Comet assay as a human biomonitoring tool: application in occupational exposure to antineoplastic drugs

Directory of Open Access Journals (Sweden)

Carina Ladeira

2015-05-01

Occupational exposure to antineoplastic drugs is associated with genotoxic effects, although comet assay analyzed parameters were higher in exposed comparing with controls, were not significant. Also the study of the susceptibility biomarkers did not show statistical significant differences, the small size of our sample hampered the finding of a possible association, let alone a causality relationship.

A cell-based high-throughput screening assay for radiation susceptibility using automated cell counting

International Nuclear Information System (INIS)

Hodzic, Jasmina; Dingjan, Ilse; Maas, Mariëlle JP; Meulen-Muileman, Ida H van der; Menezes, Renee X de; Heukelom, Stan; Verheij, Marcel; Gerritsen, Winald R; Geldof, Albert A; Triest, Baukelien van; Beusechem, Victor W van

2015-01-01

Radiotherapy is one of the mainstays in the treatment for cancer, but its success can be limited due to inherent or acquired resistance. Mechanisms underlying radioresistance in various cancers are poorly understood and available radiosensitizers have shown only modest clinical benefit. There is thus a need to identify new targets and drugs for more effective sensitization of cancer cells to irradiation. Compound and RNA interference high-throughput screening technologies allow comprehensive enterprises to identify new agents and targets for radiosensitization. However, the gold standard assay to investigate radiosensitivity of cancer cells in vitro, the colony formation assay (CFA), is unsuitable for high-throughput screening. We developed a new high-throughput screening method for determining radiation susceptibility. Fast and uniform irradiation of batches up to 30 microplates was achieved using a Perspex container and a clinically employed linear accelerator. The readout was done by automated counting of fluorescently stained nuclei using the Acumen eX3 laser scanning cytometer. Assay performance was compared to that of the CFA and the CellTiter-Blue homogeneous uniform-well cell viability assay. The assay was validated in a whole-genome siRNA library screening setting using PC-3 prostate cancer cells. On 4 different cancer cell lines, the automated cell counting assay produced radiation dose response curves that followed a linear-quadratic equation and that exhibited a better correlation to the results of the CFA than did the cell viability assay. Moreover, the cell counting assay could be used to detect radiosensitization by silencing DNA-PKcs or by adding caffeine. In a high-throughput screening setting, using 4 Gy irradiated and control PC-3 cells, the effects of DNA-PKcs siRNA and non-targeting control siRNA could be clearly discriminated. We developed a simple assay for radiation susceptibility that can be used for high-throughput screening. This will aid

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.

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.

Rapid drug susceptibility test of mycobacterium tuberculosis by bioluminescence sensor

Science.gov (United States)

Lu, Bin; Xu, Shunqing; Chen, Zifei; Zhou, Yikai

2001-09-01

With the persisting increase of drug-resistant stains of M. Tuberculosis around the world, rapid and sensitive detection of antibiotic of M. Tuberculosis is becoming more and more important. In the present study, drug susceptibility of M. tuberculosis were detected by recombination mycobacteriophage combined with bioluminescence sensor. It is based on the use of recombination mycobacteriophage which can express firefly luciferase when it infects viable mycobacteria, and can effectively produce quantifiable photon. Meanwhile, in mycobacterium cells treated with active antibiotic, no light is observed. The emitted light is recorded by a bioluminscence sensor, so the result of drug-resistant test can be determined by the naked eye. 159 stains of M. tuberculosis were applied to this test on their resistant to rifampin, streptomycin and isoniazid. It is found that the agreement of this assay with Liewenstein- Jensen slat is: rifampin 95.60 percent, isoniazid 91.82 percent, streptomycin 88.68 percent, which showed that it is a fast and practical method to scene and detect drug resistant of mycobacterium stains.

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

A method for evaluating antiviral drug susceptibility of Epstein-Barr virus

Directory of Open Access Journals (Sweden)

Charlotte A Romain

2010-01-01

Full Text Available Charlotte A Romain1, Henry H Balfour Jr1,2, Heather E Vezina1,3, Carol J Holman11Department of Laboratory Medicine and Pathology, 2Department of Pediatrics, 3Department of Experimental and Clinical Pharmacology, University of Minnesota, Minneapolis, MN, USAAbstract: We developed an in vitro Epstein-Barr virus (EBV drug susceptibility assay using P3HR1 cells or lymphoblastoid cells from subjects with infectious mononucleosis, which were grown in the presence of various concentrations of acyclovir (ACV, ganciclovir (GCV or R-9-[4-hydroxy-2-(hydroxymethylbutyl]guanine (H2G and 12-O-tetradecanoyl-phorbol-13-acetate (TPA. On day 7, total cellular DNA was extracted and EBV DNA was detected using an in-house quantitative real-time polymerase chain reaction (PCR method. All three drugs had in vitro activity against EBV in both the laboratory standard producer cell line P3HR1 and in subject-derived lymphoblastoid cell lines. The median 50% inhibitory concentrations (IC50s in P3HR1 cells were: ACV, 3.4 μM; GCV, 2.6 μM; and H2G, 2.7 μM and in 3 subject-derived cells were: ACV, 2.5 μM; GCV, 1.7 μM; and H2G, 1.9 μM. Our assay can be used to screen candidate anti-EBV drugs. Because we can measure the IC50 of patients’ strains of EBV, this assay may also be useful for monitoring viral resistance especially in immunocompomised hosts receiving antiviral drugs for prevention or treatment of EBV diseases.Keywords: Epstein-Barr virus, ganciclovir, acyclovir, valomaciclovir, H2G, antivirals

Genotyping and drug susceptibility testing of mycobacterial isolates from population-based tuberculosis prevalence survey in Ghana.

Science.gov (United States)

Addo, Kennedy Kwasi; Addo, Samuel Ofori; Mensah, Gloria Ivy; Mosi, Lydia; Bonsu, Frank Adae

2017-12-02

Mycobacterium tuberculosis complex (MTBC) and Non-tuberculosis Mycobacterium (NTM) infections differ clinically, making rapid identification and drug susceptibility testing (DST) very critical for infection control and drug therapy. This study aims to use World Health Organization (WHO) approved line probe assay (LPA) to differentiate mycobacterial isolates obtained from tuberculosis (TB) prevalence survey in Ghana and to determine their drug resistance patterns. A retrospective study was conducted whereby a total of 361 mycobacterial isolates were differentiated and their drug resistance patterns determined using GenoType Mycobacterium Assays: MTBC and CM/AS for differentiating MTBC and NTM as well MTBDRplus and NTM-DR for DST of MTBC and NTM respectively. Out of 361 isolates, 165 (45.7%) MTBC and 120 (33.2%) NTM (made up of 14 different species) were identified to the species levels whiles 76 (21.1%) could not be completely identified. The MTBC comprised 161 (97.6%) Mycobacterium tuberculosis and 4 (2.4%) Mycobacterium africanum. Isoniazid and rifampicin monoresistant MTBC isolates were 18/165 (10.9%) and 2/165(1.2%) respectively whiles 11/165 (6.7%) were resistant to both drugs. Majority 42/120 (35%) of NTM were M. fortuitum. DST of 28 M. avium complex and 8 M. abscessus complex species revealed that all were susceptible to macrolides (clarithromycin, azithromycin) and aminoglycosides (kanamycin, amikacin, and gentamicin). Our research signifies an important contribution to TB control in terms of knowledge of the types of mycobacterium species circulating and their drug resistance patterns in Ghana.

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.

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.

qPCR-High resolution melt analysis for drug susceptibility testing of Mycobacterium leprae directly from clinical specimens of leprosy patients.

Science.gov (United States)

Araujo, Sergio; Goulart, Luiz Ricardo; Truman, Richard W; Goulart, Isabela Maria B; Vissa, Varalakshmi; Li, Wei; Matsuoka, Masanori; Suffys, Philip; Fontes, Amanda B; Rosa, Patricia S; Scollard, David M; Williams, Diana L

2017-06-01

Real-Time PCR-High Resolution Melting (qPCR-HRM) analysis has been recently described for rapid drug susceptibility testing (DST) of Mycobacterium leprae. The purpose of the current study was to further evaluate the validity, reliability, and accuracy of this assay for M. leprae DST in clinical specimens. The specificity and sensitivity for determining the presence and susceptibility of M. leprae to dapsone based on the folP1 drug resistance determining region (DRDR), rifampin (rpoB DRDR) and ofloxacin (gyrA DRDR) was evaluated using 211 clinical specimens from leprosy patients, including 156 multibacillary (MB) and 55 paucibacillary (PB) cases. When comparing the results of qPCR-HRM DST and PCR/direct DNA sequencing, 100% concordance was obtained. The effects of in-house phenol/chloroform extraction versus column-based DNA purification protocols, and that of storage and fixation protocols of specimens for qPCR-HRM DST, were also evaluated. qPCR-HRM results for all DRDR gene assays (folP1, rpoB, and gyrA) were obtained from both MB (154/156; 98.7%) and PB (35/55; 63.3%) patients. All PCR negative specimens were from patients with low numbers of bacilli enumerated by an M. leprae-specific qPCR. We observed that frozen and formalin-fixed paraffin embedded (FFPE) tissues or archival Fite's stained slides were suitable for HRM analysis. Among 20 mycobacterial and other skin bacterial species tested, only M. lepromatosis, highly related to M. leprae, generated amplicons in the qPCR-HRM DST assay for folP1 and rpoB DRDR targets. Both DNA purification protocols tested were efficient in recovering DNA suitable for HRM analysis. However, 3% of clinical specimens purified using the phenol/chloroform DNA purification protocol gave false drug resistant data. DNA obtained from freshly frozen (n = 172), formalin-fixed paraffin embedded (FFPE) tissues (n = 36) or archival Fite's stained slides (n = 3) were suitable for qPCR-HRM DST analysis. The HRM-based assay was also able to

A Review of Moxifloxacin for the Treatment of Drug-Susceptible Tuberculosis.

Science.gov (United States)

Naidoo, Anushka; Naidoo, Kogieleum; McIlleron, Helen; Essack, Sabiha; Padayatchi, Nesri

2017-11-01

Moxifloxacin, an 8-methoxy quinolone, is an important drug in the treatment of multidrug-resistant tuberculosis and is being investigated in novel drug regimens with pretomanid, bedaquiline, and pyrazinamide, or rifapentine, for the treatment of drug-susceptible tuberculosis. Early results of these studies are promising. Although current evidence does not support the use of moxifloxacin in treatment-shortening regimens for drug-susceptible tuberculosis, it may be recommended in patients unable to tolerate standard first-line drug regimens or for isoniazid monoresistance. Evidence suggests that the standard 400-mg dose of moxifloxacin used in the treatment of tuberculosis may be suboptimal in some patients, leading to worse tuberculosis treatment outcomes and emergence of drug resistance. Furthermore, a drug interaction with the rifamycins results in up to 31% reduced plasma concentrations of moxifloxacin when these are combined for treatment of drug-susceptible tuberculosis, although the clinical relevance of this interaction is unclear. Moxifloxacin exhibits extensive interindividual pharmacokinetic variability. Higher doses of moxifloxacin may be needed to achieve drug exposures required for improved clinical outcomes. Further study is, however, needed to determine the safety of proposed higher doses and clinically validated targets for drug exposure to moxifloxacin associated with improved tuberculosis treatment outcomes. We discuss in this review the evidence for the use of moxifloxacin in drug-susceptible tuberculosis and explore the role of moxifloxacin pharmacokinetics, pharmacodynamics, and drug interactions with rifamycins, on tuberculosis treatment outcomes when used in first-line tuberculosis drug regimens. © 2017, The American College of Clinical Pharmacology.

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.

[Identification and drug susceptibility testing of Mycobacterium thermoresistibile and Mycobacterium elephantis isolated from a cow with mastitis].

Science.gov (United States)

Li, W B; Ji, L Y; Xu, D L; Liu, H C; Zhao, X Q; Wu, Y M; Wan, K L

2018-05-10

Objective: To understand the etiological characteristics and drug susceptibility of Mycobacterium thermoresistibile and Mycobacterium elephantis isolated from a cow with mastitis and provide evidence for the prevention and control of infectious mastitis in cows. Methods: The milk sample was collected from a cow with mastitis, which was pretreated with 4 % NaOH and inoculated with L-J medium for Mycobacterium isolation. The positive cultures were initially identified by acid-fast staining and multi-loci PCR, then Mycobacterium species was identified by the multiple loci sequence analysis (MLSA) with 16S rRNA , hsp65 , ITS and SodA genes. The drug sensitivity of the isolates to 27 antibiotics was tested by alamar blue assay. Results: Two anti-acid stain positive strains were isolated from the milk of a cow with mastitis, which were identified as non- tuberculosis mycobacterium by multi-loci PCR, and multi-loci nucleic acid sequence analysis indicated that one strain was Mycobacterium thermoresistibile and another one was Mycobacterium elephantis . The results of the drug susceptibility test showed that the two strains were resistant to most antibiotics, including rifampicin and isoniazid, but they were sensitive to amikacin, moxifloxacin, levofloxacin, ethambutol, streptomycin, tobramycin, ciprofloxacin and linezolid. Conclusions: Mycobacterium thermoresistibile and Mycobacterium elephantis were isolated in a cow with mastitis and the drug susceptibility spectrum of the pathogens were unique. The results of the study can be used as reference for the prevention and control the infection in cows.

Application of firefly luciferase assay for adenosine triphosphate (ATP) to antimicrobial drug sensitivity testing

Science.gov (United States)

Picciolo, G. L.; Tuttle, S. A.; Schrock, C. G.; Deming, J. W.; Barza, M. J.; Wienstein, L.; Chappelle, E. W.

1977-01-01

The development of a rapid method for determining microbial susceptibilities to antibiotics using the firefly luciferase assay for adenosine triphosphate (ATP) is documented. The reduction of bacterial ATP by an antimicrobial agent was determined to be a valid measure of drug effect in most cases. The effect of 12 antibiotics on 8 different bacterial species gave a 94 percent correlation with the standard Kirby-Buer-Agar disc diffusion method. A 93 percent correlation was obtained when the ATP assay method was applied directly to 50 urine specimens from patients with urinary tract infections. Urine samples were centrifuged first to that bacterial pellets could be suspended in broth. No primary isolation or subculturing was required. Mixed cultures in which one species was predominant gave accurate results for the most abundant organism. Since the method is based on an increase in bacterial ATP with time, the presence of leukocytes did not interfere with the interpretation of results. Both the incubation procedure and the ATP assays are compatible with automation.

Extended biofilm susceptibility assay for Staphylococcus aureus bovine mastitis isolates: evidence for association between genetic makeup and biofilm susceptibility.

Science.gov (United States)

Melchior, M B; van Osch, M H J; Lam, T J G M; Vernooij, J C M; Gaastra, W; Fink-Gremmels, J

2011-12-01

Staphylococcus aureus is one of the most prevalent causes of bovine mastitis. The antimicrobial treatment of this disease is currently based on antimicrobial susceptibility tests according to Clinical and Laboratory Standards Institute standards. However, various authors have shown a discrepancy between the results of this standard susceptibility test and the actual cure rate of the applied antimicrobial treatment. Increasing evidence suggests that in vivo biofilm formation by Staph. aureus, which is not assessed in the antimicrobial susceptibility tests, is associated with this problem, resulting in disappointing cure rates, especially for infections of longer duration. Previous data obtained with a limited number of strains showed that the extended biofilm antimicrobial susceptibility (EBS) assay reveals differences between strains, which cannot be derived from a standard susceptibility test or from a 24-h biofilm susceptibility test. The objective of this study was to test a collection of Staph. aureus bovine mastitis strains in the EBS assay and to model the effect of antimicrobial exposure, duration of antimicrobial exposure, and genotype profile of the strains on antimicrobial susceptibility. With the results from a previous study with the same collection of strains, the effect of genotype represented by accessory gene regulator gene (agr-type), the presence of insertional sequence 257 (IS257), intercellular adhesion (ica), and the β-lactamase (blaZ) gene were entered as explanatory factors in a logistic regression model. The agr locus of Staph. aureus controls the expression of most of the virulence factors, represses the transcription of several cell wall-associated proteins, and activates several exoproteins during the post-exponential phase. The IS257 gene has been related to biofilm formation in vitro and was found earlier in 50% of the agr-type 2 strains. The ica gene cluster encodes for the production of an extracellular polysaccharide adhesin, termed

A Microfluidic Channel Method for Rapid Drug-Susceptibility Testing of Pseudomonas aeruginosa.

Directory of Open Access Journals (Sweden)

Yoshimi Matsumoto

Full Text Available The recent global increase in the prevalence of antibiotic-resistant bacteria and lack of development of new therapeutic agents emphasize the importance of selecting appropriate antimicrobials for the treatment of infections. However, to date, the development of completely accelerated drug susceptibility testing methods has not been achieved despite the availability of a rapid identification method. We proposed an innovative rapid method for drug susceptibility testing for Pseudomonas aeruginosa that provides results within 3 h. The drug susceptibility testing microfluidic (DSTM device was prepared using soft lithography. It consisted of five sets of four microfluidic channels sharing one inlet slot, and the four channels are gathered in a small area, permitting simultaneous microscopic observation. Antimicrobials were pre-introduced into each channel and dried before use. Bacterial suspensions in cation-adjusted Mueller-Hinton broth were introduced from the inlet slot and incubated for 3 h. Susceptibilities were microscopically evaluated on the basis of differences in cell numbers and shapes between drug-treated and control cells, using dedicated software. The results of 101 clinically isolated strains of P. aeruginosa obtained using the DSTM method strongly correlated with results obtained using the ordinary microbroth dilution method. Ciprofloxacin, meropenem, ceftazidime, and piperacillin caused elongation in susceptible cells, while meropenem also induced spheroplast and bulge formation. Morphological observation could alternatively be used to determine the susceptibility of P. aeruginosa to these drugs, although amikacin had little effect on cell shape. The rapid determination of bacterial drug susceptibility using the DSTM method could also be applicable to other pathogenic species, and it could easily be introduced into clinical laboratories without the need for expensive instrumentation.

Drug susceptibility of fungi isolated from ICU patients

Science.gov (United States)

Modrzewska, Barbara D; Kurnatowska,, Anna J; Khalid, Katarzyna

Candida species can be a reason of infections associated with high morbidity and mortality. The risk of invasive candidosis for patients admitted to intensive care units (ICUs) is increased due to immunosuppressive states, prolonged length of stay, broad-spectrum antibiotics and Candida colonization. The aim of the study was to determine selected properties of fungi isolated from patients treated in the ICUs of hospitals in Lodz. The materials were collected from the oral cavity, the tracheostomy or endotracheal tube and urine from 16 children and 35 adult. In total, 127 samples were examined to differentiate the fungal strains with used morphological and biochemical methods. Candida species were isolated from adult patients (82.9%), but were not isolated from any of the children; C. albicans was the predominant fungus (61.7%), much less frequent were C. glabrata (12.8%), C. tropicalis (6.4%) and C. kefyr, C. dubliniensis (4.3% each).The susceptibility of fungi to antimycotic drugs revealed that almost all of the strains were susceptible to nystatin (97.9%) and to amphotericin B (72.3%), and resistant to fluconazole (72.3%) and ketoconazole (57.5%). No isolation of fungi from children remaining in ICU may be an evidence of high sanitary regime at these wards; fungi from the genus Candida are the etiological factors for ICU infections; 3/5 of them are caused by C. albicans, mostly of the code 2 576 174, characteristic for strains isolated from hospitalized patients; it is necessary to determine the species of the fungus and its susceptibility to drugs, which allows to conduct effective therapy; prophylactic administration of fluconazole leads to an increase in the number of strains resistant to this chemotherapeutic agent; in the antifungal local treatment, nystatin should be a drug of choice as the drug to which most fungi are susceptible.

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.

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

Directory of Open Access Journals (Sweden)

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%.

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.

Heightened vulnerability to MDR-TB epidemics after controlling drug-susceptible TB.

Directory of Open Access Journals (Sweden)

Jason D Bishai

2010-09-01

Full Text Available Prior infection with one strain TB has been linked with diminished likelihood of re-infection by a new strain. This paper attempts to determine the role of declining prevalence of drug-susceptible TB in enabling future epidemics of MDR-TB.A computer simulation of MDR-TB epidemics was developed using an agent-based model platform programmed in NetLogo (See http://mdr.tbtools.org/. Eighty-one scenarios were created, varying levels of treatment quality, diagnostic accuracy, microbial fitness cost, and the degree of immunogenicity elicited by drug-susceptible TB. Outcome measures were the number of independent MDR-TB cases per trial and the proportion of trials resulting in MDR-TB epidemics for a 500 year period after drug therapy for TB is introduced.MDR-TB epidemics propagated more extensively after TB prevalence had fallen. At a case detection rate of 75%, improving therapeutic compliance from 50% to 75% can reduce the probability of an epidemic from 45% to 15%. Paradoxically, improving the case-detection rate from 50% to 75% when compliance with DOT is constant at 75% increases the probability of MDR-TB epidemics from 3% to 45%.The ability of MDR-TB to spread depends on the prevalence of drug-susceptible TB. Immunologic protection conferred by exposure to drug-susceptible TB can be a crucial factor that prevents MDR-TB epidemics when TB treatment is poor. Any single population that successfully reduces its burden of drug-susceptible TB will have reduced herd immunity to externally or internally introduced strains of MDR-TB and can experience heightened vulnerability to an epidemic. Since countries with good TB control may be more vulnerable, their self interest dictates greater promotion of case detection and DOTS implementation in countries with poor control to control their risk of MDR-TB.

Comparative drug susceptibility study of five clonal strains of Trichomonas vaginalis in vitro

Institute of Scientific and Technical Information of China (English)

Hemantkumar Somabhai Chaudhari; Prati Pal Singh

2011-01-01

Objective: To produce comparative data on a group of Trichomonas vaginalis clonal strains with varied drug responses using identical methods and materials. Methods: Five clonal strains of Trichomonas vaginalis were isolated from reference strain using agar plate technique. The variability of growth kinetic and susceptibility of clonal strain to metronidazole, tinidazole, satranidazole and nitazoxanide were observed in 96 well microtitre plate. Results: Among these clonal strains there was a good correlation between rates of growth with the relative susceptibility of the strains to drugs in vitro. Regarding metronidazole, tinidazole and satranidazole susceptibility, different degrees of susceptibility were determined. However, no difference in nitazoxanide susceptibility was found between the clonal strain tested and a reference strain.Conclusions: This is the first description of biological variability in clonal stock of Trichomonas vaginalis. Different degrees of drug susceptibility were determined among clonal strains tested. Further studies will be necessary to ascertain the importance of this variability in clinical infection.

Polymorphism in Mitochondrial Group I Introns among Cryptococcus neoformans and Cryptococcus gattii Genotypes and Its Association with Drug Susceptibility

Directory of Open Access Journals (Sweden)

Felipe E. E. S. Gomes

2018-02-01

Full Text Available Cryptococcosis, one of the most important systemic mycosis in the world, is caused by different genotypes of Cryptococcus neoformans and Cryptococcus gattii, which differ in their ecology, epidemiology, and antifungal susceptibility. Therefore, the search for new molecular markers for genotyping, pathogenicity and drug susceptibility is necessary. Group I introns fulfill the requisites for such task because (i they are polymorphic sequences; (ii their self-splicing is inhibited by some drugs; and (iii their correct splicing under parasitic conditions is indispensable for pathogen survival. Here, we investigated the presence of group I introns in the mitochondrial LSU rRNA gene in 77 Cryptococcus isolates and its possible relation to drug susceptibility. Sequencing revealed two new introns in the LSU rRNA gene. All the introns showed high sequence similarity to other mitochondrial introns from distinct fungi, supporting the hypothesis of an ancient non-allelic invasion. Intron presence was statistically associated with those genotypes reported to be less pathogenic (p < 0.001. Further virulence assays are needed to confirm this finding. In addition, in vitro antifungal tests indicated that the presence of LSU rRNA introns may influence the minimum inhibitory concentration (MIC of amphotericin B and 5-fluorocytosine. These findings point to group I introns in the mitochondrial genome of Cryptococcus as potential molecular markers for antifungal resistance, as well as therapeutic targets.

Drug susceptibility of influenza A/H3N2 strains co-circulating during 2009 influenza pandemic: first report from Mumbai.

Science.gov (United States)

Gohil, Devanshi J; Kothari, Sweta T; Shinde, Pramod S; Chintakrindi, Anand S; Meharunkar, Rhuta; Warke, Rajas V; Kanyalkar, Meena A; Chowdhary, Abhay S; Deshmukh, Ranjana A

2015-01-01

From its first instance in 1977, resistance to amantadine, a matrix (M2) inhibitor has been increasing among influenza A/H3N2, thus propelling the use of oseltamivir, a neuraminidase (NA) inhibitor as a next line drug. Information on drug susceptibility to amantadine and neuraminidase inhibitors for influenza A/H3N2 viruses in India is limited with no published data from Mumbai. This study aimed at examining the sensitivity to M2 and NA inhibitors of influenza A/H3N2 strains isolated from 2009 to 2011 in Mumbai. Nasopharyngeal swabs positive for influenza A/H3N2 virus were inoculated on Madin-Darby canine kidney (MDCK) cell line for virus isolation. Molecular analysis of NA and M2 genes was used to detect known mutations contributing to resistance. Resistance to neuraminidase was assayed using a commercially available chemiluminescence based NA-Star assay kit. Genotypically, all isolates were observed to harbor mutations known to confer resistance to amantadine. However, no know mutations conferring resistance to NA inhibitors were detected. The mean IC50 value for oseltamivir was 0.25 nM. One strain with reduced susceptibility to the neuraminidase inhibitor (IC₅₀=4.08 nM) was isolated from a patient who had received oseltamivir treatment. Phylogenetic analysis postulate the emergence of amantadine resistance in Mumbai may be due to genetic reassortment with the strains circulating in Asia and North America. Surveillance of drug susceptibility helped us to identify an isolate with reduced sensitivity to oseltamivir. Therefore, we infer that such surveillance would help in understanding possible trends underlying the emergence of resistant variants in humans. Copyright © 2014 Elsevier B.V. All rights reserved.

Meta-analysis and genome-wide interpretation of genetic susceptibility to drug addiction

Science.gov (United States)

2011-01-01

Background Classical genetic studies provide strong evidence for heritable contributions to susceptibility to developing dependence on addictive substances. Candidate gene and genome-wide association studies (GWAS) have sought genes, chromosomal regions and allelic variants likely to contribute to susceptibility to drug addiction. Results Here, we performed a meta-analysis of addiction candidate gene association studies and GWAS to investigate possible functional mechanisms associated with addiction susceptibility. From meta-data retrieved from 212 publications on candidate gene association studies and 5 GWAS reports, we linked a total of 843 haplotypes to addiction susceptibility. We mapped the SNPs in these haplotypes to functional and regulatory elements in the genome and estimated the magnitude of the contributions of different molecular mechanisms to their effects on addiction susceptibility. In addition to SNPs in coding regions, these data suggest that haplotypes in gene regulatory regions may also contribute to addiction susceptibility. When we compared the lists of genes identified by association studies and those identified by molecular biological studies of drug-regulated genes, we observed significantly higher participation in the same gene interaction networks than expected by chance, despite little overlap between the two gene lists. Conclusions These results appear to offer new insights into the genetic factors underlying drug addiction. PMID:21999673

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.

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

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.

Molecular characterization and drug susceptibility profile of a Mycobacterium avium subspecies avium isolate from a dog with disseminated infection.

Science.gov (United States)

Armas, Federica; Furlanello, Tommaso; Camperio, Cristina; Trotta, Michele; Novari, Gianluca; Marianelli, Cinzia

2016-01-12

Mycobacterium avium complex (MAC) infections have been described in many mammalian species including humans and pets. We isolated and molecularly typed the causative agent of a rare case of disseminated mycobacteriosis in a dog. We identified the pathogen as a M. avium subspecies avium by sequencing the partial genes gyrB and rpsA. Considering the zoonotic potential of this infection, and in an attempt to ensure the most effective treatment for the animal, we also determined the drug susceptibility profile of the isolate to the most common drugs used to treat MAC disease in humans. The pathogen was tested in vitro against the macrolide clarithromycin, as well as against amikacin, ciprofloxacin, rifampicin, ethambutol and linezolid by the resazurin microdilution assay. It was found to be sensitive to all tested drugs save ethambutol. Despite the fact that the pathogen was sensitive to the therapies administered, the dog's overall clinical status worsened, and the animal died shortly after antimicrobial susceptibility results became available. Nucleotide sequencing of the embB gene, the target gene most commonly associated with ethambutol resistance, showed new missense mutations when compared to sequences available in public databases. In conclusion, we molecularly identified the MAC pathogen and determined its drug susceptibility profile in a relatively short period of time (seven days). We also characterized new genetic mutations likely to have been involved in the observed ethambutol resistance. Our results confirm the usefulness of both the gyrB and the rpsA genes as biomarkers for an accurate identification and differentiation of MAC pathogens.

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

Microbiological quality of water from the rivers of Curitiba, Paraná State, Brazil, and the susceptibility to antimicrobial drugs and pathogenicity of Escherichia coli.

Science.gov (United States)

Giowanella, Melissa; Bozza, Angela; do Rocio Dalzoto, Patricia; Dionísio, Jair Alves; Andraus, Sumaia; Guimarães, Edson Luiz Gomes; Pimentel, Ida Chapaval

2015-11-01

Water safety is determined by several markers, and Escherichia coli is one of the most important indicators of water quality. The objective of this study was to evaluate the microbiological parameters in environmental samples of fresh water from rivers of Curitiba and its metropolitan area in Paraná State, Brazil. In addition, we evaluated the pathogenicity and susceptibility to antimicrobial drugs in E. coli. These evaluations were performed by quantitative and qualitative methods employing selective media for isolating thermotolerant coliforms and biochemical tests for identifying E. coli. Pathogenic strains of E. coli were detected by PCR multiplex using specific primers. From the water samples, 494 thermotolerant coliforms were obtained, of which 96 (19.43%) isolates were characterized as E. coli. Three isolates were identified as enteroaggregative E. coli, one as enterotoxigenic E. coli, one as enteropathogenic E. coli, and two carried the Eae virulence gene. E. coli susceptibility to commonly employed antimicrobial drugs was analyzed by the disc diffusion method. The results showed 49 (51.04%) isolates resistant to all the drugs assayed, 16 (16.67%) with an intermediate resistance to all drugs, and 31 (32.29%) intermediately or fully resistant to one or more drugs tested. The highest rate of resistance was observed for tetracycline 30 μg, streptomycin 10 μg, and ceftazidime 30 μg. Detection of E. coli is associated with water contamination by fecal material from humans and warm-blooded animals. The occurrence of resistant strains can be the result of the indiscriminate use of antimicrobial drugs and poor sanitation in the areas assayed.

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...

Drug susceptibility to etravirine and darunavir among Human Immunodeficiency Virus Type 1-derived pseudoviruses in treatment-experienced patients with HIV/AIDS in South Korea.

Science.gov (United States)

Kwon, Oh-Kyung; Kim, Sung Soon; Rhee, Jee Eun; Kee, Mee-Kyung; Park, Mina; Oh, Hye-Ri; Choi, Ju-Yeon

2015-04-09

In South Korea, about 20 types of antiretroviral drugs are used in the treatment of patients with human immunodeficiency virus/acquired immune deficiency syndrome. Since 2010, raltegravir, etravirine, and darunavir have been spotlighted as new drugs for highly active antiretroviral therapy (HAART)-experienced adults with resistant HIV-1 in South Korea. In this study, we investigated potential susceptibility of pseudoviruses derived from treatment-experienced Korean patients to etravirine vs efavirenz and to darunavir vs amprenavir and indinavir using a modified single-round assay. Pseudoviruses derived from nine treatment-experienced patients infected with HIV-1 were investigated by comparison with the wild-type strain pNL4-3. The 50% inhibitory concentration (IC50) values were calculated and drug susceptibility was compared. The intensity of genotypic drug resistance was classified based on the 'SIR' interpretation of the Stanford data base. Drug susceptibility was generally higher for etravirine and darunavir compared with efavirenz, amprenavir, and indinavir in pseudoviruses derived from treatment-experienced patients. Pseudoviruses derived from patients KRB4025 and KRB8014, who exhibited long-term use of protease inhibitors, showed an outside of tested drug concentration, especially for amprenavir and indinavir. However, they exhibited a lower fold-change in resistance to darunavir. Etravirine and darunavir have been used in HAART since 2010 in South Korea. Therefore, these antiretroviral drugs together with other newly introduced antiretroviral drugs are interesting for the optimal treatment of patients with treatment failure. This study may help to find a more effective HAART in the case of HIV-1 infected patients that have difficulty being treated.

The detection and differentiation of methicillin-resistant and methicillin-susceptible Staphylococcus aureus endocarditis by using the BD GeneOhm StaphSR Assay.

Science.gov (United States)

Frey, Amy B; Wilson, Deborah A; LaSalvia, Margaret M; Tan, Carmela D; Rodriguez, E Rene; Shrestha, Nabin K; Hall, Gerri S; Procop, Gary W

2011-11-01

We use the BD GeneOhm StaphSR Assay (BD Diagnostics, Oakville, Canada) to screen for Staphylococcus aureus nasal colonization and sought to evaluate this assay for the assessment of valve specimens from patients with endocarditis. We examined 23 paired fresh and formalin-fixed, paraffin-embedded cardiac valve tissue samples, 12 of which had S aureus endocarditis, using the BD GeneOhm StaphSR Assay for the detection and differentiation of methicillin-susceptible and methicillin-resistant S aureus. This assay appropriately characterized all specimens with respect to the presence or absence of S aureus. There was an 87.5% correlation between the presence or absence of the mecA gene and the oxacillin susceptibility results for the S aureus isolates studied. The GeneOhm StaphSR assay accurately detected S aureus in cardiac valve tissue samples. Rare discordances were observed between oxacillin susceptibility status and mecA gene detection by this assay.

Iron Deprivation Affects Drug Susceptibilities of Mycobacteria Targeting Membrane Integrity

Directory of Open Access Journals (Sweden)

Rahul Pal

2015-01-01

Full Text Available Multidrug resistance (MDR acquired by Mycobacterium tuberculosis (MTB through continuous deployment of antitubercular drugs warrants immediate search for novel targets and mechanisms. The ability of MTB to sense and become accustomed to changes in the host is essential for survival and confers the basis of infection. A crucial condition that MTB must surmount is iron limitation, during the establishment of infection, since iron is required by both bacteria and humans. This study focuses on how iron deprivation affects drug susceptibilities of known anti-TB drugs in Mycobacterium smegmatis, a “surrogate of MTB.” We showed that iron deprivation leads to enhanced potency of most commonly used first line anti-TB drugs that could be reverted upon iron supplementation. We explored that membrane homeostasis is disrupted upon iron deprivation as revealed by enhanced membrane permeability and hypersensitivity to membrane perturbing agent leading to increased passive diffusion of drug and TEM images showing detectable differences in cell envelope thickness. Furthermore, iron seems to be indispensable to sustain genotoxic stress suggesting its possible role in DNA repair machinery. Taken together, we for the first time established a link between cellular iron and drug susceptibility of mycobacteria suggesting iron as novel determinant to combat MDR.

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...

Transmission of Multidrug-Resistant and Drug-Susceptible Tuberculosis within Households: A Prospective Cohort Study

Science.gov (United States)

Grandjean, Louis; Gilman, Robert H.; Martin, Laura; Soto, Esther; Castro, Beatriz; Lopez, Sonia; Coronel, Jorge; Castillo, Edith; Alarcon, Valentina; Lopez, Virginia; San Miguel, Angela; Quispe, Neyda; Asencios, Luis; Dye, Christopher; Moore, David A. J.

2015-01-01

Background The “fitness” of an infectious pathogen is defined as the ability of the pathogen to survive, reproduce, be transmitted, and cause disease. The fitness of multidrug-resistant tuberculosis (MDRTB) relative to drug-susceptible tuberculosis is cited as one of the most important determinants of MDRTB spread and epidemic size. To estimate the relative fitness of drug-resistant tuberculosis cases, we compared the incidence of tuberculosis disease among the household contacts of MDRTB index patients to that among the contacts of drug-susceptible index patients. Methods and Findings This 3-y (2010–2013) prospective cohort household follow-up study in South Lima and Callao, Peru, measured the incidence of tuberculosis disease among 1,055 household contacts of 213 MDRTB index cases and 2,362 household contacts of 487 drug-susceptible index cases. A total of 35/1,055 (3.3%) household contacts of 213 MDRTB index cases developed tuberculosis disease, while 114/2,362 (4.8%) household contacts of 487 drug-susceptible index patients developed tuberculosis disease. The total follow-up time for drug-susceptible tuberculosis contacts was 2,620 person-years, while the total follow-up time for MDRTB contacts was 1,425 person-years. Using multivariate Cox regression to adjust for confounding variables including contact HIV status, contact age, socio-economic status, and index case sputum smear grade, the hazard ratio for tuberculosis disease among MDRTB household contacts was found to be half that for drug-susceptible contacts (hazard ratio 0.56, 95% CI 0.34–0.90, p = 0.017). The inference of transmission in this study was limited by the lack of genotyping data for household contacts. Capturing incident disease only among household contacts may also limit the extrapolation of these findings to the community setting. Conclusions The low relative fitness of MDRTB estimated by this study improves the chances of controlling drug-resistant tuberculosis. However, fitter

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.

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...

Mycobacterium abscessus subsp. abscessus Lung Disease: Drug Susceptibility Testing in Sputum Culture Negative Conversion

Directory of Open Access Journals (Sweden)

Takehiko Kobayashi

2018-01-01

Full Text Available Background: Among Mycobacterium abscessus complex infections, patients with M. abscessus subsp. abscessus (MAA lung disease are difficult to treat and no standard therapy has been established. Few reports have investigated the drug susceptibility of these strains. We retrospectively investigated how in vitro drug susceptibility testing (DST of MAA affects the induction of sputum conversion using pharmacotherapy. Methods: Patients with MAA lung disease diagnosed and treated between 2010 and 2014 at our hospital were enrolled and divided into Group A (sputum conversion without relapse within 1 year and Group B (persistent positive cultured or negative conversion with relapse. MAA was identified in M. abscessus using sequence with genotyping, and DST of MAA was performed. Results: We assessed 23 patients (9 males and 14 females. There were 8 patients in Group A and 15 in Group B. Higher prevalence of susceptible isolates for clarithromycin (CAM susceptibility on day 14 was noted in Group A than in Group B (P = 0.03 and no significant difference observed in the two groups for other drugs. Conclusions: In vitro DST of MAA, especially CAM susceptibility on day 14, affected the results of negative conversion. No other drugs were found to affect sputum culture negative conversion.

Microbial sensor for drug susceptibility testing of Mycobacterium tuberculosis.

Science.gov (United States)

Zhang, Z-T; Wang, D-B; Li, C-Y; Deng, J-Y; Zhang, J-B; Bi, L-J; Zhang, X-E

2018-01-01

Drug susceptibility testing (DST) of clinical isolates of Mycobacterium tuberculosis is critical in treating tuberculosis. We demonstrate the possibility of using a microbial sensor to perform DST of M. tuberculosis and shorten the time required for DST. The sensor is made of an oxygen electrode with M. tuberculosis cells attached to its surface. This sensor monitors the residual oxygen consumption of M. tuberculosis cells after treatment with anti-TB drugs with glycerine as a carbon source. In principle, after drug pretreatment for 4-5 days, the response differences between the sensors made of drug-sensitive isolates are distinguishable from the sensors made of drug-resistant isolates. The susceptibility of the M. tuberculosis H37Ra strain, its mutants and 35 clinical isolates to six common anti-TB drugs: rifampicin, isoniazid, streptomycin, ethambutol, levofloxacin and para-aminosalicylic acid were tested using the proposed method. The results agreed well with the gold standard method (LJ) and were determined in significantly less time. The whole procedure takes approximately 11 days and therefore has the potential to inform clinical decisions. To our knowledge, this is the first study that demonstrates the possible application of a dissolved oxygen electrode-based microbial sensor in M. tuberculosis drug resistance testing. This study used the microbial sensor to perform DST of M. tuberculosis and shorten the time required for DST. The overall detection result of the microbial sensor agreed well with that of the conventional LJ proportion method and takes less time than the existing phenotypic methods. In future studies, we will build an O 2 electrode array microbial sensor reactor to enable a high-throughput drug resistance analysis. © 2017 The Authors. Journal of Applied Microbiology published by John Wiley & Sons Ltd on behalf of The Society for Applied Microbiology.

Antimicrobial drug susceptibility of Neisseria meningitidis strains isolated from carriers

Directory of Open Access Journals (Sweden)

Dayamí García

2000-06-01

Full Text Available When it is necessary to determine the susceptibility of Neisseria meningitidis (Nm strains to antimicrobial drugs, it is important to consider that it should be analyzed in a double context. One of them related to the use of drugs in a specific medical treatment; and the other; to chemoprophylatic drugs, both with the same purpose: the accurate selection of the “in vivo” antimicrobial agent. This requires the study of the sensitivity and resistance of strains isolated in both carriers and patients. With the aim of further studying the behavior of the strains that currently circulate in Cuba, an antimicrobial drug susceptibility study was conducted in 90 strains isolated from carriers during the first half of 1998. The agar dilution method was used to determine the minimum inhibitory concentrations (MICs to: penicillin, ampicillin, rifampin, sulfadiazine, chloramphenicol, ciprofloxacin, ceftriaxone, cefotaxime. The study of the three latter drugs was done for the first time in our country. The search for β- lactamase-producer strains was also performed. There was a predominance of penicillin sensitive strains (82,2% with an intermediate sensitivity to ampicillin (57,8%, while 70% of the strains were sensitive to sulfadiazine. Regarding the rest of the antimicrobial drugs, 100% of the strains were sensitive. The paper shows the MICs for each drug as well as the phenotypic characteristics of the strains with the penicillin and sulfadiazine sensitivity and resistance patterns. No β-lactamase-producer strains were found.

Bacterial isolates and drug susceptibility patterns of urinary tract ...

African Journals Online (AJOL)

Urinary tract infection (UTI) during pregnancy may cause complications such as pyelonephritis, hypertensive disease of pregnancy, anaemia, chronic renal failure, premature delivery and foetal mortality. This study aimed to identify the etiologic agents of UTI and to determine the patterns of antimicrobial drug susceptibility ...

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.

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.

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

Directory of Open Access Journals (Sweden)

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.

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.

The usefulness of microscopic observation for drug susceptibility of ...

African Journals Online (AJOL)

The usefulness of microscopic observation for drug susceptibility of. Mycobacterium tuberculosis complex in routine clinical microbiology laboratory. S.E. MSHANA1,2*, C. IMIRZALIOGLU2, E. DOMANN2 and T. CHAKRABORTY2. 1 We l Bugando Un vers ty College of Health Sc ences, P.O. Box 1464, Mwanza, Tanzan a.

[Circadian rhythm in susceptibility of mice to the anti-tumor drug carboplatin].

Science.gov (United States)

Lu, X H; Yin, L J

1994-12-01

The platinum-containing compounds has become a major chemical agent in the treatment of cancer. A circadian rhythm in the susceptibility of rodents and human being to cisplatin has been demonstrated, the maximal tolerance being found in the animal's active phase. Carboplatin is a second generation analog. Two studies were performed on mice with carboplatin under 12:12 light dark cycle to study its chronotoxicity and chronoeffectiveness. In study I, single intraperitoneal injection of 192mg/kg (LD50) carboplatin was given to four groups of mice at four different circadian stage. It was found that at 50% the overall mortality of mice, there was a mortality difference of 28% for mice receiving the drug at 9 a.m. to 71% for mice receiving drug at 9 p.m. It demonstrated that carboplatin was better tolerated in the animal's early sleep phase. In study II, S180 tumor-bearing mice were treated with 50mg/kg of carboplatin. The longest mean survival time and the lowest marrow toxicity occurred in the group which received the drug at the beginning of the sleep phase. It showed that the susceptibility of mice to carboplatin is circadian stage dependent. These data clearly demonstrate that, by timing the administration of drugs according to body rhythms, such as the host susceptibility-resistance rhythm to a drug, one can gain a therapeutic advantage over an approach which ignores such rhythms.

Drug Susceptibility of Mycobacterium tuberculosis Beijing Genotype and Association with MDR TB

Science.gov (United States)

ten Kate, Marian T.; de Knegt, Gerjo J.; Kremer, Kristin; Aarnoutse, Rob E.; Boeree, Martin J.; Verbrugh, Henri A.; van Soolingen, Dick; Bakker-Woudenberg, Irma A.J.M.

2012-01-01

To determine differences in the ability of Mycobacterium tuberculosis strains to withstand antituberculosis drug treatment, we compared the activity of antituberculosis drugs against susceptible Beijing and East-African/Indian genotype M. tuberculosis strains. Beijing genotype strains showed high rates of mutation within a wide range of drug concentrations, possibly explaining this genotype’s association with multidrug-resistant tuberculosis. PMID:22469099

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.

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.

Miltefosine and antimonial drug susceptibility of Leishmania Viannia species and populations in regions of high transmission in Colombia.

Directory of Open Access Journals (Sweden)

Olga Lucía Fernández

2014-05-01

Full Text Available Pentavalent antimonials have been the first line treatment for dermal leishmaniasis in Colombia for over 30 years. Miltefosine is administered as second line treatment since 2005. The susceptibility of circulating populations of Leishmania to these drugs is unknown despite clinical evidence supporting the emergence of resistance.In vitro susceptibility was determined for intracellular amastigotes of 245 clinical strains of the most prevalent Leishmania Viannia species in Colombia to miltefosine (HePC and/or meglumine antimoniate (Sb(V; 163, (80% were evaluated for both drugs. Additionally, susceptibility to Sb(V was examined in two cohorts of 85 L. V. panamensis strains isolated between 1980-1989 and 2000-2009 in the municipality of Tumaco. Susceptibility to each drug differed among strains of the same species and between species. Whereas 68% of L. V. braziliensis strains presented in vitro resistance to HePC, 69% were sensitive to Sb(V. Resistance to HePC and Sb(V occurred respectively, in 20% y 21% of L. panamensis strains. Only 3% of L. V. guyanensis were resistant to HePC, and none to Sb(V. Drug susceptibility differed between geographic regions and time periods. Subpopulations having disparate susceptibility to Sb(V were discerned among L. V. panamensis strains isolated during 1980-1990 in Tumaco where resistant strains belonged to zymodeme 2.3, and sensitive strains to zymodeme 2.2.Large scale evaluation of clinical strains of Leishmania Viannia species demonstrated species, population, geographic, and epidemiologic differences in susceptibility to meglumine antimoniate and miltefosine, and provided baseline information for monitoring susceptibility to these drugs. Sensitive and resistant clinical strains within each species, and zymodeme as a proxy marker of antimony susceptibility for L. V. panamensis, will be useful in deciphering factors involved in susceptibility and the distribution of sensitive and resistant populations.

A rapid method for the determination of microbial susceptibility using the firefly luciferase assay for adenosine triphosphate (ATP)

Science.gov (United States)

Vellend, H.; Tuttle, S. A.; Barza, M.; Weinstein, L.; Picciolo, G. L.; Chappelle, E. W.

1975-01-01

Luciferase assay for adenosine triphosphate (ATP) was optimized for pure bacteria in broth in order to evaluate if changes in bacterial ATP content could be used as a rapid measure of antibiotic effect on microorganisms. Broth cultures of log phase bacteria were incubated at 310 K (37 C) for 2.5 hours at antimicrobial concentrations which resulted in the best discrimination between sensitive and resistant strains. Eighty-seven strains of 11 bacterial species were studied for their susceptibility to 12 commonly used antimicrobial agents: ampicillin, Penicillin G, nafcillin, carbenicillin, cephalothin, tetracycline, erythromycin, clindamycin, gentamicin, nitrofurantoin, colistin, and chloramplenicol. The major advantage of the ATP system over existing methods of rapid microbial susceptibility testing is that the assay can be made specific for bacterial ATP.

