least two antiepileptic drugs and have been treated with SGT between April 2006 and June 2014 at the Department of Pediatric Neurology, I-Tomato Hospital, Korea. Effect was measured by the response rate, seizure-free rate, and retention rate at six months. We also checked adverse events, change in antiepileptic drugs use, and the variables related to the outcome. Results. Intent-to-treat analysis showed that, after six months, 44.4% showed a >50% seizure reduction, 24.1% including seizure-free, respectively, and 53.7% remained on SGT. Two adverse events were reported, mild skin rash and fever. Focal seizure type presented significantly more positive responses when compared with other seizure types at six months (p=0.0284, Fisher’s exact test. Conclusion. SGT is an effective treatment with excellent tolerability for drug resistant epilepsy patients. Our data provide evidence that SGT may be used as alternative treatment option when antiepileptic drug does not work in epilepsy children.

  1. Pathogenicity Assay of Vibrio harveyi in Tiger Shrimp Larvae Employing Rifampicin-Resistant as A Molecular Marker

    Directory of Open Access Journals (Sweden)

    . Widanarni

    2007-12-01

    Full Text Available Rifampicin-resistant marker was employed as a reporter to assay pathogenicity of Vibrio harveyi  in shrimp larvae.  V. harveyi M. G3 and G7 that difference not schizotyping as shown by Pulsed-Filed Gel Electrophoresis (PFGE used in this study. Spontaneous mutation was conducted to generate V. harveyi resistant to rifampicin. Two groups of shrimp post-larvae (PL5 were immersed for 30 min in 106 CFU/ml of mutants and wild type of V. harveyi, respectively; and then placed in a 2 liter shrimp rearing tank for five days. A control group was immersed in sterile seawater. Growth curve analysis and pathogenicity assay of V. harveyi  showed that each of the V. harveyi mutant exhibited almost identical profiles to that of the wild type parental strain and did not show alteration in their pathogenicity. Sample from dead shrimp larvae showed that the dead shrimp larvae were infected by V. harveyi RfR, indicated that rifampicin-resistant marker effective as a reporter to assay pathogenicity of Vibrio harveyi in shrimp larvae. Key words: shrimp larvae, Vibrio harveyi, rifampicin-resistant, molecular marker

  2. Novel diagnostics and therapeutics for drug-resistant tuberculosis.

    Science.gov (United States)

    Toosky, Melody; Javid, Babak

    2014-06-01

    Drug-resistant tuberculosis (DR-TB) is associated with increased mortality and morbidity. This is at least partly due to late diagnosis and ineffective treatment of drug-resistant status. Selective search of the literature on DR-TB supplemented by recent guidelines from the World Health Organization. Better and more rapid diagnosis of DR-TB by new techniques such as Xpert Mtb/RIF are likely to make a substantial impact on the disease. New therapeutics for DR-TB are entering, or about to enter the market for the first time in decades. It is not clear whether new treatments should be restricted for DR-TB or also used for drug-susceptible tuberculosis. With several new agents on the horizon, there is the real possibility of an entirely new regimen for tuberculosis. An inexpensive 'near-patient' diagnostic test is still needed. Optimizing new drug combination regimens in a timely manner is urgently required. © The Author 2014. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  3. Effects of combinations of chemotherapy and radiation on the emergence of drug resistant cells in 9L rat brain tumor spheroids

    International Nuclear Information System (INIS)

    Tofilon, P.J.; Arundel, C.; Vines, C.M.

    1987-01-01

    Repeated administration of antineoplastic chemotherapeutic agents is generally considered to induce and/or select for drug resistant cells. The authors recently begun to investigate whether chemotherapy interdigitated with radiation can minimize or eliminate the emergence of drug resiistent cells in 9L rat brain tumor spheroids grown from defined mixtures of cells sensitive (9L) and resistant (R/sub 3/) to BCNU. In this experimental system, the sister chromatid exchange (SCE) assay is used to quantitate the proportions of sensitive and resistant cells within the spheroids. While 9L and R/sub 3/ cell have different sensitivities to BCNU, they are equally sensitive to radiation. Mixed-cell spheroids consisting of 1% R/sub 3/ cells were treated with three doses of BCNU (10 μM) every 72 hr resulting in a shift in the 9L to R/sub 3/ ratio to greater than 50% R/sub 3/ cells. The combined protocols to be investigated will involve γ rays administered either 36 hr before or after each BCNU treatment. By initiating these combined protocols on spheroids of different sizes, the effectiveness of each protocol is evaluated with respect to the number of resistant cells present

  4. Molecular mechanisms and theranostic potential of miRNAs in drug resistance of gastric cancer.

    Science.gov (United States)

    Yang, Wanli; Ma, Jiaojiao; Zhou, Wei; Cao, Bo; Zhou, Xin; Yang, Zhiping; Zhang, Hongwei; Zhao, Qingchuan; Fan, Daiming; Hong, Liu

    2017-11-01

    Systemic chemotherapy is a curative approach to inhibit gastric cancer cells proliferation. Despite the great progress in anti-cancer treatment achieved during the last decades, drug resistance and treatment refractoriness still extensively persists. Recently, accumulating studies have highlighted the role of miRNAs in drug resistance of gastric cancers by modulating some drug resistance-related proteins and genes expression. Pre-clinical reports indicate that miRNAs might serve as ideal biomarkers and potential targets, thus holding great promise for developing targeted therapy and personalized treatment for the patients with gastric cancer. Areas covered: This review provide a comprehensive overview of the current advances of miRNAs and molecular mechanisms underlying miRNA-mediated drug resistance in gastric cancer. We particularly focus on the potential values of drug resistance-related miRNAs as biomarkers and novel targets in gastric cancer therapy and envisage the future research developments of these miRNAs and challenges in translating the new findings into clinical applications. Expert opinion: Although the concrete mechanisms of miRNAs in drug resistance of gastric cancer have not been fully clarified, miRNA may be a promising theranostic approach. Further studies are still needed to facilitate the clinical applications of miRNAs in drug resistant gastric cancer.

  5. [Morphological signs of inflammatory activity in different clinical forms of drug-resistant pulmonary tuberculosis].

    Science.gov (United States)

    Elipashev, A A; Nikolsky, V O; Shprykov, A S

    to determine whether the activity of tuberculous inflammation is associated with different clinical forms of drug-resistant pulmonary tuberculosis. The material taken from 310 patients operated on in 2010-2015 were retrospectively examined. The patients underwent economical lung resections of limited extent (typical and atypical ones of up to 3 segments) for circumscribed forms of tuberculosis with bacterial excretion. A study group consisted of 161 (51.9%) patients with drug-resistant variants of pulmonary tuberculosis. A control group included 149 (48.1%) patients with preserved susceptibility of Mycobacterium tuberculosis to anti-TB drugs. The activity of specific changes in tuberculosis was morphologically evaluated in accordance with the classification proposed by B.M. Ariel in 1998. The highest activity of fourth-to-fifth degree specific inflammation, including that outside the primary involvement focus, was obtained in the drug-resistant pulmonary tuberculosis group due to the predominance of patients with cavernous and fibrous-cavernous tuberculosis versus those in whom the susceptibility to chemotherapeutic agents was preserved. A macroscopic study showed that the primary lesion focus had a median size in one-half of the all the examinees; but large tuberculomas, caverns, and fibrous caverns over 4 cm in diameter were multiple and detected in the drug-resistant pulmonary tuberculosis group. Multidrug resistance was observed in more than 60% of the patients with fibrous-cavernous pulmonary tuberculosis, extensive drug resistance was seen in those with cavernous tuberculosis, which is an aggravating factor. The data obtained from the morphological study of the intraoperative material can specify the clinical form of tuberculosis and evaluate the efficiency of preoperative specific therapy. The highest activity of specific inflammation was observed in patients with multiple drug-resistant pulmonary tuberculosis, the prevalence of third-to-fourth degree

  6. Phenotype, Genotype, and Drug Resistance in Subtype C HIV-1 Infection.

    Science.gov (United States)

    Derache, Anne; Wallis, Carole L; Vardhanabhuti, Saran; Bartlett, John; Kumarasamy, Nagalingeswaran; Katzenstein, David

    2016-01-15

    Virologic failure in subtype C is characterized by high resistance to first-line antiretroviral (ARV) drugs, including efavirenz, nevirapine, and lamivudine, with nucleoside resistance including type 2 thymidine analog mutations, K65R, a T69del, and M184V. However, genotypic algorithms predicting resistance are mainly based on subtype B viruses and may under- or overestimate drug resistance in non-B subtypes. To explore potential treatment strategies after first-line failure, we compared genotypic and phenotypic susceptibility of subtype C human immunodeficiency virus 1 (HIV-1) following first-line ARV failure. AIDS Clinical Trials Group 5230 evaluated patients failing an initial nonnucleoside reverse-transcriptase inhibitor (NNRTI) regimen in Africa and Asia, comparing the genotypic drug resistance and phenotypic profile from the PhenoSense (Monogram). Site-directed mutagenesis studies of K65R and T69del assessed the phenotypic impact of these mutations. Genotypic algorithms overestimated resistance to etravirine and rilpivirine, misclassifying 28% and 32%, respectively. Despite K65R with the T69del in 9 samples, tenofovir retained activity in >60%. Reversion of the K65R increased susceptibility to tenofovir and other nucleosides, while reversion of the T69del showed increased resistance to zidovudine, with little impact on other NRTI. Although genotype and phenotype were largely concordant for first-line drugs, estimates of genotypic resistance to etravirine and rilpivirine may misclassify subtype C isolates compared to phenotype. © The Author 2015. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

  7. Consensus Statement on Research Definitions for Drug-Resistant Tuberculosis in Children

    OpenAIRE

    Seddon, James A.; Perez-Velez, Carlos M.; Schaaf, H. Simon; Furin, Jennifer J.; Marais, Ben J.; Tebruegge, Marc; Detjen, Anne; Hesseling, Anneke C.; Shah, Sarita; Adams, Lisa V.; Starke, Jeffrey R.; Swaminathan, Soumya; Becerra, Mercedes C.

    2013-01-01

    Few children with drug-resistant (DR) tuberculosis (TB) are identified, diagnosed, and given an appropriate treatment. The few studies that have described this vulnerable population have used inconsistent definitions. TheWorld Health Organization (WHO) definitions used for adults with DR-TB and for children with drug-susceptible TB are not always appropriate for children with DR-TB. The Sentinel Project on Pediatric Drug-Resistant Tuberculosis was formed in 2011 as a network of experts and st...

  8. Fitness trade-offs in the evolution of dihydrofolate reductase and drug resistance in Plasmodium falciparum.

    Directory of Open Access Journals (Sweden)

    Marna S Costanzo

    Full Text Available Patterns of emerging drug resistance reflect the underlying adaptive landscapes for specific drugs. In Plasmodium falciparum, the parasite that causes the most serious form of malaria, antifolate drugs inhibit the function of essential enzymes in the folate pathway. However, a handful of mutations in the gene coding for one such enzyme, dihydrofolate reductase, confer drug resistance. Understanding how evolution proceeds from drug susceptibility to drug resistance is critical if new antifolate treatments are to have sustained usefulness.We use a transgenic yeast expression system to build on previous studies that described the adaptive landscape for the antifolate drug pyrimethamine, and we describe the most likely evolutionary trajectories for the evolution of drug resistance to the antifolate chlorcycloguanil. We find that the adaptive landscape for chlorcycloguanil is multi-peaked, not all highly resistant alleles are equally accessible by evolution, and there are both commonalities and differences in adaptive landscapes for chlorcycloguanil and pyrimethamine.Our findings suggest that cross-resistance between drugs targeting the same enzyme reflect the fitness landscapes associated with each particular drug and the position of the genotype on both landscapes. The possible public health implications of these findings are discussed.

  9. Alarming levels of drug-resistant tuberculosis in HIV-infected patients in metropolitan Mumbai, India.

    Science.gov (United States)

    Isaakidis, Petros; Das, Mrinalini; Kumar, Ajay M V; Peskett, Christopher; Khetarpal, Minni; Bamne, Arun; Adsul, Balkrishna; Manglani, Mamta; Sachdeva, Kuldeep Singh; Parmar, Malik; Kanchar, Avinash; Rewari, B B; Deshpande, Alaka; Rodrigues, Camilla; Shetty, Anjali; Rebello, Lorraine; Saranchuk, Peter

    2014-01-01

    Drug-resistant tuberculosis (DR-TB) is a looming threat to tuberculosis control in India. However, no countrywide prevalence data are available. The burden of DR-TB in HIV-co-infected patients is likewise unknown. Undiagnosed and untreated DR-TB among HIV-infected patients is a major cause of mortality and morbidity. We aimed to assess the prevalence of DR-TB (defined as resistance to any anti-TB drug) in patients attending public antiretroviral treatment (ART) centers in greater metropolitan Mumbai, India. A cross-sectional survey was conducted among adults and children ART-center attendees. Smear microscopy, culture and drug-susceptibility-testing (DST) against all first and second-line TB-drugs using phenotypic liquid culture (MGIT) were conducted on all presumptive tuberculosis patients. Analyses were performed to determine DR-TB prevalence and resistance patterns separately for new and previously treated, culture-positive TB-cases. Between March 2013 and January 2014, ART-center attendees were screened during 14135 visits, of whom 1724 had presumptive TB. Of 1724 attendees, 72 (4%) were smear-positive and 202 (12%) had a positive culture for Mycobacterium tuberculosis. Overall DR-TB was diagnosed in 68 (34%, 95% CI: 27%-40%) TB-patients. The proportions of DR-TB were 25% (29/114) and 44% (39/88) among new and previously treated cases respectively. The patterns of DR-TB were: 21% mono-resistant, 12% poly-resistant, 38% multidrug-resistant (MDR-TB), 21% pre-extensively-drug-resistant (MDR-TB plus resistance to either a fluoroquinolone or second-line injectable), 6% extensively drug-resistant (XDR-TB) and 2% extremely drug-resistant TB (XDR-TB plus resistance to any group-IV/V drug). Only previous history of TB was significantly associated with the diagnosis of DR-TB in multivariate models. The burden of DR-TB among HIV-infected patients attending public ART-centers in Mumbai was alarmingly high, likely representing ongoing transmission in the community and

  10. Alarming levels of drug-resistant tuberculosis in HIV-infected patients in metropolitan Mumbai, India.

    Directory of Open Access Journals (Sweden)

    Petros Isaakidis

    Full Text Available BACKGROUND: Drug-resistant tuberculosis (DR-TB is a looming threat to tuberculosis control in India. However, no countrywide prevalence data are available. The burden of DR-TB in HIV-co-infected patients is likewise unknown. Undiagnosed and untreated DR-TB among HIV-infected patients is a major cause of mortality and morbidity. We aimed to assess the prevalence of DR-TB (defined as resistance to any anti-TB drug in patients attending public antiretroviral treatment (ART centers in greater metropolitan Mumbai, India. METHODS: A cross-sectional survey was conducted among adults and children ART-center attendees. Smear microscopy, culture and drug-susceptibility-testing (DST against all first and second-line TB-drugs using phenotypic liquid culture (MGIT were conducted on all presumptive tuberculosis patients. Analyses were performed to determine DR-TB prevalence and resistance patterns separately for new and previously treated, culture-positive TB-cases. RESULTS: Between March 2013 and January 2014, ART-center attendees were screened during 14135 visits, of whom 1724 had presumptive TB. Of 1724 attendees, 72 (4% were smear-positive and 202 (12% had a positive culture for Mycobacterium tuberculosis. Overall DR-TB was diagnosed in 68 (34%, 95% CI: 27%-40% TB-patients. The proportions of DR-TB were 25% (29/114 and 44% (39/88 among new and previously treated cases respectively. The patterns of DR-TB were: 21% mono-resistant, 12% poly-resistant, 38% multidrug-resistant (MDR-TB, 21% pre-extensively-drug-resistant (MDR-TB plus resistance to either a fluoroquinolone or second-line injectable, 6% extensively drug-resistant (XDR-TB and 2% extremely drug-resistant TB (XDR-TB plus resistance to any group-IV/V drug. Only previous history of TB was significantly associated with the diagnosis of DR-TB in multivariate models. CONCLUSION: The burden of DR-TB among HIV-infected patients attending public ART-centers in Mumbai was alarmingly high, likely representing

  11. Drug resistance pattern of M. tuberculosis in category II treatment failure pulmonary tuberculosis patients

    Directory of Open Access Journals (Sweden)

    Fahmida Rahman

    2013-01-01

    Full Text Available This study was designed to determine the extent of drug resistance of M. tuberculosis (MTB isolated from category II treatment failure pulmonary tuberculosis (PTB patients. A total of 100 Ziehl-Neelsen (Z-N smear positive category II failure PTB patients were included in this study. Sputum culture was done in Lowenstein-Jensen (L-J media. Conventional proportion method on Lowenstein-Jensen (L-J media was used to determine the drug susceptibility of M. tuberculosis to isoniazid (INH, rifampicin (RMP, ofloxacin (OFX and kanamycin (KA. Out of 100 sputum samples, a total of 87 samples were positive by culture. Drug susceptibility test (DST revealed that 82 (94.25% isolates were resistant to one or more anti -TB drugs. Resistance to isoniazide (INH, rifampicin (RMP, ofloxacin (OFX and kanamycin (KA was 94.25%, 82.75%, 29.90% and 3.45% respectively. Among these isolates, 79.31% and 3.45% isolates were multi-drug resistant (MDR and extended drug resistant (XDR M. tuberculosis respectively. High rate of anti-tubercular drug resistance was observed among the category II treatment failure TB patients. Ibrahim Med. Coll. J. 2013; 7(1: 9-11

  12. The role of miRNA regulation in cancer progression and drug resistance

    DEFF Research Database (Denmark)

    Joshi, Tejal

    RNAs in the context of cancer biology, drug resistance and disease progression. The first project described in Chapter 6 addresses the problem of tamoxifen resistance, an anti-estrogen drug that is generally highly effective in the treatment of ER-positive breast cancers. The underlying molecular mechanisms...... to the disease transformation. In summary, this thesis focuses on regulatory role of miRNAs in drug resistance and disease progression. The findings provide hints toward various biologically and perhaps therapeutically relevant gene regulatory events. This thesis demonstrates the right choice of data analysis...... for the acquired resistance to tamoxifen are not very well understood. Therefore, with the aid of miRNA and gene expression profiles for MCF7/S0.5 (tamoxifen sensitive) and three MCF7/S0.5 derived tamoxifen resistant cell lines, we obtained several miRNA-mediated regulatory events in the tamoxifen resistant cell...

  13. Clinical characteristics, drug resistance, and treatment outcomes among tuberculosis patients with diabetes in Peru.

    Science.gov (United States)

    Magee, M J; Bloss, E; Shin, S S; Contreras, C; Huaman, H Arbanil; Ticona, J Calderon; Bayona, J; Bonilla, C; Yagui, M; Jave, O; Cegielski, J P

    2013-06-01

    Diabetes is a risk factor for active tuberculosis (TB). Data are limited regarding the association between diabetes and TB drug resistance and treatment outcomes. We examined characteristics of TB patients with and without diabetes in a Peruvian cohort at high risk for drug-resistant TB. Among TB patients with diabetes (TB-DM), we studied the association between diabetes clinical/management characteristics and TB drug resistance and treatment outcomes. During 2005-2008, adults with suspected TB with respiratory symptoms in Lima, Peru, who received rapid drug susceptibility testing (DST), were prospectively enrolled and followed during treatment. Bivariate and Kaplan-Meier analyses were used to examine the relationships of diabetes characteristics with drug-resistant TB and TB outcomes. Of 1671 adult TB patients enrolled, 186 (11.1%) had diabetes. TB-DM patients were significantly more likely than TB patients without diabetes to be older, have had no previous TB treatment, and to have a body mass index (BMI) >18.5 kg/m(2) (pdiabetes, and 12% and 28%, respectively, among TB-DM patients. Among 149 TB-DM patients with DST results, 104 (69.8%) had drug-susceptible TB and 45 (30.2%) had drug-resistant TB, of whom 29 had multidrug-resistant TB. There was no association between diabetes characteristics and drug-resistant TB. Of 136 TB-DM patients with outcome information, 107 (78.7%) had a favorable TB outcome; active diabetes management was associated with a favorable outcome. Diabetes was common in a cohort of TB patients at high risk for drug-resistant TB. Despite prevalent multidrug-resistant TB among TB-DM patients, the majority had a favorable TB treatment outcome. Copyright © 2013 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

  14. New Real-Time PCR Assays for Detection of Inducible and Acquired Clarithromycin Resistance in the Mycobacterium abscessus Group.

