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Sample records for blood culture pcr

  1. Seminested PCR for detection and identification of Candida species directly from blood culture bottles.

    Science.gov (United States)

    Cerikçioğlu, Nilgün; Aksu, Burak; Dal, Tuba Demirel; Deniz, Umut; Bilgen, Hülya Selva; Ozek, Eren; Söyletir, Güner

    2010-01-01

    We investigated the performance of a seminested PCR (snPCR) assay carried out directly from overnight incubated blood culture bottles of 50 newborn intensive care unit (NICU) patients with suspected candidemia and compared these, for sensitivity, specificity and reliability with results from blood cultures. All positive blood cultures (n = 17) yielded positive results for snPCR, which detected the same Candida species, as did the yeast isolates of which 13 were C. parapsilosis and 4 were C. albicans. With both assays showing 32 negative samples and one sample positive with snPCR but negative with blood culture, sensitivity and specificity of snPCR were 100% and 97%, respectively. The patient with contradictory results exhibited a positive blood culture one week later yielding the same species as identified by snPCR. These are the first data demonstrating that snPCR from overnight blood culture bottles can be a potential tool for rapid detection and identification of Candida species, allowing follow-up of the "gold standard" blood culturing, as well.

  2. Comparison of Real-time PCR method and blood culture in diagnosis of septicemia

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    Ali Gholami

    2016-02-01

    Full Text Available Background: Bloodstream infections (BSI have a high incidence and high mortality in the worldwide. The mortality rate is variable between 20-70%. Therefore, early and timely detection of BSI agent in clinical laboratories is necessary. The aim of this study was to determine an efficient diagnostic tool to septicemia in accompany of blood culture method by Real-time PCR (using panbacterial 23S rRNA gene. Methods: This cross-sectional study was conducted in two analytical and clinical stages in Hamadan University of Medical Sciences, Iran, from October 2014 to June 2015. In analytical stage, sensitivity (by serial dilution from 104 to 1 CFU/ml and specificity of the primer were evaluated with the Staphylococcus aureus (as Gram positive indicator bacteria and Escherichia coli (as Gram-negative indicator bacteria, human genome (from Hella cell culture, Candida albicans yeast and Aspergillus fumigatus fungus. In clinical stage, 121 blood samples were collected from patients suspected to sepsis in intensive care unit (ICU from Hamadan University Hospitals. Finally, the results of Real-time PCR and blood culture methods were compared. Results: The Real-time PCR showed a sensitivity ranging from 2 to 10 target copies per reaction to the whole blood for Escherichia coli and Staphylococcus aureus respectively. The specificity of this method was evaluated and no false positive amplification was identified. 57.85% (70 cases of the samples were positive by Real-time PCR and 13.22% (16 cases of the samples were positive by blood culture. However, none of the cases that were positive by blood culture were negative in Real-time PCR. As well as, 44.62% (54 cases of cases were positive by Real-time PCR but blood culture showed no bacteria in the samples, and 42.15% (51 cases were negative by both methods. Correlation or agreement of Kappa was 0.20, that indicating poor agreement between the two methods. Conclusion: Real-time PCR is more sensitive than blood

  3. PCR amplification of Bartonella koehlerae from human blood and enrichment blood cultures

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    Breitschwerdt Edward B

    2010-08-01

    Full Text Available Abstract Background Cats appear to be the primary reservoir host for Bartonella koehlerae, an alpha Proteobacteria that is most likely transmitted among cat populations by fleas (Ctenocephalides felis. Bartonella koehlerae has caused endocarditis in a dog and in one human patient from Israel, but other clinically relevant reports involving this bacterium are lacking. Despite publication of numerous, worldwide epidemiological studies designed to determine the prevalence of Bartonella spp. bacteremia in cats, B. koehlerae has never been isolated using conventional blood agar plates. To date, successful isolation of B. koehlerae from cats and from the one human endocarditis patient has consistently required the use of chocolate agar plates. Results In this study, Bartonella koehlerae bacteremia was documented in eight immunocompetent patients by PCR amplification and DNA sequencing, either prior to or after enrichment blood culture using Bartonella alpha Proteobacteria growth medium. Presenting symptoms most often included fatigue, insomnia, joint pain, headache, memory loss, and muscle pain. Four patients were also infected with Bartonella vinsonii subsp. berkhoffii genotype II. After molecular documentation of B. koehlerae infection in these patients, a serological test was developed and serum samples were tested retrospectively. Bartonella koehlerae antibodies were not detected (titers B. koehlerae antibody titers of 1:64 or greater. Conclusions Although biased by a study population consisting of individuals with extensive arthropod and animal exposure, the results of this study suggest that B. koehlerae bacteremia is more common in immunocompetent people than has been previously suspected. Future studies should more thoroughly define modes of transmission and risk factors for acquiring infection with B. koehlerae. In addition, studies are needed to determine if B. koehlerae is a cause or cofactor in the development of arthritis, peripheral

  4. Rapid detection of Pseudomonas aeruginosa from positive blood cultures by quantitative PCR

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    Cattoir Vincent

    2010-08-01

    Full Text Available Abstract Background Pseudomonas aeruginosa is responsible for numerous bloodstream infections associated with severe adverse outcomes in case of inappropriate initial antimicrobial therapy. The present study was aimed to develop a novel quantitative PCR (qPCR assay, using ecfX as the specific target gene, for the rapid and accurate identification of P. aeruginosa from positive blood cultures (BCs. Methods Over the period August 2008 to June 2009, 100 BC bottles positive for gram-negative bacilli were tested in order to evaluate performances of the qPCR technique with conventional methods as gold standard (i.e. culture and phenotypic identification. Results Thirty-three strains of P. aeruginosa, 53 strains of Enterobactericaeae, nine strains of Stenotrophomonas maltophilia and two other gram-negative species were isolated while 3 BCs were polymicrobial including one mixture containing P. aeruginosa. All P. aeruginosa clinical isolates were detected by qPCR except a single strain in mixed culture. Performances of the qPCR technique were: specificity, 100%; positive predictive value, 100%; negative predictive value, 98.5%; and sensitivity, 97%. Conclusions This reliable technique may offer a rapid (

  5. Development and Evaluation of a Blood Culture PCR Assay for Rapid Detection of Salmonella Paratyphi A in Clinical Samples

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    Zhou, Liqing; Jones, Claire; Gibani, Malick M.; Dobinson, Hazel; Thomaides-Brears, Helena; Shrestha, Sonu; Blohmke, Christoph J.; Darton, Thomas C.; Pollard, Andrew J.

    2016-01-01

    Background Enteric fever remains an important cause of morbidity in many low-income countries and Salmonella Paratyphi A has emerged as the aetiological agent in an increasing proportion of cases. Lack of adequate diagnostics hinders early diagnosis and prompt treatment of both typhoid and paratyphoid but development of assays to identify paratyphoid has been particularly neglected. Here we describe the development of a rapid and sensitive blood culture PCR method for detection of Salmonella Paratyphi A from blood, potentially allowing for appropriate diagnosis and antimicrobial treatment to be initiated on the same day. Methods Venous blood samples from volunteers experimentally challenged orally with Salmonella Paratyphi A, who subsequently developed paratyphoid, were taken on the day of diagnosis; 10 ml for quantitative blood culture and automated blood culture, and 5 ml for blood culture PCR. In the latter assay, bacteria were grown in tryptone soy broth containing 2.4% ox bile and micrococcal nuclease for 5 hours (37°C) before bacterial DNA was isolated for PCR detection targeting the fliC-a gene of Salmonella Paratyphi A. Results An optimized broth containing 2.4% ox bile and micrococcal nuclease, as well as a PCR test was developed for a blood culture PCR assay of Salmonella Paratyphi A. The volunteers diagnosed with paratyphoid had a median bacterial burden of 1 (range 0.1–6.9) CFU/ml blood. All the blood culture PCR positive cases where a positive bacterial growth was shown by quantitative blood culture had a bacterial burden of ≥ 0.3 CFU/ ml blood. The blood culture PCR assay identified an equal number of positive cases as automated blood culture at higher bacterial loads (≥0.3 CFU/ml blood), but utilized only half the volume of specimens. Conclusions The blood culture PCR method for detection of Salmonella Paratyphi A can be completed within 9 hours and offers the potential for same-day diagnosis of enteric fever. Using 5 ml blood, it exhibited a

  6. Multiplex tandem PCR: a novel platform for rapid detection and identification of fungal pathogens from blood culture specimens.

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    Lau, Anna; Sorrell, Tania C; Chen, Sharon; Stanley, Keith; Iredell, Jonathan; Halliday, Catriona

    2008-09-01

    We describe the first development and evaluation of a rapid multiplex tandem PCR (MT-PCR) assay for the detection and identification of fungi directly from blood culture specimens that have been flagged as positive. The assay uses a short-cycle multiplex amplification, followed by 12 simultaneous PCRs which target the fungal internal transcribed spacer 1 (ITS1) and ITS2 region, elongation factor 1-alpha (EF1-alpha), and beta-tubulin genes to identify 11 fungal pathogens: Candida albicans, Candida dubliniensis, Candida glabrata, Candida guilliermondii, Candida krusei, Candida parapsilosis complex, Candida tropicalis, Cryptococcus neoformans complex, Fusarium solani, Fusarium species, and Scedosporium prolificans. The presence or absence of a fungal target was confirmed by melting curve analysis. Identification by MT-PCR correlated with culture-based identification for 44 (100%) patients. No cross-reactivity was detected in 200 blood culture specimens that contained bacteria or in 30 blood cultures without microorganisms. Fungi were correctly identified in five specimens with bacterial coinfection and in blood culture samples that were seeded with a mixture of yeast cells. The MT-PCR assay was able to provide rapid (detection and identification of fungal pathogens directly from blood culture specimens.

  7. Prospective comparison of a PCR assay and a microbiological culture technique for identification of pathogens from blood and non-blood samples in septic patients.

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    Plettig, Runa; Nowak, Andreas; Balau, Veronika; Hahnenkamp, Klaus; Usichenko, Taras

    2015-01-01

    Molecular amplification techniques are suggested to be a useful adjunct in early detection of pathogens in septic patients. The aim was to study the feasibility of a polymerase chain reaction (PCR) assay compared to the standard microbiological culture (MC) technique in identification of pathogenic microorganisms from blood and non-blood samples in septic patients. Samples for pathogen identification were taken during febrile septic episodes (SE) in 54 patients with sepsis and analyzed using both MC and PCR. Semi-automated multiplex PCR, provided by Philips Medical Systems, was able to detect nine different pathogens. The accuracy of pathogen identification using PCR vs. MC as well as the time-saving effect of PCR on the potential decision-making process for antimicrobial therapy was evaluated. In a total of 258 samples taken during 87 SE, both methods yielded more pathogens from the non-blood than blood samples (87 % vs. 45 %; p = 0.002). PCR identified more pathogens than MC in the blood samples (98 vs. 21; p technique. In the non-blood samples, PCR was comparable to that of MC.

  8. Blood culture

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    Culture - blood ... A blood sample is needed . The site where blood will be drawn is first cleaned with an antiseptic such ... organism from the skin getting into (contaminating) the blood sample and causing a false-positive result (see ...

  9. Multiplex Tandem PCR: a Novel Platform for Rapid Detection and Identification of Fungal Pathogens from Blood Culture Specimens▿

    OpenAIRE

    Lau, Anna; Sorrell, Tania C.; Chen, Sharon; Stanley, Keith; Iredell, Jonathan; Halliday, Catriona

    2008-01-01

    We describe the first development and evaluation of a rapid multiplex tandem PCR (MT-PCR) assay for the detection and identification of fungi directly from blood culture specimens that have been flagged as positive. The assay uses a short-cycle multiplex amplification, followed by 12 simultaneous PCRs which target the fungal internal transcribed spacer 1 (ITS1) and ITS2 region, elongation factor 1-α (EF1-α), and β-tubulin genes to identify 11 fungal pathogens: Candida albicans, Candida dublin...

  10. Enteroviruses in blood of patients with type 1 diabetes detected by integrated cell culture and reverse transcription quantitative real-time PCR.

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    Alidjinou, Enagnon Kazali; Sane, Famara; Lefevre, Christine; Baras, Agathe; Moumna, Ilham; Engelmann, Ilka; Vantyghem, Marie-Christine; Hober, Didier

    2017-11-01

    Enteroviruses (EV) have been associated with type 1 diabetes (T1D), but EV RNA detection has been reported in only a small proportion of T1D patients. We studied whether integrated cell culture and reverse transcription real-time PCR could improve EV detection in blood samples from patients with T1D. Blood was collected from 13 patients with T1D. The presence of EV RNA in blood was investigated by using real-time RT-PCR. In addition, plasma and white blood cells (WBC) were inoculated to BGM and Vero cell line cultures. Culture supernatants and cells collected on day 7 and day 14 were tested for EV RNA by real-time RT-PCR. Enterovirus identification was performed through sequencing of the VP4/VP2 region. Enterovirus RNA was detected in blood by using real-time RT-PCR in only one out of 13 patients. The detection of EV RNA in cultures inoculated with clinical samples (plasma and/or WBC) gave positive results in five other patients. The viral loads were low, ranging from 45 to 4420 copies/ng of total RNA. One isolate was successfully identified as coxsackievirus B1. Integrated cell culture and reverse transcription real-time PCR can improve the detection rate of EV in blood samples of patients with T1D and can be useful to investigate further the relationship between EV and the disease.

  11. Early detection and identification of commonly encountered Candida species from simulated blood cultures by using a real-time PCR-based assay.

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    Maaroufi, Younes; De Bruyne, Jean-Marc; Duchateau, Valérie; Georgala, Aspasia; Crokaert, Françoise

    2004-05-01

    In a recent study, Candida species in clinical blood samples were detected using a real-time PCR-based method (Maaroufi et al, J Clin Microbiol 2003, 41:3293-3298). For the present study, we evaluated the efficiency of this method as an adjunct to the BACTEC blood culture system to early detection of positivity and negativity of simulated low candidemias. We first established an in vitro correlation between the inoculum of the most frequently encountered Candida species and the time to positivity of these microorganisms. Then, aliquots from blood culture bottles infected with a final average candidal inoculum of 3.18 colony-forming units (CFU)/culture bottle (range, 1 to 6 CFU) were collected at increasing incubation times, and DNA was extracted and submitted to the TaqMan-based PCR assay. To optimize this assay, we evaluated the effect of adding 0.5% bovine serum albumin (BSA) to DNA extracts and found that it decreased the effects of inhibitors. Using specific probes for the tested Candida species, the PCR assay was positive on blood culture aliquots collected from the BACTEC system after a minimum culture turnaround time (TAT) of 3.11 +/- 1.24 hours. Addition of BSA to PCR reaction mixtures improves the TAT (1.84 +/- 0.41 hours). Hence, the combination of DNA "amplification" in the culture bottles by normal growth with an additional DNA amplification by PCR might be a reliable tool facilitating the early diagnosis of low candidemias.

  12. Comparison of nested, multiplex, qPCR; FISH; SeptiFast and blood culture methods in detection and identification of bacteria and fungi in blood of patients with sepsis

    OpenAIRE

    Gosiewski, Tomasz; Flis, Agnieszka; Sroka, Agnieszka; K?dzierska, Anna; Pietrzyk, Agata; K?dzierska, Jolanta; Drwi?a, Rafa?; Bulanda, Ma?gorzata

    2014-01-01

    Background Microbiological diagnosis of sepsis relies primarily on blood culture data. This study compares four diagnostic methods, i.e. those developed by us: nested, multiplex, qPCR (qPCR) and FISH with commercial methods: SeptiFast (Roche) (SF) and BacT/ALERT? 3D blood culture system (bioM?rieux). Blood samples were derived from adult patients with clinical symptoms of sepsis, according to SIRS criteria, hospitalized in the Intensive Care Unit. Results Using qPCR, FISH, SF, and culture, mi...

  13. PCR identification of bacteria in blood culture does not fit the daily workflow of a routine microbiology laboratory.

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    Karumaa, Santra; Kärpänoja, Pauliina; Sarkkinen, Hannu

    2012-03-01

    We have evaluated the GenoType blood culture assay (Hain Lifescience, Nehren, Germany) for the identification of bacteria in 233 positive blood cultures and assessed its suitability in the workflow of a routine microbiology laboratory. In 68/233 (29.2%) samples, the culture result could not be confirmed by the GenoType assay due to a lack of primers in the test, multiple organisms in the sample, or inconsistency with respect to the identification by culture. Although the GenoType blood culture assay gives satisfactory results for bacteria for which primers are available, there are difficulties in applying the test in the routine microbiology laboratory.

  14. Improved detection of Burkholderia pseudomallei from non-blood clinical specimens using enrichment culture and PCR: narrowing diagnostic gap in resource-constrained settings.

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    Tellapragada, Chaitanya; Shaw, Tushar; D'Souza, Annet; Eshwara, Vandana Kalwaje; Mukhopadhyay, Chiranjay

    2017-07-01

    To evaluate the diagnostic utility of enrichment culture and PCR for improved case detection rates of non-bacteraemic form of melioidosis in limited resource settings. Clinical specimens (n = 525) obtained from patients presenting at a tertiary care hospital of South India with clinical symptoms suggestive of community-acquired pneumonia, lower respiratory tract infections, superficial or internal abscesses, chronic skin ulcers and bone or joint infections were tested for the presence of Burkholderia pseudomallei using conventional culture (CC), enrichment culture (EC) and PCR. Sensitivity, specificity, positive and negative predictive values of CC and PCR were initially deduced using EC as the gold standard method. Further, diagnostic accuracies of all the three methods were analysed using Bayesian latent class modelling (BLCM). Detection rates of B. pseudomallei using CC, EC and PCR were 3.8%, 5.3% and 6%, respectively. Diagnostic sensitivities and specificities of CC and PCR were 71.4, 98.4% and 100 and 99.4%, respectively in comparison with EC as the gold standard test. With Bayesian latent class modelling, EC and PCR demonstrated sensitivities of 98.7 and 99.3%, respectively, while CC showed a sensitivity of 70.3% for detection of B. pseudomallei. An increase of 1.6% (95% CI: 1.08-4.32%) in the case detection rate of melioidosis was observed in the study population when EC and/or PCR were used in adjunct to the conventional culture technique. Our study findings underscore the diagnostic superiority of enrichment culture and/or PCR over conventional microbiological culture for improved case detection of melioidosis from non-blood clinical specimens. © 2017 John Wiley & Sons Ltd.

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

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    Zhu, Lingxiang; Shen, Dingxia; Zhou, Qiming; Li, Zexia; Fang, Xiangdong; Li, Quan-Zhen

    2015-01-01

    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.

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

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    Lingxiang Zhu

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

  17. Rapid Detection of blaKPC Carbapenemase Genes by Internally Controlled Real-Time PCR Assay Using Bactec Blood Culture Bottles ▿

    OpenAIRE

    Hindiyeh, Musa; Smollan, Gill; Grossman, Zehava; Ram, Daniela; Robinov, Jana; Belausov, Natasha; Ben-David, Debbie; Tal, Ilana; Davidson, Yehudit; Shamiss, Ari; Mendelson, Ella; Keller, Nathan

    2011-01-01

    Rapid detection of drug-resistant bacteria in clinical samples plays an instrumental role in patients' infection management and in implementing effective infection control policies. In the study described in this report, we validated a multiplex TaqMan real-time quantitative PCR (qPCR) assay for the detection of blaKPC genes and the human RNase P gene in Bactec blood culture bottles. The MagNA Pure LC (version 2.0) instrument was utilized to extract nucleic acids from the inoculated broth, wh...

  18. Multiplex real-time PCR and blood culture for identification of bloodstream pathogens in patients with suspected sepsis

    DEFF Research Database (Denmark)

    Westh, H; Lisby, G; Breysse, F

    2009-01-01

    species directly from blood was used, comparatively with BC, in a multicentre trial of patients with suspected bacterial or fungal sepsis. Five hundred and fifty-eight paired samples from 359 patients were evaluated. The rate of positivity was 17% for BC and 26% for SeptiFast. Ninety-six microorganisms...... in the SeptiFast master list, and six BC isolates were identified as a species not included in the SeptiFast master list. With SeptiFast, 186 microorganisms were identified, 12 of which were considered to be contaminants. Of the 174 clinically relevant microorganisms identified with SeptiFast, 50 (29%) were...... detected by BC. More than half of the remaining microorganisms identified with SeptiFast (but not isolated after BC) were also found in routine cultures of other relevant samples taken from the patients. Future clinical studies should assess whether the use of SeptiFast is of significant advantage...

  19. Evaluation of fliC-d based direct blood PCR assays for typhoid diagnosis

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    Das, Surojit; Ray, Ujjwayini; Akhter, Irfaan; Chattopadhyay, Arka; Paul, Dilip Kumar; Dutta, Shanta

    2016-01-01

    Background Typhoid cases need to be diagnosed accurately for early antibiotic therapy and reducing mortality. Identification of Salmonella Typhi (S. Typhi) in blood culture is conclusive, but has poor sensitivity. Detection of S. Typhi by PCR from blood sample has shown promise. Real-time quantitative PCR (Q-PCR) has been widely used in diagnostics for its rapidity and reliability. In the present study, the performance of molecular methods like conventional PCR (C-PCR), nested PCR (N-PCR) and...

  20. Detection of Streptococcus pneumoniae in whole blood by PCR.

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    Zhang, Y; Isaacman, D J; Wadowsky, R M; Rydquist-White, J; Post, J C; Ehrlich, G D

    1995-03-01

    Streptococcus pneumoniae is a major cause of bacteremia in both children and adults. Currently, the diagnosis of pneumococcal bacteremia relies on the isolation and identification of the bacteria from blood cultures. We have developed a sensitive assay for the detection of S. pneumoniae in whole blood by the PCR. A specific primer-probe set (JM201 and JM202 primers with JM204 probe) designed from the penicillin-binding protein 2B gene was demonstrated to reproducibly detect between 10 and 100 fg of input purified S. pneumoniae DNA. This assay system was shown to be inclusive for all strains of S. pneumoniae evaluated, including 15 different serotypes and a battery of penicillin-resistant and -sensitive strains. The specificity of this PCR-based assay was demonstrated by its inability to support amplification from a series of human, bacterial, and yeast genomic DNAs. A general specimen preparation method which should be suitable for the purification of DNA from any pathogens in whole blood was developed. With this protocol it was possible to detect S. pneumoniae-specific DNA from whole blood specimens inoculated with as little as 4 CFU/ml. Copurified human blood DNA, ranging from 0 to 4.5 micrograms per PCR, did not affect the sensitivity of S. pneumoniae detection by PCR. A blinded clinical trial was used to compare the PCR-based assay with standard microbiological blood culture for the detection of S. pneumoniae bacteremia in 36 specimens obtained from pediatric patients seen in the emergency room of Children's Hospital of Pittsburgh. With culture as the "gold standard," the PCR-based assay had a sensitivity of 80% (4 of 5 culture-positive specimens were PCR positive) and a specificity of 84% (26 of 31 culture-negative specimens were PCR negative). However, three patients whose specimens were PCR positive and culture negative had histories suggestive of bacteremia, including recent positive blood cultures, treatment with antibiotics, cellulitis, and multiple

  1. Cost-Effectiveness Analysis of Multiplex PCR with Magnetic Resonance Detection versus Empiric or Blood Culture-Directed Therapy for Management of Suspected Candidemia.

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    Walker, Brandon; Powers-Fletcher, Margaret V; Schmidt, Robert L; Hanson, Kimberly E

    2016-03-01

    Candida bloodstream infections (BSI) are associated with significant morbidity, mortality, and increased health care costs. Early treatment is essential, because delayed therapy detrimentally impacts clinical outcomes. The FDA recently approved the first culture-independent direct molecular detection method for Candida BSIs (T2Candida). The speed and sensitivity of this assay give it the potential to improve patient care, but the reagents and instrumentation are expensive. We used an analytic decision tree model to compare the cost-effectiveness of T2Candida-directed antifungal therapy (T2DT) to that of either empirical therapy (ET) or blood culture-directed therapy (BCDT). The costs included those of T2Candida testing, antifungal treatment, and hospital length of stay. The effectiveness measure was survival status at hospital discharge. T2DT was less costly and more effective than BCDT but was less costly and less effective than ET with an echinocandin (incremental cost-effectiveness ratio, $111,084 per additional survivor). One-way sensitivity analyses demonstrated that the cost-effectiveness of T2DT was highly dependent on Candida BSI prevalence and the cost of antifungal therapy and T2Candida test reagents. The use of T2DT reduced the number of unnecessarily treated patients by 98% relative to that with ET. Reduced drug exposure might lessen the possibility of drug-related adverse events and may also prevent the development of antifungal resistance or emergence of drug-resistant Candida species. The greatest benefit of T2Candida appears to be the ability to confidently withhold or stop empirical antifungal therapy in low-to-moderate-risk patients who are unlikely to benefit from treatment. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  2. Pathogens in Maternal Blood and Fetal Cord Blood Using Q-Pcr Assay

    OpenAIRE

    Guang Qiong Hou; Sidney S. Chen; Chan Ping Lee

    2006-01-01

    Objective: To evaluate the rates of infection of asymptomatic fetuses and mothers through fetal cord blood and maternal blood examination results. Materials and Methods: Quantitative PCR (Q-PCR) was used to detect pathogens in maternal peripheral blood and fetal cord blood after delivery of term pregnancy at Buddhist Tzu Chi Medical Center, Hualien, between July 2002 and June 2003. Results: We used Q-PCR to detect pathogens in 29 samples of maternal blood. The maternal hepatitis B virus...

  3. Classification of positive blood cultures

    DEFF Research Database (Denmark)

    Gradel, Kim Oren; Knudsen, Jenny Dahl; Arpi, Magnus

    2012-01-01

    ABSTRACT: BACKGROUND: Information from blood cultures is utilized for infection control, public health surveillance, and clinical outcome research. This information can be enriched by physicians assessments of positive blood cultures, which are, however, often available from selected patient groups...... or pathogens only. The aim of this work was to determine whether patients with positive blood cultures can be classified effectively for outcome research in epidemiological studies by the use of administrative data and computer algorithms, taking physicians assessments as reference. METHODS: Physicians...... assessments of positive blood cultures were routinely recorded at two Danish hospitals from 2006 through 2008. The physicians assessments classified positive blood cultures as: a) contamination or bloodstream infection; b) bloodstream infection as mono- or polymicrobial; c) bloodstream infection as community...

  4. Blood Culture Test

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    ... normal" values. By comparing your test results with reference values, you and your healthcare provider can see if ... most effective in treating the infection Often, a complete blood count (CBC) is ordered along with or prior to ...

  5. Rapid diagnosis of candidaemia by real-time PCR detection of Candida DNA in blood samples.

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    Wellinghausen, Nele; Siegel, Dunja; Winter, Juliane; Gebert, Susanne

    2009-08-01

    This study prospectively evaluated an 18S rRNA gene-targeted real-time PCR approach in comparison with standard blood culture (BC) diagnostics for rapid diagnosis of candidaemia in a large study population of 384 patients, including 902 whole blood samples from 468 infectious episodes (IEs) of 329 adults and 55 children with haematological malignancies and various forms of immunodeficiency, and intensive care unit patients. Seven out of eight BC-proven cases (87.5 %) of candidaemia and seven out of twelve BC-positive samples (58.3 %) were positive by the Candida-specific PCR. A positive PCR result was also obtained for 28/460 BC-negative samples from IEs, including 8 patients with culture-confirmed Candida infection at primary sterile body sites. Of the PCR-positive, culture-negative patients, more than 50 % received systemic antifungal therapy. In 432/460 BC-negative IEs, the Candida specific-PCR was negative, resulting in a negative predictive value of 99.8 %. In conclusion, the Candida specific-PCR approach facilitates rapid detection of Candida DNA in blood samples of patients at risk of candidaemia within a few hours. Although standard BC diagnostics appear to remain indispensable for the detection of all cases of candidaemia, this PCR assay allowed the detection of candidaemia at a mean of 3 days earlier than BC diagnostics. Thus, it enables earlier antifungal therapy for patients with suspected candidaemia and may prevent further complications.

  6. New centrifugation blood culture device.

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    Dorn, G L; Smith, K

    1978-01-01

    A single-tube blood culture device designed for centrifugation in a tabletop centrifuge is described. Reconstruction experiments using 21 different organisms and human donor blood indicate that excellent recovery can be obtained by centrifugation for 30 min at 3,000 X g. PMID:342539

  7. Comparison of whole blood, serum, and plasma for early detection of candidemia by multiplex-tandem PCR.

    Science.gov (United States)

    Lau, Anna; Halliday, Catriona; Chen, Sharon C-A; Playford, E Geoffrey; Stanley, Keith; Sorrell, Tania C

    2010-03-01

    We applied multiplex-tandem PCR (MT-PCR) to 255 EDTA whole-blood specimens, 29 serum specimens, and 24 plasma specimens from 109 patients with Candida bloodstream infection (candidemia) to determine whether a diagnosis could be expedited in comparison with the time to diagnosis by the use of standard blood culture. Overall, the MT-PCR performed better than blood culture with DNA extracted from whole blood from 52/74 (70%) patients, accelerating the time to detection (blood culture flagging) and determination of the pathogenic species (by use of the API 32C system [bioMérieux, Marcy l'Etoile, France]) by up to 4 days (mean, 2.2 days; range, 0.5 to 8 days). Candida DNA was detected more often in serum (71%) and plasma (75%) than in whole blood (54%), although relatively small numbers of serum and plasma specimens were tested. The sensitivity, specificity, positive predictive value, and negative predictive value of the assay with whole blood were 75%, 97%, 95%, and 85%, respectively. Fungal DNA was not detected by MT-PCR in 6/24 (25%) whole-blood samples drawn simultaneously with the positive blood culture sample. MT-PCR performed better with whole-blood specimens stored at -20 degrees C (75%) and when DNA was extracted within 1 week of sampling (66%). The molecular and culture identification results correlated for 61 of 66 patients (92%); one discrepant result was due to misidentification by culture. All but one sample from 53 patients who were at high risk of candidemia but did not have proven disease were negative by MT-PCR. The results demonstrate the good potential of MT-PCR to detect candidemia, to provide Candida species identification prior to blood culture positivity, and to provide improved sensitivity when applied to with serum and plasma specimens.

  8. Improved detection of Candida albicans by PCR in blood of neutropenic mice with systemic candidiasis

    NARCIS (Netherlands)

    A.J.M. Munting-van Deventer; W.H.F. Goessens (Wil); A.F. van Belkum (Alex); H.J. van Vliet; E.W.M. van Etten (Els); H.A. Verbrugh (Henri)

    1995-01-01

    textabstractA PCR using primers aimed at the multicopy gene coding for the small subunit rRNA and resulting in the synthesis of a 180-bp fragment was evaluated for its use in diagnosing invasive candidiasis in comparison with blood culture. With the use of a C.

  9. Comparison of broad range 16S rDNA PCR to conventional blood ...

    African Journals Online (AJOL)

    Comparison of broad range 16S rDNA PCR to conventional blood culture for diagnosis of sepsis in the newborn. ... The most frequently isolated microorganisms were Staphylococcus aureus (n= 17, 56.7%), followed by Coagulase negative Staphylococci (n=7, 23.3%), Escherichia coli (n= 4, 13.3%), and Candida spp. (n=2 ...

  10. Pathogens in Maternal Blood and Fetal Cord Blood Using Q-Pcr Assay

    Directory of Open Access Journals (Sweden)

    Guang Qiong Hou

    2006-06-01

    Conclusion: Our results revealed an unexpectedly high incidence of pathogens in fetal cord blood. Screening for the above pathogens in donor cord blood in cord blood banks using Q-PCR is strongly urged to decrease morbidity and mortality rates in fetal cord blood stem cell transplant recipients.

  11. A multiplex nested PCR for the detection and identification of Candida species in blood samples of critically ill paediatric patients.

    Science.gov (United States)

    Taira, Cleison Ledesma; Okay, Thelma Suely; Delgado, Artur Figueiredo; Ceccon, Maria Esther Jurfest Rivero; de Almeida, Margarete Teresa Gottardo; Del Negro, Gilda Maria Barbaro

    2014-07-21

    Nosocomial candidaemia is associated with high mortality rates in critically ill paediatric patients; thus, the early detection and identification of the infectious agent is crucial for successful medical intervention. The PCR-based techniques have significantly increased the detection of Candida species in bloodstream infections. In this study, a multiplex nested PCR approach was developed for candidaemia detection in neonatal and paediatric intensive care patients. DNA samples from the blood of 54 neonates and children hospitalised in intensive care units with suspected candidaemia were evaluated by multiplex nested PCR with specific primers designed to identify seven Candida species, and the results were compared with those obtained from blood cultures. The multiplex nested PCR had a detection limit of four Candida genomes/mL of blood for all Candida species. Blood cultures were positive in 14.8% of patients, whereas the multiplex nested PCR was positive in 24.0% of patients, including all culture-positive patients. The results obtained with the molecular technique were available within 24 hours, and the assay was able to identify Candida species with 100% of concordance with blood cultures. Additionally, the multiplex nested PCR detected dual candidaemia in three patients. Our proposed PCR method may represent an effective tool for the detection and identification of Candida species in the context of candidaemia diagnosis in children, showing highly sensitive detection and the ability to identify the major species involved in this infection.

  12. Sensitivity of PCR and real-time PCR for the diagnosis of human visceral leishmaniasis using peripheral blood

    OpenAIRE

    da Costa Lima, Manoel Sebastião; Zorzenon, Denielly Christina Rodrigues; Dorval, Maria Elizabeth Cavalheiros; Pontes, Elenir Rose Jardim Cury; Oshiro, Elisa Teruya; Cunha, Rodrigo; Andreotti, Renato; Matos, Maria de Fatima Cepa

    2013-01-01

    Objective: To evaluate the effectiveness of PCR and real-time PCR for the diagnosis of human visceral leishmaniasis using peripheral blood samples. Methods: DNA extraction was performed using Promega Wizard襅 Genomic kits. PCR employing RV1/RV2 primers yielded 1 45-bp amplicons. Real-time PCR was performed with the same primers and SYBR Green ROX Plus mix. These techniques were used to analyze 100 peripheral blood samples from patients with clinical signs of the disease. Results...

  13. Quantification and Optimization of Candida albicans DNA in Blood Samples Using Real- Time PCR

    Directory of Open Access Journals (Sweden)

    Mojtaba Nabili

    2013-10-01

    Full Text Available Background: Candida albicans (C. albicans is a major cause of candidaemia in people with impaired immunity. Blood culture is a “gold standard” for candidaemia detection but is time-consuming and relatively insensitive. We established a real-time PCR assay for C. albicans detection in blood by LightCycler PCR and melting curve analysis. Methods: Five milliliter blood samples from healthy volunteers were spiked with 100-106 C. albicans cells to determine the detection limit of our method. DNA was extracted from whole blood using glass beads and the QIAamp DNA Blood Mini Kit (Qiagen, Hilden Germany. DNA from C. albicans isolates were amplified with primers and inserted into Escherichia coli (E. coli DH5α.1 cells with the TA cloning vector (Invitrogen. The plasmid was used for standardization and optimization. A quantitative PCR assay with the LightCycler amplification and detection system based on fluorescence resonance energy transfer (FRET with two different specific probes was established. To assess the precision and reproducibility of real-time PCR the intra-assay precision was determined in six consecutive assays. Results: No cross-reactivity of the hybridization probes with the DNA of non-C. albicans species or human genomic DNA was observed, which confirmed its 100% specificity. The minimum limit detected was one C. albicans cell or 100 CFU/ml (10 fg per PCR reaction. The real-time PCR efficiency rate for Candida was high (E = 1.95. Melting curve analysis of C. albicans showed a specific melting peak temperature of 65.76 °C. Conclusion: The real-time PCR assay we developed is highly specific and sufficiently sensitive to detect the fungal load for early diagnosis of invasive candidiasis.

  14. Quantification and optimization of Candida albicans DNA in blood samples using Real- Time PCR.

    Science.gov (United States)

    Nabili, Mojtaba; Ashrafi, Mohsen; Janbabaie, Ghasem; Hedayati, Mohamad Taghi; Ali-Moghaddam, Kamran; Shokohi, Tahereh

    2013-10-01

    Candida albicans (C. albicans) is a major cause of candidaemia in people with impaired immunity. Blood culture is a "gold standard" for candidaemia detection but is time-consuming and relatively insensitive. We established a real-time PCR assay for C. albicans detection in blood by LightCycler PCR and melting curve analysis. Five milliliter blood samples from healthy volunteers were spiked with 10(0)-10(6) C. albicans cells to determine the detection limit of our method. DNA was extracted from whole blood using glass beads and the QIAamp DNA Blood Mini Kit (Qiagen, Hilden Germany). DNA from C. albicans isolates were amplified with primers and inserted into Escherichia coli (E. coli) DH5α.1 cells with the TA cloning vector (Invitrogen). The plasmid was used for standardization and optimization. A quantitative PCR assay with the LightCycler amplification and detection system based on fluorescence resonance energy transfer (FRET) with two different specific probes was established. To assess the precision and reproducibility of real-time PCR the intra-assay precision was determined in six consecutive assays. No cross-reactivity of the hybridization probes with the DNA of non-C. albicans species or human genomic DNA was observed, which confirmed its 100% specificity. The minimum limit detected was one C. albicans cell or 10(0) CFU/ml (10 fg) per PCR reaction. The real-time PCR efficiency rate for Candida was high (E = 1.95). Melting curve analysis of C. albicans showed a specific melting peak temperature of 65.76 °C. The real-time PCR assay we developed is highly specific and sufficiently sensitive to detect the fungal load for early diagnosis of invasive candidiasis.

  15. Measurement of Epstein-Barr virus DNA loads in whole blood and plasma by TaqMan PCR and in peripheral blood lymphocytes by competitive PCR.

    Science.gov (United States)

    Wadowsky, Robert M; Laus, Stella; Green, Michael; Webber, Steven A; Rowe, David

    2003-11-01

    Epstein-Barr virus (EBV) DNA load values were measured in samples of whole blood (n = 60) and plasma (n = 59) by TaqMan PCR and in samples of peripheral blood lymphocytes (PBLs) (n = 60) by competitive PCR (cPCR). The samples were obtained from 44 transplant recipients. The whole-blood and PBL loads correlated highly (r(2) > 0.900), whereas the plasma and PBL loads correlated poorly (r(2) = 0.512). Testing of whole blood by TaqMan PCR is an acceptable alternative to testing of PBLs by cPCR for quantifying EBV DNA load.

  16. Blood and dried blood spot telomere length measurement by qPCR: assay considerations.

    Directory of Open Access Journals (Sweden)

    DeAnna L Zanet

    Full Text Available Measurement of telomere length is crucial for the study of telomere maintenance and its role in molecular pathophysiology of diseases and in aging. Several methods are used to measure telomere length, the choice of which usually depends on the type and size of sample to be assayed, as well as cost and throughput considerations. The goal of this study was to investigate the factors that may influence the reliability of qPCR-based relative telomere length measurements in whole blood. Day to day intra-individual variability, types of blood anticoagulant, sample storage conditions, processing and site of blood draw were investigated. Two qPCR-based methods to measure telomere length (monoplex vs. multiplex were also investigated and showed a strong correlation between them. Freezing and thawing of the blood and storage of the blood at 4°C for up to 4 days did not affect telomere length values. Telomere lengths in dried blood spots were significantly higher than both whole blood and peripheral mononuclear blood cells, and were highly correlated with both. We found that telomere length measurements were significantly higher in dried blood spots collected directly from fingertip prick compared to dried blood spots prepared with anticoagulated whole blood collected from the finger, and non-blotted whole blood taken from both finger and arm venipuncture. This suggests that DNA from cells blotted on paper is not equivalent to that collected from venipuncture whole blood, and caution should be taken when comparing between blood sample types.

  17. Performance of PCR-REBA assay for screening and identifying pathogens directly in whole blood of patients with suspected sepsis.

    Science.gov (United States)

    Wang, H-Y; Kim, J; Kim, S; Park, S D; Kim, H Y; Choi, H K; Uh, Y; Lee, H

    2015-11-01

    Rapid and accurate identification of a broad range of bacterial and fungal pathogens is the key to successful management of patients with bloodstream infections (BSIs). The aim of this study was to evaluate the diagnostic performance of PCR-REBA Sepsis-ID test for the detection of BSIs pathogens. EDTA anticoagulated blood for REBA Sepsis-ID assay and blood culture samples from 882 patients with suspected sepsis were simultaneously collected from January 2014 to December 2014. Of 115 patients with positive blood culture, 64 (55·7%) were Gram-positive bacteria, 35 (30·4%) were Gram-negative bacteria, 1 (0·9%) was Candida albicans and 15 (13·0%) were polymicrobial infections. The concordance rate of blood culture system and PCR-REBA Sepsis ID test was 83·0% (95% confidence interval (CI), 79·8-84·8, P pathogens by PCR-REBA revealed 81·0% (95% CI, 73·4-86·8, P pathogen by bacterial 16S rRNA sequencing. Although the sensitivity for pathogen identification was not significantly different between PCR-REBA and blood culture (P = 0·5), the combination of the two methods resulted in a significantly increased rate of pathogen detection (P = 0·002). The results of this study suggested that PCR-REBA may be helpful when added to blood culture in the diagnosis and management of sepsis. PCR-REBA Sepsis-ID test is a useful tool for the rapid identification of pathogenic isolates in whole blood to ensure adequate treatment for the causative agents of BSIs. Although the cost of molecular diagnostic assays is higher than the cost of conventional methods, clinical and economic cost-benefit analysis is still needed. PCR-REBA may provide essential information for accelerating therapeutic decisions to ensure effective treatment with antibiotics in the acute phase of pathogen infection. © 2015 The Society for Applied Microbiology.

  18. Diagnosis of Carrion's disease by direct blood PCR in thin blood smear negative samples.

    Directory of Open Access Journals (Sweden)

    Juana del Valle Mendoza

    Full Text Available Bartonella bacilliformis is the etiologic agent of Carrion's disease. This disease has two well established phases, the most relevant being the so called Oroya Fever, in which B. bacilliformis infect the erythrocytes resulting in severe anemia and transient immunosuppression, with a high lethality in the absence of adequate antibiotic treatment. The presence of B. bacilliformis was studied in 113 blood samples suspected of Carrion's disease based on clinical criteria, despite the absence of a positive thin blood smear, by two different PCR techniques (using Bartonella-specific and universal 16S rRNA gene primers, and by bacterial culture. The specific 16S rRNA gene primers revealed the presence of 21 B. bacilliformis and 1 Bartonella elizabethae, while universal primers showed both the presence of 3 coinfections in which a concomitant pathogen was detected plus Bartonella, in addition to the presence of infections by other microorganisms such as Agrobacterium or Bacillus firmus. These data support the need to implement molecular tools to diagnose Carrion's disease.

  19. Investigating the impact of blood culture bundles on the incidence of blood culture contamination rates.

    Science.gov (United States)

    Murphy, Theresa; Maile, Deborah; Barsch, Tara; Jerdan, Florence

    2014-01-01

    Blood cultures are integral diagnostic procedures for identifying serious infections and selecting antimicrobials. Positive blood cultures are the initial step in attaining a conclusive diagnosis of sepsis. Relative risk is blood culture contamination, false-positive blood culture results, diagnostic error delays, treatment errors, excessive lab testing, and increased length of stay. A complicating issue is the increased use of central venous access devices (CVADs). The purpose of this descriptive, comparative study is to evaluate the effectiveness of blood culture bundles on blood cultures drawn through a CVAD and contamination rates. The study revealed a decrease in blood culture contamination rates by 61%.

  20. Comparison of Nested-PCR technique and culture method in ...

    African Journals Online (AJOL)

    USER

    2010-04-05

    Apr 5, 2010 ... tuberculosis (GUTB) compared with acid fast staining and culture method. In total ... tuberculosis; PCR, polymerase chain reaction; MTB,. Mycobacterium tuberculosis;. EPTB, extrapulmonary tuberculosis; AFB, acid-fast bacilli; SDS, sodium ..... Dingtoumda B, Diallo B, Defer MC, Sombié I, Zanetti S, Sechi LA.

  1. RAPD-PCR analysis of cultured type olives in Turkey

    African Journals Online (AJOL)

    STORAGESEVER

    2009-08-04

    Aug 4, 2009 ... The aim of this study was to detect genetic similarities and distances among cultured type olive trees by RAPD-PCR technique. Olives are raised in a high range from the Aegean, Mediterranean, Marmara and Black Sea to Southeast Anatolia regions of Turkey. Olive breeding had a rapid increase in Turkey.

  2. Sensitivity of PCR assays for murine gammaretroviruses and mouse contamination in human blood samples.

    Directory of Open Access Journals (Sweden)

    Li Ling Lee

    Full Text Available Gammaretroviruses related to murine leukemia virus (MLV have variously been reported to be present or absent in blood from chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME patients and healthy controls. Using subjects from New York State, we have investigated by PCR methods whether MLV-related sequences can be identified in nucleic acids isolated from whole blood or from peripheral blood mononuclear cells (PBMCs or following PBMC culture. We have also passaged the prostate cancer cell line LNCaP following incubation with plasma from patients and controls and assayed nucleic acids for viral sequences. We have used 15 sets of primers that can effectively amplify conserved regions of murine endogenous and exogenous retrovirus sequences. We demonstrate that our PCR assays for MLV-related gag sequences and for mouse DNA contamination are extremely sensitive. While we have identified MLV-like gag sequences following PCR on human DNA preparations, we are unable to conclude that these sequences originated in the blood samples.

  3. Evaluation of PCR methods for detection of Brucella strains from culture and tissues.

    Science.gov (United States)

    Çiftci, Alper; İça, Tuba; Savaşan, Serap; Sareyyüpoğlu, Barış; Akan, Mehmet; Diker, Kadir Serdar

    2017-04-01

    The genus Brucella causes significant economic losses due to infertility, abortion, stillbirth or weak calves, and neonatal mortality in livestock. Brucellosis is still a zoonosis of public health importance worldwide. The study was aimed to optimize and evaluate PCR assays used for the diagnosis of Brucella infections. For this aim, several primers and PCR protocols were performed and compared with Brucella cultures and biological material inoculated with Brucella. In PCR assays, genus- or species-specific oligonucleotide primers derived from 16S rRNA sequences (F4/R2, Ba148/928, IS711, BruP6-P7) and OMPs (JPF/JPR, 31ter/sd) of Brucella were used. All primers except for BruP6-P7 detected the DNA from reference Brucella strains and field isolates. In spiked blood, milk, and semen samples, F4-R2 primer-oriented PCR assays detected minimal numbers of Brucella. In spiked serum and fetal stomach content, Ba148/928 primer-oriented PCR assays detected minimal numbers of Brucella. Field samples collected from sheep and cattle were examined by bacteriological methods and optimized PCR assays. Overall, sensitivity of PCR assays was found superior to conventional bacteriological isolation. Brucella DNA was detected in 35.1, 1.1, 24.8, 5.0, and 8.0% of aborted fetus, blood, milk, semen, and serum samples by PCR assays, respectively. In conclusion, PCR assay in optimized conditions was found to be valuable in sensitive and specific detection of Brucella infections of animals.

  4. Evaluation of a PCR for detection of Actinobacillus pleuropneumoniae in mixed bacterial cultures from tonsils

    DEFF Research Database (Denmark)

    Gram, T.; Ahrens, Peter; Nielsen, J.P.

    1996-01-01

    strains of A. lignieresii. The lower detection limit of the PCR test was 10(3) A. pleuropneumoniae CFU/PCR test tube and was not affected by addition of 10(6) E. coli CFU/PCR test tube. Mixed bacterial cultures from tonsils of 101 pigs from 9 different herds were tested by culture and by PCR using four...

  5. Detection of Candida species by polymerase chain reaction (PCR) in blood samples of experimentally infected mice and patients with suspected candidemia.

    Science.gov (United States)

    Khan, Z U; Mustafa, A S

    2001-01-01

    In this study, we have established and evaluated a genus-specific polymerase chain reaction (PCR) and species-specific nested PCRs for the detection of Candida species in blood samples of neutropenic mice and patients suspected of candidemia. DNA segments of the gene encoding cytochrome P450 L1A1 were targeted for amplification by using genus and species-specific primers. As compared to the genus-specific PCR, the species-specific nested PCRs improved the sensitivity by 10 times with the detection limit Candida albicans infection in neutropenic mice, four samples were positive by genus-specific PCR and 11 were positive by species-specific nested PCR. The PCR results were correlated with culture findings obtained on blood samples. Two of the three blood culture-positive samples were positive by genus-specific PCR and all the three with species-specific nested PCR. Among 15 mice, which were negative by blood culture but had C. albicans isolated from visceral organs, 2 and 8 mice yielded positive results by genus-specific PCR and species-specific nested PCR, respectively. Consistent with the results of the animal study, species-specific nested PCR yielded much higher positivity as compared to culture (52.2% versus 21.2%) in patients suspected for candidemia. Moreover, 8 specimens which were negative for Candida by genus-specific PCR became positive by species-specific nested PCR. No correlation was apparent between PCR positivity and Candida antigen titers. The results suggest that nested PCR is a sensitive technique for the detection of Candida species from blood samples, and thus it may have application in the diagnosis of suspected cases of candidemia and candidiasis.

  6. Application of PCR-based DNA sequencing technique for the detection of Leptospira in peripheral blood of septicemia patients

    Directory of Open Access Journals (Sweden)

    Ram, S.

    2012-01-01

    Full Text Available Aim: Isolation, dark field detection and microscopic agglutination test (MAT are considered ―gold standard‖ tests for diagnosis of Leptospirosis. Several PCR assays are reported but very few have been evaluated for detection of Leptospirosis. Therefore, this study was undertaken. This study aims to design and standardize polymerase chain reaction (PCR - based DNA sequencing technique for the detection of pathogenic Leptospira from peripheral blood of patients clinically diagnosed with septicemia. Methodology and Results: Two hundred and seven (207 blood samples from patients were diagnosed with septicemia which includes 100 bacterial (other than Leptospira culture positive and 107 bacterial culture negative samples were studied. Primers for Nested PCR targeting LipL32 gene of Leptospira interrogans were designed and the specificity of primers was tested against serum samples positive/negative by either MAT or dark field microscopy. PCR amplified products were further confirmed by DNA sequencing. The standardized nPCR was sensitive and specific to Leptospira interrogans. Twenty-one (21% out of 100 culture positive blood samples, three (2.8% out of 107 culture negative samples showed nPCR positivity and were confirmed as Leptospira interrogans by DNA sequencing (p<0.001. A sensitive nPCR specific to Leptospira interrogans was developed. Conclusion, significance and impact of study: The p value (<0.001 signifies that Leptospira is commonly associated with other bacteria circulating in blood indicating that a decreased immune status is created primarily by a bacterium with enhanced possibility of development of Leptospiral infection probably be of an endogenous origin.

  7. Linking non-culturable (qPCR) and culturable enterococci densities with hydrometeorological conditions

    Science.gov (United States)

    Byappanahalli, Muruleedhara N.; Whitman, Richard L.; Shively, Dawn A.; Nevers, Meredith B.