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.

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.

Reduced susceptibility to praziquantel among naturally occurring Kenyan isolates of Schistosoma mansoni.

Directory of Open Access Journals (Sweden)

Sandra D Melman

2009-08-01

Full Text Available The near exclusive use of praziquantel (PZQ for treatment of human schistosomiasis has raised concerns about the possible emergence of drug-resistant schistosomes.We measured susceptibility to PZQ of isolates of Schistosoma mansoni obtained from patients from Kisumu, Kenya continuously exposed to infection as a consequence of their occupations as car washers or sand harvesters. We used a an in vitro assay with miracidia, b an in vivo assay targeting adult worms in mice and c an in vitro assay targeting adult schistosomes perfused from mice. In the miracidia assay, in which miracidia from human patients were exposed to PZQ in vitro, reduced susceptibility was associated with previous treatment of the patient with PZQ. One isolate ("KCW" that was less susceptible to PZQ and had been derived from a patient who had never fully cured despite multiple treatments was studied further. In an in vivo assay of adult worms, the KCW isolate was significantly less susceptible to PZQ than two other isolates from natural infections in Kenya and two lab-reared strains of S. mansoni. The in vitro adult assay, based on measuring length changes of adults following exposure to and recovery from PZQ, confirmed that the KCW isolate was less susceptible to PZQ than the other isolates tested. A sub-isolate of KCW maintained separately and tested after three years was susceptible to PZQ, indicative that the trait of reduced sensitivity could be lost if selection was not maintained.Isolates of S. mansoni from some patients in Kisumu have lower susceptibility to PZQ, including one from a patient who was never fully cured after repeated rounds of treatment administered over several years. As use of PZQ continues, continued selection for worms with diminished susceptibility is possible, and the probability of emergence of resistance will increase as large reservoirs of untreated worms diminish. The potential for rapid emergence of resistance should be an important

Evaluation of rapid radiometric method for drug susceptibility testing of Mycobacterium tuberculosis

International Nuclear Information System (INIS)

Siddiqi, S.H.; Libonati, J.P.; Middlebrook, G.

1981-01-01

A total of 106 isolates of Mycobacterium tuberculosis were tested for drug susceptibility by the conventional 7H11 plate method and by a new rapid radiometric method using special 7H12 liquid medium with 14 C-labeled substrate. Results obtained by the two methods were compared for rapidity, sensitivity, and specificity of the new test method. There was 98% overall agreement between the results obtained by the two methods. Of a total of 424 drug tests, only 8 drug results did not agree, mostly in the case of streptomycin. This new procedure was found to be rapid, with 87% of the tests results reportable within 4 days and 98% reportable within 5 days as compared to the usual 3 weeks required with the conventional indirect susceptibility test method. The results of this preliminary study indicate that the rapid radiometric method seems to have the potential for routine laboratory use and merits further investigations

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.

Microscopic observation drug-susceptibility assay vs. Xpert® MTB/RIF for the diagnosis of tuberculosis in a rural African setting: a cost-utility analysis.

Science.gov (United States)

Wikman-Jorgensen, Philip E; Llenas-García, Jara; Pérez-Porcuna, Tomàs M; Hobbins, Michael; Ehmer, Jochen; Mussa, Manuel A; Ascaso, Carlos

2017-06-01

To compare the cost-utility of microscopic observation drug-susceptibility assay (MODS) and Xpert ® MTB/RIF implementation for tuberculosis (TB) diagnosis in rural northern Mozambique. Stochastic transmission compartmental TB model from the healthcare provider perspective with parameter input from direct measurements, systematic literature reviews and expert opinion. MODS and Xpert ® MTB/RIF were evaluated as replacement test of smear microscopy (SM) or as an add-on test after a negative SM. Costs were calculated in 2013 USD, effects in disability-adjusted life years (DALY). Willingness to pay threshold (WPT) was established at once the per capita Gross National Income of Mozambique. MODS as an add-on test to negative SM produced an incremental cost-effectiveness ratio (ICER) of 5647.89USD/DALY averted. MODS as a substitute for SM yielded an ICER of 5374.58USD/DALY averted. Xpert ® MTB/RIF as an add-on test to negative SM yielded ICER of 345.71USD/DALY averted. Xpert ® MTB/RIF as a substitute for SM obtained an ICER of 122.13USD/DALY averted. TB prevalence and risk of infection were the main factors impacting MODS and Xpert ® MTB/RIF ICER in the one-way sensitivity analysis. In the probabilistic sensitivity analysis, Xpert ® MTB/RIF was most likely to have an ICER below the WPT, whereas MODS was not. Our cost-utility analysis favours the implementation of Xpert ® MTB/RIF as a replacement of SM for all TB suspects in this rural high TB/HIV prevalence African setting. © 2017 John Wiley & Sons Ltd.

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.

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

Observation of reversible, rapid changes in drug susceptibility of hypoxic tumor cells in a microfluidic device

Energy Technology Data Exchange (ETDEWEB)

Germain, Todd; Ansari, Megan; Pappas, Dimitri, E-mail: d.pappas@ttu.edu

2016-09-14

Hypoxia is a major stimulus for increased drug resistance and for survival of tumor cells. Work from our group and others has shown that hypoxia increases resistance to anti-cancer compounds, radiation, and other damage-pathway cytotoxic agents. In this work we utilize a microfluidic culture system capable of rapid switching of local oxygen concentrations to determine changes in drug resistance in prostate cancer cells. We observed rapid adaptation to hypoxia, with drug resistance to 2 μM staurosporine established within 30 min of hypoxia. Annexin-V/Sytox Green apoptosis assays over 9 h showed 78.0% viability, compared to 84.5% viability in control cells (normoxic cells with no staurosporine). Normoxic cells exposed to the same staurosporine concentration had a viability of 48.6% after 9 h. Hypoxia adaptation was rapid and reversible, with Hypoxic cells treated with 20% oxygen for 30 min responding to staurosporine with 51.6% viability after drug treatment for 9 h. Induction of apoptosis through the receptor-mediated pathway, which bypasses anti-apoptosis mechanisms induced by hypoxia, resulted in 39.4 ± 7% cell viability. The rapid reversibility indicates co-treatment of oxygen with anti-cancer compounds may be a potential therapeutic target. - Highlights: • Microfluidic system switches rapidly between normoxia and hypoxia (5 min). • Observation of rapid adaptation of PC3 cells to hypoxia and normoxia (30 min). • Drug susceptibility in tumor cells restored after chip switched to normoxia for 30 min.

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.

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.

Use of Pinus sylvestris L. (Pinaceae), Origanum vulgare L. (Lamiaceae), and Thymus vulgaris L. (Lamiaceae) essential oils and their main components to enhance itraconazole activity against azole susceptible/not-susceptible Cryptococcus neoformans strains.

Science.gov (United States)

Scalas, Daniela; Mandras, Narcisa; Roana, Janira; Tardugno, Roberta; Cuffini, Anna Maria; Ghisetti, Valeria; Benvenuti, Stefania; Tullio, Vivian

2018-05-03

Cryptococcal infections, besides being a problem for immunocompromised patients, are occasionally being a problem for immunocompetent patients. In addition, the lower susceptibility of this yeast to azoles is a growing problem in health care. To date, there are very few molecules with any activity towards Cryptococcus neoformans, leading to heightened interest in finding new alternatives or adjuvants to conventional drugs for the treatment of mycosis caused by this yeast. Since the essential oils (EOs) are considered as a potential rich source of bioactive antimicrobial compounds, we evaluated the antifungal activity of Origanum vulgare (oregano), Pinus sylvestris (pine), and Thymus vulgaris (thyme red) EOs, and their components (α-pinene, carvacrol, thymol) compared with fluconazole, itraconazole, and voriconazole, against C.neoformans clinical strains. Then, we investigated the effect of EOs and components in combination with itraconazole. EO composition was analysed by Gas chromatography-mass spectrometry (GC-MS). A broth microdilution method was used to evaluate the susceptibility of C.neoformans to azoles, EOs and components. Checkerboard tests, isobolograms and time-kill assays were carried out for combination studies. Six C.neoformans isolates were susceptible to azoles, while one C.neoformans exhibited a reduced susceptibility to all tested azole drugs. All EOs exerted a good inhibitory activity against all C.neoformans strains. Pine EO was the most effective. Among components, thymol exerted the most remarkable activity. By checkerboard testing and isobolographic analysis, combinations of itraconazole with oregano, pine, or thyme EOs, and carvacrol were found to be synergistic (FICI≤0.5) against azole susceptible C.neoformans. Regarding the azole not susceptible C.neoformans strain, the synergistic effect with itraconazole was observed with thyme EO (chemotype: thymol 26.52%; carvacrol 7.85%), and carvacrol. Time-kill assays confirmed the synergistic

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.

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.

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.

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)

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

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.

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

Directory of Open Access Journals (Sweden)

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.

Mortality among MDR-TB cases: comparison with drug-susceptible tuberculosis and associated factors.

Directory of Open Access Journals (Sweden)

Kocfa Chung-Delgado

Full Text Available An increase in multidrug-resistant tuberculosis (MDR-TB cases is evident worldwide. Its management implies a complex treatment, high costs, more toxic anti-tuberculosis drug use, longer treatment time and increased treatment failure and mortality. The aims of this study were to compare mortality between MDR and drug-susceptible cases of tuberculosis, and to determine risk factors associated with mortality among MDR-TB cases.A retrospective cohort study was performed using data from clinical records of the National Strategy for Prevention and Control of Tuberculosis in Lima, Peru. In the first objective, MDR-TB, compared to drug-susceptible cases, was the main exposure variable and time to death, censored at 180 days, the outcome of interest. For the second objective, different variables obtained from clinical records were assessed as potential risk factors for death among MDR-TB cases. Cox regression analysis was used to determine hazard ratios (HR and 95% confidence intervals (95%CI. A total of 1,232 patients were analyzed: mean age 30.9 ±14.0 years, 60.0% were males. 61 patients (5.0% died during treatment, whereas the MDR-TB prevalence was 19.2%. MDR-TB increased the risk of death during treatment (HR = 7.5; IC95%: 4.1-13.4 when compared to presumed drug-susceptible cases after controlling for potential confounders. Education level (p = 0.01, previous TB episodes (p<0.001, diabetes history (p<0.001 and HIV infection (p = 0.04 were factors associated with mortality among MDR-TB cases.MDR-TB is associated with an increased risk of death during treatment. Lower education, greater number of previous TB episodes, diabetes history, and HIV infection were independently associated with mortality among MDR-TB cases. New strategies for appropriate MDR-TB detection and management should be implemented, including drug sensitivity tests, diabetes and HIV screening, as well as guarantee for a complete adherence to therapy.

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 ...

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

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.

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.

Profiling gene expression of antimony response genes in Leishmania (Viannia) panamensis and infected macrophages and its relationship with drug susceptibility.

Science.gov (United States)

Barrera, Maria Claudia; Rojas, Laura Jimena; Weiss, Austin; Fernandez, Olga; McMahon-Pratt, Diane; Saravia, Nancy G; Gomez, Maria Adelaida

2017-12-01

The mechanisms of Leishmania resistance to antimonials have been primarily determined in experimentally derived Leishmania strains. However, their participation in the susceptibility phenotype in field isolates has not been conclusively established. Being an intracellular parasite, the activity of antileishmanials is dependent on internalization of drugs into host cells and effective delivery to the intracellular compartments inhabited by the parasite. In this study we quantified and comparatively analyzed the gene expression of nine molecules involved in mechanisms of xenobiotic detoxification and Leishmania resistance to antimonial drugs in resistant and susceptible laboratory derived and clinical L.(Viannia) panamensis strains(n=19). In addition, we explored the impact of Leishmania susceptibility to antimonials on the expression of macrophage gene products having putative functions in transport, accumulation and metabolism of antimonials. As previously shown for other Leishmania species, a trend of increased abcc3 and lower aqp-1 expression was observed in the laboratory derived Sb-resistant L.(V.) panamensis line. However, this was not found in clinical strains, in which the expression of abca2 was significantly higher in resistant strains as both, promastigotes and intracellular amastigotes. The effect of drug susceptibility on host cell gene expression was evaluated on primary human macrophages from patients with cutaneous leishmaniasis (n=17) infected ex-vivo with the matched L.(V.) panamensis strains isolated at diagnosis, and in THP-1 cells infected with clinical strains (n=6) and laboratory adapted L.(V.) panamensis lines. Four molecules, abcb1 (p-gp), abcb6, aqp-9 and mt2a were differentially modulated by drug resistant and susceptible parasites, and among these, a consistent and significantly increased expression of the xenobiotic scavenging molecule mt2a was observed in macrophages infected with Sb-susceptible L. (V.) panamensis. Our results

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 ...

In vitro antimalarial drug susceptibility in Thai border areas from 1998–2003

Directory of Open Access Journals (Sweden)

Mungthin Mathirut

2005-08-01

Full Text Available Abstract Background The Thai-Myanmar and Thai-Cambodia borders have been historically linked with the emergence and spread of Plasmodium falciparum parasites resistant to antimalarial drugs. Indeed, the areas are often described as harbouring multi-drug resistant parasites. These areas of Thailand have experienced significant changes in antimalarial drug exposure patterns over the past decade. This study describes the in vitro antimalarial susceptibility patterns of 95 laboratory-adapted P. falciparum isolates, collected between 1998 and 2003,. Methods Ninety five P. falciparum isolates were collected from five sites in Thailand between 1998 and 2003. After laboratory adaptation to in vitro culture, the susceptibility of these parasites to a range of established antimalarial drugs (chloroquine [CQ], mefloquine [MQ], quinine [QN] and dihydroartemisinin [DHA] was determined by the isotopic microtest. Results Mefloquine (MQ sensitivity remained poorest in areas previously described as MQ-resistant areas. Sensitivity to MQ of parasites from this area was significantly lower than those from areas reported to harbour moderate (p = 0.002 of low level MQ resistance (p = 000001. Importantly for all drugs tested, there was a considerable range in absolute parasite sensitivities. There was a weak, but statistically positive correlation between parasite sensitivity to CQ and sensitivity to both QN and MQ and a positive correlation between MQ and QN. In terms of geographical distribution, parasites from the Thai-Cambodia were tended to be less sensitive to all drugs tested compared to the Thai-Myanmar border. Parasite sensitivity to all drugs was stable over the 6-year collection period with the exception of QN. Conclusion This study highlights the high degree of variability in parasite drug sensitivity in Thailand. There were geographical differences in the pattern of resistance which might reflect differences in drug usage in each area. In contrast to many

Trypanosoma cruzi benznidazole susceptibility in vitro does not predict the therapeutic outcome of human Chagas disease

Directory of Open Access Journals (Sweden)

Margoth Moreno

2010-11-01

Full Text Available Therapeutic failure of benznidazole (BZ is widely documented in Chagas disease and has been primarily associated with variations in the drug susceptibility of Trypanosoma cruzi strains. In humans, therapeutic success has been assessed by the negativation of anti-T. cruzi antibodies, a process that may take up to 10 years. A protocol for early screening of the drug resistance of infective strains would be valuable for orienting physicians towards alternative therapies, with a combination of existing drugs or new anti-T. cruzi agents. We developed a procedure that couples the isolation of parasites by haemoculture with quantification of BZ susceptibility in the resultant epimastigote forms. BZ activity was standardized with reference strains, which showed IC50 to BZ between 7.6-32 µM. The assay was then applied to isolates from seven chronic patients prior to administration of BZ therapy. The IC50 of the strains varied from 15.6 ± 3-51.4 ± 1 µM. Comparison of BZ susceptibility of the pre-treatment isolates of patients considered cured by several criteria and of non-cured patients indicates that the assay does not predict therapeutic outcome. A two-fold increase in BZ resistance in the post-treatment isolates of two patients was verified. Based on the profile of nine microsatellite loci, sub-population selection in non-cured patients was ruled out.

A cost-effective smartphone-based antimicrobial susceptibility test reader for drug resistance testing (Conference Presentation)

Science.gov (United States)

Feng, Steve W.; Tseng, Derek; Di Carlo, Dino; Garner, Omai B.; Ozcan, Aydogan

2017-03-01

Antimicrobial susceptibility testing (AST) is commonly used for determining microbial drug resistance, but routine testing, which can significantly reduce the spread of multi-drug resistant organisms, is not regularly performed in resource-limited and field-settings due to technological challenges and lack of trained diagnosticians. We developed a portable cost-effective smartphone-based colorimetric 96-well microtiter plate (MTP) reader capable of automated AST without the need for a trained diagnostician. This system is composed of a smartphone used in conjunction with a 3D-printed opto-mechanical attachment, which holds a set of inexpensive light-emitting-diodes and fiber-optic cables coupled to the 96-well MTP for enabling the capture of the transmitted light through each well by the smartphone camera. Images of the MTP plate are captured at multiple exposures and uploaded to a local or remote server (e.g., a laptop) for automated processing/analysis of the results using a custom-designed smartphone application. Each set of images are combined to generate a high dynamic-range image and analyzed for well turbidity (indicative of bacterial growth), followed by interpretative analysis per plate to determine minimum inhibitory concentration (MIC) and drug susceptibility for the specific bacterium. Results are returned to the originating device within 1 minute and shown to the user in tabular form. We demonstrated the capability of this platform using MTPs prepared with 17 antibiotic drugs targeting Gram-negative bacteria and tested 82 patient isolate MTPs of Klebsiella pneumoniae, achieving well turbidity accuracy of 98.19%, MIC accuracy of 95.15%, and drug susceptibility interpretation accuracy of 99.06%, meeting the FDA defined criteria for AST.

Recapitulation of Clinical Individual Susceptibility to Drug-Induced QT Prolongation in Healthy Subjects Using iPSC-Derived Cardiomyocytes

Directory of Open Access Journals (Sweden)

Tadahiro Shinozawa

2017-02-01

Full Text Available To predict drug-induced serious adverse events (SAE in clinical trials, a model using a panel of cells derived from human induced pluripotent stem cells (hiPSCs of individuals with different susceptibilities could facilitate major advancements in translational research in terms of safety and pharmaco-economics. However, it is unclear whether hiPSC-derived cells can recapitulate interindividual differences in drug-induced SAE susceptibility in populations not having genetic disorders such as healthy subjects. Here, we evaluated individual differences in SAE susceptibility based on an in vitro model using hiPSC-derived cardiomyocytes (hiPSC-CMs as a pilot study. hiPSCs were generated from blood samples of ten healthy volunteers with different susceptibilities to moxifloxacin (Mox-induced QT prolongation. Different Mox-induced field potential duration (FPD prolongation values were observed in the hiPSC-CMs from each individual. Interestingly, the QT interval was significantly positively correlated with FPD at clinically relevant concentrations (r > 0.66 in multiple analyses including concentration-QT analysis. Genomic analysis showed no interindividual significant differences in known target-binding sites for Mox and other drugs such as the hERG channel subunit, and baseline QT ranges were normal. The results suggest that hiPSC-CMs from healthy subjects recapitulate susceptibility to Mox-induced QT prolongation and provide proof of concept for in vitro preclinical trials.

Development of an in vitro bioassay for measuring susceptibility to macrocyclic lactone anthelmintics in Dirofilaria immitis.

Science.gov (United States)

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

2013-12-01

For more than 20 years, anthelmintics of the macrocyclic lactone (ML) drug class have been widely and effectively used as preventives against the canine heartworm, Dirofilaria immitis. However, in recent years an increased number of lack of efficacy (LOE) cases are being reported, in which dogs develop mature heartworm infections despite receiving monthly prophylactic doses of ML drugs. While this situation is raising concerns that heartworms may be developing resistance to MLs, compelling evidence for this is still lacking. Resolution of this dilemma requires validated biological or molecular diagnostic assays, but, unfortunately, no such tests currently exist. To address this need, we developed and optimized a larval migration inhibition assay (LMIA) for use with D. immitis third-stage larvae. The LMIA was used to measure the in vitro dose-response of two ML drugs (ivermectin and eprinomectin) on a known ML-susceptible laboratory strain of D. immitis. A nonlinear regression model was fit to the dose-response data, from which IC50 values were calculated; the mean IC50 and 95% confidence interval for IVM was 4.56 μM (1.26-16.4 μM), greater than that for EPR at 2.02 μM (1.68-2.42 μM), and this difference was significant (p = 0.0428). The R (2) value for EPR assays (0.90) was also greater than that for IVM treatment (0.71). The consistency and reproducibility of the dose-response data obtained with this assay suggests that it may be a useful technique for investigating the relative susceptibilities to ML drugs in other D. immitis populations.

Drug susceptibility testing of Mycobacterium Avium subsp. Avium isolates from naturally infected domestic pigeons to avian tuberculosis

Directory of Open Access Journals (Sweden)

Kaveh Parvandar

2016-01-01

Conclusion: We suggest drug susceptibility testing for more nontuberculous mycobateria, particularly M. avium complex isolated from infected birds and humans, as well as molecular basics of drug sensitivity in order to detect resistance genes of pathogenic M. avium subsp. avium.

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.

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

Directory of Open Access Journals (Sweden)

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

Analyzing pepsin degradation assay conditions used for allergenicity assessments to ensure that pepsin susceptible and pepsin resistant dietary proteins are distinguishable.

Directory of Open Access Journals (Sweden)

Rong Wang

Full Text Available The susceptibility of a dietary protein to proteolytic degradation by digestive enzymes, such as gastric pepsin, provides information on the likelihood of systemic exposure to a structurally intact and biologically active macromolecule, thus informing on the safety of proteins for human and animal consumption. Therefore, the purpose of standardized in vitro degradation studies that are performed during protein safety assessments is to distinguish whether proteins of interest are susceptible or resistant to pepsin degradation via a study design that enables study-to-study comparison. Attempting to assess pepsin degradation under a wide-range of possible physiological conditions poses a problem because of the lack of robust and consistent data collected under a large-range of sub-optimal conditions, which undermines the needs to harmonize in vitro degradation conditions. This report systematically compares the effects of pH, incubation time, and pepsin-to-substrate protein ratio on the relative degradation of five dietary proteins: three pepsin susceptible proteins [ribulose 1,5-bisphosphate carboxylase-oxygenase (Rubisco, horseradish peroxidase (HRP, hemoglobin (Hb], and two pepsin resistant proteins [lipid transfer protein (LTP and soybean trypsin inhibitor (STI]. The results indicate that proteins susceptible to pepsin degradation are readily distinguishable from pepsin-resistant proteins when the reaction conditions are within the well-characterized optima for pepsin. The current standardized in vitro pepsin resistant assay with low pH and high pepsin-to-substrate ratio fits this purpose. Using non-optimal pH and/or pepsin-to-substrate protein ratios resulted in susceptible proteins no longer being reliably degraded by this stomach enzyme, which compromises the ability of this in vitro assay to distinguish between resistant and susceptible proteins and, therefore, no longer providing useful data to an overall weight-of-evidence approach to

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.

The microculture tetrazolium assay (MTA): another colorimetric method of testing Plasmodium falciparum chemosensitivity.

Science.gov (United States)

Delhaes, L; Lazaro, J E; Gay, F; Thellier, M; Danis, M

1999-01-01

Malarial lactate dehydrogenase (LDH), which uses 3-acetyl pyridine adenine dinucleotide as coenzyme in a reaction leading to the formation of pyruvate from L-lactate, may be used to study the susceptibility of Plasmodium falciparum to a drug in vitro. Several methods to determine the activity of this enzyme are available. One, the colorimetric method of Makler and colleagues, was modified slightly, by using sodium-2,3-bis-[2-methoxy-4-nitro-5-sulphophenyl]-2H-tetrazolium-5 - carboxanilide (XTT) and following the reaction by measuring the optical density at 450 nm. Using two, culture-adapted strains of P. falciparum, this LDH assay was compared with the unmodified Makler's assay and with the isotopic microtest based on the incorporation of tritium-labelled hypoxanthine. Fresh, clinical P. falciparum isolates were also tested in the presence of several drugs, including chloroquine, mefloquine, quinine, halofantrine, atovaquone and qinghaosu derivatives. The results of the three assays were correlated for all the drugs tested except atovaquone. The two enzymatic assays are non-radioactive, rapid, reliable, inexpensive to perform and semi-automatic. However, they do require an initial parasitaemia of 2% with a haematocrit of 1.8%.

Simple, direct drug susceptibility testing technique for diagnosis of drug-resistant tuberculosis in resource-poor settings.

Science.gov (United States)

Kim, C-K; Joo, Y-T; Lee, E P; Park, Y K; Kim, H-J; Kim, S J

2013-09-01

The Korean Institute of Tuberculosis, Seoul, Republic of Korea. To develop a simple, direct drug susceptibility testing (DST) technique using Kudoh-modified Ogawa (KMO) medium. The critical concentrations of isoniazid (INH), rifampicin (RMP), kanamycin (KM) and ofloxacin (OFX) for KMO medium were calibrated by comparing the minimal inhibitory concentrations (MICs) against clinical isolates of Mycobacterium tuberculosis on KMO with those on Löwenstein-Jensen (LJ). The performance of the direct KMO DST technique was evaluated on 186 smear-positive sputum specimens and compared with indirect LJ DST. Agreement of MICs on direct vs. indirect DST was high for INH, RMP and OFX. KM MICs on KMO were ∼10 g/ml higher than those on LJ. The critical concentrations of INH, RMP, OFX and KM for KMO were therefore set at 0.2, 40.0, 2.0, and 40.0 g/ml. The evaluation of direct DST of smear-positive sputum specimens showed 100% agreement with indirect LJ DST for INH and RMP. However, the respective susceptible and resistant predictive values were 98.8% and 100% for OFX, and 100% and 80% for KM. Direct DST using KMO is useful, with clear advantages of a shorter turnaround time, procedural simplicity and low cost compared to indirect DST. It may be most indicated in resource-poor settings for programmatic management of drug-resistant tuberculosis.

Fluoromycobacteriophages for Rapid, Specific, and Sensitive Antibiotic Susceptibility Testing of Mycobacterium tuberculosis

Science.gov (United States)

Piuri, Mariana; Jacobs, William R.; Hatfull, Graham F.

2009-01-01

Rapid antibiotic susceptibility testing of Mycobacterium tuberculosis is of paramount importance as multiple- and extensively- drug resistant strains of M. tuberculosis emerge and spread. We describe here a virus-based assay in which fluoromycobacteriophages are used to deliver a GFP or ZsYellow fluorescent marker gene to M. tuberculosis, which can then be monitored by fluorescent detection approaches including fluorescent microscopy and flow cytometry. Pre-clinical evaluations show that addition of either Rifampicin or Streptomycin at the time of phage addition obliterates fluorescence in susceptible cells but not in isogenic resistant bacteria enabling drug sensitivity determination in less than 24 hours. Detection requires no substrate addition, fewer than 100 cells can be identified, and resistant bacteria can be detected within mixed populations. Fluorescence withstands fixation by paraformaldehyde providing enhanced biosafety for testing MDR-TB and XDR-TB infections. PMID:19300517

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

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.

In vitro susceptibility of nematophagous fungi to antiparasitic drugs: interactions and implications for biological control

Directory of Open Access Journals (Sweden)

J. N. Vieira

Full Text Available Abstract The fast anthelmintic resistance development has shown a limited efficiency in the control of animal’s endoparasitosis and has promoted research using alternative control methods. The use of chemicals in animal anthelmintic treatment, in association with nematophagous fungi used for biological control, is a strategy that has proven to be effective in reducing the nematode population density in farm animals. This study aims to verify the in vitro susceptibility of the nematophagous fungi Arthrobotrys oligospora, Duddingtonia flagrans and Paecilomyces lilacinus against the antiparasitic drugs albendazole, thiabendazole, ivermectin, levamisole and closantel by using the Minimum Inhibitory Concentration (MIC. MICs ranged between 4.0 and 0.031 µg/mL for albendazole, thiabendazole and ivermectin, between 0.937 and 0.117 µg/mL for levamisole, and between 0.625 and 0.034 µg/mL for closantel. The results showed that all antiparasitic drugs had an in vitro inhibitory effect on nematophagous fungi, which could compromise their action as agents of biological control. D. flagrans was the most susceptible species to all drugs.

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

Science.gov (United States)

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

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

Directory of Open Access Journals (Sweden)

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.

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...

Klebsiella pneumoniae yfiRNB operon affects biofilm formation, polysaccharide production and drug susceptibility.

Science.gov (United States)

Huertas, Mónica G; Zárate, Lina; Acosta, Iván C; Posada, Leonardo; Cruz, Diana P; Lozano, Marcela; Zambrano, María M

2014-12-01

Klebsiella pneumoniae is an opportunistic pathogen important in hospital-acquired infections, which are complicated by the rise of drug-resistant strains and the capacity of cells to adhere to surfaces and form biofilms. In this work, we carried out an analysis of the genes in the K. pneumoniae yfiRNB operon, previously implicated in biofilm formation. The results indicated that in addition to the previously reported effect on type 3 fimbriae expression, this operon also affected biofilm formation due to changes in cellulose as part of the extracellular matrix. Deletion of yfiR resulted in enhanced biofilm formation and an altered colony phenotype indicative of cellulose overproduction when grown on solid indicator media. Extraction of polysaccharides and treatment with cellulase were consistent with the presence of cellulose in biofilms. The enhanced cellulose production did not, however, correlate with virulence as assessed using a Caenorhabditis elegans assay. In addition, cells bearing mutations in genes of the yfiRNB operon varied with respect to the WT control in terms of susceptibility to the antibiotics amikacin, ciprofloxacin, imipenem and meropenem. These results indicated that the yfiRNB operon is implicated in the production of exopolysaccharides that alter cell surface characteristics and the capacity to form biofilms--a phenotype that does not necessarily correlate with properties related with survival, such as resistance to antibiotics. © 2014 The Authors.

Candida Species From Eye Infections: Drug Susceptibility, Virulence Factors, and Molecular Characterization.

Science.gov (United States)

Ranjith, Konduri; Sontam, Bhavani; Sharma, Savitri; Joseph, Joveeta; Chathoth, Kanchana N; Sama, Kalyana C; Murthy, Somasheila I; Shivaji, Sisinthy

2017-08-01

To determine the type of Candida species in ocular infections and to investigate the relationship of antifungal susceptibility profile to virulence factors. Fifty isolates of yeast-like fungi from patients with keratitis, endophthalmitis, and orbital cellulitis were identified by Vitek-2 compact system and DNA sequencing of ITS1-5.8S-ITS2 regions of the rRNA gene, followed by phylogenetic analysis for phenotypic and genotypic identification, respectively. Minimum inhibitory concentration of six antifungal drugs was determined by E test/microbroth dilution methods. Phenotypic and genotypic methods were used to determine the virulence factors. Phylogenetic analysis showed the clustering of all isolates into eight distinct groups with a major cluster formed Candida parapsilosis (n = 21), which was the most common species by both Vitek 2 and DNA sequencing. Using χ2 test no significant difference was noted between the techniques except that Vitek 2 did not identify C. viswanathii, C. orthopsilosis, and two non-Candida genera. Of 43 tested Candida isolates high susceptibility to amphotericin B (39/43, 90.6%) and natamycin (43/43, 100%) was noted. While none of the isolates produced coagulase, all produced esterase and catalase. The potential to form biofilm was detected in 23/43 (53.4%) isolates. Distribution of virulence factors by heat map analysis showed difference in metabolic activity of biofilm producers from nonbiofilm producers. Identified by Vitek 2 and DNA sequencing methods C. parapsilosis was the most common species associated with eye infections. Irrespective of the virulence factors elaborated, the Candida isolates were susceptible to commonly used antifungal drugs such as amphotericin B and natamycin.

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

Drug Susceptibility Testing of 31 Antimicrobial Agents on Rapidly Growing Mycobacteria Isolates from China.

Science.gov (United States)

Pang, Hui; Li, Guilian; Zhao, Xiuqin; Liu, Haican; Wan, Kanglin; Yu, Ping

2015-01-01

Several species of rapidly growing mycobacteria (RGM) are now recognized as human pathogens. However, limited data on effective drug treatments against these organisms exists. Here, we describe the species distribution and drug susceptibility profiles of RGM clinical isolates collected from four southern Chinese provinces from January 2005 to December 2012. Clinical isolates (73) were subjected to in vitro testing with 31 antimicrobial agents using the cation-adjusted Mueller-Hinton broth microdilution method. The isolates included 55 M. abscessus, 11 M. fortuitum, 3 M. chelonae, 2 M. neoaurum, and 2 M. septicum isolates. M. abscessus (75.34%) and M. fortuitum (15.07%), the most common species, exhibited greater antibiotic resistance than the other three species. The isolates had low resistance to amikacin, linezolid, and tigecycline, and high resistance to first-line antituberculous agents, amoxicillin-clavulanic acid, rifapentine, dapsone, thioacetazone, and pasiniazid. M. abscessus and M. fortuitum were highly resistant to ofloxacin and rifabutin, respectively. The isolates showed moderate resistance to the other antimicrobial agents. Our results suggest that tigecycline, linezolid, clofazimine, and cefmetazole are appropriate choices for M. abscessus infections. Capreomycin, sulfamethoxazole, tigecycline, clofazimine, and cefmetazole are potentially good choices for M. fortuitum infections. Our drug susceptibility data should be useful to clinicians.

Drug Susceptibility Testing of 31 Antimicrobial Agents on Rapidly Growing Mycobacteria Isolates from China

Directory of Open Access Journals (Sweden)

Hui Pang

2015-01-01

Full Text Available Objectives. Several species of rapidly growing mycobacteria (RGM are now recognized as human pathogens. However, limited data on effective drug treatments against these organisms exists. Here, we describe the species distribution and drug susceptibility profiles of RGM clinical isolates collected from four southern Chinese provinces from January 2005 to December 2012. Methods. Clinical isolates (73 were subjected to in vitro testing with 31 antimicrobial agents using the cation-adjusted Mueller-Hinton broth microdilution method. The isolates included 55 M. abscessus, 11 M. fortuitum, 3 M. chelonae, 2 M. neoaurum, and 2 M. septicum isolates. Results. M. abscessus (75.34% and M. fortuitum (15.07%, the most common species, exhibited greater antibiotic resistance than the other three species. The isolates had low resistance to amikacin, linezolid, and tigecycline, and high resistance to first-line antituberculous agents, amoxicillin-clavulanic acid, rifapentine, dapsone, thioacetazone, and pasiniazid. M. abscessus and M. fortuitum were highly resistant to ofloxacin and rifabutin, respectively. The isolates showed moderate resistance to the other antimicrobial agents. Conclusions. Our results suggest that tigecycline, linezolid, clofazimine, and cefmetazole are appropriate choices for M. abscessus infections. Capreomycin, sulfamethoxazole, tigecycline, clofazimine, and cefmetazole are potentially good choices for M. fortuitum infections. Our drug susceptibility data should be useful to clinicians.

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

Rapid, low-cost fluorescent assay of β-lactamase-derived antibiotic resistance and related antibiotic susceptibility

Science.gov (United States)

Erdem, S. Sibel; Khan, Shazia; Palanisami, Akilan; Hasan, Tayyaba

2014-10-01

Antibiotic resistance (AR) is increasingly prevalent in low and middle income countries (LMICs), but the extent of the problem is poorly understood. This lack of knowledge is a critical deficiency, leaving local health authorities essentially blind to AR outbreaks and crippling their ability to provide effective treatment guidelines. The crux of the problem is the lack of microbiology laboratory capacity available in LMICs. To address this unmet need, we demonstrate a rapid and simple test of β-lactamase resistance (the most common form of AR) that uses a modified β-lactam structure decorated with two fluorophores quenched due to their close proximity. When the β-lactam core is cleaved by β-lactamase, the fluorophores dequench, allowing assay speeds of 20 min to be obtained with a simple, streamlined protocol. Furthermore, by testing in competition with antibiotics, the β-lactamase-associated antibiotic susceptibility can also be extracted. This assay can be easily implemented into standard lab work flows to provide near real-time information of β-lactamase resistance, both for epidemiological purposes as well as individualized patient care.

A multiplex single nucleotide polymorphism typing assay for detecting mutations that result in decreased fluoroquinolone susceptibility in Salmonella enterica serovars Typhi and Paratyphi A.

LENUS (Irish Health Repository)

Song, Yajun

2010-08-01

OBJECTIVES: Decreased susceptibility to fluoroquinolones has become a major problem for the successful therapy of human infections caused by Salmonella enterica, especially the life-threatening typhoid and paratyphoid fevers. METHODS: By using Luminex xTAG beads, we developed a rapid, reliable and cost-effective multiplexed genotyping assay for simultaneously detecting 11 mutations in gyrA, gyrB and parE of S. enterica serovars Typhi and Paratyphi A that result in nalidixic acid resistance (Nal(R)) and\\/or decreased susceptibility to fluoroquinolones. RESULTS: This assay yielded unambiguous single nucleotide polymorphism calls on extracted DNA from 292 isolates of Salmonella Typhi (Nal(R) = 223 and Nal(S) = 69) and 106 isolates of Salmonella Paratyphi A (Nal(R) = 24 and Nal(S) = 82). All of the 247 Nal(R) Salmonella Typhi and Salmonella Paratyphi A isolates were found to harbour at least one of the target mutations, with GyrA Phe-83 as the most common one (143\\/223 for Salmonella Typhi and 18\\/24 for Salmonella Paratyphi A). We also identified three GyrB mutations in eight Nal(S) Salmonella Typhi isolates (six for GyrB Phe-464, one for GyrB Leu-465 and one for GyrB Asp-466), and mutations GyrB Phe-464 and GyrB Asp-466 seem to be related to the decreased ciprofloxacin susceptibility phenotype in Salmonella Typhi. This assay can also be used directly on boiled single colonies. CONCLUSIONS: The assay presented here would be useful for clinical and reference laboratories to rapidly screen quinolone-resistant isolates of Salmonella Typhi and Salmonella Paratyphi A, and decipher the underlying genetic changes for epidemiological purposes.

Antifungal susceptibility testing of Aspergillus species complex in the Clinical Laboratory: how to do it, when to do it, and how to interpret it

Directory of Open Access Journals (Sweden)

Esther Manso

2014-12-01

Full Text Available The emergence of drug resistance in fungal pathogens has a profound impact on human health given limited number of antifungal drugs. Antifungal resistance in Aspergillus spp. infection can be encountered in the antifungal drug-exposed patient due to selection of intrinsically resistant species or isolates with acquired resistance belonging to species that are normally susceptible. Resistance to triazoles is not common in Aspergillus spp., however, triazole resistance in A. fumigatus appears to be increasing in several European countries in recent years and can be clinically relevant. The Clinical and Laboratory Standards Institute and European Committee on Antimicrobial Susceptibility Testing have developed breakpoints and epidemiological cutoff values that are now established for Aspergillus spp. Clinical microbiology laboratories will be employed commercial susceptibility assays, rather than reference broth microdilution methods and comparative studies are particularly important.

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.

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.

A Low Cost/Low Power Open Source Sensor System for Automated Tuberculosis Drug Susceptibility Testing

Directory of Open Access Journals (Sweden)

Kyukwang Kim

2016-06-01

Full Text Available In this research an open source, low power sensor node was developed to check the growth of mycobacteria in a culture bottle with a nitrate reductase assay method for a drug susceptibility test. The sensor system reports the temperature and color sensor output frequency change of the culture bottle when the device is triggered. After the culture process is finished, a nitrite ion detecting solution based on a commercial nitrite ion detection kit is injected into the culture bottle by a syringe pump to check bacterial growth by the formation of a pigment by the reaction between the solution and the color sensor. Sensor status and NRA results are broadcasted via a Bluetooth low energy beacon. An Android application was developed to collect the broadcasted data, classify the status of cultured samples from multiple devices, and visualize the data for the end users, circumventing the need to examine each culture bottle manually during a long culture period. The authors expect that usage of the developed sensor will decrease the cost and required labor for handling large amounts of patient samples in local health centers in developing countries. All 3D-printerable hardware parts, a circuit diagram, and software are available online.

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.

Antimicrobial susceptibility patterns of Brazilian Haemophilus parasuis field isolates

Directory of Open Access Journals (Sweden)

Michela Miani

Full Text Available ABSTRACT: Haemophilus parasuis is the etiological agent of Glässer’s disease (GD, an ubiquitous infection of swine characterized by systemic fibrinous polyserositis, polyarthritis and meningitis. Intensive use of antimicrobial agents in swine husbandries during the last years triggered the development of antibiotic resistances in bacterial pathogens. Thus, regular susceptibility testing is crucial to ensure efficacy of different antimicrobial agents to this porcine pathogen. In this study, 50 clinical isolates from South Brazilian pig herds were characterized and analyzed for their susceptibility to commonly used antibiotic. The identification and typing of clinical isolates was carried out by a modified indirect hemagglutination assay combined with a multiplex PCR. The susceptibility of each isolate was analyzed by broth microdilution method against a panel of 21 antimicrobial compounds. We found that field isolates are highly resistance to gentamycin, bacitracin, lincomycin and tiamulin, but sensitive to ampicillin, clindamycin, neomycin, penicillin, danofloxacin and enrofloxacin. Furthermore, an individual susceptibility analysis indicated that enrofloxacin is effective to treat clinical isolates with the exception of those classified as serovar 1. The results presented here firstly demonstrate the susceptibility of Brazilian clinical isolates of H. parasuis to antimicrobials widely used by swine veterinary practitioners and strengthen the need to perform susceptibility test prior to antibiotic therapy during GD outbreaks. In addition, because only six antimicrobial drugs (28.6% were found effective against field isolates, a continuous surveillance of the susceptibility profile should be of major concern to the swine industry.

Radiometric macrophage culture assay for rapid evaluation of antileprosy activity of rifampin

Energy Technology Data Exchange (ETDEWEB)

Mittal, A.; Seshadri, P.S.; Prasad, H.K.; Sathish, M.; Nath, I.

1983-10-01

The antileprosy effect of rifampin was evaluated by a newly developed rapid in vitro assay wherein 31 human-derived strains and 1 armadillo-derived strain of Mycobacterium leprae were maintained for 2 and 3 weeks, respectively, in murine and human macrophages in the presence of (3H)thymidine. Of these strains, 27 showed significant incorporation of the radiolabel in cultures of live bacilli as compared with control cultures of heat-killed bacilli of the same strain. Consistent and significant inhibition of (3H)thymidine uptake was observed in M. leprae resident cultures with 3 to 200 ng of rifampin per ml as compared with similar cultures without the drug. In general, an increase in percent inhibition was seen from 3 to 20 ng/ml, with marginal increases at 40, 50, and 100 ng/ml. M. leprae strains appear to be remarkably susceptible to this drug in the in vitro assay.

Radiometric macrophage culture assay for rapid evaluation of antileprosy activity of rifampin

International Nuclear Information System (INIS)

Mittal, A.; Seshadri, P.S.; Prasad, H.K.; Sathish, M.; Nath, I.

1983-01-01

The antileprosy effect of rifampin was evaluated by a newly developed rapid in vitro assay wherein 31 human-derived strains and 1 armadillo-derived strain of Mycobacterium leprae were maintained for 2 and 3 weeks, respectively, in murine and human macrophages in the presence of [3H]thymidine. Of these strains, 27 showed significant incorporation of the radiolabel in cultures of live bacilli as compared with control cultures of heat-killed bacilli of the same strain. Consistent and significant inhibition of [3H]thymidine uptake was observed in M. leprae resident cultures with 3 to 200 ng of rifampin per ml as compared with similar cultures without the drug. In general, an increase in percent inhibition was seen from 3 to 20 ng/ml, with marginal increases at 40, 50, and 100 ng/ml. M. leprae strains appear to be remarkably susceptible to this drug in the in vitro assay

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.

In Vitro Susceptibility of Iranian Isolates of Trichomonas vaginalis to Metronidazole

Directory of Open Access Journals (Sweden)

Mohammad MATINI

2016-03-01

Full Text Available Background: Metronidazole, a 5-nitroimidazole derivative, is the main antitrich­omonal agent of choice for treatment of trichomoniasis. Since 1962, some cases of treatment failure with metronidazole have been reported and recently drug re­sistance is now on the rise in the world. This study was aimed to determine current susceptibility of Iranian isolates of Trichomonas vaginalis to metronidazole.Methods: This study was performed on 50 T. vaginalis isolates collected from west and central areas of Iran. After axenisation of the parasites, susceptibility testing was carried out by using serial twofold dilutions of metronidazole (400 to 0.1 µg/ml. The minimum inhibitory concentration (MIC and the minimum lethal concentration (MLC of the trichomonads were determined after 48 h incubation at 35.5 °C. Drug susceptibility assays of the all isolates were carried out two times in triplicate under aerobic and anaerobic conditions. Results: Ninety-eight percent of the T. vaginalis isolates (49/50 were sensitive to metronidazole. Metronidazole resistance was defined as aerobic MIC ≥50 µg/ml, detected in one isolate. The means of aerobic MICs and MLCs and that of anaero­bic MICs of the parasites were 2.91, 1.95 and 0.28 µg/ml, respectively.Conclusion: This investigation showed in vitro low-level tolerance to metronida­zole in a few T. vaginalis isolates that may be leading to the development of drug resistance.

Species-Specific and Drug-Specific Differences in Susceptibility of Candida Biofilms to Echinocandins: Characterization of Less Common Bloodstream Isolates

Science.gov (United States)

Simitsopoulou, Maria; Peshkova, Pavla; Tasina, Efthymia; Katragkou, Aspasia; Kyrpitzi, Daniela; Velegraki, Aristea; Walsh, Thomas J.

2013-01-01

Candida species other than Candida albicans are increasingly recognized as causes of biofilm-associated infections. This is a comprehensive study that compared the in vitro activities of all three echinocandins against biofilms formed by different common and infrequently identified Candida isolates. We determined the activities of anidulafungin (ANID), caspofungin (CAS), and micafungin (MFG) against planktonic cells and biofilms of bloodstream isolates of C. albicans (15 strains), Candida parapsilosis (6 strains), Candida lusitaniae (16 strains), Candida guilliermondii (5 strains), and Candida krusei (12 strains) by XTT [2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] assay. Planktonic and biofilm MICs were defined as ≥50% fungal damage. Planktonic cells of all Candida species were susceptible to the three echinocandins, with MICs of ≤1 mg/liter. By comparison, differences in the MIC profiles of biofilms in response to echinocandins existed among the Candida species. Thus, C. lusitaniae and C. guilliermondii biofilms were highly recalcitrant to all echinocandins, with MICs of ≥32 mg/liter. In contrast, the MICs of all three echinocandins for C. albicans and C. krusei biofilms were relatively low (MICs ≤ 1 mg/liter). While echinocandins exhibited generally high MICs against C. parapsilosis biofilms, MFG exhibited the lowest MICs against these isolates (4 mg/liter). A paradoxical growth effect was observed with CAS concentrations ranging from 8 to 64 mg/liter against C. albicans and C. parapsilosis biofilms but not against C. krusei, C. lusitaniae, or C. guilliermondii. While non-albicans Candida planktonic cells were susceptible to all echinocandins, there were drug- and species-specific differences in susceptibility among biofilms of the various Candida species, with C. lusitaniae and C. guilliermondii exhibiting profiles of high MICs of the three echinocandins. PMID:23529739

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.

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...

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.

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.

ANTIFUNGAL SUSCEPTIBILITY TESTING: CURRENT ROLE FROM THE CLINICAL LABORATORY PERSPECTIVE

Directory of Open Access Journals (Sweden)

Brunella Posteraro

2014-04-01

Full Text Available Despite availability of many antifungal agents, antifungal clinical resistance occurs, perhaps as a result of an infecting organism found to be resistant in vitro to one or more antifungals tested. Thus, antifungal susceptibility testing (AFST results, if timely generated by the clinical microbiology and communicated to clinicians, can aid them in the therapeutic decision making, especially for difficult-to-treat invasive candidiasis and aspergillosis. Although recently refined AFST methods are commercially available to allow a close antifungal resistance surveillance in many clinical setting, novel assays, relying on short-time antifungal drug exposure of fungal isolates, are upcoming tools for AFST. Based on emerging technologies such as flow cytometry, MALDI-TOF mass spectrometry, and isothermal microcalorimetry, these assays could provide a reliable means for quicker and sensitive assessment of AFST.