    Science.gov (United States)

    Shallom, Shamira J; Moura, Natalia S; Olivier, Kenneth N; Sampaio, Elizabeth P; Holland, Steven M; Zelazny, Adrian M

    2015-11-01

    Members of the Mycobacterium abscessus group (MAG) cause lung, soft tissue, and disseminated infections. The oral macrolides clarithromycin and azithromycin are commonly used for treatment. MAG can display clarithromycin resistance through the inducible erm(41) gene or via acquired mutations in the rrl (23S rRNA) gene. Strains harboring a truncation or a T28C substitution in erm(41) lose the inducible resistance trait. Phenotypic detection of clarithromycin resistance requires extended incubation (14 days), highlighting the need for faster methods to detect resistance. Two real-time PCR-based assays were developed to assess inducible and acquired clarithromycin resistance and tested on a total of 90 clinical and reference strains. A SYBR green assay was designed to distinguish between a full-length and truncated erm(41) gene by temperature shift in melting curve analysis. Single nucleotide polymorphism (SNP) allele discrimination assays were developed to distinguish T or C at position 28 of erm(41) and 23S rRNA rrl gene mutations at position 2058 and/or 2059. Truncated and full-size erm(41) genes were detected in 21/90 and 69/90 strains, respectively, with 64/69 displaying T at nucleotide position 28 and 5/69 containing C at that position. Fifteen isolates showed rrl mutations conferring clarithromycin resistance, including A2058G (11 isolates), A2058C (3 isolates), and A2059G (1 isolate). Targeted sequencing and phenotypic assessment of resistance concurred with molecular assay results. Interestingly, we also noted cooccurring strains harboring an active erm(41), inactive erm(41), and/or acquired mutational resistance, as well as slowly growing MAG strains and also strains displaying an inducible resistance phenotype within 5 days, long before the recommended 14-day extended incubation. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  15. Implementation of a national anti-tuberculosis drug resistance survey in Tanzania

    Directory of Open Access Journals (Sweden)

    Mfaume Saidi M

    2008-12-01

    Full Text Available Abstract Background A drug resistance survey is an essential public health management tool for evaluating and improving the performance of National Tuberculosis control programmes. The current manuscript describes the implementation of the first national drug resistance survey in Tanzania. Methods Description of the implementation process of a national anti-tuberculosis drug resistance survey in Tanzania, in relation to the study protocol and Standard Operating Procedures. Results Factors contributing positively to the implementation of the survey were a continuous commitment of the key stakeholders, the existence of a well organized National Tuberculosis Programme, and a detailed design of cluster-specific arrangements for rapid sputum transportation. Factors contributing negatively to the implementation were a long delay between training and actual survey activities, limited monitoring of activities, and an unclear design of the data capture forms leading to difficulties in form-filling. Conclusion Careful preparation of the survey, timing of planned activities, a strong emphasis on data capture tools and data management, and timely supervision are essential for a proper implementation of a national drug resistance survey.

  16. Implementation of a national anti-tuberculosis drug resistance survey in Tanzania.

    Science.gov (United States)

    Chonde, Timothy M; Doulla, Basra; van Leth, Frank; Mfinanga, Sayoki G M; Range, Nyagosya; Lwilla, Fred; Mfaume, Saidi M; van Deun, Armand; Zignol, Matteo; Cobelens, Frank G; Egwaga, Saidi M

    2008-12-30

    A drug resistance survey is an essential public health management tool for evaluating and improving the performance of National Tuberculosis control programmes. The current manuscript describes the implementation of the first national drug resistance survey in Tanzania. Description of the implementation process of a national anti-tuberculosis drug resistance survey in Tanzania, in relation to the study protocol and Standard Operating Procedures. Factors contributing positively to the implementation of the survey were a continuous commitment of the key stakeholders, the existence of a well organized National Tuberculosis Programme, and a detailed design of cluster-specific arrangements for rapid sputum transportation. Factors contributing negatively to the implementation were a long delay between training and actual survey activities, limited monitoring of activities, and an unclear design of the data capture forms leading to difficulties in form-filling. Careful preparation of the survey, timing of planned activities, a strong emphasis on data capture tools and data management, and timely supervision are essential for a proper implementation of a national drug resistance survey.

  17. [Mechanisms of endogenous drug resistance acquisition by spontaneous chromosomal gene mutation].

    Science.gov (United States)

    Fukuda, H; Hiramatsu, K

    1997-05-01

    Endogenous resistance in bacteria is caused by a change or loss of function and generally genetically recessive. However, this type of resistance acquisition are now prevalent in clinical setting. Chromosomal genes that afford endogenous resistance are the genes correlated with the target of the drug, the drug inactivating enzymes, and permeability of the molecules including the antibacterial agents. Endogenous alteration of the drug target are mediated by the spontaneous mutation of their structural gene. This mutation provides much lower affinity of the drugs for the target. Gene expression of the inactivating enzymes, such as class C beta-lactamase, is generally regulated by regulatory genes. Spontaneous mutations in the regulatory genes cause constitutive enzyme production and provides the resistant to the agent which is usually stable for such enzymes. Spontaneous mutation in the structural gene gives the enzyme extra-spectrum substrate specificity, like ESBL (Extra-Spectrum-beta-Lactamase). Expression of structural genes encoding the permeability systems are also regulated by some regulatory genes. The spontaneous mutation of the regulatory genes reduce an amount of porin protein. This mutation causes much lower influx of the drug in the cell. Spontaneous mutation in promoter region of the structural gene of efflux protein was observed. This mutation raised the gene transcription and overproduced efflux protein. This protein progresses the drug efflux from the cell.

  18. Autoradiographic assay of mutants resistant to diphtheria toxin in mammalian cells in vitro

    International Nuclear Information System (INIS)

    Ronen, A.; Gingerich, J.D.; Duncan, A.M.V.; Heddle, J.A.

    1984-01-01

    Diptheria toxin kills mammalian cells by ribosylating elongation factor 2, a protein factor necessary for protein synthesis. The frequency of cells able to form colonies in the presence of the toxin can be used as an assay for mutation to diphtheria toxin resistance. Resistance to diphtheria toxin can also be detected autoradiographically in cells exposed to [ 3 H]leucine after treatment with the toxin. In cultures of Chinese hamster ovary cells, the frequency of such resistant cells is increased by exposure of the cells to γ-rays, ultraviolet light, ethylnitrosourea, mitomycin c, ethidium bromide, and 5-bromo-2'-deoxyuridine in a dose- and time-dependent manner. The resistant cells form discrete microcolonies if they are allowed to divide several times before intoxication which indicates that they are genuine mutants. The assay is potentially adaptable to any cell population that can be intoxicated with diphtheria toxin and labeled with [ 3 H]leucine, whether or not the cells can form colonies. It may be useful, therefore, for measuring mutation rates in slowly growing or nondividing cell populations such as breast, brain, and liver, as well as in cells that do divide but cannot be readily cloned, such as the colonic epithelium. 23 references, 6 figures

  19. Time-programmable drug dosing allows the manipulation, suppression and reversal of antibiotic drug resistance in vitro

    OpenAIRE

    Yoshida, Mari; Galiñanes Reyes, Sabrina Galiñanes; Tsuda, Soichiro; Horinouchi, Takaaki; Furusawa, Chikara; Cronin, Leroy

    2017-01-01

    Multi-drug strategies have been attempted to prolong the efficacy of existing antibiotics, but with limited success. Here we show that the evolution of multi-drug-resistant Escherichia coli can be manipulated in vitro by administering pairs of antibiotics and switching between them in ON/OFF manner. Using a multiplexed cell culture system, we find that switching between certain combinations of antibiotics completely suppresses the development of resistance to one of the antibiotics. Using thi...

  20. An ETP model (exclusion-tolerance-progression for multi drug resistance

    Directory of Open Access Journals (Sweden)

    Kannan Subburaj

    2005-04-01

    Full Text Available Abstract Background It is known that sensitivity or resistance of tumor cells to a given chemotherapeutic agent is an acquired characteristic(s, depending on the heterogeneity of the tumor mass subjected to the treatment. The clinical success of a chemotherapeutic regimen depends on the ratio of sensitive to resistant cell populations. Results Based on findings from clinical and experimental studies, a unifying model is proposed to delineate the potential mechanism by which tumor cells progress towards multi drug resistance, resulting in failure of chemotherapy. Conclusion It is suggested that the evolution of multi drug resistance is a developmentally orchestrated event. Identifying stage-specific time windows during this process would help to identify valid therapeutic targets for the effective elimination of malignancy.

  1. Motility in the L3 stage is a poor phenotype for detecting and measuring resistance to avermectin/milbemycin drugs in gastrointestinal nematodes of livestock.

    Science.gov (United States)

    George, Melissa M; Lopez-Soberal, Lorraine; Storey, Bob E; Howell, Sue B; Kaplan, Ray M

    2018-04-01

    Motility is a commonly used in vitro phenotype for assessing anthelmintic activity of candidate compounds, and for detecting anthelmintic resistance in nematodes. Third-stage larvae (L3) of parasitic nematodes are commonly used in motility-based assays because L3 are simple to obtain and can remain viable in storage for extended periods. To improve the measurement of motility of microscopic stages of nematodes, our laboratory developed the Worminator, which quantitatively measures motility of parasites. Using the Worminator, we compared the dose-response characteristics of several avermectin/milbemycin (AM) compounds using L3 from both AM-susceptible and AM-resistant Cooperia spp. (abamectin, doramectin, eprinomectin, ivermectin, moxidectin) and Haemonchus contortus (eprinomectin, ivermectin, moxidectin). Concentrations tested with the Worminator ranged from 0.156 to 40 μM. Differences in EC 50 between AM-susceptible and AM-resistant isolates of Cooperia spp. and Haemonchus contortus were small, with resistance ratios ranging from 1.00 to 1.34 for Cooperia spp., 0.99 to 1.65 for Haemonchus contortus. Larval migration inhibition assays were conducted using the same isolates and were equally ineffective for detection of resistance with resistance ratios less than 2.0. These results contrast with those of the Larval Development Assay where we obtained a resistance ratio of 16.48 using the same isolates of Haemonchus contortus. Moreover, even at the highest concentration tested (40 μM), 100% inhibition of motility was never achieved and EC 50 for Worminator assays were more than 100× higher than peak plasma levels achieved in vivo following treatment. These data demonstrate that dose-response characteristics for inhibition of motility in L3 of gastrointestinal nematodes of livestock do not significantly differ for AM-susceptible and AM-resistant isolates. These data challenge the suitability of motility as a phenotype for detecting and measuring resistance to AM

  2. European recommendations for the clinical use of HIV drug resistance testing: 2011 update

    DEFF Research Database (Denmark)

    Vandamme, Anne-Mieke; Camacho, Ricardo J; Ceccherini-Silberstein, Francesca

    2011-01-01

    , and other drug targets (integrase and envelope) if such drugs were part of the failing regimen; (iii) consider testing for CCR5 tropism at virologic failure or when a change of therapy has to be made in absence of detectable viral load, and in the latter case test DNA or last detectable plasma RNA; (iv...... the following recommendations concerning the indications for resistance testing: for HIV-1 (i) test earliest sample for protease and reverse transcriptase drug resistance in drug-naive patients with acute or chronic infection; (ii) test protease and reverse transcriptase drug resistance at virologic failure...... is needed after treatment failure. The Panel recommends genotyping in most situations, using updated and clinically evaluated interpretation systems. It is mandatory that laboratories performing HIV resistance tests take part regularly in external quality assurance programs, and that they consider storing...

  3. Vaccination of chickens against coccidiosis ameliorates drug resistance in commercial poultry production

    Directory of Open Access Journals (Sweden)

    H. David Chapman

    2014-12-01

    Full Text Available Drug resistance is a problem wherever livestock are raised under intensive conditions and drugs are used to combat parasitic infections. This is particularly true for the anticoccidial agents used for the prevention of coccidiosis caused by protozoa of the apicomplexan genus Eimeria in poultry. Resistance has been documented for all the dozen or so drugs approved for use in chickens and varying levels of resistance is present for those currently employed. A possible solution may be the introduction of drug-sensitive parasites into the houses where poultry are raised so that they may replace such drug-resistant organisms. This can be achieved by utilizing live vaccines that contain strains of Eimeria that were isolated before most anticoccidial compounds were introduced. Such strains are inherently drug-sensitive. Practical proposals to achieve this objective involve the alternation of vaccination with medication (known as rotation programs in successive flocks reared in the same poultry house. A proposal for a yearly broiler production cycle involving chemotherapy and vaccination is presented. There are few, if any, examples in veterinary parasitology where it has proved possible to restore sensitivity to drugs used to control a widespread parasite. Further research is necessary to ascertain whether this can result in sustainable and long-term control of Eimeria infections in poultry.

  4. Vaccination of chickens against coccidiosis ameliorates drug resistance in commercial poultry production

    Science.gov (United States)

    Chapman, H. David; Jeffers, Thomas K.

    2014-01-01

    Drug resistance is a problem wherever livestock are raised under intensive conditions and drugs are used to combat parasitic infections. This is particularly true for the anticoccidial agents used for the prevention of coccidiosis caused by protozoa of the apicomplexan genus Eimeria in poultry. Resistance has been documented for all the dozen or so drugs approved for use in chickens and varying levels of resistance is present for those currently employed. A possible solution may be the introduction of drug-sensitive parasites into the houses where poultry are raised so that they may replace such drug-resistant organisms. This can be achieved by utilizing live vaccines that contain strains of Eimeria that were isolated before most anticoccidial compounds were introduced. Such strains are inherently drug-sensitive. Practical proposals to achieve this objective involve the alternation of vaccination with medication (known as rotation programs) in successive flocks reared in the same poultry house. A proposal for a yearly broiler production cycle involving chemotherapy and vaccination is presented. There are few, if any, examples in veterinary parasitology where it has proved possible to restore sensitivity to drugs used to control a widespread parasite. Further research is necessary to ascertain whether this can result in sustainable and long-term control of Eimeria infections in poultry. PMID:25516830

  5. Surgery for Drug-Resistant Epilepsy in Children.

    Science.gov (United States)

    Dwivedi, Rekha; Ramanujam, Bhargavi; Chandra, P Sarat; Sapra, Savita; Gulati, Sheffali; Kalaivani, Mani; Garg, Ajay; Bal, Chandra S; Tripathi, Madhavi; Dwivedi, Sada N; Sagar, Rajesh; Sarkar, Chitra; Tripathi, Manjari

    2017-10-26

    Neurosurgical treatment may improve seizures in children and adolescents with drug-resistant epilepsy, but additional data are needed from randomized trials. In this single-center trial, we randomly assigned 116 patients who were 18 years of age or younger with drug-resistant epilepsy to undergo brain surgery appropriate to the underlying cause of epilepsy along with appropriate medical therapy (surgery group, 57 patients) or to receive medical therapy alone (medical-therapy group, 59 patients). The patients in the medical-therapy group were assigned to a waiting list for surgery. The primary outcome was freedom from seizures at 12 months. Secondary outcomes were the score on the Hague Seizure Severity scale, the Binet-Kamat intelligence quotient, the social quotient on the Vineland Social Maturity Scale, and scores on the Child Behavior Checklist and the Pediatric Quality of Life Inventory. At 12 months, freedom from seizures occurred in 44 patients (77%) in the surgery group and in 4 (7%) in the medical-therapy group (Pchildren and adolescents with drug-resistant epilepsy who had undergone epilepsy surgery had a significantly higher rate of freedom from seizures and better scores with respect to behavior and quality of life than did those who continued medical therapy alone at 12 months. Surgery resulted in anticipated neurologic deficits related to the region of brain resection. (Funded by the Indian Council of Medical Research and others; Clinical Trial Registry-India number, CTRI/2010/091/000525 .).

  6. Options for modulation of drug resistance in ovarian cancer

    NARCIS (Netherlands)

    Arts, HJG; Van der Zee, AGJ; De Jong, S; De Vries, EGE

    2000-01-01

    The objective of this paper is to present an update of mechanisms responsible for drug resistance in ovarian cancer and the possible therapeutic options to modulate this resistance using literature review with emphasis on data acquired in studies comprising ovarian tumor samples. The classic

  7. Primary drug resistance in a region with high burden of tuberculosis. A critical problem.

    Science.gov (United States)

    Villa-Rosas, Cecilia; Laniado-Laborín, Rafael; Oceguera-Palao, Lorena

    2015-01-01

    To determine rates of drug resistance in new cases of pulmonary tuberculosis in a region with a high burden of the disease. New case suspects were referred for drug susceptibility testing. 28.9% of new cases were resistant to at least one first line drug; 3.9% had a multidrug-resistant strain, 15.6% a monoresistant strain and 9.4% a polyresistant strain. Our rate of drug resistant tuberculosis in new cases is very high; this has important clinical implications, since even monoresistance can have a negative impact on the outcome of new cases treated empirically with a six month regimen.

  8. An analytical method for assessing stage-specific drug activity in Plasmodium vivax malaria: implications for ex vivo drug susceptibility testing.

    Directory of Open Access Journals (Sweden)

    Douglas H Kerlin

    Full Text Available The emergence of highly chloroquine (CQ resistant P. vivax in Southeast Asia has created an urgent need for an improved understanding of the mechanisms of drug resistance in these parasites, the development of robust tools for defining the spread of resistance, and the discovery of new antimalarial agents. The ex vivo Schizont Maturation Test (SMT, originally developed for the study of P. falciparum, has been modified for P. vivax. We retrospectively analysed the results from 760 parasite isolates assessed by the modified SMT to investigate the relationship between parasite growth dynamics and parasite susceptibility to antimalarial drugs. Previous observations of the stage-specific activity of CQ against P. vivax were confirmed, and shown to have profound consequences for interpretation of the assay. Using a nonlinear model we show increased duration of the assay and a higher proportion of ring stages in the initial blood sample were associated with decreased effective concentration (EC(50 values of CQ, and identify a threshold where these associations no longer hold. Thus, starting composition of parasites in the SMT and duration of the assay can have a profound effect on the calculated EC(50 for CQ. Our findings indicate that EC(50 values from assays with a duration less than 34 hours do not truly reflect the sensitivity of the parasite to CQ, nor an assay where the proportion of ring stage parasites at the start of the assay does not exceed 66%. Application of this threshold modelling approach suggests that similar issues may occur for susceptibility testing of amodiaquine and mefloquine. The statistical methodology which has been developed also provides a novel means of detecting stage-specific drug activity for new antimalarials.

  9. Clinical and molecular surveillance of drug resistant vivax malaria in Myanmar (2009-2016).

    Science.gov (United States)

    Nyunt, Myat Htut; Han, Jin-Hee; Wang, Bo; Aye, Khin Myo; Aye, Kyin Hla; Lee, Seong-Kyun; Htut, Ye; Kyaw, Myat Phone; Han, Kay Thwe; Han, Eun-Taek

    2017-03-16

    One of the major challenges for control and elimination of malaria is ongoing spread and emergence of drug resistance. While epidemiology and surveillance of the drug resistance in falciparum malaria is being explored globally, there are few studies on drug resistance vivax malaria. To assess the spread of drug-resistant vivax malaria in Myanmar, a multisite, prospective, longitudinal study with retrospective analysis of previous therapeutic efficacy studies, was conducted. A total of 906 from nine study sites were included in retrospective analysis and 208 from three study sites in prospective study. Uncomplicated vivax mono-infected patients were recruited and monitored with longitudinal follow-up until day 28 after treatment with chloroquine. Amplification and sequence analysis of molecular markers, such as mutations in pvcrt-O, pvmdr1, pvdhps and pvdhfr, were done in day-0 samples in prospective study. Clinical failure cases were found only in Kawthaung, southern Myanmar and western Myanmar sites within 2009-2016. Chloroquine resistance markers, pvcrt-O 'AAG' insertion and pvmdr1 mutation (Y976F) showed higher mutant rate in southern and central Myanmar than western site: 66.7, 72.7 vs 48.3% and 26.7, 17.0 vs 1.7%, respectively. A similar pattern of significantly higher mutant rate of antifolate resistance markers, pvdhps (S382A, K512M, A553G) and pvdhfr (F57L/I, S58R, T61M, S117T/N) were noted. Although clinical failure rate was low, widespread distribution of chloroquine and antifolate resistance molecular makers alert to the emergence and spread of drug resistance vivax malaria in Myanmar. Proper strategy and action plan to eliminate and contain the resistant strain strengthened together with clinical and molecular surveillance on drug resistance vivax is recommended.