    2010-01-01

    Quantitative polymerase chain reaction (qPCR) measurement of enterococci has been proposed as a rapid technique for assessment of beach water quality, but the response of qPCR results to environmental conditions has not been fully explored. Culture-based E. coli and enterococci have been used in empirical predictive models to characterize their responses to environmental conditions and to increase monitoring frequency and efficiency. This approach has been attempted with qPCR results only in few studies. During the summer of 2006, water samples were collected from two southern Lake Michigan beaches and the nearby river outfall (Burns Ditch) and were analyzed for enterococci by culture-based and non-culture-based (i.e., qPCR) methods, as well as culture-based E. coli. Culturable enterococci densities (log CFU/100 ml) for the beaches were significantly correlated with enterococci qPCR cell equivalents (CE) (R = 0.650, P N = 32). Enterococci CE and CFU densities were highest in Burns Ditch relative to the beach sites; however, only CFUs were significantly higher (P R = 0.565, P N = 32). Culturable E. coli and enterococci densities were significantly correlated (R = 0.682, P N = 32). Regression analyses suggested that enterococci CFU could be predicted by lake turbidity, Burns Ditch discharge, and wind direction (adjusted R2 = 0.608); enterococci CE was best predicted by Burns Ditch discharge and log-transformed lake turbidity × wave height (adjusted R2 = 0.40). In summary, our results show that analytically, the qPCR method compares well to the non-culture-based method for measuring enterococci densities in beach water and that both these approaches can be predicted by hydrometeorological conditions. Selected predictors and model results highlight the differences between the environmental responses of the two method endpoints and the potentially high variance in qPCR results

  8. One-step sample preparation of positive blood cultures for the direct detection of methicillin-sensitive and -resistant Staphylococcus aureus and methicillin-resistant coagulase-negative staphylococci within one hour using the automated GenomEra CDX™ PCR system.

    Science.gov (United States)

    Hirvonen, J J; von Lode, P; Nevalainen, M; Rantakokko-Jalava, K; Kaukoranta, S-S

    2012-10-01

    A method for the rapid detection of methicillin-sensitive and -resistant Staphylococcus aureus (MSSA and MRSA, respectively) and methicillin-resistant coagulase-negative staphylococci (MRCoNS) with a straightforward sample preparation protocol of blood cultures using an automated homogeneous polymerase chain reaction (PCR) assay, the GenomEra™ MRSA/SA (Abacus Diagnostica Oy, Turku, Finland), is presented. In total, 316 BacT/Alert (bioMérieux, Marcy l'Etoile, France) and 433 BACTEC (Becton Dickinson, Sparks, MD, USA) blood culture bottles were analyzed, including 725 positive cultures containing Gram-positive cocci in clusters (n = 419) and other Gram stain forms (n = 361), as well as 24 signal- and growth-negative bottles. Detection sensitivities for MSSA, MRSA, and MRCoNS were 99.4 % (158/159), 100.0 % (9/9), and 99.3 % (132/133), respectively. One false-positive MRSA result was detected from a non-staphylococci-containing bottle, yielding a specificity of 99.8 %. The lowest detectable amount of viable cells in the blood culture sample was 4 × 10(4) CFU/mL. The results were available within one hour after microbial growth detection and the two-step, time-resolved fluorometric (TRF) measurement mode employed by the GenomEra CDX™ instrument showed no interference from blood, charcoal, or culture media. The method described lacks all sample purification steps and allows reliable and simplified pathogen detection also in clinical microbiology laboratory settings without specialized molecular microbiology competence.

  9. Culture independent PCR: an alternative enzyme discovery strategy

    DEFF Research Database (Denmark)

    Jacobsen, Jonas; Lydolph, Magnus; Lange, Lene

    2005-01-01

    Degenerate primers were designed for use in a culture-independent PCR screening of DNA from composite fungal communities, inhabiting residues of corn stovers and leaves. According to similarity searches and alignments amplified clone sequences affiliated with glycosyl hydrolase family 7...... and glycosyl hydrolase family 45 though significant sequence divergence was observed. Glycosyl hydrolases from families 7 and 45 play a crucial role in biomass conversion to fuel ethanol. Research in this renewable energy source has two objectives: (i) To contribute to development of a renewable alternative...

  10. Comparison of conventional PCR, quantitative PCR, bacteriological culture and the Warthin Starry technique to detect Leptospira spp. in kidney and liver samples from naturally infected sheep from Brazil.

    Science.gov (United States)

    Fornazari, Felipe; da Silva, Rodrigo Costa; Richini-Pereira, Virginia Bodelão; Beserra, Hugo Enrique Orsini; Luvizotto, Maria Cecília Rui; Langoni, Helio

    2012-09-01

    Leptospirosis is an infectious disease of worldwide importance. The development of diagnostic techniques allows sick animals to be identified, reservoirs to be eliminated and the disease prevented and controlled. The present study aimed to compare different techniques for diagnosing leptospirosis in sheep. Samples of kidney, liver and blood were collected from 465 animals that originated from a slaughterhouse. The sera were analyzed by the Microscopic Agglutination Test (MAT), and kidney and liver samples of seropositive animals were analyzed using four techniques: bacteriological culture, the Warthin Starry (WS) technique, conventional PCR (cPCR), and quantitative PCR (qPCR). With the MAT, 21 animals were positive (4.5%) to serovars Hardjo (n=12), Hebdomadis (n=5), Sentot (n=2), Wolfii (n=1) and Shermani (n=1). Titers were 100 (n=10), 200 (n=2), 400 (n=6) and 1600 (n=3). No animal was positive by bacteriological culture; four animals were positive by the WS technique in kidney samples; six animals were positive by cPCR in kidney samples; and 11 animals were positive by qPCR, eight of which in kidney samples and three in liver. The bacterial quantification revealed a median of 4.3 bacteria/μL in liver samples and 36.6 bacteria/μL in kidney samples. qPCR presented the highest sensitivity among the techniques, followed by cPCR, the WS technique and bacteriological culture. These results indicate that sheep can carry leptospires of the Sejroe serogroup, and demonstrate the efficiency of quantitative PCR to detect Leptospira spp. in tissue samples. Published by Elsevier B.V.

  11. Real-Time PCR to Identify Staphylococci and Assay for Virulence from Blood.

    Science.gov (United States)

    Okolie, Charles E

    2017-01-01

    The genus Staphylococcus includes pathogenic and non-pathogenic facultative anaerobes. Due to the plethora of virulence factors encoded in its genome, the species Staphylococcus aureus is known to be the most pathogenic. S. aureus strains harboring genes encoding virulence and antibiotic resistance are of public health importance. In clinical samples, however, pathogenic S. aureus is often mixed with putatively less pathogenic coagulase-negative staphylococci (CoNS), both of which can harbor mecA, the genetic driver for staphylococcal methicillin-resistance. In this chapter, the detailed practical procedure for operating a real-time pentaplex PCR assay in blood cultures is described. The pentaplex real-time PCR assay simultaneously detects markers for the presence of bacteria (16S rRNA), coagulase-negative staphylococcus (cns), S. aureus (spa), Panton-Valentine leukocidin (pvl), and methicillin resistance (mecA).

  12. Prevalence of Bartonella spp. by culture, PCR and serology, in veterinary personnel from Spain

    Directory of Open Access Journals (Sweden)

    José A. Oteo

    2017-11-01

    Full Text Available Abstract Background The genus Bartonella includes fastidious, facultative intracellular bacteria mainly transmitted by arthropods and distributed among mammalian reservoirs. Bartonella spp. implicated as etiological agents of zoonoses are increasing. Apart from the classical Bartonella henselae, B. bacilliformis or B. quintana, other species (B. elizabethae, B. rochalimae, B. vinsonii arupensis and B. v. berkhoffii, B. tamiae or B. koehlerae, among others have also been associated with human and/or animal diseases. Laboratory techniques for diagnosis (culture, PCR assays and serology usually show lack of sensitivity. Since 2005, a method based on a liquid enrichment Bartonella alphaproteobacteria growth medium (BAPGM followed by PCRs for the amplification of Bartonella spp. has been developed. We aimed to assess culture, molecular and serological prevalence of Bartonella infections in companion animal veterinary personnel from Spain. Methods Each of 89 participants completed a questionnaire. Immunofluorescence assays (IFA using B. vinsonii berkhoffii (genotypes I, II and III, B. henselae, B. quintana and B. koehlerae as antigens were performed. A cut-off of 1:64 was selected as a seroreactivity titer. Blood samples were inoculated into BAPGM and subcultured onto blood agar plates. Bartonella spp. was detected using conventional and quantitative real-time PCR assays and DNA sequencing. Results Among antigens corresponding to six Bartonella spp. or genotypes, the lowest seroreactivity was found against B. quintana (11.2% and the highest, against B. v. berkhoffii genotype III (56%. A total of 27% of 89 individuals were not seroreactive to any test antigen. Bartonella spp. IFA seroreactivity was not associated with any clinical sign or symptom. DNA from Bartonella spp., including B. henselae (n = 2, B. v. berkhoffii genotypes I (n = 1 and III (n = 2, and B. quintana (n = 2 was detected in 7/89 veterinary personnel. PCR and DNA sequencing

  13. Whole blood Nested PCR and Real-time PCR amplification of Talaromyces marneffei specific DNA for diagnosis.

    Science.gov (United States)

    Lu, Sha; Li, Xiqing; Calderone, Richard; Zhang, Jing; Ma, Jianchi; Cai, Wenying; Xi, Liyan

    2016-02-01

    Talaromyces marneffei is a dimorphic pathogenic fungus, which is a life-threatening invasive mycosis in the immunocompromised host. Prompt diagnosis of T. marneffei infection remains difficult although there has been progress in attempts to expedite the diagnosis of this infection. We previously demonstrated the value of nested polymerase chain reaction (PCR) to detect T. marneffei in paraffin embedded tissue samples with high sensitivity and specificity. In this study, this assay was used to detect the DNA of T. marneffei in whole blood samples. Real-time PCR assay was also evaluated to identify T. marneffei in the same samples. Twenty out of 30 whole blood samples (67%) collected from 23 patients were found positive by using the nested PCR assay, while 23/30 (77%) samples were found positive by using the real-time PCR assay. In order to express accurately the fungal loads, we used a normalized linearized plasmid as an internal control for real-time PCR. The assay results were correlated as the initial quantity (copies/μl) with fungal burden. These data indicate that combination of nested PCR and real-time PCR assay provides an attractive alternative for identification of T. marneffei DNA in whole blood samples of HIV-infected patients. © The Author 2015. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  14. Comparison between Plasma and Whole Blood Specimens for Detection of Aspergillus DNA by PCR

    Science.gov (United States)

    Loeffler, Juergen; Hebart, Holger; Brauchle, Ulrike; Schumacher, Ulrike; Einsele, Hermann

    2000-01-01

    Ninety-six plasma and whole blood specimens from nine selected patients were analyzed for the presence of Aspergillus DNA. Nineteen specimens from three patients with proven aspergillosis were PCR positive in both materials, whereas an additional 22 were PCR positive in whole blood only. All 36 samples from six patients without signs of aspergillosis were negative in both assays. We conclude that although plasma and whole blood spiked with Aspergillus conidia showed an identical lower detection limit (10 CFU), the sensitivity of plasma PCR was lower than that of PCR performed on whole blood samples. PMID:11015412

  15. Improved blood culture identification by FilmArray in cultures from regional hospitals compared with teaching hospital cultures.

    Science.gov (United States)

    Inglis, Timothy J J; Bzdyl, Nicole; Chua, I-Ly Joanna; Urosevic, Nadezda M; Leung, Michael J; Geelhoed, Elizabeth

    2016-01-01

    Rapid identification of bacteria isolated from blood cultures by direct matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is now in wide spread use in major centres but is not yet feasible in smaller hospital laboratories. A FilmArray multiplex PCR panel for blood culture isolate identification (BCID) provides an alternative approach to near point-of-care microbial identification in regional hospitals. We assessed the accuracy and time to identification of the BCID FilmArray in a consecutive series of 149 blood cultures from 143 patients in a teaching hospital and smaller regional hospitals, currently identified by direct MALDI-TOF and proprietary molecular methods. The BCID FilmArray contained 18 of 34 species and 20 of 23 species isolated from teaching and regional hospital, respectively. Overall, 85 % of the teaching hospital and 100 % of the regional hospital monomicrobial blood cultures were identified, compared with 60 and 68 %, respectively, for direct MALDI-TOF on the same cultures. There were no incorrect results from blood cultures containing Staphylococcus aureus, streptococci, Pseudomonas aeruginosa or Enterobacteriaceae. The three discrepant results were all in mixed cultures. The mean reduction in time to identification of blood culture isolates was 53 h, which did not include the time required to transport cultures from regional centres to a central laboratory. The overall performance of the BCID FilmArray is stronger in blood cultures from smaller regional hospitals that encounter a narrower range of bacterial species dominated by the commonest species. This approach is more suited to smaller clinical laboratories than the MALDI-TOF direct method.

  16. Polymerase chain reaction and blood culture in blood donors screened by ELISA test for Chagas' disease

    Directory of Open Access Journals (Sweden)

    Andréa Tieko Kinoshita-Yanaga

    2011-03-01

    Full Text Available The objective of this study was to evaluate, through blood culture and PCR, the results of the ELISA for Chagas' disease in the screening of blood donors in the public blood-supply network of the state of Paraná, Brazil, and to map the epidemiological profile of the donors with respect to their risk of infection by Trypanosoma cruzi. The negative and positive results of the ELISA were confirmed by blood culture and PCR for 190/191 individuals (99.5%. For one individual (0.5%, the ELISA was inconclusive, blood culture and IIF were negative, and IHA and PCR positive. Three individuals (1.6% were positive for T. cruzi on all the tests. Donors were predominantly female, and natives of Paraná, of rural origin, had observed or been informed of the presence of the vector in the municipalities where they resided, had never received a blood transfusion, had donated blood 1 to 4 times, and reported no cases of Chagas' disease in their families. We concluded that PCR and blood culturing have excellent potential for confirming the results of the ELISA, and that candidate blood donors with negative or positive tests have a similar risk of infection by T. cruzi, indicating that the ELISA test is sufficiently safe for screening blood prior to use.O objetivo deste estudo foi avaliar, pela hemocultura e PCR, os resultados do teste ELISA utilizado para doença de Chagas na triagem de doadores de sangue na rede pública do Estado do Paraná, Brasil, e traçar o perfil epidemiológico dos doadores quanto ao risco de infecção pelo Trypanosoma cruzi. Os resultados negativos e positivos do ELISA foram confirmados pela hemocultura e PCR em 190/191 indivíduos (99,5%. Para um indivíduo (0,5%, o teste de ELISA foi inconclusivo, hemocultura e IFI foram negativas, HAI e PCR foram positivas. Três indivíduos (1,6% foram positivos para T. cruzi em todos os testes. A maioria dos doadores era do sexo feminino, oriundos do Estado do Paraná, de origem rural, tinham

  17. Multiplex PCR from Menstrual Blood: A Non-Invasive Cost-Effective Approach to Reduce Diagnostic Dilemma for Genital Tuberculosis.

    Science.gov (United States)

    Paine, Suman K; Basu, Analabha; Choudhury, Rajib Gon; Bhattacharya, Basudev; Chatterjee, Sidhartha; Bhattacharya, Chandra

    2018-03-16

    Genital tuberculosis (GTB) is a potent contributor to irreversible damage to the reproductive system and infertility in females. As no gold standard diagnostic tool is yet available, clinical suspicion and relatively insensitive approaches such as histopathology, laparoscopy and hysterosalpingogram are currently critical determinants in the diagnosis of GTB. Although a polymerase chain reaction (PCR)-based assay using endometrial tissue seems promising, sampling does require an invasive procedure. We hypothesized that menstrual blood may provide an alternate non-invasive source of samples for PCR-based GTB diagnosis. We enrolled 195 women with primary infertility in whom GTB was suspected. We obtained ethics committee approval from our institution and written informed consent from subjects. Endometrial tissue and menstrual blood was collected from the subjects and culture, histopathology, and multiplex PCR with both sample type was performed for each subject. The sensitivity and specificity of multiplex PCR was, respectively, 90.2 and 86.1% for menstrual blood, 95.8 and 84.3% for endometrial tissue, and 64.8 and 93.2% for histopathology staining. A strong clinical suspicion aided with multiplex PCR using menstrual blood may significantly reduce the diagnostic dilemma for GTB diagnosis in a non-invasive, sensitive, rapid, and cost-effective manner.

  18. Sensitive and rapid RT-qPCR quantification of pathogenic Candida species in human blood.

    Science.gov (United States)

    Ogata, Kiyohito; Matsuda, Kazunori; Tsuji, Hirokazu; Nomoto, Koji

    2015-10-01

    For accurate diagnosis and appropriate treatment of candidiasis, we developed a highly sensitive quantitative RT-PCR (RT-qPCR) system for five Candida species that have been reported to be the major causes of bloodstream fungal infection (Candida albicans, Candida glabrata, Candida tropicalis, Candida parapsilosis, and Candida krusei), together with a system for all pathogenic Candida species. Cells of each fungal species spiked into human peripheral blood (PB) were specifically detected at a lower detection limit of 10(0) cell/1 mL PB by this system using the newly developed specific primer sets targeting 18S or 26S rRNA of the five Candida species, together with the existing group primer set. The total count of the five Candida spp. as the sum of those obtained by using the five species primer sets was equivalent to the count obtained by using the group primer set, indicating that the group set covered the major five Candida spp. in human blood with the same degree of accuracy as the species primer sets. The RT-qPCR counts of the Candida species were in good agreement with CFU counts obtained by their culture on CHROMagar™, with a lower detection limit of 10(0)cell/mL of PB. Candida rRNA molecules were stably stored for at least 7 days at 4°C by keeping the blood specimens in an RNA stabilizing reagent. These results strongly suggest that this sensitive system is useful for accurate and rapid diagnosis of Candida bloodstream infections. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. Rapid detection of Candida albicans in clinical blood samples by using a TaqMan-based PCR assay.

    Science.gov (United States)

    Maaroufi, Younes; Heymans, Corine; De Bruyne, Jean-Marc; Duchateau, Valerie; Rodriguez-Villalobos, Hector; Aoun, Michel; Crokaert, Françoise

    2003-07-01

    We describe a rapid and reproducible PCR assay for quantitation of the Candida albicans ribosomal DNA (rDNA) in clinical blood samples based on the TaqMan principle (Applied Biosystems), in which a signal is generated by cleavage of a template-specific probe during amplification. We used two fluorogenic probes based on universal, fungus-specific primers, one for the detection of C. albicans species DNA and one for the detection of all Candida genus DNA. C. albicans blastoconidia mixed with whole blood in a titration experiment yielded a linear PCR signal over a range of 3 orders of magnitude. The TaqMan-based PCR assay for C. albicans exhibited a low limit of detection (5 CFU/ml of blood) and an excellent reproducibility (96 to 99%). While the C. albicans species-specific probe had 100% specificity for C. albicans, all Candida genus-specific probes cross-reacted with other organisms likely to coinfect patients with C. albicans infections. On the basis of these data, we determined the C. albicans loads with a species-specific probe from 122 blood samples from 61 hematology or oncology patients with clinically proven or suspected systemic Candida infections. Eleven positive samples exhibited a wide range of C. albicans loads, extending from 5 to 100,475 CFU/ml of blood. The sensitivity and specificity of the present assay were 100 and 97%, respectively, compared with the results of blood culture. These data indicate that the TaqMan-based PCR assay for quantitation of C. albicans with a species-specific probe provides an attractive alternative for the identification and quantitation of C. albicans rDNA in pure cultures and blood samples.

  20. Evaluation of a digital microfluidic real-time PCR platform to detect DNA of Candida albicans in blood.

    Science.gov (United States)

    Schell, W A; Benton, J L; Smith, P B; Poore, M; Rouse, J L; Boles, D J; Johnson, M D; Alexander, B D; Pamula, V K; Eckhardt, A E; Pollack, M G; Benjamin, D K; Perfect, J R; Mitchell, T G

    2012-09-01

    Species of Candida frequently cause life-threatening infections in neonates, transplant and intensive care unit (ICU) patients, and others with compromised host defenses. The successful management of systemic candidiasis depends upon early, rapid diagnosis. Blood cultures are the standard diagnostic method, but identification requires days and less than half of the patients are positive. These limitations may be eliminated by using real-time polymerase chain reaction (PCR) to detect Candida DNA in the blood specimens of patients at risk. Here, we optimized a PCR protocol to detect 5-10 yeasts in low volumes of simulated and clinical specimens. We also used a mouse model of systemic candidiasis and determined that candidemia is optimally detectable during the first few days after infection. However, PCR tests are often costly, labor-intensive, and inconvenient for routine use. To address these obstacles, we evaluated the innovative microfluidic real-time PCR platform (Advanced Liquid Logic, Inc.), which has the potential for full automation and rapid turnaround. Eleven and nine of 16 specimens from individual patients with culture-proven candidemia tested positive for C. albicans DNA by conventional and microfluidic real-time PCR, respectively, for a combined sensitivity of 94%. The microfluidic platform offers a significant technical advance in the detection of microbial DNA in clinical specimens.

  1. Evaluation of droplet digital PCR for quantification of residual leucocytes in red blood cell concentrates.

    Science.gov (United States)

    Doescher, A; Loges, U; Petershofen, E K; Müller, T H

    2017-11-01

    Enumeration of residual white blood cells in leucoreduced blood components is essential part of quality control. Digital PCR has substantially facilitated quantitative PCR and was thus evaluated for measurements of leucocytes. Target for quantification of leucocytes by digital droplet PCR was the blood group gene RHCE. The SPEF1 gene was added as internal control for the entire assay starting with automated DNA extraction. The sensitivity of the method was determined by serial dilutions of standard samples. Quality control samples were analysed within 24 h, 7 days and 6 months after collection. Routine samples from leucodepleted red blood cell concentrates (n = 150) were evaluated in parallel by flow-cytometry (LeucoCount) and by digital PCR. Digital PCR reliably detected at least 0·4 leucocytes per assay. The mean difference between PCR and flow-cytometric results from 150 units was -0·01 (±1·0). DNA samples were stable for up to at least six months. PCR measurement of leucocytes in samples from plasma and platelet concentrates also provided valid results in a pilot study. Droplet digital PCR to enumerate leucocytes offers an alternative for quality control of leucoreduced blood products. Sensitivity, specificity and reproducibility are comparable to flow-cytometry. The option to collect samples over an extended period of time and the automatization introduce attractive features for routine quality control. © 2017 International Society of Blood Transfusion.

  2. A novel method of selective removal of human DNA improves PCR sensitivity for detection of Salmonella Typhi in blood samples.

    Science.gov (United States)

    Zhou, Liqing; Pollard, Andrew J

    2012-07-27

    Enteric fever is a major public health problem, causing an estimated 21million new cases and 216,000 or more deaths every year. Current diagnosis of the disease is inadequate. Blood culture only identifies 45 to 70% of the cases and is time-consuming. Serological tests have very low sensitivity and specificity. Clinical samples obtained for diagnosis of enteric fever in the field generally have blood, so that even PCR-based methods, widely used for detection of other infectious diseases, are not a straightforward option in typhoid diagnosis. We developed a novel method to enrich target bacterial DNA by selective removal of human DNA from blood samples, enhancing the sensitivity of PCR tests. This method offers the possibility of improving PCR assays directly using clinical specimens for diagnosis of this globally important infectious disease. Blood samples were mixed with ox bile for selective lysis of human blood cells and the released human DNA was then digested with addition of bile resistant micrococcal nuclease. The intact Salmonella Typhi bacteria were collected from the specimen by centrifugation and the DNA extracted with QIAamp DNA mini kit. The presence of Salmonella Typhi bacteria in blood samples was detected by PCR with the fliC-d gene of Salmonella Typhi as the target. Micrococcal nuclease retained activity against human blood DNA in the presence of up to 9% ox bile. Background human DNA was dramatically removed from blood samples through the use of ox bile lysis and micrococcal nuclease for removal of mammalian DNA. Consequently target Salmonella Typhi DNA was enriched in DNA preparations and the PCR sensitivity for detection of Salmonella Typhi in spiked blood samples was enhanced by 1,000 fold. Use of a combination of selective ox-bile blood cell lysis and removal of human DNA with micrococcal nuclease significantly improves PCR sensitivity and offers a better option for improved typhoid PCR assays directly using clinical specimens in diagnosis of

  3. A Proline Racemase Based PCR for Identification of Trypanosoma vivax in Cattle Blood

    Science.gov (United States)

    Fikru, Regassa; Hagos, Ashenafi; Rogé, Stijn; Reyna-Bello, Armando; Gonzatti, Mary Isabel; Merga, Bekana; Goddeeris, Bruno Maria; Büscher, Philippe

    2014-01-01

    A study was conducted to develop a Trypanosoma vivax (T. vivax) specific PCR based on the T. vivax proline racemase (TvPRAC) gene. Forward and reverse primers were designed that bind at 764–783 bp and 983–1002 bp of the gene. To assess its specificity, TvPRAC PCR was conducted on DNA extracted from different haemotropic pathogens: T. vivax from Nigeria, Ethiopia and Venezuela, T. congolense Savannah type, T. brucei brucei, T. evansi, T. equiperdum, T. theileri, Theileria parva, Anaplasma marginale, Babesia bovis and Babesia bigemina and from bovine, goat, mouse, camel and human blood. The analytical sensitivity of the TvPRAC PCR was compared with that of the ITS-1 PCR and the 18S PCR-RFLP on a dilution series of T. vivax DNA in water. The diagnostic performance of the three PCRs was compared on 411 Ethiopian bovine blood specimens collected in a former study. TvPRAC PCR proved to be fully specific for T. vivax, irrespective of its geographical origin. Its analytical sensitivity was lower than that of ITS-1 PCR. On these bovine specimens, TvPRAC PCR detected 8.3% T. vivax infections while ITS-1 PCR and 18S PCR-RFLP detected respectively 22.6 and 6.1% T. vivax infections. The study demonstrates that a proline racemase based PCR could be used, preferably in combination with ITS-1 PCR, as a species-specific diagnostic test for T. vivax infections worldwide. PMID:24416292

  4. A proline racemase based PCR for identification of Trypanosoma vivax in cattle blood.

    Directory of Open Access Journals (Sweden)

    Regassa Fikru

    Full Text Available A study was conducted to develop a Trypanosoma vivax (T. vivax specific PCR based on the T. vivax proline racemase (TvPRAC gene. Forward and reverse primers were designed that bind at 764-783 bp and 983-1002 bp of the gene. To assess its specificity, TvPRAC PCR was conducted on DNA extracted from different haemotropic pathogens: T. vivax from Nigeria, Ethiopia and Venezuela, T. congolense Savannah type, T. brucei brucei, T. evansi, T. equiperdum, T. theileri, Theileria parva, Anaplasma marginale, Babesia bovis and Babesia bigemina and from bovine, goat, mouse, camel and human blood. The analytical sensitivity of the TvPRAC PCR was compared with that of the ITS-1 PCR and the 18S PCR-RFLP on a dilution series of T. vivax DNA in water. The diagnostic performance of the three PCRs was compared on 411 Ethiopian bovine blood specimens collected in a former study. TvPRAC PCR proved to be fully specific for T. vivax, irrespective of its geographical origin. Its analytical sensitivity was lower than that of ITS-1 PCR. On these bovine specimens, TvPRAC PCR detected 8.3% T. vivax infections while ITS-1 PCR and 18S PCR-RFLP detected respectively 22.6 and 6.1% T. vivax infections. The study demonstrates that a proline racemase based PCR could be used, preferably in combination with ITS-1 PCR, as a species-specific diagnostic test for T. vivax infections worldwide.

  5. Evaluation of Verigene Blood Culture Test Systems for Rapid Identification of Positive Blood Cultures.

    Science.gov (United States)

    Kim, Jae-Seok; Kang, Go-Eun; Kim, Han-Sung; Kim, Hyun Soo; Song, Wonkeun; Lee, Kyu Man

    2016-01-01

    The performance of molecular tests using the Verigene Gram-Positive and Gram-Negative Blood Culture nucleic acid tests (BC-GP and BC-GN, resp.; Naosphere, Northbrook, IL, USA) was evaluated for the identification of microorganisms detected from blood cultures. Ninety-nine blood cultures containing Gram-positive bacteria and 150 containing Gram-negative bacteria were analyzed using the BC-GP and BC-GN assays, respectively. Blood cultures were performed using the Bactec blood culture system (BD Diagnostic Systems, Franklin Lakes, NJ, USA) and conventional identification and antibiotic-susceptibility tests were performed using a MicroScan system (Siemens, West Sacramento, CA, USA). When a single strain of bacteria was isolated from the blood culture, Verigene assays correctly identified 97.9% (94/96) of Gram-positive bacteria and 93.8% (137/146) of Gram-negative bacteria. Resistance genes mecA and vanA were correctly detected by the BC-GP assay, while the extended-spectrum β-lactamase CTX-M and the carbapenemase OXA resistance gene were detected from 30 cases cultures by the BC-GN assay. The BC-GP and BC-GN assays showed high agreement with conventional identification and susceptibility tests. These tests are useful for rapid identification of microorganisms and the detection of clinically important resistance genes from positive Bactec blood cultures.

  6. Comparison among the Quantification of Bacterial Pathogens by qPCR, dPCR, and Cultural Methods

    Directory of Open Access Journals (Sweden)

    Matteo Ricchi

    2017-06-01

    Full Text Available The demand for rapid methods for the quantification of pathogens is increasing. Among these methods, those based on nucleic acids amplification (quantitative PCRs are the most widespread worldwide. Together with the qPCR, a new approach named digital PCR (dPCR, has rapidly gained importance. The aim of our study was to compare the results obtained using two different dPCR systems and one qPCR in the quantification of three different bacterial pathogens: Listeria monocytogenes, Francisella tularensis, and Mycobacterium avium subsp. paratuberculosis. For this purpose, three pre-existing qPCRs were used, while the same primers and probes, as well as PCR conditions, were transferred to two different dPCR systems: the QX200 (Bio-Rad and the Quant Studio 3D (Applied Biosystems. The limits of detection and limits of quantification for all pathogens, and all PCR approaches applied, were determined using genomic pure DNAs. The quantification of unknown decimal suspensions of the three bacteria obtained by the three different PCR approaches was compared through the Linear Regression and Bland and Altman analyses. Our results suggest that, both dPCRs are able to quantify the same amount of bacteria, while the comparison among dPCRs and qPCRs, showed both over and under-estimation of the bacteria present in the unknown suspensions. Our results showed qPCR over-estimated the amount of M. avium subsp. paratuberculosis and F. tularensis cells. On the contrary, qPCR, compared to QX200 dPCR, under-estimated the amount of L. monocytogenes cells. However, the maximum difference among PCRs approaches was <0.5 Log10, while cultural methods underestimated the number of bacteria by one to two Log10 for Francisella tularensis and Mycobacterium avium subsp. paratuberculosis. On the other hand, cultural and PCRs methods quantified the same amount of bacteria for L. monocytogenes, suggesting for this last pathogen, PCRs approaches can be considered as a valid alternative

  7. KNOWLEDGE, ATTITUDE AND PRACTICE OF BLOOD CULTURE ...

    African Journals Online (AJOL)

    boaz

    KNOWLEDGE, ATTITUDE AND PRACTICE OF BLOOD CULTURE: A CROSS. SECTIONAL STUDY AMONG MEDICAL DOCTORS IN A NIGERIAN. TERTIARY HOSPITAL. OJIDE, C. K.*, ONWUEZOBE, I. A., ASUQUO, E. E., OBIAGWU, C. S.. Department of Medical Microbiology and Parasitology, UUTH, Uyo, Akwa-Ibom.

  8. Detection of Mycobacterium avium subsp. paratuberculosis in Milk from Clinically Affected Cows by PCR and culture

    DEFF Research Database (Denmark)

    Giese, Steen Bjørck; Ahrens, Peter

    1999-01-01

    animals. In milk from 5 cows (all faecal culture-positive) we cultivated a few colonies of M. a. paratuberculosis (less than 100 CFU per mi). Milk samples from 2 cows were PCR-positive (both animals were faecal culture-positive, and 1 cow was milk culture positive). One cow was culture......Milk and faecal samples from cows with clinical symptoms of paratuberculosis were examined for the presence of Mycobacterium avium subsp.paratuberculosis (M. a. paratuberculosis) by culture and PCR. M. a. paratuberculosis was isolated in varied numbers from faeces or intestinal mucosa in 8 of 11......-negative on intestinal mucosa, but culture-positive in milk, and both faeces and milk were negative in culture and PCR from 2 cows. In conclusion the presence of M. a. paratuberculosis could be detected in raw milk by PCR but cultivation of milk was more sensitive in detecting the organism....

  9. MALDI-TOF MS based carbapenemase detection from culture isolates and from positive blood culture vials.

    Science.gov (United States)

    Ghebremedhin, B; Halstenbach, A; Smiljanic, M; Kaase, M; Ahmad-Nejad, P

    2016-02-02

    Antibiotic resistance in bacteria leads to massive health problems. Incidence of carbapenem and multidrug resistance in Gram-negative bacteria are increasing globally and turn out to be a very urgent challenge in health care. Resistant bacteria play an important clinical role during hospital outbreaks as well as in sepsis. Rapid diagnostic tests are necessary to provide immediate information for antimicrobial treatment and infection control measures. Our mass spectrometry-based assay was validated with 63 carbapenemase-producing Gram-negative bacterial isolates, and 35 carbapenem-resistant Gram-negative species with no carbapenemase production. These were analyzed from solid culture media and positive blood culture vials. After 4 h of incubation the carbapenemase products were analyzed with the MALDI-TOF MS. All the isolates were genotyped for carbapenemase genes by PCR and sequencing. For culture isolates the concordance of hydrolysis assay to genetic results was 98 % for OXA variants, KPC, VIM, IMP, GIM, and NDM. In contrast, only 14 of 29 Acinetobacter baumannii isolates carrying the OXA and NDM genes could be identified from blood culture. However, from blood culture vials our method allowed the detection of carbapenemases in 98 % of Pseudomonas and Enterobacteriaceae isolates harboring different genes. This MALDI-TOF MS-based assay permitted the detection of carbapenemases either from solid culture media (98-100 %) or blood culture vials (96 %) for all non-A. baumannii isolates within 4 h. In case of A. baumannii isolates the assay was highly sensitive for the detection of carbapenemases directly from solid culture media.

  10. Performance of Gram staining on blood cultures flagged negative by an automated blood culture system.

    Science.gov (United States)

    Peretz, A; Isakovich, N; Pastukh, N; Koifman, A; Glyatman, T; Brodsky, D

    2015-08-01

    Blood is one of the most important specimens sent to a microbiology laboratory for culture. Most blood cultures are incubated for 5-7 days, except in cases where there is a suspicion of infection caused by microorganisms that proliferate slowly, or infections expressed by a small number of bacteria in the bloodstream. Therefore, at the end of incubation, misidentification of positive cultures and false-negative results are a real possibility. The aim of this work was to perform a confirmation by Gram staining of the lack of any microorganisms in blood cultures that were identified as negative by the BACTEC™ FX system at the end of incubation. All bottles defined as negative by the BACTEC FX system were Gram-stained using an automatic device and inoculated on solid growth media. In our work, 15 cultures that were defined as negative by the BACTEC FX system at the end of the incubation were found to contain microorganisms when Gram-stained. The main characteristic of most bacteria and fungi growing in the culture bottles that were defined as negative was slow growth. This finding raises a problematic issue concerning the need to perform Gram staining of all blood cultures, which could overload the routine laboratory work, especially laboratories serving large medical centers and receiving a large number of blood cultures.

  11. Inhibition mechanisms of hemoglobin, immunoglobulin G, and whole blood in digital and real-time PCR.

    Science.gov (United States)

    Sidstedt, Maja; Hedman, Johannes; Romsos, Erica L; Waitara, Leticia; Wadsö, Lars; Steffen, Carolyn R; Vallone, Peter M; Rådström, Peter

    2018-04-01

    Blood samples are widely used for PCR-based DNA analysis in fields such as diagnosis of infectious diseases, cancer diagnostics, and forensic genetics. In this study, the mechanisms behind blood-induced PCR inhibition were evaluated by use of whole blood as well as known PCR-inhibitory molecules in both digital PCR and real-time PCR. Also, electrophoretic mobility shift assay was applied to investigate interactions between inhibitory proteins and DNA, and isothermal titration calorimetry was used to directly measure effects on DNA polymerase activity. Whole blood caused a decrease in the number of positive digital PCR reactions, lowered amplification efficiency, and caused severe quenching of the fluorescence of the passive reference dye 6-carboxy-X-rhodamine as well as the double-stranded DNA binding dye EvaGreen. Immunoglobulin G was found to bind to single-stranded genomic DNA, leading to increased quantification cycle values. Hemoglobin affected the DNA polymerase activity and thus lowered the amplification efficiency. Hemoglobin and hematin were shown to be the molecules in blood responsible for the fluorescence quenching. In conclusion, hemoglobin and immunoglobulin G are the two major PCR inhibitors in blood, where the first affects amplification through a direct effect on the DNA polymerase activity and quenches the fluorescence of free dye molecules, and the latter binds to single-stranded genomic DNA, hindering DNA polymerization in the first few PCR cycles. Graphical abstract PCR inhibition mechanisms of hemoglobin and immunoglobulin G (IgG). Cq quantification cycle, dsDNA double-stranded DNA, ssDNA single-stranded DNA.

  12. A duplex PCR for rapid and simultaneous detection of Brucella spp. in human blood samples.

    Science.gov (United States)

    Mirnejad, Reza; Mohamadi, Mozafar; Piranfar, Vahbeh; Mortazavi, Seied Mojtaba; Kachuei, Reza

    2013-06-01

    To design a duplex PCR for rapid and simultaneous detection of Brucella species. in human blood samples. Fifty-two peripheral bloods samples were collected from suspicious patients with brucellosis. Following DNA extraction, PCR assay were performed, using three primers that could simultaneously identify and differentiate three major species of pathogenic Brucella in humans and animals. Of the 52 peripheral bloods samples tested, 25 sample (48%) showed positive reactions in PCR. Twelve samples were positive for Brucella abortus 39 (B. abortus 39) (23%), 13 for Brucella melitensis 39 (B. melitensis 39) (25%) and 0 for Brucella ovis 39 (B. ovis 39) (0%). This work demonstrates that in case where specific primers were utilized, duplex PCR has proved to be a simple, fast, and relatively inexpensive method for simultaneous detection of important species of Brucella in clinical samples. Copyright © 2013 Hainan Medical College. Published by Elsevier B.V. All rights reserved.

  13. Comparison of conventional culture and real-time quantitative PCR ...

    African Journals Online (AJOL)

    2009-10-28

    HACCP) process in environments or during outbreak investigations this delay is a serious drawback. In recent years polymerase chain reaction (PCR) meth- ods have become the alternative to detect the presence of. Legionella in a ...

  14. [Diagnosis of chlamydia trachomatis infections in women: urinary PCR compared to cervical culture and PCR on cervical swabs in high risk females.].

    Science.gov (United States)

    Olafsson, J H; Davidsson, S; Karlsson, S M; Palsdottir, R; Steingrimsson, O

    1995-07-01

    Diagnosis of Chlamydia trachomatis infections in women has traditionally depended on cell culture or enzyme linked immunoassay. Recently Polymerase Chain Reaction (PCR) has been shown to be more sensitive than these methods when performed on endocervical swabs. A total of 203 high risk females were enrolled in a comparative study of three methods for diagnosing C. trachomatis infections: McCoy cell culture and Amplicor(R) PCR on endocervical swabs and urine. Thirty four had positive cultures, 38 positive PCR from cervix and 37 had positive PCR on urine specimens. When discrepancy occurred, the leftover Amplicor(R) specimen was retested by Roche with Amplicor(R) and a primer for the Major Outer Membrane Protein (MOMP) gene. None was false positive in cell culture or in urinary PCR but two were false positive in cervical PCR. In all three tests, 32 were positive. The sensitivity of culture was 87%, 92% in cervical PCR and 95% in urinary PCR. The specificity was 100% in both culture and urinary PCR but 98% in cervical PCR. The results show that Amplicor(R) PCR performed on female urine is more sensitive and as specific as cell culture.

  15. ABO Blood Group Genotyping by Real-time PCR in Kazakh Population

    Directory of Open Access Journals (Sweden)

    Pavel Tarlykov

    2014-12-01

    Full Text Available Introduction. ABO blood group genotyping is a new technology in hematology that helps prevent adverse transfusion reactions in patients. Identification of antigens on the surface of red blood cells is based on serology; however, genotyping employs a different strategy and is aimed directly at genes that determine the surface proteins. ABO blood group genotyping by real-time PCR has several crucial advantages over other PCR-based techniques, such as high rapidity and reliability of analysis. The purpose of this study was to examine nucleotide substitutions differences by blood types using a PCR-based method on Kazakh blood donors.Methods. The study was approved by the Ethics Committee of the National Center for Biotechnology. Venous blood samples from 369 healthy Kazakh blood donors, whose blood types had been determined by serological methods, were collected after obtaining informed consent. The phenotypes of the samples included blood group A (n = 99, B (n = 93, O (n = 132, and AB (n = 45. Genomic DNA was extracted using a salting-out method. PCR products of ABO gene were sequenced on an ABI 3730xl DNA analyzer (Applied Biosystems. The resulting nucleotide sequences were compared and aligned against reference sequence NM_020469.2. Real-time PCR analysis was performed on CFX96 Touch™ Real-Time PCR Detection System (BioRad.Results. Direct sequencing of ABO gene in 369 samples revealed that the vast majority of nucleotide substitutions that change the ABO phenotype were limited to exons 6 and 7 of the ABO gene at positions 261, 467, 657, 796, 803, 930 and 1,060. However, genotyping of only three of them (261, 796 and 803 resulted in identification of major ABO genotypes in the Kazakh population. As a result, TaqMan probe based real-time PCR assay for the specific detection of genotypes 261, 796 and 803 was developed. The assay did not take into account several other mutations that may affect the determination of blood group, because they have a

  16. PCR

    African Journals Online (AJOL)

    Jane

    2011-07-11

    Jul 11, 2011 ... then in age group 31 to 40 years, 2.25% (2/89) CMV DNA were detected by PCR and 0% was recorded in age group of above 40 years. The overall prevalence of human cytomegalovirus (HCMV) infection in 16 ..... genome revisited: Comparison with the chimpanzee cytomegalovirus genome. J. Gen. Virol.

  17. Evaluation of a PCR test for detection of treponema pallidum in swabs and blood.

    Science.gov (United States)

    Grange, P A; Gressier, L; Dion, P L; Farhi, D; Benhaddou, N; Gerhardt, P; Morini, J P; Deleuze, J; Pantoja, C; Bianchi, A; Lassau, F; Avril, M F; Janier, M; Dupin, N

    2012-03-01

    Syphilis diagnosis is based on clinical observation, serological analysis, and dark-field microscopy (DFM) detection of Treponema pallidum subsp. pallidum, the etiological agent of syphilis, in skin ulcers. We performed a nested PCR (nPCR) assay specifically amplifying the tpp47 gene of T. pallidum from swab and blood specimens. We studied a cohort of 294 patients with suspected syphilis and 35 healthy volunteers. Eighty-seven of the 294 patients had primary syphilis, 103 had secondary syphilis, 40 had latent syphilis, and 64 were found not to have syphilis. The T. pallidum nPCR results for swab specimens were highly concordant with syphilis diagnosis, with a sensitivity of 82% and a specificity of 95%. Reasonable agreement was observed between the results obtained with the nPCR and DFM methods (kappa = 0.53). No agreement was found between the nPCR detection of T. pallidum in blood and the diagnosis of syphilis, with sensitivities of 29, 18, 14.7, and 24% and specificities of 96, 92, 93, and 97% for peripheral blood mononuclear cell (PBMC), plasma, serum, and whole-blood fractions, respectively. HIV status did not affect the frequency of T. pallidum detection in any of the specimens tested. Swab specimens from mucosal or skin lesions seemed to be more useful than blood for the efficient detection of the T. pallidum genome and, thus, for the diagnosis of syphilis.

  18. Comparison of PCR, Culture, and Direct Fluorescent-Antibody Testing for Detection of Bordetella pertussis

    Science.gov (United States)

    Loeffelholz, Mike J.; Thompson, Curt J.; Long, Karla S.; Gilchrist, Mary J. R.

    1999-01-01

    We prospectively compared the performance of culture, direct fluorescent-antibody testing (DFA), and an in-house-developed PCR test targeting the repeated insertion sequence IS481 for the detection of Bordetella pertussis in nasopharyngeal swab specimens. We tested 319 consecutive paired specimens on which all three tests were performed. A total of 59 specimens were positive by one or more tests. Of these, 5 were positive by all three tests, 2 were positive by culture and PCR, 16 were positive by PCR and DFA, 28 were positive by PCR only, and 8 were positive by DFA only. Any specimen positive by culture was considered to be a true positive, as were specimens positive by both PCR and DFA. Specimens positive only by PCR or DFA were considered discrepant, and their status was resolved by review of patient histories. Patients with symptoms meeting the Centers for Disease Control and Prevention clinical case definition for pertussis and who had a specimen positive by PCR or DFA were considered to have true B. pertussis infections. Of the 28 patients positive by PCR only, 20 met the clinical case definition for pertussis, while 3 of the 8 patients positive by DFA only met the clinical case definition. After resolution of the status of discrepant specimens, the sensitivity, specificity, positive predictive value, and negative predictive value were 15.2, 100, 100, and 87.5%, respectively, for culture; 93.5, 97.1, 84.3, and 98.9%, respectively, for PCR; and 52.2, 98.2, 82.8, and 92.4%, respectively, for DFA. The actual positive predictive value of PCR was probably greater, as several PCR-positive patients who did not meet the clinical case definition had symptoms consistent with typical or atypical pertussis. PCR is a sensitive and specific method for the detection of B. pertussis. PMID:10449467

  19. The Utility of Blood Culture Fluid for the Molecular Diagnosis of Leptospira

    DEFF Research Database (Denmark)

    Dittrich, Sabine; Rudgard, William E.; Woods, Kate L.

    2016-01-01

    or direct and indirect testing combined, as the respective reference standards (specificities > 95%). Despite these promising diagnostic parameters, a subsequent prospective evaluation in a Lao hospital population (N = 352) showed that the sensitivity was very low (∼30%) compared with qPCR on venous blood...... samples. The disappointingly low sensitivity does suggest that venous blood samples are preferable for the clinical microbiology laboratory, although BCF might be an alternative if leptospirosis is only suspected postadmission after antibiotics have been used....... showing limited sensitivity. Leptospira have been shown to survive and multiply in blood culture media, and we hypothesized that extracting DNA from incubated blood culture fluid (BCF), followed by quantitative real-time polymerase chain reaction (qPCR) could improve the accuracy and speed of leptospira...

  20. Utilization of blood cultures in Danish hospitals

    DEFF Research Database (Denmark)

    Gubbels, S; Nielsen, J; Voldstedlund, M

    2015-01-01

    This national population-based study was conducted as part of the development of a national automated surveillance system for hospital-acquired bacteraemia and ascertains the utilization of blood cultures (BCs). A primary objective was to understand how local differences may affect interpretation...... older patients. BCs showed a seasonal pattern overall and for S. pneumoniae particularly. A predominance of male patients was seen for bacteraemias due to S. aureus, E. faecium and K. pneumoniae. Minor differences in BCs and positive BCs between departments of clinical microbiology underpin...

  1. A real-time PCR assay for the detection of Campylobacter jejuni in foods after enrichment culture.

    Science.gov (United States)

    Sails, Andrew D; Fox, Andrew J; Bolton, Frederick J; Wareing, David R A; Greenway, David L A

    2003-03-01

    A real-time PCR assay was developed for the quantitative detection of Campylobacter jejuni in foods after enrichment culture. The specificity of the assay for C. jejuni was demonstrated with a diverse range of Campylobacter species, related organisms, and unrelated genera. The assay had a linear range of quantification over six orders of magnitude, and the limit of detection was approximately 12 genome equivalents. The assay was used to detect C. jejuni in both naturally and artificially contaminated food samples. Ninety-seven foods, including raw poultry meat, offal, raw shellfish, and milk samples, were enriched in blood-free Campylobacter enrichment broth at 37 degrees C for 24 h, followed by 42 degrees C for 24 h. Enrichment cultures were subcultured to Campylobacter charcoal-cefoperazone-deoxycholate blood-free selective agar, and presumptive Campylobacter isolates were identified with phenotypic methods. DNA was extracted from enrichment cultures with a rapid lysis method and used as the template in the real-time PCR assay. A total of 66 samples were positive for C. jejuni by either method, with 57 samples positive for C. jejuni by subculture to selective agar medium and 63 samples positive in the real-time PCR assay. The results of both methods were concordant for 84 of the samples. The total time taken for detection from enrichment broth samples was approximately 3 h for the real-time PCR assay, with the results being available immediately at the end of PCR cycling, compared to 48 h for subculture to selective agar. This assay significantly reduces the total time taken for the detection of C. jejuni in foods and is an important model for other food-borne pathogens.

  2. Prognostic Value of RT-PCR Tyrosinase Detection in Peripheral Blood of Melanoma Patients

    Science.gov (United States)

    Carrillo, Esmeralda; Prados, José; Marchal, Juan Antonio; Boulaiz, Houria; Martínez, Antonio; Rodríguez-Serrano, Fernando; Caba, Octavio; Serrano, Salvio; Aránega, Antonia

    2006-01-01

    Malignant melanoma (MM) prognosis has been related to tumour thickness and clinical stage and metastasis risk has been associated with presence of tumour cells in peripheral blood. The aim of this study was to determine the relationship between presence of tyrosinase in peripheral blood of MM patients and their clinical prognosis. Blood samples from 58 MM patients (stage I–IV) were analysed, using RT-PCR assay to detect tyrosinase mRNA. The results showed that positive RT-PCR assay for tyrosinase were significantly associated with clinical status and tumour thickness. After a median follow-up of 24 months, RT-PCR results were found to be significant correlated with recurrence (p < 0.05) and clinical stage III (p < 0.05). Separate analysis of stage III tumours to determine the prognostic value of tyrosinase presence in peripheral blood showed an overall 24-month survival rate of 70% in the RT-PCR negative group versus 10% in the positive group (p < 0.02). These results suggest that detection of circulating melanoma cells may be especially relevant in stage III patients, in whom RT-PCR positivity defines a subpopulation at high risk of recurrence. PMID:16788251

  3. Comparison of COBAS AMPLICOR Neissefia gonorrhoeae PCR, including confirmation with N-gonorrhoeae-specific 16S rRNA PCR, with traditional culture

    NARCIS (Netherlands)

    Luijt, DS; Bos, PAJ; van Zwet, AA; Vader, PCV; Schirm, J

    A total of 3,023 clinical specimens were tested for Neisseria gonorrhoeae by using COBAS AMPLICOR (CA) PCR and confirmation of positives by N. gonorrhoeae-specific 16S rRNA PCR. The sensitivity of CA plus 16S rRNA PCR was 98.8%, compared to 68.2% for culture. Confirmation of CA positives increased

  4. QUANTIFICATION OF LISTERIA MONOCYTOGENES IN MILK BY MPN-PCR AND MPN-CULTURE METHODS

    Directory of Open Access Journals (Sweden)

    Mahzad Hosseini

    2014-10-01

    Full Text Available The aim of this study was to compare the MPN-PCR (Most Probable Number- Polymerase Chain Reaction and MPN-Culture methods in enumerating of Listeria monocytogenes in milk. In order to compare the accuracy of these methods, 103 cell/ml Listeria monocytogenes and different background bacteria which may be present in raw milk, were inoculated in sterilized milk. After preparing serial dilutions, three replicates per dilution were inoculated in tubes containing listeria enrichment broth. After 48 hours of incubation, for MPN-Culture three inoculated replicates were subcultured on Oxford agar and suspected colonies were confirmed by performing by biochemical tests. For MPN-PCR assay, the DNA extraction was performed from the three inoculated replicates which were already used for MPN-Culture and PCR assay was performed using primers specific for Listeria monocytogenes. The experiment was repeated three times and the average of enumerated bacteria was calculated by each method separately. Statistical analysis using one sample Wilcoxon signed rank test showed that enumeration by MPN-PCR method was more accurate than enumeration by MPN-Culture method. The result of this study showed that MPN-PCR method in comparision with MPN-Culture even in the presence of different background microorganisms is more rapid and reliable. It is concluded that MPN-PCR method facilitates the enumeration of Listeria monocytogenes without excessive work and could be considered as an alternative to MPN-Culture technique.