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)

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.

Characterization of drug susceptibility of Mycobacterium tuberculosis isolated from new cases of tuberculosis concurrent with HIV infection

Directory of Open Access Journals (Sweden)

G. V. Panov

2015-01-01

Full Text Available The paper characterizes drug susceptibility in Mycobacterium tuberculosis isolated from new cases of tuberculosis concurrent with HIV infection. The investigators have studied the spectrum of drug resistance in Mycobacterium tuberculosis isolated from new cases of tuberculosis concurrent with and without HIV infection (172 and 309 clinical isolates, respectively. There are differences in the rate of primary drug resistance to antituberculosis drugs in patients with and without HIV infection (59 and 43.5% of the cases, respectively. The HIV-infected have also shown high rifampicin resistance rates in Mycobacterium tuberculosis (41.7%. The reasons for these differences are as yet unknown and call for further investigation.

Activity of clinically relevant antimalarial drugs on Plasmodium falciparum mature gametocytes in an ATP bioluminescence "transmission blocking" assay.

Directory of Open Access Journals (Sweden)

Joël Lelièvre

Full Text Available BACKGROUND: Current anti-malarial drugs have been selected on the basis of their activity against the symptom-causing asexual blood stage of the parasite. Which of these drugs also target gametocytes, in the sexual stage responsible for disease transmission, remains unknown. Blocking transmission is one of the main strategies in the eradication agenda and requires the identification of new molecules that are active against gametocytes. However, to date, the main limitation for measuring the effect of molecules against mature gametocytes on a large scale is the lack of a standardized and reliable method. Here we provide an efficient method to produce and purify mature gametocytes in vitro. Based on this new procedure, we developed a robust, affordable, and sensitive ATP bioluminescence-based assay. We then assessed the activity of 17 gold-standard anti-malarial drugs on Plasmodium late stage gametocytes. METHODS AND FINDINGS: Difficulties in producing large amounts of gametocytes have limited progress in the development of malaria transmission blocking assays. We improved the method established by Ifediba and Vanderberg to obtain viable, mature gametocytes en masse, whatever the strain used. We designed an assay to determine the activity of antimalarial drugs based on the intracellular ATP content of purified stage IV-V gametocytes after 48 h of drug exposure in 96/384-well microplates. Measurements of drug activity on asexual stages and cytotoxicity on HepG2 cells were also obtained to estimate the specificity of the active drugs. CONCLUSIONS: The work described here represents another significant step towards determination of the activity of new molecules on mature gametocytes of any strain with an automated assay suitable for medium/high-throughput screening. Considering that the biology of the forms involved in the sexual and asexual stages is very different, a screen of our 2 million-compound library may allow us to discover novel anti

Development of a novel cell-based assay system EPISSAY for screening epigenetic drugs and liposome formulated decitabine

International Nuclear Information System (INIS)

Lim, Sue Ping; Callen, David F; Kumar, Raman; Akkamsetty, Yamini; Wang, Wen; Ho, Kristen; Neilsen, Paul M; Walther, Diego J; Suetani, Rachel J; Prestidge, Clive

2013-01-01

Despite the potential of improving the delivery of epigenetic drugs, the subsequent assessment of changes in their epigenetic activity is largely dependent on the availability of a suitable and rapid screening bioassay. Here, we describe a cell-based assay system for screening gene reactivation. A cell-based assay system (EPISSAY) was designed based on a silenced triple-mutated bacterial nitroreductase TMnfsB fused with Red-Fluorescent Protein (RFP) expressed in the non-malignant human breast cell line MCF10A. EPISSAY was validated using the target gene TXNIP, which has previously been shown to respond to epigenetic drugs. The potency of a epigenetic drug model, decitabine, formulated with PEGylated liposomes was also validated using this assay system. Following treatment with DNA methyltransferase (DNMT) and histone deacetylase (HDAC) inhibitors such as decitabine and vorinostat, increases in RFP expression were observed, indicating expression of RFP-TMnfsB. The EPISSAY system was then used to test the potency of decitabine, before and after PEGylated liposomal encapsulation. We observed a 50% higher potency of decitabine when encapsulated in PEGylated liposomes, which is likely to be due to its protection from rapid degradation. The EPISSAY bioassay system provides a novel and rapid system to compare the efficiencies of existing and newly formulated drugs that reactivate gene expression

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...

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

Directory of Open Access Journals (Sweden)

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.

Pathogens causing blood stream infections and their drug susceptibility profile in immunocompromised patients.

Science.gov (United States)

Fayyaz, Muhammad; Mirza, Irfan Ali; Ikram, Aamer; Hussain, Aamir; Ghafoor, Tahir; Shujat, Umer

2013-12-01

To determine the types of pathogens causing blood stream infections and their drug susceptibility profile in immunocompromised patients. Cross-sectional, observational study. Department of Microbiology, Armed Forces Institute of Pathology, Rawalpindi, from January to September 2012. Blood culture bottles received from immunocompromised patients were dealt by two methods, brain heart infusion (BHI) broth based manual method and automated BACTEC system. The samples yielding positive growth from either of two methods were further analyzed. The identification of isolates was done with the help of biochemical reactions and rapid tests. Antimicrobial susceptibility of the isolates was carried out as per recommendations of Clinical and Laboratory Standards Institute (CLSI). Out of the 938 blood culture specimens received from immunocompromised patients, 188 (20%) yielded positive growth. Out of these, 89 (47.3%) isolates were Gram positive and Gram negative each, while 10 (5.3%) isolates were fungi (Candida spp.). In case of Gram positive isolates, 75 (84.3%) were Staphylococcus spp. and 51 (67%) were Methicillin resistant. Amongst Gram negative group 49 (55.1%) isolates were of enterobacteriaceae family, while 40 (44.9%) were non-lactose fermenters (NLF). In vitro antimicrobial susceptibility of Staphylococci revealed 100% susceptibility to vancomycin and linezolid. The enterobacteriaceae isolates had better susceptibility against amikacin 85.7% compared to tigecycline 61.2% and imipenem 59.2%. For NLF, the in vitro efficacy of aminoglycosides was 72.5%. The frequency of Gram positive and Gram negative organisms causing blood stream infections in immunocompromised patients was equal. Vancomycin in case of Gram positive and amikacin for Gram negative organisms revealed better in vitro efficacy as compared to other antibiotics.

Pathogens Causing Blood Stream Infections and their Drug Susceptibility Profile in Immunocompromised Patients

International Nuclear Information System (INIS)

Fayyaz, M.; Mirza, I.A.; Ikram, A.; Hussain, A.; Ghafoor, T.; Shujat, U.

2013-01-01

Objective: To determine the types of pathogens causing blood stream infections and their drug susceptibility profile in immunocompromised patients. Study Design: Cross-sectional, observational study. Place and Duration of Study: Department of Microbiology, Armed Forces Institute of Pathology, Rawalpindi, from January to September 2012. Methodology: Blood culture bottles received from immunocompromised patients were dealt by two methods, brain heart infusion (BHI) broth based manual method and automated BACTEC system. The samples yielding positive growth from either of two methods were further analyzed. The identification of isolates was done with the help of biochemical reactions and rapid tests. Antimicrobial susceptibility of the isolates was carried out as per recommendations of Clinical and Laboratory Standards Institute (CLSI). Results: Out of the 938 blood culture specimens received from immunocompromised patients, 188 (20%) yielded positive growth. Out of these, 89 (47.3%) isolates were Gram positive and Gram negative each, while 10 (5.3%) isolates were fungi (Candida spp.). In case of Gram positive isolates, 75 (84.3%) were Staphylococcus spp. and 51 (67%) were Methicillin resistant. Amongst Gram negative group 49 (55.1%) isolates were of enterobacteriaceae family, while 40 (44.9%) were non-lactose fermenters (NLF). In vitro antimicrobial susceptibility of Staphylococci revealed 100% susceptibility to vancomycin and linezolid. The enterobacteriaceae isolates had better susceptibility against amikacin 85.7% compared to tigecycline 61.2% and imipenem 59.2%. For NLF, the in vitro efficacy of aminoglycosides was 72.5%. Conclusion: The frequency of Gram positive and Gram negative organisms causing blood stream infections in immunocompromised patients was equal. Vancomycin in case of Gram positive and amikacin for Gram negative organisms revealed better in vitro efficacy as compared to other antibiotics. (author)

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.

Directory of Open Access Journals (Sweden)

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.

A simple field kit for the determination of drug susceptibility in Plasmodium falciparum.

Science.gov (United States)

Nguyen-Dinh, P; Magloire, R; Chin, W

1983-05-01

A field kit has been developed which greatly simplifies the performance of the 48-hour in vitro test for drug resistance in Plasmodium falciparum. The kit uses an easily reconstituted lyophilized culture medium, and requires only a fingerprick blood sample. In parallel tests with 13 isolates of P. falciparum in Haiti, the new technique had a success rate equal to that of the previously described method, with comparable results in terms of parasite susceptibility in vitro to chloroquine and pyrimethamine.

Correlation between the genotoxicity endpoints measured by two different genotoxicity assays: comet assay and CBMN assay

Directory of Open Access Journals (Sweden)

Carina Ladeira

2015-06-01

The results concerning of positive findings by micronuclei and non significant ones by comet assay, are corroborated by Deng et al. (2005 study performed in workers occupationally exposed to methotrexate, also a cytostatic drug. According to Cavallo et al. (2009, the comet assay seems to be more suitable for the prompt evaluation of the genotoxic effects, for instance, of polycyclic aromatic hydrocarbons mixtures containing volatile substances, whereas the micronucleus test seems more appropriate to evaluate the effects of exposure to antineoplastic agents. However, there are studies that observed an increase in both the comet assay and the micronucleus test in nurses handling antineoplastic drugs, although statistical significance was only seen in the comet assay, quite the opposite of our results (Maluf & Erdtmann, 2000; Laffon et al. 2005.

Morphological Characteristics of Schistosoma mansoni PZQ-Resistant and -Susceptible Strains Are Different in Presence of Praziquantel

Science.gov (United States)

Pinto-Almeida, António; Mendes, Tiago; de Oliveira, Rosimeire Nunes; Corrêa, Sheila de Andrade Penteado; Allegretti, Silmara Marques; Belo, Silvana; Tomás, Ana; Anibal, Fernanda de Freitas; Carrilho, Emanuel; Afonso, Ana

2016-01-01

Schistosomiasis is one of the most common human parasitic diseases whose socioeconomic impact is only surpassed by malaria. Praziquantel (PZQ) is the only drug commercially available for the treatment of all schistosome species causing disease in humans. However, there has been stronger evidences of PZQ-resistance on Schistosoma mansoni and thus it is very important to study the phenotypic characteristics associated with it. The aim of this study was to evaluate morphological alterations in S. mansoni PZQ-resistant adult worms and eggs, by comparing a PZQ- resistant strain obtained under PZQ drug pressure with a PZQ-susceptible strain. For this, scanning electronic microscopy was used to assess tegumental responsiveness of both strains under PZQ exposure, and optical microscopy allowed the monitoring of worms and eggs in the presence of the drug. Those assays showed that PZQ-susceptible worms exposed to the drug had more severe tegumental damages than the resistant one, which had only minor alterations. Moreover, contrary to what occurred in the susceptible strain, resistant worms were viable after PZQ exposure and gradually regaining full motility after removal of the drug. Eggs from resistant strain parasites are considerably smaller than those from susceptible strain. Our results suggest that there might be a difference in the tegument composition of the resistant strain and that worms are less responsive to PZQ. Changes observed in egg morphology might imply alterations in the biology of schistosomes associated to PZQ-resistance, which could impact on transmission and pathology of the disease. Moreover, we propose a hypothetical scenario where there is a different egg tropism of the S. mansoni resistant strain. This study is the first comparing two strains that only differ in their resistance characteristics, which makes it a relevant step in the search for resistance determinants. PMID:27199925

An in vitro study of antifungal drug susceptibility of Candida species isolated from human immunodeficiency virus seropositive and human immunodeficiency virus seronegative individuals in Lucknow population Uttar Pradesh.

Science.gov (United States)

Dar, Mohammad Shafi; Sreedar, Gadiputi; Shukla, Abhilasha; Gupta, Prashant; Rehan, Ahmad Danish; George, Jiji

2015-01-01

Candidiasis is the most common opportunistic infection in human immunodeficiency virus (HIV) seropositive patients, starting from asymptomatic colonization to pathogenic forms and gradual colonization of non-albicans in patients with advanced immunosuppression leads to resistance for azole group of antifungal drugs with high rate of morbidity and mortality. To isolate the Candida species and determine of antifungal drug susceptibility against fluconazole, itraconazole, nystatin, amphotericin B, and clotrimazolein HIV seropositive and control individuals, with or without clinical oropharyngeal candidiasis (OPC). Includes samples from faucial region of 70 subjects with and without clinical candidiasis in HIV seropositive and controls were aseptically inoculated onto Sabaraud's Dextrose Agar media and yeasts were identified for the specific species by Corn Meal Agar, sugar fermentation and heat tolerance tests. Antifungal drug susceptibility of the isolated species was done against above-mentioned drugs by E-test and disc diffusion method. The commonly isolated species in HIV seropositive and controls were Candida albicans, Candida glabrata and Candida tropicalis Candida guilliermondii and Candida dubliniensis isolated only in HIV seropositive patients. Susceptibility against selected antifungal drugs was observed more in HIV-negative individuals whereas susceptible dose-dependent and resistance were predominant in HIV-positive patients. Resistance is the major problem in the therapy of OPC, especially in HIV seropositive patients due to aggressive and prolonged use of antifungal agents, therefore, our study emphasizes the need for antifungal drug susceptibility testing whenever antifungal treatment is desired, especially in HIV-infected subjects.

Drug susceptibility testing of Mycobacterium tuberculosis to fluoroquinolones

DEFF Research Database (Denmark)

Johansen, I S; Larsen, A R; Sandven, P

2003-01-01

In the first attempt to establish a quality assurance programme for susceptibility testing of Mycobacterium tuberculosis to fluoroquinolones, 20 strains with different fluoroquinolone susceptibility patterns were distributed by the Supranational Reference Laboratory in Stockholm to the other...

Microscopic observation drug susceptibility assay (MODS for early diagnosis of tuberculosis in children.

Directory of Open Access Journals (Sweden)

Dang Thi Minh Ha

2009-12-01

Full Text Available MODS is a novel liquid culture based technique that has been shown to be effective and rapid for early diagnosis of tuberculosis (TB. We evaluated the MODS assay for diagnosis of TB in children in Viet Nam. 217 consecutive samples including sputum (n = 132, gastric fluid (n = 50, CSF (n = 32 and pleural fluid (n = 3 collected from 96 children with suspected TB, were tested by smear, MODS and MGIT. When test results were aggregated by patient, the sensitivity and specificity of smear, MGIT and MODS against "clinical diagnosis" (confirmed and probable groups as the gold standard were 28.2% and 100%, 42.3% and 100%, 39.7% and 94.4%, respectively. The sensitivity of MGIT and MODS was not significantly different in this analysis (P = 0.5, but MGIT was more sensitive than MODS when analysed on the sample level using a marginal model (P = 0.03. The median time to detection of MODS and MGIT were 8 days and 13 days, respectively, and the time to detection was significantly shorter for MODS in samples where both tests were positive (P<0.001. An analysis of time-dependent sensitivity showed that the detection rates were significantly higher for MODS than for MGIT by day 7 or day 14 (P<0.001 and P = 0.04, respectively. MODS is a rapid and sensitive alternative method for the isolation of M.tuberculosis from children.

Development and characterization of a pre-treatment procedure to eliminate human monoclonal antibody therapeutic drug and matrix interference in cell-based functional neutralizing antibody assays.

Science.gov (United States)

Xu, Weifeng; Jiang, Hao; Titsch, Craig; Haulenbeek, Jonathan R; Pillutla, Renuka C; Aubry, Anne-Françoise; DeSilva, Binodh S; Arnold, Mark E; Zeng, Jianing; Dodge, Robert W

2015-01-01

Biological therapeutics can induce an undesirable immune response resulting in the formation of anti-drug antibodies (ADA), including neutralizing antibodies (NAbs). Functional (usually cell-based) NAb assays are preferred to determine NAb presence in patient serum, but are often subject to interferences from numerous serum factors, such as growth factors and disease-related cytokines. Many functional cell-based NAb assays are essentially drug concentration assays that imply the presence of NAbs by the detection of small changes in functional drug concentration. Any drug contained in the test sample will increase the total amount of drug in the assay, thus reducing the sensitivity of NAb detection. Biotin-drug Extraction with Acid Dissociation (BEAD) has been successfully applied to extract ADA, thereby removing drug and other interfering factors from human serum samples. However, to date there has been no report to estimate the residual drug level after BEAD treatment when the drug itself is a human monoclonal antibody; mainly due to the limitation of traditional ligand-binding assays. Here we describe a universal BEAD optimization procedure for human monoclonal antibody (mAb) drugs by using a LC-MS/MS method to simultaneously measure drug (a mutant human IgG4), NAb positive control (a mouse IgG), and endogenous human IgGs as an indicator of nonspecific carry-over in the BEAD eluate. This is the first report demonstrating that residual human mAb drug level in clinical sample can be measured after BEAD pre-treatment, which is critical for further BEAD procedure optimization and downstream immunogenicity testing. Copyright © 2014 Elsevier B.V. All rights reserved.

A fast, simple method for screening radiation susceptibility genes by RNA interference

International Nuclear Information System (INIS)

Tsuji, Atsushi B.; Sudo, Hitomi; Sugyo, Aya; Otsuki, Marika; Miyagishi, Makoto; Taira, Kazunari; Imai, Takashi; Harada, Yoshi-nobu

2005-01-01

Radiotherapy can cause unacceptable levels of damage to normal tissues in some cancer patients. To understand the molecular mechanisms underlying radiation-induced physiological responses, and to be able to predict the radiation susceptibility of normal tissues in individual patients, it is important to identify a comprehensive set of genes responsible for radiation susceptibility. We have developed a simple and rapid 96-well screening protocol using cell proliferation assays and RNA interference to identify genes associated with radiation susceptibility. We evaluated the performance of alamarBlue-, BrdU-, and sulforhodamine B-based cell proliferation assays using the 96-well format. Each proliferation assay detected the known radiation susceptibility gene, PRKDC. In a trial screen using 28 shRNA vectors, another known gene, CDKN1A, and one new radiation susceptibility gene, ATP5G3, were identified. Our results indicate that this method may be useful for large-scale screens designed to identify novel radiation susceptibility genes

High-throughput behavioral phenotyping of drug and alcohol susceptibility traits in the expanded panel of BXD recombinant inbred strains

Energy Technology Data Exchange (ETDEWEB)

Philip, Vivek M [ORNL; Ansah, T [University of Tennessee Health Science Center, Memphis; Blaha, C, [University of Tennessee Health Science Center, Memphis; Cook, Melloni N. [University of Memphis; Hamre, Kristin M. [University of Tennessee Health Science Center, Memphis; Lariviere, William R [University of Pittsburgh; Matthews, Douglas B [Baylor University; Goldowitz, Daniel [University of British Columbia, Vancouver; Chesler, Elissa J [ORNL

2010-01-01

Genetic reference populations, particularly the BXD recombinant inbred strains, are a valuable resource for the discovery of the bio-molecular substrates and genetic drivers responsible for trait variation and co- ariation. This approach can be profitably applied in the analysis of susceptibility and mechanisms of drug and alcohol use disorders for which many predisposing behaviors may predict occurrence and manifestation of increased preference for these substances. Many of these traits are modeled by common mouse behavioral assays, facilitating the detection of patterns and sources of genetic co-regulation of predisposing phenotypes and substance consumption. Members of the Tennessee Mouse Genome Consortium have obtained behavioral phenotype data from 260 measures related to multiple behavioral assays across several domains: self-administration, response to, and withdrawal from cocaine, MDMA, morphine and alcohol; novelty seeking; behavioral despair and related neurological phenomena; pain sensitivity; stress sensitivity; anxiety; hyperactivity; and sleep/wake cycles. All traits have been measured in both sexes and the recently expanded panel of 69 additional BXD recombinant inbred strains (N=69). Sex differences and heritability estimates were obtained for each trait, and a comparison of early (N = 32) and recent BXD RI lines was performed. Primary data is publicly available for heritability, sex difference and genetic analyses using www.GeneNetwork.org. These analyses include QTL detection and genetic analysis of gene expression. Stored results from these analyses are available at http://ontologicaldiscovery.org for comparison to other genomic analysis results. Together with the results of related studies, these data form a public resource for integrative systems genetic analysis of neurobehavioral traits.

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

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

Science.gov (United States)

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.

[Drug susceptibility test guided therapy and novel empirical quadruple therapy for Helicobacter pylori infection: a network Meta-analysis].

Science.gov (United States)

Gou, Q Y; Yu, R B; Shi, R H

2017-05-10

Objective: To compare the efficacy and the risk of adverse effect of drug susceptibility test guided therapy and novel empirical quadruple therapy for Helicobacter ( H .) pylori infection. Methods: Literature retrieval was conducted by using major databases. Related papers published up to June 2015 were considered eligible if they were randomized control trials comparing different pharmacological formulations for H. pylori infection and used in a network Meta-analysis and a single rate Meta-analysis to evaluate the relative and absolute rates of H. pylori eradication and the risk of adverse effect. The Jadad score was used to evaluate the methodological quality. Funnel plot was constructed to evaluate the risk of publication bias. Begg's rank correlation test or Egger's regression intercept test was done for the asymmetry of funnel plot. Results: Twenty randomized control trials for the treatment of 6 753 initial treated patients with H. pylori infection were included. Drug susceptibility test guided therapy was significantly superior to concomitant therapy, hybrid therapy, sequential therapy and bismuth quadruple therapy. The culture-based therapy had the highest likelihood of improving clinical efficacy, with lowest risk of adverse effect. Concomitant therapy had the highest probability of causing adverse effect despite its effectiveness. Hybrid therapy and bismuth quadruple therapy were associated with lower risk of adverse effect and higher effectiveness. Conclusion: Drug susceptibility test guided therapy showed superiority to other 4 interventions for H. pylori eradication mentioned above. Hybrid therapy and bismuth quadruple therapy might be applied in the settings where the culture-based strategy is not available.

Bacterial profile and drug susceptibility pattern of urinary tract infection in pregnant women at University of Gondar Teaching Hospital, Northwest Ethiopia.

Science.gov (United States)

Alemu, Agersew; Moges, Feleke; Shiferaw, Yitayal; Tafess, Ketema; Kassu, Afework; Anagaw, Belay; Agegn, Abebe

2012-04-25

Urinary tract infection (UTI) is a common health problem among pregnant women. Proper investigation and prompt treatment are needed to prevent serious life threatening condition and morbidity due to urinary tract infection that can occur in pregnant women. Recent report in Addis Ababa, Ethiopia indicated the prevalence of UTI in pregnant women was 11.6% and Gram negative bacteria was the predominant isolates and showed multi drug resistance. This study aimed to assess bacterial profile that causes urinary tract infection and their antimicrobial susceptibility pattern among pregnant women visiting antenatal clinic at University of Gondar Teaching Hospital, Northwest Ethiopia. A cross-sectional study was conducted at University of Gondar Teaching Hospital from March 22 to April 30, 2011. Mid stream urine samples were collected and inoculated into Cystine Lactose Electrolyte Deficient medium (CLED). Colony counts yielding bacterial growth of 105/ml of urine or more of pure isolates were regarded as significant bacteriuria for infection. Colony from CLED was sub cultured onto MacConkey agar and blood agar plates. Identification was done using cultural characteristics and a series of biochemical tests. A standard method of agar disc diffusion susceptibility testing method was used to determine susceptibility patterns of the isolates. The overall prevalence of UTI in pregnant women was 10.4%. The predominant bacterial pathogens were Escherichia coli 47.5% followed by coagulase-negative staphylococci 22.5%, Staphylococcus aureus 10%, and Klebsiella pneumoniae 10%. Gram negative isolates were resulted low susceptibility to co-trimoxazole (51.9%) and tetracycline (40.7%) whereas Gram positive showed susceptibility to ceftriaxon (84.6%) and amoxicillin-clavulanic acid (92.3%). Multiple drug resistance (resistance to two or more drugs) was observed in 95% of the isolates. Significant bacteriuria was observed in asymptomatic pregnant women. Periodic studies are recommended to

Bacterial profile and drug susceptibility pattern of urinary tract infection in pregnant women at University of Gondar Teaching Hospital, Northwest Ethiopia

Directory of Open Access Journals (Sweden)

Alemu Agersew

2012-04-01

Full Text Available Abstract Background Urinary tract infection (UTI is a common health problem among pregnant women. Proper investigation and prompt treatment are needed to prevent serious life threatening condition and morbidity due to urinary tract infection that can occur in pregnant women. Recent report in Addis Ababa, Ethiopia indicated the prevalence of UTI in pregnant women was 11.6 % and Gram negative bacteria was the predominant isolates and showed multi drug resistance. This study aimed to assess bacterial profile that causes urinary tract infection and their antimicrobial susceptibility pattern among pregnant women visiting antenatal clinic at University of Gondar Teaching Hospital, Northwest Ethiopia. Methods A cross-sectional study was conducted at University of Gondar Teaching Hospital from March 22 to April 30, 2011. Mid stream urine samples were collected and inoculated into Cystine Lactose Electrolyte Deficient medium (CLED. Colony counts yielding bacterial growth of 105/ml of urine or more of pure isolates were regarded as significant bacteriuria for infection. Colony from CLED was sub cultured onto MacConkey agar and blood agar plates. Identification was done using cultural characteristics and a series of biochemical tests. A standard method of agar disc diffusion susceptibility testing method was used to determine susceptibility patterns of the isolates. Results The overall prevalence of UTI in pregnant women was 10.4 %. The predominant bacterial pathogens were Escherichia coli 47.5 % followed by coagulase-negative staphylococci 22.5 %, Staphylococcus aureus 10 %, and Klebsiella pneumoniae 10 %. Gram negative isolates were resulted low susceptibility to co-trimoxazole (51.9 % and tetracycline (40.7 % whereas Gram positive showed susceptibility to ceftriaxon (84.6 % and amoxicillin–clavulanic acid (92.3 %. Multiple drug resistance (resistance to two or more drugs was observed in 95 % of the isolates. Conclusion

Optimization of a Fluorescence-Based Assay for Large-Scale Drug Screening against Babesia and Theileria Parasites

OpenAIRE

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-thr...

Interventions on Metabolism: Making Antibiotic-Susceptible Bacteria

Directory of Open Access Journals (Sweden)

Fernando Baquero

2017-11-01

Full Text Available Antibiotics act on bacterial metabolism, and antibiotic resistance involves changes in this metabolism. Interventions on metabolism with drugs might therefore modify drug susceptibility and drug resistance. In their recent article, Martin Vestergaard et al. (mBio 8:e01114-17, 2017, https://doi.org/10.1128/mBio.01114-17 illustrate the possibility of converting intrinsically resistant bacteria into susceptible ones. They reported that inhibition of a central metabolic enzyme, ATP synthase, allows otherwise ineffective polymyxin antibiotics to act on Staphylococcus aureus. The study of the intrinsic resistome of bacterial pathogens has shown that several metabolic genes, including multigene transcriptional regulators, contribute to antibiotic resistance. In some cases, these genes only marginally increase antibiotic resistance, but reduced levels of susceptibility might be critical in the evolution or resistance under low antibiotic concentrations or in the clinical response of highly resistant bacteria. Drug interventions on bacterial metabolism might constitute a critical adjuvant therapy in combination with antibiotics to ensure susceptibility of pathogens with intrinsic or acquired antimicrobial resistance.

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

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.

Evaluation of MGIT 960 System for the Second-Line Drugs Susceptibility Testing of Mycobacterium tuberculosis

Directory of Open Access Journals (Sweden)

Hyejin Kim

2013-01-01

Full Text Available Many laboratories validate DST of the second-line drugs by BACTEC MGIT 960 system. The objective of this study is to evaluate the critical concentration and perform DST for the 2nd line drugs. We evaluated 193 clinical strains of M. tuberculosis isolated from patients in South Korea. Testing the critical concentration of six second-line drugs was performed by MGIT 960 and compared with L-J proportion method. The critical concentration was determined to establish the most one that gave the difference between drug resistance and susceptibility in MGIT960 system. Good agreement of the following concentrations was found: Concordance was 95% for 0.5 μg/mL of moxifloxacin; 93.6%, 1.0 μg/mL of levofloxacin; 97.5%, 2.5 μg/mL of kanamycin; 90.6%, 2.5 μg/mL of capreomycin; 86.2%, 5.0 μg/mL of ethionamide; and 90.8%, 2.0 μg/mL of ρ-aminosalicylic acid. The critical concentrations of the four drugs, moxifloxacin, levofloxacin, kanamycin, and capreomycin, were concordant and reliable for testing 2nd line drug resistance. Further study of ethionamide and ρ-aminosalicylic acid is required.

Susceptibility of Aeromonas Hydophila Isolates to Antimicrobial Drugs

Directory of Open Access Journals (Sweden)

Igor Stojanov

2010-05-01

Full Text Available Aeromonas hydrophila is a microorganism widely distributed in nature: in water, soil, food. It is also part of the normal bacterial flora of many animals. As an opportune microorganism it is a secondary biological agent that contributes to the occurrence of a fish disease and its deterioration. Frequently, its presence is an indication of bad zoohygiene and zootechnical conditions in fish ponds. Reduced quality and quantity of feed, mechanical injuries, parasitosis, seasonal oscillation in temperature present some of the factors that produce favorable conditions for bacterial proliferation of aeromonas in fish ponds, so clinical symptoms of the disease occur. Aeromonas is almost always present in clinical isolates and may be unjustly accused for bad health of fish. Antibiotic therapy is applied even when the clinical findings are clear, what certainly effects the susceptibility to chemotherapeutics. The subject of our work was bacteriological examination of the material obtained from the carps with the observed skin changes and the carps without these changes. Also, antimicrobial susceptibility of Aeromonas hydrophila was tested. The aim of this research was to determined the presence of Aeromonas hydrophilia in the carp ponds and to test antibiotic susceptibility. The material consisted of the samples from the fish ponds where the carps were with and without changed skin. The method the isolation of Aeromonas hydrophila was used. The diffusion disk technique was used for testing antibiotic susceptibility. The isolates were tested for their susceptibility to Florephenikol, Flumequine, Olaqindox and Oxitetracycline. The obtained results point that antimicrobial susceptibility was the same regardless of the origin of the samples, i.e. the resistance was the same for both groups of samples (the strains isolated from the fish with skin changes and the strains from fish without changes on skin. The strains were highly resistant: 35% were resistant to

Trends of Mycobacterium bovis Isolation and First-Line Anti-tuberculosis Drug Susceptibility Profile: A Fifteen-Year Laboratory-Based Surveillance.

Science.gov (United States)

Bobadilla-del Valle, Miriam; Torres-González, Pedro; Cervera-Hernández, Miguel Enrique; Martínez-Gamboa, Areli; Crabtree-Ramirez, Brenda; Chávez-Mazari, Bárbara; Ortiz-Conchi, Narciso; Rodríguez-Cruz, Luis; Cervantes-Sánchez, Axel; Gudiño-Enríquez, Tomasa; Cinta-Severo, Carmen; Sifuentes-Osornio, José; Ponce de León, Alfredo

2015-09-01

Mycobacterium tuberculosis causes the majority of tuberculosis (TB) cases in humans; however, in developing countries, human TB caused by M. bovis may be frequent but undetected. Human TB caused by M. bovis is considered a zoonosis; transmission is mainly through consumption of unpasteurized dairy products, and it is less frequently attributed to animal-to-human or human-to-human contact. We describe the trends of M. bovis isolation from human samples and first-line drug susceptibility during a 15-year period in a referral laboratory located in a tertiary care hospital in Mexico City. Data on mycobacterial isolates from human clinical samples were retrieved from the laboratory's database for the 2000-2014 period. Susceptibility to first-line drugs: rifampin, isoniazid, streptomycin (STR) and ethambutol was determined. We identified 1,165 isolates, 73.7% were M. tuberculosis and 26.2%, M. bovis. Among pulmonary samples, 16.6% were M. bovis. The proportion of M. bovis isolates significantly increased from 7.8% in 2000 to 28.4% in 2014 (X(2)trend, ptuberculosis isolates (10.9% vs.3.4%, ptuberculosis, respectively (p = 0.637). A rising trend of primary STR monoresistance was observed for both species (3.4% in 2000-2004 vs. 7.6% in 2010-2014; p = 0.02). There is a high prevalence and a rising trend of M. bovis isolates in our region. The proportion of pulmonary M. bovis isolates is higher than in previous reports. Additionally, we report high rates of primary anti-tuberculosis resistance and secondary MDR in both M. tuberculosis and M. bovis. This is one of the largest reports on drug susceptibility of M. bovis from human samples and shows a significant proportion of first-line anti-tuberculosis drug resistance.

Trends of Mycobacterium bovis Isolation and First-Line Anti-tuberculosis Drug Susceptibility Profile: A Fifteen-Year Laboratory-Based Surveillance

Science.gov (United States)

Bobadilla-del Valle, Miriam; Torres-González, Pedro; Cervera-Hernández, Miguel Enrique; Martínez-Gamboa, Areli; Crabtree-Ramirez, Brenda; Chávez-Mazari, Bárbara; Ortiz-Conchi, Narciso; Rodríguez-Cruz, Luis; Cervantes-Sánchez, Axel; Gudiño-Enríquez, Tomasa; Cinta-Severo, Carmen; Sifuentes-Osornio, José; Ponce de León, Alfredo

2015-01-01

Background Mycobacterium tuberculosis causes the majority of tuberculosis (TB) cases in humans; however, in developing countries, human TB caused by M. bovis may be frequent but undetected. Human TB caused by M. bovis is considered a zoonosis; transmission is mainly through consumption of unpasteurized dairy products, and it is less frequently attributed to animal-to-human or human-to-human contact. We describe the trends of M. bovis isolation from human samples and first-line drug susceptibility during a 15-year period in a referral laboratory located in a tertiary care hospital in Mexico City. Methodology/Principal Findings Data on mycobacterial isolates from human clinical samples were retrieved from the laboratory’s database for the 2000–2014 period. Susceptibility to first-line drugs: rifampin, isoniazid, streptomycin (STR) and ethambutol was determined. We identified 1,165 isolates, 73.7% were M. tuberculosis and 26.2%, M. bovis. Among pulmonary samples, 16.6% were M. bovis. The proportion of M. bovis isolates significantly increased from 7.8% in 2000 to 28.4% in 2014 (X 2 trend, ptuberculosis isolates (10.9% vs.3.4%, ptuberculosis, respectively (p = 0.637). A rising trend of primary STR monoresistance was observed for both species (3.4% in 2000–2004 vs. 7.6% in 2010–2014; p = 0.02). Conclusions/Significance There is a high prevalence and a rising trend of M. bovis isolates in our region. The proportion of pulmonary M. bovis isolates is higher than in previous reports. Additionally, we report high rates of primary anti-tuberculosis resistance and secondary MDR in both M. tuberculosis and M. bovis. This is one of the largest reports on drug susceptibility of M. bovis from human samples and shows a significant proportion of first-line anti-tuberculosis drug resistance. PMID:26421930

Prediction of phospholipidosis-inducing potential of drugs by in vitro biochemical and physicochemical assays followed by multivariate analysis.

Science.gov (United States)

Kuroda, Yukihiro; Saito, Madoka

2010-03-01

An in vitro method to predict phospholipidosis-inducing potential of cationic amphiphilic drugs (CADs) was developed using biochemical and physicochemical assays. The following parameters were applied to principal component analysis, as well as physicochemical parameters: pK(a) and clogP; dissociation constant of CADs from phospholipid, inhibition of enzymatic phospholipid degradation, and metabolic stability of CADs. In the score plot, phospholipidosis-inducing drugs (amiodarone, propranolol, imipramine, chloroquine) were plotted locally forming the subspace for positive CADs; while non-inducing drugs (chlorpromazine, chloramphenicol, disopyramide, lidocaine) were placed scattering out of the subspace, allowing a clear discrimination between both classes of CADs. CADs that often produce false results by conventional physicochemical or cell-based assay methods were accurately determined by our method. Basic and lipophilic disopyramide could be accurately predicted as a nonphospholipidogenic drug. Moreover, chlorpromazine, which is often falsely predicted as a phospholipidosis-inducing drug by in vitro methods, could be accurately determined. Because this method uses the pharmacokinetic parameters pK(a), clogP, and metabolic stability, which are usually obtained in the early stages of drug development, the method newly requires only the two parameters, binding to phospholipid, and inhibition of lipid degradation enzyme. Therefore, this method provides a cost-effective approach to predict phospholipidosis-inducing potential of a drug. Copyright (c) 2009 Elsevier Ltd. All rights reserved.

Prevalence and Antibiotic susceptibility pattern of Panton-Valentine ...

African Journals Online (AJOL)

Staphylococcal cassette chromosome mec typing by two standard multiplex PCR assay, revealed an uncharacterized resistance element. Overall antibiotic susceptibility pattern showed relatively high degree of susceptibility,however 1 isolate demostrated multidrug resistant pattern, 37(74.0%)resistant to only penicillin, 5 to ...

Prediction of phenotypic susceptibility to antiretroviral drugs using physiochemical properties of the primary enzymatic structure combined with artificial neural networks

DEFF Research Database (Denmark)

Kjaer, J; Høj, L; Fox, Z

2008-01-01

OBJECTIVES: Genotypic interpretation systems extrapolate observed associations in datasets to predict viral susceptibility to antiretroviral drugs (ARVs) for given isolates. We aimed to develop and validate an approach using artificial neural networks (ANNs) that employ descriptors...

Rapid identification and susceptibility testing of uropathogenic microbes via immunosorbent ATP-bioluminescence assay on a microfluidic simulator for antibiotic therapy.

Science.gov (United States)

Dong, Tao; Zhao, Xinyan

2015-02-17

The incorporation of pathogen identification with antimicrobial susceptibility testing (AST) was implemented on a concept microfluidic simulator, which is well suited for personalizing antibiotic treatment of urinary tract infections (UTIs). The microfluidic device employs a fiberglass membrane sandwiched between two polypropylene components, with capture antibodies immobilized on the membrane. The chambers in the microfluidic device share the same geometric distribution as the wells in a standard 384-well microplate, resulting in compatibility with common microplate readers. Thirteen types of common uropathogenic microbes were selected as the analytes in this study. The microbes can be specifically captured by various capture antibodies and then quantified via an ATP bioluminescence assay (ATP-BLA) either directly or after a variety of follow-up tests, including urine culture, antibiotic treatment, and personalized antibiotic therapy simulation. Owing to the design of the microfluidic device, as well as the antibody specificity and the ATP-BLA sensitivity, the simulator was proven to be able to identify UTI pathogen species in artificial urine samples within 20 min and to reliably and simultaneously verify the antiseptic effects of eight antibiotic drugs within 3-6 h. The measurement range of the device spreads from 1 × 10(3) to 1 × 10(5) cells/mL in urine samples. We envision that the medical simulator might be broadly employed in UTI treatment and could serve as a model for the diagnosis and treatment of other diseases.

Ergothioneine Maintains Redox and Bioenergetic Homeostasis Essential for Drug Susceptibility and Virulence of Mycobacterium tuberculosis

Directory of Open Access Journals (Sweden)

Vikram Saini

2016-01-01

Full Text Available The mechanisms by which Mycobacterium tuberculosis (Mtb maintains metabolic equilibrium to survive during infection and upon exposure to antimycobacterial drugs are poorly characterized. Ergothioneine (EGT and mycothiol (MSH are the major redox buffers present in Mtb, but the contribution of EGT to Mtb redox homeostasis and virulence remains unknown. We report that Mtb WhiB3, a 4Fe-4S redox sensor protein, regulates EGT production and maintains bioenergetic homeostasis. We show that central carbon metabolism and lipid precursors regulate EGT production and that EGT modulates drug sensitivity. Notably, EGT and MSH are both essential for redox and bioenergetic homeostasis. Transcriptomic analyses of EGT and MSH mutants indicate overlapping but distinct functions of EGT and MSH. Last, we show that EGT is critical for Mtb survival in both macrophages and mice. This study has uncovered a dynamic balance between Mtb redox and bioenergetic homeostasis, which critically influences Mtb drug susceptibility and pathogenicity.

Antimicrobial drug resistance of Salmonella enterica serovar typhi in asia and molecular mechanism of reduced susceptibility to the fluoroquinolones

NARCIS (Netherlands)

Chau, Tran Thuy; Campbell, James Ian; Galindo, Claudia M.; van Minh Hoang, Nguyen; Diep, To Song; Nga, Tran Thu Thi; van Vinh Chau, Nguyen; Tuan, Phung Quoc; Page, Anne Laure; Ochiai, R. Leon; Schultsz, Constance; Wain, John; Bhutta, Zulfiqar A.; Parry, Christopher M.; Bhattacharya, Sujit K.; Dutta, Shanta; Agtini, Magdarina; Dong, Baiqing; Honghui, Yang; Anh, Dang Duc; Canh, Do Gia; Naheed, Aliya; Albert, M. John; Phetsouvanh, Rattanaphone; Newton, Paul N.; Basnyat, Buddha; Arjyal, Amit; La, Tran Thi Phi; Rang, Nguyen Ngoc; Phuong, Le Thi; van Be Bay, Phan; von Seidlein, Lorenz; Dougan, Gordon; Clemens, John D.; Vinh, Ha; Hien, Tran Tinh; Chinh, Nguyen Tran; Acosta, Camilo J.; Farrar, Jeremy; Dolecek, Christiane

2007-01-01

This study describes the pattern and extent of drug resistance in 1,774 strains of Salmonella enterica serovar Typhi isolated across Asia between 1993 and 2005 and characterizes the molecular mechanisms underlying the reduced susceptibilities to fluoroquinolones of these strains. For 1,393 serovar

Feasibility of the fluorometric microculture cytotoxicity assay (FMCA) for cytotoxic drug sensitivity testing of tumor cells from patients with acute lymphoblastic leukemia.

Science.gov (United States)

Nygren, P; Kristensen, J; Jonsson, B; Sundström, C; Lönnerholm, G; Kreuger, A; Larsson, R

1992-11-01

The automated fluorometric microculture cytotoxicity assay (FMCA) was used for chemotherapeutic drug sensitivity testing of fresh and cryopreserved tumor cells from patients with acute lymphoblastic leukemia (ALL) at diagnosis and relapse. The technique success rate was 87% for fresh and 81% for cryopreserved samples. Up to 16 different cytotoxic drugs were routinely tested, but neither asparaginase nor methotrexate produced dose-response related cell kill. FMCA data showed good correlation to the well established Disc assay and the drug sensitivity reported by the FMCA was in good agreement with known clinical activity. Samples from children and initial ALL tended to be more drug sensitive than those from adults and ALL at relapse, respectively. For 36 samples clinical outcome was correlated to the quartile position in comparison to all other samples for the most in vitro active drug actually given to the patient. For patients with samples in the first, second, third, and fourth quartiles, the probabilities of complete remission were 89, 57, 38, and 0%, respectively. Using the median value as cut-off line, the sensitivity and specificity of the assay were 87 and 62%, respectively. It is concluded that the FMCA with a minimum of effort and with high success rate report clinically relevant drug sensitivity profiles for ALL.

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.

Miniaturizing 3D assay for high-throughput drug and genetic screens for small patient-derived tumor samples (Conference Presentation)

Science.gov (United States)

Rotem, Asaf; Garraway, Levi; Su, Mei-Ju; Basu, Anindita; Regev, Aviv; Struhl, Kevin

2017-02-01

Three-dimensional growth conditions reflect the natural environment of cancer cells and are crucial to be performed at drug screens. We developed a 3D assay for cellular transformation that involves growth in low attachment (GILA) conditions and is strongly correlated with the 50-year old benchmark assay-soft agar. Using GILA, we performed high-throughput screens for drugs and genes that selectively inhibit or increase transformation, but not proliferation. This phenotypic approach is complementary to our genetic approach that utilizes single-cell RNA-sequencing of a patient sample to identify putative oncogenes that confer sensitivity to drugs designed to specifically inhibit the identified oncoprotein. Currently, we are dealing with a big challenge in our field- the limited number of cells that might be extracted from a biopsy. Small patient-derived samples are hard to test in the traditional multiwell plate and it will be helpful to minimize the culture area and the experimental system. We managed to design a suitable microfluidic device for limited number of cells and perform the assay using image analysis. We aim to test drugs on tumor cells, outside of the patient body- and recommend on the ideal treatment that is tailored to the individual. This device will help to minimize biopsy-sampling volumes and minimize interventions in the patient's tumor.

Utility of a Multiplex PCR Assay for Detecting Herpesvirus DNA in Clinical Samples

Science.gov (United States)

Druce, Julian; Catton, Mike; Chibo, Doris; Minerds, Kirsty; Tyssen, David; Kostecki, Renata; Maskill, Bill; Leong-Shaw, Wendy; Gerrard, Marie; Birch, Chris

2002-01-01

A multiplex PCR was designed to amplify herpes simplex virus types 1 and 2, cytomegalovirus, and varicella-zoster virus DNA present in a diverse range of clinical material. The susceptibility of these viruses to in vivo inhibition by at least one antiviral drug was an important consideration in their inclusion in the multiplex detection system. An aliquot of equine herpesvirus was introduced into each specimen prior to extraction and served as an indicator of potential inhibitors of the PCR and a detector of suboptimal PCR conditions. Compared to virus isolation and immunofluorescence-based antigen detection, the multiplex assay yielded higher detection rates for all viruses represented in the assay. The turnaround time for performance of the assay was markedly reduced compared to those for the other techniques used to identify these viruses. More than 21,000 tests have been performed using the assay. Overall, the multiplex PCR enabled the detection of substantially increased numbers of herpesviruses, in some cases in specimens or anatomical sites where previously they were rarely if ever identified using traditional detection methods. PMID:11980951

21 CFR 864.7525 - Heparin assay.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Heparin assay. 864.7525 Section 864.7525 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7525 Heparin assay. (a) Identification. A...

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)

Susceptibility Testing of Medically Important Parasites.

Science.gov (United States)

Genetu Bayih, Abebe; Debnath, Anjan; Mitre, Edward; Huston, Christopher D; Laleu, Benoît; Leroy, Didier; Blasco, Benjamin; Campo, Brice; Wells, Timothy N C; Willis, Paul A; Sjö, Peter; Van Voorhis, Wesley C; Pillai, Dylan R

2017-07-01

In the last 2 decades, renewed attention to neglected tropical diseases (NTDs) has spurred the development of antiparasitic agents, especially in light of emerging drug resistance. The need for new drugs has required in vitro screening methods using parasite culture. Furthermore, clinical laboratories sought to correlate in vitro susceptibility methods with treatment outcomes, most notably with malaria. Parasites with their various life cycles present greater complexity than bacteria, for which standardized susceptibility methods exist. This review catalogs the state-of-the-art methodologies used to evaluate the effects of drugs on key human parasites from the point of view of drug discovery as well as the need for laboratory methods that correlate with clinical outcomes. Copyright © 2017 American Society for Microbiology.

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.

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.

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.