  10. Sentinel surveillance of HIV-1 transmitted drug resistance, acute infection and recent infection.

    Directory of Open Access Journals (Sweden)

    Hong-Ha M Truong

    Full Text Available HIV-1 acute infection, recent infection and transmitted drug resistance screening was integrated into voluntary HIV counseling and testing (VCT services to enhance the existing surveillance program in San Francisco. This study describes newly-diagnosed HIV cases and characterizes correlates associated with infection.A consecutive sample of persons presenting for HIV VCT at the municipal sexually transmitted infections (STI clinic from 2004 to 2006 (N = 9,868 were evaluated by standard enzyme-linked immunoassays (EIA. HIV antibody-positive specimens were characterized as recent infections using a less-sensitive EIA. HIV-RNA pooled testing was performed on HIV antibody-negative specimens to identify acute infections. HIV antibody-positive and acute infection specimens were evaluated for drug resistance by sequence analysis. Multivariable logistic regression was performed to evaluate associations. The 380 newly-diagnosed HIV cases included 29 acute infections, 128 recent infections, and 47 drug-resistant cases, with no significant increases or decreases in prevalence over the three years studied. HIV-1 transmitted drug resistance prevalence was 11.0% in 2004, 13.4% in 2005 and 14.9% in 2006 (p = 0.36. Resistance to non-nucleoside reverse transcriptase inhibitors (NNRTI was the most common pattern detected, present in 28 cases of resistance (59.6%. Among MSM, recent infection was associated with amphetamine use (AOR = 2.67; p<0.001, unprotected anal intercourse (AOR = 2.27; p<0.001, sex with a known HIV-infected partner (AOR = 1.64; p = 0.02, and history of gonorrhea (AOR = 1.62; p = 0.03.New HIV diagnoses, recent infections, acute infections and transmitted drug resistance prevalence remained stable between 2004 and 2006. Resistance to NNRTI comprised more than half of the drug-resistant cases, a worrisome finding given its role as the backbone of first-line antiretroviral therapy in San Francisco as well as worldwide. The integration of HIV-1 drug

  11. Detection of expression and modulation of multidrug-resistance (MDR) and establishment of a new bioassay

    International Nuclear Information System (INIS)

    Berger, W.

    1993-08-01

    The present thesis deals with the resistance of human malignant cells against cellular toxicity of anticancer drugs, a phenomenon representing one of the major obstacles to successful chemotherapy. One mechanism underlying a cross-resistance to different drugs called multidrug resistance (MDR) is characterized by the expression of an active transport protein (P-glycoprotein), causing decreased intracellular drug retention and cytotoxicity. The main subjects of the present work were to establish different detection methods for MDR and its modulation (by substances blocking activity of P-glycoprotein) including immunological methods (immunocytochemistry, radioimmunoassay), molecular biology (slot-blot analysis, in-situ hybridization) and functional assays (drug-accumulation analysis, drug-cytotoxicity analysis). The methods were evaluated and compared using human and mouse MDR control cell lines and human tumor cell lines established in our laboratory. In cell lines derived from human melanoma - a malignancy insensitive to chemotherapy - expression of P-glycoprotein of relatively low transporting activity was detected by different methods in 8 of 33 cases. Furthermore a new sensitive in vitro assay for the functional detection of MDR was established using the biological features of cytochalasins, a microfilament disrupting substance group. These compounds were shown to be substrates for the P-glycoprotein efflux pump and their effects on cell division (blockade of cytokinesis resulting in multinucleate cells) correlated with MDR-activity of the tested cells. With this new assay P-glycoprotein activity can be demonstrated and analysed over a wide range of resistance against different cytotoxic drugs. Therefore it may by a suitable tool for research and diagnosis in the field of drug resistance

  12. Investigational drugs to treat methicillin-resistant Staphylococcus aureus

    Science.gov (United States)

    Vuong, Cuong; Yeh, Anthony J; Cheung, Gordon YC; Otto, Michael

    2016-01-01

    Introduction Staphylococcus aureus remains one of the leading causes of morbidity and mortality worldwide. This is to a large extent due to antibiotic-resistant strains, in particular methicillin-resistant S. aureus (MRSA). While the toll of invasive MRSA infections appears to decrease in U.S. hospitals, the rate of community-associated MRSA infections remains constant and there is a surge of MRSA in many other countries. This situation calls for continuing if not increased efforts to find novel strategies to combat MRSA infections. Areas covered This review will provide an overview of current investigational antibiotics in clinical development (up to phase II), and of therapeutic antibodies and alternative drugs against S. aureus in preclinical and clinical development, including a short description of the mechanism of action and a presentation of microbiological and clinical data. Expert opinion Increased recent antibiotic development efforts and results from pathogenesis research have led to several new antibiotics and alternative drugs, as well as a more informed selection of targets for vaccination efforts against MRSA. This developing portfolio of novel anti-staphylococcal drugs will hopefully provide us with additional and more efficient ways to combat MRSA infections in the near future and prevent us from running out of treatment options, even if new resistances arise. PMID:26536498

  13. DR_SEQAN: a PC/Windows-based software to evaluate drug resistance using human immunodeficiency virus type 1 genotypes

    Directory of Open Access Journals (Sweden)

    Menéndez-Arias Luis

    2006-03-01

    Full Text Available Abstract Background Genotypic assays based on DNA sequencing of part or the whole reverse transcriptase (RT- and protease (PR-coding regions of the human immunodeficiency virus type 1 (HIV-1 genome have become part of the routine clinical management of HIV-infected individuals. However, the results are difficult to interpret due to complex interactions between mutations found in viral genes. Results DR_SEQAN is a tool to analyze RT and PR sequences. The program output includes a list containing all of the amino acid changes found in the query sequence in comparison with the sequence of a wild-type HIV-1 strain. Translation of codons containing nucleotide mixtures can result in potential ambiguities or heterogeneities in the amino acid sequence. The program identifies all possible combinations of 2 or 3 amino acids that derive from translation of triplets containing nucleotide mixtures. In addition, when ambiguities affect codons relevant for drug resistance, DR_SEQAN allows the user to select the appropriate mutation to be considered by the program's drug resistance interpretation algorithm. Resistance is predicted using a rule-based algorithm, whose efficiency and accuracy has been tested with a large set of drug susceptibility data. Drug resistance predictions given by DR_SEQAN were consistent with phenotypic data and coherent with predictions provided by other publicly available algorithms. In addition, the program output provides two tables showing published drug susceptibility data and references for mutations and combinations of mutations found in the analyzed sequence. These data are retrieved from an integrated relational database, implemented in Microsoft Access, which includes two sets of non-redundant core tables (one for combinations of mutations in the PR and the other for combinations in the RT. Conclusion DR_SEQAN is an easy to use off-line application that provides expert advice on HIV genotypic resistance interpretation. It is

  14. Neurological autoantibodies in drug-resistant epilepsy of unknown cause.

    Science.gov (United States)

    Tecellioglu, Mehmet; Kamisli, Ozden; Kamisli, Suat; Yucel, Fatma Ebru; Ozcan, Cemal

    2018-03-09

    Autoimmune epilepsy is a rarely diagnosed condition. Recognition of the underlying autoimmune condition is important, as these patients can be resistant to antiepileptic drugs. To determine the autoimmune and oncological antibodies in adult drug-resistant epilepsy of unknown cause and identify the clinical, radiological, and EEG findings associated with these antibodies according to data in the literature. Eighty-two patients with drug-resistant epilepsy of unknown cause were prospectively identified. Clinical features were recorded. The levels of anti-voltage-gated potassium channel complex (anti-VGKCc), anti-thyroid peroxidase (anti-TPO), anti-nuclear antibody (ANA), anti-glutamic acid decarboxylase (anti-GAD), anti-phospholipid IgG and IgM, anti-cardiolipin IgG and IgM, and onconeural antibodies were determined. Serum antibody positivity suggesting the potential role of autoimmunity in the aetiology was present in 17 patients with resistant epilepsy (22.0%). Multiple antibodies were found in two patients (2.6%). One of these patients (1.3%) had anti-VGKCc and ANA, whereas another (1.3%) had anti-VGKCc and anti-TPO. A single antibody was present in 15 patients (19.5%). Of the 77 patients finally included in the study, 4 had anti-TPO (5.2%), 1 had anti-GAD (1.3%), 4 had anti-VGKCc (5.2%) 8 had ANA (10.3%), and 2 had onconeural antibodies (2.6%) (1 patient had anti-Yo and 1 had anti-MA2/TA). The other antibodies investigated were not detected. EEG abnormality (focal), focal seizure incidence, and frequent seizures were more common in antibody-positive patients. Autoimmune factors may be aetiologically relevant in patients with drug-resistant epilepsy of unknown cause, especially if focal seizures are present together with focal EEG abnormality and frequent seizures.

  15. Plasmid Conjugation in E. coli and Drug Resistance | Igwe ...

    African Journals Online (AJOL)

    This study aimed at determining the antibiotics susceptibility pattern of E. coli isolates claimed to be multidrug resistance using disc diffusion method. It also determined the presence of transferable resistance plasmids through conjugation and evaluated the medical significance of plasmid encoding E. coli and drug ...

  16. Mycobacterium tuberculosis drug-resistance in previously treated ...

    African Journals Online (AJOL)

    Corresponding to: Professor Lassana Sangaré, Department of Bacteriology and Virology, University Hospital Centre. Yalgado Ouedraogo, 03 BP 7022 Ouagadougou 03, Burkina Faso. E-mail: sangarel@hotmail.com. Abstract. Background: Tuberculosis drug-resistance becomes common in sub-Saharan Africa; however, ...

  17. Mechanisms of antifungal drug resistance in Candida dubliniensis.

    LENUS (Irish Health Repository)

    Coleman, David C

    2010-06-01

    Candida dubliniensis was first described in 1995 and is the most closely related species to the predominant human fungal pathogen Candida albicans. C. dubliniensis is significantly less prevalent and less pathogenic than C. albicans and is primarily associated with infections in HIV-infected individuals and other immunocompromised cohorts. The population structure of C. dubliniensis consists of three well-defined major clades and is significantly less diverse than C. albicans. The majority of C. dubliniensis isolates are susceptible to antifungal drugs commonly used to treat Candida infections. To date only two major patterns of antifungal drug resistance have been identified and the molecular mechanisms of these are very similar to the resistance mechanisms that have been described previously in C. albicans. However, significant differences are evident in the predominant antifungal drug mechanisms employed by C. dubliniensis, differences that reflect its more clonal nature, its lower prevalence and characteristics of its genome, the complete sequence of which has only recently been determined.

  18. Association between Mycobacterium tuberculosis complex phylogenetic lineage and acquired drug resistance.

    Directory of Open Access Journals (Sweden)

    Courtney M Yuen

    Full Text Available BACKGROUND: Development of resistance to antituberculosis drugs during treatment (i.e., acquired resistance can lead to emergence of resistant strains and consequent poor clinical outcomes. However, it is unknown whether Mycobacterium tuberculosis complex species and lineage affects the likelihood of acquired resistance. METHODS: We analyzed data from the U.S. National Tuberculosis Surveillance System and National Tuberculosis Genotyping Service for tuberculosis cases during 2004-2011 with assigned species and lineage and both initial and final drug susceptibility test results. We determined univariate associations between species and lineage of Mycobacterium tuberculosis complex bacteria and acquired resistance to isoniazid, rifamycins, fluoroquinolones, and second-line injectables. We used Poisson regression with backward elimination to generate multivariable models for acquired resistance to isoniazid and rifamycins. RESULTS: M. bovis was independently associated with acquired resistance to isoniazid (adjusted prevalence ratio = 8.46, 95% CI 2.96-24.14 adjusting for HIV status, and with acquired resistance to rifamycins (adjusted prevalence ratio = 4.53, 95% CI 1.29-15.90 adjusting for homelessness, HIV status, initial resistance to isoniazid, site of disease, and administration of therapy. East Asian lineage was associated with acquired resistance to fluoroquinolones (prevalence ratio = 6.10, 95% CI 1.56-23.83. CONCLUSIONS: We found an association between mycobacterial species and lineage and acquired drug resistance using U.S. surveillance data. Prospective clinical studies are needed to determine the clinical significance of these findings, including whether rapid genotyping of isolates at the outset of treatment may benefit patient management.

  19. Genome-wide analysis of multi- and extensively drug-resistant Mycobacterium tuberculosis

    KAUST Repository

    Coll, Francesc

    2018-01-16

    To characterize the genetic determinants of resistance to antituberculosis drugs, we performed a genome-wide association study (GWAS) of 6,465 Mycobacterium tuberculosis clinical isolates from more than 30 countries. A GWAS approach within a mixed-regression framework was followed by a phylogenetics-based test for independent mutations. In addition to mutations in established and recently described resistance-associated genes, novel mutations were discovered for resistance to cycloserine, ethionamide and para-aminosalicylic acid. The capacity to detect mutations associated with resistance to ethionamide, pyrazinamide, capreomycin, cycloserine and para-aminosalicylic acid was enhanced by inclusion of insertions and deletions. Odds ratios for mutations within candidate genes were found to reflect levels of resistance. New epistatic relationships between candidate drug-resistance-associated genes were identified. Findings also suggest the involvement of efflux pumps (drrA and Rv2688c) in the emergence of resistance. This study will inform the design of new diagnostic tests and expedite the investigation of resistance and compensatory epistatic mechanisms.

  20. Exploiting Drug Addiction Mechanisms to Select against MAPKi-Resistant Melanoma.

    Science.gov (United States)

    Hong, Aayoung; Moriceau, Gatien; Sun, Lu; Lomeli, Shirley; Piva, Marco; Damoiseaux, Robert; Holmen, Sheri L; Sharpless, Norman E; Hugo, Willy; Lo, Roger S

    2018-01-01

    Melanoma resistant to MAPK inhibitors (MAPKi) displays loss of fitness upon experimental MAPKi withdrawal and, clinically, may be resensitized to MAPKi therapy after a drug holiday. Here, we uncovered and therapeutically exploited the mechanisms of MAPKi addiction in MAPKi-resistant BRAF MUT or NRAS MUT melanoma. MAPKi-addiction phenotypes evident upon drug withdrawal spanned transient cell-cycle slowdown to cell-death responses, the latter of which required a robust phosphorylated ERK (pERK) rebound. Generally, drug withdrawal-induced pERK rebound upregulated p38-FRA1-JUNB-CDKN1A and downregulated proliferation, but only a robust pERK rebound resulted in DNA damage and parthanatos-related cell death. Importantly, pharmacologically impairing DNA damage repair during MAPKi withdrawal augmented MAPKi addiction across the board by converting a cell-cycle deceleration to a caspase-dependent cell-death response or by furthering parthanatos-related cell death. Specifically in MEKi-resistant NRAS MUT or atypical BRAF MUT melanoma, treatment with a type I RAF inhibitor intensified pERK rebound elicited by MEKi withdrawal, thereby promoting a cell death-predominant MAPKi-addiction phenotype. Thus, MAPKi discontinuation upon disease progression should be coupled with specific strategies that augment MAPKi addiction. Significance: Discontinuing targeted therapy may select against drug-resistant tumor clones, but drug-addiction mechanisms are ill-defined. Using melanoma resistant to but withdrawn from MAPKi, we defined a synthetic lethality between supraphysiologic levels of pERK and DNA damage. Actively promoting this synthetic lethality could rationalize sequential/rotational regimens that address evolving vulnerabilities. Cancer Discov; 8(1); 74-93. ©2017 AACR. See related commentary by Stern, p. 20 This article is highlighted in the In This Issue feature, p. 1 . ©2017 American Association for Cancer Research.

  1. Supermolecular drug challenge to overcome drug resistance in cancer cells.

    Science.gov (United States)

    Onishi, Yasuhiko; Eshita, Yuki; Ji, Rui-Cheng; Kobayashi, Takashi; Onishi, Masayasu; Mizuno, Masaaki; Yoshida, Jun; Kubota, Naoji

    2018-06-04

    Overcoming multidrug resistance (MDR) of cancer cells can be accomplished using drug delivery systems in large-molecular-weight ATP-binding cassette transporters before entry into phagolysosomes and by particle-cell-surface interactions. However, these hypotheses do not address the intratumoral heterogeneity in cancer. Anti-MDR must be related to alterations of drug targets, expression of detoxification, as well as altered proliferation. In this study, it is shown that the excellent efficacy and sustainability of anti-MDR is due to a stable ES complex because of the allosteric facilities of artificial enzymes when they are used as supramolecular complexes. The allosteric effect of supermolecular drugs can be explained by the induced-fit model and can provide stable feedback control systems through the loop transfer function of the Hill equation. Copyright © 2018 Elsevier Ltd. All rights reserved.

  2. Primary drug resistance in a region with high burden of tuberculosis. A critical problem

    Directory of Open Access Journals (Sweden)

    Cecilia Villa-Rosas

    2015-03-01

    Full Text Available Objective. To determine rates of drug resistance in new cases of pulmonary tuberculosis in a region with a high burden of the disease. Materials and methods. New case suspects were referred for drug susceptibility testing. Results. 28.9% of new cases were resistant to at least one first line drug; 3.9% had a multidrug-resistant strain, 15.6% a monoresistant strain and 9.4% a polyresistant strain. Conclusion. Our rate of drug resistant tuberculosis in new cases is very high; this has important clinical implications, since even monoresistance can have a negative impact on the outcome of new cases treated empirically with a six month regimen.

  3. Drug Resistance and the Kinetics of Metastatic Cancer

    Science.gov (United States)

    Blagoev, Krastan B.

    2012-02-01

    Most metastatic cancers after initial response to current drug therapies develop resistance to the treatment. We present cancer data and a theory that explains the observed kinetics of tumor growth in cancer patients and using a stochastic model based on this theory we relate the kinetics of tumor growth to Kaplan-Meyer survival curves. The theory points to the tumor growth rate as the most important parameter determining the outcome of a drug treatment. The overall tumor growth or decay rate is a reflection of the balance between cell division, senescence and apoptosis and we propose that the deviation of the decay rate from exponential is a measure of the emergence of drug resistance. In clinical trials the progression free survival, the overall survival, and the shape of the Kaplan-Meyer plots are determined by the tumor growth rate probability distribution among the patients in the trial. How drug treatments modify this distribution will also be described. At the end of the talk we will discuss the connection between the theory described here and the age related cancer mortality rates in the United States.

  4. Structural Biology Meets Drug Resistance: An Overview on Multidrug Resistance Transporters

    DEFF Research Database (Denmark)

    Shaheen, Aqsa; Iqbal, Mazhar; Mirza, Osman

    2017-01-01

    . Research on the underlying causes of multidrug resistance in cancerous cells and later on in infectious bacteria revealed the involvement of integral membrane transporters, capable of recognizing a broad range of structurally different molecules as substrates and exporting them from the cell using cellular...... superfamilies, viz., ATP-binding cassette superfamily, major facilitator superfamily and resistance nodulation division superfamily are presented. Further, the future role of structural biology in improving our understanding of drug-transporter interactions and in designing novel inhibitors against MDR pump...... century, mankind has become aware and confronted with the emergence of antibiotic-resistant pathogens. In parallel to the failure of antibiotic therapy against infectious pathogens, there had been continuous reports of cancerous cells not responding to chemotherapy with increase in the duration of therapy...

  5. Molecular detection of drug resistance in microbes by isotopic techniques: The IAEA experience

    International Nuclear Information System (INIS)

    Dar, L.; Boussaha, A.; Padhy, A.K.; Khan, B.

    2003-01-01

    The International Atomic Energy Agency (IAEA) supports various programmes on the uses of radionuclide techniques in the management of human communicable diseases. An important issue, being addressed through several technology transfer projects, is the detection of drug resistance in microbes by radioisotope based molecular-biology diagnostic procedures. The techniques employed include dot blot hybridisation with P-32 labelled oligonucleotide probes to detect point mutations, associated with drug resistance, in microbial genes amplified by the polymerase chain reaction (PCR). Molecular methods have been used for the detection of drug resistance in the malarial parasite, Plasmodium falciparum, and in Mycobacterium tuberculosis. Radioisotope based molecular-biology methods have been demonstrated to have comparative advantages in being sensitive, specific, cost-effective, and suitable for application to large-scale molecular surveillance for drug resistance. (author)

  6. Molecular Evidence of Drug Resistance in Asymptomatic Malaria Infections, Myanmar, 2015.

    Science.gov (United States)

    Nyunt, Myat Htut; Shein, Thinzar; Zaw, Ni Ni; Han, Soe Soe; Muh, Fauzi; Lee, Seong-Kyun; Han, Jin-Hee; Thant, Kyaw Zin; Han, Eun-Taek; Kyaw, Myat Phone

    2017-03-01

    Artemisinin resistance containment in Myanmar was initiated in 2011 after artemisinin-resistant Plasmodium falciparum malaria was reported. Molecular evidence suggests that asymptomatic malaria infections harboring drug resistance genes are present among residents of the Myanmar artemisinin resistance containment zone. This evidence supports efforts to eliminate these hidden infections.

  7. Drug Resistance and Population Structure of Mycobacterium tuberculosis Beijing Strains Isolated in Poland.

    Science.gov (United States)

    Kozińska, Monika; Augustynowicz-Kopeć, Ewa

    2015-01-01

    In total, 1095 Mycobacterium tuberculosis clinical isolates from 282 patients with drug-resistant and 813 with drug-sensitive tuberculosis (TB) in Poland during 2007-2011 were analysed. Seventy-one (6.5%) patients were found to have strains of Beijing genotype as defined by spoligotyping. The majority of patients were Polish-born; among foreign-born a large proportion came from Chechnya and Vietnam. Analysis showed strong associations between Beijing genotype infection and MDR, pre-XDR and XDR resistance, with a considerable relative risk among new patients, suggesting that this is due to increased spread of drug-resistant strains rather than acquisition of resistance during treatment.