  5. Bayesian analysis of culture and PCR methods for detection of Campylobacter spp. in broiler caecal samples.

    Science.gov (United States)

    Arnold, M E; Jones, E M; Lawes, J R; Vidal, A B; Clifton-Hadley, F A; Rodgers, J D; Powell, L F

    2015-01-01

    The objective of this study was to estimate the sensitivity and specificity of a culture method and a polymerase chain reaction (PCR) method for detection of two Campylobacter species: C. jejuni and C. coli. Data were collected during a 3-year survey of UK broiler flocks, and consisted of parallel sampling of caeca from 436 batches of birds by both PCR and culture. Batches were stratified by season (summer/non-summer) and whether they were the first depopulation of the flock, resulting in four sub-populations. A Bayesian approach in the absence of a gold standard was adopted, and the sensitivity and specificity of the PCR and culture for each Campylobacter subtype was estimated, along with the true C. jejuni and C. coli prevalence in each sub-population. Results indicated that the sensitivity of the culture method was higher than that of PCR in detecting both species when the samples were derived from populations infected with at most one species of Campylobacter. However, from a mixed population, the sensitivity of culture for detecting both C. jejuni or C. coli is reduced while PCR is potentially able to detect both species, although the total probability of correctly identifying at least one species by PCR is similar to that of the culture method.

  6. Red blood cells in cerebrospinal fluid as possible inhibitory factor for enterovirus RT-PCR

    Directory of Open Access Journals (Sweden)

    Sérgio Monteiro de Almeida

    Full Text Available ABSTRACT The presence of hemoglobin in samples are considered an important inhibitory factor for polymerase chain reaction (PCR. The aim of this study was to examine the influence of red blood cells (RBCs in cerebrospinal fluid (CSF as an inhibitory factor to reverse transcription polymerase chain reaction (RT-PCR for enteroviruses (EV. Forty-four CSF samples from patients showing characteristics of viral meningitis were assessed for EV by RT-PCR. Viral RNA extracted with guanidine isothyocianate buffer and virus detection was performed by in-house nested PCR. Positivity for EV RT-PCR was higher in CSF samples without RBCs than in samples with RBCs: 13(26% and 36(9.2%, p = 0.001. In the group with positive EV RT-PCR, the mean + SD CSF RBC was 37 ± 183 cell/mm3; the group with negative results had 580 + 2,890 cell/mm3 (p = 0.007. The acceptable upper limit for CSF RBCs that could not influence RT-PCR was 108 cells/mm3. CSF samples with negative results for EV RT-PCR have more erythrocytes.

  7. The Optimization of Molecular Detection of Clinical Isolates of Brucella in Blood Cultures by eryD Transcriptase Gene for Confirmation of Culture-Negative Samples.

    Science.gov (United States)

    Tabibnejad, Mahsa; Alikhani, Mohammad Yousef; Arjomandzadegan, Mohammad; Hashemi, Seyed Hamid; Naseri, Zahra

    2016-04-01

    Brucellosis is a zoonosis disease which is widespread across the world. The aim of the present study is the evaluation of culture-negative blood samples. A total of 100 patients with suspected brucellosis were included in this experimental study and given positive serological tests. Diagnosis was performed on patients with clinical symptoms of the disease, followed by the detection of a titer that was equal to or more than 1:160 (in endemic areas) by the standard tube agglutination method. Blood samples were cultured by a BACTEC 9050 system, and subsequently by Brucella agar. At the same time, DNA from all blood samples was extracted by Qiagen Kit Company (Qia Amp Mini Kit). A molecular assay of blood samples was carried out by detection of eryD transcriptase and bcsp 31 genes in specific double PCR reactions. The specificity of the primers was evaluated by DNA from pure and approved Brucella colonies found in the blood samples, by DNA from other bacteria, and by ordinary PCR. DNA extraction from the pure colonies was carried out by both Qiagen Kit and Chelex 100 methods; the two were compared. 39 cases (39%) had positive results when tested by the BACTEC system, and 61 cases (61%) became negative. 23 culture-positive blood samples were randomly selected for PCR reactions; all showed 491 bp for the eryD gene and 223 bp for the bcsp 31 gene. Interestingly, out of 14 culture-negative blood samples, 13 cases showed positive bonds in PCR. The specificity of the PCR method was equal to 100%. DNA extraction from pure cultures was done by both Chelex 100 and Qiagen Kit; these showed the same results for all samples. The results prove that the presented double PCR method could be used to detect positive cases from culture-negative blood samples. The Chelex 100 method is simpler and safer than the use of Qiagen Kit for DNA extraction.

  8. Molecular typing for blood group antigens within 40 minutes by direct PCR from plasma or serum

    Science.gov (United States)

    Wagner, Franz Friedrich; Flegel, Willy Albert; Bittner, Rita; Döscher, Andrea

    2016-01-01

    Determining blood group antigens by serological methods may be unreliable in certain situations, such as in patients after chronic or massive transfusion. Red cell genotyping offers a complementary approach, but current methods may take much longer than conventional serological typing, limiting their utility in urgent situations. To narrow this gap, we devised a rapid method using direct polymerase chain reaction (PCR) amplification while avoiding the DNA extraction step. DNA was amplified by PCR directly from plasma or serum of blood donors followed by a melting curve analysis in a capillary rapid-cycle PCR assay. We evaluated the single nucleotide polymorphisms underlying the clinically relevant Fya, Fyb, Jka and Jkb antigens, with our analysis being completed within 40 min of receiving a plasma or serum sample. The positive predictive value was 100% and the negative predictive value at least 84%. Direct PCR with melting point analysis allowed faster red cell genotyping to predict blood group antigens than any previous molecular method. Our assay may be used as a screening tool with subsequent confirmatory testing, within the limitations of the false-negative rate. With fast turnaround times, the rapid-cycle PCR assay may eventually be developed and applied to red cell genotyping in the hospital setting. PMID:27991657

  9. Bone Marrow Culture Vs Blood Culture in FUO

    Directory of Open Access Journals (Sweden)

    Abhimanyu Jha

    2009-04-01

    bacterial culture. The results of BMCs and BCs were compared. Results:Total 57 cases of FUO were included in the study. Male female ratio was 1.22:1. Age range was fi ve to 83 years (median 30. Duration of fever was 21 to 365 days. Bacterial growth was seen in nine cases (15.78% of BMCs and in three cases (5.26% of corresponding BCs. Fungal or myocbacterial growth was not seen. Salmonella typhi was the commonest organism isolated in BMCs (three cases followed by Staphylococcus aureus (two cases, Escherichia coli, Non fermenting Gram negative bacilli, Enterococcus species and Salmonella paratyphi–A (one case each. Two cases of Salmonella typhi and one case of Salmonella paratyphi–A were isolated in BCs. Conclusions:BMCs are more useful than BCs in evaluation of patients with FUO, especially in cases of salmonella infection and are particularly important when the patient has already taken antibiotics. In immuno-competent patients presenting with FUO, BMCs for mycobacteria or fungi is unlikely to yield any growth. Key Words: blood culture, bone marrow culture, fever of unknown origin

  10. Topography-Assisted Electromagnetic Platform for Blood-to-PCR in a Droplet

    Science.gov (United States)

    Chiou, Chi-Han; Shin, Dong Jin; Zhang, Yi; Wang, Tza-Huei

    2013-01-01

    This paper presents an electromagnetically actuated platform for automated sample preparation and detection of nucleic acids. The proposed platform integrates nucleic acid extraction using silica-coated magnetic particles with real-time polymerase chain reaction (PCR) on a single cartridge. Extraction of genomic material was automated by manipulating magnetic particles in droplets using a series of planar coil electromagnets assisted by topographical features, enabling efficient fluidic processing over a variety of buffers and reagents. The functionality of the platform was demonstrated by performing nucleic acid extraction from whole blood, followed by real-time PCR detection of KRAS oncogene. Automated sample processing from whole blood to PCR-ready droplet was performed in 15 minutes. We took a modular approach of decoupling the modules of magnetic manipulation and optical detection from the device itself, enabling a low-complexity cartridge that operates in tandem with simple external instruments. PMID:23835223

  11. Comparison of real-time quantitative PCR and culture for the diagnosis of emerging Rickettsioses.

    Directory of Open Access Journals (Sweden)

    Emmanouil Angelakis

    Full Text Available BACKGROUND: Isolation of Rickettsia species from skin biopsies may be replaced by PCR. We evaluated culture sensitivity compared to PCR based on sampling delay and previous antibiotic treatment. METHODOLOGY/PRINCIPAL FINDINGS: Skin biopsies and ticks from patients with suspected Rickettsia infection were screened for Rickettsia spp. using qPCR, and positive results were amplified and sequenced for the gltA and ompA genes. Immunofluorescence for spotted fever group rickettsial antigens was done for 79 patients. All skin biopsies and only ticks that tested positive using qPCR were cultured in human embryonic lung (HEL fibroblasts using the centrifugation-shell vial technique. Patients and ticks were classified as definitely having rickettsioses if there was direct evidence of infection with a Rickettsia sp. using culture or molecular assays or in patients if serology was positive. Data on previous antibiotic treatments were obtained for patients with rickettsiosis. Rickettsia spp. infection was diagnosed in 47 out of 145 patients (32%, 41 by PCR and 12 by culture, whereas 3 isolates were obtained from PCR negative biopsies. For 3 of the patients serology was positive although PCR and culture were negative. Rickettsia africae was the most common detected species (n = 25, [17.2%] and isolated bacterium (n = 5, [3.4%]. The probability of isolating Rickettsia spp. was 12 times higher in untreated patients and 5.4 times higher in patients from our hometown. Rickettsia spp. was amplified in 24 out of 95 ticks (25% and we isolated 7 R. slovaca and 1 R. raoultii from Dermacentor marginatus. CONCLUSIONS/SIGNIFICANCE: We found a positive correlation between the bacteria copies and the isolation success in skin biopsies and ticks. Culture remains critical for strain analysis but is less sensitive than serology and PCR for the diagnosis of a Rickettsia infection.

  12. Single-step blood direct PCR: A robust and rapid method to diagnose triplet repeat disorders.

    Science.gov (United States)

    Singh, Inder; Swarup, Vishnu; Shakya, Sunil; Goyal, Vinay; Faruq, Mohammed; Srivastava, Achal Kumar

    2017-08-15

    DNA extraction prior to polymerase chain reaction (PCR) amplification in genetic diagnoses of triplet repeat disorders (TRDs) is tedious and labour-intensive and has the limitations of sample contamination with foreign DNA, including that from preceding samples. Therefore, we aimed to develop a rapid, robust, and cost-effective method for expeditious genetic investigation of TRDs from whole blood as a DNA template. Peripheral blood samples were collected from 70 clinically suspected patients of progressive ataxia. The conventional method using genomic DNA and single-step Blood-Direct PCR (BD-PCR) method with just 2μl of whole blood sample were tested to amplify triplet repeat expansion in genes related to spinocerebellar ataxia (SCA) types 1, 2, 3, 12 and Friedreich's ataxia (FRDA). Post-PCR, the allele sizes were mapped and repeat numbers were calculated using GeneMapper and macros run in Microsoft Excel programmes. Successful amplification of target regions was achieved in all samples by both methods. The frequency of the normal and mutated allele was concordant between both methods, diagnosing 37% positive for a mutation in either of the candidate genes. The BD-PCR resulted in higher intensities of product peaks of normal and pathogenic alleles. The nearly-accurate sizing of the normal and expanded allele was achieved in a shorter time (4-5h), without DNA extraction and any risk of cross contamination, which suggests the BD-PCR to be a reliable, inexpensive, and rapid method to confirm TRDs. This technique can be introduced in routine diagnostic procedures of other tandem repeat disorders. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Rapid Diagnosis of Staphylococcal Catheter-Related Bacteraemia in Direct Blood Samples by Real-Time PCR.

    Directory of Open Access Journals (Sweden)

    Yuliya Zboromyrska

    Full Text Available Catheter-related bacteremia (CRB is an important cause of morbidity and mortality among hospitalized patients, being staphylococci the main etiologic agents. The objective of this study was to assess the use of a PCR-based assay for detection of staphylococci directly from blood obtained through the catheter to diagnose CRB caused by these microorganisms and to perform a cost-effectiveness analysis. A total of 92 patients with suspected CRB were included in the study. Samples were obtained through the catheter. Paired blood cultures were processed by standard culture methods and 4 ml blood samples were processed by GeneXpert-MRSA assay for the detection of methicillin-susceptible (MSSA or methicillin-resistant (MRSA Staphylococcus aureus, and methicillin-resistant coagulase-negative staphylococci (MR-CoNS. Sixteen CRB caused by staphylococci were diagnosed among 92 suspected patients. GeneXpert detected 14 out of 16 cases (87.5%, including 4 MSSA and 10 MR-CoNS in approximately 1 hour after specimen receipt. The sensitivity and specificity of GeneXpert were 87.5% (CI 95%: 60.4-97.8 and 92.1% (CI 95%: 83-96.7, respectively, compared with standard culture methods. The sensitivity of GeneXpert for S. aureus was 100%. Regarding a cost-effectiveness analysis, the incremental cost of using GeneXpert was of 31.1€ per patient while the incremental cost-effectiveness ratio of GeneXpert compared with blood culture alones was about 180€ per life year gained. In conclusion, GeneXpert can be used directly with blood samples obtained through infected catheters to detect S. aureus and MR-CoNS in approximately 1h after sampling. In addition, it is cost-effective especially in areas with high prevalence of staphylococcal CRB.

  14. Survival and function of phagocytes in blood culture media

    DEFF Research Database (Denmark)

    Fischer, T K; Prag, J; Kharazmi, A

    1999-01-01

    The survival and function of human phagocytes in sterile aerobic and anaerobic blood culture media were investigated using neutrophil morphology, white blood cell count in a haemoanalyser, flow cytometry, oxidative burst response, and bactericidal effect in Colorbact and Septi-Chek blood culture...

  15. [Importance of skin contamination in blood culture readings].

    Science.gov (United States)

    Kunze, M; Volkman, H; Köhler, W

    1979-11-01

    The importance of the skin contamination for the results of blood cultures was emphasized by model examinations. In the method of blood taking without previous desinfection of the skin the quota of positive blood cultures increased by the twofold to threefold per culture and test person (5.7 to 18.8% and 11.3 to 26.3%, respectively). In large-volume blood takings the contamination rate becomes smaller with increasing blood volume. The rejecting of a first blood sample is to be recommended, when the possibility is given. With an increased quantity o blood per taking by blood bactericidia a decreased contamination rate is to be expected. By the results of the examinations the necessity of a consequent desinfection of the skin is to be emphasized, also when closed systems of blood cultures are used.

  16. Diagnosis of common bacterial causes of urethritis in men by Gram stain, culture and multiplex PCR.

    Science.gov (United States)

    Jahan, F; Shamsuzzaman, S M; Akter, S

    2014-12-01

    Urethritis is one of the most important causes of morbidity and mortality in developing countries. The aim of this study was to detect common bacterial causes of urethritis in men by Gram stain, culture and multiplex PCR.185 male patients who presented at the Skin and venereal clinic of the Dhaka Medical College, Bangladesh with clinical symptoms suggestive of urethritis were enrolled in this study. Urethral discharges were tested for detection of Neisseria gonorrhoeae by Gram stain, culture and PCR. Multiplex PCR assay was done to detect DNA of Chlamydia trachomatis, Ureaplasma urealyticum and Mycoplasma genitalium. Out of 185 participants, 30.27% and 14.6% were infected by Neisseria gonorrhoeae and Chlamydia trachomatis respectively. None of the individuals was found positive for either Ureaplasma urealyticum or Mycoplasma genitalium. Among the Neisseria gonorrhoeae positive patients 27.57% were positive from Gram stain, 26.49% were culture positive, 30.27% were positive by PCR (p<0.001). 32.65% of the Neisseria gonorrhoeae isolates were penicillinase producers and 83.67% were susceptible to ceftriaxone. Considering culture as the gold standard, the sensitivity and specificity of PCR for the detection of Neisseria gonorrhoeae was 100%, and 94.85% respectively with an accuracy of 96.22%. 3.73% of the 134 smear negative and 5.15% of the 136 culture negative samples were positive by PCR. PCR was the most sensitive and rapid method for the diagnosis of urethritis. Multiplex PCR may be a useful approach to laboratory diagnosis of urethritis in men for its high sensitivity and specificity.

  17. Sensitivity of PCR IS6110 in relation to culture and staining in Pott′s disease

    Directory of Open Access Journals (Sweden)

    Manoj Kumar

    2013-01-01

    Full Text Available Background: Rapid diagnosis is essential to decrease the morbidity and mortality of Pott′s disease. The bacteriological methods are time-consuming or insensitive. Polymerase chain reaction (PCR provides a rapid diagnostic tool and hope for early diagnosis of this disease. The aim of this study was to compare and assess of a rapid and effective method among diagnostic battery (Ziehl-Neelsen (ZN microscopy, BACTEC culture and PCR of Pott′s disease. Materials and Methods: Sixty-five specimens from clinico-radiological suspected cases of Pott′s disease were included in this study. They were processed for ZN microscopy, BACTEC culture, and PCR IS6110. The tests tool′s efficiency, positive agreement Kc (Kappa coefficient, and significance level (P value were calculated for correlation between PCR and performed tests. Results: The PCR sensitivity reached to 96% and 46.3% among positive and negative specimens on ZN microscopy. Further, 94% and 36.4% sensitivity were found among positive and negative specimens by BACTEC culture. The total 38 (58.5% specimens were detected either ZN microscopy or by BACTEC culture. Thus, the overall sensitivity and specificity of PCR were 95% and 74.1%. The kappa coefficient and P value, calculated for PCR against BACTEC culture and combined results of performed bacteriological tests were (Kc=0.60, (P<0.001 and (Kc=0.70, (P<0.001, respectively. Above statistical relations showed a fair agreement with significant differences. Conclusion: The PCR IS6110 may be useful in rapid detection of clinico-radiological suspected cases of Pott′s disease and those that are negative with bacteriological methods.

  18. Comparison of multiplex real-time PCR and PCR-reverse blot hybridization assay for the direct and rapid detection of bacteria and antibiotic resistance determinants in positive culture bottles.

    Science.gov (United States)

    Wang, Hye-Young; Kim, Seoyong; Kim, Jungho; Park, Soon Deok; Kim, Hyo Youl; Uh, Young; Lee, Hyeyoung

    2016-09-01

    The aim of this study was to evaluate the performance of a commercially available multiplex real-time PCR assay and a PCR-reverse blot hybridization assay (PCR-REBA) for the rapid detection of bacteria and identification of antibiotic resistance genes directly from blood culture bottles and to compare the results of these molecular assays with conventional culture methods. The molecular diagnostic methods were used to evaluate 593 blood culture bottles from patients with bloodstream infections. The detection positivity of multiplex real-time PCR assay for Gram-positive bacteria, Gram-negative bacteria and Candida spp. was equivalent to PCR-REBA as 99.6 %, 99.1 % and 100 %, respectively. Using conventional bacterial cultures as the gold standard, the sensitivity, specificity, positive predictive value and negative predictive value of these two molecular methods were 99.5 % [95 % confidence interval (CI), 0.980-1.000; PPCR assay targeting the mecA gene to detect methicillin resistance was lower than that of the PCR-REBA method, detecting an overall positivity of 98.4 % (n=182; 95 % CI, 0.964-1.000; P<0.009) and 99.5 % (n=184; 95 % CI, 0.985-1.000; P<0.0001), respectively. The entire two methods take about 3 h, while results from culture can take up to 48-72 h. Therefore, the use of these two molecular methods was rapid and reliable for the characterization of causative pathogens in bloodstream infections.

  19. Plasmodium Detection and Differentiation by Direct-on-Blood PCR Nucleic Acid Lateral Flow Immunoassay Development, Validation, and Evaluation

    NARCIS (Netherlands)

    Roth, Johanna M.; de Bes, Laura; Sawa, Patrick; Omweri, George; Osoti, Victor; Oberheitmann, Boris; Schallig, Henk D. F. H.; Mens, Pètra F.

    2018-01-01

    Decreasing malaria transmission warrants the search for highly sensitive point-of-care diagnostics, especially in resource-limited settings. The direct-on-blood PCR nucleic add lateral flow immunoassay (db-PCR-NALFIA) is a simplified PCR-based technique with a lateral flow readout that does not

  20. Legionella detection by culture and qPCR: Comparing apples and oranges.

    Science.gov (United States)

    Whiley, Harriet; Taylor, Michael

    2016-01-01

    Legionella spp. are the causative agent of Legionnaire's disease and an opportunistic pathogen of significant public health concern. Identification and quantification from environmental sources is crucial for identifying outbreak origins and providing sufficient information for risk assessment and disease prevention. Currently there are a range of methods for Legionella spp. quantification from environmental sources, but the two most widely used and accepted are culture and real-time polymerase chain reaction (qPCR). This paper provides a review of these two methods and outlines their advantages and limitations. Studies from the last 10 years which have concurrently used culture and qPCR to quantify Legionella spp. from environmental sources have been compiled. 26/28 studies detected Legionella at a higher rate using qPCR compared to culture, whilst only one study detected equivalent levels of Legionella spp. using both qPCR and culture. Aggregating the environmental samples from all 28 studies, 2856/3967 (72%) tested positive for the presence of Legionella spp. using qPCR and 1331/3967 (34%) using culture. The lack of correlation between methods highlights the need to develop an acceptable standardized method for quantification that is sufficient for risk assessment and management of this human pathogen.

  1. Time to positivity of blood cultures in neonates.

    Science.gov (United States)

    Vamsi, Sivarama Raju; Bhat, Ramesh Y; Lewis, Leslie E; Vandana, Kalwaje E

    2014-02-01

    Blood culture reports in neonatal sepsis aid physician in either optimizing therapy or discontinuing antibiotics. We determined the time taken for neonatal blood cultures to become positive using the aerobic BacT/Alert system. Of 944 blood cultures from 816 neonates, 139 (14.7%) were positive. Growth of all definitive bacteria, 95% of possible bacteria and 84% of fungi were detected within 48 hours of incubation.

  2. Failure of PCR to Detect Treponema pallidum ssp. pertenue DNA in Blood in Latent Yaws.

    Science.gov (United States)

    Marks, Michael; Katz, Samantha; Chi, Kai-Hua; Vahi, Ventis; Sun, Yongcheng; Mabey, David C; Solomon, Anthony W; Chen, Cheng Y; Pillay, Allan

    2015-01-01

    Yaws, caused by Treponema pallidum ssp. pertenue, is a neglected tropical disease closely related to venereal syphilis and is targeted for eradication by 2020. Latent yaws represents a diagnostic challenge, and current tools cannot adequately distinguish between individuals with true latent infection and individuals who are serofast following successful treatment. PCR on blood has previously been shown to detect T. pallidum DNA in patients with syphilis, suggesting that this approach may be of value in yaws. We performed real-time PCR for Treponema pallidum ssp. pertenue on blood samples from 140 children with positive T. pallidum Particle Agglutination (TPPA) and Rapid Plasma Reagin (RPR) tests and 7 controls (negative serology), all collected as part of a prospective study of yaws in the Solomon Islands. All samples were also tested by a nested PCR for T. pallidum. 12 patients had clinical evidence of active yaws whilst 128 were considered to have latent yaws. 43 children had high titre rapid plasma reagins (RPRs) of ≥1:32. PCR testing with both assays gave negative results in all cases. It is possible that the failure to detect T. pallidum ssp. pertenue in blood reflects lower loads of organism in latent yaws compared to those in latent infection with T. pallidum ssp. pertenue, and/or a lower propensity for haematogenous dissemination in yaws than in syphilis. As the goal of the yaws control programme is eradication, a tool that can differentiate true latent infection from individuals who are serofast would be of value; however, PCR of blood is not that tool.

  3. Failure of PCR to Detect Treponema pallidum ssp. pertenue DNA in Blood in Latent Yaws.

    Directory of Open Access Journals (Sweden)

    Michael Marks

    Full Text Available Yaws, caused by Treponema pallidum ssp. pertenue, is a neglected tropical disease closely related to venereal syphilis and is targeted for eradication by 2020. Latent yaws represents a diagnostic challenge, and current tools cannot adequately distinguish between individuals with true latent infection and individuals who are serofast following successful treatment. PCR on blood has previously been shown to detect T. pallidum DNA in patients with syphilis, suggesting that this approach may be of value in yaws. We performed real-time PCR for Treponema pallidum ssp. pertenue on blood samples from 140 children with positive T. pallidum Particle Agglutination (TPPA and Rapid Plasma Reagin (RPR tests and 7 controls (negative serology, all collected as part of a prospective study of yaws in the Solomon Islands. All samples were also tested by a nested PCR for T. pallidum. 12 patients had clinical evidence of active yaws whilst 128 were considered to have latent yaws. 43 children had high titre rapid plasma reagins (RPRs of ≥1:32. PCR testing with both assays gave negative results in all cases. It is possible that the failure to detect T. pallidum ssp. pertenue in blood reflects lower loads of organism in latent yaws compared to those in latent infection with T. pallidum ssp. pertenue, and/or a lower propensity for haematogenous dissemination in yaws than in syphilis. As the goal of the yaws control programme is eradication, a tool that can differentiate true latent infection from individuals who are serofast would be of value; however, PCR of blood is not that tool.

  4. Preanalytical blood sample workup for cell-free DNA analysis using Droplet Digital PCR for future molecular cancer diagnostics

    NARCIS (Netherlands)

    van Ginkel, Joost H.; van den Broek, Daan A.; van Kuik, Joyce; Linders, Dorothé; de Weger, Roel; Willems, Stefan M.; Huibers, Manon M.H.

    2017-01-01

    In current molecular cancer diagnostics, using blood samples of cancer patients for the detection of genetic alterations in plasma (cell-free) circulating tumor DNA (ctDNA) is an emerging practice. Since ctDNA levels in blood are low, highly sensitive Droplet Digital PCR (ddPCR) can be used for

  5. Comparative analysis between RQ-PCR and digital droplet PCR of BCL2/IGH gene rearrangement in the peripheral blood and bone marrow of early stage follicular lymphoma.

    Science.gov (United States)

    Cavalli, Marzia; De Novi, Lucia Anna; Della Starza, Irene; Cappelli, Luca Vincenzo; Nunes, Vittorio; Pulsoni, Alessandro; Del Giudice, Ilaria; Guarini, Anna; Foà, Robin

    2017-05-01

    BCL2/IGH rearrangements were analysed by polymerase chain reaction (PCR) at diagnosis in paired peripheral blood (PB) and bone marrow (BM) samples from 67 patients with stage I/II follicular lymphoma (FL). Real time quantitative PCR (RQ-PCR) and digital droplet PCR (ddPCR) were performed in cases with a major breakpoint region (MBR+) at diagnosis and after localized radiotherapy and rituximab administration in order to investigate the applicability of ddPCR. The overall ddPCR/RQ-PCR concordance was 81·9% (113/138 samples) and 97·5% in the 40/138 with quantifiable disease (RQ-PCR≥10 -5 ). At baseline, ddPCR allowed the recovery of a MBR+ marker in 8/18 (44·4%) samples that resulted MBR-negative/minor cluster region-negative/minor BCL2-negative by qualitative PCR. Moreover, the tumour burden at diagnosis significantly predicted progression-free survival (PSF) only when quantified by ddPCR. Paired PB and BM samples analysis demonstrated a high concordance in the detection of BCL2/IGH+ cells by qualitative and quantitative methods; in particular, 40/62 samples were positive by ddPCR (25 PB+/BM+; 9 PB+/BM-; 6 PB-/BM+), with 34/40 (85%) identified by the study of PB only. In conclusion, in localized FL, ddPCR is a promising tool for monitoring minimal residual disease (MRD) that is at least comparable to RQ-PCR and potentially more accurate. PB is a suitable source for serial BCL2/IGH MRD assessments, regardless of the methodology utilized. © 2017 John Wiley & Sons Ltd.

  6. Miltenberger blood group typing by real-time polymerase chain reaction (qPCR) melting curve analysis in Thai population.

    Science.gov (United States)

    Vongsakulyanon, A; Kitpoka, P; Kunakorn, M; Srikhirin, T

    2015-12-01

    To develop reliable and convenient methods for Miltenberger (Mi(a) ) blood group typing. To apply real-time polymerase chain reaction (qPCR) melting curve analysis to Mi(a) blood group typing. The Mi(a) blood group is the collective set of glycophorin hybrids in the MNS blood group system. Mi(a+) blood is common among East Asians and is also found in the Thai population. Incompatible Mi(a) blood transfusions pose the risk of life-threatening haemolysis; therefore, Mi(a) blood group typing is necessary in ethnicities where the Mi(a) blood group is prevalent. One hundred and forty-three blood samples from Thai blood donors were used in the study. The samples included 50 Mi(a+) samples and 93 Mi(a-) samples, which were defined by serology. The samples were typed by Mi(a) typing qPCR, and 50 Mi(a+) samples were sequenced to identify the Mi(a) subtypes. Mi(a) subtyping qPCR was performed to define GP.Mur. Both Mi(a) typing and Mi(a) subtyping were tested on a conventional PCR platform. The results of Mi(a) typing qPCR were all concordant with serology. Sequencing of the 50 Mi(a+) samples revealed 47 GP.Mur samples and 3 GP.Hop or Bun samples. Mi(a) subtyping qPCR was the supplementary test used to further define GP.Mur from other Mi(a) subtypes. Both Mi(a) typing and Mi(a) subtyping performed well using a conventional PCR platform. Mi(a) typing qPCR correctly identified Mi(a) blood groups in a Thai population with the feasibility of Mi(a) subtype discrimination, and Mi(a) subtyping qPCR was able to further define GP.Mur from other Mi(a) subtypes. © 2015 British Blood Transfusion Society.

  7. Detection of Legionella bozemanae, a New Cause of Septic Arthritis, by PCR Followed by Specific Culture

    DEFF Research Database (Denmark)

    Just, Søren Andreas; Knudsen, John Bonde; Uldum, Søren Anker

    2012-01-01

    Legionella bozemanae is a rare isolate in clinical specimens. We describe a case of joint infection due to L. bozemanae in an immunocompromised patient with dermatomyositis. Without the use of PCR screening or culture on specialized medium, the organism would not have been detected.......Legionella bozemanae is a rare isolate in clinical specimens. We describe a case of joint infection due to L. bozemanae in an immunocompromised patient with dermatomyositis. Without the use of PCR screening or culture on specialized medium, the organism would not have been detected....

  8. Blood culture bottles are superior to conventional media for vitreous culture.

    Science.gov (United States)

    Thariya, Patsuda; Yospaiboon, Yosanan; Sinawat, Suthasinee; Sanguansak, Thuss; Bhoomibunchoo, Chavakij; Laovirojjanakul, Wipada

    2016-08-01

    To compare blood culture bottles and conventional media for the vitreous culture in patients with clinically suspected infectious endophthalmitis. Retrospective comparative study at KKU Eye Center, Khon Kaen University. There were 342 patients with clinically suspected infectious endophthalmitis participated in the study. The vitreous specimens were inoculated in both blood culture bottles and on conventional culture media (blood agar, MacConkey agar, chocolate agar, Sabouraud dextrose agar and thioglycolate broth). The number of positive culture yields in both blood culture bottles and conventional media. Positive culture yields in both methods were found in 151 eyes (49.5%). There were 136 of 151 eyes (90.1%) with positive culture in blood culture bottles, whereas 99 of 151 eyes (65.6%) yielded positive cultures in conventional media. These findings were different with a statistical significance (P culture bottles and conventional media improved the yield. Blood culture bottles are superior to conventional media for vitreous culture in clinically suspected infectious endophthalmitis. Vitreous culture using blood culture bottles should be recommended as the primary method for microbiological diagnosis. A combination of both methods further improves the positive culture yield. © 2016 Royal Australian and New Zealand College of Ophthalmologists.

  9. A new whole mitochondrial genome qPCR (WMG-qPCR) with SYBR Green®to identify phlebotomine sand fly blood meals.

    Science.gov (United States)

    Rodrigues, Ana Caroline Moura; Magalhães, Rafaela Damasceno; Romcy, Kalil Andrade Mubarac; Freitas, Jeferson Lucas Sousa; Melo, Ana Carolina Fonseca Lindoso; Rodon, Fernanda Cristina Macedo; Bevilaqua, Claudia Maria Leal; Melo, Luciana Magalhães

    2017-04-30

    Phlebotomine sand flies are blood-feeding insects of marked medical and veterinary significance. Investigations on the biology of these insects hold great importance for both ecological and epidemiological purposes. The present work describes a new approach for real-time PCR (qPCR) with SYBR Green ® , named WMG-qPCR, to identify phlebotomine blood meals. The novelty of the assay was to design primers based on the Whole Mitochondrial Genome (WMG) of the potential hosts (human, dog, cat, brown rat and chicken) aiming to amplify through qPCR the regions of mitochondrial DNA (mtDNA) which are less conserved among all species. Initially, the best method for mtDNA extraction to be applied in WMG-qPCR was determined. Afterwards, amplification specificities were accessed by cross-reaction assays with mtDNA samples from all animal species, besides phlebotomine DNA. Finally, the selected primers were also tested for their limit of DNA detection through standard curves constructed by serial dilution of blood DNA obtained for each target animal species. The WMG-qPCR was able to detect as low as 10pL of blood, equivalent to 26, 84, 130, and 320fg DNA of cat, human, dog and rat, respectively. The assay was also capable to amplify as low as 5pL of chicken blood (5pg DNA). In conclusion, WMG-qPCR seems to be a promising tool to identify phlebotomine blood meals, with high species-specificity and sensitivity. Furthermore, as no supplementary techniques are required, this new approach presents minimized costs and simplified technical-training requirements for execution. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Detection of Candida in concentrated oral rinse cultures by real-time PCR.

    Science.gov (United States)

    White, P Lewis; Williams, David W; Kuriyama, Tomoari; Samad, Shamim A; Lewis, Michael A O; Barnes, Rosemary A

    2004-05-01

    The incidence of oral candidosis has increased in recent years, largely as a result of the emergence of human immunodeficiency virus infection and the more widespread use of immunosuppressive chemotherapy. This development has been associated with a need for more reliable methods for the detection of Candida. The present study assessed the performance of a real-time PCR and two block-based PCRs for the detection of Candida in 193 concentrated oral rinse culture (CRC) specimens. A total of 102 CRC specimens were positive by culture for Candida; and 96, 90, and 75 of these were also positive by real-time, N18-specific, and internal transcribed spacer (ITS)-specific PCRs, respectively. The five false-negative results by the real-time PCR were all non-Candida albicans positive by culture. Of the 91 culture-negative CRC specimens, 20, 41, and 44 were positive by the real-time PCR and the N18- and ITS-specific PCRs, respectively. All three PCRs detected fungal DNA in 8 culture-negative CRC specimens, with a further 30 being positive by two of the three PCRs. A total of 32 CRC specimens were Candida free by all methods. In summary, a real-time PCR that provides a sensitive, specific, and rapid alternative technique for detection of Candida in the mouth is described.

  11. Interlaboratory comparison of PCR-based identification of Candida and Aspergillus DNA in spiked blood samples.

    Science.gov (United States)

    Reichard, Utz; Buchheidt, Dieter; Lass-Flörl, Cornelia; Loeffler, Juergen; Lugert, Raimond; Ruhnke, Markus; Tintelnot, Kathrin; Weig, Michael; Groß, Uwe

    2012-09-01

    Despite PCR per se being a powerful and sensitive technique, regarding the detection of fungi in patients' blood, no consensus for a standardised PCR protocol yet exists. To complement other ongoing or accomplished studies which tackle this problem, the German Reference Center for Systemic Mycoses conducted an interlaboratory comparison starting with blood samples spiked with fungal cell elements. Altogether, six laboratories using in-house PCR-protocols from Germany and Austria participated in the trial. Blood samples were spiked with vital cells of Candida albicans or Aspergillus fumigatus. Candida was used in the yeast form, whereas Aspergillus cells were either spiked as conidia or as very young germlings, also known as smoo cells. Spiked blood samples contained between 10 and 10 000 cells ml(-1). Depending on the techniques used for fungal cell disruption and DNA-amplification, detection quality was variable between laboratories, but also differed within single laboratories in different trials particularly for samples spiked with less than 100 cells ml(-1). Altogether, at least regarding the detection of A. fumigatus, two of six laboratories showed constant reliable test results also with low fungal cell number spiked samples. Protocols used by these labs do not differ substantially from others. However, as particularities, one protocol included a conventional phenol chloroform extraction during the DNA preparation process and the other included a real time PCR-protocol based on FRET probes. Other laboratory comparisons on the basis of clinical samples should follow to further evaluate the procedures. The difficulties and problems of such trials in general are discussed. © 2012 Blackwell Verlag GmbH.

  12. Multiplex real-time PCR targeting the RNase P RNA gene for detection and identification of Candida species in blood.

    Science.gov (United States)

    Innings, Asa; Ullberg, Måns; Johansson, Anders; Rubin, Carl Johan; Noreus, Niklas; Isaksson, Magnus; Herrmann, Björn

    2007-03-01

    We have developed a single-tube multiplex real-time PCR method for the detection of the eight most common Candida species causing septicemia: Candida albicans, C. dubliniensis, C. famata, C. glabrata, C. guilliermondii, C. krusei, C. parapsilosis, and C. tropicalis. The method developed targets the RNase P RNA gene RPR1. Sequences of this gene were determined for seven of the Candida species and showed surprisingly large sequence variation. C. glabrata was found to have a gene that was five times longer gene than those of the other species, and the nucleotide sequence similarity between C. krusei and C. albicans was as low as 55%. The multiplex PCR contained three probes that enabled the specific detection of C. albicans, C. glabrata, and C. krusei and a fourth probe that allowed the general detection of the remaining species. The method was able to detect 1 to 10 genome copies when the detection limit was tested repeatedly for the four species C. albicans, C. glabrata, C. krusei, and C. guilliermondii. No significant difference in the detection limit was seen when the multiplex format was compared with single-species PCR, i.e., two primers and one probe. The method detected eight clinically relevant Candida species and did not react with other tested non-Candida species or human DNA. The assay was applied to 20 blood samples from nine patients and showed a sensitivity similar to that of culture.

  13. Genotoxic damage in cultured human peripheral blood lymphocytes ...

    African Journals Online (AJOL)

    Falaq Naz

    2012-06-29

    Jun 29, 2012 ... chromatid exchanges and DNA damage as a parameter, in cultured human peripheral blood lym- phocytes. The study was ... Human lymphocyte culture. A sample of heparinized venous blood was obtained from 25 ... The fixative was removed by centrifugation and the procedure was re- peated twice.

  14. Contaminants in blood cultures: Importance, implications, interpretation and prevention.

    Science.gov (United States)

    Dargère, Sylvie; Cormier, Hélène; Verdon, Renaud

    2018-04-02

    Despite the development of new microbiological technologies, blood cultures remain the first line tool for the diagnosis of bloodstream infections. Their diagnostic value may be affected when a microorganism of questionable evidence is isolated, for example, coagulase-negative staphylococci, Bacillus spp., viridans group streptococci, Corynebacterium spp., Propionibacterium spp., and Micrococcus spp. Finally, making a correct diagnosis of pathogenicity (vs contamination) is very challenging. To review the current ways of dealing with the problem of blood culture contaminants and to provide practical suggestions to decrease blood culture contamination rates. PubMed electronic databases and existing reviews were searched up to December 2017 to retrieve relevant publications related to the topic. This review describes the burden of blood culture contamination, and analyses the main current issues and controversies in interpreting the occurrence of potential blood culture contaminants. It focuses on the best described approaches to decide whether blood culture contamination is present or not, and discusses the different strategies of prevention in adults. Each institution should have an efficient policy to prevent blood culture contamination, emphasizing the importance to follow guidelines for prescribing and collecting blood cultures. Training healthcare workers should focus on detrimental influence on patient care, and highlight the work and costs due to contaminants. The accurate differentiation of a contaminant from a true pathogen relies on a multidisciplinary approach and clinical judgment of experienced practitioners. Copyright © 2018. Published by Elsevier Ltd.

  15. Real-time PCR diagnosis of Plasmodium vivax among blood donors

    Directory of Open Access Journals (Sweden)

    Batista-dos-Santos Sergio

    2012-10-01

    Full Text Available Abstract Background When selecting blood donors in transfusion centres, one important problem is to identify, during screening, individuals with infectious diseases that can be transmitted by blood, such as malaria, especially when the parasite densities are very low. This problem is particularly severe in endemic areas, such as the Brazilian Amazon. In the present study, molecular diagnostic (real-time PCR of Plasmodium vivax was used to identify blood donors infected with malaria parasites. Methods Samples from 595 blood donors were collected in seven haemotherapy centres in northern Brazil located in areas at risk for malaria transmission, and the analyses were performed by real-time PCR with TaqMan probes on 7500 Real-Time PCR Systems, to genotype the mitochondrial DNA region specific to P. vivax. The experiment was designed for hybridization of the cytochrome c oxidase genes of the mitochondrial genome (GenBank GI63022502. The serological data were obtained using enzyme-linked immunosorbent assay - ELISA (Anti-HIV, Anti-HTLV I-II; Anti-HVC, HBsAg, Anti-HBc, Chagas disease and VDRL (Syphilis from the Blood Bank System of the Haematology and Haemotherapy Centre of Pará. Results The assay identified eight individuals in the sample (1.34% infected with P. vivax at the time of blood donation. This percentage was higher than the altered serological results (reactive or inconclusive of the prevalence of anti-HIV (0.67%, anti-hepatitis C virus (0.34%, anti-hepatitis B surface antigen (0.67%, anti-human T-lymphotropic virus I/II (1.18%, anti-Chagas disease (0.17% and syphilis (VDRL (0.50%, but not higher than anti-hepatitis B core antigen antibodies (4.37%. This result indicates the need to use more sensitive methods of diagnosing malaria in blood banks. Conclusion The real-time PCR with TaqMan probes enabled the identification of P. vivax in a high proportion of clinically healthy donors, highlighting the potential risk for transfusion

  16. Sources of blood meals of sylvatic Triatoma guasayana near Zurima, Bolivia, assayed with qPCR and 12S cloning.

    Directory of Open Access Journals (Sweden)

    David E Lucero

    2014-12-01

    Full Text Available In this study we compared the utility of two molecular biology techniques, cloning of the mitochondrial 12S ribosomal RNA gene and hydrolysis probe-based qPCR, to identify blood meal sources of sylvatic Chagas disease insect vectors collected with live-bait mouse traps (also known as Noireau traps. Fourteen T. guasayana were collected from six georeferenced trap locations in the Andean highlands of the department of Chuquisaca, Bolivia.We detected four blood meals sources with the cloning assay: seven samples were positive for human (Homo sapiens, five for chicken (Gallus gallus and unicolored blackbird (Agelasticus cyanopus, and one for opossum (Monodelphis domestica. Using the qPCR assay we detected chicken (13 vectors, and human (14 vectors blood meals as well as an additional blood meal source, Canis sp. (4 vectors.We show that cloning of 12S PCR products, which avoids bias associated with developing primers based on a priori knowledge, detected blood meal sources not previously considered and that species-specific qPCR is more sensitive. All samples identified as positive for a specific blood meal source by the cloning assay were also positive by qPCR. However, not all samples positive by qPCR were positive by cloning. We show the power of combining the cloning assay with the highly sensitive hydrolysis probe-based qPCR assay provides a more complete picture of blood meal sources for insect disease vectors.

  17. Sources of Blood Meals of Sylvatic Triatoma guasayana near Zurima, Bolivia, Assayed with qPCR and 12S Cloning

    Science.gov (United States)

    Lucero, David E.; Ribera, Wilma; Pizarro, Juan Carlos; Plaza, Carlos; Gordon, Levi W.; Peña, Reynaldo; Morrissey, Leslie A.; Rizzo, Donna M.; Stevens, Lori

    2014-01-01

    Background In this study we compared the utility of two molecular biology techniques, cloning of the mitochondrial 12S ribosomal RNA gene and hydrolysis probe-based qPCR, to identify blood meal sources of sylvatic Chagas disease insect vectors collected with live-bait mouse traps (also known as Noireau traps). Fourteen T. guasayana were collected from six georeferenced trap locations in the Andean highlands of the department of Chuquisaca, Bolivia. Methodology/Principal Findings We detected four blood meals sources with the cloning assay: seven samples were positive for human (Homo sapiens), five for chicken (Gallus gallus) and unicolored blackbird (Agelasticus cyanopus), and one for opossum (Monodelphis domestica). Using the qPCR assay we detected chicken (13 vectors), and human (14 vectors) blood meals as well as an additional blood meal source, Canis sp. (4 vectors). Conclusions/Significance We show that cloning of 12S PCR products, which avoids bias associated with developing primers based on a priori knowledge, detected blood meal sources not previously considered and that species-specific qPCR is more sensitive. All samples identified as positive for a specific blood meal source by the cloning assay were also positive by qPCR. However, not all samples positive by qPCR were positive by cloning. We show the power of combining the cloning assay with the highly sensitive hydrolysis probe-based qPCR assay provides a more complete picture of blood meal sources for insect disease vectors. PMID:25474154

  18. Sources of blood meals of sylvatic Triatoma guasayana near Zurima, Bolivia, assayed with qPCR and 12S cloning.

    Science.gov (United States)

    Lucero, David E; Ribera, Wilma; Pizarro, Juan Carlos; Plaza, Carlos; Gordon, Levi W; Peña, Reynaldo; Morrissey, Leslie A; Rizzo, Donna M; Stevens, Lori

    2014-12-01

    In this study we compared the utility of two molecular biology techniques, cloning of the mitochondrial 12S ribosomal RNA gene and hydrolysis probe-based qPCR, to identify blood meal sources of sylvatic Chagas disease insect vectors collected with live-bait mouse traps (also known as Noireau traps). Fourteen T. guasayana were collected from six georeferenced trap locations in the Andean highlands of the department of Chuquisaca, Bolivia. We detected four blood meals sources with the cloning assay: seven samples were positive for human (Homo sapiens), five for chicken (Gallus gallus) and unicolored blackbird (Agelasticus cyanopus), and one for opossum (Monodelphis domestica). Using the qPCR assay we detected chicken (13 vectors), and human (14 vectors) blood meals as well as an additional blood meal source, Canis sp. (4 vectors). We show that cloning of 12S PCR products, which avoids bias associated with developing primers based on a priori knowledge, detected blood meal sources not previously considered and that species-specific qPCR is more sensitive. All samples identified as positive for a specific blood meal source by the cloning assay were also positive by qPCR. However, not all samples positive by qPCR were positive by cloning. We show the power of combining the cloning assay with the highly sensitive hydrolysis probe-based qPCR assay provides a more complete picture of blood meal sources for insect disease vectors.

  19. Multidrug resistant Salmonellae isolated from blood culture samples ...

    African Journals Online (AJOL)

    This study investigates the prevalence of R-plasmids in Salmonella sp. isolated from blood samples of suspected typhoid patients in Warri, Nigeria. A total of 136 blood samples were collected between May and December,2009 and screened for the presence of Salmonellae using standard blood culture techniques of which ...

  20. Detection of Mycobacterium avium subsp. paratuberculosis in milk from clinically affected cows by PCR and culture

    DEFF Research Database (Denmark)

    Giese, Steen Bjørck; Ahrens, Peter

    2000-01-01

    Milk and faeces samples from cows with clinical symptoms of paratuberculosis were examined for the presence of Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) by culture and PCR. M. paratuberculosis was cultivated in variable numbers from faeces or intestinal mucosa in eight of 11...

  1. Periodontal pathogens: a quantitative comparison of anaerobic culture and real-time PCR

    NARCIS (Netherlands)

    Boutaga, Khalil; van Winkelhoff, Arie Jan; Vandenbroucke-Grauls, Christina M. J. E.; Savelkoul, Paul H. M.

    2005-01-01

    Periodontitis is a multi-factorial chronic inflammatory and destructive disease of the tooth-supporting tissues. Quantitative anaerobic culture techniques have been used for microbial diagnosis of the different forms of the disease. The aim of this study was to compare real-time PCR with

  2. Real-time PCR detection of Campylobacter spp.: A comparison toclassic culturing and enrichment

    NARCIS (Netherlands)

    Boer, P. de; Rahaoui, H.; Leer, R.J.; Montijn, R.C.; Vossen, J.M.B.M. van der

    2015-01-01

    The major disadvantage of the current gold standard for detection of the food pathogen Campylobacter, i.e. culturing, is the lengthy procedure. In this study we assessed the use of real-time PCR for detection of Campylobacter. To this end, 926 poultry samples, taken from transport containers and

  3. A comparison of the sensitivities of detection of Plasmodium falciparum gametocytes by magnetic fractionation, thick blood film microscopy, and RT-PCR

    Directory of Open Access Journals (Sweden)

    St-Pierre Tim G

    2009-05-01

    Full Text Available Abstract Background The magnetic properties of Plasmodium-infected erythrocytes have been exploited for different clinical and research purposes. A recent study in a rural clinical setting in Papua New Guinea has demonstrated that Plasmodium falciparum gametocyte detection is facilitated by magnetic deposition microscopy but no study has yet determined the relative sensitivity and limit of detection of a magnetic fractionation technique. The present study compares the detection limit and sensitivity of a technique based on the use of commercially available magnetic fractionation columns with those for thick blood film microscopy and reverse transcriptase polymerase chain reaction (RT-PCR methods. Methods Gametocyte detection in six series of dilutions of cultured P. falciparum parasites with known gametocytaemia was conducted using magnetic fractionation, thick blood film, and RT-PCR techniques. Results The preparations obtained by the magnetic fractionation method were of thin film quality allowing easy gametocyte identification by light microscopy. Magnetic fractionation had a higher sensitivity and approximately two orders of magnitude better limit of detection than thick blood film microscopy. Gametocytes were also more readily detectable on the magnetically fractionated preparations. Magnetic fractionation had a similar limit of detection to that of RT-PCR. Conclusion Magnetic fractionation is a highly sensitive and convenient method for gametocyte detection in comparison with the standard thick blood film and RT-PCR methods, and could readily be adapted to field application.

  4. Whole Blood PCR Amplification with Pfu DNA Polymerase and Its Application in Single-Nucleotide Polymorphism Analysis.

    Science.gov (United States)

    Liu, Er-Ping; Wang, Yan; He, Xiao-Hui; Guan, Jun-Jie; Wang, Jin; Qin, Zheng-Hong; Sun, Wan-Ping

    2015-11-01

    Point-of-care genetic analysis may require polymerase chain reaction (PCR) to be carried out on whole blood. However, human blood contains natural inhibitors of PCR such as hemoglobin, immunoglobulin G, lactoferrin, and proteases, as well as anticoagulant agents, including EDTA and heparin that can reduce whole blood PCR efficiency. Our purpose was to develop a highly specific, direct whole blood single-nucleotide polymorphism (SNP) analysis method based on allele-specific (AS) PCR that is mediated by Pfu DNA polymerase and phosphorothioate-modified AS primers. At high Mg(2+) concentrations, Pfu DNA polymerase efficiently amplified genomic DNA in a reaction solution containing up to 14% whole blood. Among the three anticoagulants tested, Pfu DNA polymerase showed the highest activity with sodium citrate. Meanwhile, Triton X-100 and betaine inhibited Pfu DNA polymerase activity in whole blood PCR, whereas trehalose had virtually no effect. These findings provided for the development of a low-cost, simple, and fast direct whole blood genotyping method that uses Pfu DNA polymerase combined with phosphorothioate AS primers for CYP2C9*3 and VKORC1(-1639) loci. With its high DNA amplification efficiency and tolerance of various blood conditions, Pfu DNA polymerase can be used in clinical laboratories to analyze SNPs in whole blood samples.

  5. Synovial fluid multiplex PCR is superior to culture for detection of low-virulent pathogens causing periprosthetic joint infection.