Second line drug susceptibility testing to inform the treatment of rifampin-resistant tuberculosis: a quantitative perspective

Directory of Open Access Journals (Sweden)

Emily A. Kendall

2017-03-01

Full Text Available Treatment failure and resistance amplification are common among patients with rifampin-resistant tuberculosis (TB. Drug susceptibility testing (DST for second-line drugs is recommended for these patients, but logistical difficulties have impeded widespread implementation of second-line DST in many settings. To provide a quantitative perspective on the decision to scale up second-line DST, we synthesize literature on the prevalence of second-line drug resistance, the expected clinical and epidemiologic benefits of using second-line DST to ensure that patients with rifampin-resistant TB receive effective regimens, and the costs of implementing (or not implementing second-line DST for all individuals diagnosed with rifampin-resistant TB. We conclude that, in most settings, second-line DST could substantially improve treatment outcomes for patients with rifampin-resistant TB, reduce transmission of drug-resistant TB, prevent amplification of drug resistance, and be affordable or even cost-saving. Given the large investment made in each patient treated for rifampin-resistant TB, these payoffs would come at relatively small incremental cost. These anticipated benefits likely justify addressing the real challenges faced in implementing second-line DST in most high-burden settings.

Genotyping three SNPs affecting warfarin drug response by isothermal real-time HDA assays.

Science.gov (United States)

Li, Ying; Jortani, Saeed A; Ramey-Hartung, Bronwyn; Hudson, Elizabeth; Lemieux, Bertrand; Kong, Huimin

2011-01-14

The response to the anticoagulant drug warfarin is greatly affected by genetic polymorphisms in the VKORC1 and CYP2C9 genes. Genotyping these polymorphisms has been shown to be important in reducing the time of the trial and error process for finding the maintenance dose of warfarin thus reducing the risk of adverse effects of the drug. We developed a real-time isothermal DNA amplification system for genotyping three single nucleotide polymorphisms (SNPs) that influence warfarin response. For each SNP, real-time isothermal Helicase Dependent Amplification (HDA) reactions were performed to amplify a DNA fragment containing the SNP. Amplicons were detected by fluorescently labeled allele specific probes during real-time HDA amplification. Fifty clinical samples were analyzed by the HDA-based method, generating a total of 150 results. Of these, 148 were consistent between the HDA-based assays and a reference method. The two samples with unresolved HDA-based test results were repeated and found to be consistent with the reference method. The HDA-based assays demonstrated a clinically acceptable performance for genotyping the VKORC1 -1639G>A SNP and two SNPs (430C>T and 1075A>C) for the CYP2C9 enzyme (CYP2C9*2 and CYP2C9*3), all of which are relevant in warfarin pharmacogenentics. Copyright © 2010 Elsevier B.V. All rights reserved.

Trends of Mycobacterium bovis Isolation and First-Line Anti-tuberculosis Drug Susceptibility Profile: A Fifteen-Year Laboratory-Based Surveillance.

Directory of Open Access Journals (Sweden)

Miriam Bobadilla-del Valle

2015-09-01

Full Text Available Mycobacterium tuberculosis causes the majority of tuberculosis (TB cases in humans; however, in developing countries, human TB caused by M. bovis may be frequent but undetected. Human TB caused by M. bovis is considered a zoonosis; transmission is mainly through consumption of unpasteurized dairy products, and it is less frequently attributed to animal-to-human or human-to-human contact. We describe the trends of M. bovis isolation from human samples and first-line drug susceptibility during a 15-year period in a referral laboratory located in a tertiary care hospital in Mexico City.Data on mycobacterial isolates from human clinical samples were retrieved from the laboratory's database for the 2000-2014 period. Susceptibility to first-line drugs: rifampin, isoniazid, streptomycin (STR and ethambutol was determined. We identified 1,165 isolates, 73.7% were M. tuberculosis and 26.2%, M. bovis. Among pulmonary samples, 16.6% were M. bovis. The proportion of M. bovis isolates significantly increased from 7.8% in 2000 to 28.4% in 2014 (X(2trend, p<0.001. Primary STR resistance was higher among M. bovis compared with M. tuberculosis isolates (10.9% vs.3.4%, p<0.001. Secondary multidrug resistance (MDR rates were 38.5% and 34.4% for M. bovis and M. tuberculosis, respectively (p = 0.637. A rising trend of primary STR monoresistance was observed for both species (3.4% in 2000-2004 vs. 7.6% in 2010-2014; p = 0.02.There is a high prevalence and a rising trend of M. bovis isolates in our region. The proportion of pulmonary M. bovis isolates is higher than in previous reports. Additionally, we report high rates of primary anti-tuberculosis resistance and secondary MDR in both M. tuberculosis and M. bovis. This is one of the largest reports on drug susceptibility of M. bovis from human samples and shows a significant proportion of first-line anti-tuberculosis drug resistance.

The fluorometric microculture cytotoxicity assay.

Science.gov (United States)

Lindhagen, Elin; Nygren, Peter; Larsson, Rolf

2008-01-01

The fluorometric microculture cytotoxicity assay (FMCA) is a nonclonogenic microplate-based cell viability assay used for measurement of the cytotoxic and/or cytostatic effect of different compounds in vitro. The assay is based on hydrolysis of the probe, fluorescein diacetate (FDA) by esterases in cells with intact plasma membranes. The assay is available as both a semiautomated 96-well plate setup and a 384-well plate version fully adaptable to robotics. Experimental plates are prepared with a small amount of drug solution and can be stored frozen. Cells are seeded on the plates and cell viability is evaluated after 72 h. The protocol described here is applicable both for cell lines and freshly prepared tumor cells from patients and is suitable both for screening in drug development and as a basis for a predictive test for individualization of anticancer drug therapy.

Biotransformation of albendazole and activities of selected detoxification enzymes in Haemonchus contortus strains susceptible and resistant to anthelmintics.

Science.gov (United States)

Vokřál, Ivan; Jirásko, Robert; Stuchlíková, Lucie; Bártíková, Hana; Szotáková, Barbora; Lamka, Jiří; Várady, Marián; Skálová, Lenka

2013-09-23

The increased activity of drug-metabolizing enzymes can protect helminths against the toxic effect of anthelmintics. The aim of this study was to compare the metabolism of the anthelmintic drug albendazole (ABZ) and the activities of selected biotransformation and antioxidant enzymes in three different strains of Haemonchus contortus: the ISE strain (susceptible to common anthelmintics), the BR strain (resistant to benzimidazole anthelmintics) and the WR strain (multi-resistant). H. contortus adults were collected from the abomasum of experimentally infected lambs. In vitro (subcellular fractions of H. contortus homogenate) as well as ex vivo (living nematodes cultivated in flasks with medium) experiments were performed. HPLC with spectrofluorimetric and mass-spectrometric detection was used in the analysis of ABZ metabolites. The in vitro activities of oxidation/antioxidation and conjugation enzymes toward model substrates were also assayed. The in vitro data showed significant differences between the susceptible (ISE) and resistant (BR, WR) strains regarding the activities of peroxidases, catalase and UDP-glucosyltransferases. S-oxidation of ABZ was significantly lower in BR than in the ISE strain. Ex vivo, four ABZ metabolites were identified: ABZ sulphoxide and three ABZ glucosides. In the resistant strains BR and WR, the ex vivo formation of all ABZ glucosides was significantly higher than in the susceptible ISE strain. The altered activities of certain detoxifying enzymes might partly protect the parasites against the toxic effect of the drugs as well as contribute to drug-resistance in these parasites. Copyright © 2013 Elsevier B.V. All rights reserved.

M. tuberculosis genotypic diversity and drug susceptibility pattern in HIV- infected and non-HIV-infected patients in northern Tanzania

Directory of Open Access Journals (Sweden)

van Soolingen Dick

2007-05-01

Full Text Available Abstract Background Tuberculosis (TB is a major health problem and HIV is the major cause of the increase in TB. Sub-Saharan Africa is endemic for both TB and HIV infection. Determination of the prevalence of M. tuberculosis strains and their drug susceptibility is important for TB control. TB positive culture, BAL fluid or sputum samples from 130 patients were collected and genotyped. The spoligotypes were correlated with anti-tuberculous drug susceptibility in HIV-infected and non-HIV patients from Tanzania. Results One-third of patients were TB/HIV co-infected. Forty-seven spoligotypes were identified. Fourteen isolates (10.8% had new and unique spoligotypes while 116 isolates (89.2% belonged to 33 known spoligotypes. The major spoligotypes contained nine clusters: CAS1-Kili 30.0%, LAM11- ZWE 14.6%, ND 9.2%, EAI 6.2%, Beijing 5.4%, T-undefined 4.6%, CAS1-Delhi 3.8%, T1 3.8% and LAM9 3.8%. Twelve (10.8% of the 111 phenotypically tested strains were resistant to anti-TB drugs. Eight (7.2% were monoresistant strains: 7 to isoniazid (INH and one to streptomycin. Four strains (3.5% were resistant to multiple drugs: one (0.9% was resistant to INH and streptomycin and the other three (2.7% were MDR strains: one was resistant to INH, rifampicin and ethambutol and two were resistant to all four anti-TB drugs. Mutation in the katG gene codon 315 and the rpoB hotspot region showed a low and high sensitivity, respectively, as predictor of phenotypic drug resistance. Conclusion CAS1-Kili and LAM11-ZWE were the most common families. Strains of the Beijing family and CAS1-Kili were not or least often associated with resistance, respectively. HIV status was not associated with spoligotypes, resistance or previous TB treatment.

A multiple biomarker assay for quality assessment of botanical drugs using a versatile microfluidic chip.

Science.gov (United States)

Li, Zhen-Hao; Ai, Ni; Yu, Lawrence X; Qian, Zhong-Zhi; Cheng, Yi-Yu

2017-09-25

Quality control is critical for ensuring the safety and effectiveness of drugs. Current quality control method for botanical drugs is mainly based on chemical testing. However, chemical testing alone may not be sufficient as it may not capture all constituents of botanical drugs. Therefore, it is necessary to establish a bioassay correlating with the drug's known mechanism of action to ensure its potency and activity. Herein we developed a multiple biomarker assay to assess the quality of botanicals using microfluidics, where enzyme inhibition was employed to indicate the drug's activity and thereby evaluate biological consistency. This approach was exemplified on QiShenYiQi Pills using thrombin and angiotensin converting enzyme as "quality biomarkers". Our results demonstrated that there existed variations in potency across different batches of the intermediates and preparations. Compared with chromatographic fingerprinting, the bioassay provided better discrimination ability for some abnormal samples. Moreover, the chip could function as "affinity chromatography" to identify bioactive phytochemicals bound to the enzymes. This work proposed a multiple-biomarker strategy for quality assessment of botanical drugs, while demonstrating for the first time the feasibility of microfluidics in this field.

The Calgary Biofilm Device: New Technology for Rapid Determination of Antibiotic Susceptibilities of Bacterial Biofilms

OpenAIRE

Ceri, H.; Olson, M. E.; Stremick, C.; Read, R. R.; Morck, D.; Buret, A.

1999-01-01

Determination of the MIC, based on the activities of antibiotics against planktonic bacteria, is the standard assay for antibiotic susceptibility testing. Adherent bacterial populations (biofilms) present with an innate lack of antibiotic susceptibility not seen in the same bacteria grown as planktonic populations. The Calgary Biofilm Device (CBD) is described as a new technology for the rapid and reproducible assay of biofilm susceptibilities to antibiotics. The CBD produces 96 equivalent bi...

Investigation of Susceptibility of Mycobacterium tuberculosis Complex Strains Isolated from Clinical Samples Against the First and Second-Line Anti-tuberculosis Drugs by the Sensititre MycoTB Plate Method

Directory of Open Access Journals (Sweden)

Figen KAYSERİLİ ORHAN

2018-03-01

Full Text Available Introduction: Phenotypic methods for drug susceptibility testing of Mycobacterium tuberculosis complex (MTC to second-line drugs are not yet standardized. The Sensititre MycoTB Plate is a microtiter plate containing lyophilized antibiotics and configured for determination of MIC to first and second-line anti-tuberculosis drugs. The purpose of this study is to detect the susceptibility rates of MTC strains isolated from patients’ specimens for first and second-line anti-tuberculosis drugs. Materials and Methods: This study included 50 MTC strains isolated from various clinical specimens. Out of the 50 strains, 38 were isolated from sputum, three from cerebrospinal fluid, three from bronchoalveolar lavage, and six from other samples in this study. The susceptibility of strains to anti-tuberculosis drugs were determined by the Sensititre MycoTB Plate Method. Thawed isolates were subcultured, and dilutions were inoculated into MycoTB wells. The results were read at days 7, 14 and 21. Results: At the end of study, out of 50 MTC isolates, 7 (14% showed resistance to Isoniazid (INH, 5 (10% to streptomycin (SM, 4 (8% to ethambutol (EMB, 4 (8% to ethionamide (ETH, 3 (6% to rifampicin (RIF, 3 (6% to rifabutin (RFB, 2 (4% to kanamycin (KAN, 2 (4% to ofloxacin (OFL, 2 (4% to P-aminosalicyclic acid (PAS, 1 (2% to moxiflocacin (MOX, and 1 (2% to cycloserine (CYC. All strains were found sensitive to amikacin while 2 strains (4% were identified as multidrug-resistant tuberculosis (MDR-TB. Thirty-five strains (70% were sensitive to all drugs. Extensively drug resistant tuberculosis (XDR-TB was not determined in this study. Conclusion: This is the first study that tests second line anti-tuberculosis drugs in our location and provides us valuable data regarding MDR-TB and XDR-TB rates. The Sensititre MycoTB Plate Method is a fast, reliable and practical method and can be used to determine the susceptibility of first and second-line anti-tuberculosis drugs.

Development and Validation of an Enzyme-Linked Immunosorbent Assay for the Detection of Binding Anti-Drug Antibodies against Interferon Beta

Directory of Open Access Journals (Sweden)

Kathleen Ingenhoven

2017-07-01

Full Text Available ObjectiveTo develop and validate a method for the detection of binding anti-drug antibodies (ADAs against interferon beta (IFN-β in human serum as part of a European initiative (ABIRISK aimed at the prediction and analysis of clinical relevance of anti-biopharmaceutical immunization to minimize the risk.MethodA two-tiered bridging enzyme-linked immunosorbent assay (ELISA format was selected and validated according to current recommendations. Screening assay: ADA in serum samples form complexes with immobilized IFN-β and biotinylated IFN-β, which are then detected using HRP labeled Streptavidin and TMB substrate. Confirmation assay: Screen “putative positive” samples are tested in the presence of excess drug (preincubation of sera with 0.3 µg/mL of soluble IFN-β and percentage of inhibition is calculated.ResultsThe assay is precise, and the sensitivity of the assay was confirmed to be 26 ng/mL using commercially available polyclonal rabbit antihuman IFN-β in human sera as the positive control.ConclusionAn ultrasensitive ELISA for IFN-β-binding ADA testing has been validated. This will form the basis to assess anti-biopharmaceutical immunization toward IFN-β with regards to its clinical relevance and may allow for the development of predictive tools, key aims within the ABIRISK consortium.

Flow cytometry susceptibility testing for conventional antifungal drugs and Comparison with the NCCLS Broth Macrodilution Test

Directory of Open Access Journals (Sweden)

M.J. Najafzadeh

2009-08-01

Full Text Available Introduction: During the last decade, the incidence of fungal infection has been increased in many countries. Because of the advent of resistant to antifungal agents, determination of an efficient strategic plan for treatment of fungal disease is an important issue in clinical mycology. Many methods have been introduced and developed for determination of invitro susceptibility tests. During the recent years, flow cytometry has developed to solving the problem and many papers have documented the usefulness of this technique. Materials and methods: As the first step, the invitro susceptibility of standard PTCC (Persian Type of Culture Collection strain and some clinical isolates of Candida consisting of Candida albicans, C. dubliniensis, C. glabrata, C. kefyer and C. parapsilosis were evaluated by macrodilution broth method according to NCCLS (National Committee for Clinical Laboratory Standards guidelines and flow cytometry susceptibility test. Results:  The data indicated that macro dilution broth methods and flow cytometry have the same results in determination of MIC (Minimum Inhibitory Concentration for amphotericin B, clotrimazole, fluconazole, ketoconazole and miconazole in C. albicans PTCC 5027 as well as clinical Candida isolates, such as C.albicans, C.dubliniensis, C.glabrata C.kefyr, and C.parapsilosis. Discussion: Comparing the results obtained by macrodilution broth and flow cytometry methods revealed that flow cytometry was faster. It is suggested that flow cytometry susceptibility test can be used as a powerful tool for determination of MIC and administration of the best antifungal drug in treatment of patients with Candida infections.

Environmental isolation, biochemical identification, and antifungal drug susceptibility of Cryptococcus species

Directory of Open Access Journals (Sweden)

Valter Luis Iost Teodoro

2013-12-01

Full Text Available Introduction The incidence of opportunistic fungal infections has increased in recent years and is considered an important public health problem. Among systemic and opportunistic mycoses, cryptococcosis is distinguished by its clinical importance due to the increased risk of infection in individuals infected by human immunodeficiency virus. Methods To determine the occurrence of pathogenic Cryptococcus in pigeon excrement in the City of Araraquara, samples were collected from nine environments, including state and municipal schools, abandoned buildings, parks, and a hospital. The isolates were identified using classical tests, and susceptibility testing for the antifungal drugs (fluconazole, itraconazole, voriconazole, and amphotericin B independently was also performed. After collection, the excrement samples were plated on Niger agar and incubated at room temperature. Results A total of 87 bird dropping samples were collected, and 66.6% were positive for the genus Cryptococcus. The following species were identified: Cryptococcus neoformans (17.2%, Cryptococcus gattii (5.2%, Cryptococcus ater (3.5%, Cryptococcus laurentti (1.7%, and Cryptococcus luteolus (1.7%. A total of 70.7% of the isolates were not identified to the species level and are referred to as Cryptococcus spp. throughout the manuscript. Conclusions Although none of the isolates demonstrated resistance to antifungal drugs, the identification of infested areas, the proper control of birds, and the disinfection of these environments are essential for the epidemiological control of cryptococcosis.

In Vitro Susceptibility of Various Genotypic Strains of Toxoplasma gondii to Pyrimethamine, Sulfadiazine, and Atovaquone▿

Science.gov (United States)

Meneceur, Pascale; Bouldouyre, Marie-Anne; Aubert, Dominique; Villena, Isabelle; Menotti, Jean; Sauvage, Virginie; Garin, Jean-François; Derouin, Francis

2008-01-01

Sulfadiazine, pyrimethamine, and atovaquone are widely used for the treatment of severe toxoplasmosis. Their in vitro activities have been almost exclusively demonstrated on laboratory strains belonging to genotype I. We determined the in vitro activities of these drugs against 17 strains of Toxoplasma gondii belonging to various genotypes and examined the correlations among 50% inhibitory concentrations (IC50s), growth kinetics, strain genotypes, and mutations on drug target genes. Growth kinetics were determined in THP-1 cell cultures using real-time PCR. IC50s were determined in MRC-5 cell cultures using a T. gondii-specific enzyme-linked immunosorbent assay performed on cultures. Mutations in dihydrofolate reductase (DHFR), dihydropteroate synthase (DHPS), and cytochrome b genes were determined by sequencing. Pyrimethamine IC50s ranged between 0.07 and 0.39 mg/liter, with no correlation with the strain genotype but a significant correlation with growth kinetics. Several mutations found on the DHFR gene were not linked to lower susceptibility. Atovaquone IC50s were in a narrow range of concentrations (mean, 0.06 ± 0.02 mg/liter); no mutation was found on the cytochrome b gene. IC50s for sulfadiazine ranged between 3 and 18.9 mg/liter for 13 strains and were >50 mg/liter for three strains. High IC50s were not correlated to strain genotypes or growth kinetics. A new mutation of the DHPS gene was demonstrated in one of these strains. In conclusion, we found variability in the susceptibilities of T. gondii strains to pyrimethamine and atovaquone, with no evidence of drug resistance. A higher variability was found for sulfadiazine, with a possible resistance of three strains. No relationship was found between drug susceptibility and strain genotype. PMID:18212105

Species used for drug testing reveal different inhibition susceptibility for 17beta-hydroxysteroid dehydrogenase type 1.

Directory of Open Access Journals (Sweden)

Gabriele Möller

Full Text Available Steroid-related cancers can be treated by inhibitors of steroid metabolism. In searching for new inhibitors of human 17beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD 1 for the treatment of breast cancer or endometriosis, novel substances based on 15-substituted estrone were validated. We checked the specificity for different 17beta-HSD types and species. Compounds were tested for specificity in vitro not only towards recombinant human 17beta-HSD types 1, 2, 4, 5 and 7 but also against 17beta-HSD 1 of several other species including marmoset, pig, mouse, and rat. The latter are used in the processes of pharmacophore screening. We present the quantification of inhibitor preferences between human and animal models. Profound differences in the susceptibility to inhibition of steroid conversion among all 17beta-HSDs analyzed were observed. Especially, the rodent 17beta-HSDs 1 were significantly less sensitive to inhibition compared to the human ortholog, while the most similar inhibition pattern to the human 17beta-HSD 1 was obtained with the marmoset enzyme. Molecular docking experiments predicted estrone as the most potent inhibitor. The best performing compound in enzymatic assays was also highly ranked by docking scoring for the human enzyme. However, species-specific prediction of inhibitor performance by molecular docking was not possible. We show that experiments with good candidate compounds would out-select them in the rodent model during preclinical optimization steps. Potentially active human-relevant drugs, therefore, would no longer be further developed. Activity and efficacy screens in heterologous species systems must be evaluated with caution.

Microbial receptor assay for rapid detection and identification of seven families of antimicrobial drugs in milk: collaborative study

International Nuclear Information System (INIS)

Charm, S.E.; Chi, R.

1988-01-01

A microbial competitive receptor assay for detecting residues of antibiotic families in milk was studied collaboratively by 13 laboratories. In this method, microbial cells added to a milk sample provide specific binding sites for which 14 C or 3 H labeled drug competes with drug resides in the sample. The 14 C or 3 H binding to the specific binding sites is measured in a scintillation counter and compared with a zero standard milk. If the sample is statistically different from the zero standard, it is positive. The assay takes about 15 min. The binding reaction occurs between the receptor site and the drug functional group, so all members of a drug family are detected. In this case, beta-lactams, tetracyclines, macrolides, aminoglycosides, novobiocin, chloramphenicol, and sulfonamides, including p-amino-benzoic acid (PABA) and its other analogs, are detectable. The incidence of false negative determinations among samples is about 1%; the incidence of false positives is about 3%. For negative cases, the relative standard deviations for repeatability ranged from 0 to 5% and for reproducibility from 0 to 6%. For positive cases, relative standard deviations ranged from 0 to 13% for repeatability and from 0 to 14% for reproducibility. The method has been adopted official first action

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

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.

Radioreceptor opioid assay

International Nuclear Information System (INIS)

Miller, R.J.; Chang, K.-J.

1981-01-01

A radioreceptor assay is described for assaying opioid drugs in biological fluids. The method enables the assay of total opioid activity, being specific for opioids as a class but lacking specificity within the class. A radio-iodinated opioid and the liquid test sample are incubated with an opiate receptor material. The percentage inhibition of the binding of the radio-iodinated compound to the opiate receptor is calculated and the opioid activity of the test liquid determined from a standard curve. Examples of preparing radio-iodinated opioids and assaying opioid activity are given. A test kit for the assay is described. Compared to other methods, this assay is cheap, easy and rapid. (U.K.)

A high performance liquid chromatographic assay of Mefloquine in saliva after a single oral dose in healthy adult Africans

Directory of Open Access Journals (Sweden)

Gbotosho Grace O

2012-02-01

Full Text Available Abstract Background Mefloquine-artesunate is a formulation of artemisinin based combination therapy (ACT recommended by the World Health Organization and historically the first ACT used clinically. The use of ACT demands constant monitoring of therapeutic efficacies and drug levels, in order to ensure that optimum drug exposure is achieved and detect reduced susceptibility to these drugs. Quantification of anti-malarial drugs in biological fluids other than blood would provide a more readily applicable method of therapeutic drug monitoring in developing endemic countries. Efforts in this study were devoted to the development of a simple, field applicable, non-invasive method for assay of mefloquine in saliva. Methods A high performance liquid chromatographic method with UV detection at 220 nm for assaying mefloquine in saliva was developed and validated by comparing mefloquine concentrations in saliva and plasma samples from four healthy volunteers who received single oral dose of mefloquine. Verapamil was used as internal standard. Chromatographic separation was achieved using a Hypersil ODS column. Results Extraction recoveries of mefloquine in plasma or saliva were 76-86% or 83-93% respectively. Limit of quantification of mefloquine was 20 ng/ml. Agreement between salivary and plasma mefloquine concentrations was satisfactory (r = 0.88, p Conclusion Disposition of mefloquine in saliva paralleled that in plasma, making salivary quantification of mefloquine potentially useful in therapeutic drug monitoring.

Bacteriuria and antimicrobial susceptibility pattern of bacterial ...

African Journals Online (AJOL)

Bacterial isolates and drug susceptibility patterns of urinary tract infection among ... Key words: Urinary tract infection, pregnant women, antimicrobial drug ..... and premature labour as well as adverse outcome for the unborn child (Raz, 2003).

Rapid, automated, nonradiometric susceptibility testing of Mycobacterium tuberculosis complex to four first-line antituberculous drugs used in standard short-course chemotherapy

DEFF Research Database (Denmark)

Johansen, Isik Somuncu; Thomsen, Vibeke Østergaard; Marjamäki, Merja

2004-01-01

The increasing prevalence of drug-resistant tuberculosis necessitates rapid and accurate susceptibility testing. The nonradiometric BACTEC Mycobacteria Growth Indicator Tube 960 (MGIT) system for susceptibility testing was evaluated on 222 clinical Mycobacterium tuberculosis complex isolates...... for isoniazid, rifampin, and ethambutol. Fifty-seven of the isolates were tested for pyrazinamide. Results were compared to those of radiometric BACTEC 460 system and discrepancies were resolved by the agar proportion method. We found an overall agreement of 99.0% for isoniazid, 99.5% for rifampin, 98.......2% for ethambutol, and 100% for pyrazinamide. After resolution of discrepancies, MGIT yielded no false susceptibility for rifampin and isoniazid. Although turnaround times were comparable, MGIT provides an advantage as inoculation can be done on any weekday as the growth is monitored automatically. The automated...

Distribution and antibiotic susceptibility pattern of methicillin ...

African Journals Online (AJOL)

Distribution and antibiotic susceptibility pattern of methicillin-resistant Staphylococcus aureus isolates in a university Teaching hospital in Nigeria. ... Amoxycillin clavunanic acid and ciprofloxacin were most active with MRSA isolates showing 97% and 93.9% susceptibility to the two drugs respectively. Eighteen (54.5%) ...

Species-specific identification of equine cyathostomes resistant to fenbendazole and susceptible to oxibendazole and moxidectin by macroarray probing.

Science.gov (United States)

Traversa, Donato; Iorio, Raffaella; Otranto, Domenico; Giangaspero, Annunziata; Milillo, Piermarino; Klei, Thomas R

2009-01-01

Cyathostome populations in horses on two farms located in central Italy with a history of fenbendazole (FBZ) resistance were investigated with the Faecal Egg Count Reduction Test to evaluate the susceptibility to oxibendazole and moxidectin. Faecal eggs were collected pre- and post-treatment on each farm and molecularly examined with a Reverse Line Blot (RLB) assay able to unequivocally detect and identify 13 cyathostome species. Resistance to FBZ was confirmed on both farms, while oxibendazole and moxidectin demonstrated 97% and 100% efficacy, respectively. Overall eight species of cyathostomes (Coronocyclus labiatus, Cylicocyclus ashworthi, Cylicocyclus nassatus, Cyathostomum catinatum, Cylicostephanus longibursatus, Cylicostephanus goldi, Cylicostephanus calicatus and Cylicocyclus insigne) were identified in pre-treatment samples. Coronocyclus labiatus and C. goldi were identified after treatment with FBZ while C. calicatus and C. labiatus were shown to be <100% susceptible to oxibendazole. These data confirm that resistance to benzimidazoles is established in cyathostome populations from horse farms in Italy and that they are susceptible to moxidectin. The oxibendazole has been successfully demonstrated for the first time as effective against Italian populations of cyathostomes resistant to other benzimidazoles. The RLB assay herein used showed to be useful to study the distribution of these parasitic populations at species level under field conditions and could represent a powerful tool in broader investigation of drug resistance in horse farms from several countries.

Feasibility and cost analysis of programmatic implementation of ...

African Journals Online (AJOL)

Objectives: Detection of Multi-drug resistant tuberculosis in Nigeria still remains a challenge. We evaluated the feasibility of programmatic implementation of the Microscopic-Observation Drug Susceptibility (MODS) assay, a rapid culture and drug susceptibility testing technique for drug susceptibility testing in a low resource ...

Extended spectrum of antibiotic susceptibility for tuberculosis, Djibouti.

Science.gov (United States)

Bouzid, Fériel; Astier, Hélène; Osman, Djaltou Aboubaker; Javelle, Emilie; Hassan, Mohamed Osman; Simon, Fabrice; Garnotel, Eric; Drancourt, Michel

2018-02-01

In the Horn of Africa, there is a high prevalence of tuberculosis that is reported to be partly driven by multidrug-resistant (MDR) Mycobacterium tuberculosis strictu sensu strains. We conducted a prospective study to investigate M. tuberculosis complex species causing tuberculosis in Djibouti, and their in vitro susceptibility to standard anti-tuberculous antibiotics in addition to clofazimine, minocycline, chloramphenicol and sulfadiazine. Among the 118 mycobacteria isolates from 118 successive patients with suspected pulmonary tuberculosis, 111 strains of M. tuberculosis, five Mycobacterium canettii, one 'Mycobacterium simulans' and one Mycobacterium kansasii were identified. Drug-susceptibility tests performed on the first 78 isolates yielded nine MDR M. tuberculosis isolates. All isolates were fully susceptible to clofazimine, minocycline and chloramphenicol, and 75 of 78 isolates were susceptible to sulfadiazine. In the Horn of Africa, patients with confirmed pulmonary tuberculosis caused by an in vitro susceptible strain may benefit from anti-leprosy drugs, sulfamides and phenicol antibiotics. Copyright © 2017 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.

Genetic architecture of intrinsic antibiotic susceptibility.

Directory of Open Access Journals (Sweden)

Hany S Girgis

2009-05-01

Full Text Available Antibiotic exposure rapidly selects for more resistant bacterial strains, and both a drug's chemical structure and a bacterium's cellular network affect the types of mutations acquired.To better characterize the genetic determinants of antibiotic susceptibility, we exposed a transposon-mutagenized library of Escherichia coli to each of 17 antibiotics that encompass a wide range of drug classes and mechanisms of action. Propagating the library for multiple generations with drug concentrations that moderately inhibited the growth of the isogenic parental strain caused the abundance of strains with even minor fitness advantages or disadvantages to change measurably and reproducibly. Using a microarray-based genetic footprinting strategy, we then determined the quantitative contribution of each gene to E. coli's intrinsic antibiotic susceptibility. We found both loci whose removal increased general antibiotic tolerance as well as pathways whose down-regulation increased tolerance to specific drugs and drug classes. The beneficial mutations identified span multiple pathways, and we identified pairs of mutations that individually provide only minor decreases in antibiotic susceptibility but that combine to provide higher tolerance.Our results illustrate that a wide-range of mutations can modulate the activity of many cellular resistance processes and demonstrate that E. coli has a large mutational target size for increasing antibiotic tolerance. Furthermore, the work suggests that clinical levels of antibiotic resistance might develop through the sequential accumulation of chromosomal mutations of small individual effect.

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.

Leveraging the contribution of thermodynamics in drug discovery with the help of fluorescence-based thermal shift assays.

Science.gov (United States)

Hau, Jean Christophe; Fontana, Patrizia; Zimmermann, Catherine; De Pover, Alain; Erdmann, Dirk; Chène, Patrick

2011-06-01

The development of new drugs with better pharmacological and safety properties mandates the optimization of several parameters. Today, potency is often used as the sole biochemical parameter to identify and select new molecules. Surprisingly, thermodynamics, which is at the core of any interaction, is rarely used in drug discovery, even though it has been suggested that the selection of scaffolds according to thermodynamic criteria may be a valuable strategy. This poor integration of thermodynamics in drug discovery might be due to difficulties in implementing calorimetry experiments despite recent technological progress in this area. In this report, the authors show that fluorescence-based thermal shift assays could be used as prescreening methods to identify compounds with different thermodynamic profiles. This approach allows a reduction in the number of compounds to be tested in calorimetry experiments, thus favoring greater integration of thermodynamics in drug discovery.

Comparison of constitutive and thiabendazole-induced expression of five cytochrome P450 genes in fourth-stage larvae of Haemonchus contortus isolates with different drug susceptibility identifies one gene with high constitutive expression in a multi-resistant isolate

Directory of Open Access Journals (Sweden)

Esra Yilmaz

2017-12-01

Full Text Available Benzimidazoles (BZs remain amongst the most widely used anthelmintic drug classes against gastro-intestinal nematode infections, although their efficacy is increasingly compromised by resistance. The primary underlying mechanisms for BZ resistance are single-nucleotide polymorphisms (SNPs in the isotype 1 β-tubulin gene causing the substitutions F167Y, E198A or F200Y. However, resistance is believed to be multi-genic and previous studies have shown that isolates carrying 90–100% F200Y can vary considerably in their resistance level in the egg hatch assay (EHA. Cytochrome P450 monooxygenases (CYPs are associated with drug resistance in mammals and arthropods and have been considered as mediators of anthelmintic resistance. In Caenorhabditis elegans, several members of the CYP34/35 and CYP31 families are BZ and/or xenobiotic inducible and thiabendazole (TBZ is metabolised by CYP35D1. Here, expression of all 5 CYPs closely related to the C. elegans CYP34/35 and CYP31 families was investigated in fourth-stage larvae of two susceptible and three BZ-resistant Haemonchus contortus isolates following in vitro exposure to TBZ for 3 and 6 h using real-time RT-PCR. The resistance status of all isolates was determined using EHAs and quantification of resistance-associated β-tubulin SNPs using pyrosequencing. While none of the CYPs was TBZ inducible, constitutive expression of CYP34/35 family member HCOI100383400 was significantly 2.4–3.7-fold higher in the multi-drug resistant WR isolate with the strongest BZ resistance phenotype compared to susceptible and intermediate-level BZ-resistant isolates. Although this increase is only moderate, HCOI100383400 might still be involved in high-level BZ resistance by further decreasing susceptibility in isolates already carrying 100% of a β-tubulin SNP causing BZ resistance. Lower transcript levels were observed for all CYPs in the intermediately resistant IRE isolate in comparison to the susceptible Hc

Penicillin-susceptible Staphylococcus aureus: susceptibility testing, resistance rates and outcome of infection.

Science.gov (United States)

Hagstrand Aldman, Malin; Skovby, Annette; I Påhlman, Lisa

2017-06-01

Staphylococcus aureus (SA) is an important human pathogen that causes both superficial and invasive infections. Penicillin is now rarely used in the treatment of SA infections due to widespread resistance and a concern about the accuracy of existing methods for penicillin susceptibility testing. The aims of the present study were to determine the frequency of penicillin-susceptible SA isolates from blood and wound cultures in Lund, Sweden, and to evaluate methods for penicillin testing in SA. We also wanted to investigate if penicillin-susceptible isolates are associated with higher mortality. Hundred blood culture isolates collected 2008/2009, 140 blood culture isolates from 2014/2015, and 141 superficial wound culture strains from 2015 were examined. Penicillin susceptibility was tested with disk diffusion according to EUCAST guidelines, and results were confirmed with a cloverleaf assay and PCR amplification of the BlaZ gene. Patient data for all bacteraemia cases were extracted from medical records. The disk diffusion method with assessment of both zone size and zone edge appearance had high accuracy in our study. About 57% of bacteraemia isolates from 2008/2009 were sensitive to penicillin compared to 29% in 2014/2015 (p penicillin susceptible. There was no difference in co-morbidity or mortality rates between patients with penicillin resistant and penicillin sensitive SA bacteraemia. Disk-diffusion is a simple and reliable method to detect penicillin resistance in SA, and susceptibility rates are significant. Penicillin has many theoretical advantages and should be considered in the treatment of SA bacteraemia when susceptible.

Outcomes of treatment of drug-susceptible tuberculosis at public sector primary healthcare clinics in Johannesburg, South Africa: A retrospective cohort study.

Science.gov (United States)

Budgell, E P; Evans, D; Schnippel, K; Ive, P; Long, L; Rosen, S

2016-09-05

Despite the large number of tuberculosis (TB) patients treated in South Africa (SA), there are few descriptions in the published literature of drug-susceptible TB patient characteristics, mode of diagnosis or treatment outcomes in routine public sector treatment programmes. To enhance the evidence base for public sector TB treatment service delivery, we reported the characteristics of and outcomes for a retrospective cohort of adult TB patients at public sector clinics in the Johannesburg Metropolitan Municipality (JHB), SA. We collected medical record data for a retrospective cohort of adult (≥18 years) TB patients registered between 1 April 2011 and 31 March 2012 at three public sector clinics in JHB. Data were abstracted from National TB Programme clinic cards and the TB case registers routinely maintained at study sites. We report patient characteristics, mode of diagnosis, mode of treatment supervision, treatment characteristics, HIV status and treatment outcomes for this cohort. A total of 544 patients were enrolled in the cohort. Most (86%) were new TB cases, 81% had pulmonary TB, 58% were smear-positive at treatment initiation and 71% were HIV co-infected. Among 495 patients with treatment outcomes reported, 80% (n=394) had successful outcomes, 11% (n=55) were lost to follow-up, 8% (n=40) died and 1% (n=6) failed treatment. Primary healthcare clinics in JHB are achieving relatively high rates of success in treating drug-susceptible TB. Missing laboratory results were common, including follow-up smears, cultures and drug susceptibility tests, making it difficult to assess adherence to guidelines and leaving scope for substantial improvements in record-keeping at the clinics involved.

A quantitative comet infection assay for influenza virus

Science.gov (United States)

Lindsay, Stephen M.; Timm, Andrea; Yin, John

2011-01-01

Summary The virus comet assay is a cell-based virulence assay used to evaluate an antiviral drug or antibody against a target virus. The comet assay differs from the plaque assay in allowing spontaneous flows in 6-well plates to spread virus. When implemented quantitatively the comet assay has been shown to have an order-of-magnitude greater sensitivity to antivirals than the plaque assay. In this study, a quantitative comet assay for influenza virus is demonstrated, and is shown to have a 13-fold increase in sensitivity to ribavirin. AX4 cells (MDCK cells with increased surface concentration of α2–6 sialic acid, the influenza virus receptor) have reduced the comet size variability relative to MDCK cells, making them a better host cell for use in this assay. Because of enhanced antiviral sensitivity in flow-based assays, less drug is required, which could lead to lower reagent costs, reduced cytotoxicity, and fewer false-negative drug screen results. The comet assay also serves as a readout of flow conditions in the well. Observations from comets formed at varying humidity levels indicate a role for evaporation in the mechanism of spontaneous fluid flow in wells. PMID:22155578

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.

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

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.

An HPLC-UV method for the measurement of permeability of marker drugs in the Caco-2 cell assay

Directory of Open Access Journals (Sweden)

J.M. Kratz

2011-06-01

Full Text Available The Caco-2 cell line has been used as a model to predict the in vitro permeability of the human intestinal barrier. The predictive potential of the assay relies on an appropriate in-house validation of the method. The objective of the present study was to develop a single HPLC-UV method for the identification and quantitation of marker drugs and to determine the suitability of the Caco-2 cell permeability assay. A simple chromatographic method was developed for the simultaneous determination of both passively (propranolol, carbamazepine, acyclovir, and hydrochlorothiazide and actively transported drugs (vinblastine and verapamil. Separation was achieved on a C18 column with step-gradient elution (acetonitrile and aqueous solution of ammonium acetate, pH 3.0 at a flow rate of 1.0 mL/min and UV detection at 275 nm during the total run time of 35 min. The method was validated and found to be specific, linear, precise, and accurate. This chromatographic system can be readily used on a routine basis and its utilization can be extended to other permeability models. The results obtained in the Caco-2 bi-directional transport experiments confirmed the validity of the assay, given that high and low permeability profiles were identified, and P-glycoprotein functionality was established.

Current status of therapeutic drug monitoring in Australia and New Zealand: a need for improved assay evaluation, best practice guidelines, and professional development.

Science.gov (United States)

Norris, Ross L; Martin, Jennifer H; Thompson, Erin; Ray, John E; Fullinfaw, Robert O; Joyce, David; Barras, Michael; Jones, Graham R; Morris, Raymond G

2010-10-01

The measurement of drug concentrations, for clinical purposes, occurs in many diagnostic laboratories throughout Australia and New Zealand. However, the provision of a comprehensive therapeutic drug monitoring (TDM) service requires the additional elements of pre- and postanalytical advice to ensure that concentrations reported are meaningful, interpretable, and clinically applicable to the individual patient. The aim of this project was to assess the status of TDM services in Australia and New Zealand. A range of professions involved in key aspects of TDM was surveyed by questionnaire in late 2007. Information gathered included: the list of drugs assayed; analytical methods used; interpretation services offered; interpretative methods used; and further monitoring advice provided. Fifty-seven responses were received, of which 42% were from hospitals (public and/or private); 11% a hospital (public and/or private) and pathology provider; and 47% a pathology provider only (public and/or private). Results showed that TDM is applied to a large number of different drugs. Poorly performing assay methods were used in some cases, even when published guidelines recommended alternative practices. Although there was a wide array of assays available, the evidence suggested a need for better selection of assay methods. In addition, only limited advice and/or interpretation of results was offered. Of concern, less than 50% of those providing advice on aminoglycoside dosing in adults used pharmacokinetic tools with six of 37 (16.2%) respondents using Bayesian pharmacokinetic tools, the method recommended in the Australian Therapeutic Guidelines: Antibiotic. In conclusion, the survey highlighted deficiencies in the provision of TDM services, in particular assay method selection and both quality and quantity of postanalytical advice. A range of recommendations, some of which may have international implications, are discussed. There is a need to include measures of impact on clinical

Adhesion, biofilm formation, cell surface hydrophobicity, and antifungal planktonic susceptibility: relationship among Candida spp.

OpenAIRE

Silva-Dias, Ana; Miranda, Isabel M.; Branco, Joana; Monteiro-Soares, Matilde; Pina-Vaz, Cid?lia; Rodrigues, Ac?cio G.

2015-01-01

We have performed the characterization of the adhesion profile, biofilm formation, cell surface hydrophobicity (CSH) and antifungal susceptibility of 184 Candida clinical isolates obtained from different human reservoirs. Adhesion was quantified using a flow cytometric assay and biofilm formation was evaluated using two methodologies: XTT and crystal violet assay. CSH was quantified with the microbial adhesion to hydrocarbons test while planktonic susceptibility was assessed accordingly the C...

Biomonitoring of genotoxic risk in radar facility workers: comparison of the comet assay with micronucleus assay and chromatid breakage assay

International Nuclear Information System (INIS)

Garaj-Vrhovac, V.; Kopjar, N.

2003-01-01

Genotoxic risks of occupational exposure in a radar facility were evaluated by using alkaline comet assay, micronucleus assay and chromatid breakage assay on peripheral blood leukocytes in exposed subjects and corresponding controls. Results show that occupational exposure to microwave radiation correlates with an increase of genome damage in somatic cells. The levels of DNA damage in exposed subjects determined by using alkaline comet assay were increased compared to control and showed interindividual variations. Incidence of micronuclei was also significantly increased compared to baseline control values. After short exposure of cultured lymphocytes to bleomycin, cells of occupationally exposed subjects responded with high numbers of chromatid breaks. Although the level of chromosome damage generated by bleomycin varied greatly between individuals, in exposed subjects a significantly elevated number of chromatid breaks was observed. Our results support data reported in literature indicating that microwave radiation represents a potential DNA-damaging hazard. Alkaline comet assay is confirmed as a sensitive and highly reproducible technique for detection of primary DNA damage inflicted in somatic cells. Micronucleus assay was confirmed as reliable bio-markers of effect and chromatid breakage assay as sensitive bio-marker of individual cancer susceptibility. The results obtained also confirm the necessity to improve measures and to perform accurate health surveillance of individuals occupationally exposed to microwave radiation

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

Directory of Open Access Journals (Sweden)

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

Suitability of a liquid chromatography assay of neomycin sulfate to replace the microbiological assay for neomycin in USP Monographs.

Science.gov (United States)

Hanko, Valoran P; Rohrer, Jeffrey S

2010-01-05

The current USP National Formulary contains 65 Monographs for drug formulations containing neomycin. All 65 Monographs prescribe a bioassay for neomycin assay. This bioassay, based on cell culture, is labor intensive, has poor precision, and cannot be adapted for purity or identification. High-performance anion-exchange chromatography with integrated pulsed amperometric detection (HPAE-IPAD), a liquid chromatography technique, has been shown to be suitable for neomycin purity analysis and neomycin assay of an over-the-counter first aid cream (Hanko and Rohrer [17]). Here we propose that an HPAE-IPAD assay can replace the bioassay in the 65 neomycin-containing Monographs. We applied the HPAE-IPAD assay to four neomycin-containing drug products representing the four classes of formulations found in the 65 Monographs, liquid, solid, suspension, and cream. Each drug was analyzed with two chromatography systems, and on 3 separate days. For all products, HPAE-IPAD measurements were precise and accurate with respect to the label concentrations. There was also high accuracy for spike recovery of neomycin from the four drug products throughout 70-150% of the labeled concentration. These results suggest that an HPAE-IPAD assay would be an accurate assay for neomycin, and would be faster and more precise than the current bioassay.

Development and validation of cell-based luciferase reporter gene assays for measuring neutralizing anti-drug antibodies against interferon beta

DEFF Research Database (Denmark)

Hermanrud, Christina; Ryner, Malin; Luft, Thomas

2016-01-01

a normal distribution for the majority of runs, allowing a parametric approach for cut-point calculation to be used, where NAb positive samples could be identified with 95% confidence. An analysis of means and variances indicated that a floating cut-point should be used for all assays. The assays......Neutralizing anti-drug antibodies (NAbs) against therapeutic interferon beta (IFNβ) in people with multiple sclerosis (MS) are measured with cell-based bioassays. The aim of this study was to redevelop and validate two luciferase reporter-gene bioassays, LUC and iLite, using a cut-point approach...... to identify NAb positive samples. Such an approach is favored by the pharmaceutical industry and governmental regulatory agencies as it has a clear statistical basis and overcomes the limitations of the current assays based on the Kawade principle. The work was conducted following the latest assay guidelines...

21 CFR 864.7320 - Fibrinogen/fibrin degradation products assay.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Fibrinogen/fibrin degradation products assay. 864.7320 Section 864.7320 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN....7320 Fibrinogen/fibrin degradation products assay. (a) Identification. A fibrinogen/fibrin degradation...

21 CFR 866.1620 - Antimicrobial susceptibility test disc.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Antimicrobial susceptibility test disc. 866.1620 Section 866.1620 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Diagnostic Devices § 866.1620 Antimicrobial...

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.