  8. Drug resistance in vectorborne parasites: multiple actors and scenarios for an evolutionary arms race.

    Science.gov (United States)

    Vanaerschot, Manu; Huijben, Silvie; Van den Broeck, Frederik; Dujardin, Jean-Claude

    2014-01-01

    Drug-resistant pathogens emerge faster than new drugs come out of drug discovery pipelines. Current and future drug options should therefore be better protected, requiring a clear understanding of the factors that contribute to the natural history of drug resistance. Although many of these factors are relatively well understood for most bacteria, this proves to be more complex for vectorborne parasites. In this review, we discuss considering three key models (Plasmodium, Leishmania and Schistosoma) how drug resistance can emerge, spread and persist. We demonstrate a multiplicity of scenarios, clearly resulting from the biological diversity of the different organisms, but also from the different modes of action of the drugs used, the specific within- and between-host ecology of the parasites, and environmental factors that may have direct or indirect effects. We conclude that integrated control of drug-resistant vectorborne parasites is not dependent upon chemotherapy only, but also requires a better insight into the ecology of these parasites and how their transmission can be impaired. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  9. Molecular epidemiology of malaria in Cameroon. XV. Experimental studies on serum substitutes and supplements and alternative culture media for in vitro drug sensitivity assays using fresh isolates of Plasmodium falciparum.

    Science.gov (United States)

    Basco, Leonardo K

    2003-08-01

    In vitro drug sensitivity assay is an important tool for various on-going studies aiming to establish the correlation between candidate molecular markers for drug resistance and drug response in laboratory-adapted strains and field isolates of Plasmodium falciparum. A widespread use of this technique in the field would require a suitable substitute that can replace human serum. In this study, several alternative sources of serum substitutes and supplements were evaluated for their capacity to sustain parasite growth for a single life cycle and their compatibility with in vitro assays for clinical isolates that have not been adapted to in vitro culture. Albumax, a commercial preparation of lipid-enriched bovine albumin, did not support parasite growth as much as human serum and fetal calf serum in several isolates. Other serum supplements (AmnioMax and Ultroser) supported parasite growth relatively well. The 50% inhibitory concentrations (IC50s) of chloroquine and antifolates determined with 0.05% Albumax were generally two or three times higher than with human serum. With 10% fetal calf serum, IC50s for chloroquine and antifolates were approximately two times higher and three times lower than with human serum, respectively. The use of AmnioMax and OptiMAb resulted in a greater than two-fold increase in IC50s and several uninterpretable assays. Despite possible batch-to-batch differences, fetal calf serum may be a suitable substitute for in vitro drug assays while awaiting the results of further studies on other serum substitutes and supplements.

  10. Pathogen infection distribution and drug resistance analysis of patients with severe liver disease

    Directory of Open Access Journals (Sweden)

    Xi CHEN

    2018-04-01

    Full Text Available Objective To explore the infection distribution and drug resistance of pathogens in patients with severe liver disease, and provide reference for clinical medication. Methods Retrospective analysis of the microbiological specimens from patients with severe liver disease in Department of Infection of our hospital from August 2014 to November 2016 and the drug susceptibility testing were carried out by means of K-B disc diffusion method after bacterial culturing, and the distribution and drug resistance of pathogens were analyzed. Results Totally 17 of 73 patients with severe liver disease developed hospital infection (23.3%. 104 strains of bacteria were isolated and 78 strains out of them were multidrug-resistant bacteria (75.0%. Among them, 28(26.9% strains were gram-positive coccus, mainly consisting of Staphylococcus aureus and Staphylococcus epidermidis, and 58(55.8% were gram-negative coccus, mainly composed of Escherichia coli, Klebsiella pneumonia and Acinetobacter baumannii, and 18(17.3% strains fungi. S.aureus and enterococci were resistant to penicillin, erythromycin and levofloxacin, the resistance rates were above 80.0%, but had low resistance rates to vancomycin, teicoplanin and tigecycline. The resistance rates of E.coli and K.pneumoniae to piperacillin, cefazolin and cefuroxime sodium were above 85.0%, but they had lower resistance rates to tigecycline and amikacin. Acinetobacter baumannii was 100% resistant to piperacillin and tazobactam, ceftazidime, imipenem and amikacin, but had low resistance to tigecycline and minocycline. Conclusions Multi-drug resistant bacteria are the main bacterial pathogens in patients with severe liver disease and have a high resistance rate to commonly used antibiotics, empirical treatment in the population at high risk of multidrug-resistant bacteria infections requires the use of broad-spectrum or high-grade antibiotics (e.g. carbapenems or tigecycline and drugs against specific pathogenic

  11. Streptococcus suis, an emerging drug-resistant animal and human pathogen

    Directory of Open Access Journals (Sweden)

    Claudio ePalmieri

    2011-11-01

    Full Text Available Streptococcus suis, a major porcine pathogen, has been receiving growing attention not only for its role in severe and increasingly reported infections in humans, but also for its involvement in drug resistance. Recent studies and the analysis of sequenced genomes have been providing important insights into the S. suis resistome, and have resulted in the identification of resistance determinants for tetracyclines, macrolides, aminoglycosides, chloramphenicol, antifolate drugs, streptothricin, and cadmium salts. Resistance gene-carrying genetic elements described so far include integrative and conjugative elements, transposons, genomic islands, phages, and chimeric elements. Some of these elements are similar to those reported in major streptococcal pathogens such as Streptococcus pyogenes, Streptococcus pneumoniae, and Streptococcus agalactiae and share the same chromosomal insertion sites. The available information strongly suggests that S. suis is an important antibiotic resistance reservoir that can contribute to the spread of resistance genes to the above-mentioned streptococci. S. suis is thus a paradigmatic example of possible intersections between animal and human resistomes.

  12. Drug-resistant tuberculosis in Mumbai, India: An agenda for operations research

    Science.gov (United States)

    Mistry, Nerges; Tolani, Monica; Osrin, David

    2012-01-01

    Operations research (OR) is well established in India and is also a prominent feature of the global and local agendas for tuberculosis (TB) control. India accounts for a quarter of the global burden of TB and of new cases. Multidrug-resistant TB is a significant problem in Mumbai, India’s most populous city, and there have been recent reports of totally resistant TB. Much thought has been given to the role of OR in addressing programmatic challenges, by both international partnerships and India’s Revised National TB Control Programme. We attempt to summarize the major challenges to TB control in Mumbai, with an emphasis on drug resistance. Specific challenges include diagnosis of TB and defining cure, detecting drug resistant TB, multiple sources of health care in the private, public and informal sectors, co-infection with human immunodeficiency virus (HIV) and a concurrent epidemic of non-communicable diseases, suboptimal prescribing practices, and infection control. We propose a local agenda for OR: modeling the effects of newer technologies, active case detection, and changes in timing of activities, and mapping hotspots and contact networks; modeling the effects of drug control, changing the balance of ambulatory and inpatient care, and adverse drug reactions; modeling the effects of integration of TB and HIV diagnosis and management, and preventive drug therapy; and modeling the effects of initiatives to improve infection control. PMID:24501697

  13. Global Phenotypic Characterization of Effects of Fluoroquinolone Resistance Selection on the Metabolic Activities and Drug Susceptibilities of Clostridium perfringens Strains

    Directory of Open Access Journals (Sweden)

    Miseon Park

    2014-01-01

    Full Text Available Fluoroquinolone resistance affects toxin production of Clostridium perfringens strains differently. To investigate the effect of fluoroquinolone resistance selection on global changes in metabolic activities and drug susceptibilities, four C. perfringens strains and their norfloxacin-, ciprofloxacin-, and gatifloxacin-resistant mutants were compared in nearly 2000 assays, using phenotype microarray plates. Variations among mutant strains resulting from resistance selection were observed in all aspects of metabolism. Carbon utilization, pH range, osmotic tolerance, and chemical sensitivity of resistant strains were affected differently in the resistant mutants depending on both the bacterial genotype and the fluoroquinolone to which the bacterium was resistant. The susceptibilities to gentamicin and erythromycin of all resistant mutants except one increased, but some resistant strains were less susceptible to amoxicillin, cefoxitin, ceftriaxone, chloramphenicol, and metronidazole than their wild types. Sensitivity to ethidium bromide decreased in some resistant mutants and increased in others. Microarray analysis of two gatifloxacin-resistant mutants showed changes in metabolic activities that were correlated with altered expression of various genes. Both the chemical structures of fluoroquinolones and the genomic makeup of the wild types influenced the changes found in resistant mutants, which may explain some inconsistent reports of the effects of therapeutic use of fluoroquinolones on clinical isolates of bacteria.

  14. Multi drug resistance and β-lactamase production by Klebsiella ...

    African Journals Online (AJOL)

    SERVER

    2007-08-06

    Aug 6, 2007 ... *Corresponding author. E-mail: gnsimha123@rediffmail.com. (Rice, 1999). plasmid that can be easily spread from one organisms to another (Sirot, 1995) these enzymes are capable of inactivating a variety of β-lactam drugs (Rice,. 1999). The ESBL producing organisms often show multi- drug resistant as ...

  15. Antimicrobial drug resistance in Staphylococcus aureus isolated from cattle in Brazil.

    Science.gov (United States)

    Pereira, M S; Siqueira-Júnior, J P

    1995-06-01

    Isolates of Staphylococcus aureus obtained from apparently healthy cattle in the State of Paraiba, Brazil were characterized in relation to resistance to 21 antimicrobial agents. Among the 46 isolates obtained, resistance to penicillin was most frequent, followed by resistance to cadmium, streptomycin, arsenate, tetracycline, mercury, erythromycin and kanamycin/neomycin. All isolates were susceptible to fusidic acid, ethidium bromide, cetrimide, chloramphenicol, benzalkonium chloride, doxycycline, gentamicin, methicillin, minocycline, novobiocin, rifamycin, tylosin and vancomycin. Only six isolates were susceptible to all the drugs tested. With respect to the antibiotics, multi-resistant isolates were uncommon. These results are probably a consequence of the peculiarities of local drug usage pressures. In relation to metal ions, resistance to mercury was rare while resistance to arsenate was relatively frequent, which contrasts with the situation for human Staph. aureus strains. After treatment with ethidium bromide, elimination of resistance to penicillin, tetracycline, streptomycin, erythromycin and cadmium was observed, which was consistent with the genetic determinants being plasmid-borne.

  16. Treatment of extensively drug-resistant tuberculosis and role of the pharmacist.

    Science.gov (United States)

    Mitrzyk, Beatriz Manzor

    2008-10-01

    Abstract Outbreaks of extensively drug-resistant tuberculosis (XDR-TB) in developing countries and recent headlines of an American traveling with a resistant variant of tuberculosis have brought XDR-TB into the spotlight. The World Health Organization and the United States Centers for Disease Control and Prevention have identified XDR-TB as a serious public health threat and are mandating increased efforts at control of tuberculosis. Although XDR-TB is believed to be no more infectious than other variants of tuberculosis, infection with and spread of XDR-TB are concerning because of the ineffectiveness, toxicity, and cost of the available tuberculosis treatment options. Pharmacists may not be aware of the recent trends in tuberculosis resistance or of the impact that they can have on educating the public about this disease. To gain a better understanding of this disease and the potential roles for pharmacists in public health awareness of tuberculosis and in the care of patients with and at risk for this disease, we undertook an extensive search of the Internet, including Web sites of tuberculosis advocacy groups, and of MEDLINE from January 1968-March 2008. Currently, XDR-TB infection is uncommon in the United States, but if history is any indication, there is a high potential for an outbreak or epidemic. The XDR-TB variant has emerged from mismanaging multidrug-resistant tuberculosis, treating tuberculosis with too few drugs, using less effective second-line drugs, and not educating patients about the dangers of nonadherence. With only limited hopes of a novel effective drug combination regimen, use of available antimycobacterial drugs needs to be optimized. Pharmacists can be key players in the prevention and treatment of tuberculosis by promoting adherence, assessing patients for risk factors for resistant disease, providing information about disease control and prevention, and monitoring for effectiveness, adverse effects, and drug interactions.

  17. The Emerging Role of Extracellular Vesicle-Mediated Drug Resistance in Cancers: Implications in Advanced Prostate Cancer.

    Science.gov (United States)

    Soekmadji, Carolina; Nelson, Colleen C

    2015-01-01

    Emerging evidence has shown that the extracellular vesicles (EVs) regulate various biological processes and can control cell proliferation and survival, as well as being involved in normal cell development and diseases such as cancers. In cancer treatment, development of acquired drug resistance phenotype is a serious issue. Recently it has been shown that the presence of multidrug resistance proteins such as Pgp-1 and enrichment of the lipid ceramide in EVs could have a role in mediating drug resistance. EVs could also mediate multidrug resistance through uptake of drugs in vesicles and thus limit the bioavailability of drugs to treat cancer cells. In this review, we discussed the emerging evidence of the role EVs play in mediating drug resistance in cancers and in particular the role of EVs mediating drug resistance in advanced prostate cancer. The role of EV-associated multidrug resistance proteins, miRNA, mRNA, and lipid as well as the potential interaction(s) among these factors was probed. Lastly, we provide an overview of the current available treatments for advanced prostate cancer, considering where EVs may mediate the development of resistance against these drugs.

  18. Drug ratio-dependent antagonism: a new category of multidrug resistance and strategies for its circumvention.

    Science.gov (United States)

    Harasym, Troy O; Liboiron, Barry D; Mayer, Lawrence D

    2010-01-01

    A newly identified form of multidrug resistance (MDR) in tumor cells is presented, pertaining to the commonly encountered resistance of cancer cells to anticancer drug combinations at discrete drug:drug ratios. In vitro studies have revealed that whether anticancer drug combinations interact synergistically or antagonistically can depend on the ratio of the combined agents. Failure to control drug ratios in vivo due to uncoordinated pharmacokinetics could therefore lead to drug resistance if tumor cells are exposed to antagonistic drug ratios. Consequently, the most efficacious drug combination may not occur at the typically employed maximum tolerated doses of the combined drugs if this leads to antagonistic ratios in vivo after administration and resistance to therapeutic effects of the drug combination. Our approach to systematically screen a wide range of drug ratios and concentrations and encapsulate the drug combination in a liposomal delivery vehicle at identified synergistic ratios represents a means to mitigate this drug ratio-dependent MDR mechanism. The in vivo efficacy of the improved agents (CombiPlex formulations) is demonstrated and contrasted with the decreased efficacy when drug combinations are exposed to tumor cells in vivo at antagonistic ratios.

  19. Chaetominine reduces MRP1-mediated drug resistance via inhibiting PI3K/Akt/Nrf2 signaling pathway in K562/Adr human leukemia cells

    Energy Technology Data Exchange (ETDEWEB)

    Yao, Jingyun; Wei, Xing [State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai (China); Shanghai Collaborative Innovation Center for Biomanufacturing Technology, 130 Meilong Road, Shanghai (China); Lu, Yanhua, E-mail: luyanhua@ecust.edu.cn [State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai (China); Shanghai Collaborative Innovation Center for Biomanufacturing Technology, 130 Meilong Road, Shanghai (China)

    2016-05-13

    Drug resistance limits leukemia treatment and chaetominine, a cytotoxic alkaloid that promotes apoptosis in a K562 human leukemia cell line via the mitochondrial pathway was studied with respect to chemoresistance in a K562/Adr human resistant leukemia cell line. Cytotoxicity assays indicated that K562/Adr resistance to adriamycin (ADR) did not occur in the presence of chaetominine and that chaetominine increased chemosensitivity of K562/Adr to ADR. Data show that chaetominine enhanced ADR-induced apoptosis and intracellular ADR accumulation in K562/Adr cells. Accordingly, chaetominine induced apoptosis by upregulating ROS, pro-apoptotic Bax and downregulating anti-apoptotic Bcl-2. RT-PCR and western-blot confirmed that chaetominine suppressed highly expressed MRP1 at mRNA and protein levels. But little obvious alternation of another drug transporter MDR1 mRNA was observed. Furthermore, inhibition of MRP1 by chaetominine relied on inhibiting Akt phosphorylation and nuclear Nrf2. In summary, chaetominine strongly reverses drug resistance by interfering with the PI3K/Akt/Nrf2 signaling, resulting in reduction of MRP1-mediated drug efflux and induction of Bax/Bcl-2-dependent apoptosis in an ADR-resistant K562/Adr leukemia cell line. - Highlights: • Chaetominine enhanced chemosensitivity of ADR against K562/Adr cells. • Chaetominine increased intracellular ADR levels via inhibiting MRP1. • Chaetominine induced apoptosis of K562/Adr cells through upregulation of ROS and modulation of Bax/Bcl-2. • Inhibition of MRP1 and Nrf2 by chaetominine treatment was correlative with blockade of PI3K/Akt signaling.

  20. Genotyping and drug resistance patterns of Mycobacterium tuberculosis strains observed in a tuberculosis high-burden municipality in Northeast, Brazil

    Directory of Open Access Journals (Sweden)

    Roberta dos Santos Silva Luiz

    2013-06-01

    Full Text Available OBJECTIVES: This study has used a combination of clinical information, spoligotyping, and georeferencing system to elucidate the genetic diversity of the Mycobacterium tuberculosis isolates circulating in a TB-prevalent municipality of Northeast Brazil. METHODS: A total of 115 M. tuberculosis strains were isolated from pulmonary tuberculosis patients from January 2007 to March 2008 in Fortaleza. Drug susceptibility and spoligotyping assays were performed and place of residence of the patients were georeferenced. RESULTS: Of the M. tuberculosis strains studied, 51 (44.3% isolates were resistant to at least one drug (R-TB and 64 (55.7% were sensitive to all the drugs tested (S-TB. A high frequency of resistance was found in previously treated cases (84% and among new cases (16%; p < 0.001. a total of 74 (64% isolates were grouped into 22 spoligotyped lineages, while 41 (36% isolates were identified as new. among the predominant genotypes, 33% were latim american mediterranean (lam, 12% haarlem (h, and 5% u. there was no association of geographic distribution of rt-tb patients as compared to the controls and also the geographic location to the spoligotype patterns. the geospatial analysis revealed that 24 (23% patients (hot spot zones either shared the same residence or lived in a close neighborhood of a case. among these concentration zones, the patients lived in the same residence and shared a common genotype pattern and resistance pattern. DISCUSSION: it was observed that the spoligopatterns family distribution was similar to that reported for south america, prevailing the lam and h lineages. a high rate-case among the resistant TB group occurs as a result of transmitted and acquired resistance. A more effective surveillance program is needed in order to succeed in reducing tuberculosis in Northeast Brazil.

  1. Genotyping and drug resistance patterns of Mycobacterium tuberculosis strains observed in a tuberculosis high-burden municipality in Northeast, Brazil

    Directory of Open Access Journals (Sweden)

    Roberta dos Santos Silva Luiz

    Full Text Available OBJECTIVES: This study has used a combination of clinical information, spoligotyping, and georeferencing system to elucidate the genetic diversity of the Mycobacterium tuberculosis isolates circulating in a TB-prevalent municipality of Northeast Brazil. METHODS: A total of 115 M. tuberculosis strains were isolated from pulmonary tuberculosis patients from January 2007 to March 2008 in Fortaleza. Drug susceptibility and spoligotyping assays were performed and place of residence of the patients were georeferenced. RESULTS: Of the M. tuberculosis strains studied, 51 (44.3% isolates were resistant to at least one drug (R-TB and 64 (55.7% were sensitive to all the drugs tested (S-TB. A high frequency of resistance was found in previously treated cases (84% and among new cases (16%; p < 0.001. a total of 74 (64% isolates were grouped into 22 spoligotyped lineages, while 41 (36% isolates were identified as new. among the predominant genotypes, 33% were latim american mediterranean (lam, 12% haarlem (h, and 5% u. there was no association of geographic distribution of rt-tb patients as compared to the controls and also the geographic location to the spoligotype patterns. the geospatial analysis revealed that 24 (23% patients (hot spot zones either shared the same residence or lived in a close neighborhood of a case. among these concentration zones, the patients lived in the same residence and shared a common genotype pattern and resistance pattern. DISCUSSION: it was observed that the spoligopatterns family distribution was similar to that reported for south america, prevailing the lam and h lineages. a high rate-case among the resistant TB group occurs as a result of transmitted and acquired resistance. A more effective surveillance program is needed in order to succeed in reducing tuberculosis in Northeast Brazil.