    Science.gov (United States)

    Morgenstern, Christian; Cabric, Sabrina; Perka, Carsten; Trampuz, Andrej; Renz, Nora

    2018-02-01

    Analysis of joint aspirate is the standard preoperative investigation for diagnosis of periprosthetic joint infection (PJI). We compared the diagnostic performance of culture and multiplex polymerase chain reaction (PCR) of synovial fluid for diagnosis of PJI. Patients in whom aspiration of the prosthetic hip or knee joint was performed before revision arthroplasty were prospectively included. The performance of synovial fluid culture and multiplex PCR was compared by McNemar's chi-squared test. A total of 142 patients were included, 82 with knee and 60 with hip prosthesis. PJI was diagnosed in 77 patients (54%) and aseptic failure in 65 patients (46%). The sensitivity of synovial fluid culture and PCR was 52% and 60%, respectively, showing concordant results in 116 patients (82%). In patients with PJI, PCR missed 6 high-virulent pathogens (S. aureus, streptococci, E. faecalis, E. coli) which grew in synovial fluid culture, whereas synovial fluid culture missed 12 pathogens detected by multiplex PCR, predominantly low-virulent pathogens (Cutibacterium acnes and coagulase-negative staphylococci). In patients with aseptic failure, PCR detected 6 low-virulent organisms (predominantly C. acnes). While the overall performance of synovial fluid PCR was comparable to culture, PCR was superior for detection of low-virulent bacteria such as Cutibacterium spp. and coagulase-negative staphylococci. In addition, synovial fluid culture required several days for growth, whereas multiplex PCR provided results within 5hours in an automated manner. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Comparison of culture and qPCR for the detection of Pseudomonas aeruginosa in not chronically infected cystic fibrosis patients

    Directory of Open Access Journals (Sweden)

    Boboli Hedwige

    2010-09-01

    Full Text Available Abstract Background Pseudomonas aeruginosa is the major respiratory pathogen causing severe lung infections among CF patients, leading to high morbidity and mortality. Once infection is established, early antibiotic treatment is able to postpone the transition to chronic lung infection. In order to optimize the early detection, we compared the sensitivity of microbiological culture and quantitative PCR (qPCR for the detection of P. aeruginosa in respiratory samples of not chronically infected CF patients. Results In this national study, we followed CF patients during periods between 1 to 15 months. For a total of 852 samples, 729 (86% remained P. aeruginosa negative by both culture and qPCR, whereas 89 samples (10% were positive by both culture and qPCR. Twenty-six samples were negative by culture but positive by qPCR, and 10 samples were positive by culture but remained negative by qPCR. Five of the 26 patients with a culture negative, qPCR positive sample became later P. aeruginosa positive both by culture and qPCR. Conclusion Based on the results of this study, it can be concluded that qPCR may have a predictive value for impending P. aeruginosa infection for only a limited number of patients.

  7. Comparison of broad range 16S rDNA PCR to conventional blood ...

    African Journals Online (AJOL)

    Nehal I. Draz

    2013-06-17

    Jun 17, 2013 ... Abstract Neonatal sepsis is a significant cause of morbidity and mortality in neonates. The gold standard for detecting bacterial sepsis is blood culture. However, it has low sensitivity and a report- ing delay of approximately 48–72 h. Molecular assays for the detection of bacterial DNA represent possible new ...

  8. Reduced culture time and improved isolation rate through culture of sonicate fluid in blood culture bottles.

    Science.gov (United States)

    Janz, Viktor; Trampuz, Andrej; Perka, Carsten F; Wassilew, Georgi I

    2017-08-09

    The aim of this study was to investigate if the cultivation of sonicate fluid (SFC) in blood culture bottles (BCB) leads to a higher rate of bacterial isolations than agar plate culture (APC) and to investigate whether the utilization of BCB leads to a reduction in culture time. We performed a retrospective analysis of 206 revision total knee and total hip arthroplasty patients comparing the results of both synovial fluid culture and SFC in both BCB and conventional APC. The use of BCB improved both the rate of positive bacterial isolations and reduced the culture time for synovial fluid as well as SFC. Fifty-one patients showed a bacterial isolation in SFC-APC and 101 in SFC-BCB. For synovial fluid 24 patients showed a bacterial isolation on APC and 37 showed a bacterial isolation in BCB. The synovial fluid cultures showed growth on APC after an average of 2.8 days vs. 1.8 days in BCB. The SFC-APC showed growth after an average of 4.2 days vs. 2.9 days for SFC-BCB. The culture of synovial and sonicate fluid in BCB leads to more positive bacterial isolations and quicker bacterial growth than conventional agar plate cultures.

  9. Comparison of immunochromatography, PCR and culture methods for the detection of Campylobacter bacteria.

    Science.gov (United States)

    Dey, Shuvra Kanti; Nishimura, Shuichi; Okitsu, Shoko; Hayakawa, Satoshi; Mizuguchi, Masashi; Ushijima, Hiroshi

    2012-12-01

    A total of 463 fecal specimens from human patients were tested for the presence of Camphylobacter by a commercial immunochromatography kit (ImmunoCard STAT! CAMPY), polymerase chain reaction (PCR) and conventional bacteriological (CB) methods. Using culture as the standard of reference, the ImmunoCard STAT! CAMPY assay had a sensitivity of 86% and a specificity of 100%. On the other hand, using PCR as the standard of reference, ImmunoCard STAT! CAMPY assay had a sensitivity of 90.5% and a specificity of 100%. Copyright © 2012. Published by Elsevier B.V.

  10. Laboratory Workflow Analysis of Culture of Periprosthetic Tissues in Blood Culture Bottles.

    Science.gov (United States)

    Peel, Trisha N; Sedarski, John A; Dylla, Brenda L; Shannon, Samantha K; Amirahmadi, Fazlollaah; Hughes, John G; Cheng, Allen C; Patel, Robin

    2017-09-01

    Culture of periprosthetic tissue specimens in blood culture bottles is more sensitive than conventional techniques, but the impact on laboratory workflow has yet to be addressed. Herein, we examined the impact of culture of periprosthetic tissues in blood culture bottles on laboratory workflow and cost. The workflow was process mapped, decision tree models were constructed using probabilities of positive and negative cultures drawn from our published study (T. N. Peel, B. L. Dylla, J. G. Hughes, D. T. Lynch, K. E. Greenwood-Quaintance, A. C. Cheng, J. N. Mandrekar, and R. Patel, mBio 7:e01776-15, 2016, https://doi.org/10.1128/mBio.01776-15), and the processing times and resource costs from the laboratory staff time viewpoint were used to compare periprosthetic tissues culture processes using conventional techniques with culture in blood culture bottles. Sensitivity analysis was performed using various rates of positive cultures. Annualized labor savings were estimated based on salary costs from the U.S. Labor Bureau for Laboratory staff. The model demonstrated a 60.1% reduction in mean total staff time with the adoption of tissue inoculation into blood culture bottles compared to conventional techniques (mean ± standard deviation, 30.7 ± 27.6 versus 77.0 ± 35.3 h per month, respectively; P < 0.001). The estimated annualized labor cost savings of culture using blood culture bottles was $10,876.83 (±$337.16). Sensitivity analysis was performed using various rates of culture positivity (5 to 50%). Culture in blood culture bottles was cost-effective, based on the estimated labor cost savings of $2,132.71 for each percent increase in test accuracy. In conclusion, culture of periprosthetic tissue in blood culture bottles is not only more accurate than but is also cost-saving compared to conventional culture methods. Copyright © 2017 American Society for Microbiology.

  11. The Utility of Blood Culture Fluid for the Molecular Diagnosis of Leptospira: A Prospective Evaluation.

    Science.gov (United States)

    Dittrich, Sabine; Rudgard, William E; Woods, Kate L; Silisouk, Joy; Phuklia, Weerawat; Davong, Viengmon; Vongsouvath, Manivanh; Phommasone, Koukeo; Rattanavong, Sayaphet; Knappik, Michael; Craig, Scott B; Weier, Steven L; Tulsiani, Suhella M; Dance, David A B; Newton, Paul N

    2016-04-01

    Leptospirosis is an important zoonosis worldwide, with infections occurring after exposure to contaminated water. Despite being a global problem, laboratory diagnosis remains difficult with culture results taking up to 3 months, serology being retrospective by nature, and polymerase chain reaction showing limited sensitivity. Leptospira have been shown to survive and multiply in blood culture media, and we hypothesized that extracting DNA from incubated blood culture fluid (BCF), followed by quantitative real-time polymerase chain reaction (qPCR) could improve the accuracy and speed of leptospira diagnosis. We assessed this retrospectively, using preincubated BCF of Leptospira spp. positive (N= 109) and negative (N= 63) febrile patients in Vientiane, Lao PDR. The final method showed promising sensitivities of 66% (95% confidence interval [CI]: 55-76) and 59% (95% CI: 49-68) compared with direct or direct and indirect testing combined, as the respective reference standards (specificities > 95%). Despite these promising diagnostic parameters, a subsequent prospective evaluation in a Lao hospital population (N= 352) showed that the sensitivity was very low (∼30%) compared with qPCR on venous blood samples. The disappointingly low sensitivity does suggest that venous blood samples are preferable for the clinical microbiology laboratory, although BCF might be an alternative if leptospirosis is only suspected postadmission after antibiotics have been used. © The American Society of Tropical Medicine and Hygiene.

  12. Comparison of PCR method with the culture method for identification of gonococci from endocervical swabs

    Directory of Open Access Journals (Sweden)

    Alam A

    2002-01-01

    Full Text Available Gonococcal infection remains still a major cause of morbidity among sexually active individuals. Diagnosis of the infection in a female case is more difficult than that in a male. This was a prospective study among 269 female commercial sex workers (CSWs to screen them for gonococcal infection, comparing the rapid method of identification of gonococci by polymerase chain reaction (PCR with the selective culture method. A total of 92 (34.2% CSWs were identified positive for Neisseria gonorrhoeae by combination of the two methods. The PCR method identified 87 of the specimens to harbour cppB gene of N. gonorrhoeae, whereas culture method identified 83 specimens showing colonies of gonococci. Taking into consideration of the total positive cases (92, the PCR method showed a sensitivity of 94.57%, whereas sensitivity of culture method was 90.22%. The selective culture method appears to be the most applicable in the identification of gonococci from clinical specimens, particularly in the less resourceful countries like Bangladesh.

  13. SMA Diagnosis: Detection of SMN1 Deletion with Real-Time mCOP-PCR System Using Fresh Blood DNA.

    Science.gov (United States)

    Niba, Emma Tabe Eko; Ar Rochmah, Mawaddah; Harahap, Nur Imma Fatimah; Awano, Hiroyuki; Morioka, Ichiro; Iijima, Kazumoto; Saito, Toshio; Saito, Kayoko; Takeuchi, Atsuko; Lai, Poh San; Bouike, Yoshihiro; Nishio, Hisahide; Shinohara, Masakazu

    2017-12-18

    Spinal muscular atrophy (SMA) is one of the most common autosomal recessive disorders. The symptoms are caused by defects of lower motor neurons in the spinal cord. More than 95% of SMA patients are homozygous for survival motor neuron 1 (SMN1) deletion. We previously developed a screening system for SMN1 deletion based on a modified competitive oligonucleotide priming-PCR (mCOP-PCR) technique using dried blood spot (DBS) on filter paper. This system is convenient for mass screening in the large population and/or first-tier diagnostic method of the patients in the remote areas. However, this system was still time-consuming and effort-taking, because it required pre-amplification procedure to avoid non-specific amplification and gel-electrophoresis to detect the presence or absence of SMN1 deletion. When the fresh blood samples are used instead of DBS, or when the gel-electrophoresis is replaced by real-time PCR, we may have a simpler and more rapid diagnostic method for SMA. To establish a simpler and more rapid diagnostic method of SMN1 deletion using fresh blood DNA. DNA samples extracted from fresh blood and stored at 4 ℃ for 1 month. The samples were assayed using a real-time mCOP-PCR system without pre-amplification procedures. DNA samples had already been genotyped by PCR-restriction fragment length polymorphism (PCR-RFLP), showing the presence or absence of SMN1 exon 7. The DNA samples were directly subjected to the mCOP-PCR step. The amplification of mCOP-PCR was monitored in a real-time PCR apparatus. The genotyping results of the real-time mCOP-PCR system using fresh blood DNA were completely matched with those of PCR-RFLP. In this real-time mCOP-PCR system using fresh blood-DNA, it took only four hours from extraction of DNA to detection of the presence or absence of SMN1 deletion, while it took more than 12 hours in PCR-RFLP. Our real-time mCOP-PCR system using fresh blood DNA was rapid and accurate, suggesting it may be useful for the first

  14. Blood cultures in emergency medical admissions: a key patient cohort.

    Science.gov (United States)

    Chotirmall, Sanjay H; Callaly, Elizabeth; Lyons, Judith; O'Connell, Brian; Kelleher, Mary; Byrne, Declan; O'Riordan, Deirdre; Silke, Bernard

    2016-02-01

    Blood cultures are performed in the emergency room when sepsis is suspected, and a cohort of patients is thereby identified. The present study investigated the outcomes (mortality and length of hospital stay) in this group following an emergency medical admission. Prospective assessment of all emergency medical admissions presenting to the emergency department at St James's Hospital, Dublin, over an 11-year period (2002-2012) was carried out. Outcomes including 30-day in-hospital mortality and length of stay were explored in the context of an admission blood culture. Generalized estimating equations, logistic or zero-truncated Poisson multivariate models were used, with adjustment for confounding variables including illness severity, comorbidity, and chronic disabling disease, to assess the effect of an urgent blood culture on mortality and length of stay. A total of 60 864 episodes were recorded in 35 168 patients admitted over the time period assessed. Patients more likely to undergo blood cultures in the emergency department were male, younger, and had more comorbidity. Univariate and multivariate analyses showed that those who had a blood culture, irrespective of result, had increased mortality and a longer in-hospital stay. This was highest for those with a positive culture, irrespective of the organism isolated. A clinical decision to request a blood culture identified a subset of emergency admissions with markedly worse outcomes. This patient cohort warrants close monitoring in the emergency setting.

  15. Comparison of Simplexa HSV 1 & 2 PCR with culture, immunofluorescence, and laboratory-developed TaqMan PCR for detection of herpes simplex virus in swab specimens.

    Science.gov (United States)

    Gitman, Melissa R; Ferguson, David; Landry, Marie L

    2013-11-01

    The Simplexa HSV 1 & 2 direct PCR assay was compared with conventional cell culture, cytospin-enhanced direct fluorescent antibody (DFA), and a laboratory-developed real-time TaqMan PCR (LDT HSV PCR) using extracted nucleic acid for the detection of herpes simplex virus (HSV) in dermal, genital, mouth, ocular, and other swab samples. One hundred seventy-one swabs were tested prospectively, and 58 were positive for HSV (34 HSV-1 and 24 HSV-2). Cytospin-DFA detected 50 (86.2%), conventional cell culture 51 (87.9%), Simplexa direct 55 (94.8%), and LDT HSV PCR 57 (98.3%) of 58 true positives. Simplexa direct detected more positives than DFA and culture, but the differences were not significant (P = 0.0736 and P = 0.3711, respectively, by the McNemar test). Samples that were positive by all methods (n = 48) were strong positives (LDT cycle threshold [CT] value, 14.4 to 26.1). One strongly positive sample was falsely negative by LDT HSV PCR due to a failure of TaqMan probe binding. Three samples falsely negative by Simplexa direct had high CT values by LDT HSV PCR (LDT CT, 35.8 to 38.2). Omission of the DNA extraction step by Simplexa direct led to a drop in sensitivity compared to the sensitivity of LDT HSV PCR using extracted samples (94.8% versus 98.3%, respectively), but the difference was not significant (P = 0.6171). Simplexa HSV 1 & 2 direct PCR was the most expensive but required the least training of the assays used, had the lowest hands-on time and fastest assay time (75 min, versus 3 h by LDT HSV PCR), and provided the HSV type.

  16. Prevalence of periodontopathogenic bacteria in patients suffering from periodontitis using culture and PCR methods

    Directory of Open Access Journals (Sweden)

    Amir Aliramezani

    2012-01-01

    Full Text Available Background and Aims: Periodontitis is one of the most common oral diseases with the various incidence rates in different populations. A number of bacteria are considered as the major etiologic agents of periodontitis. The aim of the present study was to determine the prevalence of periodontopathogen bacteria in patients using both PCR and culture techniques.Materials and Methods: In this study, one-hundred patients (including 62 females and 38 males with an average age of 49±11.5 years with adult periodontitis referred to periodontics department of School of Dentistry/Tehran University of Medical Sciences were investigated. The samples were taken and sent immediately to the laboratory for culture and molecular evaluation. The PCR was performed using specific primers and the statistical analysis of data was performed using SPSS statistic software and McNemar test.Results: The results demonstrated that the total detection rate in culture method was 64%. The rate of Aggregatibacter actinomycetemcomitans (Aa was 28% which was significantly higher than that of Porphyromonas gingivalis (Pg (6% and Prevotella intermedia (Pi (3%. 27% of cases showed mixed bacterial growth. 65% of patients were positive using molecular method. The rate of Aa (30% was significantly higher than that of Pg (7% and Pi (5%. The mixed PCR positive rate containing of Aa, Pg and Pi was (23%.Conclusion: In this study, it was found that most of the bacteria isolated using culture and molecular methods were Aa, Pg and Pi, respectively. Although the detection frequencies of both techniques were similar, the specificity, sensitivity and bacterial detection speed of the PCR technique is obviously higher. Therefore, the use of molecular techniques is strongly recommended. However, both techniques seem to be suitable for microbiological diagnostics.

  17. Detection of Trichomonas Vaginalis in Vaginal Speciemens from Women by Wet Mount, Culture and PCR

    Directory of Open Access Journals (Sweden)

    Gulnaz Culha

    2014-03-01

    Full Text Available Aim: Trichomoniasis, a sexually transmitted infection (STI caused by Trichomonas vaginalis, affects 180 million people worldwide and causes significant morbidity. Infection with T. vaginalis has been associated with vaginitis, exocervicitis, and urethritis in women. Material and Method: In this study, we aim to investigate the presence of T. vaginalis by using three different methods for comparing the results. Two hundred T. vaginalis isolates taken from swap samples were collected in Medical Faculty, Department of Gynecology, Mustafa Kemal University Polyclinic, and examined genotypically and phenotypically to identify T. vaginalis in Parasitology Department. This research is unique in terms of its contribution to patient treatment, being the first molecular study in Turkey/Hatay to determine Trichomonas (TV genes stemming from Trichomonas vaginalis strains. Result: 56 out of 200 patients examined were identified as positive and 24 (42.8% of these were identified through microscopy, 18 (32,1% with culture and 24 (42,8% with PCR. The number of those identified through all these methods is 14 (25%. In this study, difference was calculated using three methods (p=0.022 with Cochran%u2019s Q test. When compared with McNemar two by two, no superiority in T. vaginalis diagnosis was found between microscopy and culture (p=0.5, microscopy and PCR (p=0.063, or culture and PCR (p=0.25 methods. Discussion: Culture method is not used in routine laboratory procedures and has contamination risk. PCR method shows directly the parasite of DNAs, and so it is thought to be more reliable compared to the other two methods.

  18. Detection of Candida in Concentrated Oral Rinse Cultures by Real-Time PCR

    OpenAIRE

    White, P. Lewis; Williams, David W.; Kuriyama, Tomoari; Samad, Shamim A.; Lewis, Michael A. O.; Barnes, Rosemary A.

    2004-01-01

    The incidence of oral candidosis has increased in recent years, largely as a result of the emergence of human immunodeficiency virus infection and the more widespread use of immunosuppressive chemotherapy. This development has been associated with a need for more reliable methods for the detection of Candida. The present study assessed the performance of a real-time PCR and two block-based PCRs for the detection of Candida in 193 concentrated oral rinse culture (CRC) specimens. A total of 102...

  19. Investigation of Legionella Contamination in Bath Water Samples by Culture, Amoebic Co-Culture, and Real-Time Quantitative PCR Methods.

    Science.gov (United States)

    Edagawa, Akiko; Kimura, Akio; Kawabuchi-Kurata, Takako; Adachi, Shinichi; Furuhata, Katsunori; Miyamoto, Hiroshi

    2015-10-19

    We investigated Legionella contamination in bath water samples, collected from 68 bathing facilities in Japan, by culture, culture with amoebic co-culture, real-time quantitative PCR (qPCR), and real-time qPCR with amoebic co-culture. Using the conventional culture method, Legionella pneumophila was detected in 11 samples (11/68, 16.2%). Contrary to our expectation, the culture method with the amoebic co-culture technique did not increase the detection rate of Legionella (4/68, 5.9%). In contrast, a combination of the amoebic co-culture technique followed by qPCR successfully increased the detection rate (57/68, 83.8%) compared with real-time qPCR alone (46/68, 67.6%). Using real-time qPCR after culture with amoebic co-culture, more than 10-fold higher bacterial numbers were observed in 30 samples (30/68, 44.1%) compared with the same samples without co-culture. On the other hand, higher bacterial numbers were not observed after propagation by amoebae in 32 samples (32/68, 47.1%). Legionella was not detected in the remaining six samples (6/68, 8.8%), irrespective of the method. These results suggest that application of the amoebic co-culture technique prior to real-time qPCR may be useful for the sensitive detection of Legionella from bath water samples. Furthermore, a combination of amoebic co-culture and real-time qPCR might be useful to detect viable and virulent Legionella because their ability to invade and multiply within free-living amoebae is considered to correlate with their pathogenicity for humans. This is the first report evaluating the efficacy of the amoebic co-culture technique for detecting Legionella in bath water samples.

  20. Investigation of Legionella Contamination in Bath Water Samples by Culture, Amoebic Co-Culture, and Real-Time Quantitative PCR Methods

    Directory of Open Access Journals (Sweden)

    Akiko Edagawa

    2015-10-01

    Full Text Available We investigated Legionella contamination in bath water samples, collected from 68 bathing facilities in Japan, by culture, culture with amoebic co-culture, real-time quantitative PCR (qPCR, and real-time qPCR with amoebic co-culture. Using the conventional culture method, Legionella pneumophila was detected in 11 samples (11/68, 16.2%. Contrary to our expectation, the culture method with the amoebic co-culture technique did not increase the detection rate of Legionella (4/68, 5.9%. In contrast, a combination of the amoebic co-culture technique followed by qPCR successfully increased the detection rate (57/68, 83.8% compared with real-time qPCR alone (46/68, 67.6%. Using real-time qPCR after culture with amoebic co-culture, more than 10-fold higher bacterial numbers were observed in 30 samples (30/68, 44.1% compared with the same samples without co-culture. On the other hand, higher bacterial numbers were not observed after propagation by amoebae in 32 samples (32/68, 47.1%. Legionella was not detected in the remaining six samples (6/68, 8.8%, irrespective of the method. These results suggest that application of the amoebic co-culture technique prior to real-time qPCR may be useful for the sensitive detection of Legionella from bath water samples. Furthermore, a combination of amoebic co-culture and real-time qPCR might be useful to detect viable and virulent Legionella because their ability to invade and multiply within free-living amoebae is considered to correlate with their pathogenicity for humans. This is the first report evaluating the efficacy of the amoebic co-culture technique for detecting Legionella in bath water samples.

  1. Blood Meal Identification in Field-Captured Sand flies: Comparison of PCR-RFLP and ELISA Assays

    Directory of Open Access Journals (Sweden)

    N Maleki-Ravasan

    2009-07-01

    Full Text Available   Abstract Background: We aimed to develop a PCR-RFLP assay based on available sequences of putative vertebrate hosts to iden­tify blood meals ingested by field female sand fly in the northwest of Iran. In addition, the utility of PCR-RFLP was compared with ELISA as a standard method."nMethods: This experimental study was performed in the Insect Molecular Biology Laboratory of School of Public Health, Tehran University of Medical Sciences, Iran in 2006-2007. For PCR-RFLP a set of conserved vertebrate prim­ers were used to amplify a part of the host mitochondrial cytochrome b (cyt b gene followed by digestion of the PCR products by Hae III enzyme."nResults: The PCR-RFLP and ELISA assays revealed that 34% and 27% of field-collected sand flies had fed on hu­mans, respectively. Additionally, PCR-RFLP assays could reveal specific host DNA as well as the components of mixed blood meals. Results of PCR-RFLP assay showed that the sand flies had fed on cow (54%, human (10%, dog (4%, human and cow (21%, dog and cow (14%, and human and dog (3%. "nConclusion: The results can provide a novel method for rapid diagnosis of blood meal taken by sandflies. The advan­tages and limitations of PCR and ELISA assays are discussed.

  2. Blood Meal Identification in Field-Captured Sand flies: Comparison of PCR-RFLP and ELISA Assays

    Science.gov (United States)

    Maleki-Ravasan, N; Oshaghi, MA; Javadian, E; Rassi, Y; Sadraei, J; Mohtarami, F

    2009-01-01

    Background We aimed to develop a PCR-RFLP assay based on available sequences of putative vertebrate hosts to identify blood meals ingested by field female sand fly in the northwest of Iran. In addition, the utility of PCR-RFLP was compared with ELISA as a standard method. Methods: This experimental study was performed in the Insect Molecular Biology Laboratory of School of Public Health, Tehran University of Medical Sciences, Iran in 2006–2007. For PCR-RFLP a set of conserved vertebrate primers were used to amplify a part of the host mitochondrial cytochrome b (cyt b) gene followed by digestion of the PCR products by Hae III enzyme. Results: The PCR-RFLP and ELISA assays revealed that 34% and 27% of field-collected sand flies had fed on humans, respectively. Additionally, PCR-RFLP assays could reveal specific host DNA as well as the components of mixed blood meals. Results of PCR-RFLP assay showed that the sand flies had fed on cow (54%), human (10%), dog (4%), human and cow (21%), dog and cow (14%), and human and dog (3%). Conclusion: The results can provide a novel method for rapid diagnosis of blood meal taken by sandflies. The advantages and limitations of PCR and ELISA assays are discussed. PMID:22808367

  3. Blood Meal Identification in Field-Captured Sand flies: Comparison of PCR-RFLP and ELISA Assays

    Directory of Open Access Journals (Sweden)

    N Maleki-Ravasan

    2009-06-01

    Full Text Available  Background: We aimed to develop a PCR-RFLP assay based on available sequences of putative vertebrate hosts to iden­tify blood meals ingested by field female sand fly in the northwest of Iran. In addition, the utility of PCR-RFLP was compared with ELISA as a standard method.Methods: This experimental study was performed in the Insect Molecular Biology Laboratory of School of Public Health, Tehran University of Medical Sciences, Iran in 2006-2007. For PCR-RFLP a set of conserved vertebrate prim­ers were used to amplify a part of the host mitochondrial cytochrome b (cyt b gene followed by digestion of the PCR products by Hae III enzyme.Results: The PCR-RFLP and ELISA assays revealed that 34% and 27% of field-collected sand flies had fed on hu­mans, respectively. Additionally, PCR-RFLP assays could reveal specific host DNA as well as the components of mixed blood meals. Results of PCR-RFLP assay showed that the sand flies had fed on cow (54%, human (10%, dog (4%, human and cow (21%, dog and cow (14%, and human and dog (3%. Conclusion: The results can provide a novel method for rapid diagnosis of blood meal taken by sandflies. The advan­tages and limitations of PCR and ELISA assays are discussed.

  4. Positive blood culture with Plasmodium falciparum : Case report

    NARCIS (Netherlands)

    De Vries, Jutte J. C.; Van Assen, Sander; Mulder, André B.; Kampinga, Greetje A.

    2007-01-01

    An adult traveler presented with fever and malaise after returning from Sierra Leone. Young trophozoites of Plasmodium falciparum were seen in a blood smear, with parasitemia being 10%. Moreover, blood cultures drawn on admission signaled as "positive" after 1 day of incubation, but no bacteria were

  5. COMPARISION OF THE UTILITY OF CONVENTIONAL CULTURE VERSUS MULTIPLEX PCR IN THE DIAGNOSIS AND MANAGEMENT OF SEPSIS AND MENINGITIS

    Directory of Open Access Journals (Sweden)

    Harish

    2015-11-01

    Full Text Available OBJECTIVE: Sepsis and meningitis are few of the most important causes of morbidity and mortality. Even so, establishing a microbial diagnosis for is still arduous and is often achieved in only half of cases by conventional culture techniques. This study was designed to compare the multiplex PCR method with the traditional culture method in sepsis and meningitis. The other aim was to evaluate the reliability of multiplex PCR method. METHODS: Forty - four patients with symptoms of sepsis and meningitis were included in the study. Both culture and multiplex PCR methods were performed for the isolation of most commonly seen pathogen, from bronchoalveolar lavage (BAL and cerebrospinal fluid (CSF samples. RESULTS: The conventional culture method detected at least one bact erial isolation in 19 patients. Whereas, the number for multiplex PCR was 44 (100%. The pathogens most commonly detected by PCR were Pseudomonas, Candida, S. pneumonia and CMV. In terms of detection of multiple pathogens, multiplex PCR was significantly e fficient than conventional culture (p<0.05. CONCLUSION: The traditional methods, such as culture are often inadequate in detection of the pathogens in sample from patients of sepsis and meningitis. Multiplex PCR assays proved highly sensitive and rapid. Widespread use of PCR methods will not only provide the immediate and appropriate ''agent specific antibiotic treatment'' of sepsis and meningitis, it will also contribute to a reduction in antibiotic resistance

  6. Volume of blood submitted for culture from neonates.

    OpenAIRE

    Neal, P R; Kleiman, M B; Reynolds, J K; Allen, S D; Lemons, J A; Yu, P L

    1986-01-01

    We prospectively examined 298 sets (298 aerobic, 299 anaerobic, and 73 resin cultures) of blood cultures from 161 critically ill newborns. The attending physicians were unaware of the study. The mean blood volume per patient (aerobic and anaerobic) was 1.05 (range, 0.11 to 3.04) ml. The mean blood volume per aerobic bottle was 0.53 (range, 0.01 to 1.90) ml. Among aerobic samples 2.7% were less than or equal to 0.1 ml, 16% were less than or equal to 0.3 ml, 33% were less than or equal to 0.4 m...

  7. The use of culture, pooled samples and PCR for identification of herds infected with Brachyspira hyodysenteriae.

    Science.gov (United States)

    Fellström, C; Zimmerman, U; Aspan, A; Gunnarsson, A

    2001-06-01

    The sensitivity of culturing Brachyspira hyodysenteriae was determined after sampling with swabs from porcine fecal specimens inoculated with tenfold dilutions of a field strain of these microbes. After storage of swabs, Brachyspira hyodysenteriae was recovered throughout the first 3 weeks after inoculation from feces with more than 140 cells/g. Viable spirochetes could still be recovered after up to 83 days of storage from feces, with 1.4 x 10(6) cells or more per gram. Culture for Brachyspira spp. was performed on 285 rectal swabs, which were pooled in batches of five. The number of pooled samples positive for B. hyodysenteriae corresponded with the sum results of individual analysis of the corresponding collections of five samples. A PCR system based on the tlyA gene of B. hyodysenteriae was developed and tested on primary cultures of pooled samples. The results of the PCR assay showed a 97% correlation with the culture results. The prevalence of Brachyspira spp. was determined in five swine herds and found to be highest among breeding gilts and boars aged 13-16 weeks and among 6-12-week-old weaned pigs. In contrast, Brachyspira spp. were only rarely found in sows, which may reflect the development of immunity by adult pigs to all species of the genus.

  8. Purification and microRNA profiling of exosomes derived from blood and culture media.

    Science.gov (United States)

    McDonald, Marguerite K; Capasso, Kathryn E; Ajit, Seena K

    2013-06-14

    Stable miRNAs are present in all body fluids and some circulating miRNAs are protected from degradation by sequestration in small vesicles called exosomes. Exosomes can fuse with the plasma membrane resulting in the transfer of RNA and proteins to the target cell. Their biological functions include immune response, antigen presentation, and intracellular communication. Delivery of miRNAs that can regulate gene expression in the recipient cells via blood has opened novel avenues for target intervention. In addition to offering a strategy for delivery of drugs or RNA therapeutic agents, exosomal contents can serve as biomarkers that can aid in diagnosis, determining treatment options and prognosis. Here we will describe the procedure for quantitatively analyzing miRNAs and messenger RNAs (mRNA) from exosomes secreted in blood and cell culture media. Purified exosomes will be characterized using western blot analysis for exosomal markers and PCR for mRNAs of interest. Transmission electron microscopy (TEM) and immunogold labeling will be used to validate exosomal morphology and integrity. Total RNA will be purified from these exosomes to ensure that we can study both mRNA and miRNA from the same sample. After validating RNA integrity by Bioanalyzer, we will perform a medium throughput quantitative real time PCR (qPCR) to identify the exosomal miRNA using Taqman Low Density Array (TLDA) cards and gene expression studies for transcripts of interest. These protocols can be used to quantify changes in exosomal miRNAs in patients, rodent models and cell culture media before and after pharmacological intervention. Exosomal contents vary due to the source of origin and the physiological conditions of cells that secrete exosomes. These variations can provide insight on how cells and systems cope with stress or physiological perturbations. Our representative data show variations in miRNAs present in exosomes purified from mouse blood, human blood and human cell culture media

  9. Development and comparison of a real-time PCR assay for detection of Dichelobacter nodosus with culturing and conventional PCR: harmonisation between three laboratories

    DEFF Research Database (Denmark)

    Frosth, Sara; Slettemeås, Jannice S.; Jørgensen, Hannah J.

    2012-01-01

    BACKGROUND: Ovine footrot is a contagious disease with worldwide occurrence in sheep. The main causative agent is the fastidious bacterium Dichelobacter nodosus. In Scandinavia, footrot was first diagnosed in Sweden in 2004 and later also in Norway and Denmark. Clinical examination of sheep feet...... was tested using 55 bacterial and two fungal strains. To evaluate the sensitivity and harmonisation of results between different laboratories, aliquots of a single DNA preparation were analysed at three Scandinavian laboratories. The developed real-time PCR assay was compared to culturing by analysing 126...... laboratories and corresponds to approximately three copies of the D. nodosus genome per reaction. The assay showed 100% inclusivity and 100% exclusivity for the strains tested. The real-time PCR assay found 54.8% more positive samples than by culturing and 8% more than conventional PCR. CONCLUSIONS...

  10. Comparison of the sensitivity of culture, PCR and quantitative real-time PCR for the detection of Pseudomonas aeruginosa in sputum of cystic fibrosis patients

    Directory of Open Access Journals (Sweden)

    De Vos Daniel

    2009-11-01

    Full Text Available Abstract Background Pseudomonas aeruginosa is the major pathogen involved in the decline of lung function in cystic fibrosis (CF patients. Early aggressive antibiotic therapy has been shown to be effective in preventing chronic colonization. Therefore, early detection is important and sensitive detection methods are warranted. In this study, we used a dilution series of P. aeruginosa positive sputa, diluted in a pool of P. aeruginosa negative sputa, all from CF patients - to mimick as closely as possible the sputa sent to routine laboratories - to compare the sensitivity of three culture techniques versus that of two conventional PCR formats and four real-time PCR formats, each targeting the P. aeruginosa oprL gene. In addition, we compared five DNA-extraction protocols. Results In our hands, all three culture methods and the bioMérieux easyMAG Nuclisens protocol Generic 2.0.1, preceded by proteinase K pretreatment and followed by any of the 3 real-time PCR formats with probes were most sensitive and able to detect P. aeruginosa up to 50 cfu/ml, i.e. the theoretical minimum of one cell per PCR mixture, when taking into account the volumes used in this study of sample for DNA-extraction, of DNA-elution and of DNA-extract in the PCR mixture. Conclusion In this study, no difference in sensitivity could be found for the detection of P. aeruginosa from sputum between microbiological culture and optimized DNA-extraction and real-time PCR. The results also indicate the importance of the optimization of the DNA-extraction protocol and the PCR format.

  11. PCR-based detection of a rare linear DNA in cell culture

    Directory of Open Access Journals (Sweden)

    Saveliev Sergei V.

    2002-01-01

    Full Text Available The described method allows for detection of rare linear DNA fragments generated during genomic deletions. The predicted limit of the detection is one DNA molecule per 107 or more cells. The method is based on anchor PCR and involves gel separation of the linear DNA fragment and chromosomal DNA before amplification. The detailed chemical structure of the ends of the linear DNA can be defined with the use of additional PCR-based protocols. The method was applied to study the short-lived linear DNA generated during programmed genomic deletions in a ciliate. It can be useful in studies of spontaneous DNA deletions in cell culture or for tracking intracellular modifications at the ends of transfected DNA during gene therapy trials.

  12. Preanalytical blood sample workup for cell-free DNA analysis using Droplet Digital PCR for future molecular cancer diagnostics.

    Science.gov (United States)

    van Ginkel, Joost H; van den Broek, Daan A; van Kuik, Joyce; Linders, Dorothé; de Weger, Roel; Willems, Stefan M; Huibers, Manon M H

    2017-10-01

    In current molecular cancer diagnostics, using blood samples of cancer patients for the detection of genetic alterations in plasma (cell-free) circulating tumor DNA (ctDNA) is an emerging practice. Since ctDNA levels in blood are low, highly sensitive Droplet Digital PCR (ddPCR) can be used for detecting rare mutational targets. In order to perform ddPCR on blood samples, a standardized procedure for processing and analyzing blood samples is necessary to facilitate implementation into clinical practice. Therefore, we assessed the technical sample workup procedure for ddPCR on blood plasma samples. Blood samples from healthy individuals, as well as lung cancer patients were analyzed. We compared different methods and protocols for sample collection, storage, centrifugation, isolation, and quantification. Cell-free DNA (cfDNA) concentrations of several wild-type targets and BRAF and EGFR-mutant ctDNA concentrations quantified by ddPCR were primary outcome measurements. Highest cfDNA concentrations were measured in blood collected in serum tubes. No significant differences in cfDNA concentrations were detected between various time points of up to 24 h until centrifugation. Highest cfDNA concentrations were detected after DNA isolation with the Quick cfDNA Serum & Plasma Kit, while plasma isolation using the QIAamp Circulating Nucleic Acid Kit yielded the most consistent results. DdPCR results on cfDNA are highly dependent on multiple factors during preanalytical sample workup, which need to be addressed during the development of this diagnostic tool for cancer diagnostics in the future. © 2017 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

  13. Development of a PCR Assay to Detect Low Level Trypanosoma cruzi in Blood Specimens Collected with PAXgene Blood DNA Tubes for Clinical Trials Treating Chagas Disease.

    Science.gov (United States)

    Wei, Bo; Chen, Lei; Kibukawa, Miho; Kang, John; Waskin, Hetty; Marton, Matthew

    2016-12-01

    Chagas disease is caused by the parasitic infection of Trypanosoma cruzi (T. cruzi). The STOP CHAGAS clinical trial was initiated in 2011 to evaluate posaconazole in treating Chagas disease, with treatment success defined as negative qualitative PCR results of detecting the parasites in blood specimens collected post-treatment. PAXgene Blood DNA tubes were utilized as a simple procedure to collect and process blood specimens. However, the PAXgene blood specimens challenged published T. cruzi PCR methods, resulting in poor sensitivity and reproducibility. To accurately evaluate the treatment efficacy of the clinical study, we developed and validated a robust PCR assay for detecting low level T. cruzi in PAXgene blood specimens. The assay combines a new DNA extraction method with a custom designed qPCR assay, resulting in limit of detection of 0.005 and 0.01 fg/μl for K98 and CL Brener, two representative strains of two of T. cruzi's discrete typing units. Reliable qPCR standard curves were established for both strains to measure parasite loads, with amplification efficiency ≥ 90% and the lower limit of linearity ≥ 0.05 fg/μl. The assay successfully analyzed the samples collected from the STOP CHAGAS study and may prove useful for future global clinical trials evaluating new therapies for asymptomatic chronic Chagas disease.

  14. Direct testing of blood cultures for detection of streptococcal antigens.

    Science.gov (United States)

    Wetkowski, M A; Peterson, E M; de la Maza, L M

    1982-07-01

    A direct, rapid, and simple method for the detection of streptococcal antigens of Lancefield groups A, B, C, D, and G from blood cultures was developed by using a coagglutination test. Fifty-five clinical specimens and 117 simulated blood cultures containing gram-positive cocci were tested. Out of 6,261 clinical blood cultures screened, 55 cultures from 53 patients were positive, with organisms resembling streptococci, by Gram stain. Of these cultures, 78% (43 of 55) were pure cultures of streptococci, and 22% (12 of 55) were mixed with at least one other organism. Of the 43 pure cultures only, correct reactions were obtained (grouping correctly or giving no cross-reactions, or both) with 86% (37 of 43) of the isolates, 12% (5 of 43) exhibited cross-reactions, and 2% (1 of 43) gave false-negative reactions. All of the cross-reacting isolates were Streptococcus pneumoniae, which reacted with the group C reagent, and the false-negative reaction occurred with a Streptococcus bovis isolate. However, by using a direct modified bile solubility test, the correct identification of the S. pneumoniae isolates was obtained. Therefore, by using the modified bile solubility test in conjunction with the direct grouping method, 98% (42 of 43) of the isolates in pure culture could be identified accurately and rapidly after the detection of a positive Gram stain. Correct grouping reactions were obtained with 83% (10 of 12) of the mixed blood cultures, and false-negative results occurred with 17% (2 of 12) of them. Both cultures contained an enterococcus and a gram-negative rod. Of the 117 simulated blood cultures, there was only one incorrect grouping reaction; this occurred with an S. bovis isolate that cross-reacted with the group C reagent. The direct grouping reaction was positive when blood cultures contained a minimum of 1 x 10(8) to 8 x 10(8) colony-forming units per ml. In general, this procedure provided information on the identification of the organism 24 h earlier than

  15. Antibiotic susceptibility testing of grown blood cultures by combining culture and real-time polymerase chain reaction is rapid and effective.

    Directory of Open Access Journals (Sweden)

    Judith Beuving

    Full Text Available BACKGROUND: Early administration of appropriate antibiotic therapy in bacteraemia patients dramatically reduces mortality. A new method for RApid Molecular Antibiotic Susceptibility Testing (RAMAST that can be applied directly to positive blood cultures was developed and evaluated. METHODOLOGY/PRINCIPAL FINDINGS: Growth curves and antibiotic susceptibility of blood culture isolates (Staphylococcus aureus, enterococci and (facultative aerobic gram-negative rods were determined by incubating diluted blood cultures with and without antibiotics, followed by a quantitative universal 16S PCR to detect the presence or absence of growth. Testing 114 positive blood cultures, RAMAST showed an agreement with microbroth dilution of 96.7% for gram-negative rods, with a minor error (false-susceptibility with a intermediate resistant strain rate of 1.9%, a major error (false resistance rate of 0.8% and a very major error (false susceptibility rate of 0.6%. Agreement for S. aureus was 97.9%, with a very major error rate of 2.1%. Enterococcus species showed 95.0% agreement, with a major error rate of 5.0%. These agreements are comparable with those of the Phoenix system. Starting from a positive blood culture, the test was completed within 9 hours. CONCLUSIONS/SIGNIFICANCE: This new rapid method for antibiotic susceptibility testing can potentially provide accurate results for most relevant bacteria commonly isolated from positive blood cultures in less time than routine methods.

  16. Enumeration of viable non-culturable Vibrio cholerae using propidium monoazide combined with quantitative PCR.

    Science.gov (United States)

    Wu, Bin; Liang, Weili; Kan, Biao

    2015-08-01

    The well-known human pathogenic bacterium, Vibrio cholerae, can enter a physiologically viable but non-culturable (VBNC) state under stress conditions. The differentiation of VBNC cells and nonviable cells is essential for both disease prevention and basic research. Among all the methods for detecting viability, propidium monoazide (PMA) combined with real-time PCR is popular because of its specificity, sensitivity, and speed. However, the effect of PMA treatment is not consistent and varies among different species and conditions. In this study, with an initial cell concentration of 1×10(8) CFU/ml, time and dose-effect relationships of different PMA treatments were evaluated via quantitative real-time PCR using live cell suspensions, dead cell suspensions and VBNC cell suspensions of V. cholerae O1 El Tor strain C6706. The results suggested that a PMA treatment of 20 μM PMA for 20 min was optimal under our conditions. This treatment maximized the suppression of the PCR signal from membrane-compromised dead cells but had little effect on the signal from membrane-intact live cells. In addition to the characteristics of PMA treatment itself, the initial concentration of the targeted bacteria showed a significant negative influence on the stability of PMA-PCR assay in this study. We developed a strategy that mimicked a 1×10(8) CFU/ml cell concentration with dead bacteria of a different bacterial species, the DNA of which cannot be amplified using the real time PCR primers. With this strategy, our optimal approach successfully overcame the impact of low cell density and generated stable and reliable results for counting viable cells of V. cholerae in the VBNC state. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. Detection of Bordetella pertussis from Clinical Samples by Culture and End-Point PCR in Malaysian Patients.

    Science.gov (United States)

    Ting, Tan Xue; Hashim, Rohaidah; Ahmad, Norazah; Abdullah, Khairul Hafizi

    2013-01-01

    Pertussis or whooping cough is a highly infectious respiratory disease caused by Bordetella pertussis. In vaccinating countries, infants, adolescents, and adults are relevant patients groups. A total of 707 clinical specimens were received from major hospitals in Malaysia in year 2011. These specimens were cultured on Regan-Lowe charcoal agar and subjected to end-point PCR, which amplified the repetitive insertion sequence IS481 and pertussis toxin promoter gene. Out of these specimens, 275 were positive: 4 by culture only, 6 by both end-point PCR and culture, and 265 by end-point PCR only. The majority of the positive cases were from ≤3 months old patients (77.1%) (P 0.05). Our study showed that the end-point PCR technique was able to pick up more positive cases compared to culture method.

  18. Identification of blood meals in field captured sand flies by a PCR-RFLP approach based on cytochrome b gene.

    Science.gov (United States)

    González, Estela; Gállego, Montserrat; Molina, Ricardo; Abras, Alba; Alcover, M Magdalena; Ballart, Cristina; Fernández, Anna; Jiménez, Maribel

    2015-12-01

    Leishmaniasis is a vector-borne disease transmitted by phlebotomine sand flies. Information about blood meal preferences in sand flies is essential to understand the epidemiology of the disease to adopt control measures. In previous studies, a polymerase chain reaction (PCR) of 359bp fragment of the conserved gene cytochrome b (cyt b) and further sequencing were applied in the study of blood meal sources in sand flies collected in the area of a leishmaniasis outbreak in southwest Madrid, Spain, providing significant information about blood meal preferences in the focus. In this work, a PCR-restriction fragment length polymorphism (RFLP) targeting a fragment of 359bp of vertebrate cyt b gene was developed. Restriction endonucleases HaeIII and HinfI generated specific patterns consistent with the blood meal sources found in sand flies. The protocol has been validated with twenty six engorged females collected in the field with CDC traps. Blood meals from nine vertebrates were identified based on PCR-cyt b and sequencing-human, dog, cat, horse, hare, rabbit, sheep, goat and chicken - and mixed blood meals (sheep/human; sheep/goat) - and successfully distinguished by PCR-RFLP. Therefore, this approach is an efficient and reliable alternative method to be applied in entomological surveys. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. Application of PCR-based DNA sequencing technique for the detection of Leptospira in peripheral blood of septicemia patients

    OpenAIRE

    Ram, S.; Vimalin, J.M.; Jambulingam, M.; Tiru, V.; Gopalakrishnan, R.K.; Madhavan, H.N.

    2012-01-01

    Aim: Isolation, dark field detection and microscopic agglutination test (MAT) are considered ―gold standard‖ tests for diagnosis of Leptospirosis. Several PCR assays are reported but very few have been evaluated for detection of Leptospirosis. Therefore, this study was undertaken. This study aims to design and standardize polymerase chain reaction (PCR) - based DNA sequencing technique for the detection of pathogenic Leptospira from peripheral blood of patients clinically diagnosed with septi...

  20. Effects of bacterial contamination of media on the diagnosis of Tritrichomonas foetus by culture and real-time PCR.

    Science.gov (United States)

    Clothier, Kristin A; Villanueva, Michelle; Torain, Andrea; Hult, Cynthia; Wallace, Rachel

    2015-03-15

    The venereal pathogen Tritrichomonas foetus causes early embryonic death and abortion in cattle. With no approved treatment, control involves detection of infected animals and their removal from the herd. Culture is the traditional diagnostic method; standard media are formulated to support protozoal growth while suppressing competing organisms which may prevent microscopic recognition of T. foetus. Real-time PCR increases diagnostic sensitivity and specificity over culture but requires intact T. foetus DNA for detection. The purposes of this study were 1) to evaluate the effects of resident preputial bacteria that are not suppressed by antimicrobials in a commercial culture medium (InPouch™) on T. foetus detection by culture and PCR, and 2) to determine the performance of a laboratory-prepared culture medium on T. foetus detection by culture and PCR in samples with and without this bacterial contamination. A known concentration of one of three different strains of T. foetus inoculated into InPouch™ (IP) or modified Diamonds-Plastridge media (DPM) were co-incubated with a smegma culture media (CONTAM) for 24h and examined microscopically for the presence of identifiable T. foetus. PCR was performed on IP samples to determine if CONTAM also affected T. foetus DNA detection. A PCR protocol was then validated in DPM that performed similarly to the established IP PCR method. IP and DPM with CONTAM were spiked with serial dilutions that mimic field infections of one of four T. foetus strains and evaluated by real-time PCR; cycles to threshold (Ct) values and "positive" classification were compared between media. T. foetus motility and morphology as well as media pH were severely altered in IP samples with CONTAM compared to those without as well as to DPM medium with and without CONTAM (Pmedia interfere with T. foetus identification by culture and PCR and adversely affect diagnostic sensitivity for this fastidious pathogen. Copyright © 2015 Elsevier B.V. All rights

  1. Optimized quantification of fragmented, free circulating DNA in human blood plasma using a calibrated duplex real-time PCR.

    Directory of Open Access Journals (Sweden)

    Martin Horlitz

    Full Text Available BACKGROUND: Duplex real-time PCR assays have been widely used to determine amounts and concentrations of free circulating DNA in human blood plasma samples. Circulatory plasma DNA is highly fragmented and hence a PCR-based determination of DNA concentration may be affected by the limited availability of full-length targets in the DNA sample. This leads to inaccuracies when counting PCR target copy numbers as whole genome equivalents. METHODOLOGY/PRINCIPAL FINDINGS: A model system was designed allowing for assessment of bias in a duplex real-time PCR research assay. We collected blood plasma samples from male donors in pools of 6 to 8 individuals. Circulatory plasma DNA was extracted and separated by agarose gel electrophoresis. Separated DNA was recovered from the gel in discrete size fractions and analyzed with different duplex real-time PCR Taqman assays detecting a Y chromosome-specific target and an autosomal target. The real-time PCR research assays used differed significantly in their ability to determine the correct copy number ratio of 0.5 between Y chromosome and autosome targets in DNA of male origin. Longer PCR targets did not amplify quantitatively in circulatory DNA, due to limited presence of full-length target sequence in the sample. CONCLUSIONS: PCR targets of the same small size are preferred over longer targets when comparing fractional circulatory DNA concentrations by real-time PCR. As an example, a DYS14/18S duplex real-time PCR research assay is presented that correctly measures the fractional concentration of male DNA in a male/female mixture of circulatory, fragmented DNA.