Evaluation of rapid MTT tube method for detection of drug susceptibility of mycobacterium tuberculosis to rifampicin and isoniazid

Directory of Open Access Journals (Sweden)

Raut U

2008-01-01

Full Text Available Purpose: To evaluate MTT method for detection of drug resistance to rifampicin and isoniazid in M.tuberculosis . This method utilises the ability of viable mycobacterial cells to reduce MTT( 3-4,5-dimethylthiazol-2-yl-2, 5-diphenyl tetrazolium bromide. Methods: The method was standardised with known resistant and sensitive strains of M.tuberculosis and was then extended to 50 clinical isolates. An inoculum of 10 7 cfu/mL was prepared in Middlebrook 7H9 medium supplemented with oleic acid, albumin, dextrose and catalase. For each drug three tubes were used, one with INH(0.2μg/mL or RIF(1μg/mL, another as inoculum control and third as blank control. These were incubated at 37°C for four and seven days respectively for RIF and INH after which MTT assay was performed. Results were read visually and by colorimeter at 570 nm. Relative optical density unit (RODU of 0.2 was taken as cut off. Results were compared with drug sensitivity obtained by proportion method using LJ medium. Results: For rifampicin, concordance with proportion method was 90% by visual and 94% by RODU. Sensitivity and specificity was 86.8% and 100% respectively by visual method and 95.2% and 87.5% respectively by RODU. For Isoniazid, concordance was 94% and sensitivity and specificity was 94.7 and 91.7% respectively by both visual and RODU. Conclusions: MTT assay proved to be rapid and cheap method for performing drug sensitivity of M.tuberculosis

A High Throughput, 384-Well, Semi-Automated, Hepatocyte Intrinsic Clearance Assay for Screening New Molecular Entities in Drug Discovery.

Science.gov (United States)

Heinle, Lance; Peterkin, Vincent; de Morais, Sonia M; Jenkins, Gary J; Badagnani, Ilaria

2015-01-01

A high throughput, semi-automated clearance screening assay in hepatocytes was developed allowing a scientist to generate data for 96 compounds in one week. The 384-well format assay utilizes a Thermo Multidrop Combi and an optimized LC-MS/MS method. The previously reported LCMS/ MS method reduced the analytical run time by 3-fold, down to 1.2 min injection-to-injection. The Multidrop was able to deliver hepatocytes to 384-well plates with minimal viability loss. Comparison of results from the new 384-well and historical 24-well assays yielded a correlation of 0.95. In addition, results obtained for 25 marketed drugs with various metabolism pathways had a correlation of 0.75 when compared with literature values. Precision was maintained in the new format as 8 compounds tested in ≥39 independent experiments had coefficients of variation ≤21%. The ability to predict in vivo clearances using the new stability assay format was also investigated using 22 marketed drugs and 26 AbbVie compounds. Correction of intrinsic clearance values with binding to hepatocytes (in vitro data) and plasma (in vivo data) resulted in a higher in vitro to in vivo correlation when comparing 22 marketed compounds in human (0.80 vs 0.35) and 26 AbbVie Discovery compounds in rat (0.56 vs 0.17), demonstrating the importance of correcting for binding in clearance studies. This newly developed high throughput, semi-automated clearance assay allows for rapid screening of Discovery compounds to enable Structure Activity Relationship (SAR) analysis based on high quality hepatocyte stability data in sufficient quantity and quality to drive the next round of compound synthesis.

Expression of Plasmodium vivax crt-o Is Related to Parasite Stage but Not Ex Vivo Chloroquine Susceptibility.

Science.gov (United States)

Pava, Zuleima; Handayuni, Irene; Wirjanata, Grennady; To, Sheren; Trianty, Leily; Noviyanti, Rintis; Poespoprodjo, Jeanne Rini; Auburn, Sarah; Price, Ric N; Marfurt, Jutta

2016-01-01

Chloroquine (CQ)-resistant Plasmodium vivax is present in most countries where P. vivax infection is endemic, but the underlying molecular mechanisms responsible remain unknown. Increased expression of P. vivax crt-o (pvcrt-o) has been correlated with in vivo CQ resistance in an area with low-grade resistance. We assessed pvcrt-o expression in isolates from Papua (Indonesia), where P. vivax is highly CQ resistant. Ex vivo drug susceptibilities to CQ, amodiaquine, piperaquine, mefloquine, and artesunate were determined using a modified schizont maturation assay. Expression levels of pvcrt-o were measured using a novel real-time quantitative reverse transcription-PCR method. Large variations in pvcrt-o expression were observed across the 51 isolates evaluated, with the fold change in expression level ranging from 0.01 to 59 relative to that seen with the P. vivax β-tubulin gene and from 0.01 to 24 relative to that seen with the P. vivax aldolase gene. Expression was significantly higher in isolates with the majority of parasites at the ring stage of development (median fold change, 1.7) compared to those at the trophozoite stage (median fold change, 0.5; P determinant of ex vivo drug susceptibility. A comprehensive transcriptomic approach is warranted for an in-depth investigation of the role of gene expression levels and P. vivax drug resistance. Copyright © 2015 Pava et al.

In vitro susceptibility of Borrelia burgdorferi isolates to three antibiotics commonly used for treating equine Lyme disease.

Science.gov (United States)

Caol, Sanjie; Divers, Thomas; Crisman, Mark; Chang, Yung-Fu

2017-09-29

Lyme disease in humans is predominantly treated with tetracycline, macrolides or beta lactam antibiotics that have low minimum inhibitory concentrations (MIC) against Borrelia burgdorferi. Horses with Lyme disease may require long-term treatment making frequent intravenous or intramuscular treatment difficult and when administered orally those drugs may have either a high incidence of side effects or have poor bioavailability. The aim of the present study was to determine the in vitro susceptibility of three B. burgdorferi isolates to three antibiotics of different classes that are commonly used in practice for treating Borrelia infections in horses. Broth microdilution assays were used to determine minimum inhibitory concentration of three antibiotics (ceftiofur sodium, minocycline and metronidazole), for three Borrelia burgdorferi isolates. Barbour-Stoner-Kelly (BSK K + R) medium with a final inoculum of 10 6 Borrelia cells/mL and incubation periods of 72 h were used in the determination of MICs. Observed MICs indicated that all isolates had similar susceptibility to each drug but susceptibility to the tested antimicrobial agents varied; ceftiofur sodium (MIC = 0.08 μg/ml), minocycline hydrochloride (MIC = 0.8 μg/ml) and metronidazole (MIC = 50 μg/ml). The MIC against B. burgorferi varied among the three antibiotics with ceftiofur having the lowest MIC and metronidazole the highest MIC. The MIC values observed for ceftiofur in the study fall within the range of reported serum and tissue concentrations for the drug metabolite following ceftiofur sodium administration as crystalline-free acid. Minocycline and metronidazole treatments, as currently used in equine practice, could fall short of attaining MIC concentrations for B. burgdorferi.

In planta assay of hygromycin susceptibility and mutant screening in rice at heading stage

International Nuclear Information System (INIS)

Fu Haowei; Li Youfa; Ma Xinghua; Shen Shengquan; Shu Xiaoli; Shu Qingyao; Chen Yang

2012-01-01

Cells and tissue of plants carrying the hygromycin phosphotransferase gene (HPT) are tolerant to antibiotic hygromycin and hence become the common selection gene for genetic transformation of crop plants, particularly monocots. The present study aimed at establishing a rapid method for in planta screening of hygromycin tolerant plants in transgenic breeding. At heading stage transgenic and conventional rice plants were sprayed with hygromycin solutions of different concentrations (25 ∼ 100 mg/L) and toxic symptoms were observed in the following days. It was observed that yellow-brown necrotic spots appeared in the leaves and grains of conventional rice varieties after foliar spraying of 50 mg/L hygromycin, and the more spots were occurred with the higher hygromycin solution. There were 21.1 and 19.2 spots per cm 2 on flag leaves of indica varieties Jiayou 99 and C10, respectively; while 27.6% and 23.5% grains were yellow-brown in the varieties, respectively. The corresponding data for two japonica varieties Jiayou 5 and R5 were 11.8 and 10.7 for leave spots and 11.2% and 11.6% for yellow-brown grains, respectively. These results indicated that the two indica varieties are more sensitive to hygromycin than the two japonica varieties. In the treatment of hygromycin with above levels, the transgenic rice line KMD1 with the HPT gene showed no toxic symptoms on leaf or panicles. The γ-rays treated M 2 population of KMD1 was sprayed with 100 mg/L hygromycin solution at heading stage and 42 plants were screened out with toxic symptoms in leaves and panicles form 120000 plants. Among the 42 hygromycin susceptible plants, 14 plants with leaves available for in vitro feeding assay were subjected to the feeding of rice striped steam borers [Chilo supperssalis (Walker)] and all showed high resistance. The above results showed that spraying of 100 mg/L hygromycin solution at heading stage would lead to visually apparent toxic symptoms in susceptibility rice plants and hence

A Prospective Study of Tuberculosis Drug Susceptibility in Sabah, Malaysia, and an Algorithm for Management of Isoniazid Resistance

Science.gov (United States)

Rashid Ali, Muhammad Redzwan S.; Parameswaran, Uma; William, Timothy; Bird, Elspeth; Wilkes, Christopher S.; Lee, Wai Khew; Yeo, Tsin Wen; Anstey, Nicholas M.; Ralph, Anna P.

2015-01-01

Introduction. The burden of tuberculosis is high in eastern Malaysia, and rates of Mycobacterium tuberculosis drug resistance are poorly defined. Our objectives were to determine M. tuberculosis susceptibility and document management after receipt of susceptibility results. Methods. Prospective study of adult outpatients with smear-positive pulmonary tuberculosis (PTB) in Sabah, Malaysia. Additionally, hospital clinicians accessed the reference laboratory for clinical purposes during the study. Results. 176 outpatients were enrolled; 173 provided sputum samples. Mycobacterial culture yielded M. tuberculosis in 159 (91.9%) and nontuberculous Mycobacterium (NTM) in three (1.7%). Among outpatients there were no instances of multidrug resistant M. tuberculosis (MDR-TB). Seven people (4.5%) had isoniazid resistance (INH-R); all were switched to an appropriate second-line regimen for varying durations (4.5–9 months). Median delay to commencement of the second-line regimen was 13 weeks. Among 15 inpatients with suspected TB, 2 had multidrug resistant TB (one extensively drug resistant), 2 had INH-R, and 4 had NTM. Conclusions. Current community rates of MDR-TB in Sabah are low. However, INH-resistance poses challenges, and NTM is an important differential diagnosis in this setting, where smear microscopy is the usual diagnostic modality. To address INH-R management issues in our setting, we propose an algorithm for the treatment of isoniazid-resistant PTB. PMID:25838829

Radioreceptor assay: theory and applications to pharmacology

International Nuclear Information System (INIS)

Perret, G.; Simon, P.

1984-01-01

The aim of the first part of this work is to present the theory of the radioreceptor assay and to compare it to the other techniques of radioanalysis (radioimmunoassay, competitive protein binding assays). The technology of the radioreceptor assay is then presented and its components (preparation of the receptors, radioligand, incubation medium) are described. The analytical characteristics of the radioreceptor assay (specificity, sensitivity, reproductibility, accuracy) and the pharmacological significance of the results are discussed. The second part is devoted to the description of the radioreceptor assays of some pharmacological classes (neuroleptics, tricyclic antidepressants, benzodiazepines, β-blockers, anticholinergic drugs) and to their use in therapeutic drug monitoring. In conclusion, by their nature, radioreceptor assays are highly sensitive, reliable, precise, accurate and simple to perform. Their chief disadvantage relates to specificity, since any substance having an appreciable affinity to the receptor site will displace the specifically bound radioligand. Paradoxically in some cases, this lack of specificity may be advantageous in that it allows for the detection of not only the apparent compound but of active metabolites and endogenous receptor agonists as well and in that radioreceptors assays can be devised for a whole pharmacological class and not only for one drug as it is the case for classical physico-chemical techniques. For all these reasons future of radioreceptor assay in pharmacology appears promising [fr

Assay method for organic calcium antagonist drugs and a kit for such an assay

International Nuclear Information System (INIS)

Snyder, S. H.; Gould, R. J.

1985-01-01

A method for measuring the level of organic calcium antagonist drug in a body fluid comprises preparing a mixture of a radioactive calcium antagonist drug, a body fluid containing a calcium antagonist drug and a calcium antagonist receptor material, measuring the radioactivity of the radioactive calcium antagonist drug bound to said calcium antagonist receptor material and deriving the concentration of the calcium antagonist drug in the body fluid from a standard curve indicating the concentration of calcium antagonist drug versus inhibition of binding of said radioactive calcium antagonist drug to said receptor sites caused by the calcium antagonist drug in said body fluid. A kit for measuring the level of an organic calcium drug comprises a receptacle containing a radioactive calcium antagonist drug, a calcium antagonist receptor material and a standard amount of a nonradioactive calcium antagonist drug

Antimicrobial Drug Resistance of Salmonella enterica Serovar Typhi in Asia and Molecular Mechanism of Reduced Susceptibility to the Fluoroquinolones▿

OpenAIRE

Chau, Tran Thuy; Campbell, James Ian; Galindo, Claudia M.; Van Minh Hoang, Nguyen; Diep, To Song; Nga, Tran Thu Thi; Van Vinh Chau, Nguyen; Tuan, Phung Quoc; Page, Anne Laure; Ochiai, R. Leon; Schultsz, Constance; Wain, John; Bhutta, Zulfiqar A.; Parry, Christopher M.; Bhattacharya, Sujit K.

2007-01-01

This study describes the pattern and extent of drug resistance in 1,774 strains of Salmonella enterica serovar Typhi isolated across Asia between 1993 and 2005 and characterizes the molecular mechanisms underlying the reduced susceptibilities to fluoroquinolones of these strains. For 1,393 serovar Typhi strains collected in southern Vietnam, the proportion of multidrug resistance has remained high since 1993 (50% in 2004) and there was a dramatic increase in nalidixic acid resistance between ...

A high content, high throughput cellular thermal stability assay for measuring drug-target engagement in living cells.

Science.gov (United States)

Massey, Andrew J

2018-01-01

Determining and understanding drug target engagement is critical for drug discovery. This can be challenging within living cells as selective readouts are often unavailable. Here we describe a novel method for measuring target engagement in living cells based on the principle of altered protein thermal stabilization / destabilization in response to ligand binding. This assay (HCIF-CETSA) utilizes high content, high throughput single cell immunofluorescent detection to determine target protein levels following heating of adherent cells in a 96 well plate format. We have used target engagement of Chk1 by potent small molecule inhibitors to validate the assay. Target engagement measured by this method was subsequently compared to target engagement measured by two alternative methods (autophosphorylation and CETSA). The HCIF-CETSA method appeared robust and a good correlation in target engagement measured by this method and CETSA for the selective Chk1 inhibitor V158411 was observed. However, these EC50 values were 23- and 12-fold greater than the autophosphorylation IC50. The described method is therefore a valuable advance in the CETSA method allowing the high throughput determination of target engagement in adherent cells.

A Cardiac Cell Outgrowth Assay for Evaluating Drug Compounds Using a Cardiac Spheroid-on-a-Chip Device

Directory of Open Access Journals (Sweden)

Jonas Christoffersson

2018-05-01

Full Text Available Three-dimensional (3D models with cells arranged in clusters or spheroids have emerged as valuable tools to improve physiological relevance in drug screening. One of the challenges with cells cultured in 3D, especially for high-throughput applications, is to quickly and non-invasively assess the cellular state in vitro. In this article, we show that the number of cells growing out from human induced pluripotent stem cell (hiPSC-derived cardiac spheroids can be quantified to serve as an indicator of a drug’s effect on spheroids captured in a microfluidic device. Combining this spheroid-on-a-chip with confocal high content imaging reveals easily accessible, quantitative outgrowth data. We found that effects on outgrowing cell numbers correlate to the concentrations of relevant pharmacological compounds and could thus serve as a practical readout to monitor drug effects. Here, we demonstrate the potential of this semi-high-throughput “cardiac cell outgrowth assay” with six compounds at three concentrations applied to spheroids for 48 h. The image-based readout complements end-point assays or may be used as a non-invasive assay for quality control during long-term culture.

In vitro susceptibility to ST-246 and Cidofovir corroborates the phylogenetic separation of Brazilian Vaccinia virus into two clades.

Science.gov (United States)

Pires, Mariana A; Rodrigues, Nathália F S; de Oliveira, Danilo B; de Assis, Felipe L; Costa, Galileu B; Kroon, Erna G; Mota, Bruno E F

2018-04-01

The Orthopoxvirus (OPV) genus of the Poxviridae family contains several human pathogens, including Vaccinia virus (VACV), which have been implicating in outbreaks of a zoonotic disease called Bovine Vaccinia in Brazil. So far, no approved treatment exists for OPV infections, but ST-246 and Cidofovir (CDV) are now in clinical development. Therefore, the objective of this work was to evaluate the susceptibility of five strains of Brazilian VACV (Br-VACV) to ST-246 and Cidofovir. The susceptibility of these strains to both drugs was evaluated by plaque reduction assay, extracellular virus's quantification in the presence of ST-246 and one-step growth curve in cells treated with CDV. Besides that, the ORFs F13L and E9L were sequenced for searching of polymorphisms associated with drug resistance. The effective concentration of 50% (EC 50 ) from both drugs varies significantly for different strains (from 0.0054 to 0.051 μM for ST-246 and from 27.14 to 61.23 μM for CDV). ST-246 strongly inhibits the production of extracellular virus for all isolates in concentrations as low as 0.1 μM and it was observed a relevant decrease of progeny production for all Br-VACV after CDV treatment. Sequencing of the F13L and E9L ORFs showed that Br-VACV do not present the polymorphism(s) associated with resistance to ST-246 and CDV. Taken together, our results showed that ST-246 and CDV are effective against diverse, wild VACV strains and that the susceptibility of Br-VACV to these drugs mirrored the phylogenetic split of these isolates into two groups. Thus, both ST-246 and CDV are of great interest as compounds to treat individuals during Bovine Vaccinia outbreaks in Brazil. Copyright © 2018 Elsevier B.V. All rights reserved.

Susceptibility profiles of Nocardia isolates based on current taxonomy.

Science.gov (United States)

Schlaberg, Robert; Fisher, Mark A; Hanson, Kimberley E

2014-01-01

The genus Nocardia has undergone rapid taxonomic expansion in recent years, and an increasing number of species are recognized as human pathogens. Many established species have predictable antimicrobial susceptibility profiles, but sufficient information is often not available for recently described organisms. Additionally, the effectiveness of sulfonamides as first-line drugs for Nocardia has recently been questioned. This led us to review antimicrobial susceptibility patterns for a large number of molecularly identified clinical isolates. Susceptibility results were available for 1,299 isolates representing 39 different species or complexes, including 11 that were newly described, during a 6-year study period. All tested isolates were susceptible to linezolid. Resistance to trimethoprim-sulfamethoxazole (TMP-SMX) was rare (2%) except among Nocardia pseudobrasiliensis (31%) strains and strains of the N. transvalensis complex (19%). Imipenem susceptibility varied for N. cyriacigeorgica and N. farcinica, as did ceftriaxone susceptibility of the N. nova complex. Resistance to more than one of the most commonly used drugs (amikacin, ceftriaxone, TMP-SMX, and imipenem) was highest for N. pseudobrasiliensis (100%), N. transvalensis complex (83%), N. farcinica (68%), N. puris (57%), N. brasiliensis (51%), N. aobensis (50%), and N. amikacinitolerans (43%). Thus, while antimicrobial resistance can often be predicted, susceptibility testing should still be considered when combination therapy is warranted, for less well characterized species or those with variable susceptibility profiles, and for patients with TMP-SMX intolerance.

Reliability of the MODS assay decentralisation process in three health regions in Peru

Science.gov (United States)

Mendoza, A.; Castillo, E.; Gamarra, N.; Huamán, T.; Perea, M.; Monroi, Y.; Salazar, R.; Coronel, J.; Acurio, M.; Obregón, G.; Roper, M.; Bonilla, C.; Asencios, L.; Moore, D. A. J.

2011-01-01

OBJECTIVE To deliver rapid isoniazid (INH) and rifampicin (RMP) drug susceptibility testing (DST) close to the patient, we designed a decentralisation process for the microscopic observation drug susceptibility (MODS) assay in Peru and evaluated its reliability. METHODS After 2 weeks of training, laboratory staff processed ≥120 consecutive sputum samples each in three regional laboratories. Samples were processed in parallel with MODS testing at an expert laboratory. Blinded paired results were independently analysed by the Instituto Nacional de Salud (INS) according to predetermined criteria: concordance for culture, DST against INH and RMP and diagnosis of multidrug-resistant t uberculosis (MDR-TB) ≥ 95%, McNemar's P > 0.05, kappa index (κ) ≥ 0.75 and contamination 1–4%. Sensitivity and specificity for MDR-TB were calculated. RESULTS The accreditation process for Callao (126 samples, 79.4% smear-positive), Lima Sur (n = 130, 84%) and Arequipa (n = 126, 80%) took respectively 94, 97 and 173 days. Pre-determined criteria in all regional laboratories were above expected values. The sensitivity and specificity for detecting MDR-TB in regional laboratories were >95%, except for sensitivity in Lima Sur, which was 91.7%. Contamination was 1.0–2.3%. Mean delay to positive MODS results was 9.9–12.9 days. CONCLUSION Technology transfer of MODS was reliable, effective and fast, enabling the INS to accredit regional laboratories swiftly. PMID:21219684

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.

Development of a solid-phase assay for measurement of proteolytic enzyme activity

International Nuclear Information System (INIS)

Varani, J.; Johnson, K.; Kaplan, J.

1980-01-01

A solid-phase, plate assay was developed for the measurement of proteolytic enzyme activity. In this assay procedure, radiolabeled substrates were dried onto the surface of microtiter wells. Following drying, the wells were washed two times with saline to remove the nonadherent substrate. When proteolytic enzymes were added to the wells, protein hydrolysis occurred, releasing radioactivity into the supernatant fluid. The amount of protein hydrolysis that occurred was reflected by the amount of radioactivity in the supernatant fluid. When 125 I-hemoglobin was used as the substrate, it was as susceptible to hydrolysis by trypsin in the solid-phase assay as it was in solution in a standard assay procedure. Protease activity from a variety of sources (including from viable cells as well as from extracellular sources) were also able to hydrolyze the hemoglobin on the plate. 125 I-Labeled serum albumen, fibrinogen, and rat pulmonary basement membrane were also susceptible to hydrolysis by trypsin in the solid phase. When [ 14 C]elastin was dried onto the plate, it behaved in a similar manner to elastin in solution. It was resistant to hydrolysis by nonspecific proteases such as trypsin and chymotrypsin but was highly susceptible to hydrolysis by elastase. The solid-phase plate assay has several features which recommended it for routine use. It is as sensitive as standard tube assays (and much more sensitive than routinely used colormetric assays). It is quick and convenient; there are no precipitation, centrifugation, or filtration steps. In addition, very small volumes of radioactive wastes are generated. Another advantage of the solid-phase plate assay is the resistance of the dried substrates to spontaneous breakdown and to microbial contamination. Finally, this assay is suitable for use with viable cells as well as for extracellular proteases

Radioreceptor assay for oxyphenonium

International Nuclear Information System (INIS)

Ensing, K.; Zeeuw, R.A. de

1984-01-01

The development of a radioreceptor assay for the quaternary anticholinergic drug, oxyphenonium, in plasma is reported. It is based on competition between this drug and 3 H-dexetimide for binding to muscarinic receptors. After ion pair extraction and reextraction, the drug can be determined in plasma at concentrations down to a value of 100 pg/ml. This permits pharmacokinetic studies to be made after inhalation of oxyphenonium. (author)

Interdependence of initial cell density, drug concentration and exposure time revealed by real-time impedance spectroscopic cytotoxicity assay

DEFF Research Database (Denmark)

Caviglia, Claudia; Zor, Kinga; Canepa, Silvia

2015-01-01

We investigated the combined effect of the initial cell density (12 500, 35 000, 75 000, and 100 000 cells cm−2) and concentration of the anti-cancer drug doxorubicin on HeLa cells by performing timedependent cytotoxicity assays using real-time electrochemical impedance spectroscopy. A correlation...... between the rate of cell death and the initial cell seeding density was found at 2.5 μM doxorubicin concentration, whereas this was not observed at 5 or 100 μM. By sensing the changes in the cell–substrate interaction using impedance spectroscopy under static conditions, the onset of cytotoxicity...... was observed 5 h earlier than when using a standard colorimetric end-point assay (MTS) which measures changes in the mitochondrial metabolism. Furthermore, with the MTS assay no cytotoxicity was observed after 15 h of incubation with 2.5 μM doxorubicin, whereas the impedance showed at this time point cell...

Silver and Nitrate Oppositely Modulate Antimony Susceptibility through Aquaglyceroporin 1 in Leishmania (Viannia) Species.

Science.gov (United States)

Andrade, Juvana M; Baba, Elio H; Machado-de-Avila, Ricardo A; Chavez-Olortegui, Carlos; Demicheli, Cynthia P; Frézard, Frédéric; Monte-Neto, Rubens L; Murta, Silvane M F

2016-08-01

Antimony (Sb) resistance in leishmaniasis chemotherapy has become one of the major challenges to the control of this spreading worldwide public health problem. Since the plasma membrane pore-forming protein aquaglyceroporin 1 (AQP1) is the major route of Sb uptake in Leishmania, functional studies are relevant to characterize drug transport pathways in the parasite. We generated AQP1-overexpressing Leishmania guyanensis and L. braziliensis mutants and investigated their susceptibility to the trivalent form of Sb (Sb(III)) in the presence of silver and nitrate salts. Both AQP1-overexpressing lines presented 3- to 4-fold increased AQP1 expression levels compared with those of their untransfected counterparts, leading to an increased Sb(III) susceptibility of about 2-fold. Competition assays using silver nitrate, silver sulfadiazine, or silver acetate prior to Sb(III) exposure increased parasite growth, especially in AQP1-overexpressing mutants. Surprisingly, Sb(III)-sodium nitrate or Sb(III)-potassium nitrate combinations showed significantly enhanced antileishmanial activities compared to those of Sb(III) alone, especially against AQP1-overexpressing mutants, suggesting a putative nitrate-dependent modulation of AQP1 activity. The intracellular level of antimony quantified by graphite furnace atomic absorption spectrometry showed that the concomitant exposure to Sb(III) and nitrate favors antimony accumulation in the parasite, increasing the toxicity of the drug and culminating with parasite death. This is the first report showing evidence of AQP1-mediated Sb(III) susceptibility modulation by silver in Leishmania and suggests the potential antileishmanial activity of the combination of nitrate salts and Sb(III). Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Antifungal susceptibility testing of Candida in the Clinical Laboratory: how to do it, when to do it, and how to interpret it

Directory of Open Access Journals (Sweden)

Esther Manso

2014-06-01

Full Text Available Significant changes in the management of fungaemia have occurred in the last decade with increased use of fluconazole prophylaxis, of empirical treatment and of echinocandins as first-line agents for documented disease. The emergence of drug resistance in fungal pathogens has a profound impact on human health given limited number of antifungal drugs. Antifungal resistance in Candida may be either intrinsic or acquired and may be encountered in the antifungal drug exposed but also the antifungal drug naïve patient The variation in resistance rates between centers emphasizes that it is essential to have knowledge of the local Candida species distribution and antifungal resistance rates to guide initial therapy for Candida BSI. Moreover, all Candida isolates from blood and normally sterile sites should be identified to the species level. The Clinical and Laboratory Standards Institute and European Committee on Antimicrobial Susceptibility Testing have developed breakpoints and epidemiological cutoff values that are now established for Candida spp. Clinical microbiology laboratories will be employed commercial susceptibility assays, rather than reference broth microdilution methods and comparative studies are particularly important. Vitek 2®, Etest® and Sensititre YeastOne® provided a high degree of essential agreement and comparable sensitivity and specificity to BMD-RPMI for identifying resistance to azole and echinocandins in Candida spp.

A polychromatic turbidity microplate assay to distinguish discovery stage drug molecules with beneficial precipitation properties.

Science.gov (United States)

Morrison, John; Nophsker, Michelle; Elzinga, Paul; Donoso, Maria; Park, Hyunsoo; Haskell, Roy

2017-10-05

A material sparing microplate screening assay was developed to evaluate and compare the precipitation of discovery stage drug molecules as a function of time, concentration and media composition. Polychromatic turbidity time course profiles were collected for cinnarizine, probucol, dipyridamole as well as BMS-932481, and compared with turbidity profiles of monodisperse particle size standards. Precipitation for select sample conditions were further characterized at several time points by size, morphology, amount and form via laser diffraction, microscopy, size based particle counting and X-ray diffraction respectively. Wavelength dependent turbidity was found indicative of nanoprecipitate, while wavelength independent turbidity was consistent with larger microprecipitate formation. A transition from wavelength dependent to wavelength independent turbidity occurred for nanoparticle to microparticle growth, and a decrease in wavelength independent turbidity correlated with continued growth in size of microparticles. Other sudden changes in turbidity signal over time such as rapid fluctuation, a decrease in slope or a sharp inversion were correlated with very large or aggregated macro-precipitates exceeding 100μm in diameter, a change in the rate of precipitate formation or an amorphous to crystalline form conversion respectively. The assay provides an effective method to efficiently monitor and screen the precipitation fates of drug molecules, even during the early stages of discovery with limited amounts of available material. This capability highlights molecules with beneficial precipitation properties that are able to generate and maintain solubility enabling amorphous or nanoparticle precipitates. Copyright © 2017 Elsevier B.V. All rights reserved.

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.

21 CFR 864.7425 - Carboxyhemoglobin assay.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Carboxyhemoglobin assay. 864.7425 Section 864.7425 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7425 Carboxyhemoglobin...

21 CFR 864.7250 - Erythropoietin assay.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Erythropoietin assay. 864.7250 Section 864.7250 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7250 Erythropoietin...

21 CFR 864.7490 - Sulfhemoglobin assay.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Sulfhemoglobin assay. 864.7490 Section 864.7490 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7490 Sulfhemoglobin...

A yeast-based assay identifies drugs that interfere with immune evasion of the Epstein-Barr virus

Directory of Open Access Journals (Sweden)

Cécile Voisset

2014-04-01

Full Text Available Epstein-Barr virus (EBV is tightly associated with certain human cancers, but there is as yet no specific treatment against EBV-related diseases. The EBV-encoded EBNA1 protein is essential to maintain viral episomes and for viral persistence. As such, EBNA1 is expressed in all EBV-infected cells, and is highly antigenic. All infected individuals, including individuals with cancer, have CD8+ T cells directed towards EBNA1 epitopes, yet the immune system fails to detect and destroy cells harboring the virus. EBV immune evasion depends on the capacity of the Gly-Ala repeat (GAr domain of EBNA1 to inhibit the translation of its own mRNA in cis, thereby limiting the production of EBNA1-derived antigenic peptides presented by the major histocompatibility complex (MHC class I pathway. Here we establish a yeast-based assay for monitoring GAr-dependent inhibition of translation. Using this assay we identify doxorubicin (DXR as a compound that specifically interferes with the GAr effect on translation in yeast. DXR targets the topoisomerase-II–DNA complexes and thereby causes genomic damage. We show, however, that the genotoxic effect of DXR and various analogs thereof is uncoupled from the effect on GAr-mediated translation control. This is further supported by the observation that etoposide and teniposide, representing another class of topoisomerase-II–DNA targeting drugs, have no effect on GAr-mediated translation control. DXR and active analogs stimulate, in a GAr-dependent manner, EBNA1 expression in mammalian cells and overcome GAr-dependent restriction of MHC class I antigen presentation. These results validate our approach as an effective high-throughput screening assay to identify drugs that interfere with EBV immune evasion and, thus, constitute candidates for treating EBV-related diseases, in particular EBV-associated cancers.

A yeast-based assay identifies drugs that interfere with immune evasion of the Epstein-Barr virus.

Science.gov (United States)

Voisset, Cécile; Daskalogianni, Chrysoula; Contesse, Marie-Astrid; Mazars, Anne; Arbach, Hratch; Le Cann, Marie; Soubigou, Flavie; Apcher, Sébastien; Fåhraeus, Robin; Blondel, Marc

2014-04-01

Epstein-Barr virus (EBV) is tightly associated with certain human cancers, but there is as yet no specific treatment against EBV-related diseases. The EBV-encoded EBNA1 protein is essential to maintain viral episomes and for viral persistence. As such, EBNA1 is expressed in all EBV-infected cells, and is highly antigenic. All infected individuals, including individuals with cancer, have CD8(+) T cells directed towards EBNA1 epitopes, yet the immune system fails to detect and destroy cells harboring the virus. EBV immune evasion depends on the capacity of the Gly-Ala repeat (GAr) domain of EBNA1 to inhibit the translation of its own mRNA in cis, thereby limiting the production of EBNA1-derived antigenic peptides presented by the major histocompatibility complex (MHC) class I pathway. Here we establish a yeast-based assay for monitoring GAr-dependent inhibition of translation. Using this assay we identify doxorubicin (DXR) as a compound that specifically interferes with the GAr effect on translation in yeast. DXR targets the topoisomerase-II-DNA complexes and thereby causes genomic damage. We show, however, that the genotoxic effect of DXR and various analogs thereof is uncoupled from the effect on GAr-mediated translation control. This is further supported by the observation that etoposide and teniposide, representing another class of topoisomerase-II-DNA targeting drugs, have no effect on GAr-mediated translation control. DXR and active analogs stimulate, in a GAr-dependent manner, EBNA1 expression in mammalian cells and overcome GAr-dependent restriction of MHC class I antigen presentation. These results validate our approach as an effective high-throughput screening assay to identify drugs that interfere with EBV immune evasion and, thus, constitute candidates for treating EBV-related diseases, in particular EBV-associated cancers.

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.

Further characterization of porcine Brachyspira hyodysenteriae isolates with decreased susceptibility to tiamulin.

Science.gov (United States)

Karlsson, M; Aspán, A; Landén, A; Franklin, A

2004-04-01

Brachyspira hyodysenteriae is the causative agent of swine dysentery, a severe diarrhoeal disease in pigs. Few drugs are available to treat the disease, owing to both antimicrobial resistance and withdrawal of drugs authorized for use in pigs. Tiamulin is the drug of choice in many countries, but isolates with decreased susceptibility have recently been reported. The mechanism of tiamulin resistance in B. hyodysenteriae is not known and this facet is essential to understand the dissemination of the trait. To study the resistance epidemiology of B. hyodysenteriae, further characterization of a set of isolates from Germany (n = 16) and the UK (n = 6) with decreased susceptibility to tiamulin was performed. The relatedness between the isolates was studied by comparing PFGE patterns, and the in vitro susceptibility to five other antimicrobials (aivlosin, doxycycline, salinomycin, chloramphenicol and avilamycin) was also determined. For comparison of the antimicrobial-susceptibility pattern, Swedish (n = 20) and British (n = 4) tiamulin-susceptible isolates were tested. The German isolates represented several different PFGE patterns, indicating that tiamulin usage has been sufficient to select clones with decreased tiamulin susceptibility at different farms in Germany. The PFGE pattern for the six British isolates with decreased tiamulin susceptibility was identical to that of the German isolates, and they had a similar antimicrobial-susceptibility pattern, except for resistance to aivlosin, which was only found in a few German isolates. No other co-resistance with tiamulin was found.

Evaluation of semisolid agar method for antifungal susceptibility test of T. rubrum

Directory of Open Access Journals (Sweden)

Sultana Razia

2016-08-01

Full Text Available Background: With increasing fungal disease many newer antifungal drugs are available with different spectrum of activ­ity. Antifungal susceptibility test will help clinicians for selection of effective drug and thereby treatment of patient. Objective: The study was undertaken to perform a simple screening drug susceptibility test of T. rnbrum by Semi Solid Agar Antifungal Susceptibility (SAAS Method: Perfonnance of susceptibility method was assessed by comparing the MICs of three commonly prescribed antifungal agents namely- tluconazole (FCZ, itraconazole (ITZ and terbinafine (TER to the CLSI (Clinical and Laboratory Standard Institute recommended M-38, a broth microdilution method. Results: In SAAS method, among twenty nine T. rubrum, twenty five (86.2% were susceptible (MIC range 0.5-64 µg/ml to Fluconazole (FCZ and four (13.7% were resistant (MIC value >64 µg/ml. In broth microdilution method, among twenty nine T. rubrum, twenty six (89.6% were susceptible (MIC range 0.3-64 µg/ml to FCZ and three (10.3% were resistant (MIC value >64 µg/ml. In case of both ITZ and TER, all were susceptible (MIC range 0.3-64 µg/ml to both methods. The SAAS method demonstrated the susceptibility pattern of T. rubrum against FCZ, ITZ and TER usually within 72 to 96 hours after organism isolation and results were concordance with the results of CLSI broth microdilution method. Conclusion: Though it is a newer method with proper standardization of the test method, SAAS method is simple and easily applicable screening method for susceptibility testing of antifungal agents against dermatophytes in any microbiology laboratories.

Correlation of the microculture-kinetic drug-induced apoptosis assay with patient outcomes in initial treatment of adult acute myelocytic leukemia.

Science.gov (United States)

Strickland, Stephen A; Raptis, Anastasios; Hallquist, Allan; Rutledge, James; Chernick, Michael; Perree, Mathieu; Talbott, Mahsa S; Presant, Cary A

2013-03-01

Overall survival (OS) with acute myeloid leukemia (AML) remains poor. Determining prognostic factors will help in selecting patients for appropriate treatments. Our aim was to determine whether the level of drug-induced apoptosis (chemosensitivity) demonstrated by the microculture-kinetic drug-induced apoptosis (MiCK) assay significantly predicted outcomes after standard AML induction therapy. A total of 109 patients with untreated AML had blood and/or bone marrow aspirate samples analyzed for anthracycline-induced apoptosis using the MiCK assay. The amount of apoptosis observed over 48 h was determined and expressed as kinetic units of apoptosis (KU). Complete remission (CR) was significantly higher (72%) in patients with high idarubicin-induced apoptosis >3 KU compared to patients with apoptosis ≤ 3 KU (p = 0.01). Multivariate analysis showed the only significant variables to be idarubicin-induced apoptosis and karyotype. Median overall survival of patients with idarubicin-induced apoptosis >3 KU was 16.1 months compared to 4.5 months in patients with apoptosis ≤ 3 KU (p = 0.004). Multivariate analysis showed the only significant variable to be idarubicin-induced apoptosis. Chemotherapy-induced apoptosis measured by the MiCK assay demonstrated significant correlation with outcomes and appears predictive of complete remission and overall survival for patients receiving standard induction chemotherapy.

A comprehensive review of the published assays for the quantitation of the immunosuppressant drug mycophenolic acid and its glucuronidated metabolites in biological fluids.

Science.gov (United States)

Syed, Muzeeb; Srinivas, Nuggehally R

2016-05-01

Therapeutic use of mycophenolic acid (MPA) is steadily on the rise in combination with other immunosuppressant drugs in transplantation patients. The biotransformation of MPA resulted in the formation of glucuronide metabolites, MPAG and AcMPAG. There are a plethora of assays validated for the analysis of MPA alone or with MPAG/AcMPAG in various biological specimens including plasma/serum, urine, ultrafiltrate, saliva, PBMC, dried blood spots, tissue extract, tumor biopsies and vitreous humor. Based on the need for experimental work, a proper choice of the assay and internal standard may be made using the choices in the literature. While the chemical methods involving high-performance liquid chromatography (HPLC) or LC coupled with triple quadrupole mass spectrometry (LC-MS/MS) are popular, enzymatic assays, in spite of their higher bias, have been used for the routine drug monitoring of MPA. The objectives of the present review are: (a) to provide a focused systematic compilation of the HPLC or LC-MS/MS methods for MPA, MPAG and/or AcMPAG published in the last decade (2005 to current) to enable visual comparison of the methods; (b) to compare and contrast a few enzymatic assays with those of the chemical methods; and (c) to discuss relevant issues/limitations and perspectives on select assays under various subheadings. Copyright © 2016 John Wiley & Sons, Ltd.

[Antimicrobial susceptibility and drug-resistance genes of Yersinia spp. of retailed poultry in 4 provinces of China].

Science.gov (United States)

Peng, Z X; Zou, M Y; Xu, J; Guan, W Y; Li, Y; Liu, D R; Zhang, S S; Hao, Q; Yan, S F; Wang, W; Yu, D M; Li, F Q

2018-04-06

Objective: To monitor the antimicrobial resistance and drug-resistance genes of Yersinia enterocolitis , Y. intermedia and Y. frederiksenii recovered from retailed fresh poultry of 4 provinces of China. Methods: The susceptibility of 25 isolated Yersinia spp. to 14 classes and 25 kinds of antibiotics was determined by broth microdilution method according to CLSI (Clinical and Laboratory Standards Institute). The antibiotic resistance genes were predicted with antibiotic resistance genes database (ARDB) using whole genome sequences of Yersinia spp. Results: In all 22 Y. enterocolitis tested, 63.7% (14 isolates), 22.8% (5 isolates), 4.6% and 4.6% of 1 isolates exhibited the resistance to cefoxitin, ampicillin-sulbactam, nitrofurantoin and trimethoprim-sulfamethoxazole, respectively. All the 25 isolates were multi-drug resistant to more than 3 antibiotics, while 64.0% of isolates were resistant to more than 4 antibiotics. A few Y. enterocolitis isolates of this study were intermediate to ceftriaxone and ciprofloxacin. Most Yersinia spp. isolates contained antibiotic resistance genes mdtG, ksgA, bacA, blaA, rosAB and acrB , and 5 isolates recovered from fresh chicken also contained dfrA 1, catB 2 and ant 3 ia . Conclusion: The multi-drug resistant Yersinia spp. isolated from retailed fresh poultry is very serious in the 4 provinces of China, and their contained many kinds of drug-resistance genes.

An in vitro larval migration assay for assessing anthelmintic activity of different drug classes against Ascaris suum

DEFF Research Database (Denmark)

Zhao, Jianguo; Williams, Andrew R; Hansen, Tina Vicky Alstrup

2017-01-01

and agar gel larval migration assay to test the effect of benzimidazole and tetrahydropyrimidin/imidazothiazole anthelmintics against nine isolates of A. suum collected from locations in China and Denmark. Drugs tested were thiabendazole, fenbendazole, mebendazole, levamisole, and pyrantel. The percentages...

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.

Plaque assay for human coronavirus NL63 using human colon carcinoma cells

Directory of Open Access Journals (Sweden)

Drosten Christian

2008-11-01

Full Text Available Abstract Background Coronaviruses cause a broad range of diseases in animals and humans. Human coronavirus (hCoV NL63 is associated with up to 10% of common colds. Viral plaque assays enable the characterization of virus infectivity and allow for purifying virus stock solutions. They are essential for drug screening. Hitherto used cell cultures for hCoV-NL63 show low levels of virus replication and weak and diffuse cytopathogenic effects. It has not yet been possible to establish practicable plaque assays for this important human pathogen. Results 12 different cell cultures were tested for susceptibility to hCoV-NL63 infection. Human colon carcinoma cells (CaCo-2 replicated virus more than 100 fold more efficiently than commonly used African green monkey kidney cells (LLC-MK2. CaCo-2 cells showed cytopathogenic effects 4 days post infection. Avicel, agarose and carboxymethyl-cellulose overlays proved suitable for plaque assays. Best results were achieved with Avicel, which produced large and clear plaques from the 4th day of infection. The utility of plaque assays with agrose overlay was demonstrated for purifying virus, thereby increasing viral infectivity by 1 log 10 PFU/mL. Conclusion CaCo-2 cells support hCoV-NL63 better than LLC-MK2 cells and enable cytopathogenic plaque assays. Avicel overlay is favourable for plaque quantification, and agarose overlay is preferred for plaque purification. HCoV-NL63 virus stock of increased infectivity will be beneficial in antiviral screening, animal modelling of disease, and other experimental tasks.

Influence of doxorubicin on fluconazole susceptibility and efflux pump gene expression of Candida dubliniensis.

LENUS (Irish Health Repository)

Schulz, Bettina

2012-05-01

The effect of doxorubicin (DOX) on the fluconazole (FLU) susceptibility of C. dubliniensis was investigated. Isolates were exposed to DOX and FLU in a chequerboard assay and resistance gene expressions were analysed after DOX exposure. The susceptibility of the yeast to FLU was decreased in the presence of DOX in the chequerboard assay with FIC indices suggesting an antagonistic effect. Gene expression analyses showed an overexpression of CdCDR2. Hence, DOX was found to have an impact on resistance mechanisms in C. dubliniensis isolates.

Assay system

International Nuclear Information System (INIS)

Patzke, J.B.; Rosenberg, B.J.

1984-01-01

The accuracy of assays for monitoring concentrations of basic drugs in biological fluids containing a 1 -acid glycoproteins, such as blood (serum or plasma), is improved by the addition of certain organic phosphate compounds to minimize the ''protein effect.'' Kits containing the elements of the invention are also disclosed

Susceptibility of Selected Multi-Drug Resistant Clinical Isolates to ...

African Journals Online (AJOL)

2018-03-01

Mar 1, 2018 ... under the terms of the Creative Commons. Attribution ... relies on traditional medicine for their primary ... Collection and identification of plant materials: ..... versus carbapenem- susceptible Gram-negative microorganisms: Risk.

Polymyxins: Antimicrobial susceptibility concerns and therapeutic options

Directory of Open Access Journals (Sweden)

V Balaji

2011-01-01

Full Text Available The increasing prevalence of multidrug-resistant nosocomial pathogens such as Acinetobacter baumannii, Pseudomonas aeruginosa and Klebsiella pneumoniae poses a great challenge to the treating physicians. The paucity of newer effective antimicrobials has led to renewed interest in the polymyxin group of drugs, as a last resort for treatment of gram-negative bacterial infections. There is a dearth of information on the pharmacological properties of colistin, leading to difficulties in selecting the right dose, dosing interval, and route of administration for treatment, especially in critically-ill patients. The increasing use of colistin over the last few years necessitates the need for accurate and reliable in vitro susceptibility testing methods. Development of heteroresistant strains as a result of colistin monotherapy is also a growing concern. There is a compelling need from the clinicians to provide options for probable and possible colistin combination therapy for multidrug-resistant bacterial infections in the ICU setting. Newer combination drug synergy determination tests are being developed and reported. There are no standardized recommendations from antimicrobial susceptibility testing reference agencies for the testing and interpretation of these drug combinations. Comparison and analysis of these reported methodologies may help to understand and assist the microbiologist to choose the best method that produces accurate results at the earliest. This will help clinicians to select the appropriate combination therapy. In this era of multidrug resistance it is important for the microbiology laboratory to be prepared, by default, to provide timely synergistic susceptibility results in addition to routine susceptibility, if warranted. Not as a favour or at request, but as a responsibility.

Different neuraminidase inhibitor susceptibilities of human H1N1, H1N2, and H3N2 influenza A viruses isolated in Germany from 2001 to 2005/2006.

Science.gov (United States)

Bauer, Katja; Richter, Martina; Wutzler, Peter; Schmidtke, Michaela

2009-04-01

In the flu season 2005/2006 amantadine-resistant human influenza A viruses (FLUAV) of subtype H3N2 circulated in Germany. This raises questions on the neuraminidase inhibitor (NAI) susceptibility of FLUAV. To get an answer, chemiluminescence-based neuraminidase inhibition assays were performed with 51 H1N1, H1N2, and H3N2 FLUAV isolated in Germany from 2001 to 2005/2006. According to the mean IC(50) values (0.38-0.91 nM for oseltamivir and 0.76-1.13 nM for zanamivir) most H1N1 and H3N2 FLUAV were NAI-susceptible. But, about four times higher zanamivir concentrations were necessary to inhibit neuraminidase activity of H1N2 viruses. Two H1N1 isolates were less susceptible to both drugs in NA inhibition as well as virus yield reduction assays. Results from sequence analysis of viral hemagglutinin and neuraminidase genes and evolutionary analysis of N2 gene revealed (i) different subclades for N2 in H1N2 and H3N2 FLUAV that could explain the differences in zanamivir susceptibility among these viruses and (ii) specific amino acid substitutions in the neuraminidase segment of the two less NAI-susceptible H1N1 isolates. One H3N2 was isolate proved to be a mixture of a NA deletion mutant and full-length NA viruses.

Drugs and Young People

Science.gov (United States)

Drug abuse is a serious public health problem. It affects almost every community and family in some way. Drug abuse in children and teenagers may pose a ... of young people may be more susceptible to drug abuse and addiction than adult brains. Abused drugs ...