  2. The Pathway Analysis of Micrornas Regulated Drug-Resistant Responses in HeLa Cells.

    Science.gov (United States)

    Yang, Yubo; Dai, Cuihong; Cai, Zhipeng; Hou, Aiju; Cheng, Dayou; Wu, Guanying; Li, Jing; Cui, Jie; Xu, Dechang

    2016-03-01

    Chemotherapy is the main strategy in the treatment of cancer; however, the development of drug-resistance is the obstacle in long-term treatment of cervical cancer. Cisplatin is one of the most common drugs used in cancer therapy. Recently, accumulating evidence suggests that miRNAs are involved in various bioactivities in oncogenesis. It is not unexpected that miRNAs play a key role in acquiring of drug-resistance in the progression of tumor. In this study, we induced and maintained four levels of cisplatin-resistant HeLa cell lines (HeLa/CR1, HeLa/CR2, HeLa/CR3, and HeLa/CR4). According to the previous studies and existing evidence, we selected five miRNAs (miR-183, miR-182, miR-30a, miR-15b, and miR-16) and their potential target mRNAs as our research targets. The real-time RT-PCR was adopted to detect the relative expression of miRNAs and their mRNAs. The results show that miR-182 and miR-15b were up-regulated in resistant cell lines, while miR-30a was significantly down-regulated. At the same time, their targets are related to drug resistance. Compared to their parent HeLa cell line, the expression of selected miRNAs in resistant cell lines altered. The alteration suggests that HeLa cell drug resistance is associated with distinct miRNAs, which indicates that miRNAs may be one of the therapy targets in the treatment of cervical cancer by sensitizing cell to chemotherapy. We suggested a possible network diagram based on the existing theory and the preliminary results of candidate miRNAs and their targets in HeLa cells during development of drug resistance.

  3. Overcoming multidrug resistance in 2D and 3D culture models by controlled drug chitosan-graft poly(caprolactone)-based nanoparticles.

    Science.gov (United States)

    Shi, Wei-Bin; Le, Van-Minh; Gu, Chun-Hua; Zheng, Yuan-Hong; Lang, Mei-Dong; Lu, Yan-Hua; Liu, Jian-Wen

    2014-04-01

    The principal limitations of chemotherapy are dose-limiting systemic toxicity and the development of multidrug-resistant phenotypes. The aim of this study was to investigate the efficiency of a new sustained drug delivery system based on chitosan and ε-caprolactone to overcome multidrug resistance in monolayer and drug resistance associated with the three-dimensional (3D) tumor microenvironment in our established 3D models. The 5-fluorouracil (5-FU)-loaded nanoparticles (NPs) were characterized by transmission electron microscope and dynamic light scattering, and its released property was determined at different pH values. 5-FU/NPs exhibited well-sustained release properties and markedly enhanced the cytotoxicity of 5-FU against HCT116/L-OHP or HCT8/VCR MDR cells in two-dimensional (2D) and its parental cells in 3D collagen gel culture with twofold to threefold decrease in the IC50 values, as demonstrated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, Hoechst/propidium iodide staining and flow cytometry analysis. Furthermore, the possible mechanism was explored by high-performance liquid chromatography and rhodamine 123 accumulation experiment. Overall, the results demonstrated that 5-FU/NPs increase intracellular concentration of 5-FU and enhance its anticancer efficiency by inducing apoptosis. It was suggested that this novel NPs are a promising carrier to decrease toxic of 5-FU and has the potential to reverse the forms of both intrinsic and acquired drug resistance in 2D and 3D cultures. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.

  4. Assessment of clinical risk factors for drug-resistant epilepsy in children and teenagers

    Directory of Open Access Journals (Sweden)

    Marta Kasprzyk

    2014-09-01

    Full Text Available Introduction: Epilepsy is one of the most common neurological illnesses occurring in children. In approximately 20–30% of cases it is drug-resistant. Aim of the research: To assess the already-known risk factors, analyse the rarely described ones, and find new causes of epilepsy drug resistance in children, taking into account the level of impact of each factor. Material and methods : The study comprised 152 of all 383 children hospitalised in 2012 at the Neurology Department of the Polish Mother’s Memorial Hospital in Lodz due to epilepsy. Based on medical documentation, neurological examination, and our own questionnaire, we divided patients into two groups: drug-resistant epilepsy or drug-sensitive epilepsy. We compared the type, level of influence, and prevalence of different factors. For statistical analysis, the 2 test was used. Statistical significance was set at p < 0.05. Results: Drug-resistant epilepsy was found in 64 patients (42.1%, and drug-sensitive epilepsy was found in 88 patients (57.9%. Factors that were most probable to cause drug resistance included: high prevalence of seizures (Cramer’s V = 0.66, type of epileptic syndrome (V = 0.62, psychomotor developmental delay (V = 0.62, and occurrence of status epilepticus (V = 0.6. Factors such as infections of CNS in early childhood, repeated severe infections of airways in childhood, and mother’s infectious diseases with high fever during pregnancy were rare or non occurring (Cramer’s V = 0.41, 0.32, and 0.31, respectively. Conclusions : The study confirmed the previously known causes of drug resistance and indicated the significance of underestimated inflammatory and infectious factors involving pyrexia, in children and also in mothers during pregnancy.

  5. Development of novel, 384-well high-throughput assay panels for human drug transporters: drug interaction and safety assessment in support of discovery research.

    Science.gov (United States)

    Tang, Huaping; Shen, Ding Ren; Han, Yong-Hae; Kong, Yan; Balimane, Praveen; Marino, Anthony; Gao, Mian; Wu, Sophie; Xie, Dianlin; Soars, Matthew G; O'Connell, Jonathan C; Rodrigues, A David; Zhang, Litao; Cvijic, Mary Ellen

    2013-10-01

    Transporter proteins are known to play a critical role in affecting the overall absorption, distribution, metabolism, and excretion characteristics of drug candidates. In addition to efflux transporters (P-gp, BCRP, MRP2, etc.) that limit absorption, there has been a renewed interest in influx transporters at the renal (OATs, OCTs) and hepatic (OATPs, BSEP, NTCP, etc.) organ level that can cause significant clinical drug-drug interactions (DDIs). Several of these transporters are also critical for hepatobiliary disposition of bilirubin and bile acid/salts, and their inhibition is directly implicated in hepatic toxicities. Regulatory agencies took action to address transporter-mediated DDI with the goal of ensuring drug safety in the clinic and on the market. To meet regulatory requirements, advanced bioassay technology and automation solutions were implemented for high-throughput transporter screening to provide structure-activity relationship within lead optimization. To enhance capacity, several functional assay formats were miniaturized to 384-well throughput including novel fluorescence-based uptake and efflux inhibition assays using high-content image analysis as well as cell-based radioactive uptake and vesicle-based efflux inhibition assays. This high-throughput capability enabled a paradigm shift from studying transporter-related issues in the development space to identifying and dialing out these concerns early on in discovery for enhanced mechanism-based efficacy while circumventing DDIs and transporter toxicities.

  6. Genetic Indicators of Drug Resistance in the Highly Repetitive Genome of Trichomonas vaginalis.

    Science.gov (United States)

    Bradic, Martina; Warring, Sally D; Tooley, Grace E; Scheid, Paul; Secor, William E; Land, Kirkwood M; Huang, Po-Jung; Chen, Ting-Wen; Lee, Chi-Ching; Tang, Petrus; Sullivan, Steven A; Carlton, Jane M

    2017-06-01

    Trichomonas vaginalis, the most common nonviral sexually transmitted parasite, causes ∼283 million trichomoniasis infections annually and is associated with pregnancy complications and increased risk of HIV-1 acquisition. The antimicrobial drug metronidazole is used for treatment, but in a fraction of clinical cases, the parasites can become resistant to this drug. We undertook sequencing of multiple clinical isolates and lab derived lines to identify genetic markers and mechanisms of metronidazole resistance. Reduced representation genome sequencing of ∼100 T. vaginalis clinical isolates identified 3,923 SNP markers and presence of a bipartite population structure. Linkage disequilibrium was found to decay rapidly, suggesting genome-wide recombination and the feasibility of genetic association studies in the parasite. We identified 72 SNPs associated with metronidazole resistance, and a comparison of SNPs within several lab-derived resistant lines revealed an overlap with the clinically resistant isolates. We identified SNPs in genes for which no function has yet been assigned, as well as in functionally-characterized genes relevant to drug resistance (e.g., pyruvate:ferredoxin oxidoreductase). Transcription profiles of resistant strains showed common changes in genes involved in drug activation (e.g., flavin reductase), accumulation (e.g., multidrug resistance pump), and detoxification (e.g., nitroreductase). Finally, we identified convergent genetic changes in lab-derived resistant lines of Tritrichomonas foetus, a distantly related species that causes venereal disease in cattle. Shared genetic changes within and between T. vaginalis and Tr. foetus parasites suggest conservation of the pathways through which adaptation has occurred. These findings extend our knowledge of drug resistance in the parasite, providing a panel of markers that can be used as a diagnostic tool. © The Author 2017. Published by Oxford University Press on behalf of the Society for

  7. The Prevalence of Drug-Resistant Tuberculosis in Mainland China: An Updated Systematic Review and Meta-Analysis.

    Science.gov (United States)

    Duan, Qionghong; Chen, Zi; Chen, Cong; Zhang, Zhengbin; Lu, Zhouqin; Yang, Yalong; Zhang, Lin

    2016-01-01

    In recent years, drug resistant tuberculosis (DR-TB) particularly the emergence of multi-drug-resistant tuberculosis (MDR-TB) has become a major public health issue. The most recent study regarding the prevalence of drug-resistant tuberculosis in mainland China was a meta-analysis published in 2011, and the subjects from the included studies were mostly enrolled before 2008, thus making it now obsolete. Current data on the national prevalence of DR-TB is needed. This review aims to provide a comprehensive and up-to-date assessment of the status of DR-TB epidemic in mainland China. A systematic review and meta-analysis of studies regarding the prevalence of drug-resistant tuberculosis in mainland China was performed. Pubmed/MEDLINE, EMBASE, the Cochrane central database, the Chinese Biomedical Literature Database and the China National Knowledge Infrastructure Database were searched for studies relevant to drug-resistant tuberculosis that were published between January 1, 2012 and May 18, 2015. Comprehensive Meta-Analysis (V2.2, Biostat) software was used to analyse the data. A total of fifty-nine articles, published from 2012 to 2015, were included in our review. The result of this meta-analysis demonstrated that among new cases, the rate of resistance to any drug was 20.1% (18.0%-22.3%; n/N = 7203/34314) and among retreatment cases, the rate was 49.8% (46.0%-53.6%; n/N = 4155/8291). Multi-drug resistance among new and retreatment cases was 4.8% (4.0%-5.7%; n/N = 2300/42946) and 26.3% (23.1%-29.7%; n/N = 3125/11589) respectively. The results were significantly heterogeneous (pdrug resistance patterns were found by subgroup analysis according to geographic areas, subject enrolment time, and methods of drug susceptibility test (DST). The prevalence of resistance to any drug evidently dropped for both new and retreatment cases, and multi-drug resistance declined among new cases but became more prevalent among retreatment cases compared to the data before 2008

  8. [Antituberculosis-drug resistance in the border of Brazil with Paraguay and Bolivia].

    Science.gov (United States)

    Marques, Marli; Cunha, Eunice Atsuko Totumi; Evangelista, Maria do Socorro Nantua; Basta, Paulo Cesar; Marques, Ana Maria Campos; Croda, Julio; de Andrade, Sonia Maria Oliveira

    2017-04-20

    To estimate the rate of drug resistance among pulmonary tuberculosis (PTB) cases in the state of Mato Grosso do Sul, Brazil, and specifically in the border areas with Paraguay and Bolivia, as well as to identify associated risk factors. The present cross-sectional, epidemiological study focused on PTB cases recorded between January 2007 and December 2010 in the State Reportable Disease Information System with results of susceptibility tests to rifampicin, isoniazid, ethambutol, and streptomycin. Dependent variables were development of resistance to a single drug or any combination of drugs. Independent variables were being a new or treated case, living in border areas, presence/absence of diabetes, and history of alcoholism. There were 789 TBP cases with susceptibility testing. The following characteristics were associated with resistance: treated case (P = 0.0001), border region (P = 0.0142), alcoholism (P = 0.0451), and diabetes (P = 0.0708). The rates of combined, primary, and acquired resistance for the state were 16.3%, 10.6%, and 39.0%, vs. 22.3%, 19.2%, and 37.5% for the border region. The rates of combined, primary, and acquired multidrug resistance for the state were 1.8%, 0.6%, and 6.3%, vs. 3.1%, 1.2%, and 12.5% for the border region. In the border region, the state should investigate drug resistance in all patients with respiratory symptoms, determine the pattern of resistance in confirmed cases, adopt directly observed treatment for cases of PTB, and develop health actions together with neighboring countries. Across the state, the levels of acquired resistance should be monitored, with investigation of resistance in all treated cases and implementation of directly observed treatment especially among patients with diabetes or alcoholism.

  9. The Role of ABC Proteins in Drug Resistant Breast Cancer Cells

    Science.gov (United States)

    2008-04-01

    called the Plasmodium falciparum Chloroquine Transporter (PfCRT). While PfCRT is known to be the main molecular determinant of chloroquine resistance...proteins (such as human P-glycoprotein) and labeled PfCRT with a photoaffinity drug analogue . A manuscript is currently in preparation detailing my results...directly responsible for drug response, the Plasmodium falciparum Chloroquine Resistance Transporter (PfCRT) (Fidock et al 2000). While not a member of

  10. Exploring Culturally Specific Drug Resistance Strategies of Hawaiian Youth in Rural Communities

    Science.gov (United States)

    Okamoto, Scott K.; Po'a-Kekuawela, Ka'ohinani; Chin, Coralee I. H.; Nebre, La Risa H.; Helm, Susana

    2010-01-01

    This qualitative study examined the drug resistance strategies of Hawaiian youth residing in rural communities in Hawai'i. Forty seven youth participated in 14 focus groups which focused on the social and environmental context of drug use for these youth. The findings indicated that there were 47 references to resistance strategies used in drug…

  11. Characterization of extensively drug-resistant Mycobacterium tuberculosis in Nepal.

    Science.gov (United States)

    Poudel, Ajay; Maharjan, Bhagwan; Nakajima, Chie; Fukushima, Yukari; Pandey, Basu D; Beneke, Antje; Suzuki, Yasuhiko

    2013-01-01

    The emergence of extensively drug-resistant tuberculosis (XDR-TB) has raised public health concern for global control of TB. Although molecular characterization of drug resistance-associated mutations in multidrug-resistant isolates in Nepal has been made, mutations in XDR isolates and their genotypes have not been reported previously. In this study, we identified and characterized 13 XDR Mycobacterium tuberculosis isolates from clinical isolates in Nepal. The most prevalent mutations involved in rifampicin, isoniazid, ofloxacin, and kanamycin/capreomycin resistance were Ser531Leu in rpoB gene (92.3%), Ser315Thr in katG gene (92.3%), Asp94Gly in gyrA gene (53.9%) and A1400G in rrs gene (61.5%), respectively. Spoligotyping and multilocus sequence typing revealed that 69% belonged to Beijing family, especially modern types. Further typing with 26-loci variable number of tandem repeats suggested the current spread of XDR M. tuberculosis. Our result highlights the need to reinforce the TB policy in Nepal with regard to control and detection strategies. Copyright © 2012 Elsevier Ltd. All rights reserved.

  12. A magnetic bead-based ligand binding assay to facilitate human kynurenine 3-monooxygenase drug discovery.

    Science.gov (United States)

    Wilson, Kris; Mole, Damian J; Homer, Natalie Z M; Iredale, John P; Auer, Manfred; Webster, Scott P

    2015-02-01

    Human kynurenine 3-monooxygenase (KMO) is emerging as an important drug target enzyme in a number of inflammatory and neurodegenerative disease states. Recombinant protein production of KMO, and therefore discovery of KMO ligands, is challenging due to a large membrane targeting domain at the C-terminus of the enzyme that causes stability, solubility, and purification difficulties. The purpose of our investigation was to develop a suitable screening method for targeting human KMO and other similarly challenging drug targets. Here, we report the development of a magnetic bead-based binding assay using mass spectrometry detection for human KMO protein. The assay incorporates isolation of FLAG-tagged KMO enzyme on protein A magnetic beads. The protein-bound beads are incubated with potential binding compounds before specific cleavage of the protein-compound complexes from the beads. Mass spectrometry analysis is used to identify the compounds that demonstrate specific binding affinity for the target protein. The technique was validated using known inhibitors of KMO. This assay is a robust alternative to traditional ligand-binding assays for challenging protein targets, and it overcomes specific difficulties associated with isolating human KMO. © 2014 Society for Laboratory Automation and Screening.

  13. The human multidrug resistance-associated protein MRP is a plasma membrane drug-efflux pump

    NARCIS (Netherlands)

    Zaman, G. J.; Flens, M. J.; van Leusden, M. R.; de Haas, M.; Mülder, H. S.; Lankelma, J.; Pinedo, H. M.; Scheper, R. J.; Baas, F.; Broxterman, H. J.

    1994-01-01

    The multidrug-resistance associated protein MRP is a 180- to 195-kDa membrane protein associated with resistance of human tumor cells to cytotoxic drugs. We have investigated how MRP confers drug resistance in SW-1573 human lung carcinoma cells by generating a subline stably transfected with an

  14. Multi-drug resistant Ewingella Americana

    International Nuclear Information System (INIS)

    Bukhari, Syed Z.; Ashshi, Ahmad M.; Hussain, Waleed M.; Fatani, Mohammad I.

    2008-01-01

    We report a case of pneumonia due to multi-drug resistant Ewingella Americana in a young patient admitted in the Intensive Care Unit of Hera General Hospital, Makkah, Saudi Arabia with severe head injury in a road traffic accident. He was an Indonesian pilgrim who had traveled to the Kingdom of Saudi Arabia to perform Hajj in December 2007. Ewingella Americana was identified to be the pathogen of pneumonia with clinical signs and symptoms along with positive radiological findings. (author)

  15. GENETIC DIVERSITY OF DRUG RESISTANT STRAINS OF MYCOBACTERIUM TUBERCULOSIS IN OMSK REGION

    Directory of Open Access Journals (Sweden)

    O. A. Pаsechnik

    2017-01-01

    Full Text Available The article presents the investigation results of the specific epidemic situation on tuberculous infection in Omsk Region in 2006-2015 and molecular genetic features of M. tuberculosis strains with multiple drug resistance circulating in this region. Bacteriological, molecular genetic methods, VNTR-typing were used as well as descriptive techniques of the epidemiological process. Tuberculosis prevalence made 269.2 per 100,000 population. There is an increase in those with bacillary excretion among new cases of respiratory tuberculosis from 39.8% to 53.4%. Drug resistance was detected in 48.0% of new cases. Among drug resistance patterns, MDR made 57%, and extensive drug resistance (XDR increased from 2.5 to 7.0%. In 2015 prevalence of XDR tuberculosis made 8.9 per 100,000 population in Omsk Region. When performing VNTR-typing of 77 samples of M. tuberculosis DNA with MDR, 27 genetic types were identified. The population of MDR strain of M. tuberculosis is heterogeneous and presented by strains of various genetic families -Beijing, LAM, S,Haarlem,Uganda. The investigation showed that isolates ofBeijing family prevailed (76.6%.