  2. Detection of newly produced T and B lymphocytes by digital PCR in blood stored dry on nylon flocked swabs.

    Science.gov (United States)

    Tessitore, Marion Vaglio; Sottini, Alessandra; Roccaro, Aldo M; Ghidini, Claudia; Bernardi, Simona; Martellosio, Giovanni; Serana, Federico; Imberti, Luisa

    2017-04-05

    A normal number of T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs) is considered a biomarker for adequate new T- and B-cell production. In newborns, detection of TRECs and KRECs by real time PCR from dried blood spotted on filter paper is used for the screening of severe immunodeficiency. In adults, elderly and during diseases, where the number of TRECs is lower than in newborns and children, a large amount of DNA and a sensitive method of amplification are necessary to identify newly produced lymphocytes. DNA was prepared from blood of 203 healthy adults (range: 18-91 years old) absorbed for 10 s on flocked swabs and let to dry, or from peripheral blood mononuclear cells. DNA was subjected to digital PCR and to well established conventional real time PCR-based method using TREC- and KREC-specific primers and probes. The number of TRECs and KRECs was expressed per mL of blood. Statistical analysis was performed by nested ANOVA, Pearson coefficient of determination, and by linear regression tests. The novel method for the storage of dried blood on nylon flocked swabs and the use of digital PCR allow quantification of TRECs and KRECs with high degree of sensitivity, specificity, accuracy, and precision. TRECs and KRECs were amplified by digital PCR in all tested blood samples, including those obtained from elderly individuals (>70 years old) and that were negative by real time PCR. Furthermore, values of TRECs and KRECs obtained by digital PCR were in the range of those acquired by real time PCR. Our findings demonstrate that DNA isolation from dried blood on flocked swabs followed by digital PCR-based analysis represents a useful tool for studying new lymphocyte production in adults and elderly individuals. This suggests the potential use of the methodology when monitoring of clinical variables is limited by the number of molecules that can be amplified and detected, such as in patients with immunodeficiency or under

  3. Radiometric detection of yeasts in blood cultures of cancer patients

    International Nuclear Information System (INIS)

    Hopfer, R.L.; Orengo, A.; Chesnut, S.; Wenglar, M.

    1980-01-01

    During a 12-month period, 19,457 blood cultures were collected. Yeasts were isolated from 193 cultures derived from 76 cancer patients. Candida albicans or Candida tropicalis accounted for 79% of isolates. Of the three methods compared, the radiometric method required 2.9 days to become positive, blind subculture required 2.6 days, and Gram stains required 1 day. However, the radiometric method was clearly superior in detecting positive cultures, since 73% of all cultures were first detected radiometrically, 22% were detected by subculture, and only 5% were detected by Gram stain. Although 93% of the isolates were detected by aerobic culture, five (7%) isolates were obtained only from anaerobic cultures. Seven days of incubation appear to be sufficient for the radiometric detection of yeasts

  4. Detection of Mycoplasma synoviae infection in broiler breeder farms of Tehran province using PCR and culture methods

    Directory of Open Access Journals (Sweden)

    Elhamnia, F.

    2010-07-01

    Full Text Available Mycoplasma synoviae (MS is an important avian pathogen that can cause both respiratory disease and joint inflammation synovitis in poultry, inducing economic losses to the Iranian chicken industry especially breeder farms. The aim of this study was to use the MS specific PCR and culture methods in order to detect of M. synoviae from breeder farms where located in Tehran province. A total of 475 samples including choanal cleft, trachea, ovary and /or joint cavities from 23 broiler breeder farms of Tehran area were collected. Samples were cultured in PPLO broth media supplemented for MS isolation. The bacteria DNAs were extracted by phenol/chloroform method. Specific published primers amplify a 207 bp region of the 16S rRNA gene of MS were used for PCR method. Out of 475 samples, 146 cultures were shown positive and typical Mycoplasma colonies, 85 samples were also identified MS based on agglutination test with specific MS antiserum and the PCR method. A total of 122 samples, a band with 207 bp was shown as MS specific PCR product in electrophoresis. In addition to these 85 samples that were positives in both culture and PCR, 37 samples that had not grown in Mycoplasma media were positive in MS specific PCR. A total of 292 samples were negatives in both culture and PCR methods. 122 positive samples out of 475 samples (25.7% were belonged to 7 breeder farms (30.4%. On conclusions, the MS infection of broiler breeder farms of Tehran area was confirmed truly. From the results, as the PCR method reduces the time consuming, an effectiveness and efficient for detection of M. synoviae infection of chicken breeder. It is then suggested that the PCR method could be an alternative method for culturing.

  5. Porphyromonas gingivalis, Porphyromonas endodontalis, Prevotella intermedia and Prevotella nigrescens in endodontic lesions detected by culture and by PCR.

    Science.gov (United States)

    Gomes, B P F A; Jacinto, R C; Pinheiro, E T; Sousa, E L R; Zaia, A A; Ferraz, C C R; Souza-Filho, F J

    2005-08-01

    he aim of this study was to investigate the presence of four black-pigmented bacteria, Porphyromonas gingivalis, Porphyromonas endodontalis, Prevotella intermedia and Prevotella nigrescens, in endodontic infections by culture and polymerase chain reaction (PCR) analyses. Microbial samples were obtained from 50 teeth with untreated necrotic pulps (primary infection) and from 50 teeth with failing endodontic treatment (secondary infection). Microbiological strict anaerobic techniques were used for serial dilution, plating, incubation, and identification. For PCR detection, the samples were analyzed using species-specific primers of 16S rDNA and the downstream intergenic spacer region. Culture and PCR detected the test species in 13/100 and 50/100 of the study teeth, respectively. The organisms were cultured from 11/50 (22%) of primarily infected root canal samples and from 2/50 (4%) of secondary root canal samples. PCR detection identified the target species in 32/50 (64%) and 18/50 (36%) of primary and secondary infections, respectively. P. gingivalis was rarely isolated by culture methods (1%), but was the most frequently identified test species by PCR (38%). Similarly, P. endodontalis was not recovered by culture from any tooth studied, but was detected by PCR in 25% of the sampled teeth. PCR-based identification also showed higher detection rates of P. intermedia (33%) and P. nigrescens (22%) than culture (13%). In conclusion, P. gingivalis, P. endodontalis, P. intermedia, and P. nigrescens were identified more frequently in teeth with necrotic pulp than in teeth with failing endodontic treatment. Also, a higher frequency of black-pigmented species was detected by PCR than by culture.

  6. A rapid, highly sensitive and culture-free detection of pathogens from blood by positive enrichment.

    Science.gov (United States)

    Vutukuru, Manjula Ramya; Sharma, Divya Khandige; Ragavendar, M S; Schmolke, Susanne; Huang, Yiwei; Gumbrecht, Walter; Mitra, Nivedita

    2016-12-01

    Molecular diagnostics is a promising alternative to culture based methods for the detection of bloodstream infections, notably due to its overall lower turnaround time when starting directly from patient samples. Whole blood is usually the starting diagnostic sample in suspected bloodstream infections. The detection of low concentrations of pathogens in blood using a molecular assay necessitates a fairly high starting volume of blood sample in the range of 5-10mL. This large volume of blood sample has a substantial accompanying human genomic content that interferes with pathogen detection. In this study, we have established a workflow using magnetic beads coated with Apolipoprotein H that makes it possible to concentrate pathogens from a 5.0mL whole blood sample, thereby enriching pathogens from whole blood background and also reducing the sample volume to ~200μL or less. We have also demonstrated that this method of enrichment allows detection of 1CFU/mL of Escherichia coli, Enterococcus gallinarum and Candida tropicalis from 5mL blood using quantitative PCR; a detection limit that is not possible in unenriched samples. The enrichment method demonstrated here took 30min to complete and can be easily integrated with various downstream molecular and microbiological techniques. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. A Real-Time PCR Assay Based on 5.8S rRNA Gene (5.8S rDNA) for Rapid Detection of Candida from Whole Blood Samples.

    Science.gov (United States)

    Guo, Yi; Yang, Jing-Xian; Liang, Guo-Wei

    2016-06-01

    The prevalence of Candida in bloodstream infections (BSIs) has increased. To date, the identification of Candida in BSIs still mainly relies on blood culture and serological tests, but they have various limitations. Therefore, a real-time PCR assay for the detection of Candida from whole blood is presented. The unique primers/probe system was designed on 5.8S rRNA gene (5.8S rDNA) of Candida genus. The analytical sensitivity was determined by numbers of positive PCRs in 12 repetitions. At the concentration of 10(1) CFU/ml blood, positive PCR rates of 100 % were obtained for C. albicans, C. parapsilosis, C. tropicalis, and C. krusei. The detection rate for C. glabrata was 75 % at 10(1) CFU/ml blood. The reaction specificity was 100 % when evaluating the assay using DNA samples from clinical isolates and human blood. The maximum CVs of intra-assay and inter-assay for the detection limit were 1.22 and 2.22 %, respectively. To assess the clinical applicability, 328 blood samples from 82 patients were prospectively tested and real-time PCR results were compared with results from blood culture. Diagnostic sensitivity of the PCR was 100 % using as gold standard blood culture, and specificity was 98.4 %. Our data suggest that the developed assay can be used in clinical laboratories as an accurate and rapid screening test for the Candida from whole blood. Although further evaluation is warranted, our assay holds promise for earlier diagnosis of candidemia.

  8. Real-time PCR detection of Campylobacter spp.: A comparison to classic culturing and enrichment.

    Science.gov (United States)

    de Boer, P; Rahaoui, H; Leer, R J; Montijn, R C; van der Vossen, J M B M

    2015-10-01

    The major disadvantage of the current gold standard for detection of the food pathogen Campylobacter, i.e. culturing, is the lengthy procedure. In this study we assessed the use of real-time PCR for detection of Campylobacter. To this end, 926 poultry samples, taken from transport containers and broiler caeca in The Netherlands in 2007, were subjected to three different real-time PCR detection methods: one targeting the Campylobacter jejuni hipO gene, one targeting the Campylobacter coli glyA gene, and one generically targeting Campylobacter spp. 16S rDNA sequence. The PCR results from the three different PCR protocols were compared to the work of Nauta et al. (2009) who analyzed the same set of samples collected from 62 broiler flocks by means of enrichment culturing. The results indicate that the generic 16S campylobacter PCR detection is equally reliable but much faster (4 h instead of ≥2 days) than detection by means of culturing. Moreover, PCR detection targeting the hipO and the glyA gene provide the possibility of C. jejuni and C. coli species discrimination. The generic Campylobacter spp. PCR analysis also confirmed the high incidence of Campylobacter spp. in poultry samples (∼90%) and the species specific PCR showed the simultaneous presence of C. jejuni and C. coli in ∼24% of the samples. Furthermore, the results from the three PCR analyses suggested the occurrence of alternative Campylobacter species in almost 10% of the samples. The campylobacter PCR detection methods reported here can replace traditional culturing because of being quicker and more reliable. Copyright © 2015 Elsevier Ltd. All rights reserved.

  9. Innovation for reducing blood culture contamination: initial specimen diversion technique.

    Science.gov (United States)

    Patton, Richard G; Schmitt, Timothy

    2010-12-01

    We hypothesized that diversion of the first milliliter of venipuncture blood-the initial specimen diversion technique (ISDT)-would eliminate incompletely sterilized fragments of skin from the culture specimen and significantly reduce our blood culture contamination rate (R). We studied our hypothesis prospectively beginning with our control culture (C) definition: one venipuncture with two sequentially obtained specimens, 10 ml each, the first specimen (M1) for aerobic and the second (M2) for anaerobic media. The test ISDT culture (D) was identical, with the exception that each was preceded by diverting a 1-ml sample (DS) from the same venipuncture. During the first of two sequential 9-month periods, we captured D versus C data (n=3,733), where DMXR and CMXR are R for D and C specimens. Our hypothesis predicted DS would divert soiled skin fragments from DM1, and therefore, CM1R would be significantly greater than DM1R. This was confirmed by CM1R (30/1,061 [2.8%]) less DM1R (37/2,672 [1.4%]; P=0.005), which equals 1.4%. For the second 9-month follow-up period, data were compiled for all cultures (n=4,143), where ADMXR is R for all (A) diversion specimens, enabling comparison to test ISDT. Our hypothesis predicted no significant differences for test ISDT versus all ISDT. This was confirmed by DM1R (37/2,672 [1.4%]) versus ADM1R (42/4,143 [1.0%]; P=0.17) and DM2R (21/2,672 [0.80%]) versus ADM2R (39/4,143 [0.94%]; P=0.50). We conclude that our hypothesis is valid: venipuncture needles soil blood culture specimens with unsterilized skin fragments and increase R, and ISDT significantly reduces R from venipuncture-obtained blood culture specimens.

  10. Relationship and variation of qPCR and culturable enterococci estimates in ambient surface waters are predictable

    Science.gov (United States)

    Whitman, Richard L.; Ge, Zhongfu; Nevers, Meredith B.; Boehm, Alexandria B.; Chern, Eunice C.; Haugland, Richard A.; Lukasik, Ashley M.; Molina, Marirosa; Przybyla-Kelly, Kasia; Shively, Dawn A.; White, Emily M.; Zepp, Richard G.; Byappanahalli, Muruleedhara N.

    2010-01-01

    The quantitative polymerase chain reaction (qPCR) method provides rapid estimates of fecal indicator bacteria densities that have been indicated to be useful in the assessment of water quality. Primarily because this method provides faster results than standard culture-based methods, the U.S. Environmental Protection Agency is currently considering its use as a basis for revised ambient water quality criteria. In anticipation of this possibility, we sought to examine the relationship between qPCR-based and culture-based estimates of enterococci in surface waters. Using data from several research groups, we compared enterococci estimates by the two methods in water samples collected from 37 sites across the United States. A consistent linear pattern in the relationship between cell equivalents (CCE), based on the qPCR method, and colony-forming units (CFU), based on the traditional culturable method, was significant (P 10CFU > 2.0/100 mL) while uncertainty increases at lower CFU values. It was further noted that the relative error in replicated qPCR estimates was generally higher than that in replicated culture counts even at relatively high target levels, suggesting a greater need for replicated analyses in the qPCR method to reduce relative error. Further studies evaluating the relationship between culture and qPCR should take into account analytical uncertainty as well as potential differences in results of these methods that may arise from sample variability, different sources of pollution, and environmental factors.

  11. Inhibition of pneumococcal autolysis in lysis-centrifugation blood culture.

    OpenAIRE

    Lehtonen, O P

    1986-01-01

    The recovery of Streptococcus pneumoniae from the Isolator lysis-centrifugation blood culture has been low in many studies. The poor survival of pneumococci was not due to toxicity of the Isolator medium but to autolysis before plating. This autolysis was completely inhibited by adding 10 mM phosphorylcholine to the Isolator medium.

  12. Inoculation ofperitoneal dialysate fluid into blood culture bottles im ...

    African Journals Online (AJOL)

    Abstract The aiIn of the study was to detennine if direct inoculation of peritoneal fluid into Bactec blood culture bottles would improve the positive bacteriological yield compared with conventional techniques in continuous ambulatory peritoneal dialysis (CAPD) patients with peritonitis. All patients presenting with suspected ...

  13. Inoculation of peritoneal dialysate fluid into blood culture bottles ...

    African Journals Online (AJOL)

    The aim of the study was to determine if direct inoculation of peritoneal fluid into Bactec blood culture bottles would improve the positive bacteriological yield compared with conventional techniques in continuous ambulatory peritoneal dialysis (CAPD) patients with peritonitis. All patients presenting with suspected peritonitis ...

  14. Avian malaria in Brazilian passerine birds: parasitism detected by nested PCR using DNA from stained blood smears.

    Science.gov (United States)

    Ribeiro, S F; Sebaio, F; Branquinho, F C S; Marini, M A; Vago, A R; Braga, E M

    2005-03-01

    The microscopical examination of Giemsa-stained thin blood smears and a nested PCR were performed to detect avian Plasmodium in 275 passerine birds from small and large fragments of Atlantic Forest, Minas Gerais, Brazil. The 275 blood smears were used both for the microscopical examination and nested PCR providing the DNA template used for the reactions. The sensitivity of the nested PCR assay was higher than that observed for blood smears through microscopical examination. High prevalence (39.6%) of Plasmodium infections was detected by nested PCR while the microscopical examination detected only 16.5 % positive birds. Poor agreement was observed between the results of the two different tests. The PCR data obtained were correlated to the forest fragment size of the Atlantic Forest and also correlated to the biological characteristics of the birds (nest type construction, diet, participation in mixed-species flocks, age and sex). Birds captured in the large forest areas were more infected than birds captured in the small areas (51.9 % and 28.5 %, respectively). Diet and participation in mixed-species flocks were correlated to the Plasmodium parasitism. The insectivorous birds and those that participated in mixed-species flocks were more frequently infected (47% and 41.5%, respectively) than the other groups.

  15. Survival and function of phagocytes in blood culture media

    DEFF Research Database (Denmark)

    Fischer, T K; Prag, J; Kharazmi, A

    1999-01-01

    The survival and function of human phagocytes in sterile aerobic and anaerobic blood culture media were investigated using neutrophil morphology, white blood cell count in a haemoanalyser, flow cytometry, oxidative burst response, and bactericidal effect in Colorbact and Septi-Chek blood culture...... media and Bact/Alert. When comparing agitation to stationary incubation no difference in phagocytic activity was found. The methods showed the same trends demonstrating that the phagocytes' viability and activity were prolonged by oxygen and shortened by anaerobic conditions and sodium polyethanol...... sulfonate (SPS). Best preserved activity and viability were found in the aerobic media containing less than 0.5 g/l SPS, in which significant phagocyte oxidative burst and bactericidal activity were found up to 4 days after inoculation. Considering that the majority of bacteremias are due to aerobic...

  16. Comparison of culture and PCR methods in the diagnosis of bacterial meningitis.

    Science.gov (United States)

    Başpınar, Emel Ödemiş; Dayan, Saim; Bekçibaşı, Muhammed; Tekin, Recep; Ayaz, Celal; Deveci, Özcan; Hoşoğlu, Salih

    Our aim in this study is to compare the standard culture method with the multiplex PCR and the Speed-Oligo ® Bacterial Meningitis Test (SO-BMT) - a hybridization-based molecular test method - during the CSF examination of the patients with the pre-diagnosis of acute bacterial meningitis. For the purposes of this study, patients with acute bacterial meningitis treated at the Dicle University Medical Faculty Hospital, Infectious Diseases and Clinical Microbiology Clinic between December 2009 and April 2012 were retrospectively evaluated. The diagnosis of bacterial meningitis was made based on the clinical findings, laboratory test anomalies, CSF analysis results, and the radiological images. Growth was observed in the CSF cultures of 10 out of the 57 patients included in the study (17.5%) and Streptococcus pneumoniae was isolated in all of them. The CSF samples of 34 patients (59.6%) were positive according to the SO-BMT and S. pneumoniae was detected in 33 of the samples (97.05%), while Neisseria meningitidis was found in 1 sample (2.95%). In a total of 10 patients, S. pneumoniae was both isolated in the CSF culture and detected in the SO-BMT. The culture and the SO-BMT were negative in 23 of the CSF samples. There was no sample in which the CSF culture was positive although the SO-BMT was negative. While SO-BMT seems to be a more efficient method than bacterial culturing to determine the pathogens that most commonly cause bacterial meningitis in adults, further studies conducted on larger populations are needed in order to assess its efficiency and uses. Copyright © 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

  17. Decreased mortality associated with prompt Gram staining of blood cultures.

    Science.gov (United States)

    Barenfanger, Joan; Graham, Donald R; Kolluri, Lavanya; Sangwan, Gaurav; Lawhorn, Jerry; Drake, Cheryl A; Verhulst, Steven J; Peterson, Ryan; Moja, Lauren B; Ertmoed, Matthew M; Moja, Ashley B; Shevlin, Douglas W; Vautrain, Robert; Callahan, Charles D

    2008-12-01

    Gram stains of positive blood cultures are the most important factor influencing appropriate therapy. The sooner appropriate therapy is initiated, the better. Therefore, it is reasonable to expect that the sooner Gram stains are performed, the better. To determine the value of timely Gram stains and whether improvement in Gram stain turnaround time (TAT) is feasible, we compared data for matched pairs of patients with cultures processed promptly ( or =1 hour TAT) and then monitored TAT by control charting.In 99 matched pairs, average difference in time to detection of positive blood cultures within a pair of patients was less than 0.1 hour. For the less than 1 hour TAT group, the average TAT and crude mortality were 0.1 hour and 10.1%, respectively; for the 1 hour or longer TAT group, they were 3.3 hours and 19.2%, respectively (P Gram stains.

  18. Should blood cultures be performed in terminally Ill cancer patients?

    Directory of Open Access Journals (Sweden)

    Nobuhiro Asai

    2015-04-01

    Full Text Available Background: No evidence-based guidelines or protocols to treat the infection-related symptoms in cancer patients with terminal stages have been established. Materials and Methods: We retrospectively analyzed all the patients with terminal stage cancer who died between April 2009 and March 2010. The patients' background, the prevalence of infection and clinical outcomes, pathogens isolated, antibiotics used, and whether blood cultures and some of examinations were performed or not were evaluated. Results: A total of 62 (44 males and 18 females patients were included in this study. The median age was 73 years (35-98 years. The most common cancer was that of the lung (n =59, 95.2%. A total of 32 patients were diagnosed with the following infections: Infection of respiratory tract in 27 (84.4%, of urinary tract in 4 (12.5%, and cholangitis in 1 (3.1%. Two cases (6.3% had pneumonia complicated with urinary tract infection. Blood cultures and antibiotic therapies were performed in 28 and 30 cases, respectively. Four (14.3% positive cultures were isolated from the blood obtained from 28 individual patients. As for clinical course, 3 (10% of them experienced improved symptoms after antibiotic therapy. Twenty-seven (90% patients were not confirmed as having any symptom improvement. Conclusions: Blood cultures and antibiotic therapy were limited, and might not be effective in terminally ill cancer patients with lung cancer. We suggest that administering an antibiotic therapy without performing a blood culture would be one of choices in those with respiratory tract infections if patients' life expectancy is short.

  19. Real-time PCR versus viral culture on urine as a gold standard in the diagnosis of congenital cytomegalovirus infection

    NARCIS (Netherlands)

    de Vries, Jutte J. C.; van der Eijk, Annemiek A.; Wolthers, Katja C.; Rusman, Lisette G.; Pas, Suzan D.; Molenkamp, Richard; Claas, Eric C.; Kroes, Aloys C. M.; Vossen, Ann C. T. M.

    2012-01-01

    Background: Cytomegalovirus (CMV) infection is the most common cause of congenital infection. Whereas CMV PCR has replaced viral culture and antigen detection in immunocompromised patients because of higher sensitivity, viral culture of neonatal urine is still referred to as the gold standard in the

  20. Comparison of PCR methods and culture for the detection of Borrelia spp. in patients with erythema migrans.

    Science.gov (United States)

    Cerar, T; Ruzić-Sabljić, E; Glinsek, U; Zore, A; Strle, F

    2008-07-01

    The sensitivities of two PCR assays and culture were compared for the detection of Borrelia spp. in skin specimens of 150 patients with typical erythema migrans. In addition, the accuracy of the methods for the identification of Borrelia spp. was compared by analysing culture isolates and material obtained directly from skin biopsy specimens. Borrelia burgdorferi sensu lato was isolated from 73 (49%) of 150 skin biopsy specimens. Using a nested PCR targeting the rrf-rrl region and a PCR targeting the flagellin gene, 107 (71%) and 36 (24%) specimens, respectively, were positive. With both PCRs, positive results were more frequent with culture-positive samples (67/73 (92%) and 24/73 (33%) for the nested and flagellin PCRs, respectively) than with culture-negative samples (40/77 (52%) and 12/77 (16%) for nested and flagellin PCR, respectively). Pulsed-field gel electrophoresis after MluI restriction identified 69/73 (95%) isolates, of which 58/69 (84%) were Borrelia afzelii and 11/69 (16%) were Borrelia garinii. After MseI restriction of PCR products amplified from the intergenic rrf-rrl region, B. afzelii was identified in 73/107 (68%) samples, B. garinii in 22/107 (21%) samples, and both species in 11/107 (10%) samples. The corresponding results for culture-positive specimens were 41/69 (59%), 14/69 (20%), and 7/69 (10%). Comparison of the results for specimens positive according to both approaches revealed complete uniformity in 80% of the cases. Overall, nested PCR was the most sensitive method for the demonstration of Borrelia spp. in erythema migrans skin lesions, followed by culture and PCR targeting the flagellin gene. The congruence of identification results obtained by analyzing culture isolates and material obtained directly from skin biopsies was relatively high but incomplete.

  1. Detection of bacterial species involved in perimplantitis concerned with cultural and RT-PCR

    Directory of Open Access Journals (Sweden)

    Marcello Gatti

    2010-06-01

    Full Text Available Dental implants offer new treatment options for edentulous either partially or completely, now represent a viable alternative to conventional fixed protheses. Dental implants are colonized by a flora dominated by Gram-positive facultative aerobic, while in patients with bone loss and formation of pockets peri-implant diseases was found a significant difference in the composition of microflora, bacteria, Gram-negative anaerobes in particular Fusobacterium spp., Treponema denticola (Spirochetes, Tannerella forsythensis, Aggregatibacter actinomycetemcomitans, Prevotella intermedia as interim black-pigmented bacteria, Porphyromonas gingivalis, often in high concentrations. Aims. The purpose of this study was to identify those at risk of perimplantitis using 2 techniques: RT-PCR examination of trade and culture. The results were compared taking into consideration the advantages and disadvantages of both methods. Materials and methods.We studied 24 patients (14 women and 10 men, aged, women between 43 and 76 years, with an average of 63.8 + / - 10.9 years, men between 45 and 88 years with a average of 64.3 years + / - 12.5 years. Was performed a double levy of sub-gingival plaque at multiple sites that had an implant CAL (clinical attachment level> 4mm in order to assess the microbiological identification with the two techniques: Examining culture and Real-Time PCR of Commerce ( Gum-Sunstar that identifies 4 bacterial species: A. actinomycetemcomitans (A.a., P.gingivalis (P.g., T.forsythensis (T.f., and T.denticola (T.d.. Results. All patients studied were positive to both tests with charger high: the consideration of tenure, with CFU / ml > 105, was positive in 66.6% of samples by:T.f., and P.g., in 12.5% for A.a., while T.d. not been sought by examining culture, the RT-PCR was positive, with high loads, in 95.8% of samples for T.f., in 79.1% for P.g., in 12.5% for A.a. and 20.8% for T.d.The test crop showed the presence of even P.intermedia in 91

  2. Genetic diversity of PCR-positive, culture-negative and culture-positive Mycobacterium ulcerans isolated from Buruli ulcer patients in Ghana.

    Directory of Open Access Journals (Sweden)

    Heather Williamson

    Full Text Available Culture of Mycobacterium ulcerans from Buruli ulcer patients has very low sensitivity. Thus confirmation of M. ulcerans infection is primarily based on PCR directed against IS2404. In this study we compare the genotypes obtained by variable number of tandem repeat analysis of DNA from IS2404-PCR positive cultures with that obtained from IS2404 positive, culture-negative tissue. A significantly greater genetic heterogeneity was found among culture-negative samples compared with that found in cultured strains but a single genotype is over-represented in both sample sets. This study provides evidence that both the focal location of bacteria in a lesion as well as differences in the ability to culture a particular genotype may underlie the low sensitivity of culture. Though preliminary, data from this work also suggests that mycobacteria previously associated with fish disease (M. pseudoshottsii may be pathogenic for humans.

  3. DNA from pre-erythrocytic stage malaria parasites is detectable by PCR in the faeces and blood of hosts.

    Science.gov (United States)

    Abkallo, Hussein M; Liu, Weimin; Hokama, Sarina; Ferreira, Pedro E; Nakazawa, Shusuke; Maeno, Yoshimasa; Quang, Nguyen T; Kobayashi, Nobuyuki; Kaneko, Osamu; Huffman, Michael A; Kawai, Satoru; Marchand, Ron P; Carter, Richard; Hahn, Beatrice H; Culleton, Richard

    2014-06-01

    Following the bite of an infective mosquito, malaria parasites first invade the liver where they develop and replicate for a number of days before being released into the bloodstream where they invade red blood cells and cause disease. The biology of the liver stages of malaria parasites is relatively poorly understood due to the inaccessibility of the parasites to sampling during this phase of their life cycle. Here we report the detection in blood and faecal samples of malaria parasite DNA throughout their development in the livers of mice and before the parasites begin their growth in the blood circulation. It is shown that parasite DNA derived from pre-erythrocytic stage parasites reaches the faeces via the bile. We then show that different primate malaria species can be detected by PCR in blood and faecal samples from naturally infected captive macaque monkeys. These results demonstrate that pre-erythrocytic parasites can be detected and quantified in experimentally infected animals. Furthermore, these results have important implications for both molecular epidemiology and phylogenetics of malaria parasites. In the former case, individuals who are malaria parasite negative by microscopy, but PCR positive for parasite DNA in their blood, are considered to be "sub-microscopic" blood stage parasite carriers. We now propose that PCR positivity is not necessarily an indicator of the presence of blood stage parasites, as the DNA could derive from pre-erythrocytic parasites. Similarly, in the case of molecular phylogenetics based on DNA sequences alone, we argue that DNA amplified from blood or faeces does not necessarily come from a parasite species that infects the red blood cells of that particular host. Copyright © 2014 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

  4. Diagnosis of Trichomonas vaginalis from vaginal specimens by wet mount microscopy, in pouch TV culture system, and PCR

    Directory of Open Access Journals (Sweden)

    Madhumati J Patil

    2012-01-01

    Full Text Available Background: In recent years, trichomoniasis has emerged as the most common sexually transmitted disease and limited data are available on the effective screening technique for the diagnosis of Trichomonas vaginalis. Aim : The aim was to compare and evaluate different diagnostic methods like wet mount microscopy, In Pouch TV culture, and Polymerase chain reaction (PCR to establish which method or combination of methods was most effective for detection of Trichomonas vaginalis in vaginal swab specimens. Settings and Design : This is a cross-sectional study. Materials and Methods : A total of 200 patients complaining of vaginal discharge were included in the study. Three vaginal swabs were screened for trichomoniasis by wet mount microscopy, In Pouch TV culture system and PCR, using TVK3 and TVK7 specific primers. Results : Of the 200 cases studied, 36 (18% were positive by wet mount microscopy, 44 (22% by In Pouch TV culture system and 60(30% by PCR. Sensitivity and specificity of wet mount were 60% and 100%, respectively, whereas sensitivity and specificity of the In Pouch TV culture system were 73.33% and 100%, respectively when compared to PCR. Conclusion : Comparison of different methods showed that at least two techniques, such as wet mount microscopy and culture have a better chance of detection of T. vaginalis infection. Diagnosis of trichomoniasis by PCR was found to be highly specific and sensitive, but its availability and cost effectiveness limit its use in routine diagnostic laboratories.

  5. Effective PCR-based detection of Naegleria fowleri from cultured sample and PAM-developed mouse.

    Science.gov (United States)

    Kang, Heekyoung; Seong, Gi-Sang; Sohn, Hae-Jin; Kim, Jong-Hyun; Lee, Sang-Eun; Park, Mi Yeoun; Lee, Won-Ja; Shin, Ho-Joon

    2015-10-01

    Increasing numbers of Primary Amoebic Meningoencephalitis (PAM) cases due to Naegleria fowleri are becoming a serious issue in subtropical and tropical countries as a Neglected Tropical Disease (NTD). To establish a rapid and effective diagnostic tool, a PCR-based detection technique was developed based on previous PCR methods. Four kinds of primer pairs, Nfa1, Nae3, Nf-ITS, and Naegl, were employed in the cultured amoebic trophozoites and a mouse with PAM experimentally developed by N. fowleri inoculation (PAM-mouse). For the extraction of genomic DNA from N. fowleri trophozoites (1×10(6)), simple boiling with 10μl of PBS (pH 7.4) at 100°C for 30min was found to be the most rapid and efficient procedure, allowing amplification of 2.5×10(2) trophozoites using the Nfa-1 primer. The primers Nfa1 and Nae3 amplified only N. fowleri DNA, whereas the ITS primer detected N. fowleri and N. gruberi DNA. Using the PAM-mouse brain tissue, the Nfa1 primer was able to amplify the N. fowleri DNA 4 days post infection with 1ng/μl of genomic DNA being detectable. Using the PAM-mouse CSF, amplification of the N. fowleri DNA with the Nae3 primer was possible 5 days post infection showing a better performance than the Nfa1 primer at day 6. Copyright © 2015 Elsevier GmbH. All rights reserved.

  6. Detection of Tilapia Lake Virus in Clinical Samples by Culturing and Nested Reverse Transcription-PCR.

    Science.gov (United States)

    Kembou Tsofack, Japhette Esther; Zamostiano, Rachel; Watted, Salsabeel; Berkowitz, Asaf; Rosenbluth, Ezra; Mishra, Nischay; Briese, Thomas; Lipkin, W Ian; Kabuusu, Richard M; Ferguson, Hugh; Del Pozo, Jorge; Eldar, Avi; Bacharach, Eran

    2017-03-01

    Tilapia are an important group of farmed fish that serve as a significant protein source worldwide. In recent years, substantial mortality of wild tilapia has been observed in the Sea of Galilee and in commercial ponds in Israel and Ecuador. We have identified the etiological agent of these mass die-offs as a novel orthomyxo-like virus and named it tilapia lake virus (TiLV). Here, we provide the conditions for efficient isolation, culturing, and quantification of the virus, including the use of susceptible fish cell lines. Moreover, we describe a sensitive nested reverse transcription-PCR (RT-PCR) assay allowing the rapid detection of TiLV in fish organs. This assay revealed, for the first time to our knowledge, the presence of TiLV in diseased Colombian tilapia, indicating a wider distribution of this emerging pathogen and stressing the risk that TiLV poses for the global tilapia industry. Overall, the described procedures should provide the tilapia aquaculture industry with important tools for the detection and containment of this pathogen. Copyright © 2017 American Society for Microbiology.

  7. Highly Sensitive Bacteriophage-Based Detection of Brucella abortus in Mixed Culture and Spiked Blood

    Science.gov (United States)

    Sergueev, Kirill V.; Filippov, Andrey A.; Nikolich, Mikeljon P.

    2017-01-01

    For decades, bacteriophages (phages) have been used for Brucella species identification in the diagnosis and epidemiology of brucellosis. Traditional Brucella phage typing is a multi-day procedure including the isolation of a pure culture, a step that can take up to three weeks. In this study, we focused on the use of brucellaphages for sensitive detection of the pathogen in clinical and other complex samples, and developed an indirect method of Brucella detection using real-time quantitative PCR monitoring of brucellaphage DNA amplification via replication on live Brucella cells. This assay allowed the detection of single bacteria (down to 1 colony-forming unit per milliliter) within 72 h without DNA extraction and purification steps. The technique was equally efficient with Brucella abortus pure culture and with mixed cultures of B. abortus and α-proteobacterial near neighbors that can be misidentified as Brucella spp., Ochrobactrum anthropi and Afipia felis. The addition of a simple short sample preparation step enabled the indirect phage-based detection of B. abortus in spiked blood, with the same high sensitivity. This indirect phage-based detection assay enables the rapid and sensitive detection of live B. abortus in mixed cultures and in blood samples, and can potentially be applied for detection in other clinical samples and other complex sample types. PMID:28604602

  8. Highly Sensitive Bacteriophage-Based Detection of Brucella abortus in Mixed Culture and Spiked Blood

    Directory of Open Access Journals (Sweden)

    Kirill V. Sergueev

    2017-06-01

    Full Text Available For decades, bacteriophages (phages have been used for Brucella species identification in the diagnosis and epidemiology of brucellosis. Traditional Brucella phage typing is a multi-day procedure including the isolation of a pure culture, a step that can take up to three weeks. In this study, we focused on the use of brucellaphages for sensitive detection of the pathogen in clinical and other complex samples, and developed an indirect method of Brucella detection using real-time quantitative PCR monitoring of brucellaphage DNA amplification via replication on live Brucella cells. This assay allowed the detection of single bacteria (down to 1 colony-forming unit per milliliter within 72 h without DNA extraction and purification steps. The technique was equally efficient with Brucella abortus pure culture and with mixed cultures of B. abortus and α-proteobacterial near neighbors that can be misidentified as Brucella spp., Ochrobactrum anthropi and Afipia felis. The addition of a simple short sample preparation step enabled the indirect phage-based detection of B. abortus in spiked blood, with the same high sensitivity. This indirect phage-based detection assay enables the rapid and sensitive detection of live B. abortus in mixed cultures and in blood samples, and can potentially be applied for detection in other clinical samples and other complex sample types.

  9. Highly Sensitive Bacteriophage-Based Detection of Brucella abortus in Mixed Culture and Spiked Blood.

    Science.gov (United States)

    Sergueev, Kirill V; Filippov, Andrey A; Nikolich, Mikeljon P

    2017-06-10

    For decades, bacteriophages (phages) have been used for Brucella species identification in the diagnosis and epidemiology of brucellosis. Traditional Brucella phage typing is a multi-day procedure including the isolation of a pure culture, a step that can take up to three weeks. In this study, we focused on the use of brucellaphages for sensitive detection of the pathogen in clinical and other complex samples, and developed an indirect method of Brucella detection using real-time quantitative PCR monitoring of brucellaphage DNA amplification via replication on live Brucella cells. This assay allowed the detection of single bacteria (down to 1 colony-forming unit per milliliter) within 72 h without DNA extraction and purification steps. The technique was equally efficient with Brucella abortus pure culture and with mixed cultures of B . abortus and α-proteobacterial near neighbors that can be misidentified as Brucella spp., Ochrobactrum anthropi and Afipia felis . The addition of a simple short sample preparation step enabled the indirect phage-based detection of B . abortus in spiked blood, with the same high sensitivity. This indirect phage-based detection assay enables the rapid and sensitive detection of live B . abortus in mixed cultures and in blood samples, and can potentially be applied for detection in other clinical samples and other complex sample types.

  10. Blood culture cross contamination associated with a radiometric analyzer

    International Nuclear Information System (INIS)

    Griffin, M.R.; Miller, A.D.; Davis, A.C.

    1982-01-01

    During a 9-day period in August 1980 in a New Jersey hospital, three pairs of consecutively numbered blood cultures from different patients were identified as positive for the same organism, for each pair, both cultures were positive in the same atmosphere, both organisms had the same sensitivities, and the second of each pair grew at least 2 days after the first and was the only positive blood culture obtained from the patient. When the hospital laboratory discontinued use of its radiometric culture analyzer for 15 days, no more consecutive pairs of positive cultures occurred. Subsequent use of the machine for 9 days with a new power unit but the original circuit boards resulted in one more similar consecutive pair (Staphylococcus epidermidis). After replacement of the entire power unit, there were no further such pairs. Examination of the machine by the manufacturer revealed a defective circuit board which resulted in inadequate needle sterilization. Laboratories which utilize radiometric analyzers should be aware of the potential for cross contamination. Recognition of such events requires alert microbiologists and infection control practitioners and a record system in the bacteriology laboratory designed to identify such clusters

  11. Seminested PCR for Diagnosis of Candidemia: Comparison with Culture, Antigen Detection, and Biochemical Methods for Species Identification

    OpenAIRE

    Ahmad, Suhail; Khan, Zaiba; Mustafa, Abu S.; Khan, Zia U.

    2002-01-01

    The rapid detection and identification of Candida species in clinical laboratories are extremely important for the management of patients with hematogenous candidiasis. The presently available culture and biochemical methods for detection and species identification of Candida are time-consuming and lack the required sensitivity and specificity. In this study, we have established a seminested PCR (snPCR) using universal and species-specific primers for detection of Candida species in serum spe...

  12. Comparison of culture and qPCR for the detection of Pseudomonas aeruginosa in not chronically infected cystic fibrosis patients

    OpenAIRE

    Deschaght, Pieter; Schelstraete, Petra; Lopes dos Santos Santiago, Guido; Van Simaey, Leen; Haerynck, Filomeen; Van daele, Sabine; De Wachter, Elke; Malfroot, Anne; Lebecque, Patrick; Knoop, Christiane; Casimir, Georges; Boboli, Hedwige; Pierart, Fr?d?ric; Desager, Kristine; Vaneechoutte, Mario

    2010-01-01

    Abstract Background Pseudomonas aeruginosa is the major respiratory pathogen causing severe lung infections among CF patients, leading to high morbidity and mortality. Once infection is established, early antibiotic treatment is able to postpone the transition to chronic lung infection. In order to optimize the early detection, we compared the sensitivity of microbiological culture and quantitative PCR (qPCR) for the detection of P. aeruginosa in respiratory samples of not chronically infecte...

  13. Detection of Herpesviridae in whole blood by multiplex PCR DNA-based microarray analysis after hematopoietic stem cell transplantation.

    Science.gov (United States)

    Debaugnies, France; Busson, Laurent; Ferster, Alina; Lewalle, Philippe; Azzi, Nadira; Aoun, Mickael; Verhaegen, Godelieve; Mahadeb, Bhavna; de Marchin, Jérôme; Vandenberg, Olivier; Hallin, Marie

    2014-07-01

    Viral infections are important causes of morbidity and mortality in patients after hematopoietic stem cell transplantation. The monitoring by PCR of Herpesviridae loads in blood samples has become a critical part of posttransplant follow-up, representing mounting costs for the laboratory. In this study, we assessed the clinical performance of the multiplex PCR DNA microarray Clart Entherpex kit for detection of cytomegalovirus (CMV), Epstein-Barr virus (EBV), and human herpesvirus 6 (HHV-6) as a screening test for virological follow-up. Two hundred fifty-five blood samples from 16 transplanted patients, prospectively tested by routine PCR assays, were analyzed by microarray. Routine PCR detected single or multiple viruses in 42% and 10% of the samples, respectively. Microarray detected single or multiple viruses in 34% and 18% of the samples, respectively. Microarray results correlated well with CMV and EBV detections by routine PCR (kappa tests = 0.79 and 0.78, respectively), whereas a weak correlation was observed with HHV-6 (0.43). HHV-7 was also detected in 48 samples by microarray. In conclusion, the microarray is a reliable screening assay for a posttransplant virological follow-up to detect CMV and EBV infections in blood. However, positive samples must be subsequently confirmed and viral loads must be quantified by PCR assays. Limitations were identified regarding HHV-6 detection. Although it is promising, is easy to use as a first-line test, and allows a reduction in the cost of analysis without undue delay in the reporting of the final quantitative result to the clinician, some characteristics of this microarray should be improved, particularly regarding quality control and the targeted virus panel, such that it could then be used as a routine test. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  14. [Fungal composition in massa medicata fermentata based on culture dependent method and independent PCR-SSCP technique].

    Science.gov (United States)

    Chen, Juan; Jiao, Xiao-lin; Yang, Chun-yong; Song, Mei-fang; Gao, Wei-wei

    2014-11-01

    To analyze the fungal composition in Massa Medicata Fermentata based on culture dependent method and independent PCR-SSCP technique. Fungi were directly isolated from Massa Medicata Fermentata samples. The obtained strains were identified according to morphology and DNA sequence. Meanwhile the total fungal DNA was extracted from Massa Medicata Fermentata samples, the cultural independent PCR-SSCP technique based on β-tubulin gene were used to identify the mycobiota. According to cultural method, Aspergillus flavus and Rhizopus oryzae were present in Massa Medicata Fermentata samples, while A. flavus and A. niger were present in fried Massa Medicata Fermentata samples. In contrast, 5 species were obtained by PCR-SSCP technique, A. flavus was overlapped with fungal taxa derived from culture dependent method; A. ambiguu and A. s ivoriensis were dominant with relative abundance of 57% and 35% respectively, while the relative abundance of A. flavus was as low as 4%. None species was obtained from fried Massa Medicata Fermentata samples. PCR-SSCP based on β-tubulin gene could distinguish fungi into species, culture dependent method combined with culture independent method could better understand the fungal composition associated with Massa Medicata Fermentata fermentation.

  15. Comparison of culture and acid-fast bacilli stain to PCR for detection of Mycobacterium tuberculosis in clinical samples.

    Science.gov (United States)

    Aslanzadeh, J; de la Viuda, M; Fille, M; Smith, W B; Namdari, H

    1998-08-01

    The major drawback in effective use of polymerase chain reaction (PCR) for detecting Mycobacterium tuberculosis (MTB) in clinical samples is the presence of PCR inhibitors and unique cell components of the organism that complicate DNA extraction and subsequent PCR amplification. A PCR assay with a unique multistep DNA extraction method that minimizes these problems was compared in a prospective study to acid-fast bacilli stain (AFBS) and culture for detecting MTB in clinical samples. A total of 254 clinical specimens in two separate studies were processed for MTB by these techniques. While PCR and culture were 100% sensitive and specific, culture required up to 8 weeks of incubation and additional time to perform biochemical testing to identify the isolated micro-organism. Acid-fast bacilli stain had a specificity of about 87% and did not differentiate among Mycobacterial species. In contrast, the results from PCR were available within 48 h and did not require additional testing to attain a final result. Polymerase chain reaction was highly reliable for detection and confirmation and interpretation of positive AFBS results. The assay was easy to perform with a turn around time of about 2 days.

  16. Comparative analysis of cultural isolation and PCR based assay for detection of Campylobacter jejuni in food and faecal samples

    Directory of Open Access Journals (Sweden)

    Harkanwaldeep Singh

    2011-03-01

    Full Text Available In the present study, the efficacy of polymerase chain reaction (PCR based on mapA gene of C. jejuni was tested for detection of Campylobacter jejuni in naturally infected as well as spiked faecal and food samples of human and animal origin. Simultaneously, all the samples were subjected to the cultural isolation of organism and biochemical characterization. The positive samples resulted in the amplification of a DNA fragment of size ~589 bp in PCR assay whereas the absence of such amplicon in DNA extracted from E. coli, Listeria, Salmonella and Staphylococcus confirmed the specificity of the primers. Of randomly collected 143 faecal samples comprising human diarrheic stools (43, cattle diarrheic faeces (48 and poultry faecal swabs (52 only 4, 3 and 8, respectively, could be detected by isolation whereas 6, 3 and 10, respectively, were found positive by PCR. However, among food samples viz. beef (30, milk (35, cheese (30, only one beef sample was detected both by culture as well as PCR. Additionally, PCR was found to be more sensitive for C. jejuni detection in spiked faecal and food samples (96.1% each as relative to culture isolation which could detect the organism in 86.7% and 80% samples, respectively. The results depicted the superior efficacy of PCR for rapid screening of samples owing to its high sensitivity, specificity and automation potential.

  17. Comparative analysis of cultural isolation and PCR based assay for detection of campylobacter jejuni in food and faecal samples.

    Science.gov (United States)

    Singh, Harkanwaldeep; Rathore, R S; Singh, Satparkash; Cheema, Pawanjit Singh

    2011-01-01

    In the present study, the efficacy of polymerase chain reaction (PCR) based on mapA gene of C. jejuni was tested for detection of Campylobacter jejuni in naturally infected as well as spiked faecal and food samples of human and animal origin. Simultaneously, all the samples were subjected to the cultural isolation of organism and biochemical characterization. The positive samples resulted in the amplification of a DNA fragment of size ~589 bp in PCR assay whereas the absence of such amplicon in DNA extracted from E. coli, Listeria, Salmonella and Staphylococcus confirmed the specificity of the primers. Of randomly collected 143 faecal samples comprising human diarrheic stools (43), cattle diarrheic faeces (48) and poultry faecal swabs (52) only 4, 3 and 8, respectively, could be detected by isolation whereas 6, 3 and 10, respectively, were found positive by PCR. However, among food samples viz. beef (30), milk (35), cheese (30), only one beef sample was detected both by culture as well as PCR. Additionally, PCR was found to be more sensitive for C. jejuni detection in spiked faecal and food samples (96.1% each) as relative to culture isolation which could detect the organism in 86.7% and 80% samples, respectively. The results depicted the superior efficacy of PCR for rapid screening of samples owing to its high sensitivity, specificity and automation potential.

  18. First Report of Clostridium lavalense Isolated in Human Blood Cultures

    Directory of Open Access Journals (Sweden)

    Richard Garceau

    2016-01-01

    Full Text Available An 88-year-old man was admitted to the hospital with worsening malaise, fever, and weakness. Anaerobic blood culture bottles revealed the presence of an anaerobic, Gram-positive sporulated bacillus. Empirical antibiotherapy with intravenous piperacillin-tazobactam was initiated. The patient defervesced after four days and was switched to oral amoxicillin on his 6th day of antibiotic therapy and later discharged from the hospital. Four months later, he had recovered. The bacterium was initially identified as Clostridium butyricum using anaerobic manual identification panel. 16S rRNA gene sequence and phylogenetic analysis showed the bacterium to be Clostridium lavalense, a recently described species with no previously published case of isolation in human diagnostic samples so far. This is the first report of Clostridium lavalense isolation from human blood cultures. Further studies are needed in order to elucidate the role of Clostridium lavalense in human disease and its virulence factors.

  19. Should varicella-zoster virus culture be eliminated? A comparison of direct immunofluorescence antigen detection, culture, and PCR, with a historical review.

    Science.gov (United States)

    Wilson, D A; Yen-Lieberman, B; Schindler, S; Asamoto, K; Schold, J D; Procop, G W

    2012-12-01

    A comparison of direct fluorescent-antibody assay (DFA), culture, and two PCR assays disclosed sensitivities of 87.8%, 46.3%, and 97.6% and 100%, respectively. We reviewed 1,150 results for clinical specimens submitted for DFA and culture and found that only 17 were culture positive/DFA negative. The incremental cost to detect these 17 positives was $3,078/specimen.

  20. Accuracy of real-time PCR, Gram stain and culture for Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae meningitis diagnosis.

    Science.gov (United States)

    Wu, Henry M; Cordeiro, Soraia M; Harcourt, Brian H; Carvalho, Mariadaglorias; Azevedo, Jailton; Oliveira, Tainara Q; Leite, Mariela C; Salgado, Katia; Reis, Mitermayer G; Plikaytis, Brian D; Clark, Thomas A; Mayer, Leonard W; Ko, Albert I; Martin, Stacey W; Reis, Joice N

    2013-01-22

    Although cerebrospinal fluid (CSF) culture is the diagnostic reference standard for bacterial meningitis, its sensitivity is limited, particularly when antibiotics were previously administered. CSF Gram staining and real-time PCR are theoretically less affected by antibiotics; however, it is difficult to evaluate these tests with an imperfect reference standard. CSF from patients with suspected meningitis from Salvador, Brazil were tested with culture, Gram stain, and real-time PCR using S. pneumoniae, N. meningitidis, and H. influenzae specific primers and probes. An antibiotic detection disk bioassay was used to test for the presence of antibiotic activity in CSF. The diagnostic accuracy of tests were evaluated using multiple methods, including direct evaluation of Gram stain and real-time PCR against CSF culture, evaluation of real-time PCR against a composite reference standard, and latent class analysis modeling to evaluate all three tests simultaneously. Among 451 CSF specimens, 80 (17.7%) had culture isolation of one of the three pathogens (40 S. pneumoniae, 36 N. meningitidis, and 4 H. influenzae), and 113 (25.1%) were real-time PCR positive (51 S. pneumoniae, 57 N. meningitidis, and 5 H. influenzae). Compared to culture, real-time PCR sensitivity and specificity were 95.0% and 90.0%, respectively. In a latent class analysis model, the sensitivity and specificity estimates were: culture, 81.3% and 99.7%; Gram stain, 98.2% and 98.7%; and real-time PCR, 95.7% and 94.3%, respectively. Gram stain and real-time PCR sensitivity did not change significantly when there was antibiotic activity in the CSF. Real-time PCR and Gram stain were highly accurate in diagnosing meningitis caused by S. pneumoniae, N. meningitidis, and H. influenzae, though there were few cases of H. influenzae. Furthermore, real-time PCR and Gram staining were less affected by antibiotic presence and might be useful when antibiotics were previously administered. Gram staining, which is

  1. Evaluating a commercial PCR assay against bacterial culture for diagnosing Streptococcus uberis and Staphylococcus aureus throughout lactation.