Design, development, and validation of a high-throughput drug-screening assay for targeting of human leukemia

Science.gov (United States)

Karjalainen, Katja; Pasqualini, Renata; Cortes, Jorge E.; Kornblau, Steven M.; Lichtiger, Benjamin; O'Brien, Susan; Kantarjian, Hagop M.; Sidman, Richard L.; Arap, Wadih; Koivunen, Erkki

2015-01-01

Background We introduce an ex vivo methodology to perform drug library screening against human leukemia. Method Our strategy relies on human blood or bone marrow cultures under hypoxia; under these conditions, leukemia cells deplete oxygen faster than normal cells, causing a hemoglobin oxygenation shift. We demonstrate several advantages: (I) partial recapitulation of the leukemia microenvironment, (ii) use of native hemoglobin oxygenation as real-time sensor/reporter, (iii) cost-effectiveness, (iv) species-specificity, and (v) format that enables high-throughput screening. Results As a proof-of-concept, we screened a chemical library (size ∼20,000) against human leukemia cells. We identified 70 compounds (“hit” rate=0.35%; Z-factor=0.71) with activity; we examined 20 to find 18 true-positives (90%). Finally, we show that carbonohydraxonic diamide group-containing compounds are potent anti-leukemia agents that induce cell death in leukemia cells and patient-derived samples. Conclusions This unique functional assay can identify novel drug candidates as well as find future applications in personalized drug selection for leukemia patients. PMID:24496871

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.

Assaying of drugs in body fluids

International Nuclear Information System (INIS)

Braestrup, C.; Squires, R.F.

1981-01-01

The invention provides, in general terms, a process for determining the concentration of a psychotropically active benzodiazepine drug in blood or other body fluid or urine, including bringing a sample of the fluid or urine into contact with brain tissue and with tritium labelled molecules of a benzodiazepine which can bind reversibly to receptors of the brain tissue to induce binding of molecules of the unlabelled drug and of the tritium labelled benzodiazepine to the receptors, and determining the radioactivity of the brain tissue, preferably by scintillation counting. (author)

The Ussing Chamber Assay to Study Drug Metabolism and Transport in the Human Intestine.

Science.gov (United States)

Kisser, Beatrice; Mangelsen, Eva; Wingolf, Caroline; Partecke, Lars Ivo; Heidecke, Claus-Dieter; Tannergren, Christer; Oswald, Stefan; Keiser, Markus

2017-06-22

The Ussing chamber is an old but still powerful technique originally designed to study the vectorial transport of ions through frog skin. This technique is also used to investigate the transport of chemical agents through the intestinal barrier as well as drug metabolism in enterocytes, both of which are key determinants for the bioavailability of orally administered drugs. More contemporary model systems, such as Caco-2 cell monolayers or stably transfected cells, are more limited in their use compared to the Ussing chamber because of differences in expression rates of transporter proteins and/or metabolizing enzymes. While there are limitations to the Ussing chamber assay, the use of human intestinal tissue remains the best laboratory test for characterizing the transport and metabolism of compounds following oral administration. Detailed in this unit is a step-by-step protocol for preparing human intestinal tissue, for designing Ussing chamber experiments, and for analyzing and interpreting the findings. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

Antiretroviral neuropenetration scores better correlate with cognitive performance of HIV-infected patients after accounting for drug susceptibility.

Science.gov (United States)

Fabbiani, Massimiliano; Grima, Pierfrancesco; Milanini, Benedetta; Mondi, Annalisa; Baldonero, Eleonora; Ciccarelli, Nicoletta; Cauda, Roberto; Silveri, Maria C; De Luca, Andrea; Di Giambenedetto, Simona

2015-01-01

The aim of the study was to explore how viral resistance and antiretroviral central nervous system (CNS) penetration could impact on cognitive performance of HIV-infected patients. We performed a multicentre cross-sectional study enrolling HIV-infected patients undergoing neuropsychological testing, with a previous genotypic resistance test on plasma samples. CNS penetration-effectiveness (CPE) scores and genotypic susceptibility scores (GSS) were calculated for each regimen. A composite score (CPE-GSS) was then constructed. Factors associated with cognitive impairment were investigated by logistic regression analysis. A total of 215 patients were included. Mean CPE was 7.1 (95% CI 6.9, 7.3) with 206 (95.8%) patients showing a CPE≥6. GSS correction decreased the CPE value in 21.4% (mean 6.5, 95% CI 6.3, 6.7), 26.5% (mean 6.4, 95% CI 6.1, 6.6) and 24.2% (mean 6.4, 95% CI 6.2, 6.6) of subjects using ANRS, HIVDB and REGA rules, respectively. Overall, 66 (30.7%) patients were considered cognitively impaired. No significant association could be demonstrated between CPE and cognitive impairment. However, higher GSS-CPE was associated with a lower risk of cognitive impairment (CPE-GSSANRS odds ratio 0.75, P=0.022; CPE-GSSHIVDB odds ratio 0.77, P=0.038; CPE-GSSREGA odds ratio 0.78, P=0.038). Overall, a cutoff of CPE-GSS≥5 seemed the most discriminatory according to each different interpretation system. GSS-corrected CPE score showed a better correlation with neurocognitive performance than the standard CPE score. These results suggest that antiretroviral drug susceptibility, besides drug CNS penetration, can play a role in the control of HIV-associated neurocognitive disorders.

Species distribution and drug susceptibility of candida in clinical isolates from a tertiary care centre at Indore

Directory of Open Access Journals (Sweden)

N Pahwa

2014-01-01

Full Text Available Background: The incidence of fungal infections has increased significantly, contributing to morbidity and mortality. This is caused by an alarming increase in infections with multi-drug resistant bacteria leading to overuse of broad-spectrum antimicrobials, which lead to overgrowth of Candida, thus enhancing its opportunity to cause disease. Candida are major human fungal pathogens that cause both mucosal and deep tissue infections. Objective : The aim of our study was to identify the distribution of Candida species among clinical isolates and their sensitivity pattern for common antifungal drugs. Materials and Methods : Two hundred and thirty-seven different clinical isolates of Candida were collected from patients visiting to a tertiary care centre of Indore from 2010 to 2012. Identification of Candida species as well as antifungal sensitivity testing was performed with Vitek2 Compact (Biomerieux France using vitek 2 cards for identification of yeast and yeast like organisms (ID-YST cards. Antifungal susceptibility testing was performed with Vitek2 "Fungal Susceptibility Card (AST YS01 kits respectively. Results : We found that the non-albicans Candida were more prevalent than Candida albicans in paediatric (60 year patients than other age group (4-18, 19-60 years patients and also in intensive care unit (ICU patients as compared to out patient department (OPD patients. Resistance rates for amphotericin B, fluconazole, flucytosine, itraconazole, and voriconazole were 2.9%, 5.9%, 0.0%, 4.2% and 2.5%%, respectively. All the strains of C. krusei were found resistant to fluconazole with intermediate sensitivity to flucytosine. Conclusion: Species-level identification of Candida and their antifungal sensitivity testing should be performed to achieve better clinical results.

Determination of Nitrate Reductase Assay Depending on the Microbial Growth

International Nuclear Information System (INIS)

El-Kabbany, H.M.

2012-01-01

A rapid micro-dilution assay for determination of the antimicrobial susceptibility of different bacterial isolates was developed. This assay is based on the ability of the most of viable organisms to reduce nitrates. The MIC or MBC could be determined by nitrate reductase (NR) only after 30 to 90 min of incubation depending on the behaviour of microbial growth. Bacterial viability is detected by a positive nitrite reduction rather than visible turbidity. The nitrate reduction assay was compared with standard micro-assay using 250 isolates of different taxa against 10 antibiotics belonging to different classes. An excellent agreement of 82.5 % was found between the two methods and only 17.5 % of 1794 trials showed difference in the determined MIC by tow-dilution interval above or below the MIC determined by the turbidimetric method under the same test conditions. However, the nitrate reduction assay was more rapid and sensitive in detecting viable bacteria and so, established an accurate estimate of the minimal inhibitory concentration (MIC) or the minimal bacterial concentration (MBC). The nitrate reduction assay offers the additional advantage that it could be used to determine the MBC without having to subculture the broth. 232 cases of resistance were detected by NR and 4 different media were tested for susceptibility test. The bacterial isolates were exposed to ultra violet (UV) light for different period

Clinical Usefulness of the Histoculture Drug Response Assay for Prostate Cancer and Benign Prostate Hypertrophy (BPH).

Science.gov (United States)

Hoffman, Robert M

2018-01-01

The histoculture drug response assay (HDRA) has been adapted to determine androgen sensitivity in Gelfoam histoculture of human benign prostatic tissue as well as prostate cancer. Gelfoam histoculture was used to measure androgen-independent and androgen-dependent growth of benign and malignant prostate tissue. The androgen-sensitivity index was significantly higher in 23 paired specimens of prostate cancer compared to benign prostate hypertrophy (BPH). Genistein decreased the androgen-sensitivity index of BPH and prostate cancer in Gelfoam ® histoculture in a dose-dependent manner.

Optimizing the HRP-2 In Vitro Malaria Drug Susceptibility Assay Using a Reference Clone to Improve Comparisons of Plasmodium falciparum Field Isolates

Science.gov (United States)

2012-09-13

6. Desjardins RE, Canfield CJ, Haynes JD, Chulay JD: Quantitative assessment of antimalarial activity in vitro by a semiautomated microdilution...vitro antimalarial drug efficacy testing and application to clinical isolates. Antimicrob Agents Chemother 2007, 51:1172–1178. 12. Akala HM, Eyase FL...Cheruiyot AC, Omondi AA, Ogutu BR, Waters NC, Johnson JD, Polhemus ME, Schnabel DC, Walsh DS: Antimalarial drug sensitivity profile of western Kenya

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

Inducing optimal substitution between antibiotics under open access to the resource of antibiotic susceptibility.

Science.gov (United States)

Herrmann, Markus; Nkuiya, Bruno

2017-06-01

This paper designs a bio-economic model to examine the use of substitute antibiotic drugs (analogs) sold by an industry that has open access to the resource of the antibiotic class's susceptibility (treatment effectiveness). Antibiotics are characterized by different expected recovery rates and production costs, which in conjunction with the class's treatment susceptibility determines their relative effectiveness. Our analysis reveals that the high-quality antibiotic drug loses its comparative advantage over time making the low-quality drug the treatment of last resort in the market equilibrium and the social optimum when antibiotic susceptibility cannot replenish. However, when antibiotic susceptibility is renewable, both antibiotics may be used in the long run, and the comparative advantage of the high-quality drug may be restored in the social optimum that allows lowering infection in the long run. We develop the optimal tax/subsidy scheme that would induce antibiotic producers under open access to behave optimally and account for the social cost of infection and value of antibiotic susceptibility. We show that the welfare loss associated with the uncorrected open-access allocation is highest; when the resource of antibiotic susceptibility is non-renewable, high morbidity costs are incurred by individuals, and low social discount rates apply. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Mechanism and kinetics of the loss of poorly soluble drugs from liposomal carriers studied by a novel flow field-flow fractionation-based drug release-/transfer-assay.

Science.gov (United States)

Hinna, Askell Hvid; Hupfeld, Stefan; Kuntsche, Judith; Bauer-Brandl, Annette; Brandl, Martin

2016-06-28

Liposomes represent a versatile drug formulation approach e.g. for improving the water-solubility of poorly soluble drugs but also to achieve drug targeting and controlled release. For the latter applications it is essential that the drug remains associated with the liposomal carrier during transit in the vascular bed. A range of in vitro test methods has been suggested over the years for prediction of the release of drug from liposomal carriers. The majority of these fail to give a realistic prediction for poorly water-soluble drugs due to the intrinsic tendency of such compounds to remain associated with liposome bilayers even upon extensive dilution. Upon i.v. injection, in contrast, rapid drug loss often occurs due to drug transfer from the liposomal carriers to endogenous lipophilic sinks such as lipoproteins, plasma proteins or membranes of red blood cells and endothelial cells. Here we report on the application of a recently introduced in vitro predictive drug transfer assay based on incubation of the liposomal drug carrier with large multilamellar liposomes, the latter serving as a biomimetic model sink, using flow field-flow fractionation as a tool to separate the two types of liposomes. By quantifying the amount of drug remaining associated with the liposomal drug carrier as well as that transferred to the acceptor liposomes at distinct times of incubation, both the kinetics of drug transfer and release to the water phase could be established for the model drug p-THPP (5,10,15,20-tetrakis(4-hydroxyphenyl)21H,23H-porphine). p-THPP is structurally similar to temoporfin, a photosensitizer which is under clinical evaluation in a liposomal formulation. Mechanistic insights were gained by varying the donor-to-acceptor lipid mass ratio, size and lamellarity of the liposomes. Drug transfer kinetics from one liposome to another was found rate determining as compared to redistribution from the outermost to the inner concentric bilayers, such that the overall

Detection of induced male germline mutation: Correlations and comparisons between traditional germline mutation assays, transgenic rodent assays and expanded simple tandem repeat instability assays

Energy Technology Data Exchange (ETDEWEB)

Singer, Timothy M. [Mutagenesis Section, Environmental and Occupational Toxicology Division, Safe Environments Programme, 0803A, Health Canada, Ottawa, Ont., K1A 0K9 (Canada); Department of Biology, Carleton University, 1125 Colonel By Drive, Ottawa, Ont., K1S 5B6 (Canada); Lambert, Iain B. [Department of Biology, Carleton University, 1125 Colonel By Drive, Ottawa, Ont., K1S 5B6 (Canada); Williams, Andrew [Biostatistics and Epidemiology Division, Safe Environments Programme, 6604B, Health Canada, Ottawa, Ont., K1A 0K9 (Canada); Douglas, George R. [Mutagenesis Section, Environmental and Occupational Toxicology Division, Safe Environments Programme, 0803A, Health Canada, Ottawa, Ont., K1A 0K9 (Canada); Yauk, Carole L. [Mutagenesis Section, Environmental and Occupational Toxicology Division, Safe Environments Programme, 0803A, Health Canada, Ottawa, Ont., K1A 0K9 (Canada)]. E-mail: carole_yauk@hc-sc.gc.ca

2006-06-25

Several rodent assays are capable of monitoring germline mutation. These include traditional assays, such as the dominant lethal (DL) assay, the morphological specific locus (SL) test and the heritable translocation (HT) assay, and two assays that have been developed more recently-the expanded simple tandem repeat (ESTR) and transgenic rodent (TGR) mutation assays. In this paper, we have compiled the limited amount of experimental data that are currently available to make conclusions regarding the comparative ability of the more recently developed assays to detect germline mutations induced by chemical and radiological agents. The data suggest that ESTR and TGR assays are generally comparable with SL in detecting germline mutagenicity induced by alkylating agents and radiation, though TGR offered less sensitivity than ESTR in some cases. The DL and HT assays detect clastogenic events and are most susceptible to mutations arising in post-spermatogonial cells, and they may not provide the best comparisons with TGR and ESTR instability. The measurement of induced ESTR instability represents a relatively sensitive method of identifying agents causing germline mutation in rodents, and may also be useful for bio-monitoring exposed individuals in the human population. Any future use of the TGR and ESTR germline mutation assays in a regulatory testing context will entail more robust and extensive characterization of assay performance. This will require substantially more data, including experiments measuring multiple endpoints, a greatly expanded database of chemical agents and a focus on characterizing stage-specific activity of mutagens in these assays, preferably by sampling epididymal sperm exposed at defined pre-meiotic, meiotic and post-meiotic stages of development.

Detection of induced male germline mutation: Correlations and comparisons between traditional germline mutation assays, transgenic rodent assays and expanded simple tandem repeat instability assays

International Nuclear Information System (INIS)

Singer, Timothy M.; Lambert, Iain B.; Williams, Andrew; Douglas, George R.; Yauk, Carole L.

2006-01-01

Several rodent assays are capable of monitoring germline mutation. These include traditional assays, such as the dominant lethal (DL) assay, the morphological specific locus (SL) test and the heritable translocation (HT) assay, and two assays that have been developed more recently-the expanded simple tandem repeat (ESTR) and transgenic rodent (TGR) mutation assays. In this paper, we have compiled the limited amount of experimental data that are currently available to make conclusions regarding the comparative ability of the more recently developed assays to detect germline mutations induced by chemical and radiological agents. The data suggest that ESTR and TGR assays are generally comparable with SL in detecting germline mutagenicity induced by alkylating agents and radiation, though TGR offered less sensitivity than ESTR in some cases. The DL and HT assays detect clastogenic events and are most susceptible to mutations arising in post-spermatogonial cells, and they may not provide the best comparisons with TGR and ESTR instability. The measurement of induced ESTR instability represents a relatively sensitive method of identifying agents causing germline mutation in rodents, and may also be useful for bio-monitoring exposed individuals in the human population. Any future use of the TGR and ESTR germline mutation assays in a regulatory testing context will entail more robust and extensive characterization of assay performance. This will require substantially more data, including experiments measuring multiple endpoints, a greatly expanded database of chemical agents and a focus on characterizing stage-specific activity of mutagens in these assays, preferably by sampling epididymal sperm exposed at defined pre-meiotic, meiotic and post-meiotic stages of development

Distribution, detection of enterotoxigenic strains and antimicrobial drug susceptibility patterns of Bacteroides fragilis group in diarrheic and non-diarrheic feces from Brazilian infants

Directory of Open Access Journals (Sweden)

Débora Paula Ferreira

2010-10-01

Full Text Available Despite the importance of gastrointestinal diseases and their global distribution, affecting millions of individuals around the world, the role and antimicrobial susceptibility patterns of anaerobic bacteria such as those in the Bacteroides fragilis group (BFG are still unclear in young children. This study investigated the occurrence and distribution of species in the BFG and enterotoxigenic strains in the fecal microbiota of children and their antimicrobial susceptibility patterns. Diarrheic (n=110 and non-diarrheic (n=65 fecal samples from children aged 0-5 years old were evaluated. BFG strains were isolated and identified by conventional biochemical, physiological and molecular approaches. Alternatively, bacteria and enterotoxigenic strains were detected directly from feces by molecular biology. Antimicrobial drug susceptibility patterns were determined by the agar dilution method according to the guidelines for isolated bacteria. BFG was detected in 64.3% of the fecal samples (55% diarrheic and 80.4% non-diarrheic, and 4.6% were enterotoxigenic. Antimicrobial resistance was observed against ampicillin, ampicillin/sulbactam, piperacillin/tazobactam, meropenem, ceftriaxone, clindamycin and chloramphenicol. The data show that these bacteria are prevalent in fecal microbiota at higher levels in healthy children. The molecular methodology was more effective in identifying the B. fragilis group when compared to the biochemical and physiological techniques. The observation of high resistance levels stimulates thoughts about the indiscriminate use of antimicrobial drugs in early infancy. Further quantitative studies are needed to gain a better understanding of the role of these bacteria in acute diarrhea in children.

Observation of reversible, rapid changes in drug susceptibility of hypoxic tumor cells in a microfluidic device.

Science.gov (United States)

Germain, Todd; Ansari, Megan; Pappas, Dimitri

2016-09-14

Hypoxia is a major stimulus for increased drug resistance and for survival of tumor cells. Work from our group and others has shown that hypoxia increases resistance to anti-cancer compounds, radiation, and other damage-pathway cytotoxic agents. In this work we utilize a microfluidic culture system capable of rapid switching of local oxygen concentrations to determine changes in drug resistance in prostate cancer cells. We observed rapid adaptation to hypoxia, with drug resistance to 2 μM staurosporine established within 30 min of hypoxia. Annexin-V/Sytox Green apoptosis assays over 9 h showed 78.0% viability, compared to 84.5% viability in control cells (normoxic cells with no staurosporine). Normoxic cells exposed to the same staurosporine concentration had a viability of 48.6% after 9 h. Hypoxia adaptation was rapid and reversible, with Hypoxic cells treated with 20% oxygen for 30 min responding to staurosporine with 51.6% viability after drug treatment for 9 h. Induction of apoptosis through the receptor-mediated pathway, which bypasses anti-apoptosis mechanisms induced by hypoxia, resulted in 39.4 ± 7% cell viability. The rapid reversibility indicates co-treatment of oxygen with anti-cancer compounds may be a potential therapeutic target. Copyright © 2016 Elsevier B.V. All rights reserved.

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.

Highly sensitive bacterial susceptibility test against penicillin using parylene-matrix chip.

Science.gov (United States)

Park, Jong-Min; Kim, Jo-Il; Song, Hyun-Woo; Noh, Joo-Yoon; Kang, Min-Jung; Pyun, Jae-Chul

2015-09-15

This work presented a highly sensitive bacterial antibiotic susceptibility test through β-lactamase assay using Parylene-matrix chip. β-lactamases (EC 3.5.2.6) are an important family of enzymes that confer resistance to β-lactam antibiotics by catalyzing the hydrolysis of these antibiotics. Here we present a highly sensitive assay to quantitate β-lactamase-mediated hydrolysis of penicillin into penicilloic acid. Typically, MALDI-TOF mass spectrometry has been used to quantitate low molecular weight analytes and to discriminate them from noise peaks of matrix fragments that occur at low m/z ratios (m/ztest was carried out using Parylene-matrix chip and MALDI-TOF mass spectrometry. The Parylene-matrix chip was successfully used to quantitate penicillin (m/z: [PEN+H](+)=335.1 and [PEN+Na](+)=357.8) and penicilloic acid (m/z: [PA+H](+)=353.1) in a β-lactamase assay with minimal interference of low molecular weight noise peaks. The β-lactamase assay was carried out with an antibiotic-resistant E. coli strain and an antibiotic-susceptible E. coli strain, revealing that the minimum number of E. coli cells required to screen for antibiotic resistance was 1000 cells for the MALDI-TOF mass spectrometry/Parylene-matrix chip assay. Copyright © 2015 Elsevier B.V. All rights reserved.

Phosphorylcholine impairs susceptibility to biofilm formation of hydrogel contact lenses.

Science.gov (United States)

Selan, Laura; Palma, Stefano; Scoarughi, Gian Luca; Papa, Rosanna; Veeh, Richard; Di Clemente, Daniele; Artini, Marco

2009-01-01

To compare silicone-hydrogel, poly(2-hydroxyethyl methacrylate) (pHEMA), and phosphorylcholine-coated (PC-C) contact lenses in terms of their susceptibility to biofilm formation by Staphylococcus epidermidis and Pseudomonas aeruginosa. Laboratory investigation. Biofilm formation on colonized test lenses was evaluated with confocal microscopy and in vitro antibiotic susceptibility assays. The results of the latter assays were compared with those performed on planktonic cultures of the same organism. For both microorganisms, sessile colonies on silicone-hydrogel and pHEMA lenses displayed lower antibiotic susceptibility than their planktonic counterparts. In contrast, the susceptibility of cultures growing on PC-C lenses was comparable with that for planktonic cultures. In particular, minimum inhibitory concentration for Tazocin (piperacillin plus tazobactam; Wyeth Pharmaceuticals, Aprilia, Italy; S. epidermidis) and gentamicin (P. aeruginosa) was identical, either in the presence of PC-C support or in planktonic cultures (Tazocin, aeruginosa) was two-fold higher for PC-C lenses (0.4 mug/ml) with respect to planktonic cultures (0.2 mug/ml). Confocal microscopy of lenses colonized for 24 hours with P. aeruginosa green fluorescent protein-expressing cells revealed a sessile colonization on silicone-hydrogel lens and a few isolated bacterial cells scattered widely over the surface of the PC-C lens. An increase in antibiotic susceptibility of bacterial cultures was associated with diminished bacterial adhesion. Our results indicate that PC-C lenses seem to be more resistant than silicone-hydrogel and pHEMA lenses to bacterial adhesion and colonization. This feature may facilitate their disinfection.

Optimization of the THP-1 activation assay to detect pharmaceuticals with potential to cause immune mediated drug reactions.

Science.gov (United States)

Corti, Daniele; Galbiati, Valentina; Gatti, Nicolò; Marinovich, Marina; Galli, Corrado L; Corsini, Emanuela

2015-10-01

Despite important impacts of systemic hypersensitivity induced by pharmaceuticals, for such endpoint no reliable preclinical approaches are available. We previously established an in vitro test to identify contact and respiratory allergens based on interleukin-8 (IL-8) production in THP-1 cells. Here, we challenged it for identification of pharmaceuticals associated with systemic hypersensitivity reactions, with the idea that drug sensitizers share common mechanisms of cell activation. Cells were exposed to drugs associated with systemic hypersensitivity reactions (streptozotocin, sulfamethoxazole, neomycin, probenecid, clonidine, procainamide, ofloxacin, methyl salicylate), while metformin was used as negative drug. Differently to chemicals, drugs tested were well tolerated, except clonidine and probenecid, with no signs of cytotoxicity up to 1-2mg/ml. THP-1 activation assay was adjusted, and conditions, that allow identification of all sensitizing drugs tested, were established. Next, using streptozotocin and selective inhibitors of PKC-β and p38 MAPK, two pathways involved in chemical allergen-induced cell activation, we tested the hypothesis that similar pathways were also involved in drug-induced IL-8 production and CD86 upregulation. Results indicated that drugs and chemical allergens share similar activation pathways. Finally, we made a structure-activity hypothesis related to hypersensitivity reactions, trying to individuate structural requisite that can be involved in immune mediated adverse reactions. Copyright © 2015 Elsevier Ltd. All rights reserved.

Genetic susceptibility of newborn daughters to oxidative stress

DEFF Research Database (Denmark)

Decordier, Ilse; De Bont, Kelly; De Bock, Kirsten

2007-01-01

A central question in risk assessment is whether newborns' susceptibility to mutagens is different from that of adults. Therefore we investigated whether genotype and/or the DNA strand break repair phenotype in combination with the MN assay would allow estimation of the relative sensitivity of a ...

The Role of Abcb5 Alleles in Susceptibility to Haloperidol-Induced Toxicity in Mice and Humans

Science.gov (United States)

Zheng, Ming; Zhang, Haili; Dill, David L.; Clark, J. David; Tu, Susan; Yablonovitch, Arielle L.; Tan, Meng How; Zhang, Rui; Rujescu, Dan; Wu, Manhong; Tessarollo, Lino; Vieira, Wilfred; Gottesman, Michael M.; Deng, Suhua; Eberlin, Livia S.; Zare, Richard N.; Billard, Jean-Martin; Gillet, Jean-Pierre; Li, Jin Billy; Peltz, Gary

2015-01-01

Background We know very little about the genetic factors affecting susceptibility to drug-induced central nervous system (CNS) toxicities, and this has limited our ability to optimally utilize existing drugs or to develop new drugs for CNS disorders. For example, haloperidol is a potent dopamine antagonist that is used to treat psychotic disorders, but 50% of treated patients develop characteristic extrapyramidal symptoms caused by haloperidol-induced toxicity (HIT), which limits its clinical utility. We do not have any information about the genetic factors affecting this drug-induced toxicity. HIT in humans is directly mirrored in a murine genetic model, where inbred mouse strains are differentially susceptible to HIT. Therefore, we genetically analyzed this murine model and performed a translational human genetic association study. Methods and Findings A whole genome SNP database and computational genetic mapping were used to analyze the murine genetic model of HIT. Guided by the mouse genetic analysis, we demonstrate that genetic variation within an ABC-drug efflux transporter (Abcb5) affected susceptibility to HIT. In situ hybridization results reveal that Abcb5 is expressed in brain capillaries, and by cerebellar Purkinje cells. We also analyzed chromosome substitution strains, imaged haloperidol abundance in brain tissue sections and directly measured haloperidol (and its metabolite) levels in brain, and characterized Abcb5 knockout mice. Our results demonstrate that Abcb5 is part of the blood-brain barrier; it affects susceptibility to HIT by altering the brain concentration of haloperidol. Moreover, a genetic association study in a haloperidol-treated human cohort indicates that human ABCB5 alleles had a time-dependent effect on susceptibility to individual and combined measures of HIT. Abcb5 alleles are pharmacogenetic factors that affect susceptibility to HIT, but it is likely that additional pharmacogenetic susceptibility factors will be discovered

The role of Abcb5 alleles in susceptibility to haloperidol-induced toxicity in mice and humans.

KAUST Repository

Zheng, Ming

2015-02-03

We know very little about the genetic factors affecting susceptibility to drug-induced central nervous system (CNS) toxicities, and this has limited our ability to optimally utilize existing drugs or to develop new drugs for CNS disorders. For example, haloperidol is a potent dopamine antagonist that is used to treat psychotic disorders, but 50% of treated patients develop characteristic extrapyramidal symptoms caused by haloperidol-induced toxicity (HIT), which limits its clinical utility. We do not have any information about the genetic factors affecting this drug-induced toxicity. HIT in humans is directly mirrored in a murine genetic model, where inbred mouse strains are differentially susceptible to HIT. Therefore, we genetically analyzed this murine model and performed a translational human genetic association study.A whole genome SNP database and computational genetic mapping were used to analyze the murine genetic model of HIT. Guided by the mouse genetic analysis, we demonstrate that genetic variation within an ABC-drug efflux transporter (Abcb5) affected susceptibility to HIT. In situ hybridization results reveal that Abcb5 is expressed in brain capillaries, and by cerebellar Purkinje cells. We also analyzed chromosome substitution strains, imaged haloperidol abundance in brain tissue sections and directly measured haloperidol (and its metabolite) levels in brain, and characterized Abcb5 knockout mice. Our results demonstrate that Abcb5 is part of the blood-brain barrier; it affects susceptibility to HIT by altering the brain concentration of haloperidol. Moreover, a genetic association study in a haloperidol-treated human cohort indicates that human ABCB5 alleles had a time-dependent effect on susceptibility to individual and combined measures of HIT. Abcb5 alleles are pharmacogenetic factors that affect susceptibility to HIT, but it is likely that additional pharmacogenetic susceptibility factors will be discovered.ABCB5 alleles alter susceptibility to

The Predictive Role of Difficulties in Emotion Regulation and Self-Control with Susceptibility to Addiction in Drug-Dependent Individuals

Directory of Open Access Journals (Sweden)

Mahmoud Shirazi

2015-06-01

Full Text Available Objective: The present study aimed to examine the predictive role of difficulties in emotion regulation and self-control in potential for addiction among drug-dependent individuals. Method: This was a correlational study which falls within the category of descriptive studies. The statistical population of the current study included all patients under treatment in outpatient health centers in Bam, among whom 315 individuals were selected through cluster sampling method as the participants of the study. Difficulties in Emotion Regulation Scale, Self-Control Scale, and Addiction Susceptibility Questionnaire were used for data collection purposes. Results: The results indicated that difficulties engaging in goal directed behavior, impulse control difficulties, lack of emotional awareness, and lack of emotional clarity (dimensions of difficulties in emotion regulation had a significant positive correlation with potential for addiction. In addition, there was a negative significant relationship between self-control and potential for addiction among drug-dependent individuals. Conclusion: In addition to common methods of abstinence from drug dependence, teaching self-control and emotional control techniques to addicted patients can help them reduce their dependence.

Rapid genotyping assays for the 4-base pair deletion of canine MDR1/ABCB1 gene and low frequency of the mutant allele in Border Collie dogs.

Science.gov (United States)

Mizukami, Keijiro; Chang, Hye-Sook; Yabuki, Akira; Kawamichi, Takuji; Hossain, Mohammad A; Rahman, Mohammad M; Uddin, Mohammad M; Yamato, Osamu

2012-01-01

P-glycoprotein, encoded by the MDR1 or ABCB1 gene, is an integral component of the blood-brain barrier as an efflux pump for xenobiotics crucial in limiting drug uptake into the central nervous system. Dogs homozygous for a 4-base pair deletion of the canine MDR1 gene show altered expression or function of P-glycoprotein, resulting in neurotoxicosis after administration of the substrate drugs. In the present study, the usefulness of microchip electrophoresis for genotyping assays detecting this deletion mutation was evaluated. Mutagenically separated polymerase chain reaction (MS-PCR) and real-time PCR assays were newly developed and evaluated. Furthermore, a genotyping survey was carried out in a population of Border Collies dogs in Japan to determine the allele frequency in this breed. Microchip electrophoresis showed advantages in detection sensitivity and time saving over other modes of electrophoresis. The MS-PCR assay clearly discriminated all genotypes. Real-time PCR assay was most suitable for a large-scale survey due to its high throughput and rapidity. The genotyping survey demonstrated that the carrier and mutant allele frequencies were 0.49% and 0.25%, respectively, suggesting that the mutant allele frequency in Border Collies is markedly low compared to that in the susceptible dog breeds such as rough and smooth Collies.

21 CFR 864.7470 - Glycosylated hemoglobin assay.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Glycosylated hemoglobin assay. 864.7470 Section 864.7470 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7470...

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.

Development and validation of cell-based luciferase reporter gene assays for measuring neutralizing anti-drug antibodies against interferon beta.

Science.gov (United States)

Hermanrud, Christina; Ryner, Malin; Luft, Thomas; Jensen, Poul Erik; Ingenhoven, Kathleen; Rat, Dorothea; Deisenhammer, Florian; Sørensen, Per Soelberg; Pallardy, Marc; Sikkema, Dan; Bertotti, Elisa; Kramer, Daniel; Creeke, Paul; Fogdell-Hahn, Anna

2016-03-01

Neutralizing anti-drug antibodies (NAbs) against therapeutic interferon beta (IFNβ) in people with multiple sclerosis (MS) are measured with cell-based bioassays. The aim of this study was to redevelop and validate two luciferase reporter-gene bioassays, LUC and iLite, using a cut-point approach to identify NAb positive samples. Such an approach is favored by the pharmaceutical industry and governmental regulatory agencies as it has a clear statistical basis and overcomes the limitations of the current assays based on the Kawade principle. The work was conducted following the latest assay guidelines. The assays were re-developed and validated as part of the "Anti-Biopharmaceutical Immunization: Prediction and analysis of clinical relevance to minimize the risk" (ABIRISK) consortium and involved a joint collaboration between four academic laboratories and two pharmaceutical companies. The LUC assay was validated at Innsbruck Medical University (LUCIMU) and at Rigshospitalet (LUCRH) Copenhagen, and the iLite assay at Karolinska Institutet, Stockholm. For both assays, the optimal serum sample concentration in relation to sensitivity and recovery was 2.5% (v/v) in assay media. A Shapiro-Wilk test indicated a normal distribution for the majority of runs, allowing a parametric approach for cut-point calculation to be used, where NAb positive samples could be identified with 95% confidence. An analysis of means and variances indicated that a floating cut-point should be used for all assays. The assays demonstrated acceptable sensitivity for being cell-based assays, with a confirmed limit of detection in neat serum of 1519 ng/mL for LUCIMU, 814 ng/mL for LUCRH, and 320 ng/mL for iLite. Use of the validated cut-point assay, in comparison with the previously used Kawade method, identified 14% more NAb positive samples. In conclusion, implementation of the cut-point design resulted in increased sensitivity to detect NAbs. However, the clinical significance of these low

Antimicrobial susceptibility among clinical Nocardia species identified by multilocus sequence analysis.

Science.gov (United States)

McTaggart, Lisa R; Doucet, Jennifer; Witkowska, Maria; Richardson, Susan E

2015-01-01

Antimicrobial susceptibility patterns of 112 clinical isolates, 28 type strains, and 9 reference strains of Nocardia were determined using the Sensititre Rapmyco microdilution panel (Thermo Fisher, Inc.). Isolates were identified by highly discriminatory multilocus sequence analysis and were chosen to represent the diversity of species recovered from clinical specimens in Ontario, Canada. Susceptibility to the most commonly used drug, trimethoprim-sulfamethoxazole, was observed in 97% of isolates. Linezolid and amikacin were also highly effective; 100% and 99% of all isolates demonstrated a susceptible phenotype. For the remaining antimicrobials, resistance was species specific with isolates of Nocardia otitidiscaviarum, N. brasiliensis, N. abscessus complex, N. nova complex, N. transvalensis complex, N. farcinica, and N. cyriacigeorgica displaying the traditional characteristic drug pattern types. In addition, the antimicrobial susceptibility profiles of a variety of rarely encountered species isolated from clinical specimens are reported for the first time and were categorized into four additional drug pattern types. Finally, MICs for the control strains N. nova ATCC BAA-2227, N. asteroides ATCC 19247(T), and N. farcinica ATCC 23826 were robustly determined to demonstrate method reproducibility and suitability of the commercial Sensititre Rapmyco panel for antimicrobial susceptibility testing of Nocardia spp. isolated from clinical specimens. The reported values will facilitate quality control and standardization among laboratories. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

21 CFR 864.7415 - Abnormal hemoglobin assay.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Abnormal hemoglobin assay. 864.7415 Section 864.7415 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7415 Abnormal...

21 CFR 864.7375 - Glutathione reductase assay.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Glutathione reductase assay. 864.7375 Section 864.7375 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7375 Glutathione...

21 CFR 864.7060 - Antithrombin III assay.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Antithrombin III assay. 864.7060 Section 864.7060 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7060 Antithrombin III...

21 CFR 864.7455 - Fetal hemoglobin assay.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Fetal hemoglobin assay. 864.7455 Section 864.7455 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7455 Fetal hemoglobin...

Kinase profiling of liposarcomas using RNAi and drug screening assays identified druggable targets

Directory of Open Access Journals (Sweden)

Deepika Kanojia

2017-11-01

Full Text Available Abstract Background Liposarcoma, the most common soft tissue tumor, is understudied cancer, and limited progress has been made in the treatment of metastatic disease. The Achilles heel of cancer often is their kinases that are excellent therapeutic targets. However, very limited knowledge exists of therapeutic critical kinase targets in liposarcoma that could be potentially used in disease management. Methods Large RNAi and small-molecule tyrosine kinase inhibitor screens were performed against the proliferative capacity of liposarcoma cell lines of different subtypes. Each small molecule inhibitor was either FDA approved or in a clinical trial. Results Screening assays identified several previously unrecognized targets including PTK2 and KIT in liposarcoma. We also observed that ponatinib, multi-targeted tyrosine kinase inhibitor, was the most effective drug with anti-growth effects against all cell lines. In vitro assays showed that ponatinib inhibited the clonogenic proliferation of liposarcoma, and this anti-growth effect was associated with apoptosis and cell cycle arrest at the G0/G1 phase as well as a decrease in the KIT signaling pathway. In addition, ponatinib inhibited in vivo growth of liposarcoma in a xenograft model. Conclusions Two large-scale kinase screenings identified novel liposarcoma targets and a FDA-approved inhibitor, ponatinib with clear anti-liposarcoma activity highlighting its potential therapy for treatment of this deadly tumor.

Comparison of cell-based and non-cell-based assay platforms for the detection of clinically relevant anti-drug neutralizing antibodies for immunogenicity assessment of therapeutic proteins.

Science.gov (United States)

Hu, Jenny; Wala, Iwona; Han, Hong; Nagatani, Janice; Barger, Troy; Civoli, Francesca; Kaliyaperumal, Arunan; Zhuang, Yao; Gupta, Shalini

2015-04-01

Anti-drug neutralizing antibodies (NAbs) formed due to unwanted immunogenicity of a therapeutic protein point towards a mature immune response. NAb detection is important in interpreting the therapeutic's efficacy and safety in vivo. In vitro cell-based NAb assays provide a physiological system for NAb detection, however are complex assays. Non-cell-based competitive ligand binding (CLB) approaches are also employed for NAb detection. Instead of cells, CLB assays use soluble receptor and conjugated reagents and are easier to perform, however have reduced physiological relevance. The aim of this study was to compare the performance of CLB assays to established cell-based assays to determine the former's ability to detect clinically relevant NAbs towards therapeutics that (i) acted as an agonist or (ii) acted as antagonists by binding to a target receptor. We performed a head-to-head comparison of the performance of cell-based and CLB NAb assays for erythropoietin (EPO) and two anti-receptor monoclonal antibodies (AMG-X and AMG 317). Clinically relevant NAb-positive samples identified previously by a cell-based assay were assessed in the corresponding CLB format(s). A panel of 12 engineered fully human anti-EPO monoclonal antibodies (MAbs) was tested in both EPO NAb assay formats. Our results showed that the CLB format was (i) capable of detecting human anti-EPO MAbs of differing neutralizing capabilities and affinities and (ii) provided similar results as the cell-based assay for detecting NAbs in patient samples. The cell-based and CLB assays also behaved comparably in detecting NAbs in clinical samples for AMG-X. In the case of anti-AMG 317 NAbs, the CLB format failed to detect NAbs in more than 50% of the tested samples. We conclude that assay sensitivity, drug tolerance and the selected assay matrix played an important role in the inability of AMG 317 CLB assays to detect clinically relevant NAbs. Copyright © 2015 Elsevier B.V. All rights reserved.

"From safe source to safe sink" development of colorimetric assay for gabapentin in bulk drug and capsules using naturally derived genipin.

Science.gov (United States)

Winotapun, Weerapath; Kongpakwattana, Khachen; Dejpittayanunt, Sirirat; Pathomcharoensukchai, Suwaparp; Suksaran, Udomluck; Nuntharatanapong, Nopparat; Rojanarata, Theerasak

2012-09-15

A novel colorimetric assay for gabapentin in bulk drug and capsules has been developed via a safety-and-sustainability concerning concept. The method relied on the reaction of primary amino group of drug with non-toxic and eco-friendly genipin in totally aqueous medium to form the blue product which was subsequently measured by visible spectrophotometry at 590 nm. Under the optimized conditions, Beer's law was obeyed in the concentration range of 0.15-0.50 mM (r(2)=0.9998). It was accurate, precise and insensitive to the interferences from all related compounds specified in the United States Pharmacopeia as well as commonly used excipients. Furthermore, it gave the assay results in agreement with the pharmacopeial chromatographic method. Owing to the environmental concern and responsibility, a fast and facile method was also proposed for the treatment of waste generated from the assay based on the decoloration by using gypsum as a cheap and commonly available adsorbent. After the treatment, more than 95% of the initial blue product was removed from the waste solution and the treated waste was proven to be safe for aquatic organisms, as studied in brine shrimp and guppy fishes. Therefore, this work not only reports for the first time the application of naturally derived genipin to drug analysis, but also presents a new and contemporary paradigm that illustrates the fully benign-by-design development of the analytical methodologies in the era of Green Chemistry, starting from the safe source of reagents toward the safe sink when waste is released into the environment. Copyright © 2012 Elsevier B.V. All rights reserved.

Etest Cannot Be Recommended for In Vitro Susceptibility Testing of Mucorales

OpenAIRE

Caramalho, Rita; Maurer, Elisabeth; Binder, Ulrike; Araújo, Ricardo; Dolatabadi, Somayeh; Lass-Flörl, Cornelia; Lackner, Michaela

2015-01-01

Amphotericin B and posaconazole susceptibility patterns were determined for the most prevalent Mucorales, following EUCAST (European Committee on Antimicrobial Susceptibility Testing) broth microdilution guidelines. In parallel, Etest was performed and evaluated against EUCAST. The overall agreement of MICs gained with Etest and EUCAST was 75.1%; therefore, Etest cannot be recommended for antifungal susceptibility testing of Mucorales. Amphotericin B was the most active drug against Mucorales...

Culture and drug susceptibility testing among previously treated tuberculosis patients in the Dominican Republic, 2014

Directory of Open Access Journals (Sweden)

Katia J. Romero Mercado

Full Text Available ABSTRACT Multidrug-resistant tuberculosis (MDR-TB is a major public health concern that threatens global progress toward effective TB control. The risk of MDR-TB is increased in patients who have received previous TB treatment. This article describes the performance of culture and drug susceptibility testing (DST in patients registered as previously treated TB patients in the Dominican Republic in 2014, based on operational research that followed a retrospective cohort design and used routine program data. Under the current system of TB culturing and DST, the majority of patients with previously treated TB do not undergo DST, and those who do often experience considerable delay in obtaining their results. The lack of DST and delay in receiving DST results leads to underestimation of the number of MDR-TB cases and hinders the timely initiation of MDR-TB treatment.

Drugs + HIV, Learn the Link

Medline Plus

Full Text Available ... risk for getting HIV. Drug and alcohol intoxication affect judgment and can lead to unsafe sexual practices, ... effects of drugs. Drug misuse and addiction can affect a person's overall health, thereby altering susceptibility to ...

21 CFR 866.1700 - Culture medium for antimicrobial susceptibility tests.

Science.gov (United States)

2010-04-01

... tests. 866.1700 Section 866.1700 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Diagnostic Devices § 866.1700 Culture medium for antimicrobial susceptibility tests. (a) Identification. A culture medium for...

Development of an in vitro cytochrome P450 cocktail inhibition assay for assessing the inhibition risk of drugs of abuse.

Science.gov (United States)

Dinger, Julia; Meyer, Markus R; Maurer, Hans H

2014-10-01

Drugs of abuse are not tested for cytochrome P450 (CYP) inhibition potential before distribution. Therefore, a cocktail assay should be developed for testing the inhibition potential for all relevant CYPs. The following CYP test substrates and selective inhibitors were incubated in pooled human liver microsomes: phenacetin (alpha-naphthoflavone for CYP1A2), coumarin (tranylcypromine, CYP2A6), bupropion (sertraline, CYP2B6), amodiaquine (trimethoprim, CYP2C8), diclofenac (sulfaphenazole, CYP2C9), omeprazole (fluconazole, CYP2C19), dextromethorphan (quinidine, CYP2D6), chlorzoxazone (clomethiazole, CYP2E1), testosterone (verapamil, CYP3A). Samples were analyzed after protein precipitation using a Thermo Fisher Q-Exactive LC-high-resolution-MS/MS. The IC50 values were calculated by plotting the concentration of the formed metabolite, relative to the control sample, over the logarithm of the inhibitor concentration. They were determined either for single substrate or the cocktail incubation. Unfortunately, the cocktail assay had to be split because of interferences during incubation caused by substrates or metabolites, but the mixture of both incubates could be analyzed in one analytical run. The IC50 values determined in the single substrate or both cocktail incubations were comparable among themselves and with published data. In conclusion, the new inhibition cocktail assay was reproducible and applicable for testing the inhibition potential of drugs of abuse as exemplified for 2,5-dimethoxy-4-iodo-amfetamine (DOI). Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Rapid antibiotic susceptibility testing of Mycobacterium tuberculosis : Its utility in resource poor settings

Directory of Open Access Journals (Sweden)

Poojary A

2006-01-01

Full Text Available Purpose: To compare the rapid colorimetric nitrate reductase based antibiotic susceptibility (CONRAS test performed on Mycobacterium tuberculosis isolates with the conventional method i.e., the proportion method. Methods: One hundred clinical isolates of M. tuberculosis were tested for susceptibility to isoniazid (INH and rifampicin (RIF by the conventional proportion method and CONRAS in Middlebrook 7H9 liquid medium enriched with growth supplements (MB7H9S. Results: The performance of the CONRAS test was evaluated using proportion method as the gold standard. The sensitivity (ability to detect true drug resistance and specificity (ability to detect true drug susceptibility of the CONRAS test to INH was 93.75 and 98.52% and for RIF it was 96.10 and 100% respectively. The mean time for reporting was 6.3 days and the test showed excellent reproducibility. The kappa (k value for INH was 0.92 and for RIF was 0.99, indicating excellent agreement between the two methods. Conclusions: CONRAS test is a rapid and reliable method of drug susceptibility for M. tuberculosis.