  16. Low-cost ultra-wide genotyping using Roche/454 pyrosequencing for surveillance of HIV drug resistance.

    Directory of Open Access Journals (Sweden)

    Dawn M Dudley

    Full Text Available Great efforts have been made to increase accessibility of HIV antiretroviral therapy (ART in low and middle-income countries. The threat of wide-scale emergence of drug resistance could severely hamper ART scale-up efforts. Population-based surveillance of transmitted HIV drug resistance ensures the use of appropriate first-line regimens to maximize efficacy of ART programs where drug options are limited. However, traditional HIV genotyping is extremely expensive, providing a cost barrier to wide-scale and frequent HIV drug resistance surveillance.We have developed a low-cost laboratory-scale next-generation sequencing-based genotyping method to monitor drug resistance. We designed primers specifically to amplify protease and reverse transcriptase from Brazilian HIV subtypes and developed a multiplexing scheme using multiplex identifier tags to minimize cost while providing more robust data than traditional genotyping techniques. Using this approach, we characterized drug resistance from plasma in 81 HIV infected individuals collected in São Paulo, Brazil. We describe the complexities of analyzing next-generation sequencing data and present a simplified open-source workflow to analyze drug resistance data. From this data, we identified drug resistance mutations in 20% of treatment naïve individuals in our cohort, which is similar to frequencies identified using traditional genotyping in Brazilian patient samples.The developed ultra-wide sequencing approach described here allows multiplexing of at least 48 patient samples per sequencing run, 4 times more than the current genotyping method. This method is also 4-fold more sensitive (5% minimal detection frequency vs. 20% at a cost 3-5× less than the traditional Sanger-based genotyping method. Lastly, by using a benchtop next-generation sequencer (Roche/454 GS Junior, this approach can be more easily implemented in low-resource settings. This data provides proof-of-concept that next

  17. Effect of repetitive transcranial magnetic stimulation in drug resistant depressed patients

    International Nuclear Information System (INIS)

    Chung, Yong An; Yoo, Ie Ryung; Kang, Bong Joo; Chae, Jeong Ho; Lee, Hye Won; Moon, Hyun Jin; Kim, Sung Hoon; Sohn, Hyung Sun; Chung, Soo Kyo

    2007-01-01

    Repetitive transcranial magnetic stimulation (rTMS) has recently been clinically applied in the treatment of drug resistant depressed patients. There are mixed findings about the efficacy of rTMS on depression. Furthermore, the influence of rTMS on the physiology of the brain is not clear. We prospectively evaluated changes of regional cerebral blood flow (rCBF) between pre- and post-rTMS treatment in patients with drug resistant depression. Twelve patients with drug-resistant depression (7 male, 5 female; age range; 19∼ 52 years; mean age: 29.3 ± 9.3 years) were given rTMS on right prefrontal lobe with low frequency (1 Hz) and on left prefrontal lobe with high frequency (20 Hz), with 20-minute-duration each day for 3 weeks. Tc-99m ECD brain perfusion SPECT was obtained before and after rTMS treatment. The changes of cerebral perfusion were analyzed using statistical parametric mapping (SPM; t=3.14, uncorrected ρ < 0.01, voxel = 100). Following areas showed significant increase in rCBF after 3 weeks rTMS treatment: the cingulate gyrus, fusiform gyrus of right temporal lobe, precuneus, and left lateral globus pallidus. Significant decrement was noted in the precental and middle frontal gyrus of right frontal lobe, and fusiform gyrus of left occipital lobe. Low-frequency rTMS on the right prefrontal cortex and high-frequency rTMS on the left prefrontal cortex for 3 weeks as an add-on regimen have increased and decreased rCBF in the specific brain regions in drug-resistant depressed patients. Further analyses correlating clinical characteristics and treatment paradigm with functional imaging data may be helpful in clarifying the pathophysiology of drug-resistant patients

  18. Effect of repetitive transcranial magnetic stimulation in drug resistant depressed patients

    Energy Technology Data Exchange (ETDEWEB)

    Chung, Yong An; Yoo, Ie Ryung; Kang, Bong Joo; Chae, Jeong Ho; Lee, Hye Won; Moon, Hyun Jin; Kim, Sung Hoon; Sohn, Hyung Sun; Chung, Soo Kyo [The Catholic University of Korea, Seoul (Korea, Republic of)

    2007-02-15

    Repetitive transcranial magnetic stimulation (rTMS) has recently been clinically applied in the treatment of drug resistant depressed patients. There are mixed findings about the efficacy of rTMS on depression. Furthermore, the influence of rTMS on the physiology of the brain is not clear. We prospectively evaluated changes of regional cerebral blood flow (rCBF) between pre- and post-rTMS treatment in patients with drug resistant depression. Twelve patients with drug-resistant depression (7 male, 5 female; age range; 19{approx} 52 years; mean age: 29.3 {+-} 9.3 years) were given rTMS on right prefrontal lobe with low frequency (1 Hz) and on left prefrontal lobe with high frequency (20 Hz), with 20-minute-duration each day for 3 weeks. Tc-99m ECD brain perfusion SPECT was obtained before and after rTMS treatment. The changes of cerebral perfusion were analyzed using statistical parametric mapping (SPM; t=3.14, uncorrected {rho} < 0.01, voxel = 100). Following areas showed significant increase in rCBF after 3 weeks rTMS treatment: the cingulate gyrus, fusiform gyrus of right temporal lobe, precuneus, and left lateral globus pallidus. Significant decrement was noted in the precental and middle frontal gyrus of right frontal lobe, and fusiform gyrus of left occipital lobe. Low-frequency rTMS on the right prefrontal cortex and high-frequency rTMS on the left prefrontal cortex for 3 weeks as an add-on regimen have increased and decreased rCBF in the specific brain regions in drug-resistant depressed patients. Further analyses correlating clinical characteristics and treatment paradigm with functional imaging data may be helpful in clarifying the pathophysiology of drug-resistant patients.

  19. What is the mechanism for persistent coexistence of drug-susceptible and drug-resistant strains of Streptococcus pneumoniae?

    Science.gov (United States)

    Colijn, Caroline; Cohen, Ted; Fraser, Christophe; Hanage, William; Goldstein, Edward; Givon-Lavi, Noga; Dagan, Ron; Lipsitch, Marc

    2010-01-01

    The rise of antimicrobial resistance in many pathogens presents a major challenge to the treatment and control of infectious diseases. Furthermore, the observation that drug-resistant strains have risen to substantial prevalence but have not replaced drug-susceptible strains despite continuing (and even growing) selective pressure by antimicrobial use presents an important problem for those who study the dynamics of infectious diseases. While simple competition models predict the exclusion of one strain in favour of whichever is ‘fitter’, or has a higher reproduction number, we argue that in the case of Streptococcus pneumoniae there has been persistent coexistence of drug-sensitive and drug-resistant strains, with neither approaching 100 per cent prevalence. We have previously proposed that models seeking to understand the origins of coexistence should not incorporate implicit mechanisms that build in stable coexistence ‘for free’. Here, we construct a series of such ‘structurally neutral’ models that incorporate various features of bacterial spread and host heterogeneity that have been proposed as mechanisms that may promote coexistence. We ask to what extent coexistence is a typical outcome in each. We find that while coexistence is possible in each of the models we consider, it is relatively rare, with two exceptions: (i) allowing simultaneous dual transmission of sensitive and resistant strains lets coexistence become a typical outcome, as does (ii) modelling each strain as competing more strongly with itself than with the other strain, i.e. self-immunity greater than cross-immunity. We conclude that while treatment and contact heterogeneity can promote coexistence to some extent, the in-host interactions between strains, particularly the interplay between coinfection, multiple infection and immunity, play a crucial role in the long-term population dynamics of pathogens with drug resistance. PMID:19940002

  20. Novel Phenotypic Outcomes Identified for a Public Collection of Approved Drugs from a Publicly Accessible Panel of Assays.

    Directory of Open Access Journals (Sweden)

    Jonathan A Lee

    Full Text Available Phenotypic assays have a proven track record for generating leads that become first-in-class therapies. Whole cell assays that inform on a phenotype or mechanism also possess great potential in drug repositioning studies by illuminating new activities for the existing pharmacopeia. The National Center for Advancing Translational Sciences (NCATS pharmaceutical collection (NPC is the largest reported collection of approved small molecule therapeutics that is available for screening in a high-throughput setting. Via a wide-ranging collaborative effort, this library was analyzed in the Open Innovation Drug Discovery (OIDD phenotypic assay modules publicly offered by Lilly. The results of these tests are publically available online at www.ncats.nih.gov/expertise/preclinical/pd2 and via the PubChem Database (https://pubchem.ncbi.nlm.nih.gov/ (AID 1117321. Phenotypic outcomes for numerous drugs were confirmed, including sulfonylureas as insulin secretagogues and the anti-angiogenesis actions of multikinase inhibitors sorafenib, axitinib and pazopanib. Several novel outcomes were also noted including the Wnt potentiating activities of rotenone and the antifolate class of drugs, and the anti-angiogenic activity of cetaben.

  1. characterization of drug resistant pseudomonas aeruginosa and ...

    African Journals Online (AJOL)

    Abstract: Lizards as well as some other reptiles have been known to carry pathogenic bacteria organisms as well as drug resistant pathogens. Despite the fact that they remain asymptomatic in many cases, they nevertheless play significant roles in the epidemiology of these pathogens through their dissemination to the ...

  2. Comparative evaluation of GenoType MTBDRplus line probe assay with solid culture method in early diagnosis of multidrug resistant tuberculosis (MDR-TB at a tertiary care centre in India.

    Directory of Open Access Journals (Sweden)

    Raj N Yadav

    Full Text Available The objectives of the study were to compare the performance of line probe assay (GenoType MTBDRplus with solid culture method for an early diagnosis of multidrug resistant tuberculosis (MDR-TB, and to study the mutation patterns associated with rpoB, katG and inhA genes at a tertiary care centre in north India.In this cross-sectional study, 269 previously treated sputum-smear acid-fast bacilli (AFB positive MDR-TB suspects were enrolled from January to September 2012 at the All India Institute of Medical Sciences hospital, New Delhi. Line probe assay (LPA was performed directly on the sputum specimens and the results were compared with that of conventional drug susceptibility testing (DST on solid media [Lowenstein Jensen (LJ method].DST results by LPA and LJ methods were compared in 242 MDR-TB suspects. The LPA detected rifampicin (RIF resistance in 70 of 71 cases, isoniazid (INH resistance in 86 of 93 cases, and MDR-TB in 66 of 68 cases as compared to the conventional method. Overall (rifampicin, isoniazid and MDR-TB concordance of the LPA with the conventional DST was 96%. Sensitivity and specificity were 98% and 99% respectively for detection of RIF resistance; 92% and 99% respectively for detection of INH resistance; 97% and 100% respectively for detection of MDR-TB. Frequencies of katG gene, inhA gene and combined katG and inhA gene mutations conferring all INH resistance were 72/87 (83%, 10/87 (11% and 5/87 (6% respectively. The turnaround time of the LPA test was 48 hours.The LPA test provides an early diagnosis of monoresistance to isoniazid and rifampicin and is highly sensitive and specific for an early diagnosis of MDR-TB. Based on these findings, it is concluded that the LPA test can be useful in early diagnosis of drug resistant TB in high TB burden countries.

  3. Clinical implications of molecular drug resistance testing for Mycobacterium tuberculosis: a TBNET/RESIST-TB consensus statement

    NARCIS (Netherlands)

    Domínguez, J.; Boettger, E. C.; Cirillo, D.; Cobelens, F.; Eisenach, K. D.; Gagneux, S.; Hillemann, D.; Horsburgh, R.; Molina-Moya, B.; Niemann, S.; Tortoli, E.; Whitelaw, A.; Lange, C.

    2016-01-01

    The emergence of drug-resistant strains of Mycobacterium tuberculosis is a challenge to global tuberculosis (TB) control. Although culture-based methods have been regarded as the gold standard for drug susceptibility testing (DST), molecular methods provide rapid information on mutations in the M.

  4. Monitoring of drug resistance amplification and attenuation with the use of tetracycline-resistant bacteria during wastewater treatment

    Science.gov (United States)

    Harnisz, Monika; Korzeniewska, Ewa; Niestępski, Sebastian; Osińska, Adriana; Nalepa, Beata

    2017-11-01

    The objective of this study was to monitor changes (amplification or attenuation) in antibiotic resistance during wastewater treatment based on the ecology of tetracycline-resistant bacteria. The untreated and treated wastewater were collected in four seasons. Number of tetracycline-(TETR) and oxytetracycline-resistant (OTCR) bacteria, their qualitative composition, minimum inhibitory concentrations (MICs), sensitivity to other antibiotics, and the presence of tet (A, B, C, D, E) resistance genes were determined. TETR and OTCR counts in untreated wastewater were 100 to 1000 higher than in treated effluent. OTCR bacterial counts were higher than TETR populations in both untreated and treated wastewater. TETR isolates were not dominated by a single bacterial genus or species, whereas Aeromonas hydrophila and Aeromonas sobria were the most common in OTCR isolates. The treatment process attenuated the drug resistance of TETR bacteria and amplified the resistance of OTCR bacteria. In both microbial groups, the frequency of tet(A) gene increased in effluent in comparison with untreated wastewater. Our results also indicate that treated wastewater is a reservoir of multiple drug-resistant bacteria as well as resistance determinants which may pose a health hazard for humans and animals when released to the natural environment.

  5. Antimicrobial activity of the bioactive components of essential oils from Pakistani spices against Salmonella and other multi-drug resistant bacteria

    Science.gov (United States)

    2013-01-01

    drug resistant clinical and soil bacterial strains. Cinnamaldehyde was identified as the most active antimicrobial component present in the cinnamon essential oil which acted as a strong inhibitory agent in MIC assay against the tested bacteria. The results indicate that essential oils from Pakistani spices can be pursued against multidrug resistant bacteria. PMID:24119438

  6. 78 FR 24425 - Assay Migration Studies for In Vitro Diagnostic Devices; Guidance for Industry and Food and Drug...

    Science.gov (United States)

    2013-04-25

    ... DEPARTMENT OF HEALTH AND HUMAN SERVICES Food and Drug Administration [Docket No. FDA-2008-D-0642] Assay Migration Studies for In Vitro Diagnostic Devices; Guidance for Industry and Food and Drug Administration Staff; Availability AGENCY: Food and Drug Administration, HHS. ACTION: Notice. SUMMARY: The Food...

  7. Comparison of Tetrazolium Salt Assays for Evaluation of Drug Activity against Leishmania spp.

    Science.gov (United States)

    Ginouves, Marine; Carme, Bernard; Couppie, Pierre

    2014-01-01

    In French Guiana, leishmaniasis is an essentially cutaneous infection. It constitutes a major public health problem, with a real incidence of 0.2 to 0.3%. Leishmania guyanensis is the causal species most frequently encountered in French Guiana. The treatment of leishmaniasis is essentially drug based, but the therapeutic compounds available have major side effects (e.g., liver damage and diabetes) and must be administered parenterally or are costly. The efficacy of some of these agents has declined due to the emergence of resistance in certain strains of Leishmania. There is currently no vaccine against leishmaniasis, and it is therefore both necessary and urgent to identify new compounds effective against Leishmania. The search for new drugs requires effective tests for evaluations of the leishmanicidal activity of a particular molecule or extract. Microculture tetrazolium assays (MTAs) are colorimetric tests based on the use of tetrazolium salts. We compared the efficacies of three tetrazolium salts—3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT), and 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-8)—for quantification of the promastigotes of various species of Leishmania. We found that the capacity of Leishmania to metabolize a tetrazolium salt depended on the salt used and the species of Leishmania. WST-8 was the tetrazolium salt best metabolized by L. guyanensis and gave the best sensitivity. PMID:24719447

  8. A Novel Multiplex HRM Assay to Detect Clopidogrel Resistance.

    Science.gov (United States)

    Zhang, Lichen; Ma, Xiaowei; You, Guoling; Zhang, Xiaoqing; Fu, Qihua

    2017-11-22

    Clopidogrel is an antiplatelet medicine used to prevent blood clots in patients who have had a heart attack, stroke, or other symptoms. Variability in the clinical response to clopidogrel treatment has been attributed to genetic factors. In particular, five SNPs of rs4244285, rs4986893, rs12248560, rs662 and rs1045642 have been associated with resistance to clopidogrel therapy in Chinese population. This work involves the development of a multiplex high-resolution melting (HRM) assay to genotype all five of these loci in 2 tubes. Amplicons corresponding to distinct SNPs in a common tube were designed with the aid of uMelt prediction software to have different melting temperatures Tm by addition of a GC-rich tail to the 5' end of the certain primers. Two kinds of commercial methods, Digital Fluorescence Molecular Hybridization (DFMH) and Sanger sequencing, were used as a control. Three hundred sixteen DFMH pretested samples from consecutive acute coronary syndrome patients were used for a blinded study of multiplex HRM. The sensitivity of HRM was 100% and the specificity was 99.93% reflecting detection of variants other than the known resistance SNPs. Multiplex HRM is an effective closed-tube, highly accurate, fast, and inexpensive method for genotyping the 5 clopidogrel resistance associated SNPs.

  9. [Survey on the transmission of HIV drug resistance in Kunming, Yunnan province in 2010].

    Science.gov (United States)

    Chen, Min; Ma, Yan-ling; Chu, Cheng-xia; Xing, Hui; Xu, Yan-sheng; Su, Ying-zhen; Yang, Ying; Chen, Hui-chao; Luo, Hong-bing; Jia, Man-hong; Lu, Lin

    2012-01-01

    To study the HIV drug resistance (HIVDR) transmission in Kunming city of Yunnan province in 2010. Referring to the guidelines for HIV drug resistance threshold survey (HIVDR-TS) set by WHO, 62 plasma samples of recently reported HIV-infected individuals who were older than 25 years of age, were collected from January to August 2010. Genotyping of pol genetic mutations associated with HIVDR with reverse transcriptional PCR was performed and the prevalence of HIV-1 drug resistance transmission was evaluated. Of the 62 plasma samples, 54 were successfully sequenced and genotyped on pol sequence. Based on the pol sequences, HIV subtypes including CRF08_BC (53.2%), CRF07_BC (25.5%), CRF01_AE (19.1%) and C (2.1%) were identified. According to the time of sampling, the first 47 sequenced samples were used for drug resistance prevalence analysis. A protease inhibitor (PI) relative mutation was found in one sample. Based on the WHO standard, the prevalence of transmitted HIV-1 drug resistance was scientific management to AIDS patients seemed to be quite important.

  10. Tenofovir alafenamide demonstrates broad cross-genotype activity against wild-type HBV clinical isolates and maintains susceptibility to drug-resistant HBV isolates in vitro.

    Science.gov (United States)

    Liu, Yang; Miller, Michael D; Kitrinos, Kathryn M

    2017-03-01

    Tenofovir alafenamide (TAF) is a novel prodrug of tenofovir (TFV). This study evaluated the antiviral activity of TAF against wild-type genotype A-H HBV clinical isolates as well as adefovir-resistant, lamivudine-resistant, and entecavir-resistant HBV isolates. Full length HBV genomes or the polymerase/reverse transcriptase (pol/RT) region from treatment-naïve patients infected with HBV genotypes A-H were amplified and cloned into an expression vector under the control of a CMV promoter. In addition, 11 drug resistant HBV constructs were created by site-directed mutagenesis of a full length genotype D construct. Activity of TAF was measured by transfection of each construct into HepG2 cells and assessment of HBV DNA levels following treatment across a range of TAF concentrations. TAF activity in vitro was similar against wild-type genotype A-H HBV clinical isolates. All lamivudine- and entecavir-resistant isolates and 4/5 adefovir-resistant isolates were found to be sensitive to inhibition by TAF in vitro as compared to the wild-type isolate. The adefovir-resistant isolate rtA181V + rtN236T exhibited low-level reduced susceptibility to TAF. TAF is similarly active in vitro against wild-type genotype A-H HBV clinical isolates. The TAF sensitivity results for all drug-resistant isolates are consistent with what has been observed with the parent drug TFV. The in vitro cell-based HBV phenotyping assay results support the use of TAF in treatment of HBV infected subjects with diverse HBV genotypes, in both treatment-naive and treatment-experienced HBV infected patients. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. The potential impact of density dependent fecundity on the use of the faecal egg count reduction test for detecting drug resistance in human hookworms.

    Directory of Open Access Journals (Sweden)

    Andrew C Kotze

    from a FECRT, as these cases may have the greatest potential to increase their egg output upon removal of density dependent constraints. The potential impact of worm reproductive biology on the utility of the FECRT as a resistance detection tool highlights the need to develop new drug resistance monitoring methods which examine either direct drug effects on isolated worms with in vitro phenotypic assays, or changes in worm genotypes.

  12. Efficacy of verapamil as an adjunctive treatment in children with drug-resistant epilepsy

    DEFF Research Database (Denmark)

    Nicita, Francesco; Spalice, Alberto; Papetti, Laura

    2014-01-01

    Verapamil, a voltage-gated calcium channel blocker, has been occasionally reported to have some effect on reducing seizure frequency in drug-resistant epilepsy or status epilepticus. We aimed to investigate the efficacy of verapamil as add-on treatment in children with drug-resistant epilepsy....

  13. Optimization of a Fluorescence-Based Assay for Large-Scale Drug Screening against Babesia and Theileria Parasites.

    Science.gov (United States)

    Rizk, Mohamed Abdo; El-Sayed, Shimaa Abd El-Salam; Terkawi, Mohamed Alaa; Youssef, Mohamed Ahmed; El Said, El Said El Shirbini; Elsayed, Gehad; El-Khodery, Sabry; El-Ashker, Maged; Elsify, Ahmed; Omar, Mosaab; Salama, Akram; Yokoyama, Naoaki; Igarashi, Ikuo

    2015-01-01

    A rapid and accurate assay for evaluating antibabesial drugs on a large scale is required for the discovery of novel chemotherapeutic agents against Babesia parasites. In the current study, we evaluated the usefulness of a fluorescence-based assay for determining the efficacies of antibabesial compounds against bovine and equine hemoparasites in in vitro cultures. Three different hematocrits (HCTs; 2.5%, 5%, and 10%) were used without daily replacement of the medium. The results of a high-throughput screening assay revealed that the best HCT was 2.5% for bovine Babesia parasites and 5% for equine Babesia and Theileria parasites. The IC50 values of diminazene aceturate obtained by fluorescence and microscopy did not differ significantly. Likewise, the IC50 values of luteolin, pyronaridine tetraphosphate, nimbolide, gedunin, and enoxacin did not differ between the two methods. In conclusion, our fluorescence-based assay uses low HCT and does not require daily replacement of culture medium, making it highly suitable for in vitro large-scale drug screening against Babesia and Theileria parasites that infect cattle and horses.