    Science.gov (United States)

    Steele, N M; Williamson, J H; Thresher, R; Laven, R A; Hillerton, J E

    2017-05-01

    The performance of a commercial, real-time PCR assay was compared with traditional bacterial culture for the identification of Streptococcus uberis and Staphylococcus aureus in bovine milk collected at different stages of lactation. Initial validation tests using fresh and frozen quarter milk samples identified factors that affected the success of the PCR. Therefore, the standard protocol was adjusted for samples collected at the first milking postpartum (colostrum) and from clinical mastitis cases. The adjustment involved PCR testing both undiluted and diluted (1 in 10 with sterile water) DNA extracts. The performance comparison between culture and the PCR assay used milk samples collected aseptically from individual quarters of mixed-age spring-calving dairy cows, during early, mid, and late lactation. Bacterial culture results were used to select a subset of samples for PCR testing (n = 315) that represented quarters with a current or prior Strep. uberis or Staph. aureus infection. Compared with culture, PCR had a sensitivity of 86.8% and specificity of 87.7% for detecting Strep. uberis (kappa = 0.74) and 96.4% and 99.7%, respectively, for detecting Staph. aureus (kappa = 0.96). The dilution of DNA extracts for colostrum and clinical samples increased the relative sensitivity from 79.2% to 86.8% for Strep. uberis detection and from 92.9% to 96.4% for Staph. aureus, presumably through diluting unidentified PCR inhibitors. The sensitivity for detecting Strep. uberis using PCR, relative to culture, was similar throughout lactation (85-89%), whereas relative specificity was lowest immediately postcalving (64%) but improved in mid and late lactation (98%). Specificity estimates for samples collected in early lactation can be optimized by reducing the cutoff cycle threshold (Ct) value from the recommended value of 37 to 34. Although using this value improved specificity (77%), it reduced test sensitivity (77%). The PCR assay lacked agreement with culture in early

  2. Comparative study of Gram stain, potassium hydroxide smear, culture and nested PCR in the diagnosis of fungal keratitis.

    Science.gov (United States)

    Badiee, Parisa; Nejabat, Mahmood; Alborzi, Abdolvahab; Keshavarz, Fatemeh; Shakiba, Elaheh

    2010-01-01

    This study seeks to evaluate the efficacy and practicality of the molecular method, compared to the standard microbiological techniques for diagnosing fungal keratitis (FK). Patients with eye findings suspected of FK were enrolled for cornea sampling. Scrapings from the affected areas of the infected corneas were obtained and were divided into two parts: one for smears and cultures, and the other for nested PCR analysis. Of the 38 eyes, 28 were judged to have fungal infections based on clinical and positive findings in the culture, smear and responses to antifungal treatment. Potassium hydroxide, Gram staining, culture and nested PCR results (either positive or negative) matched in 76.3, 42.1, 68.4 and 81.6%, respectively. PCR is a sensitive method but due to the lack of sophisticated facilities in routine laboratory procedures, it can serve only complementarily and cannot replace conventional methods. Copyright © 2010 S. Karger AG, Basel.

  3. [Molecular biological sepsis diagnostic using multiplex PCR in surgical intensive care as suitable alternative to conventional microbial culture - a representative overview].

    Science.gov (United States)

    Lodes, U; Lippert, H; Meyer, F

    2011-04-01

    Sepsis causes a substantial rate of morbidity and mortality in intensive care patients, which is, in particular, triggered by an inadequate antimicrobial treatment from the beginning. Conventional microbiological standard procedures cannot provide valuable information on bacterial or fungal species of sepsis-relevant microbes within the first hours of a developing sepsis. However, multiplex PCR (PCR-M) focussing on the spectrum of the most relevant sepsis-associated microbes can considerably shorten the time period; the analytical tests have been standardised and, subsequently, inaugurated into clinical practice; they also have thus been available since 2005. Interestingly, in the surgical field an appropriate summary and concluding recommendation have been lacking so far. AIM, MATERIAL AND METHODS: A compact short overview based on a characteristic selection of relevant references from the literature is given on the commercially available sepsis-associated multiplex-PCR methods, reflecting critically the time point of inauguration, clinical value and future perspectives including our own experiences from clinical practice and medical studies. Multiplex PCR in adult sepsis patients yielded in a range from 13.7 to 39.9 % of positive findings, whereas conventional blood cultures only range from 8 to 29.9 %. From 8 to 16.9 % of all investigations performed prompted us to a change of the antimicrobial treatment by using a positive PCR-M finding. A prospective study (end-point, reduction of sepsis-associated mortality) has not yet been initiated. Positive PCR-M findings correlate with an increased morbidity and mortality as well as clinical and laboratory sepsis parameters. Recent studies have aimed for a comparison of PCR-M on sepsis-associated microbes with regard to specificity and sensitivity with the current "gold standard", conventional blood culture. A few studies wrongly claimed to compare the methods because of the difference in the procedures; in addition

  4. International study to evaluate PCR methods for detection of Trypanosoma cruzi DNA in blood samples from Chagas disease patients.

    Directory of Open Access Journals (Sweden)

    Alejandro G Schijman

    Full Text Available BACKGROUND: A century after its discovery, Chagas disease still represents a major neglected tropical threat. Accurate diagnostics tools as well as surrogate markers of parasitological response to treatment are research priorities in the field. The purpose of this study was to evaluate the performance of PCR methods in detection of Trypanosoma cruzi DNA by an external quality evaluation. METHODOLOGY/FINDINGS: An international collaborative study was launched by expert PCR laboratories from 16 countries. Currently used strategies were challenged against serial dilutions of purified DNA from stocks representing T. cruzi discrete typing units (DTU I, IV and VI (set A, human blood spiked with parasite cells (set B and Guanidine Hidrochloride-EDTA blood samples from 32 seropositive and 10 seronegative patients from Southern Cone countries (set C. Forty eight PCR tests were reported for set A and 44 for sets B and C; 28 targeted minicircle DNA (kDNA, 13 satellite DNA (Sat-DNA and the remainder low copy number sequences. In set A, commercial master mixes and Sat-DNA Real Time PCR showed better specificity, but kDNA-PCR was more sensitive to detect DTU I DNA. In set B, commercial DNA extraction kits presented better specificity than solvent extraction protocols. Sat-DNA PCR tests had higher specificity, with sensitivities of 0.05-0.5 parasites/mL whereas specific kDNA tests detected 5.10(-3 par/mL. Sixteen specific and coherent methods had a Good Performance in both sets A and B (10 fg/µl of DNA from all stocks, 5 par/mL spiked blood. The median values of sensitivities, specificities and accuracies obtained in testing the Set C samples with the 16 tests determined to be good performing by analyzing Sets A and B samples varied considerably. Out of them, four methods depicted the best performing parameters in all three sets of samples, detecting at least 10 fg/µl for each DNA stock, 0.5 par/mL and a sensitivity between 83.3-94.4%, specificity of 85

  5. International Study to Evaluate PCR Methods for Detection of Trypanosoma cruzi DNA in Blood Samples from Chagas Disease Patients

    Science.gov (United States)

    Schijman, Alejandro G.; Bisio, Margarita; Orellana, Liliana; Sued, Mariela; Duffy, Tomás; Mejia Jaramillo, Ana M.; Cura, Carolina; Auter, Frederic; Veron, Vincent; Qvarnstrom, Yvonne; Deborggraeve, Stijn; Hijar, Gisely; Zulantay, Inés; Lucero, Raúl Horacio; Velazquez, Elsa; Tellez, Tatiana; Sanchez Leon, Zunilda; Galvão, Lucia; Nolder, Debbie; Monje Rumi, María; Levi, José E.; Ramirez, Juan D.; Zorrilla, Pilar; Flores, María; Jercic, Maria I.; Crisante, Gladys; Añez, Néstor; De Castro, Ana M.; Gonzalez, Clara I.; Acosta Viana, Karla; Yachelini, Pedro; Torrico, Faustino; Robello, Carlos; Diosque, Patricio; Triana Chavez, Omar; Aznar, Christine; Russomando, Graciela; Büscher, Philippe; Assal, Azzedine; Guhl, Felipe; Sosa Estani, Sergio; DaSilva, Alexandre; Britto, Constança; Luquetti, Alejandro; Ladzins, Janis

    2011-01-01

    Background A century after its discovery, Chagas disease still represents a major neglected tropical threat. Accurate diagnostics tools as well as surrogate markers of parasitological response to treatment are research priorities in the field. The purpose of this study was to evaluate the performance of PCR methods in detection of Trypanosoma cruzi DNA by an external quality evaluation. Methodology/Findings An international collaborative study was launched by expert PCR laboratories from 16 countries. Currently used strategies were challenged against serial dilutions of purified DNA from stocks representing T. cruzi discrete typing units (DTU) I, IV and VI (set A), human blood spiked with parasite cells (set B) and Guanidine Hidrochloride-EDTA blood samples from 32 seropositive and 10 seronegative patients from Southern Cone countries (set C). Forty eight PCR tests were reported for set A and 44 for sets B and C; 28 targeted minicircle DNA (kDNA), 13 satellite DNA (Sat-DNA) and the remainder low copy number sequences. In set A, commercial master mixes and Sat-DNA Real Time PCR showed better specificity, but kDNA-PCR was more sensitive to detect DTU I DNA. In set B, commercial DNA extraction kits presented better specificity than solvent extraction protocols. Sat-DNA PCR tests had higher specificity, with sensitivities of 0.05–0.5 parasites/mL whereas specific kDNA tests detected 5.10−3 par/mL. Sixteen specific and coherent methods had a Good Performance in both sets A and B (10 fg/µl of DNA from all stocks, 5 par/mL spiked blood). The median values of sensitivities, specificities and accuracies obtained in testing the Set C samples with the 16 tests determined to be good performing by analyzing Sets A and B samples varied considerably. Out of them, four methods depicted the best performing parameters in all three sets of samples, detecting at least 10 fg/µl for each DNA stock, 0.5 par/mL and a sensitivity between 83.3–94.4%, specificity of 85–95

  6. "Campylobacter upsaliensis" isolated from blood cultures of pediatric patients.

    OpenAIRE

    Lastovica, A J; Le Roux, E; Penner, J L

    1989-01-01

    Seventeen campylobacters isolated from cultures of blood samples of 16 bacteremic patients were susceptible to cephalothin and were either catalase negative or had only weak catalase activity (CNW strains) and were classified as "Campylobacter upsaliensis" (K. Sandstedt and J. Ursing, XIV Int. Congr. Microbiol., p. B8-17, 1986). All of the patients had predisposing conditions, and 10 patients were less than or equal to 12 months old, indicating that "C. upsaliensis" is an opportunistic pathog...

  7. A charcoal- and blood-free enrichment broth for isolation and PCR detection of Campylobacter jejuni and Campylobacter coli in chicken.

    Science.gov (United States)

    Solís-Soto, Luisa Y; García, Santos; Wesley, Irene; Heredia, Norma

    2011-02-01

    The microaerophilic nature of Campylobacter and its requirement of ∼5% O(2) for growth have complicated its recovery from foods. The addition to the enrichment media of oxygen quenchers such as charcoal or blood could interfere with PCR for its detection. In this study, a two-step simple aerobic method for Campylobacter detection is proposed. A modification of the Tran blood-free enrichment broth (BFEB), in which charcoal was excluded from the medium (M-BFEB), was compared with the original formulation and other enrichment broths. Campylobacter jejuni and Campylobacter coli were screened by PCR directly from the enrichment media. Various levels of pure cultures of C. jejuni and C. coli combined with Escherichia coli were inoculated into Preston, Bolton, BFEB, and the modified BFEB (M-BFEB). In addition, Campylobacter was inoculated onto retail purchased chicken skin and recovery was quantified. Rates of recovery after 24 to 48 h of enrichment at 42 °C under aerobic incubation for BFEB and M-BFEB and microaerobic incubations for Preston and Bolton broths were determined. Overall, our results indicated that the most sensitive medium was Bolton's, followed by either BFEB or M-BFEB; the least sensitive was Preston's. M-BFEB was directly coupled to a PCR assay to detect Campylobacter, avoiding intermediate plating. Campylobacter was detected in the presence of up to 10(8) E. coli cells per ml. M-BFEB facilitated detection of both C. jejuni and C. coli artificially inoculated onto chicken skin samples. M-BFEB coupled to PCR is a rapid and attractive alternative for isolation and identification of C. coli and C. jejuni from poultry. Copyright ©, International Association for Food Protection

  8. Microscopy, culture, and quantitative real-time PCR examination confirm internalization of mycobacteria in plants.

    Science.gov (United States)

    Kaevska, M; Lvoncik, S; Slana, I; Kulich, P; Kralik, P

    2014-07-01

    The environment is a reservoir of nontuberculous mycobacteria and is considered a source of infection for animals and humans. Mycobacteria can persist in different types of environments for a relatively long time. We have studied their possible internalization into plant tissue through intact, as well as damaged, root systems of different types of plants grown in vitro and under field conditions. The substrate into which plants were seeded was previously contaminated with different strains of Mycobacterium avium (10(8) to 10(10) cells/g of soil) and feces from animals with paratuberculosis. We detected M. avium subsp. avium, hominissuis, and paratuberculosis in the stems and leaves of the plants by both culture and real-time quantitative PCR. The presence of mycobacteria in the plant tissues was confirmed by microscopy. The concentration of mycobacteria found inside plant tissue was several orders of magnitude lower (up to 10(4) cells/g of tissue) than the initial concentration of mycobacteria present in the culture medium or substrate. These findings led us to the hypothesis that plants may play a role in the spread and transmission of mycobacteria to other organisms in the environment. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  9. Detection of Angiostrongylus cantonensis in the Blood and Peripheral Tissues of Wild Hawaiian Rats (Rattus rattus by a Quantitative PCR (qPCR Assay.

    Directory of Open Access Journals (Sweden)

    Susan I Jarvi

    Full Text Available The nematode Angiostrongylus cantonensis is a rat lungworm, a zoonotic pathogen that causes human eosinophilic meningitis and ocular angiostrongyliasis characteristic of rat lungworm (RLW disease. Definitive diagnosis is made by finding and identifying A. cantonensis larvae in the cerebral spinal fluid or by using a custom immunological or molecular test. This study was conducted to determine if genomic DNA from A. cantonensis is detectable by qPCR in the blood or tissues of experimentally infected rats. F1 offspring from wild rats were subjected to experimental infection with RLW larvae isolated from slugs, then blood or tissue samples were collected over multiple time points. Blood samples were collected from 21 rats throughout the course of two trials (15 rats in Trial I, and 6 rats in Trial II. In addition to a control group, each trial had two treatment groups: the rats in the low dose (LD group were infected by approximately 10 larvae and the rats in the high dose (HD group were infected with approximately 50 larvae. In Trial I, parasite DNA was detected in cardiac bleed samples from five of five LD rats and five of five HD rats at six weeks post-infection (PI, and three of five LD rats and five of five HD rats from tail tissue. In Trial II, parasite DNA was detected in peripheral blood samples from one of two HD rats at 53 minutes PI, one of two LD rats at 1.5 hours PI, one of two HD rats at 18 hours PI, one of two LD rats at five weeks PI and two of two at six weeks PI, and two of two HD rats at weeks five and six PI. These data demonstrate that parasite DNA can be detected in peripheral blood at various time points throughout RLW infection in rats.

  10. Misinterpretation of Gram Stain from the Stationary Growth Phase of Positive Blood Cultures forBrucellaandAcinetobacterSpecies.

    Science.gov (United States)

    Bazzi, Ali M; Al-Tawfiq, Jaffar A; Rabaan, Ali A

    2017-01-01

    Acinetobacter baumannii and Brucella species are Gram-negative organisms that are vulnerable to misinterpretation as Gram-positive or Gram-variable in blood cultures. We assess the random errors in gram stain interpretation to reduce the likelihood of such errors and therefore patient harm. Aerobic and anaerobic blood cultures from two patients in an acute care facility in Saudi Arabia were subjected to preliminary Gram-staining. In case 1, VITEK-2 Anaerobe Identification, repeat Gram staining from a blood agar plate, Remel BactiDrop™ Oxidase test, Urea Agar urease test and real-time PCR were used to confirm presence of Brucella and absence of Coryneform species. In case 2, repeat Gram- staining from the plate and the vials, VITEK-2 Gram-Negative Identification, real-time PCR and subculture on to Columbia agar, blood agar, and MacConkey agar were carried out to identify A. baumannii . In case 1, initially pleomorphic Gram-positive bacteria were identified. Coryneform species were suspected. Tiny growth was observed after 24 h on blood agar plates, and good growth by 48 h. Presence of Brucella species was ultimately confirmed. In case 2, preliminary Gram-stain results suggested giant Gram-positive oval cocci. Further testing over 18-24 h identified A. baumannii . Oxidase test from the plate and urease test from the culture vial is recommended after apparent identification of pleomorphic Gram-positive bacilli from blood culture, once tiny growth is observed, to distinguish Brucella from Corynebacterium species. If giant Gram-positive oval cocci are indicated by preliminary Gram-staining, it is recommended that the Gram stain be repeated from the plate after 4-6 h, or culture should be tested in Triple Sugar Iron (TSI) medium and the Gram stain repeated after 2-4 h incubation.

  11. Detection of Campylobacter in Stool and Determination of Significance by Culture, Enzyme Immunoassay, and PCR in Developing Countries

    Science.gov (United States)

    Platts-Mills, James A.; Liu, Jie; Gratz, Jean; Mduma, Esto; Amour, Caroline; Swai, Ndealilia; Taniuchi, Mami; Begum, Sharmin; Peñataro Yori, Pablo; Tilley, Drake H.; Lee, Gwenyth; Shen, Zeli; Whary, Mark T.; Fox, James G.; McGrath, Monica; Kosek, Margaret; Haque, Rashidul

    2014-01-01

    Campylobacter is a common bacterial enteropathogen that can be detected in stool by culture, enzyme immunoassay (EIA), or PCR. We compared culture for C. jejuni/C. coli, EIA (ProSpecT), and duplex PCR to distinguish Campylobacter jejuni/C. coli and non-jejuni/coli Campylobacter on 432 diarrheal and matched control stool samples from infants in a multisite longitudinal study of enteric infections in Tanzania, Bangladesh, and Peru. The sensitivity and specificity of culture were 8.5% and 97.6%, respectively, compared with the results of EIA and 8.7% and 98.0%, respectively, compared with the results of PCR for C. jejuni/C. coli. Most (71.6%) EIA-positive samples were positive by PCR for C. jejuni/C. coli, but 27.6% were positive for non-jejuni/coli Campylobacter species. Sequencing of 16S rRNA from 53 of these non-jejuni/coli Campylobacter samples showed that it most closely matched the 16S rRNA of C. hyointestinalis subsp. lawsonii (56%), C. troglodytis (33%), C. upsaliensis (7.7%), and C. jejuni/C. coli (2.6%). Campylobacter-negative stool spiked with each of the above-mentioned Campylobacter species revealed reactivity with EIA. PCR detection of Campylobacter species was strongly associated with diarrhea in Peru (odds ratio [OR] = 3.66, P < 0.001) but not in Tanzania (OR = 1.56, P = 0.24) or Bangladesh (OR = 1.13, P = 0.75). According to PCR, Campylobacter jejuni/C. coli infections represented less than half of all infections with Campylobacter species. In sum, in infants in developing country settings, the ProSpecT EIA and PCR for Campylobacter reveal extremely high rates of positivity. We propose the use of PCR because it retains high sensitivity, can ascertain burden, and can distinguish between Campylobacter infections at the species level. PMID:24452175

  12. Detection of Campylobacter in stool and determination of significance by culture, enzyme immunoassay, and PCR in developing countries.

    Science.gov (United States)

    Platts-Mills, James A; Liu, Jie; Gratz, Jean; Mduma, Esto; Amour, Caroline; Swai, Ndealilia; Taniuchi, Mami; Begum, Sharmin; Peñataro Yori, Pablo; Tilley, Drake H; Lee, Gwenyth; Shen, Zeli; Whary, Mark T; Fox, James G; McGrath, Monica; Kosek, Margaret; Haque, Rashidul; Houpt, Eric R

    2014-04-01

    Campylobacter is a common bacterial enteropathogen that can be detected in stool by culture, enzyme immunoassay (EIA), or PCR. We compared culture for C. jejuni/C. coli, EIA (ProSpecT), and duplex PCR to distinguish Campylobacter jejuni/C. coli and non-jejuni/coli Campylobacter on 432 diarrheal and matched control stool samples from infants in a multisite longitudinal study of enteric infections in Tanzania, Bangladesh, and Peru. The sensitivity and specificity of culture were 8.5% and 97.6%, respectively, compared with the results of EIA and 8.7% and 98.0%, respectively, compared with the results of PCR for C. jejuni/C. coli. Most (71.6%) EIA-positive samples were positive by PCR for C. jejuni/C. coli, but 27.6% were positive for non-jejuni/coli Campylobacter species. Sequencing of 16S rRNA from 53 of these non-jejuni/coli Campylobacter samples showed that it most closely matched the 16S rRNA of C. hyointestinalis subsp. lawsonii (56%), C. troglodytis (33%), C. upsaliensis (7.7%), and C. jejuni/C. coli (2.6%). Campylobacter-negative stool spiked with each of the above-mentioned Campylobacter species revealed reactivity with EIA. PCR detection of Campylobacter species was strongly associated with diarrhea in Peru (odds ratio [OR] = 3.66, P PCR, Campylobacter jejuni/C. coli infections represented less than half of all infections with Campylobacter species. In sum, in infants in developing country settings, the ProSpecT EIA and PCR for Campylobacter reveal extremely high rates of positivity. We propose the use of PCR because it retains high sensitivity, can ascertain burden, and can distinguish between Campylobacter infections at the species level.

  13. Comparison of a PCR-Based Method with Culture and Direct Examination for Diagnosis of Acanthamoeba keratitis

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    S Farnia

    2009-05-01

    Full Text Available "nBackground: The aim was to compare three different methods (direct examination, culture and PCR meth­ods for the diagnosis of Acanthamoeba keratitis (AK in corneal scrapes."nMethods: Twenty eight corneal scrapes and contact lenses were collected from keratitis patients and re­ferred to the De­partment of Medical Parasitology and Mycology, School of Public Health, Tehran Univer­sity of Medical Sci­ences. Corneal scrapes were divided in three parts for direct examination, culture on non-nutrient agar and PCR analysis. PCR analysis was also performed using a 18S rRNA gene primer pair (DF3 region. DF3 (Diagnostic frag­ment 3 is a region of the nuclear small subunit ribosomal RNA gene which is specific for detecting Acan­thamoeba strains."nResults:  Acanthamoeba was the causative agent of keratitis in 50% of the patients. Direct smear of all pre­pared corneal scrapes in AK patients was negative and culture was positive in only 14.3% of the isolates. PCR analysis was positive in 71.4% of AK patients. These three methods were negative in corneal scrapes of non-AK patients. The sensitivity and specificity of PCR technique for the detection of Acanthamoeba sp. were calculated as 71.4% and 100%, respectively."nConclusion: According to high sensitivity and specificity of PCR-based method, this study confirmed that PCR using 18S rRNA gene primers (DF3 region is more useful for detecting AK cases compare to culture and direct microscopy methods.

  14. Development of a nested PCR assay to detect equine infectious anemia proviral DNA from peripheral blood of naturally infected horses.

    Science.gov (United States)

    Dong, Jian-Bao; Zhu, Wei; Cook, Frank R; Goto, Yoshitaka; Horii, Yoichiro; Haga, Takeshi

    2012-11-01

    Equine infectious anemia (EIA) has posed a major challenge and caused significant losses to the equine industry worldwide. PCR detection methods have considerable potential as an adjunct to conventional serological diagnostic techniques. However, most published PCR methods, including that recommended by the OIE, were designed using laboratory-adapted virus strains and do not function with field isolates of EIA virus (EIAV). In the present study, a nested PCR assay for detection of EIAV proviral DNA in peripheral blood cells of naturally infected horses was developed. Primer sets were designed based on conserved 5' regions of the viral genome extending from the LTR to the tat gene. Preliminary studies demonstrated that the method has a detection limit of 10 genomic copies and, when applied to a naturally EIAV-infected feral horse population, shows 100 % correlation with conventional serological diagnostic techniques. This assay provides a powerful new tool in the control of EIAV.

  15. Role of microbiological culture and polymerase chain reaction (PCR) of actinomyces in medication-related osteonecrosis of the jaw (MRONJ).

    Science.gov (United States)

    Panya, Sappasith; Fliefel, Riham; Probst, Florian; Tröltzsch, Matthias; Ehrenfeld, Michael; Schubert, Sören; Otto, Sven

    2017-03-01

    We hypothesized that local infection plays a critical role in the pathogenesis of medication-related osteonecrosis of the jaw (MRONJ). Recent developments in molecular methods have revolutionized new approaches for the rapid detection of microorganisms including those difficult to culture. The aim of our study is to identify the bacterial profiles in MRONJ by microbiological culture and polymerase chain reactions (PCR). A retrospective analysis was performed on MRONJ patients from 2008 to 2014. The bacterial profile from MRONJ bone samples was determined using microbiological culture and PCR. Ninety five patients fulfilled the inclusion criteria with mean age of 69.85 ± 8.71 years. A female predilection was detected. The mandible was more commonly affected than maxilla. Tooth extraction was the frequent triggering factor. Breast cancer was the primary cause for administration and intravenous bisphosphonates were the most commonly administrated antiresorptive drugs. The majority of patients were classified as stage 2. Posterior teeth were most commonly affected. Based on bone culture results, the most common microorganism were both actinomyces and mixed flora. PCR confirmed the presence of actinomyces in 55 patients. Our data suggest that PCR might be an innovative method for detection of microorganisms difficult to culture using traditional microbiological techniques. Copyright © 2017 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.

  16. Improved Diagnosis of Prosthetic Joint Infection by Culturing Periprosthetic Tissue Specimens in Blood Culture Bottles

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    Trisha N. Peel

    2016-01-01

    Full Text Available Despite known low sensitivity, culture of periprosthetic tissue specimens on agars and in broths is routine. Culture of periprosthetic tissue samples in blood culture bottles (BCBs is potentially more convenient, but it has been evaluated in a limited way and has not been widely adopted. The aim of this study was to compare the sensitivity and specificity of inoculation of periprosthetic tissue specimens into blood culture bottles with standard agar and thioglycolate broth culture, applying Bayesian latent class modeling (LCM in addition to applying the Infectious Diseases Society of America (IDSA criteria for prosthetic joint infection. This prospective cohort study was conducted over a 9-month period (August 2013 to April 2014 at the Mayo Clinic, Rochester, MN, and included all consecutive patients undergoing revision arthroplasty. Overall, 369 subjects were studied; 117 (32% met IDSA criteria for prosthetic joint infection, and 82% had late chronic infection. Applying LCM, inoculation of tissues into BCBs was associated with a 47% improvement in sensitivity compared to the sensitivity of conventional agar and broth cultures (92.1 versus 62.6%, respectively; this magnitude of change was similar when IDSA criteria were applied (60.7 versus 44.4%, respectively; P = 0.003. The time to microorganism detection was shorter with BCBs than with standard media (P < 0.0001, with aerobic and anaerobic BCBs yielding positive results within a median of 21 and 23 h, respectively. Results of our study demonstrate that the semiautomated method of periprosthetic tissue culture in blood culture bottles is more sensitive than and as specific as agar and thioglycolate broth cultures and yields results faster.

  17. Detection of Mycoplasma hyopneumoniae by ELISA and nested PCR from blood samples and nasal swabs from pigs in Slovakia

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    Marián Prokeš

    2012-01-01

    Full Text Available The aim of our study was to map the situation of swine mycoplasmoses on four farms in the region of Eastern Slovakia. The primary agent of Enzootic pneumonia of swine is Mycoplasma hyopneumoniae. After reviewing the health status of conventional herds and evaluation of clinical symptoms, paired samples of nasal swabs and venous blood samples were collected from 38 pigs with clinical signs of respiratory disease. Nasal swab samples were tested by nested PCR, while blood samples were used to detect antibodies against M. hyopneumoniae by blocking ELISA. The presence of M. hyopneumoniae was confirmed by nested PCR in four pigs (10.5% and by blocking ELISA in 16 pigs (42.1% of all four farms. This work presents for the first time comparison of different methods to diagnose M. hyopneumoniae infection on pig farms in Eastern Slovakia.

  18. Molecular diagnosis of Salmonella typhi and its virulence in suspected typhoid blood samples through nested multiplex PCR.

    Science.gov (United States)

    Prabagaran, Solai Ramatchandirane; Kalaiselvi, Vellingiri; Chandramouleeswaran, Naganathan; Deepthi, Krishnan Nair Geetha; Brahmadathan, Kootallur Narayanan; Mani, Mariappa

    2017-08-01

    A nested multiplex polymerase chain reaction (PCR) based diagnosis was developed for the detection of virulent Salmonella typhi in the blood specimens from patients suspected for typhoid fever. After the Widal test, two pairs of primers were used for the detection of flagellin gene (fliC) of S. typhi. Among them, those positive for fliC alone were subjected to identification of genes in Via B operon of Salmonella Pathogenesity Island (SPI-7) where four primer pairs were used to detect tviA and tviB genes. Among 250 blood samples tested, 115 were positive by fliC PCR; 22 of these were negative for tviA and tviB. Hence, the method described here can be used to diagnose the incidence of Vi-negative serovar typhi especially in endemic regions where the Vi vaccine is administered. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Comparison of culture, single and multiplex real-time PCR for detection of Sabin poliovirus shedding in recently vaccinated Indian children.

    Science.gov (United States)

    Giri, Sidhartha; Rajan, Anand K; Kumar, Nirmal; Dhanapal, Pavithra; Venkatesan, Jayalakshmi; Iturriza-Gomara, Miren; Taniuchi, Mami; John, Jacob; Abraham, Asha Mary; Kang, Gagandeep

    2017-08-01

    Although, culture is considered the gold standard for poliovirus detection from stool samples, real-time PCR has emerged as a faster and more sensitive alternative. Detection of poliovirus from the stool of recently vaccinated children by culture, single and multiplex real-time PCR was compared. Of the 80 samples tested, 55 (68.75%) were positive by culture compared to 61 (76.25%) and 60 (75%) samples by the single and one step multiplex real-time PCR assays respectively. Real-time PCR (singleplex and multiplex) is more sensitive than culture for poliovirus detection in stool, although the difference was not statistically significant. © 2017 Wiley Periodicals, Inc.

  20. Staphylococci with markers of antibiotic resistance collected from blood cultures

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    Vittorio Focarelli

    2012-06-01

    Full Text Available Introduction: Blood culture is still the gold standard for the detection of the causative agent of sepsis. Especially in intensive care patients and those with vascular catheters, the most common organisms isolated are coagulase-negative staphylococci (CoNS and Staphylococcus aureus, both characterized by multidrug resistance. Purposes of our work are the study of the incidence of markers of resistance in staphylococci and evaluation of potential changes over the years. Materials and methods: In the period January 2008-June 2011 5239 blood cultures were analyzed.They were mainly obtained from the departments of Intensive Care, Cardiology, Hematology, General Medicine, Emergency Medicine, Infectious Diseases, Oncology, Pulmonology and Pediatric Hematoncology. The vials containing the blood were incubated in the BACTEC 9120 automated tool of Becton Dickinson and susceptibility testing performed with the Phoenix instrument of the same company. Results:Within a total of 5239 blood cultures, 3967 (75.7% were negative and 1272 (24.3% positive. Fungi were isolated in 6.2% (79 of the positive ones, Gram-negative bacteria in 24.6% (313 and Gram-positive bacteria in 69.2% (880. Within the latter, 187 (21.2% were not staphylococcal isolates, 693 (78.8% were stafiloccocci mainly represented by S. epidermidis, S. aureus, S. hominis, S. haemolyticus and S. saprophyticus. Of the 693 staphylococcal isolates, 436 (62.9% were b lactamase producers, and between them 336 (77.1% were methicillin resistant, while only 3 of 436 (0.69% were S. aureus resistant to vancomycin as well.The incidence of markers of resistance was very high, especially in patients in intensive care and cardiac surgery, who are usually subjected to combined antibiotic therapy. In the three years studied there were no statistically significant differences in the resistance of staphylococci. Conclusions: The data show an alarming high number of multi-resistant staphylococci, which is often a

  1. Survey of blood cultures methods in Italy in 2010

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    Antonio Goglio

    2011-09-01

    Full Text Available Sepsis is a serious clinical condition, associated with high mortality despite advanced modern medical treatment. Traditionally, the detection and identification of bacteria and fungi circulating in the blood-stream is based on blood cultures. A number of factors influence the yield of blood culture, most of them concerning the microbiologist skill and the laboratory organization. In order to collect information about the practices and procedures used for the detection of microrganisms in blood cultures in the italian laboratory (lab, an e-mail with the invitation to participate in the survey was sent to 2000 members of the Italian Association of Clinical Microbiology. Responses were received from 100 lab, located from all over the country (in 18/20 italian regions. The results presented hereby concern specimen collection, culture techniques, rapid identification and susceptibility testing, laboratory organization, relationships with physicians. In summary, most lab use automated systems (96%, the bottles are incubated immediately during public holidays in 72/96 lab (75% and in 49/97 lab at night (50.5%, the lenght of incubation was 5 or 7 days in 93% of the lab, although it is common to extend the incubation period when brucellosis (74 lab, endocarditis (49 lab, systemic mycosis (33 lab is suspected. A wide variety of media are employed for subcultures. All lab process the positive bottles at least once a day, while only in 42 of 81 (51.9% lab the positive blood are processed on holiday. Communication between clinicians and microbiologist include: distribution of specimen collection guidelines (96/100 lab, availability to microbiologist of patients’ clinical situation (77/96 lab, 80.2%, and adding to report the microbiologist’ suggestion (75/98 lab, 76.5%. The results, compared with those collected with a similar questionnaire in 2001, show a greater adherence to guidelines: the number of bottles examined by lab yearly is almost doubled

  2. Identify of Granulicatella adiacens from blood cultures of a patient bearer of prosthetic valve

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    Raffaele Gargiulo

    2012-03-01

    Full Text Available The clinical case studied concerns a woman 81 years old, with a history of prosthetic valve and mitral insufficiency, admitted to internal medicine ward of NOCSAE hospital as a result of a recurrent fever. Due to the suspicion of endocarditis and with the aim to identify the presence of aerobic/anaerobic microorganisms, two set of blood cultures collected within 24 hours were sent to the Laboratory of microbiology. All the bottles were incubated into the Bact-Alert 3D System (bioMérieux. After an 19 hours incubation time, the samples were identified as positive by the automated system; consequently they cultured on a blood agar and selective media, according to our laboratory operational protocol. In the same time Gram stain of the cultural broth revealed the presence of Gram positive cocci arranged in chains different in length. Since there wasn’t an evident microbial growth on solid media after 24-48 hours of incubation, a new culture was carried out on blood and chocolate agar after the addition of Staphylococcus aureus ATCC 25923. After 24 hours of incubation it was possible appreciate the growth of tiny colonies around the S. aureus ones. These colonies were identified by Vitek2 and Api Rapid 32 Strep (bioMérieux as Granulicatella adiacens. The results were confirmed by PCR and sequencing of the groESL gene. MIC values obtained by the means of E-test (bioMérieux were: 0.016mg/L for penicillin, 0.125mg/L for cefotaxime, 1mg/L for both vancomicin and levofloxacin. Resistance was observed for cloramphenicol (MIC=16mg/L. The timely communication of these findings, supported by clinical data like the appearance of vegetation on mitral valve highlighted by trans-oesophageal echocardiography, allowed to establish an adequate antibiotic therapy, rapid resolution of fever and normalisation of inflammatory parameters.

  3. DETECTION OF BIOFILM PRODUCTION IN BLOOD CULTURE ISOLATES OF STAPHYLOCOCCI

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    Gupta Puja, Gupta Pratima, Mittal Garima, Agarwal RK, Goyal Rohit

    2015-01-01

    Full Text Available Background: Biofilm producing bacteria which are inherently resistant to antibiotics and disinfectants are widely associated with implant associated infections. Staphylococcus is the most commonly associated pathogens with bloodstream infection. Aims: The current study was conducted to detect biofilm production in Staphylococci isolated from blood culture specimens. Materials and Methods: 70 clinically significant staphylococcal isolates from blood culture were screened for biofilm production by Tissue culture plate (TCP method, Tube method (TM and Congo red agar (CRA method and their antibiotic susceptibility profile was studied. Results: 59 out of 70 staphylococcal isolates were positive by TCP, out of these 21.4% staphylococci were high biofilm producers, 62.8% staphylococci were moderate biofilm producers and 15.8% were non-biofilm producers. Maximum resistance was observed in biofilm producers to cotrimoxazole (74.5% and erythromycin (62.7% and none were resistant to vancomycin and linezolid. Out of total 59 biofilm producers, 20.3 % (12 were methicillin resistant and all these were S. aureus isolates. 19% (1 out of total 11 biofilm non-producers were methicillin resistant. Conclusion: Biofilm production was seen to be a major virulence factor in most of the staphylococcal isolates obtained from patients with signs and symptoms of septicaemia. S. aureus was found to be the major pathogen and timely detection of biofilm producing phenotype should be carried out using a simple and reproducible method, TCP which is both qualitative and quantitative.

  4. Isolation and detection of Listeria monocytogenes in poultry meat by standard culture methods and PCR

    Science.gov (United States)

    Kureljušić, J.; Rokvić, N.; Jezdimirović, N.; Kureljušić, B.; Pisinov, B.; Karabasil, N.

    2017-09-01

    Listeria is the genus of a bacteria found in soil and water and some animals, including poultry and cattle. It can be present in raw milk and food made from raw milk. It can also live in food processing plants and contaminate a variety of processed meats. Microscopically, Listeria species appear as small, Gram-positive rods, which are sometimes arranged in short chains. In direct smears, they can be coccoid, so they can be mistaken for streptococci. Longer cells can resemble corynebacteria. Flagella are produced at room temperature but not at 37°C. Haemolytic activity on blood agar has been used as a marker to distinguish Listeria monocytogenes among other Listeria species, but it is not an absolutely definitive criterion. Further biochemical characterization is necessary to distinguish between the different Listeria species. The objective of this study was to detect, isolate and identify Listeria monocytogenes from poultry meat. Within a period of six months from January to June 2017, a total of 15 samples were collected. Three samples were positive for the presence of Listeria monocytogenes. Biochemical and microbiological tests as well as PCR technique using specific primers were used to confirm L. Monocytogenes in the samples.

  5. Obtaining blood cultures by venipuncture versus from central lines: impact on blood culture contamination rates and potential effect on central line-associated bloodstream infection reporting.

    Science.gov (United States)

    Boyce, John M; Nadeau, Jacqueline; Dumigan, Diane; Miller, Debra; Dubowsky, Cindy; Reilly, Lenore; Hannon, Carla V

    2013-10-01

    Reduce the frequency of contaminated blood cultures that meet National Healthcare Safety Network definitions for a central line-associated bloodstream infection (CLABSI). An observational study. A 500-bed university-affiliated hospital. A new blood culture policy discouraged drawing blood samples from central lines. Phlebotomists were reeducated regarding aseptic technique when obtaining blood samples by venipuncture. The intravenous therapy team was taught how to draw blood samples by venipuncture and served as a backup when phlebotomists were unable to obtain blood samples. A 2-nurse protocol and a special supply kit for obtaining blood samples from catheters were developed. Rates of blood culture contamination were monitored by the microbiology laboratory. The proportion of blood samples obtained for culture from central lines decreased from 10.9% during January-June 2010 to 0.4% during July-December 2012 (P < .001). The proportion of blood cultures that were contaminated decreased from 84 (1.6%) of 5,274 during January-June 2010 to 21 (0.5%) of 4,245 during January-June 2012 (P < .001). Based on estimated excess hospital costs of $3,000 per contaminated blood culture, the reduction in blood culture contaminants yielded an estimated annualized savings of $378,000 in 2012 when compared to 2010. In mid-2010, 3 (30%) of 10 reported CLABSIs were suspected to represent blood culture contamination compared with none of 6 CLABSIs reported from mid-November 2010 through June 2012 (P = 0.25). Multiple interventions resulted in a reduction in blood culture contamination rates and substantial cost savings to the hospital, and they may have reduced the number of reportable CLABSIs.

  6. Survey of Naegleria from Taiwan recreational waters using culture enrichment combined with PCR.

    Science.gov (United States)

    Huang, Shih-Wei; Hsu, Bing-Mu

    2011-08-01

    Naegleria is a free-living amoeba. Pathogenic Naegleria may pose a health risk to people who come in contact with recreational waters. Here, we used Naegleria culture enrichment with PCR to identify the Naegleria species and investigated the distribution of Naegleria spp. in recreational waters including spring water, stream water and raw domestic water in central and southern Taiwan. In this study, Naegleria spp. were detected in 19 (17.8%) of the water samples. The occurrence of Naegleria in raw domestic water was 28.6%, higher than in stream water (14.7%) and in spring water (6.5%). The most frequently identified species exhibiting the closest phylogenetic relationships to the isolates were N. australiensis (n=4) and N. canariensis (n=4), followed by N. clarki (n=3) and N. philippinensis (n=3); N. americana (n=2). N. lovaniensis, N. dobsoni, and N. gruberi were each detected once. The pathogenic species N. fowleri was not detected, probably due to the low incubation temperature; however, the isolates exhibiting the closest phylogenetic relationships to the pathogenic species in mice of PAM, N. australiensis and N. philippinensis, were found. Results of this survey suggest the distribution of Naegleria spp. excluding N. fowleri in recreational waters. It should be considered a potential threat for health associated with human activities in recreational waters. Copyright © 2011 Elsevier B.V. All rights reserved.

  7. Reliability of direct sensitivity determination of blood cultures

    International Nuclear Information System (INIS)

    Noman, F.; Ahmed, A.

    2008-01-01

    The aim of this study was to evaluate the error in interpreting antimicrobial sensitivity by direct method when compared to standard method and find out if specific antibiotic-organism combination had more discrepancies. All blood culture samples received at Microbiology Laboratory from 1st July 2006 to 31st August 2006 were ncluded in the study. All samples were inoculated in automated blood culture system BACTEC 9240 which contained enriched Soybean-Casein Digest broth with CO/sub 2/. Once positive, bottles were removed from system; gram staining of the positive broths was done. Susceptibility test was performed from positive broth, on MHA (Mueller-Hinton Agar), with antibiotics panel according to gram stain result. All positive broths were also sub-cultured on blood agar, chocolate agar and McConkey agar for only gram-negative rods. Next day, the zone sizes of all antibiotics were recorded using measuring scale and at the same time susceptibility test was repeated from isolated colonies from subcultures, with inoculums prepared of McFarland 0.5 standard 0.2 Staphylococcus aureus (ATCC 29213); E.coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 27853) were included as quality control strain. Zone sizes were interpreted as sensitive (S), resistant (R) and intermediate (I) according to CLSI recommendation. Two results were compared and recorded. Out of a total 1083 combinations, zone diameters by standard method were either equal or greater than direct zone diameter (never smaller). Most of the discrepancies were in b-lactam/b-lactamase combinations and aminoglycosides. While reporting these groups of antibiotics with direct sensitivity test, one should be cautious. These are the major antibiotic used for life-threatening infections. In case of being heavy/lighter standard inoculums or marginal zones, repeating with standard method should be preferred to minimize the chances of error. (author)

  8. Detection of cytomegalovirus in blood donors by PCR using the digene SHARP signal system assay: effects of sample preparation and detection methodology.

    OpenAIRE

    Krajden, M; Shankaran, P; Bourke, C; Lau, W

    1996-01-01

    Cytomegalovirus (CMV) is an important cause of transfusion-associated morbidity and mortality; however, only 0.4 to 12% of the blood products obtained from seropositive blood donors transmit infection. The effects of three commercially available whole-blood sample preparation kits on the detection of CMV PCR products by a semiquantitative adaptation of the Digene SHARP Signal System Assay (DSSSA) in samples from volunteer blood donors was assessed. Of 101 samples from seropositive blood donor...

  9. Detection of Trypanosoma cruzi DNA in blood by PCR is associated with Chagas cardiomyopathy and disease severity.

    Science.gov (United States)

    Sabino, E C; Ribeiro, A L; Lee, T H; Oliveira, C L; Carneiro-Proietti, A B; Antunes, A P; Menezes, M M; Ianni, B M; Salemi, V M; Nastari, L; Fernandes, F; Sachdev, V; Carrick, D M; Deng, X; Wright, D; Gonçalez, T T; Murphy, E L; Custer, B; Busch, M P

    2015-04-01

    The significance of detection of Trypanosoma cruzi DNA in blood of antibody-positive patients for risk of development of Chagas heart disease is not well established. The objective of this study was to compare detection of T. cruzi DNA with known clinical and laboratory markers of Chagas cardiomyopathy (CC) severity. This is a case-control study nested within a retrospective cohort developed in Brazil to understand the natural history of Chagas disease. The study enrolled 499 T. cruzi seropositive blood donors (SP-BD) and 488 frequency matched seronegative control donors (SN-BD) who had donated between 1996 and 2002, and 101 patients with clinically diagnosed CC. In 2008-2010 all enrolled subjects underwent a health questionnaire, medical examination, electrocardiograms and echocardiograms and polymerase chain reaction (PCR) analyses. A blinded panel of three cardiologists adjudicated the outcome of CC. Trypanosoma cruzi kinetoplast minicircle sequences were amplified by real-time PCR using an assay with a sensitivity of one parasite per 20 mL of blood. All testing was performed on coded samples. Rates of PCR detection of T. cruzi DNA were significantly (P = 0.003) higher in CC patients and SP-BD diagnosed with CC (79/105 [75.2 %]) compared with SP-BD without CC (143/279 [51.3%]). The presence of parasitaemia was significantly associated with known markers of disease progression such as QRS and QT interval duration, lower left ventricular ejection fraction, higher left ventricular index mass, and elevated troponin and NTpro-BNP levels. Trypanosoma cruzi PCR positivity is associated with presence and severity of cardiomyopathy, suggesting a direct role of parasite persistence in disease pathogenesis. © 2015 The Authors. European Journal of Heart Failure © 2015 European Society of Cardiology.

  10. Evaluating genomic DNA extraction methods from human whole blood using endpoint and real-time PCR assays.

    Science.gov (United States)

    Koshy, Linda; Anju, A L; Harikrishnan, S; Kutty, V R; Jissa, V T; Kurikesu, Irin; Jayachandran, Parvathy; Jayakumaran Nair, A; Gangaprasad, A; Nair, G M; Sudhakaran, P R

    2017-02-01

    The extraction of genomic DNA is the crucial first step in large-scale epidemiological studies. Though there are many popular DNA isolation methods from human whole blood, only a few reports have compared their efficiencies using both end-point and real-time PCR assays. Genomic DNA was extracted from coronary artery disease patients using solution-based conventional protocols such as the phenol-chloroform/proteinase-K method and a non-phenolic non-enzymatic Rapid-Method, which were evaluated and compared vis-a-vis a commercially available silica column-based Blood DNA isolation kit. The appropriate method for efficiently extracting relatively pure DNA was assessed based on the total DNA yield, concentration, purity ratios (A 260 /A 280 and A 260 /A 230 ), spectral profile and agarose gel electrophoresis analysis. The quality of the isolated DNA was further analysed for PCR inhibition using a murine specific ATP1A3 qPCR assay and mtDNA/Y-chromosome ratio determination assay. The suitability of the extracted DNA for downstream applications such as end-point SNP genotyping, was tested using PCR-RFLP analysis of the AGTR1-1166A>C variant, a mirSNP having pharmacogenetic relevance in cardiovascular diseases. Compared to the traditional phenol-chloroform/proteinase-K method, our results indicated the Rapid-Method to be a more suitable protocol for genomic DNA extraction from human whole blood in terms of DNA quantity, quality, safety, processing time and cost. The Rapid-Method, which is based on a simple salting-out procedure, is not only safe and cost-effective, but also has the added advantage of being scaled up to process variable sample volumes, thus enabling it to be applied in large-scale epidemiological studies.

  11. A Proline Racemase Based PCR for Identification of Trypanosoma vivax in Cattle Blood

    OpenAIRE

    Fikru, Regassa; Hagos, Ashenafi; Rogé, Stijn; Reyna-Bello, Armando; Gonzatti, Mary Isabel; Merga, Bekana; Goddeeris, Bruno Maria; Büscher, Philippe

    2014-01-01

    A study was conducted to develop a Trypanosoma vivax (T. vivax) specific PCR based on the T. vivax proline racemase (TvPRAC) gene. Forward and reverse primers were designed that bind at 764-783 bp and 983-1002 bp of the gene. To assess its specificity, TvPRAC PCR was conducted on DNA extracted from different haemotropic pathogens: T. vivax from Nigeria, Ethiopia and Venezuela, T. congolense Savannah type, T. brucei brucei, T. evansi, T. equiperdum, T. theileri, Theileria parva, Anaplasma marg...

  12. [Comparison study on polymerase chain reaction (PCR) and standard culture technique in detecting mycobacterium tuberculosis to diagnose of joint tuberculosis].

    Science.gov (United States)

    Sun, Yong-sheng; Wen, Jian-min; Lü, Wei-xin; Lou, Si-quan; Jiao, Chang-geng; Yang, Su-min; Xu, Hai-bin; Duan, Yong-zhuang

    2009-07-01

    To study the role of PCR technique in detection of mycobacterium tuberculosis in the samples from joint tuberculosis, and to evaluate the clinical value of PCR in diagnosis of joint tuberculosis. From June 1993 to August 2001, PCR was used to detect DNA of mycobacterium tuberculosis, and the standard culture was applied to detect mycobacterium tuberculosis. Mycobacterium tuberculosis were respectively blindly by the two techniques in the samples obtained from 95 patients with joint tuberculosis (55 males and 40 females, the age ranging from 2 to 75 years, with an average of 34 years). The positive rate of mycobacterium tuberculosis detection was calculated. In the detection of mycobacterium tuberculosis, positive rate was 82% (78/95) in PCR technique, and 16% (15/95) in standard culture technique. There were statistical differences between the two groups (chi2=67, Ptechnique is a rapid, simple, sensitive and specific method for detection of mycobacterium tuberculosis in the samples of joint tuberculosis, showing more marked advantages than the standard culture technique. It is valuable in the early rapid diagnosis and differential diagnosis of joint tuberculosis.

  13. Evaluation of Reference Genes for Quantitative Real-Time PCR in Oil Palm Elite Planting Materials Propagated by Tissue Culture

    Science.gov (United States)

    Chan, Pek-Lan; Rose, Ray J.; Abdul Murad, Abdul Munir; Zainal, Zamri; Leslie Low, Eng-Ti; Ooi, Leslie Cheng-Li; Ooi, Siew-Eng; Yahya, Suzaini; Singh, Rajinder

    2014-01-01

    Background The somatic embryogenesis tissue culture process has been utilized to propagate high yielding oil palm. Due to the low callogenesis and embryogenesis rates, molecular studies were initiated to identify genes regulating the process, and their expression levels are usually quantified using reverse transcription quantitative real-time PCR (RT-qPCR). With the recent release of oil palm genome sequences, it is crucial to establish a proper strategy for gene analysis using RT-qPCR. Selection of the most suitable reference genes should be performed for accurate quantification of gene expression levels. Results In this study, eight candidate reference genes selected from cDNA microarray study and literature review were evaluated comprehensively across 26 tissue culture samples using RT-qPCR. These samples were collected from two tissue culture lines and media treatments, which consisted of leaf explants cultures, callus and embryoids from consecutive developmental stages. Three statistical algorithms (geNorm, NormFinder and BestKeeper) confirmed that the expression stability of novel reference genes (pOP-EA01332, PD00380 and PD00569) outperformed classical housekeeping genes (GAPDH, NAD5, TUBULIN, UBIQUITIN and ACTIN). PD00380 and PD00569 were identified as the most stably expressed genes in total samples, MA2 and MA8 tissue culture lines. Their applicability to validate the expression profiles of a putative ethylene-responsive transcription factor 3-like gene demonstrated the importance of using the geometric mean of two genes for normalization. Conclusions Systematic selection of the most stably expressed reference genes for RT-qPCR was established in oil palm tissue culture samples. PD00380 and PD00569 were selected for accurate and reliable normalization of gene expression data from RT-qPCR. These data will be valuable to the research associated with the tissue culture process. Also, the method described here will facilitate the selection of appropriate

  14. Evaluation of reference genes for quantitative real-time PCR in oil palm elite planting materials propagated by tissue culture.