Agar dilution method for susceptibility testing of Neisseria gonorrhoeae

Directory of Open Access Journals (Sweden)

Marta C de Castillo

1996-12-01

Full Text Available The antibiotic susceptibilities of Neisseria gonorrhoeae isolates obtained from patients attending a clinic for sexually transmitted diseases in Tucumán, Argentina, were determined by the agar dilution method (MIC. 3.5% of the isolates produced ²-lactamase. A total of 96.5% of ²-lactamase negative isolates tested were susceptible to penicillin (MIC < 2 µgml-1; 14.03% of the tested isolates were resistant to tetracycline (MIC < 2 µgml-1, and 98% of the tested isolates were susceptible to spectinomycin (MIC < 64 µgml-1. The MICs for 95% of the isolates, tested for other drugs were: < 2 µgml-1 for cefoxitin, < 0.06 µgml-1 for cefotaxime, < 0.25 µgml-1 for norfloxacin, < 10 µgml-1 for cephaloridine, < 10 µgml-1 for cephalexin, and < 50 µgml-1 for kanamycin. Antibiotic resistance among N. gonorrhoeae isolates from Tucumán, Argentina, appeared to be primarily limited to penicillin and tetracycline, which has been a general use against gonorrhoeae in Tucumán since 1960. Periodic monitoring of the underlying susceptibility profiles of the N. gonorrhoeae strains prevalent in areas of frequent transmission may provide clues regarding treatment options and emerging of drug resistance.

A systematic study of the effect of low pH acid treatment on anti-drug antibodies specific for a domain antibody therapeutic: Impact on drug tolerance, assay sensitivity and post-validation method assessment of ADA in clinical serum samples.

Science.gov (United States)

Kavita, Uma; Duo, Jia; Crawford, Sean M; Liu, Rong; Valcin, Joan; Gleason, Carol; Dong, Huijin; Gadkari, Snaehal; Dodge, Robert W; Pillutla, Renuka C; DeSilva, Binodh S

2017-09-01

We developed a homogeneous bridging anti-drug antibody (ADA) assay on an electro chemiluminescent immunoassay (ECLIA) platform to support the immunogenicity evaluation of a dimeric domain antibody (dAb) therapeutic in clinical studies. During method development we evaluated the impact of different types of acid at various pH levels on polyclonal and monoclonal ADA controls of differing affinities and on/off rates. The data shows for the first time that acids of different pH can have a differential effect on ADA of various affinities and this in turn impacts assay sensitivity and drug tolerance as defined by these surrogate controls. Acid treatment led to a reduction in signal of intermediate and low affinity ADA, but not high affinity or polyclonal ADA. We also found that acid pretreatment is a requisite for dissociation of drug bound high affinity ADA, but not for low affinity ADA-drug complexes. Although we were unable to identify an acid that would allow a 100% retrieval of ADA signal post-treatment, use of glycine pH3.0 enabled the detection of low, intermediate and high affinity antibodies (Abs) to various extents. Following optimization, the ADA assay method was validated for clinical sample analysis. Consistencies within various parameters of the clinical data such as dose dependent increases in ADA rates and titers were observed, indicating a reliable ADA method. Pre- and post-treatment ADA negative or positive clinical samples without detectable drug were reanalyzed in the absence of acid treatment or presence of added exogenous drug respectively to further assess the effectiveness of the final acid treatment procedure. The overall ADA results indicate that assay conditions developed and validated based on surrogate controls sufficed to provide a reliable clinical data set. The effect of low pH acid treatment on possible pre-existing ADA or soluble multimeric target in normal human serum was also evaluated, and preliminary data indicate that acid type and

The susceptibility of circulating human influenza viruses to tizoxanide, the active metabolite of nitazoxanide.

Science.gov (United States)

Tilmanis, Danielle; van Baalen, Carel; Oh, Ding Yuan; Rossignol, Jean-Francois; Hurt, Aeron C

2017-11-01

Nitazoxanide is a thiazolide compound that was originally developed as an anti-parasitic agent, but has recently been repurposed for the treatment of influenza virus infections. Thought to exert its anti-influenza activity via the inhibition of hemagglutinin maturation and intracellular trafficking in infected cells, the effectiveness of nitazoxanide in treating patients with non-complicated influenza is currently being assessed in phase III clinical trials. Here, we describe the susceptibility of 210 seasonal influenza viruses to tizoxanide, the active circulating metabolite of nitazoxanide. An optimised cell culture-based focus reduction assay was used to determine the susceptibility of A(H1N1)pdm09, A(H3N2), and influenza B viruses circulating in the southern hemisphere from the period March 2014 to August 2016. Tizoxanide showed potent in vitro antiviral activity against all influenza viruses tested, including neuraminidase inhibitor-resistant viruses, allowing the establishment of a baseline level of susceptibility for each subtype. Median EC 50 values (±IQR) of 0.48 μM (0.33-0.71), 0.62 μM (0.56-0.75), 0.66 μM (0.62-0.69), and 0.60 μM (0.51-0.67) were obtained for A(H1N1)pdm09, A(H3N2), B(Victoria lineage), and B(Yamagata lineage) influenza viruses respectively. There was no significant difference in the median baseline tizoxanide susceptibility for each influenza subtype tested. This is the first report on the susceptibility of circulating viruses to tizoxanide. The focus reduction assay format described is sensitive, robust, and less laborious than traditional cell based antiviral assays, making it highly suitable for the surveillance of tizoxanide susceptibility in circulating seasonal influenza viruses. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Antifungal susceptibility profiles of 1698 yeast reference strains revealing potential emerging human pathogens.

Directory of Open Access Journals (Sweden)

Marie Desnos-Ollivier

Full Text Available New molecular identification techniques and the increased number of patients with various immune defects or underlying conditions lead to the emergence and/or the description of novel species of human and animal fungal opportunistic pathogens. Antifungal susceptibility provides important information for ecological, epidemiological and therapeutic issues. The aim of this study was to assess the potential risk of the various species based on their antifungal drug resistance, keeping in mind the methodological limitations. Antifungal susceptibility profiles to the five classes of antifungal drugs (polyens, azoles, echinocandins, allylamines and antimetabolites were determined for 1698 yeast reference strains belonging to 992 species (634 Ascomycetes and 358 Basidiomycetes. Interestingly, geometric mean minimum inhibitory concentrations (MICs of all antifungal drugs tested were significantly higher for Basidiomycetes compared to Ascomycetes (p<0.001. Twenty four strains belonging to 23 species of which 19 were Basidiomycetes seem to be intrinsically "resistant" to all drugs. Comparison of the antifungal susceptibility profiles of the 4240 clinical isolates and the 315 reference strains belonging to 53 shared species showed similar results. Even in the absence of demonstrated in vitro/in vivo correlation, knowing the in vitro susceptibility to systemic antifungal agents and the putative intrinsic resistance of yeast species present in the environment is important because they could become opportunistic pathogens.

21 CFR 866.3305 - Herpes simplex virus serological assays.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Herpes simplex virus serological assays. 866.3305... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3305 Herpes simplex virus serological assays. (a) Identification. Herpes simplex virus serological assays are devices...

Spectrum and the In Vitro Antifungal Susceptibility Pattern of Yeast Isolates in Ethiopian HIV Patients with Oropharyngeal Candidiasis.

Science.gov (United States)

Moges, Birhan; Bitew, Adane; Shewaamare, Aster

2016-01-01

Background. In Ethiopia, little is known regarding the distribution and the in vitro antifungal susceptibility profile of yeasts. Objective. This study was undertaken to determine the spectrum and the in vitro antifungal susceptibility pattern of yeasts isolated from HIV infected patients with OPC. Method. Oral pharyngeal swabs taken from oral lesions of study subjects were inoculated onto Sabouraud Dextrose Agar. Yeasts were identified by employing conventional test procedures and the susceptibility of yeasts to antifungal agents was evaluated by disk diffusion assay method. Result. One hundred and fifty-five yeast isolates were recovered of which 91 isolates were from patients that were not under HAART and 64 were from patients that were under HAART. C. albicans was the most frequently isolated species followed by C. glabrata, C. tropicalis, C. krusei, C. kefyr, Cryptococcus laurentii, and Rhodotorula species. Irrespective of yeasts isolated and identified, 5.8%, 5.8%, 12.3%, 8.4%, 0.6%, and 1.3% of the isolates were resistant to amphotericin B, clotrimazole, fluconazole, ketoconazole, miconazole, and nystatin, respectively. Conclusion. Yeast colonization rate of 69.2% and 31% resistance to six antifungal agents was documented. These highlight the need for nationwide study on the epidemiology of OPC and resistance to antifungal drugs.

Spectrum and the In Vitro Antifungal Susceptibility Pattern of Yeast Isolates in Ethiopian HIV Patients with Oropharyngeal Candidiasis

Directory of Open Access Journals (Sweden)

Birhan Moges

2016-01-01

Full Text Available Background. In Ethiopia, little is known regarding the distribution and the in vitro antifungal susceptibility profile of yeasts. Objective. This study was undertaken to determine the spectrum and the in vitro antifungal susceptibility pattern of yeasts isolated from HIV infected patients with OPC. Method. Oral pharyngeal swabs taken from oral lesions of study subjects were inoculated onto Sabouraud Dextrose Agar. Yeasts were identified by employing conventional test procedures and the susceptibility of yeasts to antifungal agents was evaluated by disk diffusion assay method. Result. One hundred and fifty-five yeast isolates were recovered of which 91 isolates were from patients that were not under HAART and 64 were from patients that were under HAART. C. albicans was the most frequently isolated species followed by C. glabrata, C. tropicalis, C. krusei, C. kefyr, Cryptococcus laurentii, and Rhodotorula species. Irrespective of yeasts isolated and identified, 5.8%, 5.8%, 12.3%, 8.4%, 0.6%, and 1.3% of the isolates were resistant to amphotericin B, clotrimazole, fluconazole, ketoconazole, miconazole, and nystatin, respectively. Conclusion. Yeast colonization rate of 69.2% and 31% resistance to six antifungal agents was documented. These highlight the need for nationwide study on the epidemiology of OPC and resistance to antifungal drugs.

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.

Microfluidics for Antibiotic Susceptibility and Toxicity Testing

Directory of Open Access Journals (Sweden)

Jing Dai

2016-10-01

Full Text Available The recent emergence of antimicrobial resistance has become a major concern for worldwide policy makers as very few new antibiotics have been developed in the last twenty-five years. To prevent the death of millions of people worldwide, there is an urgent need for a cheap, fast and accurate set of tools and techniques that can help to discover and develop new antimicrobial drugs. In the past decade, microfluidic platforms have emerged as potential systems for conducting pharmacological studies. Recent studies have demonstrated that microfluidic platforms can perform rapid antibiotic susceptibility tests to evaluate antimicrobial drugs’ efficacy. In addition, the development of cell-on-a-chip and organ-on-a-chip platforms have enabled the early drug testing, providing more accurate insights into conventional cell cultures on the drug pharmacokinetics and toxicity, at the early and cheaper stage of drug development, i.e., prior to animal and human testing. In this review, we focus on the recent developments of microfluidic platforms for rapid antibiotics susceptibility testing, investigating bacterial persistence and non-growing but metabolically active (NGMA bacteria, evaluating antibiotic effectiveness on biofilms and combinatorial effect of antibiotics, as well as microfluidic platforms that can be used for in vitro antibiotic toxicity testing.

SEIIrR: Drug abuse model with rehabilitation

Science.gov (United States)

Sutanto, Azizah, Afina; Widyaningsih, Purnami; Saputro, Dewi Retno Sari

2017-05-01

Drug abuse in the world quite astonish and tend to increase. The increase and decrease on the number of drug abusers showed a pattern of spread that had the same characteristics with patterns of spread of infectious disease. The susceptible infected removed (SIR) and susceptible exposed infected removed (SEIR) epidemic models for infectious disease was developed to study social epidemic. In this paper, SEIR model for disease epidemic was developed to study drug abuse epidemic with rehabilitation treatment. The aims of this paper were to analogize susceptible exposed infected isolated recovered (SEIIrR) model on the drug abusers, to determine solutions of the model, to determine equilibrium point, and to do simulation on β. The solutions of SEIIrR model was determined by using fourth order of Runge-Kutta algorithm, equilibrium point obtained was free-drug equilibrium point. Solutions of SEIIrR showed that the model was able to suppress the spread of drug abuse. The increasing value of contact rate was not affect the number of infected individuals due to rehabilitation treatment.

Medical Devices; Immunology and Microbiology Devices; Classification of the Assayed Quality Control Material for Clinical Microbiology Assays. Final order.

Science.gov (United States)

2017-07-27

The Food and Drug Administration (FDA, Agency, or we) is classifying the assayed quality control material for clinical microbiology assays into class II (special controls). The special controls that will apply to the device are identified in this order and will be part of the codified language for the assayed quality control material for clinical microbiology assays' classification. The Agency is classifying the device into class II (special controls) to provide a reasonable assurance of safety and effectiveness of the device.

Electrochemical studies of nevirapine, an anti-HIV drug, and its assay in tablets and biological samples

Directory of Open Access Journals (Sweden)

JALDAPPAGARI SEETHARAMAPPA

2012-06-01

Full Text Available The electrochemical oxidation of nevirapine, an anti-HIV drug, at a glassy carbon electrode has been studied by voltammetric techniques. Nevirapine showed one well defined irreversible oxidation peak with a potential of 0.749 V in phosphate buffer at pH 10. The effects of different electrolytes, pH and scan rate on the electrochemical behaviour of nevira¬pine were examined to determine the optimum reaction conditions. The oxidation peak current was found to vary linearly with the concentration of nevirapine in the range of 5.0 – 350 µM. The limit of detection and limit of quantification values were calculated and found to be 1.026 µM and 3.420 µM, respectively. The low relative standard deviation values of inter-day and intra-day assays highlighted the good reproducibility of the proposed m¬ethod for assay of nevirapine. Further, a sensitive and accurate differential pulse voltammetric method was developed for the determination of nevirapine concentrations in pharma¬ceutical formulations.

MS transport assays for γ-aminobutyric acid transporters--an efficient alternative for radiometric assays.

Science.gov (United States)

Schmitt, Sebastian; Höfner, Georg; Wanner, Klaus T

2014-08-05

Transport assays for neurotransmitters based on radiolabeled substrates are widely spread and often indispensable in basic research and the drug development process, although the use of radioisotopes is inherently coupled to issues concerning radioactive waste and safety precautions. To overcome these disadvantages, we developed mass spectrometry (MS)-based transport assays for γ-aminobutyric acid (GABA), which is the major inhibitory neurotransmitter in the central nervous system (CNS). These "MS Transport Assays" provide all capabilities of [(3)H]GABA transport assays and therefore represent the first substitute for the latter. The performance of our approach is demonstrated for GAT1, the most important GABA transporter (GAT) subtype. As GABA is endogenously present in COS-7 cells employed as hGAT1 expression system, ((2)H6)GABA was used as a substrate to differentiate transported from endogenous GABA. To record transported ((2)H6)GABA, a highly sensitive, short, robust, and reliable HILIC-ESI-MS/MS quantification method using ((2)H2)GABA as an internal standard was developed and validated according to the Center for Drug Evaluation and Research (CDER) guidelines. Based on this LC-MS quantification, a setup to characterize hGAT1 mediated ((2)H6)GABA transport in a 96-well format was established, that enables automated processing and avoids any sample preparation. The K(m) value for ((2)H6)GABA determined for hGAT1 is in excellent agreement with results obtained from [(3)H]GABA uptake assays. In addition, the established assay format enables efficient determination of the inhibitory potency of GAT1 inhibitors, is capable of identifying those inhibitors transported as substrates, and furthermore allows characterization of efflux. The approach described here combines the strengths of LC-MS/MS with the high efficiency of transport assays based on radiolabeled substrates and is applicable to all GABA transporter subtypes.

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

Directory of Open Access Journals (Sweden)

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.

[Susceptibilities of Escherichia coli, Salmonella and Staphylococcus aureus isolated from animals to ofloxacin and commonly used antimicrobial agents].

Science.gov (United States)

Takahashi, I; Yoshida, T; Higashide, Y; Sakano, T

1990-01-01

Susceptibilities of Escherichia coli, Salmonella and Staphylococcus aureus isolated from chickens, pigs and cattle to ofloxacin (OFLX) and commonly used antimicrobial agents were investigated. 1. E. coli (28 isolates) demonstrated the highest level of susceptibility of OFLX (MIC 0.10-0.39 micrograms/ml for all the isolates) among all the test drugs. Commonly used antimicrobial agents to which these isolates responded with relatively high susceptibilities (MIC50 0.78-6.25 micrograms/ml) included oxolinic acid (OXA), ampicillin (ABPC), kanamycin (KM) and chloramphenicol (CP) with their MIC50 values in the increasing order as above. Drugs to which these isolates responded with moderate to weak susceptibilities (MIC50 25 approximately greater than 800 micrograms/ml) were doxycycline (DOXY), streptomycin (SM), spectinomycin (SPCM) and sulfadimethoxine (SDMX) in the increasing order of MIC50. E. coli isolates with resistances to all the test drugs other than OFLX and OXA amounted to 7.1-57.1% of the isolates examined and 20 isolates (71.4%) in total. 2. Susceptibilities to OFLX and 4 existing pyridonecarboxylic acid derivatives of E. coli (48 samples) isolated recently from diarrheal pigs were compared. When evaluated in terms of MIC50, the values of OFLX and norfloxacin were both 0.10 micrograms/ml. The values increased by differences of 0.39-3.13 micrograms/ml in an order of OXA, pipemidic acid and nalidixic acid. 3. Salmonella (28 isolates) demonstrated the highest level of susceptibility to OFLX (MIC 0.20-0.39 micrograms/ml for all the isolates) among all the test drugs. The drugs to which these isolates responded with relatively high to moderate susceptibilities (MIC50 0.78-12.5 micrograms/ml) included ABPC, OXA, DOXY, KM, CP and SM with their MIC50 values increasing in this order. The drugs to which the isolates responded with low susceptibilities (MIC50 above 100 micrograms/ml) were SPCM and SDMX. Of all the 28 Salmonella isolates tested, 7.1-32.1% were resistant

The Candida albicans Ddr48 protein is essential for filamentation, stress response, and confers partial antifungal drug resistance.

Science.gov (United States)

Dib, Leila; Hayek, Peter; Sadek, Helen; Beyrouthy, Berna; Khalaf, Roy A

2008-06-01

Candida albicans is a dimorphic pathogenic fungus that causes mucosal and systemic infections. C. albicans pathogenicity is attributed to its ability to exist in different morphologic states and to respond to stress by up regulating several key genes. DDR48 is a stress-associated gene involved in DNA repair and in response to antifungal drug exposure. One allele of DDR48 was knocked out by homologous recombination that inserted a marker cassette in its position. Furthermore, reintroducing DDR48 on a plasmid created a revertant strain. Strains were grown on filamentation inducing and noninducing media, subjected to an oxidative stress challenge, injected into mice to assess virulence, and assayed for antifungal susceptibility by the E-test method. DDR48 was found to be haploid insufficient and possibly essential, since only a heterozygote, but not a homozygous, null mutant was generated. The mutant was filamentation defective on all hyphal media tested including serum and corn meal agar. Discrepancies in drug resistance profiles also were present: compared with the parental strain, DDR48/ddr48 heterozygote strain was susceptible in a dose-dependent manner to itraconazole and fluconazole and susceptible to ketoconazole. The mutant also appeared to be hypersensitive to a potentially lethal hydrogen peroxide challenge. However, no reduction in virulence of the mutant was observed. The present findings provide evidence that DDR48 is essential for filamentation, stress response, and possibly viability of C. albicans, making it a prime target for antifungal drug design.

PknB remains an essential and a conserved target for drug development in susceptible and MDR strains of M. Tuberculosis.

Science.gov (United States)

Gupta, Anamika; Pal, Sudhir K; Pandey, Divya; Fakir, Najneen A; Rathod, Sunita; Sinha, Dhiraj; SivaKumar, S; Sinha, Pallavi; Periera, Mycal; Balgam, Shilpa; Sekar, Gomathi; UmaDevi, K R; Anupurba, Shampa; Nema, Vijay

2017-08-18

The Mycobacterium tuberculosis (M.tb) protein kinase B (PknB) which is now proved to be essential for the growth and survival of M.tb, is a transmembrane protein with a potential to be a good drug target. However it is not known if this target remains conserved in otherwise resistant isolates from clinical origin. The present study describes the conservation analysis of sequences covering the inhibitor binding domain of PknB to assess if it remains conserved in susceptible and resistant clinical strains of mycobacteria picked from three different geographical areas of India. A total of 116 isolates from North, South and West India were used in the study with a variable profile of their susceptibilities towards streptomycin, isoniazid, rifampicin, ethambutol and ofloxacin. Isolates were also spoligotyped in order to find if the conservation pattern of pknB gene remain consistent or differ with different spoligotypes. The impact of variation as found in the study was analyzed using Molecular dynamics simulations. The sequencing results with 115/116 isolates revealed the conserved nature of pknB sequences irrespective of their susceptibility status and spoligotypes. The only variation found was in one strains wherein pnkB sequence had G to A mutation at 664 position translating into a change of amino acid, Valine to Isoleucine. After analyzing the impact of this sequence variation using Molecular dynamics simulations, it was observed that the variation is causing no significant change in protein structure or the inhibitor binding. Hence, the study endorses that PknB is an ideal target for drug development and there is no pre-existing or induced resistance with respect to the sequences involved in inhibitor binding. Also if the mutation that we are reporting for the first time is found again in subsequent work, it should be checked with phenotypic profile before drawing the conclusion that it would affect the activity in any way. Bioinformatics analysis in our study

Co-trimoxazole susceptibility tests improved with separate trimethoprim and sulfamethoxazole disks.

OpenAIRE

Amyes, S G

1981-01-01

It is impossible to test accurately bacterial susceptibility to the trimethoprim-sulfamethoxazole combination co-trimoxazole with a single combined susceptibility disk. However, a variety of factors still affect the result even when separate trimethoprim and sulfamethoxazole disks are used. Experiments with separate disks showed that the optimum conditions for testing the susceptibilities of enterobacteria to these drugs were to flood-seed an agar plate with an inoculum of 10(4) to 10(5) orga...

Drug susceptibility of Mycobacterium tuberculosis in a rural area of Bangladesh and its relevance to the national treatment regimens.

Science.gov (United States)

Van Deun, A; Aung, K J; Chowdhury, S; Saha, S; Pankaj, A; Ashraf, A; Rigouts, L; Fissette, K; Portaels, F

1999-02-01

Greater Mymensingh District, a rural area of Bangladesh, at the start of the National Tuberculosis Programme (NTP). To determine the prevalence of initial and acquired drug resistance of Mycobacterium tuberculosis, and to assess the appropriateness of the NTP's standard regimens. Sampling of pre-treatment sputum from all newly registered smear-positive cases in five centres covering the area. Culture and susceptibility testing in a supra-national reference laboratory. Initial resistance to isoniazid (H) was 5.4%, and to rifampicin (R) 0.5%. Acquired H and R resistance were 25.9% and 7.4%, respectively. Multidrug resistance (MDR) was observed in one new case only and in 5.6% of previously treated patients. Changing the present NTP indication for retreatment regimen to one month of previous H intake would increase coverage of H-resistant cases from 52% to 89%, adding 6% to drug costs. The prevalence of drug resistance is surprisingly low in Bangladesh, but could rise with improving economic conditions. The NTP regimens for smear-positive cases are appropriate, all the more so since the human immunodeficiency virus is virtually absent. Indications for the retreatment regimen should be extended to include all patients treated for at least one month with any drug. The NTP regimen for smear-negative cases runs the risk of leading to MDR under present field conditions.

Evolving BioAssay Ontology (BAO): modularization, integration and applications.

Science.gov (United States)

Abeyruwan, Saminda; Vempati, Uma D; Küçük-McGinty, Hande; Visser, Ubbo; Koleti, Amar; Mir, Ahsan; Sakurai, Kunie; Chung, Caty; Bittker, Joshua A; Clemons, Paul A; Brudz, Steve; Siripala, Anosha; Morales, Arturo J; Romacker, Martin; Twomey, David; Bureeva, Svetlana; Lemmon, Vance; Schürer, Stephan C

2014-01-01

The lack of established standards to describe and annotate biological assays and screening outcomes in the domain of drug and chemical probe discovery is a severe limitation to utilize public and proprietary drug screening data to their maximum potential. We have created the BioAssay Ontology (BAO) project (http://bioassayontology.org) to develop common reference metadata terms and definitions required for describing relevant information of low-and high-throughput drug and probe screening assays and results. The main objectives of BAO are to enable effective integration, aggregation, retrieval, and analyses of drug screening data. Since we first released BAO on the BioPortal in 2010 we have considerably expanded and enhanced BAO and we have applied the ontology in several internal and external collaborative projects, for example the BioAssay Research Database (BARD). We describe the evolution of BAO with a design that enables modeling complex assays including profile and panel assays such as those in the Library of Integrated Network-based Cellular Signatures (LINCS). One of the critical questions in evolving BAO is the following: how can we provide a way to efficiently reuse and share among various research projects specific parts of our ontologies without violating the integrity of the ontology and without creating redundancies. This paper provides a comprehensive answer to this question with a description of a methodology for ontology modularization using a layered architecture. Our modularization approach defines several distinct BAO components and separates internal from external modules and domain-level from structural components. This approach facilitates the generation/extraction of derived ontologies (or perspectives) that can suit particular use cases or software applications. We describe the evolution of BAO related to its formal structures, engineering approaches, and content to enable modeling of complex assays and integration with other ontologies and

21 CFR 864.7500 - Whole blood hemoglobin assays.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Whole blood hemoglobin assays. 864.7500 Section 864.7500 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7500 Whole...

21 CFR 864.7040 - Adenosine triphosphate release assay.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Adenosine triphosphate release assay. 864.7040 Section 864.7040 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7040...

Drugs + HIV, Learn the Link

Medline Plus

Full Text Available ... effects of drugs. Drug misuse and addiction can affect a person's overall health, thereby altering susceptibility to HIV and progression of ... drugs and alcohol even once can have serious health consequences. We also work to reach parents and teachers—influential figures in ...

A comprehensive review of the published assays for the quantitation of the immunosuppressant drug mycophenolic acid and its glucuronidated metabolites in biological fluids

DEFF Research Database (Denmark)

Syed, Muzeeb; Srinivas, Nuggehally R

2016-01-01

Therapeutic use of mycophenolic acid (MPA) is steadily on the rise in combination with other immunosuppressant drugs in transplantation patients. The biotransformation of MPA resulted in the formation of glucuronide metabolites, MPAG and AcMPAG. There are a plethora of assays validated for the an...

Independency of Fe ions in hemoglobin on immunomagnetic reduction assay

International Nuclear Information System (INIS)

Yang, S.Y.; Lan, C.B.; Chen, C.H.; Horng, H.E.; Hong, Chin-Yih; Yang, H.C.; Lai, Y.K.; Lin, Y.H.; Teng, K.S.

2009-01-01

Immunomagnetic reduction (IMR), which involves measuring the reduction in the ac magnetic susceptibility of magnetic reagents, is due to the association between bio-functionalized magnetic nanoparticles and target bio-molecules. This has been demonstrated for assaying proteins in solutions free of Fe ions, such as serum. In this work, the validity of IMR assay for samples rich in Fe ions like hemoglobin (Hb) is investigated. According to the results, there is no magnetic signal contributed by Fe-ion-rich Hb. Furthermore, the results show a high sensitivity in assaying hemoglobin A1c (HbA1c) by using IMR.

Independency of Fe ions in hemoglobin on immunomagnetic reduction assay

Energy Technology Data Exchange (ETDEWEB)

Yang, S.Y. [MagQu Co. Ltd., Sindian City, Taipei County 231, Taiwan (China); Institute of Electro-optical Science and Technology, National Taiwan Normal University, Taipei 116, Taiwan (China); Lan, C.B.; Chen, C.H. [Institute of Electro-optical Science and Technology, National Taiwan Normal University, Taipei 116, Taiwan (China); Horng, H.E. [Institute of Electro-optical Science and Technology, National Taiwan Normal University, Taipei 116, Taiwan (China)], E-mail: phyfv001@scc.ntnu.edu.tw; Hong, Chin-Yih [Department of Mechanical Engineering, Nan-Kai University of Technology, Nantau County, Taiwan (China)], E-mail: cyhong@nkut.edu.tw; Yang, H.C. [Department of Physics, National Taiwan University, Taipei 106, Taiwan (China)], E-mail: hcyang@phys.ntu.edu.tw; Lai, Y.K. [College of Life Sciences, National Tsing Hua University, Hsinchu City 300, Taiwan (China); Department of Bioresources, Da-Yeh University, Changhua 515, Taiwan (China); Lin, Y.H.; Teng, K.S. [Apex Biotechnology Co. Ltd., Hsinchu City 300, Taiwan (China)

2009-10-15

Immunomagnetic reduction (IMR), which involves measuring the reduction in the ac magnetic susceptibility of magnetic reagents, is due to the association between bio-functionalized magnetic nanoparticles and target bio-molecules. This has been demonstrated for assaying proteins in solutions free of Fe ions, such as serum. In this work, the validity of IMR assay for samples rich in Fe ions like hemoglobin (Hb) is investigated. According to the results, there is no magnetic signal contributed by Fe-ion-rich Hb. Furthermore, the results show a high sensitivity in assaying hemoglobin A1c (HbA1c) by using IMR.

Diagnostic performance of automated liquid culture and molecular line probe assay in smear-negative pulmonary tuberculosis.

Science.gov (United States)

Kotwal, Aarti; Biswas, Debasis; Raghuvanshi, Shailendra; Sindhwani, Girish; Kakati, Barnali; Sharma, Shweta

2017-04-01

The diagnosis of smear-negative pulmonary tuberculosis (PTB) is particularly challenging, and automated liquid culture and molecular line probe assays (LPA) may prove particularly useful. The objective of our study was to evaluate the diagnostic potential of automated liquid culture (ALC) technology and commercial LPA in sputum smear-negative PTB suspects. Spot sputum samples were collected from 145 chest-symptomatic smear-negative patients and subjected to ALC, direct drug susceptibility test (DST) testing and LPA, as per manufacturers' instructions. A diagnostic yield of 26.2% was observed among sputum smear-negative TB suspects with 47.4% of the culture isolates being either INH- and/or rifampicin-resistant. Complete agreement was observed between the results of ALC assay and LPA except for two isolates which demonstrated sensitivity to INH and rifampicin at direct DST but were rifampicin-resistant in LPA. Two novel mutations were also detected among the multidrug isolates by LPA. In view of the diagnostic challenges associated with the diagnosis of TB in sputum smear-negative patients, our study demonstrates the applicability of ALC and LPA in establishing diagnostic evidence of TB.

Drugs + HIV, Learn the Link

Medline Plus

Full Text Available ... HIV or transmitting it to someone else. Biological effects of drugs. Drug misuse and addiction can affect a person's overall health, thereby altering susceptibility to HIV and progression of ...

Temporal and seasonal changes of genetic polymorphisms associated with altered drug susceptibility to chloroquine, lumefantrine and quinine in Guinea-Bissau between 2003 and 2012

DEFF Research Database (Denmark)

Jovel, Irina Tatiana; Kofoed, Poul-Erik; Rombo, Lars

2015-01-01

BACKGROUND: Guinea-Bissau, West-Africa introduced artemether-lumefantrine in 2008 but quinine has also been commonly prescribed for treatment of uncomplicated Plasmodium falciparum malaria. An efficacious high-dose chloroquine treatment regimen was used previously. Temporal and seasonal changes...... of genetic polymorphisms associated with altered drug susceptibility to chloroquine, lumefantrine and quinine are described. METHODS: Pfcrt K76T, pfmdr1 gene copy numbers, N86Y, Y184F and 1034-1246 sequences were determined using PCR-based methods. Blood samples came from virtually all (n=1806) children aged.......001). CONCLUSIONS: Following the discontinuation of an effective chloroquine regimen highly artemether-lumefantrine susceptible P. falciparum (with pfcrt 76T) accumulated possibly due to suboptimal use of quinine and despite a fitness cost linked to 76T....

Radioimmunoassay and radioenzymatic assay of a new aminoglycoside antibiotic, netilmicin

International Nuclear Information System (INIS)

Broughton, A.; Strong, J.E.; Pickering, L.K.; Knight, J.; Bodey, G.P.

1978-01-01

A radioimmunoassay and a radioenzymatic assay for netilmicin, a new aminoglycoside, were developed in our laboratories to assist in the study of the pharmacology of the drug and establish values for use in its monitoring. The assays are sensitive, precise, and rapid, giving results that correlate (r = 0.90) with each other and with those of a microbiological assay in which Klebsiella pneumoniae is used as the test organism. Preliminary pharmacological studies show the drug to have a biological half-life of 135 min, which is comparable to that for other aminoglycosides

21 CFR 864.7400 - Hemoglobin A2 assay.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Hemoglobin A2 assay. 864.7400 Section 864.7400 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7400 Hemoglobin A2...

Flow cytometric assay for analysis of cytotoxic effects of potential drugs on human peripheral blood leukocytes

Science.gov (United States)

Nieschke, Kathleen; Mittag, Anja; Golab, Karolina; Bocsi, Jozsef; Pierzchalski, Arkadiusz; Kamysz, Wojciech; Tarnok, Attila

2014-03-01

Toxicity test of new chemicals belongs to the first steps in the drug screening, using different cultured cell lines. However, primary human cells represent the human organism better than cultured tumor derived cell lines. We developed a very gentle toxicity assay for isolation and incubation of human peripheral blood leukocytes (PBL) and tested it using different bioactive oligopeptides (OP). Effects of different PBL isolation methods (red blood cell lysis; Histopaque isolation among others), different incubation tubes (e.g. FACS tubes), anticoagulants and blood sources on PBL viability were tested using propidium iodide-exclusion as viability measure (incubation time: 60 min, 36°C) and flow cytometry. Toxicity concentration and time-depended effects (10-60 min, 36 °C, 0-100 μg /ml of OP) on human PBL were analyzed. Erythrocyte lysis by hypotonic shock (dH2O) was the fastest PBL isolation method with highest viability (>85%) compared to NH4Cl-Lysis (49%). Density gradient centrifugation led to neutrophil granulocyte cell loss. Heparin anticoagulation resulted in higher viability than EDTA. Conical 1.5 mL and 2 mL micro-reaction tubes (both polypropylene (PP)) had the highest viability (99% and 97%) compared to other tubes, i.e. three types of 5.0 mL round-bottom tubes PP (opaque-60%), PP (blue-62%), Polystyrene (PS-64%). Viability of PBL did not differ between venous and capillary blood. A gentle reproducible preparation and analytical toxicity-assay for human PBL was developed and evaluated. Using our assay toxicity, time-course, dose-dependence and aggregate formation by OP could be clearly differentiated and quantified. This novel assay enables for rapid and cost effective multiparametric toxicological screening and pharmacological testing on primary human PBL and can be adapted to high-throughput-screening.°z

Antibacterial effects of Apis mellifera and stingless bees honeys on susceptible and resistant strains of Escherichia coli, Staphylococcus aureus and Klebsiella pneumoniae in Gondar, Northwest Ethiopia.

Science.gov (United States)

Ewnetu, Yalemwork; Lemma, Wossenseged; Birhane, Nega

2013-10-19

Honey is a natural substance produced by honeybees and has nutritional and therapeutic uses. In Ethiopia, honeys are used traditionally to treat wounds, respiratory infections and diarrhoea. Recent increase of drug resistant bacteria against the existing antibiotics forced investigators to search for alternative natural remedies and evaluate their potential use on scientific bases. Thus, the aim of this study was to evaluate the antibacterial effects of different types of honeys in Ethiopia which are used traditionally to treat different types of respiratory and gastrointestinal infections. Mueller Hinton agar (70191) diffusion and nutrient broth culture medium assays were performed to determine susceptibility of Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922) and resistant clinical isolates (Methicillin resistant Staphylococcus aureus(MRSA), Escherichia coli(R) and Klebsiella pneumoniae (R), using honeys of Apis mellifera and stingless bees in northern and north western Ethiopia. Honey of the stingless bees produced the highest mean inhibition (22.27 ± 3.79 mm) compared to white honey (21.0 ± 2.7 mm) and yellow honey (18.0 ± 2.3 mm) at 50% (v/v) concentration on all the standard and resistant strains. Stingless bees honey was found to have Minimum Inhibitory Concentration (MIC) of 6.25% (6.25 mg/ml) for 80% of the test organisms compared to 40% for white and yellow Apis mellifera honeys. All the honeys were found to have minimum bactericidal concentration (MBC) of 12.5% (12.5 mg/ml) against all the test organisms. Staphylococcus aureus (ATCC 25923) was susceptible to amoxicillin, methicillin, kanamycine, tetracycline, and vancomycine standard antibiotic discs used for susceptibility tests. Similarly, Escherichia coli (ATCC 25922) was found susceptible for kanamycine, tetracycline and vancomycine. Escherichia coli (ATCC 25922) has not been tested for amoxicillin ampicillin and methicillin. The susceptibility tests performed against

Generation of pH responsive fluorescent nano capsules through simple steps for the oral delivery of low pH susceptible drugs

Science.gov (United States)

Radhakumary, Changerath; Sreenivasan, Kunnatheeri

2016-11-01

pH responsive nano capsules are promising as it can encapsulate low pH susceptible drugs like insulin and guard them from the hostile environments in the intestinal tract. The strong acidity of the gastro-intestinal tract and the presence of proteolytic enzymes are the tumbling blocks for the design of drug delivery vehicles through oral route for drugs like insulin. Nano capsules are normally built over templates which are subsequently removed by further steps. Such processes are complex and often lead into deformed and collapsed capsules. In this study, we choose calcium carbonate (CaCO3) nano particles to serve as template. Over CaCO3 nanoparticles, silica layers were built followed by polymethacrylic acid chains to acquire pH responsiveness. During the polymerization process of the methacrylic acid, the calcium carbonate core particles were dissolved leading to the formation of nano hollow capsules having a size that ranges from 225 to 246 nm and thickness from 19 to 58 nm. The methodology is simple and devoid of additional steps. The nano shells exhibited 80% release of the loaded model drug, insulin at pH 7.4 while at pH 2.0 the capsules nearly stopped the release of the drug. Polymethacrylic acid shows pH responsive swelling behavior that it swells at intestinal pH (7.0-7.5) and shrinks at gastric pH (˜2.0) thus enabling the safe unloading of the drug from the nano capsules.

Antimicrobial Disk Susceptibility Testing of Leptospira spp. Using Leptospira Vanaporn Wuthiekanun (LVW) Agar.

Science.gov (United States)

Wuthiekanun, Vanaporn; Amornchai, Premjit; Langla, Sayan; White, Nicholas J; Day, Nicholas P J; Limmathurotsakul, Direk; Peacock, Sharon J

2015-08-01

Leptospira Vanaporn Wuthiekanun (LVW) agar was used to develop a disk diffusion assay for Leptospira spp. Ten pathogenic Leptospira isolates were tested, all of which were susceptible to 17 antimicrobial agents (amoxicillin/clavulanic acid, amoxicillin, azithromycin, cefoxitin, ceftazidime, ceftriaxone, chloramphenicol, ciprofloxacin, clindamycin, doripenem, doxycycline, gentamicin, linezolid, nitrofurantoin, penicillin, piperacillin/tazobactam, and tetracycline). All 10 isolates had no zone of growth inhibition for four antimicrobials (fosfomycin, nalidixic acid, rifampicin, and trimethoprim/sulfamethoxazole). Of the ten Leptospira, seven had a growth inhibition zone of ≤ 21 mm for aztreonam, the zone diameter susceptibility break point for Enterobacteriaceae. This assay could find utility as a simple screening method during the epidemiological surveillance of antimicrobial resistance in Leptospira spp. © The American Society of Tropical Medicine and Hygiene.

Multiplexing a high-throughput liability assay to leverage efficiencies.

Science.gov (United States)

Herbst, John; Anthony, Monique; Stewart, Jeremy; Connors, David; Chen, Taosheng; Banks, Martyn; Petrillo, Edward W; Agler, Michele

2009-06-01

In order to identify potential cytochrome P-450 3A4 (drug-metabolizing enzyme) inducers at an early stage of the drug discovery process, a cell-based transactivation high-throughput luciferase reporter assay for the human pregnane X receptor (PXR) in HepG2 cells has been implemented and multiplexed with a viability end point for data interpretation, as part of a Lead Profiling portfolio of assays. As a routine part of Lead Profiling operations, assays are periodically evaluated for utility as well as for potential improvements in technology or process. We used a recent evaluation of our PXR-transactivation assay as a model for the application of Lean Thinking-based process analysis to lab-bench assay optimization and automation. This resulted in the development of a 384-well multiplexed homogeneous assay simultaneously detecting PXR transactivation and HepG2 cell cytotoxicity. In order to multiplex fluorescent and luminescent read-outs, modifications to each assay were necessary, which included optimization of multiple assay parameters such as cell density, plate type, and reagent concentrations. Subsequently, a set of compounds including known cytotoxic compounds and PXR inducers were used to validate the multiplexed assay. Results from the multiplexed assay correlate well with those from the singleplexed assay formats measuring PXR transactivation and viability separately. Implementation of the multiplexed assay for routine compound profiling provides improved data quality, sample conservation, cost savings, and resource efficiencies.

Antifungal susceptibilities of Candida species isolated from the patients with vaginal candidiasis.

Science.gov (United States)

Nagashima, Masahito; Yamagishi, Yuka; Mikamo, Hiroshige

2016-02-01

There have been the current Japanese data on susceptibility testing for Candida isolates from vaginal candidiasis. The in vitro activities of therapeutic antifungal drugs for vulvovaginal candidiasis (VVC); miconazole (MCZ), itraconazole (ITCZ), fluconazole (FLCZ), clotrimazole (CTZ), oxiconazole (OCZ), isoconazole (ICZ) and bifonazole (BFZ) against vaginal isolates. Fifty-four strains Candida albicans and 19 strains of Candida glabrata were evaluated using a broth microdilution method specified by Clinical Laboratories Standard Institute (CLSI) document M27-A3. The MIC90 of each drug, MCZ, ITCZ, FLCZ, CTZ, OCZ, ICZ and BFZ, against C. albicans and C. glabrata isolates were 0.25, 0.12, 1, 0.06, 0.12, 0.12 and 1 μg/ml and 1, 1, 8, 0.5, 0.25, 0.5 and 1 μg/ml respectively. The activities of these drugs, except for BFZ, against C. glabrata were lower than that of C. albicans. There was one azole-resistant isolate in C. glabrata of which MIC of FLCZ is > 64 μg/ml and this isolate had cross resistance to other antifungal drugs tested. These results suggest that antifungal drugs for treatment of VVC continues to have potent antifungal activities against C. albicans and C. glabrata isolates from vaginitis. CTZ, OCZ and ICZ susceptibility of FLCZ low susceptibility C. glabrata are relatively higher than MCZ, ITCZ and FLCZ. Copyright © 2015. Published by Elsevier Ltd.

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.

Bacterial profile and drug susceptibility pattern of urinary tract infection in pregnant women at Tikur Anbessa Specialized Hospital Addis Ababa, Ethiopia.

Science.gov (United States)

Assefa, Addisu; Asrat, Daniel; Woldeamanuel, Yimtubezinash; G/Hiwot, Yirgu; Abdella, Ahmed; Melesse, Tadele

2008-07-01

Urinary tract infection (UTI) is a common complication of pregnancy. It may be symptomatic or asymptomatic. The aim of this cross sectional study was to identify bacterial agents and their antibiotic susceptibility pattern isolated from pregnant women with UTI attending antenatal clinic of Tikur Anbessa Specialized Hospital (TASH). Four hundred and fourteen pregnant women with asymptomatic UTI (n = 369) and symptomatic UTI (n = 45) were investigated for urinary tract infection from January to March 2005. The age range of both groups was 18 to 44 years. Bacteriological screening of mid-stream urine specimens revealed that 39/369 (10.6%) and 9/45 (20%) had significant bacteriuria in asymptomatic and symptomatic group, respectively (p = 0.10). The overall prevalence of urinary tract infection was 48/414 (11.6%). The bacterial pathogens isolated were predominantly E. coil (44%), followed by S. aureus (20%), coagulase-negative staphylococci (16%), and K. pneumoniae (8%). Others found in small in number included P. mirabilis, P. aeruginosa, Enterococcus spp. and non-Group A-beta hemolytic Streptococcus, this accounted 2% for each. The gram positive and negative bacteria accounted 40% and 60% respectively. The susceptibility pattern for gram-negative bacteria showed that most of the isolates (> 65% of the strains) were sensitive to amoxicillin-clavulanic acid (70%), chloramphenicol (83.3%), gentamicin (93.3%), kanamycin (93.3%), nitrofurantoin (87.7%) and trimethoprim-sulphamethoxazole (73.3%). Among the gram-positives, more than 60% of the isolates were sensitive to amoxicillin-clavulanic acid (100%), cephalothin (95%), chloramphenicol (70%), erythromycin (80%), gentamicin (85%), methicillin (83.3%), nitrofurantoin (100%) and trimethoprim-sulphamethoxazole (65%). Generally, amoxicillin-clavulanic acid, chloramphenicol, gentamicin, nitrofurantoin and trimethoprim-sulphamethoxazole were effective at least in 70% of the isolates. Multiple drug resistance (resistance two or

A Multi-Modality CMOS Sensor Array for Cell-Based Assay and Drug Screening.

Science.gov (United States)

Chi, Taiyun; Park, Jong Seok; Butts, Jessica C; Hookway, Tracy A; Su, Amy; Zhu, Chengjie; Styczynski, Mark P; McDevitt, Todd C; Wang, Hua

2015-12-01

In this paper, we present a fully integrated multi-modality CMOS cellular sensor array with four sensing modalities to characterize different cell physiological responses, including extracellular voltage recording, cellular impedance mapping, optical detection with shadow imaging and bioluminescence sensing, and thermal monitoring. The sensor array consists of nine parallel pixel groups and nine corresponding signal conditioning blocks. Each pixel group comprises one temperature sensor and 16 tri-modality sensor pixels, while each tri-modality sensor pixel can be independently configured for extracellular voltage recording, cellular impedance measurement (voltage excitation/current sensing), and optical detection. This sensor array supports multi-modality cellular sensing at the pixel level, which enables holistic cell characterization and joint-modality physiological monitoring on the same cellular sample with a pixel resolution of 80 μm × 100 μm. Comprehensive biological experiments with different living cell samples demonstrate the functionality and benefit of the proposed multi-modality sensing in cell-based assay and drug screening.

Drugs + HIV, Learn the Link

Medline Plus

Full Text Available ... HIV or transmitting it to someone else. Biological effects of drugs. Drug misuse and addiction can affect a person's overall health, thereby altering susceptibility to HIV and progression of AIDS. Drugs of abuse and HIV both affect the brain. Research has shown that HIV causes greater injury ...

Electrochemical studies of ropinirole, an anti-Parkinson's disease drug

Indian Academy of Sciences (India)

trochemical techniques have application to drug-protein. ∗. For correspondence ... drug bioavailability and toxicity tests. ... analysis ranging from the assay of drugs in bulk form, ... stability-indicating assays.13,14 Separation and quantifi-.

Towards an in vitro model of Plasmodium hypnozoites suitable for drug discovery.