  14. Optimization of a Fluorescence-Based Assay for Large-Scale Drug Screening against Babesia and Theileria Parasites.

    Directory of Open Access Journals (Sweden)

    Mohamed Abdo Rizk

    Full Text Available A rapid and accurate assay for evaluating antibabesial drugs on a large scale is required for the discovery of novel chemotherapeutic agents against Babesia parasites. In the current study, we evaluated the usefulness of a fluorescence-based assay for determining the efficacies of antibabesial compounds against bovine and equine hemoparasites in in vitro cultures. Three different hematocrits (HCTs; 2.5%, 5%, and 10% were used without daily replacement of the medium. The results of a high-throughput screening assay revealed that the best HCT was 2.5% for bovine Babesia parasites and 5% for equine Babesia and Theileria parasites. The IC50 values of diminazene aceturate obtained by fluorescence and microscopy did not differ significantly. Likewise, the IC50 values of luteolin, pyronaridine tetraphosphate, nimbolide, gedunin, and enoxacin did not differ between the two methods. In conclusion, our fluorescence-based assay uses low HCT and does not require daily replacement of culture medium, making it highly suitable for in vitro large-scale drug screening against Babesia and Theileria parasites that infect cattle and horses.

  15. Direct nitrate reductase assay versus microscopic observation drug susceptibility test for rapid detection of MDR-TB in Uganda.

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    Freddie Bwanga

    Full Text Available The most common method for detection of drug resistant (DR TB in resource-limited settings (RLSs is indirect susceptibility testing on Lowenstein-Jensen medium (LJ which is very time consuming with results available only after 2-3 months. Effective therapy of DR TB is therefore markedly delayed and patients can transmit resistant strains. Rapid and accurate tests suitable for RLSs in the diagnosis of DR TB are thus highly needed. In this study we compared two direct techniques--Nitrate Reductase Assay (NRA and Microscopic Observation Drug Susceptibility (MODS for rapid detection of MDR-TB in a high burden RLS. The sensitivity, specificity, and proportion of interpretable results were studied. Smear positive sputum was collected from 245 consecutive re-treatment TB patients attending a TB clinic in Kampala, Uganda. Samples were processed at the national reference laboratory and tested for susceptibility to rifampicin and isoniazid with direct NRA, direct MODS and the indirect LJ proportion method as reference. A total of 229 specimens were confirmed as M. tuberculosis, of these interpretable results were obtained in 217 (95% with either the NRA or MODS. Sensitivity, specificity and kappa agreement for MDR-TB diagnosis was 97%, 98% and 0.93 with the NRA; and 87%, 95% and 0.78 with the MODS, respectively. The median time to results was 10, 7 and 64 days with NRA, MODS and the reference technique, respectively. The cost of laboratory supplies per sample was low, around 5 USD, for the rapid tests. The direct NRA and MODS offered rapid detection of resistance almost eight weeks earlier than with the reference method. In the study settings, the direct NRA was highly sensitive and specific. We consider it to have a strong potential for timely detection of MDR-TB in RLS.

  16. Treatment of Multidrug-Resistant Leukemia Cells by Novel Artemisinin-, Egonol-, and Thymoquinone-Derived Hybrid Compounds

    Directory of Open Access Journals (Sweden)

    Lisa Gruber

    2018-04-01

    Full Text Available Two major obstacles for successful cancer treatment are the toxicity of cytostatics and the development of drug resistance in cancer cells during chemotherapy. Acquired or intrinsic drug resistance is responsible for almost 90% of treatment failure. For this reason, there is an urgent need for new anticancer drugs with improved efficacy against cancer cells, and with less toxicity on normal cells. There are impressive examples demonstrating the success of natural plant compounds to fight cancer, such as Vinca alkaloids, taxanes, and anthracyclines. Artesunic acid (ARTA, a drug for malaria treatment, also exerts cytotoxic activity towards cancer cells. Multidrug resistance often results from drug efflux pumps (ABC-transporters that reduce intracellular drug levels. Hence, it would be interesting to know, whether ARTA could overcome drug resistance of tumor cells, and in what way ABC-transporters are involved. Different derivatives showing improved features concerning cytotoxicity and pharmacokinetic behavior have been developed. Considering both drug sensitivity and resistance, we chose a sensitive and a doxorubicin-resistant leukemia cell line and determined the killing effect of ARTA on these cells. Molecular docking and doxorubicin efflux assays were performed to investigate the interaction of the derivatives with P-glycoprotein. Using single-cell gel electrophoresis (alkaline comet assay, we showed that the derivatives of ARTA induce DNA breakage and accordingly programmed cell death, which represents a promising strategy in cancer treatment. ARTA activated apoptosis in cancer cells by the iron-mediated generation of reactive oxygen species (ROS. In conclusion, ARTA derivatives may bear the potential to be further developed as anticancer drugs.

  17. Consensus Statement on Research Definitions for Drug-Resistant Tuberculosis in Children.

    Science.gov (United States)

    Seddon, James A; Perez-Velez, Carlos M; Schaaf, H Simon; Furin, Jennifer J; Marais, Ben J; Tebruegge, Marc; Detjen, Anne; Hesseling, Anneke C; Shah, Sarita; Adams, Lisa V; Starke, Jeffrey R; Swaminathan, Soumya; Becerra, Mercedes C

    2013-06-01

    Few children with drug-resistant (DR) tuberculosis (TB) are identified, diagnosed, and given an appropriate treatment. The few studies that have described this vulnerable population have used inconsistent definitions. The World Health Organization (WHO) definitions used for adults with DR-TB and for children with drug-susceptible TB are not always appropriate for children with DR-TB. The Sentinel Project on Pediatric Drug-Resistant Tuberculosis was formed in 2011 as a network of experts and stakeholders in childhood DR-TB. An early priority was to establish standardized definitions for key parameters in order to facilitate study comparisons and the development of an evidence base to guide future clinical management. This consensus statement proposes standardized definitions to be used in research. In particular, it suggests consistent terminology, as well as definitions for measures of exposure, drug resistance testing, previous episodes and treatment, certainty of diagnosis, site and severity of disease, adverse events, and treatment outcome. © The Author 2013. Published by Oxford University Press on behalf of the Pediatric Infectious Diseases Society.

  18. Acid-fast bacilli culture positivity and drug resistance in abdominal tuberculosis in Mumbai, India.

    Science.gov (United States)

    Samant, Hrishikesh; Desai, Devendra; Abraham, Philip; Joshi, Anand; Gupta, Tarun; Rodrigues, Camilla; George, Siji

    2014-09-01

    Culture positivity for Mycobacterium tuberculosis complex (MTB) in abdominal tuberculosis (TB) using Lowenstein Jensen medium and Bactec system varies from 25 % to 36 %. Data on the prevalence of drug resistance in primary abdominal TB is scant. Our aim was to study the acid-fast bacilli (AFB) culture positivity rate in primary abdominal TB using Bactec Mycobacterial Growth Indicator Tubes (MGIT) system and the prevalence of drug resistance in these patients. Records of patients with abdominal TB (diagnosed on clinical features, endoscopy, histology, microbiology) seen during the period 2008 to 2013 were retrieved from the Gastroenterology and Microbiology departments. Patients with extra-abdominal TB (five pulmonary, two nodal), adnexal (one), and HIV (one) were excluded from analysis. Of 61 patients, 31 (50.8 %) had a positive AFB culture. In the 30 culture-negative patients, histology showed non-caseating granulomas in 25 patients. Drug sensitivity pattern was analyzed in 18 patients; resistance was detected in eight (14.3 % of all patients and 44.4 % of patients in whom drug sensitivity was done) including three (5.4 % of all subjects and 16.6 % in whom drug sensitivity was available) who were multidrug-resistant. The rate of AFB culture positivity in primary abdominal TB was 50.8 % using Bactec MGIT. Likelihood of drug resistance was seen in 14.3 %, of whom 5.4 % were multidrug-resistant.

  19. Potential of berberine to enhance antimicrobial activity of commonly used antibiotics for dairy cow mastitis caused by multiple drug-resistant Staphylococcus epidermidis infection.

    Science.gov (United States)

    Zhou, X; Yang, C; Li, Y; Liu, X; Wang, Y

    2015-08-19

    Berberine is a plant alkaloid with antimicrobial activity against a variety of microorganisms. In this study, the antimicrobial properties of berberine against multi-drug resistant field isolates of Staphylococcus epidermidis were investigated using berberine alone or in combination with a commonly used antibiotics in veterinary clinics, including penicillin, lincomycin, and amoxicillin. The results indicated that the minimum inhibitory concentrations of berberine, penicillin, lincomycin, and amoxicillin against field S. epidermidis isolates were 2-512, 0.8-213, 0.4-1024, and 0.4-256 mg/mL, respectively. Furthermore, the synergistic effects of antimicrobial activity against these multi-drug resistant isolates were observed when the berberine was combined with penicillin, lincomycin, or amoxicillin; no antagonistic effect of the combination was detected in any of the clinical isolates. These observations were further confirmed using a time-killing assay, in which a combination of 2 agents yielded a greater than 2.03-2.44 log10 decrease in colony-forming unit/mL compared with each agent alone. These findings suggest that berberine is a promising compound for preventing and treating multi-drug resistant S. epidermidis infected mastitis in dairy cows either alone or in combination with other commonly used antibiotics, such as penicillin, lincomycin, and amoxicillin.

  20. Use of Lot Quality Assurance Sampling to Ascertain Levels of Drug Resistant Tuberculosis in Western Kenya.

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    Julia Jezmir

    Full Text Available To classify the prevalence of multi-drug resistant tuberculosis (MDR-TB in two different geographic settings in western Kenya using the Lot Quality Assurance Sampling (LQAS methodology.The prevalence of drug resistance was classified among treatment-naïve smear positive TB patients in two settings, one rural and one urban. These regions were classified as having high or low prevalence of MDR-TB according to a static, two-way LQAS sampling plan selected to classify high resistance regions at greater than 5% resistance and low resistance regions at less than 1% resistance.This study classified both the urban and rural settings as having low levels of TB drug resistance. Out of the 105 patients screened in each setting, two patients were diagnosed with MDR-TB in the urban setting and one patient was diagnosed with MDR-TB in the rural setting. An additional 27 patients were diagnosed with a variety of mono- and poly- resistant strains.Further drug resistance surveillance using LQAS may help identify the levels and geographical distribution of drug resistance in Kenya and may have applications in other countries in the African Region facing similar resource constraints.

  1. Use of Lot Quality Assurance Sampling to Ascertain Levels of Drug Resistant Tuberculosis in Western Kenya.

    Science.gov (United States)

    Jezmir, Julia; Cohen, Ted; Zignol, Matteo; Nyakan, Edwin; Hedt-Gauthier, Bethany L; Gardner, Adrian; Kamle, Lydia; Injera, Wilfred; Carter, E Jane

    2016-01-01

    To classify the prevalence of multi-drug resistant tuberculosis (MDR-TB) in two different geographic settings in western Kenya using the Lot Quality Assurance Sampling (LQAS) methodology. The prevalence of drug resistance was classified among treatment-naïve smear positive TB patients in two settings, one rural and one urban. These regions were classified as having high or low prevalence of MDR-TB according to a static, two-way LQAS sampling plan selected to classify high resistance regions at greater than 5% resistance and low resistance regions at less than 1% resistance. This study classified both the urban and rural settings as having low levels of TB drug resistance. Out of the 105 patients screened in each setting, two patients were diagnosed with MDR-TB in the urban setting and one patient was diagnosed with MDR-TB in the rural setting. An additional 27 patients were diagnosed with a variety of mono- and poly- resistant strains. Further drug resistance surveillance using LQAS may help identify the levels and geographical distribution of drug resistance in Kenya and may have applications in other countries in the African Region facing similar resource constraints.

  2. Simple strategy to assess linezolid exposure in patients with multi-drug-resistant and extensively-drug-resistant tuberculosis.

    Science.gov (United States)

    Kamp, Jasper; Bolhuis, Mathieu S; Tiberi, Simon; Akkerman, Onno W; Centis, Rosella; de Lange, Wiel C; Kosterink, Jos G; van der Werf, Tjip S; Migliori, Giovanni B; Alffenaar, Jan-Willem C

    2017-06-01

    Linezolid is used increasingly for the treatment of multi-drug-resistant (MDR) and extensively-drug-resistant (XDR) tuberculosis (TB). However, linezolid can cause severe adverse events, such as peripheral and optical neuropathy or thrombocytopenia related to higher drug exposure. This study aimed to develop a population pharmacokinetic model to predict the area under the concentration curve (AUC) for linezolid using a limited number of blood samples. Data from patients with MDR-/XDR-TB who received linezolid and therapeutic drug monitoring as part of their TB treatment were used. Mw\\Pharm 3.82 (Mediware, Zuidhorn, The Netherlands) was used to develop a population pharmacokinetic model and limited sampling strategy (LSS) for linezolid. LSS was evaluated over a time span of 6 h. Blood sampling directly before linezolid administration and 2 h after linezolid administration were considered to be the most clinically relevant sampling points. The model and LSS were evaluated by analysing the correlation between AUC 12h,observed and AUC 12h,estimated . In addition, LSS was validated with an external group of patients with MDR-/XDR-TB from Sondalo, Italy. Fifty-two pharmacokinetic profiles were used to develop the model. Thirty-three profiles with a 300 mg dosing regimen and 19 profiles with a 600 mg dosing regimen were obtained. Model validation showed prediction bias of 0.1% and r 2 of 0.99. Evaluation of the most clinically relevant LSS showed prediction bias of 4.8% and r 2 of 0.97. The root mean square error corresponding to the most relevant LSS was 6.07%. The developed LSS could be used to enable concentration-guided dosing of linezolid. Copyright © 2017 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.

  3. Different frequencies of drug resistance mutations among HIV-1 subtypes circulating in China: a comprehensive study.

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    Hongshuai Sui

    Full Text Available The rapid spreading of HIV drug resistance is threatening the overall success of free HAART in China. Much work has been done on drug-resistant mutations, however, most of which were based on subtype B. Due to different genetic background, subtypes difference would have an effect on the development of drug-resistant mutations, which has already been proved by more and more studies. In China, the main epidemic subtypes are CRF07_BC, CRF08_BC, Thai B and CRF01_AE. The depiction of drug resistance mutations in those subtypes will be helpful for the selection of regimens for Chinese. In this study, the distributions difference of amino acids at sites related to HIV drug resistance were compared among subtype B, CRF01_AE, CRF07_BC and CRF08_BC strains prevalent in China. The amino acid composition of sequences belonging to different subtypes, which were obtained from untreated and treated individuals separately, were also compared. The amino acids proportions of 19 sites in RT among subtype B, CRF01_AE and CRF08_BC have significant difference in drug resistance groups (chi-square test, p<0.05. Genetic barriers analysis revealed that sites 69, 138, 181, 215 and 238 were significantly different among subtypes (Kruskal Wallis test, p<0.05. All subtypes shared three highest prevalent drug resistance sites 103, 181 and 184 in common. Many drug resistant sites in protease show surprising high proportions in almost all subtypes in drug-naïve patients. This is the first comprehensive study in China on different development of drug resistance among different subtypes. The detailed data will lay a foundation for HIV treatment regimens design and improve HIV therapy in China.

  4. Low-abundance HIV drug-resistant viral variants in treatment-experienced persons correlate with historical antiretroviral use.

    Science.gov (United States)

    Le, Thuy; Chiarella, Jennifer; Simen, Birgitte B; Hanczaruk, Bozena; Egholm, Michael; Landry, Marie L; Dieckhaus, Kevin; Rosen, Marc I; Kozal, Michael J

    2009-06-29

    It is largely unknown how frequently low-abundance HIV drug-resistant variants at levels under limit of detection of conventional genotyping (<20% of quasi-species) are present in antiretroviral-experienced persons experiencing virologic failure. Further, the clinical implications of low-abundance drug-resistant variants at time of virologic failure are unknown. Plasma samples from 22 antiretroviral-experienced subjects collected at time of virologic failure (viral load 1380 to 304,000 copies/mL) were obtained from a specimen bank (from 2004-2007). The prevalence and profile of drug-resistant mutations were determined using Sanger sequencing and ultra-deep pyrosequencing. Genotypes were interpreted using Stanford HIV database algorithm. Antiretroviral treatment histories were obtained by chart review and correlated with drug-resistant mutations. Low-abundance drug-resistant mutations were detected in all 22 subjects by deep sequencing and only in 3 subjects by Sanger sequencing. In total they accounted for 90 of 247 mutations (36%) detected by deep sequencing; the majority of these (95%) were not detected by standard genotyping. A mean of 4 additional mutations per subject were detected by deep sequencing (p<0.0001, 95%CI: 2.85-5.53). The additional low-abundance drug-resistant mutations increased a subject's genotypic resistance to one or more antiretrovirals in 17 of 22 subjects (77%). When correlated with subjects' antiretroviral treatment histories, the additional low-abundance drug-resistant mutations correlated with the failing antiretroviral drugs in 21% subjects and correlated with historical antiretroviral use in 79% subjects (OR, 13.73; 95% CI, 2.5-74.3, p = 0.0016). Low-abundance HIV drug-resistant mutations in antiretroviral-experienced subjects at time of virologic failure can increase a subject's overall burden of resistance, yet commonly go unrecognized by conventional genotyping. The majority of unrecognized resistant mutations correlate with

  5. Low-abundance HIV drug-resistant viral variants in treatment-experienced persons correlate with historical antiretroviral use.

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    Thuy Le

    Full Text Available BACKGROUND: It is largely unknown how frequently low-abundance HIV drug-resistant variants at levels under limit of detection of conventional genotyping (<20% of quasi-species are present in antiretroviral-experienced persons experiencing virologic failure. Further, the clinical implications of low-abundance drug-resistant variants at time of virologic failure are unknown. METHODOLOGY/PRINCIPAL FINDINGS: Plasma samples from 22 antiretroviral-experienced subjects collected at time of virologic failure (viral load 1380 to 304,000 copies/mL were obtained from a specimen bank (from 2004-2007. The prevalence and profile of drug-resistant mutations were determined using Sanger sequencing and ultra-deep pyrosequencing. Genotypes were interpreted using Stanford HIV database algorithm. Antiretroviral treatment histories were obtained by chart review and correlated with drug-resistant mutations. Low-abundance drug-resistant mutations were detected in all 22 subjects by deep sequencing and only in 3 subjects by Sanger sequencing. In total they accounted for 90 of 247 mutations (36% detected by deep sequencing; the majority of these (95% were not detected by standard genotyping. A mean of 4 additional mutations per subject were detected by deep sequencing (p<0.0001, 95%CI: 2.85-5.53. The additional low-abundance drug-resistant mutations increased a subject's genotypic resistance to one or more antiretrovirals in 17 of 22 subjects (77%. When correlated with subjects' antiretroviral treatment histories, the additional low-abundance drug-resistant mutations correlated with the failing antiretroviral drugs in 21% subjects and correlated with historical antiretroviral use in 79% subjects (OR, 13.73; 95% CI, 2.5-74.3, p = 0.0016. CONCLUSIONS/SIGNIFICANCE: Low-abundance HIV drug-resistant mutations in antiretroviral-experienced subjects at time of virologic failure can increase a subject's overall burden of resistance, yet commonly go unrecognized by conventional

  6. Venetoclax (ABT-199) Might Act as a Perpetrator in Pharmacokinetic Drug-Drug Interactions.

    Science.gov (United States)

    Weiss, Johanna; Gajek, Thomas; Köhler, Bruno Christian; Haefeli, Walter Emil

    2016-02-24

    Venetoclax (ABT-199) represents a specific B-cell lymphoma 2 (Bcl-2) inhibitor that is currently under development for the treatment of lymphoid malignancies. So far, there is no published information on its interaction potential with important drug metabolizing enzymes and drug transporters, or its efficacy in multidrug resistant (MDR) cells. We therefore scrutinized its drug-drug interaction potential in vitro. Inhibition of cytochrome P450 enzymes (CYPs) was quantified by commercial kits. Inhibition of drug transporters (P-glycoprotein (P-gp, ABCB1), breast cancer resistance protein (BCRP), and organic anion transporting polypeptides (OATPs)) was evaluated by the use of fluorescent probe substrates. Induction of drug transporters and drug metabolizing enzymes was quantified by real-time RT-PCR. The efficacy of venetoclax in MDR cells lines was evaluated with proliferation assays. Venetoclax moderately inhibited P-gp, BCRP, OATP1B1, OATP1B3, CYP3A4, and CYP2C19, whereas CYP2B6 activity was increased. Venetoclax induced the mRNA expression of CYP1A1, CYP1A2, UGT1A3, and UGT1A9. In contrast, expression of ABCB1 was suppressed, which might revert tumor resistance towards antineoplastic P-gp substrates. P-gp over-expression led to reduced antiproliferative effects of venetoclax. Effective concentrations for inhibition and induction lay in the range of maximum plasma concentrations of venetoclax, indicating that it might act as a perpetrator drug in pharmacokinetic drug-drug interactions.