    Science.gov (United States)

    Chan, Pek-Lan; Rose, Ray J; Abdul Murad, Abdul Munir; Zainal, Zamri; Low, Eng-Ti Leslie; Ooi, Leslie Cheng-Li; Ooi, Siew-Eng; Yahya, Suzaini; Singh, Rajinder

    2014-01-01

    The somatic embryogenesis tissue culture process has been utilized to propagate high yielding oil palm. Due to the low callogenesis and embryogenesis rates, molecular studies were initiated to identify genes regulating the process, and their expression levels are usually quantified using reverse transcription quantitative real-time PCR (RT-qPCR). With the recent release of oil palm genome sequences, it is crucial to establish a proper strategy for gene analysis using RT-qPCR. Selection of the most suitable reference genes should be performed for accurate quantification of gene expression levels. In this study, eight candidate reference genes selected from cDNA microarray study and literature review were evaluated comprehensively across 26 tissue culture samples using RT-qPCR. These samples were collected from two tissue culture lines and media treatments, which consisted of leaf explants cultures, callus and embryoids from consecutive developmental stages. Three statistical algorithms (geNorm, NormFinder and BestKeeper) confirmed that the expression stability of novel reference genes (pOP-EA01332, PD00380 and PD00569) outperformed classical housekeeping genes (GAPDH, NAD5, TUBULIN, UBIQUITIN and ACTIN). PD00380 and PD00569 were identified as the most stably expressed genes in total samples, MA2 and MA8 tissue culture lines. Their applicability to validate the expression profiles of a putative ethylene-responsive transcription factor 3-like gene demonstrated the importance of using the geometric mean of two genes for normalization. Systematic selection of the most stably expressed reference genes for RT-qPCR was established in oil palm tissue culture samples. PD00380 and PD00569 were selected for accurate and reliable normalization of gene expression data from RT-qPCR. These data will be valuable to the research associated with the tissue culture process. Also, the method described here will facilitate the selection of appropriate reference genes in other oil palm

  15. Evaluation of reference genes for quantitative real-time PCR in oil palm elite planting materials propagated by tissue culture.

    Directory of Open Access Journals (Sweden)

    Pek-Lan Chan

    Full Text Available BACKGROUND: The somatic embryogenesis tissue culture process has been utilized to propagate high yielding oil palm. Due to the low callogenesis and embryogenesis rates, molecular studies were initiated to identify genes regulating the process, and their expression levels are usually quantified using reverse transcription quantitative real-time PCR (RT-qPCR. With the recent release of oil palm genome sequences, it is crucial to establish a proper strategy for gene analysis using RT-qPCR. Selection of the most suitable reference genes should be performed for accurate quantification of gene expression levels. RESULTS: In this study, eight candidate reference genes selected from cDNA microarray study and literature review were evaluated comprehensively across 26 tissue culture samples using RT-qPCR. These samples were collected from two tissue culture lines and media treatments, which consisted of leaf explants cultures, callus and embryoids from consecutive developmental stages. Three statistical algorithms (geNorm, NormFinder and BestKeeper confirmed that the expression stability of novel reference genes (pOP-EA01332, PD00380 and PD00569 outperformed classical housekeeping genes (GAPDH, NAD5, TUBULIN, UBIQUITIN and ACTIN. PD00380 and PD00569 were identified as the most stably expressed genes in total samples, MA2 and MA8 tissue culture lines. Their applicability to validate the expression profiles of a putative ethylene-responsive transcription factor 3-like gene demonstrated the importance of using the geometric mean of two genes for normalization. CONCLUSIONS: Systematic selection of the most stably expressed reference genes for RT-qPCR was established in oil palm tissue culture samples. PD00380 and PD00569 were selected for accurate and reliable normalization of gene expression data from RT-qPCR. These data will be valuable to the research associated with the tissue culture process. Also, the method described here will facilitate the selection

  16. "DRUG RESISTANCE PATTERN IN ISOLATED BACTERIA FROM BLOOD CULTURES"

    Directory of Open Access Journals (Sweden)

    A. Sobhani

    2004-05-01

    Full Text Available Bacteremia is an important infectious disease which may lead to death. Common bacteria and pattern of antibiotic resistance in different communities are different and understanding these differences is important. In the present study, relative frequency and pattern of drug resistance have been examined in bacteria isolated from blood cultures in Razi Hospital laboratory. The method of the study was descriptive. Data collection was carried out retrospectively. Total sample consisted of 311 positive blood cultures from 1999 to 2001. Variables under study were bacterial strains, antibiotics examined in antibiogram, microbial resistance, and patients' age and sex. The most common isolated bacteria were Salmonella typhi (22.2% and the least common ones were Citrobacter (1.6%. The highest antibiotic resistance was seen against amoxicillin (88.4%. The proportion of males to females was1: 1/1 and the most common age group was 15-44 (47.3%. Common bacteria and pattern of antibiotic resistance were different in some areas and this subject requires further studies in the future.

  17. A Multicenter Evaluation of Blood Culture Practices, Contamination Rates, and the Distribution of Causative Bacteria

    OpenAIRE

    Altindis, Mustafa; Koroglu, Mehmet; Demiray, Tayfur; Dal, Tuba; Ozdemir, Mehmet; Sengil, Ahmet Zeki; Atasoy, Ali Riza; Do?an, Metin; Cicek, Aysegul Copur; Ece, Gulfem; Kaya, Selcuk; Iraz, Meryem; Gultepe, Bilge Sumbul; Temiz, Hakan; Kandemir, Idris

    2016-01-01

    Background: The prognostic value of blood culture testing in the diagnosis of bacteremia is limited by contamination. Objectives: In this multicenter study, the aim was to evaluate the contamination rates of blood cultures as well as the parameters that affect the culture results. Materials and Methods: Sample collection practices and culture data obtained from 16 university/research hospitals were retrospectively evaluated. A total of 214,340 blood samples from 43,254 patients admitted to th...

  18. Effect of volume of blood cultured on detection of Streptococcus viridans bacteraemia.

    Science.gov (United States)

    Shanson, D C; Thomas, F; Wilson, D

    1984-01-01

    Fifty eight patients undergoing dental extraction each had 45 ml blood collected. This was divided into 30 ml and 15 ml blood samples for culture. The 30 ml sample was inoculated into 120 ml nutrient broth with 0.05% liquoid and the 15 ml sample into 60 ml of identical broth so that the final dilution of blood in broth was always 1/5. Bacteraemia due to viridans streptococci was found in 27 and 15 patients by culturing the 30 ml and 15 ml blood samples respectively. Only one further case of streptococcal bacteraemia was detected by culture of the total volume of blood collected (45 ml) rather than culture of the 30 ml blood sample alone. These findings suggest that the culture of 30 ml blood results in the detection of up to 80% more blood cultures yielding Streptococcus viridans than the culture of only 15 ml blood. The collection of more than 30 ml blood for each culture is unlikely to prove worthwhile. It is suggested that 30 ml rather than 15 ml blood is probably the optimal volume of blood for each culture of S viridans when patients with suspected infective endocarditis are investigated. PMID:6373833

  19. The IRIDICA BAC BSI Assay: Rapid, Sensitive and Culture-Independent Identification of Bacteria and Candida in Blood

    Science.gov (United States)

    Rothman, Richard E.; Peterson, Stephen; Carroll, Karen C.; Zhang, Sean X.; Avornu, Gideon D.; Rounds, Megan A.; Carolan, Heather E.; Toleno, Donna M.; Moore, David; Hall, Thomas A.; Massire, Christian; Richmond, Gregory S.; Gutierrez, Jose R.; Sampath, Rangarajan; Ecker, David J.; Blyn, Lawrence B.

    2016-01-01

    Bloodstream infection (BSI) and sepsis are rising in incidence throughout the developed world. The spread of multi-drug resistant organisms presents increasing challenges to treatment. Surviving BSI is dependent on rapid and accurate identification of causal organisms, and timely application of appropriate antibiotics. Current culture-based methods used to detect and identify agents of BSI are often too slow to impact early therapy and may fail to detect relevant organisms in many positive cases. Existing methods for direct molecular detection of microbial DNA in blood are limited in either sensitivity (likely the result of small sample volumes) or in breadth of coverage, often because the PCR primers and probes used target only a few specific pathogens. There is a clear unmet need for a sensitive molecular assay capable of identifying the diverse bacteria and yeast associated with BSI directly from uncultured whole blood samples. We have developed a method of extracting DNA from larger volumes of whole blood (5 ml per sample), amplifying multiple widely conserved bacterial and fungal genes using a mismatch- and background-tolerant PCR chemistry, and identifying hundreds of diverse organisms from the amplified fragments on the basis of species-specific genetic signatures using electrospray ionization mass spectrometry (PCR/ESI-MS). We describe the analytical characteristics of the IRIDICA BAC BSI Assay and compare its pre-clinical performance to current standard-of-care methods in a collection of prospectively collected blood specimens from patients with symptoms of sepsis. The assay generated matching results in 80% of culture-positive cases (86% when common contaminants were excluded from the analysis), and twice the total number of positive detections. The described method is capable of providing organism identifications directly from uncultured blood in less than 8 hours. Disclaimer: The IRIDICA BAC BSI Assay is not available in the United States. PMID:27384540

  20. The IRIDICA BAC BSI Assay: Rapid, Sensitive and Culture-Independent Identification of Bacteria and Candida in Blood.

    Science.gov (United States)

    Metzgar, David; Frinder, Mark W; Rothman, Richard E; Peterson, Stephen; Carroll, Karen C; Zhang, Sean X; Avornu, Gideon D; Rounds, Megan A; Carolan, Heather E; Toleno, Donna M; Moore, David; Hall, Thomas A; Massire, Christian; Richmond, Gregory S; Gutierrez, Jose R; Sampath, Rangarajan; Ecker, David J; Blyn, Lawrence B

    2016-01-01

    Bloodstream infection (BSI) and sepsis are rising in incidence throughout the developed world. The spread of multi-drug resistant organisms presents increasing challenges to treatment. Surviving BSI is dependent on rapid and accurate identification of causal organisms, and timely application of appropriate antibiotics. Current culture-based methods used to detect and identify agents of BSI are often too slow to impact early therapy and may fail to detect relevant organisms in many positive cases. Existing methods for direct molecular detection of microbial DNA in blood are limited in either sensitivity (likely the result of small sample volumes) or in breadth of coverage, often because the PCR primers and probes used target only a few specific pathogens. There is a clear unmet need for a sensitive molecular assay capable of identifying the diverse bacteria and yeast associated with BSI directly from uncultured whole blood samples. We have developed a method of extracting DNA from larger volumes of whole blood (5 ml per sample), amplifying multiple widely conserved bacterial and fungal genes using a mismatch- and background-tolerant PCR chemistry, and identifying hundreds of diverse organisms from the amplified fragments on the basis of species-specific genetic signatures using electrospray ionization mass spectrometry (PCR/ESI-MS). We describe the analytical characteristics of the IRIDICA BAC BSI Assay and compare its pre-clinical performance to current standard-of-care methods in a collection of prospectively collected blood specimens from patients with symptoms of sepsis. The assay generated matching results in 80% of culture-positive cases (86% when common contaminants were excluded from the analysis), and twice the total number of positive detections. The described method is capable of providing organism identifications directly from uncultured blood in less than 8 hours. The IRIDICA BAC BSI Assay is not available in the United States.

  1. The IRIDICA BAC BSI Assay: Rapid, Sensitive and Culture-Independent Identification of Bacteria and Candida in Blood.

    Directory of Open Access Journals (Sweden)

    David Metzgar

    Full Text Available Bloodstream infection (BSI and sepsis are rising in incidence throughout the developed world. The spread of multi-drug resistant organisms presents increasing challenges to treatment. Surviving BSI is dependent on rapid and accurate identification of causal organisms, and timely application of appropriate antibiotics. Current culture-based methods used to detect and identify agents of BSI are often too slow to impact early therapy and may fail to detect relevant organisms in many positive cases. Existing methods for direct molecular detection of microbial DNA in blood are limited in either sensitivity (likely the result of small sample volumes or in breadth of coverage, often because the PCR primers and probes used target only a few specific pathogens. There is a clear unmet need for a sensitive molecular assay capable of identifying the diverse bacteria and yeast associated with BSI directly from uncultured whole blood samples. We have developed a method of extracting DNA from larger volumes of whole blood (5 ml per sample, amplifying multiple widely conserved bacterial and fungal genes using a mismatch- and background-tolerant PCR chemistry, and identifying hundreds of diverse organisms from the amplified fragments on the basis of species-specific genetic signatures using electrospray ionization mass spectrometry (PCR/ESI-MS. We describe the analytical characteristics of the IRIDICA BAC BSI Assay and compare its pre-clinical performance to current standard-of-care methods in a collection of prospectively collected blood specimens from patients with symptoms of sepsis. The assay generated matching results in 80% of culture-positive cases (86% when common contaminants were excluded from the analysis, and twice the total number of positive detections. The described method is capable of providing organism identifications directly from uncultured blood in less than 8 hours.The IRIDICA BAC BSI Assay is not available in the United States.

  2. [Clinical evaluation of the application of gene chip for identifying pathogens in blood cultures].

    Science.gov (United States)

    Li, Lin-hai; Cheng, Ying; Chen, Li-dan; Huang, Xiao-yan; Shi, Yu-ling; He, Jie-jing; Wang, Lu-xia

    2009-10-01

    To explore the feasibility of using gene chip method to identify pathogens in blood cultures. Clinical blood samples were obtained and cultured using an automated blood culture system. A gene chip diagnostic kit was used to detect the pathogenic bacteria in these blood cultures following the procedures of target gene extraction and amplification, hybridization and result analysis. The conventional method was also used to isolate and identify the bacteria from the clinical blood cultures, and the results of the two methods were compared. In the 86 clinical blood samples, 74 were positive and 12 negative according to the conventional method, while 48 were positive and 38 negative as found by the gene chip method, showing significant differences in the results (Ppathogens in clinical blood cultures and awaits further improvement.

  3. Comparison of QIAsymphony Automated and QIAamp Manual DNA Extraction Systems for Measuring Epstein-Barr Virus DNA Load in Whole Blood Using Real-Time PCR

    OpenAIRE

    Laus, Stella; Kingsley, Lawrence A.; Green, Michael; Wadowsky, Robert M.

    2011-01-01

    Automated and manual extraction systems have been used with real-time PCR for quantification of Epstein-Barr virus [human herpesvirus 4 (HHV-4)] DNA in whole blood, but few studies have evaluated relative performances. In the present study, the automated QIAsymphony and manual QIAamp extraction systems (Qiagen, Valencia, CA) were assessed using paired aliquots derived from clinical whole-blood specimens and an in-house, real-time PCR assay. The detection limits using the QIAsymphony and QIAam...

  4. Reagent deposition for rapid multiplex pathogen identification in human blood culture samples

    DEFF Research Database (Denmark)

    Mogensen, Klaus Bo; Machado, Ana Manuel; Dufva, Martin

    2014-01-01

    Blood stream infections led to 135,000 deads annually in EU and fast treatment significantly increases the survival rate. This condition is diagnosed by means of blood cultures (19 Mill blood cultures are drawn annually in EU). In this work, a multiplex peptide nucleic acid / fluorescence in...

  5. Comparison of DNA extraction methods used to detect bacterial and yeast DNA from spiked whole blood by real-time PCR.

    Science.gov (United States)

    Dalla-Costa, Libera M; Morello, Luis G; Conte, Danieli; Pereira, Luciane A; Palmeiro, Jussara K; Ambrosio, Altair; Cardozo, Dayane; Krieger, Marco A; Raboni, Sonia M

    2017-09-01

    Sepsis is the leading cause of death in intensive care units (ICUs) worldwide and its diagnosis remains a challenge. Blood culturing is the gold standard technique for blood stream infection (BSI) identification. Molecular tests to detect pathogens in whole blood enable early use of antimicrobials and affect clinical outcomes. Here, using real-time PCR, we evaluated DNA extraction using seven manual and three automated commercially available systems with whole blood samples artificially contaminated with Escherichia coli, Staphylococcus aureus, and Candida albicans, microorganisms commonly associated with BSI. Overall, the commercial kits evaluated presented several technical limitations including long turnaround time and low DNA yield and purity. The performance of the kits was comparable for detection of high microorganism loads (10 6 CFU/mL). However, the detection of lower concentrations was variable, despite the addition of pre-processing treatment to kits without such steps. Of the evaluated kits, the UMD-Universal CE IVD kit generated a higher quantity of DNA with greater nucleic acid purity and afforded the detection of the lowest microbial load in the samples. The inclusion of pre-processing steps with the kit seems to be critical for the detection of microorganism DNA directly from whole blood. In conclusion, future application of molecular techniques will require overcoming major challenges such as the detection of low levels of microorganism nucleic acids amidst the large quantity of human DNA present in samples or differences in the cellular structures of etiological agents that can also prevent high-quality DNA yields. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. Detection of fungal DNA in lysis-centrifugation blood culture for the diagnosis of invasive candidiasis in neonatal patients.

    Science.gov (United States)

    Trovato, L; Betta, P; Romeo, M G; Oliveri, S

    2012-03-01

    We report data concerning the detection of fungal DNA directly from lysis-centrifugation blood culture to assess its value in the detection of fungaemia in 86 of the 347 patients admitted to the neonatal intensive-care unit between January 2009 and December 2010. The sensitivity and specificity of the PCR were 87.5% and 98.5%, respectively, with a positive predictive value of 93.3% and a negative predictive value of 97.1%. Detection of fungal DNA directly from blood culture Isolator 1.5 microbial tubes, without prior cultivation, is a promising approach for the rapid detection of Candida spp. in neonates with suspected candidaemia. © 2011 The Authors. Clinical Microbiology and Infection © 2011 European Society of Clinical Microbiology and Infectious Diseases.

  7. Use of culture, PCR analysis, and DNA microarrays for detection of Campylobacter jejuni and Campylobacter coli from chicken feces

    DEFF Research Database (Denmark)

    Keramas, Georgios; Bang, Dang Duong; Lund, Marianne

    2004-01-01

    A DNA microarray for detection of Campylobacter spp. was recently developed and applied to detect Campylobacter spp. directly from chicken feces. Sixty-five pooled chicken cloacal swab samples from 650 individual broiler chickens were included in the study. The results of Campylobacter sp....... detection obtained with DNA microarrays were compared to those obtained by conventional culture and gel electrophoresis. By conventional culture, 60% of the samples were positive for either Campylobacter jejuni or Campylobacter coli. By PCR and capillary electrophoresis, 95% of the samples were positive...... for Campylobacter spp., whereas with DNA microarrays all samples were positive for Campylobacter spp. By application of DNA microarray analysis, the isolates in 4 samples (6%) could not be identified to the species level, whereas by PCR-capillary electrophoresis, the isolates in 12 samples (19%) remained...

  8. Culture-independent qunatification of Salmonella enterica in carcass gauze swabs by flotation prior to real-time PCR

    DEFF Research Database (Denmark)

    Löfström, Charlotta; Schelin, Jenny; Norling, Börje

    2010-01-01

    To facilitate quantitative risk assessment in the meat production chain, there is a need for culture-independent quantification methods. The aim of this study was to evaluate the use of flotation, a non-destructive sample preparation method based on traditional buoyant density centrifugation......, for culture-independent quantification of intact Salmonella in pig carcass gauze swabs (100 cm2) prior to quantitative PCR (qPCR). A novel approach was investigated, excluding the homogenization step prior to flotation, to improve the detection limit and speed up the quantification procedure. The buoyant...... density of two Salmonella strains in different growth conditions was determined to be 1.065 – 1.092 g/ml. Based on these data, an optimal discontinuous flotation with three different density layers, ~1.200, 1.102 and 1.055 g/ml, was designed for extracting intact Salmonella cells from pig carcass swabs...

  9. Culture-independent quantification of Salmonella enterica in carcass gauze swabs by flotation prior to real-time PCR

    DEFF Research Database (Denmark)

    Löfström, Charlotta; Schelin, Jenny; Norling, Börje

    2011-01-01

    To facilitate quantitative risk assessment in the meat production chain, there is a need for culture-independent quantification methods. The aim of this study was to evaluate the use of flotation, a non-destructive sample preparation method based on traditional buoyant density centrifugation......, for culture-independent quantification of intact Salmonella in pig carcass gauze swabs (100 cm2) prior to quantitative PCR (qPCR). A novel approach was investigated, excluding the homogenization step prior to flotation, to improve the detection limit and speed up the quantification procedure. The buoyant...... density of two Salmonella strains in different growth conditions was determined to be 1.065 – 1.092 g/ml. Based on these data, an optimal discontinuous flotation with three different density layers, 1.200, 1.102 and 1.055 g/ml, was designed for extracting intact Salmonella cells from pig carcass swabs...

  10. Incidence of Listeria species in bovine, ovine, caprine, camel and water buffalo milk using cultural method and the PCR assay

    OpenAIRE

    Rahimi, Ebrahim; Momtaz, Hassan; Behzadnia, Asma; Baghbadorani, Zeinab Torki

    2014-01-01

    Objective: To determine the prevalence rate of Listeria species in bovine, ovine, caprine, camel and water buffalo milk in Iran. Methods: From September 2010 to December 2011 a total of 260 bulk milk samples including 85 bovine, 37 camel, 34 water buffalo, 56 ovine and 48 caprine bulk milk samples were collected from commercial dairy herds, in Fars and Khuzestan provinces, Iran and were evaluated for the presence of Listeria species using cultural method and the PCR assay. R...

  11. A Real-Time PCR Assay for the Detection of Campylobacter jejuni in Foods after Enrichment Culture

    OpenAIRE

    Sails, Andrew D.; Fox, Andrew J.; Bolton, Frederick J.; Wareing, David R. A.; Greenway, David L. A.

    2003-01-01

    A real-time PCR assay was developed for the quantitative detection of Campylobacter jejuni in foods after enrichment culture. The specificity of the assay for C. jejuni was demonstrated with a diverse range of Campylobacter species, related organisms, and unrelated genera. The assay had a linear range of quantification over six orders of magnitude, and the limit of detection was approximately 12 genome equivalents. The assay was used to detect C. jejuni in both naturally and artificially cont...

  12. Diagnostic yield and clinical impact of routine cell culture for respiratory viruses among children with a negative multiplex RT-PCR result.

    Science.gov (United States)

    AlGhounaim, M; Xiao, Y; Caya, C; Papenburg, J

    2017-09-01

    Polymerase chain reaction (PCR) is the reference standard for respiratory virus testing. However, cell culture may still have added value in identifying viruses not detected by PCR. We aimed to estimate the yield and clinical impact of routine respiratory virus culture among children with a negative PCR result. A retrospective cohort study was performed from Jan. 2013 to Sept. 2015. Respiratory samples from hospitalized or immunocompromised patients culture monolayers if they tested negative by a PCR assay for 12 respiratory viruses. We studied patients with a respiratory specimen negative by PCR and positive by culture. Duplicates and samples of sold services were excluded. Data on demographics, clinical history, laboratory findings, and patient management were collected from patients' charts. Descriptive and multivariate statistics were performed. Overall, 4638 PCR-negative samples were inoculated in cell culture. Of those, 196 (4.2%) were cell culture positive, and 144 met study inclusion criteria. Most subjects (81.9%) were hospitalized. Mean age was 2.4±3.4years. The viruses most frequently isolated were cytomegalovirus (33.3%) and enteroviruses (19.4%). Cell culture results prompted a change in management in 5 patients (3.5%), all of whom had acyclovir initiated for localized HSV-1 infection. Four of these had skin or mucosal lesions that could be sampled to establish a diagnosis. In children, routine viral culture on respiratory specimens that were negative by PCR has low yield and minimal clinical impact. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Comparison of three PCR-based assays for the non-invasive diagnosis of malaria: detection of Plasmodium parasites in blood and saliva.

    Science.gov (United States)

    Singh, R; Singh, D P; Gupta, R; Savargaonkar, D; Singh, O P; Nanda, N; Bhatt, R M; Valecha, N

    2014-09-01

    The conventional molecular diagnosis of malaria uses 18S rRNA-based PCR assay employing blood samples. This assay presents limitation in terms of long turnaround time and increased chances of false-positive results. Here, we evaluated one-step singleplex or multiplex PCR assay based on high copy species-specific consensus repeat sequences (CRS) along with standard 18S rRNA nested PCR (18S n-PCR) assay to detect P. falciparum and P. vivax infection using blood and saliva samples from Indian febrile patients. Out of 327 patients, 187 were found to be positive for malaria parasites by microscopic examination of peripheral blood smears. Among these 130 were P. vivax and 57 were P. falciparum cases. The 18S n-PCR assay and CRS PCR assay identified 186 out of 187 cases (99.4 %). Multiplex CRS PCR assay detected Plasmodium in 176 out of 187 cases (94.1 %). Both singleplex and multiplex CRS PCR assay identified 6 mixed infection cases, while 18S n-PCR assay detected 10 mixed infection cases of P. vivax and P. falciparum, which were not recognized by microscopy. Non-invasive Plasmodium detection rate with DNA derived from saliva samples was highest for 18S n-PCR (87.36 %), followed by singleplex CRS (81 %) and multiplex CRS PCR assay (70.5 %). Specificity for P. vivax and P. falciparum detection for all assays was 98.48 % and 100 % respectively. Detection rate for P. vivax in saliva correlated with parasite density for CRS target-based assays. The species-specific CRS PCR, either as a singleplex or multiplex assay, can have an impact on diagnosis and epidemiological studies in malaria.

  14. Diagnosis of Neisseria gonorrhoeae among pregnant women by culture method and PCR on cppB gene

    Directory of Open Access Journals (Sweden)

    Jalal Mardaneh

    2013-11-01

    Full Text Available Background: Neisseria gonorrhoeae is a human obligate pathogen and the etiological agent of gonorrhea. Health irreparable complications resulting from gonorrhea disease occur mainly in pregnant women and neonates. Aim of this study was diagnosis of Neisseria gonorrhoeae among pregnant women with using culture and molecular method by amplification of cppB gene with PCR. Material and Methods: In this cross-sectional study, two endocervical swab specimens were obtained from 1100 pregnant women who referred to Shiraz Hospitals. Culture on nonselective and selective media and nucleic acid amplification test (NAAT were performed for detection of Neisseria gonorrhoeae cppB gene. Results: All endocervical swabs cultures on selective and nonselective media were negative for Neisseria gonorrhoeae. Among examined endocervical swabs, 13samples (1.18% were positive by nucleic acid amplification of Neisseria gonorrgoeae cppB gene. Conclusion: Negative results of culture and positive results of PCR in this study indicate that however culture is gold standard method for detection of Neisseria gonorrhoeae but due to bacterial autolysis, poor sampling techniques and improper specimen storage and transport, its value decline as compared with Nucleic acid amplification test (NAAT.

  15. Prevalence of methicillin-resistant Staphylococcus aureus based on culture and PCR in inpatients at a tertiary care center in Tokyo, Japan.

    Science.gov (United States)

    Taguchi, Hirokazu; Matsumoto, Tetsuya; Ishikawa, Hiroki; Ohta, Shoichi; Yukioka, Tetsuo

    2012-10-01

    We investigated active screening for colonization with methicillin-resistant Staphylococcus aureus (MRSA) on admission and weekly follow-up surveillance after admission to a tertiary care center (TCC) between June 2007 and 31 December 2007. Eleven percent (30/267) of patients were found to be positive for MRSA by polymerase chain reaction (PCR) and/or culture on admission; 5% (12/267) became positive during the TCC stay. The major primary diagnoses in MRSA-positive patients were pneumonia and cerebrovascular diseases. Twenty-two (52%) of 42 patients were found to be MRSA positive by both PCR and culture, compared with 19 (45%) of 42 who were PCR positive and culture negative. These findings suggest that active surveillance with PCR is highly sensitive and useful for the detection of MRSA colonization. To our knowledge, this is the first report of active surveillance of MRSA by PCR and bacterial culture in critically ill inpatients in Japan.

  16. DEVELOPMENT OF REAL-TIME MULTIPLEX PCR FOR THE QUANTITATIVE DETERMINATION OF TREC'S AND KREC'S IN WHOLE BLOOD AND IN DRIED BLOOD SPOTS

    Directory of Open Access Journals (Sweden)

    M. A. Gordukova

    2015-01-01

    Full Text Available Primary immunodeficiencies (PID such as severe combined immunodeficiency (SCID and X-linked agammaglobulinemia are characterized by the lack of functional Tand B-cells, respectively. Without early diagnosis and prompt treatment children with PID suffer from severe infectious diseases, leading to their death or disability. Our purpose was developing of simple, inexpensive, high throughput technique based on the quantitative determination of TREC and KREC molecules by real-time PCR, and its validation in a group of children with a verified diagnosis of SCID and X-linked agammaglobulinemia.In this study, we developed and validated multiplex real-time PCR for the TREC’s and KREC’s quantitative analysis. We have shown that linear range of Ct changes depending on the concentrations of targets with a correlation coefficient R2 not worse than 0.98 was observed at concentrations from 109 to 5 × 104 copies per ml. The lowest amount of targets reliably detected in a reaction volume was 10 TREC’s copies, 5 KREC ‘s copies and 5 copies of internal control (IL17RA. We determined the age-depended reference values of TRECs and KRECs in whole blood in 29 boys and 27 girls with normal immunological parameters. The normal cut-offs for TRECs and KRECs were defined in dry blood spots depending on the method of extraction.The proposed method showed 100% diagnostic sensitivity and specificity in the studied group. The method can be proposed as a screening tool for the diagnosis of SCID and X-linked agammaglobulinemia both in whole blood and in the dry blood spots. The further investigation is required with larger number of samples. 

  17. Comparison of the sensitivity of typhi dot test with blood culture in typhoid

    International Nuclear Information System (INIS)

    Rizvi, Q.

    2006-01-01

    To evaluate the sensitivity of Typhi Dot test in comparison to Blood Culture for the diagnosis of Typhoid Fever in our setup. Fifty patients who fulfilled the clinical criteria of having Typhoid Fever. The data of all the patients was documented, and they were submitted to the Typhi Dot and Blood Culture tests, apart from other routine investigations. Out of the total 50 patients, 47(94%) had their Blood Culture positive for Typhoid bacillus, while in 49 (98%) the Typhi Dot test was positive. Two patients which were found positive on Typhi dot test, gave negative results on Blood Culture. One patient with the signs and symptoms of Typhoid Fever was found neither positive on Typhi Dot test nor upon Blood Culture. There was no significant difference between the results of Blood Culture and Typhi Dot test in the diagnosis of Typhoid Fever. However, Typhi Dot has the advantages of being less expensive and quicker in giving results with excellent sensitivity. (author)

  18. Colistin resistance among blood culture isolates at a tertiary care centre in Hungary.

    Science.gov (United States)

    Juhász, Emese; Iván, Miklós; Pintér, Eszter; Pongrácz, Júlia; Kristóf, Katalin

    2017-12-01

    The emergence of colistin resistance has been detected worldwide in recent years. Whilst colistin susceptibility has been tested in carbapenem resistant Enterobacteriaceae as well as multidrug-resistant Pseudomonas spp. and Acinetobacter spp. during routine laboratory practice, the overall rate of colistin resistance was unknown in our centre. The aim of this retrospective study was to reveal the prevalence of colistin resistance among clinically significant blood culture isolates in two different periods (2010-2011 and 2016) in our laboratory. Consecutive non-duplicate strains (n=776) were screened for colistin resistance using agar plates containing 4mg/L colistin. Strains cultured on colistin-containing plates were further examined. Minimum inhibitory concentrations (MICs) of colistin-tolerant subcultures and original cultures were determined in parallel by the broth microdilution method. Screening for mcr-1-mediated colistin resistance was performed by PCR. The rate of colistin resistance was 0.6%, 1.3% and 2.6% in Enterobacteriaceae, Pseudomonas spp. and Acinetobacter spp., respectively; colistin-resistant subpopulations were found in 17%, 27% and 20% of isolates, respectively, with low frequency. Seven colistin-resistant strains were found, among which was an mcr-1-positive Escherichia coli isolated from a blood sample of a haemato-oncology patient in 2011. All Stenotrophomonas maltophilia isolates were resistant to colistin. The low prevalence of colistin resistance was in accordance with European data. The prevalence of heteroresistance was significantly higher, but the clinical significance of the phenomenon is unclear. We have identified the first mcr-1-positive E. coli strain in Hungary. mcr-1 has been in Hungary since 2011 but has not yet expanded. Copyright © 2017 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.

  19. Developing high throughput quantitative PCR assays for diagnosing Ikeda and other Theileria orientalis types common to New Zealand in bovine blood samples.

    Science.gov (United States)

    Pulford, D J; Gias, E; Bueno, I M; McFadden, Amj

    2016-01-01

    To develop rapid, quantitative PCR (qPCR) assays using high resolution melt (HRM) analysis and type-specific TaqMan assays for identifying the prevalent types of Theileria orientalis found in New Zealand cattle; and to evaluate their analytical and diagnostic characteristics compared with other assays for T. orientalis. Nucleotide sequences aligned with T. orientalis Buffeli, Chitose and Ikeda types, obtained from DNA extracted from blood samples from infected cattle, were used to design HRM and type-specific probe-based qPCR assays. The three type-specific assays were also incorporated into a single-tube multiplex qPCR assay. These assays were validated using DNA extracted from blood samples from cattle in herds with or without clinical signs of T. orientalis infection, other veterinary laboratory samples, as well as plasmids containing T. orientalis type-specific sequences. Diagnostic specificity (DSp) and sensitivity (DSe) estimates for the qPCR assays were compared to blood smear piroplasm results, and other PCR assays for T. orientalis. Copy number estimates of Ikeda DNA in blood were determined from cattle exhibiting anaemia using the Ikeda-specific qPCR assay. The T. orientalis type-specific and the HRM qPCR assays displayed 100% analytical specificity. The Ikeda-specific qPCR assay exhibited linearity (R(2) = 0.997) with an efficiency of 94.3%. Intra-assay CV were ≤0.08 and inter-assay CV were ≤0.095. For blood samples from cows with signs of infection with T. orientalis, the DSp and DSe of the multiplex probe qPCR assay were 93 and 96%, respectively compared with blood smears, and 97 and 100%, respectively compared with conventional PCR assays. For the Ikeda-specific qPCR assay, the number of positive samples (n=66) was slightly higher than a conventional PCR assay (n=64). The concentration of Ikeda genomes in blood samples from 41 dairy cows with signs of infection with T. orientalis ranged between 5.6 × 10(4) and 3.3 × 10(6) genomes per

  20. Assessment of real-time PCR cycle threshold values in Microsporum canis culture-positive and culture-negative cats in an animal shelter: a field study.

    Science.gov (United States)

    Jacobson, Linda S; McIntyre, Lauren; Mykusz, Jenny

    2018-02-01

    Objectives Real-time PCR provides quantitative information, recorded as the cycle threshold (Ct) value, about the number of organisms detected in a diagnostic sample. The Ct value correlates with the number of copies of the target organism in an inversely proportional and exponential relationship. The aim of the study was to determine whether Ct values could be used to distinguish between culture-positive and culture-negative samples. Methods This was a retrospective analysis of Ct values from dermatophyte PCR results in cats with suspicious skin lesions or suspected exposure to dermatophytosis. Results One hundred and thirty-two samples were included. Using culture as the gold standard, 28 were true positives, 12 were false positives and 92 were true negatives. The area under the curve for the pretreatment time point was 96.8% (95% confidence interval [CI] 94.2-99.5) compared with 74.3% (95% CI 52.6-96.0) for pooled data during treatment. Before treatment, a Ct cut-off of value between culture-positive and culture-negative samples during treatment. Ct values prior to treatment differed significantly between the true-positive and false-positive groups ( P = 0.0056). There was a significant difference between the pretreatment and first and second negative culture time points ( P = 0.0002 and P values for true positives and true negatives, and for pre- and intra-treatment time points. Conclusions and relevance Ct values had limited usefulness for distinguishing between culture-positive and culture-negative cases when field study samples were analyzed. In addition, Ct values were less reliable than fungal culture for determining mycological cure.

  1. Optimization of conditions to extract high quality DNA for PCR analysis from whole blood using SDS-proteinase K method

    Directory of Open Access Journals (Sweden)

    Wajhul Qamar

    2017-11-01

    Full Text Available In case of studies associated with human genetics, genomics, and pharmacogenetics the genomic DNA is extracted from the buccal cells, whole blood etc. Several methods are exploited by the researchers to extract DNA from the whole blood. One of these methods, which utilizes cell lysis and proteolytic properties of sodium dodecyl sulfate (SDS and proteinase K respectively, might also be called SDS-PK method. It does not include any hazardous chemicals such as phenol or chloroform and is inexpensive. However, several researchers report the same method with different formulas and conditions. During our experiments with whole blood DNA extraction we experienced problems such as protein contamination, DNA purity and yield when followed some SDS-PK protocols reported elsewhere. A260/A280 and A260/A230 ratios along with PCR amplification give a clear idea about the procedure that was followed to extract the DNA. In an effort to increase the DNA purity from human whole blood, we pointed out some steps of the protocol that play a crucial role in determining the extraction of high quality DNA.

  2. Socio-cultural barriers to voluntary blood donation for obstetric use ...

    African Journals Online (AJOL)

    'Not being strong enough' and 'not having enough blood' were the two major reasons for declining blood donation, while loss of manhood/libido and exposure of blood to witchcraft were the other reasons given. Respondents' level of awareness of HIV/AIDS was appreciable. Socio-cultural barriers to voluntary blood ...

  3. Mycobacterium tuberculosis bacteremia detected by the Isolator lysis-centrifugation blood culture system.

    OpenAIRE

    Kiehn, T E; Gold, J W; Brannon, P; Timberger, R J; Armstrong, D

    1985-01-01

    Mycobacterium tuberculosis was detected by the Isolator lysis-centrifugation blood culture system from the blood of a patient with tuberculosis of the breast. The organism also grew on conventional laboratory media inoculated with pleural fluid from the patient.

  4. Comparison of the EntericBio multiplex PCR system with routine culture for detection of bacterial enteric pathogens.

    LENUS (Irish Health Repository)

    O'Leary, James

    2009-11-01

    The EntericBio system uses a multiplex PCR assay for the simultaneous detection of Campylobacter spp., Salmonella enterica, Shigella spp., and Escherichia coli O157 from feces. It combines overnight broth enrichment with PCR amplification and detection by hybridization. An evaluation of this system was conducted by comparing the results obtained with the system with those obtained by routine culture, supplemented with alternative PCR detection methods. In a study of 773 samples, routine culture and the EntericBio system yielded 94.6 and 92.4% negative results, respectively. Forty-two samples had positive results by culture, and all of these were positive with the EntericBio system. This system detected an additional 17 positive samples (Campylobacter spp., n = 12; Shigella spp., n = 1; E. coli O157, n = 4), but the results for 5 samples (Campylobacter spp., n = 2; Shigella spp., n = 1; E. coli O157, n = 2) could not be confirmed. The target for Shigella spp. detected by the EntericBio system is the ipaH gene, and the molecular indication of the presence of Shigella spp. was investigated by sequence analysis, which confirmed that the ipaH gene was present in a Klebsiella pneumoniae isolate from the patient. The sensitivity, specificity, positive predictive value, and negative predictive value were 100%, 99.3%, 91.5%, and 100%, respectively. Turnaround times were significantly reduced with the EntericBio system, and a result was available between 24 and 32 h after receipt of the sample in the laboratory. In addition, the amount of laboratory waste was significantly reduced by use of this system. In summary, the EntericBio system proved convenient to use, more sensitive than the conventional culture used in this study, and highly specific; and it generated results significantly faster than routine culture for the pathogens tested.

  5. Prospective European-wide multicentre study on a blood based real-time PCR for the diagnosis of acute schistosomiasis

    Directory of Open Access Journals (Sweden)

    Wichmann Dominic

    2013-01-01

    Full Text Available Abstract Background Acute schistosomiasis constitutes a rare but serious condition in individuals experiencing their first prepatent Schistosoma infection. To circumvent costly and time-consuming diagnostics, an early and rapid diagnosis is required. So far, classic diagnostic tools such as parasite microscopy or serology lack considerable sensitivity at this early stage of Schistosoma infection. To validate the use of a blood based real-time polymerase chain reaction (PCR test for the detection of Schistosoma DNA in patients with acute schistosomiasis who acquired their infection in various endemic regions we conducted a European-wide prospective study in 11 centres specialized in travel medicine and tropical medicine. Methods Patients with a history of recent travelling to schistosomiasis endemic regions and freshwater contacts, an episode of fever (body temperature ≥38.5°C and an absolute or relative eosinophil count of ≥700/μl or 10%, were eligible for participation. PCR testing with DNA extracted from serum was compared with results from serology and microscopy. Results Of the 38 patients with acute schistosomiasis included into the study, PCR detected Schistosoma DNA in 35 patients at initial presentation (sensitivity 92%. In contrast, sensitivity of serology (enzyme immunoassay and/or immunofluorescence assay or parasite microscopy was only 70% and 24%, respectively. Conclusion For the early diagnosis of acute schistosomiasis, real-time PCR for the detection of schistosoma DNA in serum is more sensitive than classic diagnostic tools such as serology or microscopy, irrespective of the region of infection. Generalization of the results to all Schistosoma species may be difficult as in the study presented here only eggs of S. mansoni were detected by microscopy. A minimum amount of two millilitre of serum is required for sufficient diagnostic accuracy.

  6. A real time PCR assay on blood for diagnosis of invasive candidiasis in immunocompromised patient

    Directory of Open Access Journals (Sweden)

    Mohsen Ashrafi

    2015-01-01

    Results: From 2009 to 2011, 72 patients with hematologic malignancies and bone marrow transplant recipients were evaluated for IC. The female to male ratio was 27:45; the mean age was 32.1 years. The most common malignancy in this patient was acute myeloid leukemia (AML (27.8% and acute lymphoblastic leukemia (ALL (26.4%. Out of 72 patients, 11 patients (15.3% had positive real time PCR /probe results. Based on the melting temperature (Tm analysis, 5 (45.4% C. krusei, 3 (27.2% C. tropicalis, 2 (18.1% C. parapsilosis and 1 C. albicans (9% were identified. According to the revised EORTC / MSG, 1 patient (9% and 10 patients (91% were defined as proven and possible groups of IC, respectively. The mortality rate in proven and possible IC patient was found 54.5%. Conclusion: The established Real-time PCR/FRET probe assay is an appropriate diagnostic tool for the detection of Candida species DNA and the management of patients suffering from hematologic malignancies and bone marrow recipient are at risk for IC.

  7. Quantification of fungal abundance on cultural heritage using real time PCR targeting the β-actin gene.

    Science.gov (United States)

    Ettenauer, Jörg; Piñar, Guadalupe; Tafer, Hakim; Sterflinger, Katja

    2014-01-01

    The traditional methodology used for the identification of microbes colonizing our cultural heritage was the application of cultivation methods and/or microscopy. This approach has many advantages, as living microorganisms may be obtained for physiological investigations. In addition, these techniques allow the quantitative and qualitative assessment of the investigated environment. Quantitative analyses are done by plate count and the determination of abundance by the colony forming unit (CFU). Nevertheless, these techniques have many drawbacks that lead to an underestimation of the cell numbers and do not provide a comprehensive overview of the composition of the inhabiting microbiota. In the last decades, several molecular techniques have been developed enabling many advantages over the cultivation approach. Mainly PCR-based, fingerprinting techniques allow a qualitative detection and identification of the microbiota. In this study, we developed a real time PCR method as a simple, rapid and reliable tool to detect and quantify fungal abundance using the β-actin gene, which is known to appear as a single-copy gene in fungi. To this end, five different indoor thermal insulation materials applied for historical buildings that were previously tested for their bio-susceptibility against various fungi were subjected to qPCR analyses. The obtained results were compared with those obtained from a previous study investigating the bio-susceptibility of the insulation materials using classical cultivation experiments. Both results correlated well, revealing that Perlite plaster was the most suitable insulation material, showing the lowest fungal CFU and qPCR values. In contrast, insulations made of wood showed to be not recommendable from the microbiological point of view. In addition, the potential of qPCR was tested in other materials of cultural heritage, as old parchments, showing to be a suitable method for measuring fungal abundance in these delicate materials.

  8. Blood culture collection technique and pneumococcal surveillance in Malawi during the four year period 2003–2006: an observational study

    Directory of Open Access Journals (Sweden)

    Zijlstra Eduard E

    2008-10-01

    Full Text Available Abstract Background Blood culture surveillance will be used for assessing the public health effectiveness of pneumococcal conjugate vaccines in Africa. Between 2003 and 2006 we assessed blood culture outcome and performance in adult patients in the central public hospital in Blantyre, Malawi, before and after the introduction of a dedicated nurse led blood culture team. Methods A prospective observational study. Results Following the introduction of a specialised blood culture team in 2005, the proportion of contaminated cultures decreased (19.6% in 2003 to 5.0% in 2006, blood volume cultured increased and pneumococcal recovery increased significantly from 2.8% of all blood cultures to 6.1%. With each extra 1 ml of blood cultured the odds of recovering a pneumococcus increased by 18%. Conclusion Standardisation and assessment of blood culture performance (blood volume and contamination rate should be incorporated into pneumococcal disease surveillance activities where routine blood culture practice is constrained by limited resources.

  9. Time-to-Detection Comparison for a Novel Blood Culture System Using Simulated Blood Cultures: DLTM versus BacT/ALERTTM and BACTECTM.

    Science.gov (United States)

    Demiray, Tayfur; Koroglu, Mehmet; Altindis, Mustafa

    2016-09-01

    Automated blood culture systems are routinely used in microbiology laboratories to isolate bacteria and fungi causing bloodstream infections. A novel automated blood culture system, DL-Bt112TM (DL) was compared with the BACTEC 9050TM (BCT) and BacT/Alert 3DTM (B3D) systems for time-to-detection (TTD) using 10 different clinical bacteria that commonly cause bloodstream infections. Simulated blood cultures were used to compare the three automated blood culture systems. Blood drawn from healthy donors was inoculated with known concentrations of 10 different species of commonly isolated bacteria and analysed using the automated systems. TTD values for the three systems were recorded and analysed. Significant differences in the TTD were observed among the three systems. The DL system exhibited the longest detection time of all the systems (p significance difference was observed between the BCT and the B3D systems when overall TTD values were evaluated; however, the BCT system yielded significantly better results than did the B3D system for the Gram-positive bacteria. TTD values were longer for the DL system than for the two commonly used blood culture systems when tested on simulated blood cultures. Thus, clinical laboratories considering the DL system should take its long TTD into consideration.

  10. Development of quantitative PCR and metagenomics-based approaches for strain quantification of a defined mixed-strain starter culture.

    Science.gov (United States)

    Johansen, Pernille; Vindeløv, Jannik; Arneborg, Nils; Brockmann, Elke

    2014-05-01

    Although the strain composition of mixed cultures may hugely affect production of various fermented foods, such as e.g. cheese, tools for investigating it have so far been limited. In this study, two new approaches for quantification of seven Lactococcus lactis subsp. cremoris strains (S1-S7) in a defined mixed-strain starter culture were developed and verified. By mapping NGS reads from 47 sequenced L. lactis strains to de novo assembly contigs of the seven strains, two strain-specific sequence regions (SEQ1 and SEQ2) were identified for each strain for qPCR primer design (A1 and A2). The qPCR assays amplified their strain-specific sequence region target efficiently. Additionally, high reproducibility was obtained in a validation sample containing equal amounts of the seven strains, and assay-to-assay coefficients of variance (CVs) for six (i.e. S1, S2, S4-S7) of the seven strains correlated to the inter-plate CVs. Hence, at least for six strains, the qPCR assay design approach was successful. The metagenomics-based approach quantified the seven strains based on average coverage of SEQ1 and SEQ2 by mapping sequencing reads from the validation sample to the strain-specific sequence regions. Average coverages of the SEQ1 and SEQ2 in the metagenomics data showed CVs of ≤17.3% for six strains (i.e. S1-S4, S6, S7). Thus, the metagenomics-based quantification approach was considered successful for six strains, regardless of the strain-specific sequence region used. When comparing qPCR- and metagenomics-based quantifications of the validation sample, the identified strain-specific sequence regions were considered suitable and applicable for quantification at a strain level of defined mixed-strain starter cultures. Copyright © 2014 Elsevier GmbH. All rights reserved.

  11. Comparison of Performance Characteristics of Aspergillus PCR in Testing a Range of Blood-Based Samples in Accordance with International Methodological Recommendations

    Science.gov (United States)

    Springer, Jan; Hamilton, Shanna; Michel, Denise; Barnes, Rosemary A.; Einsele, Hermann; Löffler, Juergen

    2016-01-01

    Standardized methodologies for the molecular detection of invasive aspergillosis (IA) have been established by the European Aspergillus PCR Initiative for the testing of whole blood, serum, and plasma. While some comparison of the performance of Aspergillus PCR when testing these different sample types has been performed, no single study has evaluated all three using the recommended protocols. Standardized Aspergillus PCR was performed on 423 whole-blood pellets (WBP), 583 plasma samples, and 419 serum samples obtained from hematology patients according to the recommendations. This analysis formed a bicenter retrospective anonymous case-control study, with diagnosis according to the revised European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) consensus definitions (11 probable cases and 36 controls). Values for clinical performance using individual and combined samples were calculated. For all samples, PCR positivity was significantly associated with cases of IA (for plasma, P = 0.0019; for serum, P = 0.0049; and for WBP, P = 0.0089). Plasma PCR generated the highest sensitivity (91%); the sensitivities for serum and WBP PCR were 80% and 55%, respectively. The highest specificity was achieved when testing WBP (96%), which was significantly superior to the specificities achieved when testing serum (69%, P = 0.0238) and plasma (53%, P = 0.0002). No cases were PCR negative in all specimen types, and no controls were PCR positive in all specimens. This study confirms that Aspergillus PCR testing of plasma provides robust performance while utilizing commercial automated DNA extraction processes. Combining PCR testing of different blood fractions allows IA to be both confidently diagnosed and excluded. A requirement for multiple PCR-positive plasma samples provides similar diagnostic utility and is technically less demanding. Time

  12. Real-time PCR strategy for the identification of Trypanosoma cruzi discrete typing units directly in chronically infected human blood.

    Science.gov (United States)

    Muñoz-San Martín, Catalina; Apt, Werner; Zulantay, Inés

    2017-04-01

    The protozoan Trypanosoma cruzi is the causative agent of Chagas disease, a major public health problem in Latin America. This parasite has a complex population structure comprised by six or seven major evolutionary lineages (discrete typing units or DTUs) TcI-TcVI and TcBat, some of which have apparently resulted from ancient hybridization events. Because of the existence of significant biological differences between these lineages, strain characterization methods have been essential to study T. cruzi in its different vectors and hosts. However, available methods can be laborious and costly, limited in resolution or sensitivity. In this study, a new genotyping strategy by real-time PCR to identify each of the six DTUs in clinical blood samples have been developed and evaluated. Two nuclear (SL-IR and 18S rDNA) and two mitochondrial genes (COII and ND1) were selected to develop original primers. The method was evaluated with eight genomic DNA of T. cruzi populations belonging to the six DTUs, one genomic DNA of Trypanosoma rangeli, and 53 blood samples from individuals with chronic Chagas disease. The assays had an analytical sensitivity of 1-25fg of DNA per reaction tube depending on the DTU analyzed. The selectivity of trials with 20fg/μL of genomic DNA identified each DTU, excluding non-targets DTUs in every test. The method was able to characterize 67.9% of the chronically infected clinical samples with high detection of TcII followed by TcI. With the proposed original genotyping methodology, each DTU was established with high sensitivity after a single real-time PCR assay. This novel protocol reduces carryover contamination, enables detection of each DTU independently and in the future, the quantification of each DTU in clinical blood samples. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Antibiotic Use in Thailand: Quantifying Impact on Blood Culture Yield and Estimates of Pneumococcal Bacteremia Incidence

    OpenAIRE

    Rhodes, Julia; Hyder, Joseph A.; Peruski, Leonard F.; Fisher, Cindy; Jorakate, Possawat; Kaewpan, Anek; Dejsirilert, Surang; Thamthitiwat, Somsak; Olsen, Sonja J.; Dowell, Scott F.; Chantra, Somrak; Tanwisaid, Kittisak; Maloney, Susan A.; Baggett, Henry C.