Science.gov (United States)

Dembele, Laurent; Gego, Audrey; Zeeman, Anne-Marie; Franetich, Jean-François; Silvie, Olivier; Rametti, Armelle; Le Grand, Roger; Dereuddre-Bosquet, Nathalie; Sauerwein, Robert; van Gemert, Geert-Jan; Vaillant, Jean-Christophe; Thomas, Alan W; Snounou, Georges; Kocken, Clemens H M; Mazier, Dominique

2011-03-31

Amongst the Plasmodium species in humans, only P. vivax and P. ovale produce latent hepatic stages called hypnozoites, which are responsible for malaria episodes long after a mosquito bite. Relapses contribute to increased morbidity, and complicate malaria elimination programs. A single drug effective against hypnozoites, primaquine, is available, but its deployment is curtailed by its haemolytic potential in glucose-6-phosphate dehydrogenase deficient persons. Novel compounds are thus urgently needed to replace primaquine. Discovery of compounds active against hypnozoites is restricted to the in vivo P. cynomolgi-rhesus monkey model. Slow growing hepatic parasites reminiscent of hypnozoites had been noted in cultured P. vivax-infected hepatoma cells, but similar forms are also observed in vitro by other species including P. falciparum that do not produce hypnozoites. P. falciparum or P. cynomolgi sporozoites were used to infect human or Macaca fascicularis primary hepatocytes, respectively. The susceptibility of the slow and normally growing hepatic forms obtained in vitro to three antimalarial drugs, one active against hepatic forms including hypnozoites and two only against the growing forms, was measured. The non-dividing slow growing P. cynomolgi hepatic forms, observed in vitro in primary hepatocytes from the natural host Macaca fascicularis, can be distinguished from similar forms seen in P. falciparum-infected human primary hepatocytes by the differential action of selected anti-malarial drugs. Whereas atovaquone and pyrimethamine are active on all the dividing hepatic forms observed, the P. cynomolgi slow growing forms are highly resistant to treatment by these drugs, but remain susceptible to primaquine. Resistance of the non-dividing P. cynomolgi forms to atovaquone and pyrimethamine, which do not prevent relapses, strongly suggests that these slow growing forms are hypnozoites. This represents a first step towards the development of a practical medium

Towards an in vitro model of Plasmodium hypnozoites suitable for drug discovery.

Directory of Open Access Journals (Sweden)

Laurent Dembele

2011-03-01

Full Text Available Amongst the Plasmodium species in humans, only P. vivax and P. ovale produce latent hepatic stages called hypnozoites, which are responsible for malaria episodes long after a mosquito bite. Relapses contribute to increased morbidity, and complicate malaria elimination programs. A single drug effective against hypnozoites, primaquine, is available, but its deployment is curtailed by its haemolytic potential in glucose-6-phosphate dehydrogenase deficient persons. Novel compounds are thus urgently needed to replace primaquine. Discovery of compounds active against hypnozoites is restricted to the in vivo P. cynomolgi-rhesus monkey model. Slow growing hepatic parasites reminiscent of hypnozoites had been noted in cultured P. vivax-infected hepatoma cells, but similar forms are also observed in vitro by other species including P. falciparum that do not produce hypnozoites.P. falciparum or P. cynomolgi sporozoites were used to infect human or Macaca fascicularis primary hepatocytes, respectively. The susceptibility of the slow and normally growing hepatic forms obtained in vitro to three antimalarial drugs, one active against hepatic forms including hypnozoites and two only against the growing forms, was measured.The non-dividing slow growing P. cynomolgi hepatic forms, observed in vitro in primary hepatocytes from the natural host Macaca fascicularis, can be distinguished from similar forms seen in P. falciparum-infected human primary hepatocytes by the differential action of selected anti-malarial drugs. Whereas atovaquone and pyrimethamine are active on all the dividing hepatic forms observed, the P. cynomolgi slow growing forms are highly resistant to treatment by these drugs, but remain susceptible to primaquine.Resistance of the non-dividing P. cynomolgi forms to atovaquone and pyrimethamine, which do not prevent relapses, strongly suggests that these slow growing forms are hypnozoites. This represents a first step towards the development of a

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.

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.

Calibration and LOD/LOQ estimation of a chemiluminescent hybridization assay for residual DNA in recombinant protein drugs expressed in E. coli using a four-parameter logistic model.

Science.gov (United States)

Lee, K R; Dipaolo, B; Ji, X

2000-06-01

Calibration is the process of fitting a model based on reference data points (x, y), then using the model to estimate an unknown x based on a new measured response, y. In DNA assay, x is the concentration, and y is the measured signal volume. A four-parameter logistic model was used frequently for calibration of immunoassay when the response is optical density for enzyme-linked immunosorbent assay (ELISA) or adjusted radioactivity count for radioimmunoassay (RIA). Here, it is shown that the same model or a linearized version of the curve are equally useful for the calibration of a chemiluminescent hybridization assay for residual DNA in recombinant protein drugs and calculation of performance measures of the assay.

Lack of cross-reactivity of Ambien (zolpidem) with drugs in standard urine drug screens.

Science.gov (United States)

Piergies, A A; Sainati, S; Roth-Schechter, B

1997-04-01

To determine in healthy volunteers (men and women; 18 to 40 years old) the potential cross-reactivity of Ambien (zolpidem) and/or its metabolites with drugs that are screened by the Syva EMIT II and the Abbott ADx urine drug screens assays. Open-label, fixed-treatment sequence of 1 night each of treatment with zolpidem (10 mg) and temazepam (15 mg). Clinical Pharmacology Unit within a teaching hospital. Over a 24-hour period, presence or absence of positive results on the Syva EMIT II or the Abbott ADx urine drug assay system, each performed at two different laboratory assay sites. Following ingestion of zolpidem, no subject had any positive response in either laboratory to the Syva EMIT II or the Abbott ADx urine drug screen assays at 0, 4, 8, 12, and 24 hours postdose. During the same time period, all subjects had measurable zolpidem plasma concentrations at 1.5 and 8 hours postdose, with mean concentrations of 115.2 ng/mL and 30.1 ng/mL, respectively (in agreement with its half-life of 2.5 hours). The positive response rate at 10 hours after ingestion of Restoril (temazepam) among the four laboratory/assay combinations ranged from 36.8% to 73.7%, a range that is within the reported response rates for these tests. These data indicate that zolpidem will not cross-react in standard urine drug screens with benzodiazepines, opiates, barbiturates, cocaine, cannabinoids, or amphetamines.

21 CFR 862.1660 - Quality control material (assayed and unassayed).

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Quality control material (assayed and unassayed... Test Systems § 862.1660 Quality control material (assayed and unassayed). (a) Identification. A quality... that may arise from reagent or analytical instrument variation. A quality control material (assayed and...

Etest cannot be recommended for in vitro susceptibility testing of mucorales.

Science.gov (United States)

Caramalho, Rita; Maurer, Elisabeth; Binder, Ulrike; Araújo, Ricardo; Dolatabadi, Somayeh; Lass-Flörl, Cornelia; Lackner, Michaela

2015-01-01

Amphotericin B and posaconazole susceptibility patterns were determined for the most prevalent Mucorales, following EUCAST (European Committee on Antimicrobial Susceptibility Testing) broth microdilution guidelines. In parallel, Etest was performed and evaluated against EUCAST. The overall agreement of MICs gained with Etest and EUCAST was 75.1%; therefore, Etest cannot be recommended for antifungal susceptibility testing of Mucorales. Amphotericin B was the most active drug against Mucorales species in vitro, while the activities of posaconazole were more restricted. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Phosphodiesterase (PDE5) inhibition assay for rapid detection of erectile dysfunction drugs and analogs in sexual enhancement products.

Science.gov (United States)

Santillo, Michael F; Mapa, Mapa S T

2018-02-28

Products marketed as dietary supplements for sexual enhancement are frequently adulterated with phosphodiesterase-5 (PDE5) inhibitors, which are erectile dysfunction drugs or their analogs that can cause adverse health effects. Due to widespread adulteration, a rapid screening assay was developed to detect PDE5 inhibitors in adulterated products. The assay employs fluorescence detection and is based on measuring inhibition of PDE5 activity, the pharmacological mechanism shared among the adulterants. Initially, the assay reaction scheme was established and characterized, followed by analysis of 9 representative PDE5 inhibitors (IC 50 , 0.4-4.0 ng mL -1 ), demonstrating sensitive detection in matrix-free solutions. Next, dietary supplements serving as matrix blanks (n = 25) were analyzed to determine matrix interference and establish a threshold value; there were no false positives. Finally, matrix blanks were spiked with 9 individual PDE5 inhibitors, along with several mixtures. All 9 adulterants were successfully detected (≤ 5 % false negative rate; n = 20) at a concentration of 1.00 mg g -1 , which is over 5 times lower than concentrations commonly encountered in adulterated products. A major distinction of the PDE5 inhibition assay is the ability to detect adulterants without prior knowledge of their chemical structures, demonstrating a broad-based detection capability that can address a continuously evolving threat of new adulterants. The PDE5 inhibition assay can analyze over 40 samples simultaneously within 15 minutes and involves a single incubation step and simple data analysis, all of which are advantageous for combating the widespread adulteration of sex-enhancement products. Published 2018. This article is a U.S. Government work and is in the public domain in the USA.

Repurposing of Aspirin and Ibuprofen as Candidate Anti-Cryptococcus Drugs

Science.gov (United States)

Ogundeji, Adepemi O.; Pohl, Carolina H.

2016-01-01

The usage of fluconazole and amphotericin B in clinical settings is often limited by, among other things, drug resistance development and undesired side effects. Thus, there is a constant need to find new drugs to better manage fungal infections. Toward this end, the study described in this paper considered the repurposing of aspirin (acetylsalicylic acid) and ibuprofen as alternative drugs to control the growth of cryptococcal cells. In vitro susceptibility tests, including a checkerboard assay, were performed to assess the response of Cryptococcus neoformans and Cryptococcus gattii to the above-mentioned anti-inflammatory drugs. Next, the capacity of these two drugs to induce stress as well as their mode of action in the killing of cryptococcal cells was determined. The studied fungal strains revealed a response to both aspirin and ibuprofen that was dose dependent, with ibuprofen exerting greater antimicrobial action. More importantly, the MICs of these drugs did not negatively (i) affect growth or (ii) impair the functioning of macrophages; rather, they enhanced the ability of these immune cells to phagocytose cryptococcal cells. Ibuprofen was also shown to act in synergy with fluconazole and amphotericin B. The treatment of cryptococcal cells with aspirin or ibuprofen led to stress induction via activation of the high-osmolarity glycerol (HOG) pathway, and cell death was eventually achieved through reactive oxygen species (ROS)-mediated membrane damage. The presented data highlight the potential clinical application of aspirin and ibuprofen as candidate anti-Cryptococcus drugs. PMID:27246782

Ertapenem susceptibility of extended spectrum beta-lactamase-producing organisms

Directory of Open Access Journals (Sweden)

Selby Edward B

2007-06-01

Full Text Available Abstract Background Infections caused by multiply drug resistant organisms such as extended spectrum beta-lactamase (ESBL-producing Escherichia coli and Klebsiella pneumoniae are increasing. Carbapenems (imipenem and meropenem are the antibiotics commonly used to treat these agents. There is limited clinical data regarding the efficacy of the newest carbapenem, ertapenem, against these organisms. Ertapenem susceptibility of ESBL-producing E. coli and K. pneumoniae clinical isolates were evaluated and compared to imipenem to determine if imipenem susceptibility could be used as a surrogate for ertapenem susceptibility. Methods 100 ESBL isolates (n = 34 E. coli and n = 66 K. pneumoniae collected from 2005–2006 clinical specimens at WRAMC were identified and tested for susceptibility by Vitek Legacy [bioMerieux, Durham, NC]. Ertapenem susceptibility was performed via epsilometer test (E-test [AB Biodisk, Solna, Sweden]. Results 100% of ESBL isolates tested were susceptible to ertapenem. 100% of the same isolates were also susceptible to imipenem. Conclusion These results, based on 100% susceptibility, suggest that ertapenem may be an alternative to other carbapenems for the treatment of infections caused by ESBL-producing E. coli and K. pneumoniae. Clinical outcomes studies are needed to determine if ertapenem is effective for the treatment of infection caused by these organisms. However, due to lack of resistant isolates, we are unable to conclude whether imipenem susceptibility accurately predicts ertapenem susceptibility.

Prevalence and drug susceptibility pattern of group B Streptococci (GBS) among pregnant women attending antenatal care (ANC) in Nekemte Referral Hospital (NRH), Nekemte, Ethiopia.

Science.gov (United States)

Mengist, Hylemariam Mihiretie; Zewdie, Olifan; Belew, Adugna; Dabsu, Regea

2017-08-10

The main objective of this study was to determine the prevalence and drug susceptibility pattern of group B Streptococci (GBS) among pregnant women. The specific objectives include; (1) To determine the prevalence of GBS colonization among pregnant women (2) To determine the drug susceptibility pattern of GBS among pregnant women and (3) To identify associated risk factors with GBS colonization among pregnant women. The median age of the participants was 24.5 years (range 16-38) and 86% participants were urban residents. The total prevalence of maternal GBS colonization from vaginal swab culture was 12.2% (22/180). The prevalence of GBS colonization rate was significantly higher in those pregnant women above 37 weeks of gestation [AOR, 95% CI 2.1 (1.2, 11.6), P = 0.03] and married ones [AOR, 95% CI 3.2 (1.8, 11.6), P < 0.021]. Twenty (91%) of GBS isolates were sensitive to vancomycin and the highest resistance was observed against penicillin G (77.3%). The prevalence of GBS colonization in this study was significantly high and differed by gestational age and marital status. None of the GBS isolates were resistant to vancomycin but higher resistance was shown against Penicillin G.

Applicability of the comet assay in evaluation of DNA damage in healthcare providers' working with antineoplastic drugs: a systematic review and meta-analysis.

Science.gov (United States)

Zare Sakhvidi, Mohammad Javad; Hajaghazadeh, Mohammad; Mostaghaci, Mehrdad; Mehrparvar, Amir Houshang; Zare Sakhvidi, Fariba; Naghshineh, Elham

2016-01-01

Unintended occupational exposure to antineoplastic drugs (ANDs) may occur in medical personnel. Some ANDs are known human carcinogens and exposure can be monitored by genotoxic biomarkers. To evaluate the obstacles to obtaining conclusive results from a comet assay test to determine DNA damage among AND exposed healthcare workers. We systematically reviewed studies that used alkaline comet assay to determine the magnitude and significance of DNA damage among health care workers with potential AND exposure. Fifteen studies were eligible for review and 14 studies were used in the meta-analysis. Under random effect assumption, the estimated standardized mean difference (SMD) in the DNA damage of health care workers was 1.93 (95% CI: 1.15-2.71, p comet moment, I2 test results, as a measure of heterogeneity, dropped to zero. Heterogeneity analysis showed that date of study publication was a possible source of heterogeneity (B = -0.14; p comet assay methodological variables, and exposure characteristics may be responsible for heterogenic data from comet assay studies and interfere with obtaining conclusive results. Lack of quantitative environmental exposure measures and variation in comet assay protocols across studies are important obstacles in generalization of results.

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.

Miniaturized Antimicrobial Susceptibility Test by Combining Concentration Gradient Generation and Rapid Cell Culturing

Directory of Open Access Journals (Sweden)

Samuel C. Kim

2015-10-01

Full Text Available Effective treatment of bacterial infection relies on timely diagnosis and proper prescription of antibiotic drugs. The antimicrobial susceptibility test (AST is one of the most crucial experimental procedures, providing the baseline information for choosing effective antibiotic agents and their dosages. Conventional methods, however, require long incubation times or significant instrumentation costs to obtain test results. We propose a lab-on-a-chip approach to perform AST in a simple, economic, and rapid manner. Our assay platform miniaturizes the standard broth microdilution method on a microfluidic device (20 × 20 mm that generates an antibiotic concentration gradient and delivers antibiotic-containing culture media to eight 30-nL chambers for cell culture. When tested with 20 μL samples of a model bacterial strain (E. coli ATCC 25922 treated with ampicillin or streptomycin, our method allows for the determination of minimum inhibitory concentrations consistent with the microdilution test in three hours, which is almost a factor of ten more rapid than the standard method.

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.

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.

21 CFR 866.3310 - Hepatitis A virus (HAV) serological assays.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Hepatitis A virus (HAV) serological assays. 866... Hepatitis A virus (HAV) serological assays. (a) Identification. HAV serological assays are devices that consist of antigens and antisera for the detection of hepatitis A virus-specific IgM, IgG, or total...

Standardized comparison of the relative impacts of HIV-1 reverse transcriptase (RT) mutations on nucleoside RT inhibitor susceptibility.

Science.gov (United States)

Melikian, George L; Rhee, Soo-Yon; Taylor, Jonathan; Fessel, W Jeffrey; Kaufman, David; Towner, William; Troia-Cancio, Paolo V; Zolopa, Andrew; Robbins, Gregory K; Kagan, Ron; Israelski, Dennis; Shafer, Robert W

2012-05-01

Determining the phenotypic impacts of reverse transcriptase (RT) mutations on individual nucleoside RT inhibitors (NRTIs) has remained a statistical challenge because clinical NRTI-resistant HIV-1 isolates usually contain multiple mutations, often in complex patterns, complicating the task of determining the relative contribution of each mutation to HIV drug resistance. Furthermore, the NRTIs have highly variable dynamic susceptibility ranges, making it difficult to determine the relative effect of an RT mutation on susceptibility to different NRTIs. In this study, we analyzed 1,273 genotyped HIV-1 isolates for which phenotypic results were obtained using the PhenoSense assay (Monogram, South San Francisco, CA). We used a parsimonious feature selection algorithm, LASSO, to assess the possible contributions of 177 mutations that occurred in 10 or more isolates in our data set. We then used least-squares regression to quantify the impact of each LASSO-selected mutation on each NRTI. Our study provides a comprehensive view of the most common NRTI resistance mutations. Because our results were standardized, the study provides the first analysis that quantifies the relative phenotypic effects of NRTI resistance mutations on each of the NRTIs. In addition, the study contains new findings on the relative impacts of thymidine analog mutations (TAMs) on susceptibility to abacavir and tenofovir; the impacts of several known but incompletely characterized mutations, including E40F, V75T, Y115F, and K219R; and a tentative role in reduced NRTI susceptibility for K64H, a novel NRTI resistance mutation.

An LC-MS Assay with Isocratic Separation and On-Line Solid Phase Extraction to Improve the Routine Therapeutic Drug Monitoring of Busulfan in Plasma

Directory of Open Access Journals (Sweden)

Ialongo Cristiano

2017-04-01

Full Text Available Background: Busulfan (Bu requires therapeutic drug monitoring (TDM in subjects undergoing a conditioning regimen for hematopoietic stem cell transplantation (HSCT. To speed up the procedure and increase reproducibility, we improved our routine LC-MS/MS assay using the on-line solid-phase extraction (SPE of samples.

21 CFR 864.7100 - Red blood cell enzyme assay.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Red blood cell enzyme assay. 864.7100 Section 864.7100 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7100 Red blood cell...

Development of assay platforms for in vitro screening of Treg modulating potential of pharmacological compounds

DEFF Research Database (Denmark)

Pedersen, Anders Elm; Holmstrøm, Kim; Jørgensen, Flemming

2015-01-01

that investigates Treg modulation by current drugs. For such research as well as for novel cell based therapies based on Treg infusions, rapid in vitro assays as well as functional assays based on inhibitory capacity of Tregs are required. Here, we report on such assays using highly pure fluorescence-activated cell...... and TNF-α. In conclusion, these assays have the potential for use in pharmacological screening and discovery in relation to drug development in immunology....

In Vitro Susceptibility Test of Different Clinical Isolates against Ceftriaxone

Directory of Open Access Journals (Sweden)

Syed Hakim Masood

2010-06-01

Full Text Available Objectives: Because of the prevailing penicillin resistance in microorganisms, broad spectrum cephalosporins are used empirically specially in developing countries. The aim of this study is to determine the susceptibility pattern of different gram positive and gram negative pathogens against third generation cephalosporin-ceftriaxone to explore the existing effectiveness of this antibiotic.Methods: 180 clinical isolates of different gram positive and gram negative pathogens including P.mirabilis, S. typhi P.aeruginosa, E. coli, S. aureus and Klebsiella were collected from blood and urine samples of in-patients. 30 isolates of all species were tested against each of six brands of ceftriaxone using in vitro sensitivity tests by disc diffusion method (NCCLS criteria. The susceptibility limit was ≥21 mm zone of inhibition, while moderately susceptible was considered at 20-14 mm, and those isolates which showed >13 mm or no zone of inhibition were resistant to this antibacterial drug.Results: Ceftriaxone was found most effective against S. aureus. While 96.1% of the isolates showed susceptibility towards ceftriaxone, followed by E. coli (95%, P. aeruginosa (92.7%, K. pneumonia (89.4% and S. typhi (87.2%. P. mirabilis showed lowest susceptibility amongst all the test organisms (83.8%.Conclusion: Ceftriaxone can be used as a drug of choice in infections caused by S. aureus, E. coli, P. aurigenosa, K. pneumonia and S. typhi. However, it should be used with other antimicrobial agents in order to increase its effectiveness against P. mirabilis.

Rapid bacterial antibiotic susceptibility test based on simple surface-enhanced Raman spectroscopic biomarkers

Science.gov (United States)

Liu, Chia-Ying; Han, Yin-Yi; Shih, Po-Han; Lian, Wei-Nan; Wang, Huai-Hsien; Lin, Chi-Hung; Hsueh, Po-Ren; Wang, Juen-Kai; Wang, Yuh-Lin

2016-03-01

Rapid bacterial antibiotic susceptibility test (AST) and minimum inhibitory concentration (MIC) measurement are important to help reduce the widespread misuse of antibiotics and alleviate the growing drug-resistance problem. We discovered that, when a susceptible strain of Staphylococcus aureus or Escherichia coli is exposed to an antibiotic, the intensity of specific biomarkers in its surface-enhanced Raman scattering (SERS) spectra drops evidently in two hours. The discovery has been exploited for rapid AST and MIC determination of methicillin-susceptible S. aureus and wild-type E. coli as well as clinical isolates. The results obtained by this SERS-AST method were consistent with that by the standard incubation-based method, indicating its high potential to supplement or replace existing time-consuming methods and help mitigate the challenge of drug resistance in clinical microbiology.

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

Development and Validation of an Enzyme-Linked Immunosorbent Assay for the Detection of Binding Anti-Drug Antibodies against Interferon Beta

DEFF Research Database (Denmark)

Ingenhoven, Kathleen; Kramer, Daniel; Jensen, Poul Erik Hyldgaard

2017-01-01

to be 26 ng/mL using commercially available polyclonal rabbit antihuman IFN-β in human sera as the positive control. CONCLUSION: An ultrasensitive ELISA for IFN-β-binding ADA testing has been validated. This will form the basis to assess anti-biopharmaceutical immunization toward IFN-β with regards to its......OBJECTIVE: To develop and validate a method for the detection of binding anti-drug antibodies (ADAs) against interferon beta (IFN-β) in human serum as part of a European initiative (ABIRISK) aimed at the prediction and analysis of clinical relevance of anti-biopharmaceutical immunization...... to minimize the risk. METHOD: A two-tiered bridging enzyme-linked immunosorbent assay (ELISA) format was selected and validated according to current recommendations. Screening assay: ADA in serum samples form complexes with immobilized IFN-β and biotinylated IFN-β, which are then detected using HRP labeled...

Comparative susceptibility of veliger larvae of four bivalve mollusks to a Vibrio alginolyticus strain.

Science.gov (United States)

Luna-González, A; Maeda-Martínez, A N; Sainz, J C; Ascencio-Valle, F

2002-06-03

The susceptibility of 7 d old veliger larvae of the scallops Argopecten ventricosus and Nodipecten subnodosus, the penshell Atrina maura, and the Pacific oyster Crassostrea gigas to a pathogenic strain of Vibrio alginolyticus was investigated by challenging the larvae with different bacterial concentrations in a semi-static assay. The results indicate that the larvae of the 2 scallop species are more susceptible to the V. alginolyticus strain than those of the oyster and the penshell. Signs of the disease were similar to bacillary necrosis described in previous work. Interspecies differences in susceptibility to pathogens are discussed.

Host Phenology and Leaf Effects on Susceptibility of California Bay Laurel to Phytophthora ramorum.

Science.gov (United States)

Johnston, Steven F; Cohen, Michael F; Torok, Tamas; Meentemeyer, Ross K; Rank, Nathan E

2016-01-01

Spread of the plant pathogen Phytophthora ramorum, causal agent of the forest disease sudden oak death, is driven by a few competent hosts that support spore production from foliar lesions. The relationship between traits of a principal foliar host, California bay laurel (Umbellularia californica), and susceptibility to P. ramorum infection were investigated with multiple P. ramorum isolates and leaves collected from multiple trees in leaf-droplet assays. We examined whether susceptibility varies with season, leaf age, or inoculum position. Bay laurel susceptibility was highest during spring and summer and lowest in winter. Older leaves (>1 year) were more susceptible than younger ones (8 to 11 months). Susceptibility was greater at leaf tips and edges than the middle of the leaf. Leaf surfaces wiped with 70% ethanol were more susceptible to P. ramorum infection than untreated leaf surfaces. Our results indicate that seasonal changes in susceptibility of U. californica significantly influence P. ramorum infection levels. Thus, in addition to environmental variables such as temperature and moisture, variability in host plant susceptibility contributes to disease establishment of P. ramorum.

21 CFR 864.7360 - Erythrocytic glucose-6-phosphate dehydrogenase assay.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Erythrocytic glucose-6-phosphate dehydrogenase assay. 864.7360 Section 864.7360 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages...

Secondary metabolite profiles and antifungal drug susceptibility of Aspergillus fumigatus and closely related species, Aspergillus lentulus, Aspergillus udagawae, and Aspergillus viridinutans.

Science.gov (United States)

Tamiya, Hiroyuki; Ochiai, Eri; Kikuchi, Kazuyo; Yahiro, Maki; Toyotome, Takahito; Watanabe, Akira; Yaguchi, Takashi; Kamei, Katsuhiko

2015-05-01

The incidence of Aspergillus infection has been increasing in the past few years. Also, new Aspergillus fumigatus-related species, namely Aspergillus lentulus, Aspergillus udagawae, and Aspergillus viridinutans, were shown to infect humans. These fungi exhibit marked morphological similarities to A. fumigatus, albeit with different clinical courses and antifungal drug susceptibilities. The present study used liquid chromatography/time-of-flight mass spectrometry to identify the secondary metabolites secreted as virulence factors by these Aspergillus species and compared their antifungal susceptibility. The metabolite profiles varied widely among A. fumigatus, A. lentulus, A. udagawae, and A. viridinutans, producing 27, 13, 8, and 11 substances, respectively. Among the mycotoxins, fumifungin, fumiquinazoline A/B and D, fumitremorgin B, gliotoxin, sphingofungins, pseurotins, and verruculogen were only found in A. fumigatus, whereas auranthine was only found in A. lentulus. The amount of gliotoxin, one of the most abundant mycotoxins in A. fumigatus, was negligible in these related species. In addition, they had decreased susceptibility to antifungal agents such as itraconazole and voriconazole, even though metabolites that were shared in the isolates showing higher minimum inhibitory concentrations than epidemiological cutoff values were not detected. These strikingly different secondary metabolite profiles may lead to the development of more discriminative identification protocols for such closely related Aspergillus species as well as improved treatment outcomes. Copyright © 2015 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

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)

A quantitative reverse-transcriptase PCR assay for the assessment of drug activities against intracellular Theileria annulata schizonts

Directory of Open Access Journals (Sweden)

Isabel Hostettler

2014-12-01

Full Text Available Intracellular schizonts of the apicomplexans Theileria annulata and Theileria parva immortalize bovine leucocytes thereby causing fatal immunoproliferative diseases. Buparvaquone, a hydroxynaphthoquinone related to parvaquone, is the only drug available against Theileria. The drug is only effective at the onset of infection and emerging resistance underlines the need for identifying alternative compounds. Current drug assays employ monitoring of proliferation of infected cells, with apoptosis of the infected host cell as a read-out, but it is often unclear whether active compounds directly impair the viability of the parasite or primarily induce host cell death. We here report on the development of a quantitative reverse transcriptase real time PCR method based on two Theileria genes, tasp and tap104, which are both expressed in schizonts. Upon in vitro treatment of T. annulata infected bovine monocytes with buparvaquone, TaSP and Tap104 mRNA expression levels significantly decreased in relation to host cell actin already within 4 h of drug exposure, while significant differences in host cell proliferation were detectable only after 48–72 h. TEM revealed marked alterations of the schizont ultrastructure already after 2 h of buparvaquone treatment, while the host cell remained unaffected. Expression of TaSP and Tap104 proteins showed a marked decrease only after 24 h. Therefore, the analysis of expression levels of mRNA coding for TaSP and Tap104 allows to directly measuring impairment of parasite viability. We subsequently applied this method using a series of compounds affecting different targets in other apicomplexan parasites, and show that monitoring of TaSP- and Tap104 mRNA levels constitutes a suitable tool for anti-theilerial drug development.

Genetic Susceptibility to Head and Neck Squamous Cell Carcinoma

Energy Technology Data Exchange (ETDEWEB)

Lacko, Martin [Department of Otorhinolaryngology—Head and Neck Surgery, Maastricht University Medical Center, Maastricht (Netherlands); Braakhuis, Boudewijn J.M. [Department of Otolaryngology—Head and Neck Surgery, VU University Medical Center, Amsterdam (Netherlands); Sturgis, Erich M. [Department of Head and Neck Surgery and Department of Epidemiology, The University of Texas MD Anderson Cancer Center, Houston, Texas (United States); Boedeker, Carsten C. [Department of Otorhinolaryngology—Head and Neck Surgery, Albert-Ludwigs-University, Freiburg, Germany and Department of Otorhinolaryngology - Head and Neck Surgery, HELIOS Hanseklinikum Stralsund, Stralsund (Germany); Suárez, Carlos [Department of Otolaryngology, Hospital Universitario Central de Asturias, Oviedo (Spain); Instituto Universitario de Oncología del Principado de Asturias, Oviedo (Spain); Rinaldo, Alessandra; Ferlito, Alfio [ENT Clinic, University of Udine, Udine (Italy); Takes, Robert P., E-mail: robert.takes@radboudumc.nl [Department of Otolaryngology—Head and Neck Surgery, Radboud University Nijmegen Medical Center, Nijmegen (Netherlands)

2014-05-01

Head-and-neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, and its incidence is growing. Although environmental carcinogens and carcinogenic viruses are the main etiologic factors, genetic predisposition obviously plays a risk-modulating role, given that not all individuals exposed to these carcinogens experience the disease. This review highlights some aspects of genetic susceptibility to HNSCC: among others, genetic polymorphisms in biotransformation enzymes, DNA repair pathway, apoptotic pathway, human papillomavirus-related pathways, mitochondrial polymorphisms, and polymorphism related to the bilirubin-metabolized pathway. Furthermore, epigenetic variations, familial forms of HNSCC, functional assays for HNSCC risk assessment, and the implications and perspectives of research on genetic susceptibility in HNSCC are discussed.

Genetic Susceptibility to Head and Neck Squamous Cell Carcinoma

International Nuclear Information System (INIS)

Lacko, Martin; Braakhuis, Boudewijn J.M.; Sturgis, Erich M.; Boedeker, Carsten C.; Suárez, Carlos; Rinaldo, Alessandra; Ferlito, Alfio; Takes, Robert P.

2014-01-01

Head-and-neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, and its incidence is growing. Although environmental carcinogens and carcinogenic viruses are the main etiologic factors, genetic predisposition obviously plays a risk-modulating role, given that not all individuals exposed to these carcinogens experience the disease. This review highlights some aspects of genetic susceptibility to HNSCC: among others, genetic polymorphisms in biotransformation enzymes, DNA repair pathway, apoptotic pathway, human papillomavirus-related pathways, mitochondrial polymorphisms, and polymorphism related to the bilirubin-metabolized pathway. Furthermore, epigenetic variations, familial forms of HNSCC, functional assays for HNSCC risk assessment, and the implications and perspectives of research on genetic susceptibility in HNSCC are discussed

Human In Silico Drug Trials Demonstrate Higher Accuracy than Animal Models in Predicting Clinical Pro-Arrhythmic Cardiotoxicity.

Science.gov (United States)

Passini, Elisa; Britton, Oliver J; Lu, Hua Rong; Rohrbacher, Jutta; Hermans, An N; Gallacher, David J; Greig, Robert J H; Bueno-Orovio, Alfonso; Rodriguez, Blanca

2017-01-01

(fast/late Na + and Ca 2+ currents) exhibit high susceptibility to depolarization abnormalities. Repolarization abnormalities in silico predict clinical risk for all compounds with 89% accuracy. Drug-induced changes in biomarkers are in overall agreement across different assays: in silico AP duration changes reflect the ones observed in rabbit QT interval and hiPS-CMs Ca 2+ -transient, and simulated upstroke velocity captures variations in rabbit QRS complex. Our results demonstrate that human in silico drug trials constitute a powerful methodology for prediction of clinical pro-arrhythmic cardiotoxicity, ready for integration in the existing drug safety assessment pipelines.

Human In Silico Drug Trials Demonstrate Higher Accuracy than Animal Models in Predicting Clinical Pro-Arrhythmic Cardiotoxicity

Directory of Open Access Journals (Sweden)

Elisa Passini

2017-09-01

(fast/late Na+ and Ca2+ currents exhibit high susceptibility to depolarization abnormalities. Repolarization abnormalities in silico predict clinical risk for all compounds with 89% accuracy. Drug-induced changes in biomarkers are in overall agreement across different assays: in silico AP duration changes reflect the ones observed in rabbit QT interval and hiPS-CMs Ca2+-transient, and simulated upstroke velocity captures variations in rabbit QRS complex. Our results demonstrate that human in silico drug trials constitute a powerful methodology for prediction of clinical pro-arrhythmic cardiotoxicity, ready for integration in the existing drug safety assessment pipelines.

Applicability of the comet assay in evaluation of DNA damage in healthcare providers’ working with antineoplastic drugs: a systematic review and meta-analysis

Science.gov (United States)

Zare Sakhvidi, Mohammad Javad; Hajaghazadeh, Mohammad; Mostaghaci, Mehrdad; Mehrparvar, Amir houshang; Zare Sakhvidi, Fariba; Naghshineh, Elham

2016-01-01

Background Unintended occupational exposure to antineoplastic drugs (ANDs) may occur in medical personnel. Some ANDs are known human carcinogens and exposure can be monitored by genotoxic biomarkers. Objective To evaluate the obstacles to obtaining conclusive results from a comet assay test to determine DNA damage among AND exposed healthcare workers. Methods We systematically reviewed studies that used alkaline comet assay to determine the magnitude and significance of DNA damage among health care workers with potential AND exposure. Fifteen studies were eligible for review and 14 studies were used in the meta-analysis. Results Under random effect assumption, the estimated standardized mean difference (SMD) in the DNA damage of health care workers was 1.93 (95% CI: 1.15–2.71, p comet moment, I2 test results, as a measure of heterogeneity, dropped to zero. Heterogeneity analysis showed that date of study publication was a possible source of heterogeneity (B = −0.14; p comet assay methodological variables, and exposure characteristics may be responsible for heterogenic data from comet assay studies and interfere with obtaining conclusive results. Lack of quantitative environmental exposure measures and variation in comet assay protocols across studies are important obstacles in generalization of results. PMID:27110842

Susceptibility of pathogenic and nonpathogenic Naegleria ssp

International Nuclear Information System (INIS)

Whiteman, L.Y.

1988-01-01

The susceptibility of four species of Naegleria amoebae to complement-mediated lysis was determined. The amoebicidal activity of normal human serum (NHS) and normal guinea pig serum (NGPS) for Naegleria amoebae was measured by an in vitro cytotoxicity assay. Release of radioactivity from amoebae labeled with 3 H-uridine and visual observation with a compound microscope were used as indices of lysis. Susceptibility or resistance to complement-mediated lysis in vitro correlated with the in vivo pathogenic potential. Nonpathogenic Naegleria amoebae were lysed at a faster rate and at higher cell concentrations than were pathogenic amoebae. Electrophoretic analysis of NHS incubated with pathogenic or nonpathogenic Naegleria spp. demonstrated that amoebae activate the complement cascade resulting in the production of C3 and C5 complement cleavage products. Treatment with papain or trypsin for 1 h, but not with sialidase, increase the susceptibility of highly pathogenic, mouse-passaged N. fowleri to lysis. Treatment with actinomycin D, cycloheximide or various protease inhibitors for 4 h did not increase susceptibility to lysis. Neither a repair process involving de novo protein synthesis nor a complement-inactivating protease appear to account for the increase resistance of N. fowleri amoebae to complement-mediated lysis. A binding study with 125 I radiolabeled C9 indicated that the terminal complement component does not remain stably bound to the membrane of pathogenic amoebae

Has introduction of rapid drug susceptibility testing at diagnosis impacted treatment outcomes among previously treated tuberculosis patients in Gujarat, India?

Directory of Open Access Journals (Sweden)

Paresh Dave

Full Text Available Revised National TB Control Programme (RNTCP in India recommends that all previously-treated TB (PT patients are offered drug susceptibility testing (DST at diagnosis, using rapid diagnostics and screened out for rifampicin resistance before being treated with standardized, eight-month, retreatment regimen. This is intended to improve the early diagnosis of rifampicin resistance and its appropriate management and improve the treatment outcomes among the rest of the patients. In this state-wide study from Gujarat, India, we assess proportion of PT patients underwent rapid DST at diagnosis and the impact of this intervention on their treatment outcomes.This is a retrospective cohort study involving review of electronic patient-records maintained routinely under RNTCP. All PT patients registered for treatment in Gujarat during January-June 2013 were included. Information on DST and treatment outcomes were extracted from 'presumptive DR-TB patient register' and TB treatment register respectively. We performed a multivariate analysis to assess if getting tested is independently associated with unfavourable outcomes (death, loss-to-follow-up, failure, transfer out.Of 5,829 PT patients, 5306(91% were tested for drug susceptibility with rapid diagnostics. Overall, 71% (4,113 TB patients were successfully treated - 72% among tested versus 60% among non-tested. Patients who did not get tested at diagnosis had a 34% higher risk of unsuccessful outcomes as compared to those who got tested (aRR - 1.34; 95% CI 1.20-1.50 after adjusting for age, sex, HIV status and type of TB. Unfavourable outcomes (particularly failure and switched to category IV were higher among INH-resistant patients (39% as compared to INH-sensitive (29%.Offering DST at diagnosis improved the treatment outcomes among PT patients. However, even among tested, treatment outcomes remained suboptimal and were related to INH resistance and high loss-to-follow-up. These need to be addressed

Advances in establishment and analysis of three-dimensional tumor spheroid-based functional assays for target validation and drug evaluation

Directory of Open Access Journals (Sweden)

Vinci Maria

2012-03-01

Full Text Available Abstract Background There is overwhelming evidence that in vitro three-dimensional tumor cell cultures more accurately reflect the complex in vivo microenvironment than simple two-dimensional cell monolayers, not least with respect to gene expression profiles, signaling pathway activity and drug sensitivity. However, most currently available three-dimensional techniques are time consuming and/or lack reproducibility; thus standardized and rapid protocols are urgently needed. Results To address this requirement, we have developed a versatile toolkit of reproducible three-dimensional tumor spheroid models for dynamic, automated, quantitative imaging and analysis that are compatible with routine high-throughput preclinical studies. Not only do these microplate methods measure three-dimensional tumor growth, but they have also been significantly enhanced to facilitate a range of functional assays exemplifying additional key hallmarks of cancer, namely cell motility and matrix invasion. Moreover, mutual tissue invasion and angiogenesis is accommodated by coculturing tumor spheroids with murine embryoid bodies within which angiogenic differentiation occurs. Highly malignant human tumor cells were selected to exemplify therapeutic effects of three specific molecularly-targeted agents: PI-103 (phosphatidylinositol-3-kinase (PI3K-mammalian target of rapamycin (mTOR inhibitor, 17-N-allylamino-17-demethoxygeldanamycin (17-AAG (heat shock protein 90 (HSP90 inhibitor and CCT130234 (in-house phospholipase C (PLCγ inhibitor. Fully automated analysis using a Celigo cytometer was validated for tumor spheroid growth and invasion against standard image analysis techniques, with excellent reproducibility and significantly increased throughput. In addition, we discovered key differential sensitivities to targeted agents between two-dimensional and three-dimensional cultures, and also demonstrated enhanced potency of some agents against cell migration

Human neuron-astrocyte 3D co-culture-based assay for evaluation of neuroprotective compounds.

Science.gov (United States)

Terrasso, Ana Paula; Silva, Ana Carina; Filipe, Augusto; Pedroso, Pedro; Ferreira, Ana Lúcia; Alves, Paula Marques; Brito, Catarina

Central nervous system drug development has registered high attrition rates, mainly due to the lack of efficacy of drug candidates, highlighting the low reliability of the models used in early-stage drug development and the need for new in vitro human cell-based models and assays to accurately identify and validate drug candidates. 3D human cell models can include different tissue cell types and represent the spatiotemporal context of the original tissue (co-cultures), allowing the establishment of biologically-relevant cell-cell and cell-extracellular matrix interactions. Nevertheless, exploitation of these 3D models for neuroprotection assessment has been limited due to the lack of data to validate such 3D co-culture approaches. In this work we combined a 3D human neuron-astrocyte co-culture with a cell viability endpoint for the implementation of a novel in vitro neuroprotection assay, over an oxidative insult. Neuroprotection assay robustness and specificity, and the applicability of Presto Blue, MTT and CytoTox-Glo viability assays to the 3D co-culture were evaluated. Presto Blue was the adequate endpoint as it is non-destructive and is a simpler and reliable assay. Semi-automation of the cell viability endpoint was performed, indicating that the assay setup is amenable to be transferred to automated screening platforms. Finally, the neuroprotection assay setup was applied to a series of 36 test compounds and several candidates with higher neuroprotective effect than the positive control, Idebenone, were identified. The robustness and simplicity of the implemented neuroprotection assay with the cell viability endpoint enables the use of more complex and reliable 3D in vitro cell models to identify and validate drug candidates. Copyright © 2016 Elsevier Inc. All rights reserved.

Molecular identification, antifungal susceptibility profile, and biofilm formation of clinical and environmental Rhodotorula species isolates.

Science.gov (United States)

Nunes, Jorge Meneses; Bizerra, Fernando César; Ferreira, Renata Carmona E; Colombo, Arnaldo Lopes

2013-01-01

Rhodotorula species are emergent fungal pathogens capable of causing invasive infections, primarily fungemia. They are particularly problematic in immunosuppressed patients when using a central venous catheter. In this study, we evaluated the species distribution of 51 clinical and 8 environmental Rhodotorula species isolates using the ID32C system and internal transcribed spacer (ITS) sequencing. Antifungal susceptibility testing and biofilm formation capability using a crystal violet staining assay were performed. Using ITS sequencing as the gold standard, the clinical isolates were identified as follows: 44 R. mucilaginosa isolates, 2 R. glutinis isolates, 2 R. minuta isolates, 2 R. dairenensis isolates, and 1 Rhodosporidium fluviale isolate. The environmental isolates included 7 R. mucilaginosa isolates and 1 R. slooffiae isolate. Using the ID32C system, along with a nitrate assimilation test, only 90.3% of the isolates tested were correctly identified. In the biofilm formation assay, R. mucilaginosa and R. minuta exhibited greater biofilm formation ability compared to the other Rhodotorula species; the clinical isolates of R. mucilaginosa showed greater biofilm formation compared to the environmental isolates (P = 0.04). Amphotericin B showed good in vitro activity (MIC ≤ 1 μg/ml) against planktonic cells, whereas voriconazole and posaconazole showed poor activity (MIC(50)/MIC(90), 2/4 μg/ml). Caspofungin and fluconazole MICs were consistently high for all isolates tested (≥64 μg/ml and ≥ 4 μg/ml, respectively). In this study, we emphasized the importance of molecular methods to correctly identify Rhodotorula species isolates and non-R. mucilaginosa species in particular. The antifungal susceptibility profile reinforces amphotericin B as the antifungal drug of choice for the treatment of Rhodotorula infections. To our knowledge, this is the first study evaluating putative differences in the ability of biofilm formation among different Rhodotorula

Antifungals susceptibility pattern of Candida spp . isolated from ...

African Journals Online (AJOL)

The highest susceptibility of the isolates was seen for nystatin 62 (83.78%), ketoconazole 61 (82.43%) and fluconazole 60 (81.08%). Conclusion: Despite the noticeable resistance of Candida spp. isolates to miconazole and itraconazole, the results indicate that nystatin, ketoconazole and fluconazole are the drugs of choice ...

Moving beyond the comprehensive in vitro proarrhythmia assay: Use of human-induced pluripotent stem cell-derived cardiomyocytes to assess contractile effects associated with drug-induced structural cardiotoxicity.

Science.gov (United States)

Yang, Xi; Papoian, Thomas

2018-02-27

Drug-induced cardiotoxicity is a potentially severe side effect that can adversely affect myocardial contractility through structural or electrophysiological changes in cardiomyocytes. Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are a promising human cardiac in vitro model system to assess both proarrhythmic and non-proarrhythmic cardiotoxicity of new drug candidates. The scalable differentiation of hiPSCs into cardiomyocytes provides a renewable cell source that overcomes species differences present in current animal models of drug toxicity testing. The Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative represents a paradigm shift for proarrhythmic risk assessment, and hiPSC-CMs are an integral component of that paradigm. The recent advancements in hiPSC-CMs will not only impact safety decisions for possible drug-induced proarrhythmia, but should also facilitate risk assessment for non-proarrhythmic cardiotoxicity, where current non-clinical approaches are limited in detecting this risk before initiation of clinical trials. Importantly, emerging evidence strongly suggests that the use of hiPSC-CMs with cardiac physiological relevant measurements in vitro improves the detection of structural cardiotoxicity. Here we review high-throughput drug screening using the hiPSC-CM model as an experimentally feasible approach to assess potential contractile and structural cardiotoxicity in early phase drug development. We also suggest that the assessment of structural cardiotoxicity can be added to electrophysiological tests in the same platform to complement the Comprehensive in vitro Proarrhythmia Assay for regulatory use. Ideally, application of these novel tools in early drug development will allow for more reliable risk assessment and lead to more informed regulatory decisions in making safe and effective drugs available to the public. Published 2018. This article is a U.S. Government work and is in the public domain in the USA.

Conjugation of metronidazole with dextran: a potential pharmaceutical strategy to control colonic distribution of the anti-amebic drug susceptible to metabolism by colonic microbes.

Science.gov (United States)

Kim, Wooseong; Yang, Yejin; Kim, Dohoon; Jeong, Seongkeun; Yoo, Jin-Wook; Yoon, Jeong-Hyun; Jung, Yunjin

2017-01-01

Metronidazole (MTDZ), the drug of choice for the treatment of protozoal infections such as luminal amebiasis, is highly susceptible to colonic metabolism, which may hinder its conversion from a colon-specific prodrug to an effective anti-amebic agent targeting the entire large intestine. Thus, in an attempt to control the colonic distribution of the drug, a polymeric colon-specific prodrug, MTDZ conjugated to dextran via a succinate linker (Dex-SA-MTDZ), was designed. Upon treatment with dextranase for 8 h, the degree of Dex-SA-MTDZ depolymerization (%) with a degree of substitution (mg of MTDZ bound in 100 mg of Dex-SA-MTDZ) of 7, 17, and 30 was 72, 38, and 8, respectively, while that of dextran was 85. Depolymerization of Dex-SA-MTDZ was found to be necessary for the release of MTDZ, because dextranase pretreatment ensures that de-esterification occurs between MTDZ and the dextran backbone. In parallel, Dex-SA-MTDZ with a degree of substitution of 17 was found not to release MTDZ upon incubation with the contents of the small intestine and stomach of rats, but it released MTDZ when incubated with rat cecal contents (including microbial dextranases). Moreover, Dex-SA-MTDZ exhibited prolonged release of MTDZ, which contrasts with drug release by small molecular colon-specific prodrugs, MTDZ sulfate and N -nicotinoyl-2-{2-(2-methyl-5-nitroimidazol-1-yl)ethyloxy}-d,l-glycine. These prodrugs were eliminated very rapidly, and no MTDZ was detected in the cecal contents. Consistent with these in vitro results, we found that oral gavage of Dex-SA-MTDZ delivered MTDZ (as MTDZ conjugated to [depolymerized] dextran) to the distal colon. However, upon oral gavage of the small molecular prodrugs, no prodrugs were detected in the distal colon. Collectively, these data suggest that dextran conjugation is a potential pharmaceutical strategy to control the colonic distribution of drugs susceptible to colonic microbial metabolism.

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

Science.gov (United States)