  7. High prevalence of multidrug-resistant tuberculosis among patients with rifampicin resistance using GeneXpert Mycobacterium tuberculosis/rifampicin in Ghana.

    Science.gov (United States)

    Boakye-Appiah, Justice K; Steinmetz, Alexis R; Pupulampu, Peter; Ofori-Yirenkyi, Stephen; Tetteh, Ishmael; Frimpong, Michael; Oppong, Patrick; Opare-Sem, Ohene; Norman, Betty R; Stienstra, Ymkje; van der Werf, Tjip S; Wansbrough-Jones, Mark; Bonsu, Frank; Obeng-Baah, Joseph; Phillips, Richard O

    2016-06-01

    Drug-resistant strains of tuberculosis (TB) represent a major threat to global TB control. In low- and middle-income countries, resource constraints make it difficult to identify and monitor cases of resistance using drug susceptibility testing and culture. Molecular assays such as the GeneXpert Mycobacterium tuberculosis/rifampicin may prove to be a cost-effective solution to this problem in these settings. The objective of this study is to evaluate the use of GeneXpert in the diagnosis of pulmonary TB since it was introduced into two tertiary hospitals in Ghana in 2013. A 2-year retrospective audit of clinical cases involving patients who presented with clinically suspected TB or documented TB not improving on standard therapy and had samples sent for GeneXpert testing. GeneXpert identified 169 cases of TB, including 17 cases of rifampicin-resistant TB. Of the seven cases with final culture and drug susceptibility testing results, six demonstrated further drug resistance and five of these were multidrug-resistant TB. These findings call for a scale-up of TB control in Ghana and provide evidence that the expansion of GeneXpert may be an optimal means to improve case finding and guide treatment of drug-resistant TB in this setting. Copyright © 2016. Published by Elsevier Ltd.

  8. Effects of 5-fluorouracil on biological characteristics and drug resistance mechanisms of liver cancer cell line PLC/RAF/5

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    CHENG Kangwen

    2015-09-01

    Full Text Available ObjectiveTo study the changes in biological characteristics of a liver cancer cell line PLC/RAF/5 after repeated exposure to a chemotherapy drug, 5-fluorouraci (5-FU, and to investigate the relationship between drug-resistant liver cancer cells and liver cancer stem cells. MethodsA low concentration of 5-FU (1 μg/ml was used to treat the human liver cancer cell line PLC/RAF/5 repeatedly to establish the PLC/RAF/5/5-FU cell line. Morphological differences between the two types of cells were observed. The inhibitory effects of different concentrations of 5-FU (0, 0.25, 0.5, 1, 1.5, and 2 μg/ml on the proliferation of the two types of cells were determined using the CCK-8 assay. Apoptosis of the two types of cells after exposure to different concentrations of 5-FU (0.5, 1, and 2 μg/ml for 48 h was analyzed using flow cytometry. The proportions of side population cells in both types of cells were measured using flow cytometry. The colony-forming ability was compared between the two types of cells by the plate colony-forming assay. The expression of Bax, Bcl-2, ABCG2, and FoxM1 proteins in both types of cells was examined by Western blot. Between-group comparison was performed by t test. ResultsThe PLC/RAF/5/5-FU cell line was successfully established using the chemotherapy drug 5-FU. Compared with the PLC/RAF/5 cells, the PLC/RAF/5/5-FU cells had a larger volume, fewer protrusions, a changed shape of a long shuttle, and enhanced refractivity. Moreover, compared with the parent cells, the PLC/RAF/5/5-FU cells had a significantly lower sensitivity to the inhibitory effect of 5-FU on proliferation, a significantly lower proportion of cells at the G0/G1 phase of the cell cycle, significantly higher proportions of cells at the S and G2/M phases, significantly higher resistance to apoptosis, a significantly higher proportion of side population cells, and significantly enhanced proliferation (P<0.05. According to the results of Western blot assay, the

  9. Drug resistance-related mutations in multidrug-resistant Mycobacterium tuberculosis isolates from diverse geographical regions

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    Senia Rosales-Klintz

    2012-01-01

    Conclusion: This study confirms that there are significant geographical differences in the distribution of resistance-related mutations and suggests that an increased understanding of such differences in the specific distribution of resistance conferring mutations is crucial for development of new, generally applicable, molecular tools for rapid diagnosis of drug-resistant TB. The fact that a narrower distribution of mutations in high MDR-TB prevalence settings was seen suggests that much of the problems in these settings can be a result of an ongoing transmission of certain MDR-TB strains.

  10. HIV drug resistance in infants increases with changing prevention of mother-to-child transmission regimens.

    Science.gov (United States)

    Poppe, Lisa K; Chunda-Liyoka, Catherine; Kwon, Eun H; Gondwe, Clement; West, John T; Kankasa, Chipepo; Ndongmo, Clement B; Wood, Charles

    2017-08-24

    The objectives of this study were to determine HIV drug resistance (HIVDR) prevalence in Zambian infants upon diagnosis, and to determine how changing prevention of mother-to-child transmission (PMTCT) drug regimens affect drug resistance. Dried blood spot (DBS) samples from infants in the Lusaka District of Zambia, obtained during routine diagnostic screening, were collected during four different years representing three different PMTCT drug treatment regimens. DNA extracted from dried blood spot samples was used to sequence a 1493 bp region of the reverse transcriptase gene. Sequences were analyzed via the Stanford HIVDRdatabase (http://hivdb.standford.edu) to screen for resistance mutations. HIVDR in infants increased from 21.5 in 2007/2009 to 40.2% in 2014. Nonnucleoside reverse transcriptase inhibitor resistance increased steadily over the sampling period, whereas nucleoside reverse transcriptase inhibitor resistance and dual class resistance both increased more than threefold in 2014. Analysis of drug resistance scores in each group revealed increasing strength of resistance over time. In 2014, children with reported PMTCT exposure, defined as infant prophylaxis and/or maternal treatment, showed a higher prevalence and strength of resistance compared to those with no reported exposure. HIVDR is on the rise in Zambia and presents a serious problem for the successful lifelong treatment of HIV-infected children. PMTCT affects both the prevalence and strength of resistance and further research is needed to determine how to mitigate its role leading to resistance.

  11. Drug resistant Salmonella in broiler chicken sold at local market in ...

    African Journals Online (AJOL)

    user

    2015-10-28

    Oct 28, 2015 ... Key words: Antibiogram, Salmonellosis, PCR, broiler chicken, drug resistance. ... of zoonotic origin and have gained their resistance in an animal host ..... dynamics of Salmonella enterica serotypes in commercial egg and.

  12. Drug-resistant tuberculosis among HIV-infected patients starting antiretroviral therapy in Durban, South Africa.

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    Jeffrey K Hom

    Full Text Available To estimate the prevalence of drug-resistant tuberculosis (TB and describe the resistance patterns in patients commencing antiretroviral therapy (ART in an HIV clinic in Durban, South Africa.Cross-sectional cohort study.Consecutive HIV-infected adults (≥ 18y/o initiating HIV care were enrolled from May 2007-May 2008, regardless of signs or symptoms of active TB. Prior TB history and current TB treatment status were self-reported. Subjects expectorated sputum for culture (MGIT liquid and 7H11 solid medium. Positive cultures were tested for susceptibility to first- and second-line anti-tuberculous drugs. The prevalence of drug-resistant TB, stratified by prior TB history and current TB treatment status, was assessed.1,035 subjects had complete culture results. Median CD4 count was 92/µl (IQR 42-150/µl. 267 subjects (26% reported a prior history of TB and 210 (20% were receiving TB treatment at enrollment; 191 (18% subjects had positive sputum cultures, among whom the estimated prevalence of resistance to any antituberculous drug was 7.4% (95% CI 4.0-12.4. Among those with prior TB, the prevalence of resistance was 15.4% (95% CI 5.9-30.5 compared to 5.2% (95% CI 2.1-8.9 among those with no prior TB. 5.1% (95% CI 2.4-9.5 had rifampin or rifampin plus INH resistance.The prevalence of TB resistance to at least one drug was 7.4% among adults with positive TB cultures initiating ART in Durban, South Africa, with 5.1% having rifampin or rifampin plus INH resistance. Improved tools for diagnosing TB and drug resistance are urgently needed in areas of high HIV/TB prevalence.

  13. Improved Tumor-Specific Drug Accumulation by Polymer Therapeutics with pH-Sensitive Drug Release Overcomes Chemotherapy Resistance.

    Science.gov (United States)

    Heinrich, Anne-Kathrin; Lucas, Henrike; Schindler, Lucie; Chytil, Petr; Etrych, Tomáš; Mäder, Karsten; Mueller, Thomas

    2016-05-01

    The success of chemotherapy is limited by poor selectivity of active drugs combined with occurrence of tumor resistance. New star-like structured N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer-based drug delivery systems containing doxorubicin attached via a pH-sensitive hydrazone bond were designed and investigated for their ability to overcome chemotherapy resistance. These conjugates combine two strategies to achieve a high drug concentration selectively at the tumor site: (I) high accumulation by passive tumor targeting based on enhanced permeability and retention effect and (II) pH-sensitive site-specific drug release due to an acidic tumor microenvironment. Mice bearing doxorubicin-resistant xenograft tumors were treated with doxorubicin, PBS, poly HPMA (pHPMA) precursor or pHPMA-doxorubicin conjugate at different equivalent doses of 5 mg/kg bodyweight doxorubicin up to a 7-fold total dose using different treatment schedules. Intratumoral drug accumulation was analyzed by fluorescence imaging utilizing intrinsic fluorescence of doxorubicin. Free doxorubicin induced significant toxicity but hardly any tumor-inhibiting effects. Administering at least a 3-fold dose of pHPMA-doxorubicin conjugate was necessary to induce a transient response, whereas doses of about 5- to 6-fold induced strong regressions. Tumors completely disappeared in some cases. The onset of response was differential delayed depending on the tumor model, which could be ascribed to distinct characteristics of the microenvironment. Further fluorescence imaging-based analyses regarding underlying mechanisms of the delayed response revealed a related switch to a more supporting intratumoral microenvironment for effective drug release. In conclusion, the current study demonstrates that the concept of tumor site-restricted high-dose chemotherapy is able to overcome therapy resistance. Mol Cancer Ther; 15(5); 998-1007. ©2016 AACR. ©2016 American Association for Cancer Research.

  14. Enzymatic assays for detecting lactose and sucrose in urine to reveal intravenous drug abuse with emphasis on buprenorphine.

    Science.gov (United States)

    Keltanen, T; Mariottini, C; Walta, A M; Rahikainen, A L; Ojanperä, I

    2017-06-01

    Buprenorphine and methadone are commonly used medications for opioid maintenance treatment (OMT), using sublingual and oral administration, respectively. Although beneficial for OMT, these drugs can also be abused by intravenous administration. In intravenous abuse cases, the adjuvants lactose and sucrose are excreted in urine without hydrolysis to monosaccharides, since there are no disaccharidases in the blood. We validated enzymatic methods for the analysis of lactose and sucrose in urine. The analytical performance of both assays was considered appropriate for detecting intravenous drug abuse. The principle was proven by analyzing 93 postmortem (PM) urine samples for lactose, following comprehensive toxicological drug screening. In addition, 32 clinical urine samples from potential drug abusers were analyzed to assess the effect of PM changes on the assay. The mean level of lactose was low in clinical samples and relatively low in PM samples in which no drugs were found. Markedly elevated levels were seen in many of the buprenorphine positive samples, suggesting intravenous administration. Enzymatic methods could provide a simple and cost effective way to assess the intravenous administration of OMT drugs or drugs of abuse. Very high levels of glucose in urine may interfere with the assays. Furthermore, other causes for elevated urine disaccharides, such as hypolactasia and increased intestinal permeability, need to be considered in the interpretation of the results. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  15. Predicted levels of HIV drug resistance

    DEFF Research Database (Denmark)

    Cambiano, Valentina; Bertagnolio, Silvia; Jordan, Michael R

    2014-01-01

    -term effects. METHODS: The previously validated HIV Synthesis model was calibrated to South Africa. Resistance was modeled at the level of single mutations, transmission potential, persistence, and effect on drug activity. RESULTS: We estimate 652 000 people (90% uncertainty range: 543 000-744 000) are living...... are maintained, in 20 years' time HIV incidence is projected to have declined by 22% (95% confidence interval, CI -23 to -21%), and the number of people carrying NNRTI resistance to be 2.9-fold higher. If enhancements in diagnosis and retention in care occur, and ART is initiated at CD4 cell count less than 500......  cells/μl, HIV incidence is projected to decline by 36% (95% CI: -37 to -36%) and the number of people with NNRTI resistance to be 4.1-fold higher than currently. Prevalence of people with viral load more than 500  copies/ml carrying NRMV is not projected to differ markedly according to future ART...

  16. The Second Zambian National Tuberculosis Drug Resistance survey - a comparison of conventional and molecular methods.

    Science.gov (United States)

    Kapata, Nathan; Mbulo, Grace; Cobelens, Frank; de Haas, Petra; Schaap, Ab; Mwamba, Pike; Mwanza, Winnie; Muvwimi, Mweemba; Muyoyeta, Monde; Moyo, Maureen; Mulenga, Lutinala; Grobusch, Martin P; Godfrey-Faussett, Peter; Ayles, Helen

    2015-11-01

    The prevalence of MDR-TB in Zambia was estimated to be 1.8% in 2001. A second drug resistance survey was conducted in 2008 to determine trends; the use of the Genotype MTBDRplus assay was applied to compare results to the gold standard. A two-stage cluster sampling, with health facilities as primary sampling units. Processed sputum specimens were inoculated on solid media for culture; heat-inactivated bacterial suspensions from sputum samples were tested on a commercial line probe assay for the identification of rifampicin and isoniazid resistance. A total of 917 patients with TB were enrolled and 883 (96.3%) analysed. A total of 574 (65%) had LJ results and 824 (93.3%) had results from MTBDRplus assay. The median age was 32, and 63.3% were males. MDR-TB according to LJ-based DST was 1.1% (CI 0.1-2.4) whereas according to MDTBDRplus assay was 1.6% (CI 0.6-2.6). Isoniazid monoresistance in new cases was 2.4% (CI 0.613-4.26) based on LJ results and 5.0% (CI 3.2-6.7) based on the MTBDRplus; in retreatment cases, it was 4.4% (CI 0.3-8.6) and 2.40% (CI <0.1-5.1) on LJ and MTBDRplus, respectively. Rifampicin monoresistance in new cases was 0.1% (CI <0.1-0.4) based on LJ and 0.6% (CI 0.01-1.1) based on the MTBDRplus; in retreatment cases, it was 0% (CI 0-3.8) and 1.8% (CI <0.1-4.0) on LJ and MTBDRplus, respectively. There were no XDR-TB cases found and no association between MDR-TB and HIV. There was no increase in MDR-TB prevalence in Zambia from 2001 to 2008; results from the two methods were similar. Molecular methods were quicker and simpler to use. © 2015 John Wiley & Sons Ltd.

  17. Genetic diversity of Plasmodium falciparum and distribution of drug resistance haplotypes in Yemen.

    Science.gov (United States)

    Al-Hamidhi, Salama; Mahdy, Mohammed A K; Al-Hashami, Zainab; Al-Farsi, Hissa; Al-mekhlafi, Abdulsalam M; Idris, Mohamed A; Beja-Pereira, Albano; Babiker, Hamza A

    2013-07-15

    Despite evident success of malaria control in many sites in the Arabian Peninsula, malaria remains endemic in a few spots, in Yemen and south-west of Saudi Arabia. In addition to local transmission, imported malaria sustains an extra source of parasites that can challenge the strengths of local control strategies. This study examined the genetic diversity of Plasmodium falciparum in Yemen and mutations of drug resistant genes, to elucidate parasite structure and distribution of drug resistance genotypes in the region. Five polymorphic loci (MSP-2, Pfg377 and three microsatellites on chromosome 8) not involved in anti-malarial drug resistance, and four drug resistant genes (pfcrt, pfmdr1, dhfr and dhps) were genotyped in 108 P. falciparum isolates collected in three sites in Yemen: Dhamar, Hodeidah and Taiz. High diversity was seen in non-drug genes, pfg377 (He = 0.66), msp-2 (He = 0.80) and three microsatellites on chr 8, 7.7 kb (He = 0.88), 4.3 kb (He = 0.77) and 0.8 kb (He = 0.71). There was a high level of mixed-genotype infections (57%), with an average 1.8 genotypes per patient. No linkage disequilibrium was seen between drug resistant genes and the non-drug markers (p Yemen is indicative of a large parasite reservoir, which represents a challenge to control efforts. The presence of two distinct pfcrt genotype, CVIET and SVMNT, suggests that chloroquine resistance can possibly be related to a migratory path from Africa and Asia. The absence of the triple mutant dhfr genotype (IRN) and dhps mutations supports the use of artesunate + sulphadoxine-pyrimethamine as first-line therapy. However, the prevalent pfmdr1 genotype NFSND [21%] has previously been associated with tolerance/resistance response to artemisinin combination therapy (ACT). Regular surveys are, therefore, important to monitor spread of pfmdr1 and dhfr mutations and response to ACT.

  18. Stability Analysis of an HIV/AIDS Dynamics Model with Drug Resistance

    Directory of Open Access Journals (Sweden)

    Qianqian Li

    2012-01-01

    Full Text Available A mathematical model of HIV/AIDS transmission incorporating treatment and drug resistance was built in this study. We firstly calculated the threshold value of the basic reproductive number (R0 by the next generation matrix and then analyzed stability of two equilibriums by constructing Lyapunov function. When R0<1, the system was globally asymptotically stable and converged to the disease-free equilibrium. Otherwise, the system had a unique endemic equilibrium which was also globally asymptotically stable. While an antiretroviral drug tried to reduce the infection rate and prolong the patients’ survival, drug resistance was neutralizing the effects of treatment in fact.

  19. Identification of a Non-Gatekeeper Hot Spot for Drug-Resistant Mutations in mTOR Kinase.

    Science.gov (United States)

    Wu, Tzung-Ju; Wang, Xiaowen; Zhang, Yanjie; Meng, Linghua; Kerrigan, John E; Burley, Stephen K; Zheng, X F Steven

    2015-04-21

    Protein kinases are therapeutic targets for human cancer. However, "gatekeeper" mutations in tyrosine kinases cause acquired clinical resistance, limiting long-term treatment benefits. mTOR is a key cancer driver and drug target. Numerous small-molecule mTOR kinase inhibitors have been developed, with some already in human clinical trials. Given our clinical experience with targeted therapeutics, acquired drug resistance in mTOR is thought likely, but not yet documented. Herein, we describe identification of a hot spot (L2185) for drug-resistant mutations, which is distinct from the gatekeeper site, and a chemical scaffold refractory to drug-resistant mutations. We also provide new insights into mTOR kinase structure and function. The hot spot mutations are potentially useful as surrogate biomarkers for acquired drug resistance in ongoing clinical trials and future treatments and for the design of the next generation of mTOR-targeted drugs. Our study provides a foundation for further research into mTOR kinase function and targeting. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  20. Elucidating the Interdependence of Drug Resistance from Combinations of Mutations.

    Science.gov (United States)

    Ragland, Debra A; Whitfield, Troy W; Lee, Sook-Kyung; Swanstrom, Ronald; Zeldovich, Konstantin B; Kurt-Yilmaz, Nese; Schiffer, Celia A

    2017-11-14

    HIV-1 protease is responsible for the cleavage of 12 nonhomologous sites within the Gag and Gag-Pro-Pol polyproteins in the viral genome. Under the selective pressure of protease inhibition, the virus evolves mutations within (primary) and outside of (secondary) the active site, allowing the protease to process substrates while simultaneously countering inhibition. The primary protease mutations impede inhibitor binding directly, while the secondary mutations are considered accessory mutations that compensate for a loss in fitness. However, the role of secondary mutations in conferring drug resistance remains a largely unresolved topic. We have shown previously that mutations distal to the active site are able to perturb binding of darunavir (DRV) via the protein's internal hydrogen-bonding network. In this study, we show that mutations distal to the active site, regardless of context, can play an interdependent role in drug resistance. Applying eigenvalue decomposition to collections of hydrogen bonding and van der Waals interactions from a series of molecular dynamics simulations of 15 diverse HIV-1 protease variants, we identify sites in the protease where amino acid substitutions lead to perturbations in nonbonded interactions with DRV and/or the hydrogen-bonding network of the protease itself. While primary mutations are known to drive resistance in HIV-1 protease, these findings delineate the significant contributions of accessory mutations to resistance. Identifying the variable positions in the protease that have the greatest impact on drug resistance may aid in future structure-based design of inhibitors.