    2010-01-01

    No studies have quantified the impact of pre-culture antibiotic use on the recovery of individual blood-borne pathogens or on population-level incidence estimates for Streptococcus pneumoniae. We conducted bloodstream infection surveillance in Thailand during November 2005?June 2008. Pre-culture antibiotic use was assessed by reported use and by serum antimicrobial activity. Of 35,639 patient blood cultures, 27% had reported pre-culture antibiotic use and 24% (of 24,538 tested) had serum anti...

  14. High-throughput qRT-PCR validation of blood microRNAs in non-small cell lung cancer.

    Science.gov (United States)

    Leidinger, Petra; Brefort, Thomas; Backes, Christina; Krapp, Medea; Galata, Valentina; Beier, Markus; Kohlhaas, Jochen; Huwer, Hanno; Meese, Eckart; Keller, Andreas

    2016-01-26

    Validation of biomarkers is essential to advance the translational process to clinical application. Although there exists an increasing number of reports on small non-coding RNAs (microRNAs) as minimally-invasive markers from blood, serum or plasma, just a limited number is verified in follow-up studies. We used qRT-PCR to evaluate a known miRNA signature measured from blood that allowed for separation between patients with non-small cell lung cancer (NSCLC), COPD and healthy controls.From the data of our previous microarray studies we selected a panel of 235 miRNAs related to lung cancer and COPD. We observed a high concordance between the AUC values of our initial microarray screening and the qRT-PCR data (correlation of 0.704, p < 10-16). Overall, 90.3% of markers were successfully validated. Among the top markers that were concordant between both studies we found hsa-miR-20b-5p, hsa-miR-20a-5p, hsa-miR-17-5p, and hsa-miR-106a-5p. The qRT-PCR analysis also confirmed that non-small cell lung cancer patients could be accurately differentiated from unaffected controls: a subset of five markers was sufficient to separate NSCLC patients from unaffected controls with accuracy of 94.5% (specificity and sensitivity of 98% and 91%). Beyond differentiation from controls, we also succeeded in separating NSCLC patients from patients with COPD. MiRNAs that were identified as relevant for the separation between lung cancer and COPD by both qRT-PCR and the array-based studies included hsa-miR-26a-5p, hsa-miR-328-3p and hsa-miR-1224-3p. Although for differentiation between NSCLC patients from COPD patients more markers were required, still high accuracy rates were obtained (5 markers: 78.8%; 10 markers: 83.9%; 50 markers: 87.6%).

  15. Legionella Bozemanae, a New Cause of Septic Arthritis diagnosed by 16S PCR followed by specific culture

    DEFF Research Database (Denmark)

    Just, Søren Andreas; Bonde Knudsen, John; Skov, Marianne Nielsine

    This is the first report ever to demonstrate that L. Bozemanae can colonize synovial joints leading to infectious arthritis. L. Bozemanae is a rare Legionella species, earlier described as a cause of cavitating lung infections with up to 40% mortality (2). L. Bozemanae is missed by standard cultu...... cultures, however, the case was diagnosed by combining 16S PCR and sequencing followed by culture under specific conditions, a method which may help in the diagnosis of septic arthritis caused by unusual pathogens not detected by standard culture.......This is the first report ever to demonstrate that L. Bozemanae can colonize synovial joints leading to infectious arthritis. L. Bozemanae is a rare Legionella species, earlier described as a cause of cavitating lung infections with up to 40% mortality (2). L. Bozemanae is missed by standard...

  16. PCR-based allelic discrimination for glucose-6-phosphate dehydrogenase (G6PD) deficiency in Ugandan umbilical cord blood.

    Science.gov (United States)

    Hsu, Jennifer; Fink, Deanna; Langer, Erica; Carter, Michelle L; Bengo, Derrik; Ndidde, Susan; Slusher, Tina; Ross, Julie A; Lund, Troy C

    2014-02-01

    Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common X-linked disorder in the world. G6PD deficiency puts children at risk for hyperbilirubinemia and kernicterus during the newborn period and an increased risk of severe hemolysis after exposure to many antimalarial medications. A laboratory diagnosis of G6PD deficiency is rare in the developing world due to limited resources. We developed a TaqMan-based allele-specific assay to rapidly determine rates of G6PD deficiency contributing alleles (G202A and A376G) in East Africa. We tested umbilical cord blood from 100 Ugandan newborns and found that the overall allele frequency of G202A was .13 and A376G was .32. The overall incidence of G6PD A- (G202A/A376G) was 6%; all A- variants were males. There was no correlation between G6PD deficiency and umbilical cord blood hemoglobin, white blood count, platelet count, or other hematologic parameters. Allele-specific PCR can serve as a rapid method to determine specific G6PD deficiency allele frequencies in a given population and as a diagnostic tool in a hospital setting in which laboratory resources are present.

  17. Analysis of performance of a PCR-based assay to detect DNA of Aspergillus fumigatus in whole blood and serum: a comparative study with clinical samples.

    Science.gov (United States)

    Bernal-Martínez, Leticia; Gago, Sara; Buitrago, María J; Gomez-Lopez, Alicia; Rodríguez-Tudela, Juan L; Cuenca-Estrella, Manuel

    2011-10-01

    The performance of a real-time PCR-based assay was retrospectively analyzed (according to European Organization for Research and Treatment of Cancer/Mycosis Study Group criteria) in the samples of patients with invasive aspergillosis. A total of 711 serial samples (356 whole-blood and 355 serum samples) from 38 adult patients were analyzed. The Aspergillus fumigatus PCR assay results were positive for 89 of 356 (25%) whole-blood samples and 90 of 355 (25.35%) serum samples. Positive PCR results were seen in 29 of 31 (93.5%) patients for which serum was analyzed and in 31 of 33 (93.9%) cases with whole-blood specimens. Both blood and serum samples were available in 26 cases, and significant differences were not observed in this subgroup of cases. The average number of threshold cycles (C(T)) for positive blood samples was 37.6, and the average C(T) for serum was 37.4. The DNA concentration ranged between 2 and 50 fg per μl of sample, with average DNA concentrations of 10.2 and 11.7 fg in positive blood and serum samples, respectively (P > 0.01). The performance of this PCR-based quantitative assay was similar for both serum and blood samples. We recommend serum samples as the most convenient hematological sample to use for Aspergillus DNA quantification when serial determinations are done.

  18. Comparison of DNA probe, PCR amplification, ELISA and culture methods for the rapid detection of Salmonella in poultry

    International Nuclear Information System (INIS)

    Qasem, J.A.; Al-Mouqati, S.; Rajkumar, G.

    2005-01-01

    The identification of Salmonella spp. from poultry meat was studied by comparing bacterial detection using the Gene-Trak colorimetric hybridization method, a PCR amplification kit and an Enzyme Linked Immunosorbent Assay (ELISA), and these methods were compared with the conventional methodology proposed by the United States Food and Drug Administration (US FDA) for detection of Salmonella in food samples. Forty positive and negative samples were studied. The three methods yielded similar results with levels of Salmonella greater than 10 CFU per sample, even when the samples were highly contaminated with competing bacteria. In contrast, 20 CFU of seed inoculum per sample was the lowest level of Salmonella detectable with all three methods and the standard culture method. The detection limits of the PCR and ELISA assays were 5 CFU/g after enrichment at 37 deg. C for 6 and 9 hours, respectively. Compared with conventional bacteriology, all three methods here demonstrated high sensitivity and specificity for Salmonella. (author)

  19. Comparison of the Bactec Peds Plus pediatric blood culture vial with Roche pediatric Septi-Chek for blood cultures from pediatric patients.

    OpenAIRE

    Welby, P L; Zusag, T M; Storch, G A

    1992-01-01

    Equal volumes of blood from 4,112 blood cultures were inoculated into one Bactec Peds Plus bottle and one Roche Septi-Chek bottle. There were no significant differences in the recoveries of bloodstream pathogens. Initial detection occurred earlier with Bactec Peds Plus, while growth on solid media occurred earlier with Roche Septi-Chek.

  20. Detection of Toxoplasma gondii DNA in peripheral blood and aqueous humor of patients with Toxoplasmic active focal necrotizing retinochoroiditis using real-time PCR

    Directory of Open Access Journals (Sweden)

    Fabio Felipe dos Santos

    2015-12-01

    Full Text Available ABSTRACT Purpose: To evaluate the ability of real-time quantitative PCR (qPCR for detectingToxoplasma gondii DNA in the peripheral blood and aqueous humor of patients with toxoplasmic active focal necrotizing retinochoroiditis. Methods: Fifty-five patients with infectious uveitis seen from 2009 to 2013 at the Department of Ophthalmology and Visual Sciences of the Federal University of São Paulo were enrolled in this study. Forty-three patients had toxoplasmic active focal necrotizing retinochoroiditis, and the remaining 12 had non-toxoplasmic infectious uveitis and served as controls. qPCR analysis forT. gondii DNA was performed on the patients' peripheral blood and aqueous humor samples. Results: The qPCR was positive for T. gondii DNA in 37.21% (16/43 of the aqueous humor samples and 2.33% (1/43 of the peripheral blood samples; further, 16.27% (7/43 of the patients had positive results in both their blood and aqueous humor samples. Conclusion: qPCR was able to detect T. gondii DNA in patients with toxoplasmic active focal necrotizing retinochoroiditis in the blood as well as the aqueous humor and can help with the diagnosis of the disease.

  1. Blood culture bottles are superior to lysis-centrifugation tubes for bacteriological diagnosis of spontaneous bacterial peritonitis.

    OpenAIRE

    Siersema, P D; de Marie, S; van Zeijl, J H; Bac, D J; Wilson, J H

    1992-01-01

    The conventional method of ascitic fluid culturing was compared with the bedside inoculation of ascites into blood culture bottles and into lysis-centrifugation tubes. The conventional culture method was compared with the blood culture bottle method in 31 episodes of spontaneous bacterial peritonitis (SBP). Cultures were positive with the conventional culture method in 11 (35%) episodes and with the blood culture bottle method in 26 (84%) episodes (P less than 0.001). The lysis-centrifugation...

  2. Detection of Campylobacter jejuni by culture and real-time PCR in a French cohort of patients with Guillain-Barre syndrome.

    Science.gov (United States)

    Sivadon-Tardy, Valérie; Orlikowski, David; Porcher, Raphael; Ronco, Esthel; Caudie, Christiane; Roussi, Jacqueline; Fauchère, Jean-Louis; Mégraud, Francis; Tabor, Helen; Sharshar, Tarek; Annane, Djillali; Raphaël, Jean-Claude; Gaillard, Jean-Louis

    2010-06-01

    Bacteriological culture and real-time PCR (RT-PCR) were used to detect Campylobacter jejuni in fecal samples from a French cohort of 237 patients with Guillain-Barré syndrome (GBS). We provide evidence that diverse serotypes and genotypes of C. jejuni are a major trigger of GBS in France.

  3. Comparison of EBV DNA viral load in whole blood, plasma, B-cells and B-cell culture supernatant.

    Science.gov (United States)

    Ouedraogo, David Eric; Bollore, Karine; Viljoen, Johannes; Foulongne, Vincent; Reynes, Jacques; Cartron, Guillaume; Vendrell, Jean-Pierre; Van de Perre, Philippe; Tuaillon, Edouard

    2014-05-01

    Epstein-Barr virus (EBV) genome quantitation in whole blood is used widely for therapeutic monitoring of EBV-associated disorders in immunosuppressed individuals and in patients with EBV-associated lymphoma. However, the most appropriate biological material to be used for EBV DNA quantitation remains a subject of debate. This study compare the detection rate and levels of EBV DNA from whole blood, plasma, enriched B-cells, and B-cell short-term culture supernatant using quantitative real-time PCR. Samples were collected from 33 subjects with either HIV infection or B-cell lymphoma. Overall, EBV DNA was detected in 100% of enriched B-cell samples, in 82% of B-cell culture supernatants, in 57% of plasma, and 42% of whole blood samples. A significant correlation for EBV viral load was found between enriched B-cell and B-cell culture supernatant material (ρ = 0.92; P < 0.0001), but no significant correlation existed between EBV DNA levels in whole blood and enriched B-cells (ρ = -0.02; P = 0.89), whole blood and plasma (ρ = 0.24; P = 0.24), or enriched B-cells and plasma (ρ = 0.08; P = 0.77). Testing of enriched B-cells appeared to be the most sensitive method for detection of EBV DNA as well as for exploration of the cellular reservoir. Quantitation of EBV DNA in plasma and B-cell culture supernatant may be of interest to assess EBV reactivation dynamics and response to treatment as well as to decipher EBV host-pathogen interactions in various clinical scenarios. © 2013 Wiley Periodicals, Inc.

  4. Incidence of Listeria species in bovine, ovine, caprine, camel and water buffalo milk using cultural method and the PCR assay

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    Ebrahim Rahimi

    2014-02-01

    Full Text Available Objective: To determine the prevalence rate of Listeria species in bovine, ovine, caprine, camel and water buffalo milk in Iran. Methods: From September 2010 to December 2011 a total of 260 bulk milk samples including 85 bovine, 37 camel, 34 water buffalo, 56 ovine and 48 caprine bulk milk samples were collected from commercial dairy herds, in Fars and Khuzestan provinces, Iran and were evaluated for the presence of Listeria species using cultural method and the PCR assay. Results: Using cultural method, 19 samples (7.3% were positive for Listeria spp. The highest prevalence of Listeria was found in raw water buffalo milk (11.8%, followed by raw bovine milk (10.6%, raw ovine milk (7.1%, and raw caprine milk (4.2% samples. All 37 camel milk samples from 20 camel breeding farms were negative for Listeria spp. The overall prevalence of Listeria was 7.3%, in which Listeria innocua was the most recovered species (4.2%; the remaining isolates were Listeria monocytogenes (1.9%, Listeria ivanovii (0.08% and Listeria seeligari (0.04%. The PCR assay could identify 8 Listeria-contaminated milk samples that were negative using the cultural method. Conclusions: The results presented in this study indicate the potential risk of infection with Listeria in people consuming raw and unpasteurized milk.

  5. Women with symptoms of a urinary tract infection but a negative urine culture: PCR-based quantification of Escherichia coli suggests infection in most cases.

    Science.gov (United States)

    Heytens, S; De Sutter, A; Coorevits, L; Cools, P; Boelens, J; Van Simaey, L; Christiaens, T; Vaneechoutte, M; Claeys, G

    2017-09-01

    Our objective was to examine whether or not women with symptoms of a urinary tract infection but with a negative culture (20%-30%) do have an infection. We performed quantitative PCR (qPCR) for Escherichia coli and Staphylococcus saprophyticus, on top of a standard culture, in urine samples from 220 women with dysuria and/or frequency and/or urgency and from 86 women without symptoms. For symptomatic women, qPCR was also carried out for four sexually transmitted agents. In the symptomatic group, 80.9% (178/220) of the urine cultures were positive for any uropathogen and 95.9% (211/220) were E. coli qPCR-positive. For the control group, cultures for E. coli and E. coli qPCR were positive in, respectively, 10.5% (9/86) and 11.6% (10/86). In the symptomatic group, qPCR yielded 19 positive samples for S. saprophyticus qPCR, one positive sample for Mycoplasma genitalium and one for Trichomonas vaginalis. These findings suggest that almost all women with typical urinary complaints and a negative culture still have an infection with E. coli. Copyright © 2017 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

  6. Clinical impact of preincubation of blood cultures at 37 degrees C.

    NARCIS (Netherlands)

    Velden, L.B. van der; Vos, F.J.; Mouton, J.W.; Sturm, P.D.J.

    2011-01-01

    The effect of immediate incubation of blood cultures at 37 degrees C on the turnaround time and the impact of Gram stain results on antimicrobial management were investigated. During a 6-month period, blood cultures collected at the emergency department outside laboratory operating hours were

  7. Effect of the initial specimen diversion technique on blood culture contamination rates.

    Science.gov (United States)

    Binkhamis, Khalifa; Forward, Kevin

    2014-03-01

    The initial specimen diversion technique (ISDT) was first described by Patton and Schmitt (J. Clin. Microbiol. 48:4501-4503, 2010, doi:10.1128/JCM.00910-10). This study looked at the effect of implementation of the ISDT on blood culture contamination rates at our center. We found a reduction of 30.34% in potential blood culture contaminants.

  8. Use of PCR and culture to detect Helicobacter pylori in naturally infected cats following triple antimicrobial therapy.

    Science.gov (United States)

    Perkins, S E; Yan, L L; Shen, Z; Hayward, A; Murphy, J C; Fox, J G

    1996-01-01

    Helicobacter pylori causes gastritis and peptic ulcers and is linked to gastric cancer. Domestic cats from a commercial source were found to be naturally infected with H. pylori, and studies were undertaken to eradicate H. pylori from infected cats by using triple antimicrobial therapy. Eight cats infected with H. pylori were used in the study. Six cats received a 21-day course of oral amoxicillin, metronidazole, and omeprazole, and two cats served as controls. Two weeks and 4 weeks posttreatment (p.t.), all six treated cats were negative at several sites (saliva, gastric juice, and gastric mucosa) for H. pylori by culture. However, as determined by PCR with primers specific for the 26-kDa product, the majority of cats at 2 and 4 weeks p.t. had gastric fluid samples which were positive for H. pylori and three of three cats at 2 weeks p.t. had dental plaque which was positive for H. pylori. At 6 weeks p.t., all six cats had H. pylori-negative cultures for samples from several gastric sites taken at necropsy, and only one cat had H. pylori cultured from gastric juice. PCR analysis revealed that five of six cats had H. pylori DNA amplification products from plaque, saliva, and/or gastric fluid samples. Negative bacterial cultures for cats for which there was demonstrable PCR amplification of H. pylori DNA may reflect the inability of in vitro culture techniques to isolate small numbers of H. pylori organisms, focal colonization at sites not cultured, or a failure of the antibiotics to successfully eradicate H. pylori from extragastric sites which allowed subsequent recolonization of the stomach after cessation of therapy. Alternatively, the treatment strategy may have induced in vivo viable but nonculturable coccoid forms of H. pylori. The H. pylori cat model should allow further studies to test these hypotheses as well as the efficacies of other combined therapeutic regimens. Also, because 100% of these cats were naturally infected with H.pylori, this model should

  9. Real-time polymerase chain reaction with melting analysis of positive blood culture specimens in bloodstream infections: diagnostic value and turnaround time.

    Science.gov (United States)

    Angeletti, Silvia; Gherardi, Giovanni; De Florio, Lucia; Avola, Alessandra; Crea, Francesca; Riva, Elisabetta; Vitali, Massimiliano Andrea; Galluzzo, Sara; Dicuonzo, Giordano

    2013-01-01

    A Real-time polymerase chain reaction (PCR) with melting analysis was devised to target bacterial and fungal genes together with the most prevalent antimicrobial resistance genes in 250 positive blood culture broths. This method allowed the blood culture cultivated pathogens to be classified into clinically relevant groups such as Enterobacteriaceae, oxidase-positive bacilli, oxidase-positive coccobacilli, S. aureus and yeast. Enterococci and streptococci could be distinguished from CoNS only by the Gram stain. Gram-positive bacilli were discriminated from Gram-positive cocci by Gram stain. Furthermore, the most important antimicrobial resistant genes such as mecA, vanA, bla TEM , bla SHV and bla CTX-M could be identified. All results were obtained with a turnaround time of three hours from the moment of blood culture positivity compared to 24-72 hours for phenotypic methods. In conclusion, the proposed approach can allow the clinician to implement proper early management of sepsis patients.

  10. Rapid Identification of Pathogenic Fungi Directly from Cultures by Using Multiplex PCR

    OpenAIRE

    Luo, Guizhen; Mitchell, Thomas G.

    2002-01-01

    A multiplex PCR method was developed to identify simultaneously multiple fungal pathogens in a single reaction. Five sets of species-specific primers were designed from the internal transcribed spacer (ITS) regions, ITS1 and ITS2, of the rRNA gene to identify Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis, and Aspergillus fumigatus. Another set of previously published ITS primers, CN4 and CN5, were used to identify Cryptococcus neoformans. Three sets of primers w...

  11. Blood culture in India: A proposal for a national programme for early detection of sepsis

    Directory of Open Access Journals (Sweden)

    Bhattacharya S

    2005-01-01

    Full Text Available Septicaemia is a major contributor of mortality. Blood culture is the essential investigation for the management of sepsis. Due to lack of resources blood culture is an irregularly used investigation in India. A three-tier level of development is being proposed to develop the blood culture based national programme for early detection of sepsis. The plan envisages the establishment of manual blood culture based elementary system in the health centre and district hospital level (Level 1, direct Gram stain and direct antibiotic sensitivity testing from the "positive" blood culture broths at the medical college hospital level (Level 2 and development of automated methods, enhancement of quality control and safety measures, clinical liaison and re-orientation of microbiology training at the tertiary care centre level (Level 3.

  12. Genetic screening of spinal muscular atrophy using a real-time modified COP-PCR technique with dried blood-spot DNA.

    Science.gov (United States)

    Ar Rochmah, Mawaddah; Harahap, Nur Imma Fatimah; Niba, Emma Tabe Eko; Nakanishi, Kenta; Awano, Hiroyuki; Morioka, Ichiro; Iijima, Kazumoto; Saito, Toshio; Saito, Kayoko; Lai, Poh San; Takeshima, Yasuhiro; Takeuchi, Atsuko; Bouike, Yoshihiro; Okamoto, Maya; Nishio, Hisahide; Shinohara, Masakazu

    2017-10-01

    Spinal muscular atrophy (SMA) is a common neuromuscular disorder caused by mutations in SMN1. More than 95% of SMA patients carry homozygous SMN1 deletion. SMA is the leading genetic cause of infant death, and has been considered an incurable disease. However, a recent clinical trial with an antisense oligonucleotide drug has shown encouraging clinical efficacy. Thus, early and accurate detection of SMN1 deletion may improve prognosis of many infantile SMA patients. A total of 88 DNA samples (37 SMA patients, 12 carriers and 39 controls) from dried blood spots (DBS) on filter paper were analyzed. All participants had previously been screened for SMN genes by PCR restriction fragment length polymorphism (PCR-RFLP) using DNA extracted from freshly collected blood. DNA was extracted from DBS that had been stored at room temperature (20-25°C) for 1week to 5years. To ensure sufficient quality and quantity of DNA samples, target sequences were pre-amplified by conventional PCR. Real-time modified competitive oligonucleotide priming-PCR (mCOP-PCR) with the pre-amplified PCR products was performed for the gene-specific amplification of SMN1 and SMN2 exon 7. Compared with PCR-RFLP using DNA from freshly collected blood, results from real-time mCOP-PCR using DBS-DNA for detection of SMN1 exon 7 deletion showed a sensitivity of 1.00 (CI [0.87, 1.00])] and specificity of 1.00 (CI [0.90, 1.00]), respectively. We combined DNA extraction from DBS on filter paper, pre-amplification of target DNA, and real-time mCOP-PCR to specifically detect SMN1 and SMN2 genes, thereby establishing a rapid, accurate, and high-throughput system for detecting SMN1-deletion with practical applications for newborn screening. Copyright © 2017 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.

  13. A Comparative Study of Blood Culture Sampling from Umbilical Catheter Line versus Peripheral Site

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    Abdolkarim Hamedi

    2010-08-01

    Full Text Available Neonatal sepsis is an important cause of death and morbidity in newborns and is diagnosed by isolation of organism in blood culture. In several reports,reliablity of blood cultures were done from umbi lical catheters,have been demonstrated. The objective of the present study was to determine,wether an inde welling umbilical catheter, could be an alternative site for blood culture. In a prospective study over 6 months during 2006,141 paired blood cultures from 134 infant,were done simultaneously from peripheral site and umbilical catheter (mostly U. V. C,during the first four days of life. Majority of these infants were preterm and admitted to NICU for special care. these infants had indwelling umbilical line and had indication of sepsis workup. A total of 141 pairs of blood cultures were obtained from 134 infants. In 16 infants blood culture pairs were positive for one organism in both peripheral vein and umbilical site. 71. 6% of total cultures (n=11pairs were negative in boths site. A total of 22 pairs were positive in one site only,with 5 positive from peripheral vein only and the other 17 from umblical site. Two pairs were positve in boths site with two different organism. In over all 16 infant (11%of blood were considered to be contaminated. Contamination rate were 2. 4% and 9. 2% for peripheral and umbilical catheter site. Contamination rate increased after 48 hours of age in umbilical catheter. The result showed that after 2 days contamination rate for blood culture taken from catheter line increased and specifity decreased. We recommended that blood culture via umblical catheter in first 2 days in sick neonates with indwelling catheter can be a alternate site of blood culture sampelling.

  14. Evaluation of Phadebact and Streptex Kits for rapid grouping of streptococci directly from blood cultures.

    OpenAIRE

    Wellstood, S

    1982-01-01

    The Phadebact Streptococcus Test and the Streptex Test kits were evaluated for grouping streptococci directly from blood cultures. Pellets of bacteria obtained from centrifuged samples of positive blood cultures were inoculated into Todd-Hewitt broth for 2- and 4-h Phadebact tests and into pronase for Streptex tests. Hemolysis was determined after pipetting a portion of each pellet into cuts made in blood agar plates incubated anaerobically for 2 to 6 h. Serological groups were also determine...

  15. Antibody levels correlate with detection of Trypanosoma cruzi DNA by sensitive PCR assays in seropositive blood donors and possible resolution of infection over time

    Science.gov (United States)

    Sabino, E.C.; Lee, T.H.; Montalvo, L.; Nguyen, M.L.; Leiby, D.A.; Carrick, D.M.; Otani, M.M.; Vinelli, E.; Wright, D.; Stramer, S.L.; Busch, M.

    2013-01-01

    Background The clinical significance of anti-T. cruzi low-level reactive samples is incompletely understood. PCR-positive rates and antibody levels among seropositive blood donors in three countries are described. Methods Follow-up whole blood and plasma samples were collected from T. cruzi-seropositive donors from 2008-2010 in the US (n=195) and Honduras (n=58). Also 143 samples from Brazil in 1996-2002, originally positive by three serological assays, were available and paired with contemporary follow-up samples from these donors. All samples were retested with the FDA-approved Ortho ELISA. PCR assays were performed on coded sample panels by two laboratories (BSRI and ARC) that amplified kinetoplast minicircle DNA sequences of T. cruzi. Results PCR testing at BSRI yielded slightly higher overall sensitivity and specificity (33% and 98%) compared with the ARC lab (28% and 94%). Among seropositive donors, PCR-positive rates varied by country (p<0.0001) for the BSRI laboratory: Brazil (57%), Honduras (32%) and the US (14%). ELISA signal/cutoff (S/CO) ratios were significantly higher for PCR-positive compared to PCR-negative donors (p<0.05 for all comparisons). Additionally, PCR-negative Brazilian donors exhibited greater frequencies of antibody decline over time versus PCR-positive donors (p=0.003). Conclusion For all three countries, persistent DNA positivity correlated with higher ELISA S/CO values, suggesting that high-level seroreactivity reflects chronic parasitemia. The higher rate of PCR positivity for Brazilian donors was likely attributable to required reactivity on three assays available a decade ago. Significant S/CO declines in 10% of the PCR-negative Brazilian donors may indicate seroreversion following parasite clearance in the absence of treatment. PMID:23002996

  16. Taqman real-time PCR detects Avipoxvirus DNA in blood of Hawai'i 'amakihi (Hemignathus virens.

    Directory of Open Access Journals (Sweden)

    Margaret E M Farias

    Full Text Available BACKGROUND: Avipoxvirus sp. is a significant threat to endemic bird populations on several groups of islands worldwide, including Hawai'i, the Galapagos Islands, and the Canary Islands. Accurate identification and genotyping of Avipoxvirus is critical to the study of this disease and how it interacts with other pathogens, but currently available methods rely on invasive sampling of pox-like lesions and may be especially harmful in smaller birds. METHODOLOGY/PRINCIPAL FINDINGS: Here, we present a nested TaqMan Real-Time PCR for the detection of the Avipoxvirus 4b core protein gene in archived blood samples from Hawaiian birds. The method was successful in amplifying Avipoxvirus DNA from packed blood cells of one of seven Hawaiian honeycreepers with confirmed Avipoxvirus infections and 13 of 28 Hawai'i 'amakihi (Hemignathus virens with suspected Avipoxvirus infections based on the presence of pox-like lesions. Mixed genotype infections have not previously been documented in Hawai'i but were observed in two individuals in this study. CONCLUSIONS/SIGNIFICANCE: We anticipate that this method will be applicable to other closely related strains of Avipoxvirus and will become an important and useful tool in global studies of the epidemiology of Avipoxvirus.

  17. Detection of hydrogen peroxide-producing Lactobacillus species in the vagina: a comparison of culture and quantitative PCR among HIV-1 seropositive women

    Directory of Open Access Journals (Sweden)

    Balkus Jennifer E

    2012-08-01

    Full Text Available Abstract Background The presence of hydrogen peroxide (H2O2 producing Lactobacillus in the vagina may play a role in controlling genital HIV-1 shedding. Sensitive molecular methods improve our ability to characterize the vaginal microbiota; however, they cannot characterize phenotype. We assessed the concordance of H2O2-producing Lactobacillus detected by culture with quantitative PCR (qPCR detection of Lactobacillus species commonly assumed to be H2O2-producers. Methods Samples were collected as part of a prospective cohort study of HIV-1 seropositive US women. Cervicovaginal lavage specimens were tested for L. crispatus and L. jensenii using 16S rRNA gene qPCR assays. Vaginal swabs were cultured for Lactobacillus and tested for H2O2-production. We calculated a kappa statistic to assess concordance between culture and qPCR. Results Culture and qPCR results were available for 376 visits from 57 women. Lactobacilli were detected by culture at 308 (82% visits, of which 233 of 308 (76% produced H2O2. L. crispatus and/or L. jensenii were detected at 215 (57% visits. Concordance between detection of L. crispatus and/or L. jensenii by qPCR and H2O2-producing Lactobacillus by culture was 75% (kappa = 0.45. Conclusions Among HIV-1 seropositive women, there was a moderate level of concordance between H2O2-producing Lactobacillus detected by culture and the presence of L. crispatus and/or L. jensenii by qPCR. However, one-quarter of samples with growth of H2O2-producing lactobacilli did not have L. crispatus or L. jensenii detected by qPCR. This discordance may be due to the presence of other H2O2-producing Lactobacillus species.

  18. Comparison of false-negative rates and limits of detection following macrofoam-swab sampling of Bacillus anthracis surrogates via Rapid Viability PCR and plate culture.

    Science.gov (United States)

    Hutchison, J R; Piepel, G F; Amidan, B G; Hess, B M; Sydor, M A; Deatherage Kaiser, B L

    2018-01-21

    We evaluated the effects of Bacillus anthracis surrogates, low surface concentrations, surface materials and assay methods on false-negative rate (FNR) and limit of detection (LOD 95 ) for recovering Bacillus spores using a macrofoam-swab sampling procedure. Bacillus anthracis Sterne or Bacillus atrophaeus Nakamura spores were deposited over a range of low target concentrations (2-500 per coupon) onto glass, stainless steel, vinyl tile and plastic. Samples were assayed using a modified Rapid Viability-PCR (mRV-PCR) method and the traditional plate culture method to obtain FNR and LOD 95 results. Mean FNRs tended to be lower for mRV-PCR compared to culturing, and increased as spore concentration decreased for all surface materials. Surface material, but not B. anthracis surrogate, influenced FNRs with the mRV-PCR method. The mRV-PCR LOD 95 was lowest for glass and highest for vinyl tile. LOD 95 values overall were lower for mRV-PCR than for the culture method. This study adds to the limited data on FNR and LOD 95 for mRV-PCR and culturing methods with low concentrations of B. anthracis sampled from various surface materials by the CDC macrofoam-swab method. These are key inputs for planning characterization and clearance studies for low contamination levels of B. anthracis. © 2018 The Society for Applied Microbiology.

  19. Comparison of false-negative rates and limits of detection following macrofoam-swab sampling of Bacillus anthracis surrogates via Rapid Viability PCR and plate culture

    Energy Technology Data Exchange (ETDEWEB)

    Hutchison, Janine R.; Piepel, Gregory F.; Amidan, Brett G.; Hess, Becky M.; Sydor, Michael A.; Kaiser, Brooke LD

    2018-03-13

    Aims: We evaluated the effects of Bacillus anthracis surrogates, low surface concentrations, surface materials, and assay methods on false-negative rate (FNR) and limit of detection (LOD95) for recovering Bacillus spores using a macrofoam-swab sampling procedure. Methods and Results: Bacillus anthracis Sterne or Bacillus atrophaeus Nakamura spores were deposited over a range of low target concentrations (2 – 500 coupon-1) onto glass, stainless steel, vinyl tile, and plastic. Samples were assayed using a modified Rapid Viability-PCR (mRV-PCR) method and the traditional plate culture method to obtain FNR and LOD95 results. Conclusions: Mean FNRs tended to be lower for mRV-PCR compared to culturing, and increased as spore concentration decreased for all surface materials. Surface material, but not B. anthracis surrogate, influenced FNRs with the mRV-PCR method. The mRV-PCR LOD95 was lowest for glass and highest for vinyl tile. LOD95 values overall were lower for mRV-PCR than for the culture method. Significance and Impact of Study: This study adds to the limited data on FNR and LOD95 for mRV-PCR and culturing methods with low concentrations of B. anthracis sampled from various surface materials by the CDC macrofoam-swab method. These are key inputs for planning characterization and clearance studies for low contamination levels of B. anthracis.

  20. Beyond Blood Culture and Gram Stain Analysis: A Review of Molecular Techniques for the Early Detection of Bacteremia in Surgical Patients.

    Science.gov (United States)

    Scerbo, Michelle H; Kaplan, Heidi B; Dua, Anahita; Litwin, Douglas B; Ambrose, Catherine G; Moore, Laura J; Murray, Col Clinton K; Wade, Charles E; Holcomb, John B

    2016-06-01

    Sepsis from bacteremia occurs in 250,000 cases annually in the United States, has a mortality rate as high as 60%, and is associated with a poorer prognosis than localized infection. Because of these high figures, empiric antibiotic administration for patients with systemic inflammatory response syndrome (SIRS) and suspected infection is the second most common indication for antibiotic administration in intensive care units (ICU)s. However, overuse of empiric antibiotics contributes to the development of opportunistic infections, antibiotic resistance, and the increase in multi-drug-resistant bacterial strains. The current method of diagnosing and ruling out bacteremia is via blood culture (BC) and Gram stain (GS) analysis. Conventional and molecular methods for diagnosing bacteremia were reviewed and compared. The clinical implications, use, and current clinical trials of polymerase chain reaction (PCR)-based methods to detect bacterial pathogens in the blood stream were detailed. BC/GS has several disadvantages. These include: some bacteria do not grow in culture media; others do not GS appropriately; and cultures can require up to 5 d to guide or discontinue antibiotic treatment. PCR-based methods can be potentially applied to detect rapidly, accurately, and directly microbes in human blood samples. Compared with the conventional BC/GS, particular advantages to molecular methods (specifically, PCR-based methods) include faster results, leading to possible improved antibiotic stewardship when bacteremia is not present.

  1. Procalcitonin as a marker of Candida species detection by blood culture and polymerase chain reaction in septic patients.

    Science.gov (United States)

    Cortegiani, Andrea; Russotto, Vincenzo; Montalto, Francesca; Foresta, Grazia; Accurso, Giuseppe; Palmeri, Cesira; Raineri, Santi Maurizio; Giarratano, Antonino

    2014-02-21

    The aim of our study is to test procalcitonin (PCT) as surrogate marker of identification of Candida spp. by blood culture (BC) and real-time-polymerase chain reaction (PCR), whether alone or in association with bacteria, in septic patients. We performed a single-centre retrospective study. We reviewed the clinical charts of patients with a diagnosis of severe sepsis or septic shock treated at our general intensive care unit from March 2009 to March 2013. We analysed all diagnostic episodes consisting of BC, real-time PCR assay and dosage of PCT. We registered age, sex, white blood count, sequential organ failure assessment score and type of admission between medical or surgical. When inclusion criteria were met more than once, we registered the new diagnostic episode as subsequent diagnostic episode. The diagnostic performance of PCT to predict Candida spp. identification alone or in mixed infections by either BC or PCR was tested using the receiver-operative characteristic curve. Logistic regression was constructed using presence of Candida spp. as the dependent variable. A total of 260 diagnostic episodes met the inclusion criteria. According to BC results classification, a significantly lower value of PCT was observed in Candida spp. BSI (0.99 ng/ml, 0.86 - 1.34) than in BSI caused by bacteria (16.7 ng/ml, 7.65 - 50.2) or in mixed infections (4.76 ng/ml, 2.98 - 6.08). Similar findings were observed considering PCR results. A cut-off of ≤ 6.08 ng/ml for PCT yielded a sensitivity of 86.8%, a specificity of 87.4%, a positive predictive value of 63.9%, a negative predictive value (NPV) of 96.3% and an area under the curve of 0.93 for Candida spp. identification by BC. A similar high NPV for a cut-off ≤ 6.78 ng/ml was observed considering the classification of diagnostic episodes according to PCR results, with an AUC of 0.85. A subsequent diagnostic episode was independently associated with Candida spp. detection either by BC or PCR. PCT could

  2. EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. Part III. Virus Detection by RT-qPCR

    Science.gov (United States)

    EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. The viral ribonucleic acid (RNA) from water sample concentrates is extracted and tested for enterovirus and norovirus RNA using reverse transcription-quantitative PCR (RT-qPCR). V...

  3. Analytical Validation of Quantitative Real-Time PCR Methods for Quantification of Trypanosoma cruzi DNA in Blood Samples from Chagas Disease Patients.

    Science.gov (United States)

    Ramírez, Juan Carlos; Cura, Carolina Inés; da Cruz Moreira, Otacilio; Lages-Silva, Eliane; Juiz, Natalia; Velázquez, Elsa; Ramírez, Juan David; Alberti, Anahí; Pavia, Paula; Flores-Chávez, María Delmans; Muñoz-Calderón, Arturo; Pérez-Morales, Deyanira; Santalla, José; Marcos da Matta Guedes, Paulo; Peneau, Julie; Marcet, Paula; Padilla, Carlos; Cruz-Robles, David; Valencia, Edward; Crisante, Gladys Elena; Greif, Gonzalo; Zulantay, Inés; Costales, Jaime Alfredo; Alvarez-Martínez, Miriam; Martínez, Norma Edith; Villarroel, Rodrigo; Villarroel, Sandro; Sánchez, Zunilda; Bisio, Margarita; Parrado, Rudy; Maria da Cunha Galvão, Lúcia; Jácome da Câmara, Antonia Cláudia; Espinoza, Bertha; Alarcón de Noya, Belkisyole; Puerta, Concepción; Riarte, Adelina; Diosque, Patricio; Sosa-Estani, Sergio; Guhl, Felipe; Ribeiro, Isabela; Aznar, Christine; Britto, Constança; Yadón, Zaida Estela; Schijman, Alejandro G

    2015-09-01

    An international study was performed by 26 experienced PCR laboratories from 14 countries to assess the performance of duplex quantitative real-time PCR (qPCR) strategies on the basis of TaqMan probes for detection and quantification of parasitic loads in peripheral blood samples from Chagas disease patients. Two methods were studied: Satellite DNA (SatDNA) qPCR and kinetoplastid DNA (kDNA) qPCR. Both methods included an internal amplification control. Reportable range, analytical sensitivity, limits of detection and quantification, and precision were estimated according to international guidelines. In addition, inclusivity and exclusivity were estimated with DNA from stocks representing the different Trypanosoma cruzi discrete typing units and Trypanosoma rangeli and Leishmania spp. Both methods were challenged against 156 blood samples provided by the participant laboratories, including samples from acute and chronic patients with varied clinical findings, infected by oral route or vectorial transmission. kDNA qPCR showed better analytical sensitivity than SatDNA qPCR with limits of detection of 0.23 and 0.70 parasite equivalents/mL, respectively. Analyses of clinical samples revealed a high concordance in terms of sensitivity and parasitic loads determined by both SatDNA and kDNA qPCRs. This effort is a major step toward international validation of qPCR methods for the quantification of T. cruzi DNA in human blood samples, aiming to provide an accurate surrogate biomarker for diagnosis and treatment monitoring for patients with Chagas disease. Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

  4. Analytical Validation of Quantitative Real-Time PCR Methods for Quantification of Trypanosoma cruzi DNA in Blood Samples from Chagas Disease Patients

    Science.gov (United States)

    Ramírez, Juan Carlos; Cura, Carolina Inés; Moreira, Otacilio da Cruz; Lages-Silva, Eliane; Juiz, Natalia; Velázquez, Elsa; Ramírez, Juan David; Alberti, Anahí; Pavia, Paula; Flores-Chávez, María Delmans; Muñoz-Calderón, Arturo; Pérez-Morales, Deyanira; Santalla, José; Guedes, Paulo Marcos da Matta; Peneau, Julie; Marcet, Paula; Padilla, Carlos; Cruz-Robles, David; Valencia, Edward; Crisante, Gladys Elena; Greif, Gonzalo; Zulantay, Inés; Costales, Jaime Alfredo; Alvarez-Martínez, Miriam; Martínez, Norma Edith; Villarroel, Rodrigo; Villarroel, Sandro; Sánchez, Zunilda; Bisio, Margarita; Parrado, Rudy; Galvão, Lúcia Maria da Cunha; da Câmara, Antonia Cláudia Jácome; Espinoza, Bertha; de Noya, Belkisyole Alarcón; Puerta, Concepción; Riarte, Adelina; Diosque, Patricio; Sosa-Estani, Sergio; Guhl, Felipe; Ribeiro, Isabela; Aznar, Christine; Britto, Constança; Yadón, Zaida Estela; Schijman, Alejandro G.

    2015-01-01

    An international study was performed by 26 experienced PCR laboratories from 14 countries to assess the performance of duplex quantitative real-time PCR (qPCR) strategies on the basis of TaqMan probes for detection and quantification of parasitic loads in peripheral blood samples from Chagas disease patients. Two methods were studied: Satellite DNA (SatDNA) qPCR and kinetoplastid DNA (kDNA) qPCR. Both methods included an internal amplification control. Reportable range, analytical sensitivity, limits of detection and quantification, and precision were estimated according to international guidelines. In addition, inclusivity and exclusivity were estimated with DNA from stocks representing the different Trypanosoma cruzi discrete typing units and Trypanosoma rangeli and Leishmania spp. Both methods were challenged against 156 blood samples provided by the participant laboratories, including samples from acute and chronic patients with varied clinical findings, infected by oral route or vectorial transmission. kDNA qPCR showed better analytical sensitivity than SatDNA qPCR with limits of detection of 0.23 and 0.70 parasite equivalents/mL, respectively. Analyses of clinical samples revealed a high concordance in terms of sensitivity and parasitic loads determined by both SatDNA and kDNA qPCRs. This effort is a major step toward international validation of qPCR methods for the quantification of T. cruzi DNA in human blood samples, aiming to provide an accurate surrogate biomarker for diagnosis and treatment monitoring for patients with Chagas disease. PMID:26320872

  5. A cytolethal distending toxin gene-based multiplex PCR assay for detection of Campylobacter spp. in stool specimens and comparison with culture method.

    Science.gov (United States)

    Shiramaru, Sachi; Asakura, Masahiro; Inoue, Haruna; Nagita, Akira; Matsuhisa, Akio; Yamasaki, Shinji

    2012-07-01

    In this study, we evaluated the applicability of cytolethal distending toxin (cdt) gene-based species-specific multiplex PCR for the direct detection and identification of Campylobacter jejuni, C. coli and C. fetus from stool specimens of patients with gastroenteritis in comparison to culture methods. A total of 711 stool specimens were examined for the isolation or detection of campylobacters by using Skirrow's selective agar culture plates, a filtration method and the multiplex PCR assay. Forty-one and 36 C. jejuni strains were isolated by culture and filtration methods, respectively. In addition, 2 and 3 C. coli strains were isolated by Skirrow and the filtration methods, respectively. However, when the multiplex PCR was employed, the cdtB genes of C. jejuni and C. coli were detected in 45 and 4 stool samples, respectively, and 9 C. jejuni PCR-positive samples by multiplex PCR were negative by culture method. Sequence analysis of the PCR products obtained from 8 stool specimens from which campylobacters were not isolated by culture method but the sequences exactly matched with that of the cdtB gene of C. jejuni strain 81-176. None of the remaining stool samples which were culture negative for campylobacters produced any amplicon. Stool samples were defined as Campylobacter-positive if detected by any method. The sensitivity of the multiplex PCR was 83%, which was higher than Skirrow (74%) and filtration method (66%). These data indicate that cdtB gene-based multiplex PCR is a rapid and more sensitive method to identify the most important species of Campylobacter for human diseases. (248).

  6. Storage time of platelet concentrates and risk of a positive blood culture

    DEFF Research Database (Denmark)

    Kreuger, Aukje L; Rostgaard, Klaus; Middelburg, Rutger A

    2018-01-01

    AND METHODS: We performed a nationwide cohort study among PLT transfusion recipients in Denmark between 2010 and 2012, as recorded in the Scandinavian Donations and Transfusions (SCANDAT2) database. Linking with a nationwide database on blood cultures (MiBa), we compared the incidence of a positive blood......BACKGROUND: Concern of transfusion-transmitted bacterial infections has been the major hurdle to extend shelf life of platelet (PLT) concentrates. We aimed to investigate the association between storage time and risk of positive blood cultures at different times after transfusion. STUDY DESIGN......) of a positive blood culture the day after transfusion of at least one old PLT concentrate was 0.77 (95% confidence interval [CI], 0.54-1.09) compared to transfusion of fresh PLT concentrates. The incidence rate of a positive blood culture was lower the day after receiving one old compared to one fresh PLT...

  7. Study on chromosome aberrations test determinated by micro-whole blood culture in vacuum blood collection tube

    International Nuclear Information System (INIS)

    Zhong Zhihong; Han Fang'an; Ge Qinjuan; Wu Xiao; Chen Juan

    2006-01-01

    Objective: To develop an easier and efficient method of culturing the chromosome and analyzing the aberrations in peripheral lymphocytes. Methods: Micro whole was cultured for 54 hours in home-made vacuum blood collection tube, and then collection, slice-making, microscopy detection for the chromosome aberrations was done. The difference of the results was analysed by comparing with the common method. Results: For 60 radiologists and 30 contrasts, the chromosome aberrations in peripheral lymphocytes were examed by this system, the lymphocytes and chromosome were clear and alive and easier to analyse. Compared with the common method, there was no significantly difference between the two analyzing results. Conclusion: The chromosome aberrations test by micro whole blood culture in vacuum blood collection tube is easier and efficient, and is worthy of being widely popularized. (authors)

  8. The impact of overcrowding on the bacterial contamination of blood cultures in the ED.

    Science.gov (United States)

    Lee, Ching-Chi; Lee, Nan-Yao; Chuang, Ming-Che; Chen, Po-Lin; Chang, Chia-Ming; Ko, Wen-Chien

    2012-07-01

    This study aims to determine the risk factors associated with the bacterial contamination of blood cultures among adults visiting the emergency department (ED). Clinical variables and medical records of adults with bacterial growth of blood cultures in the ED as well as the degree of ED crowding, between August 2007 and July 2008, were prospectively collected. Of the 11 491 adults who underwent blood culture sampling, the medical records of 558 (4.86%) eligible patients with bacterial growth in their blood cultures were analyzed. Most patients (366, or 3.19%) had true bacteremia, whereas 192 (1.67%) were regarded as contaminated. In multivariate analyses, ED overcrowding (scoring was based on a National Emergency Department Overcrowding Study [NEDOCS] score ≥ 100 points) was independently associated with blood culture contamination (odds ratio [OR], 1.58; P = .04). In contrast, other medical comorbidities, such as liver cirrhosis (OR, 0.31; P = .02), thrombocytopenia (100 mg/L; OR, 0.24; P overcrowded (60-100), overcrowded (100-140), severely overcrowded (140-180), and dangerously overcrowded (180-200), there was a strong correlation between blood culture contamination rates and the degrees of ED crowding (γ = 0.99, P overcrowding may have an adverse impact on the quality of clinical care, including increasing the risk of blood culture contamination. Copyright © 2012 Elsevier Inc. All rights reserved.

  9. New findings about trichodysplasia spinulosa-associated polyomavirus (TSPyV)--novel qPCR detects TSPyV-DNA in blood samples.

    Science.gov (United States)

    Urbano, Paulo R; Nali, Luiz H S; Bicalho, Camila S; Pierrotti, Ligia C; David-Neto, Elias; Pannuti, Cláudio S; Romano, Camila M

    2016-02-01

    A new real-time PCR assay for trichodysplasia spinulosa-associated polyomavirus (TSPyV) DNA detection was designed, and blood samples from kidney transplant recipients and healthy individuals were screened. TSPyV-DNA was not detected in blood from healthy individuals, but 26.8% of kidney recipients presented TSPyV-DNA. This is the first report of TSPyV viremia. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. Development of a PCR test for identification of Haemophilus somnus in pure and mixed cultures

    DEFF Research Database (Denmark)

    Angen, Øystein; Ahrens, Peter; Tegtmeier, Conny

    1998-01-01

    Based on the 16S rRNA sequences of a collection of well-characterized strains of Haemophilus somnus a set of primers was selected as candidates for a species-specific PCR test. All investigated H. somnus strains were found positive in the test, including 12 strains earlier found to represent H....... somnus by DNA-DNA hybridization as well as representatives of the 16 ribotypes previously described within this species. The specificity of the test was evaluated on a broad collection of strains within the family Pasteurellaceae and on other Gram positive and negative species. None of these strains gave...... for identification of bacteria belonging to this phenotypically heterogeneous and often slow growing species....

  11. Detection, identification and genotyping of Borrellia spp. in rodents in Slovenia by PCR and culture.

    Science.gov (United States)

    Cerar, Tjaša; Korva, Miša; Avšič-Županc, Tatjana; Ružić-Sabljić, Eva

    2015-08-08

    Borrelia burgdorferi sensu lato, the agent of Lyme borreliosis, is mainly maintained in natural foci through the transmission cycles of competent tick vectors (Ixodes sp.) and a vertebrate reservoir. Specific rodents have been identified as the principal reservoir of Borrelia burgdorferi sensu lato in Europe. Borrelia miyamotoi is the only relapsing fever spirochete transmitted by the same tick. The aim of the present study was to perform an epidemiological survey to determine the presence of B. burgdorferi sensu lato in rodents occurring in Slovenia and to explore the presence of Borrelia miyamotoi. The study was performed in two parts, retrospective and prospective; a total of 297 rodents was analyzed. Detection and identification of borrelia was performed by molecular methods and additionally in the prospective study by isolation and genotyping (MluI-LRFP and MLST). During the prospective part of the study, borrelia was isolated from 2/46 (4.3 %) lung specimens and from 10/46 (21.7 %) heart specimens of rodents. All isolated strains were identified as B. afzelii subtype Mla1, and MLST analysis revealed 5 distinct sequence types. Borrelia DNA was successfully detected by one or other of the PCR methods in 18/46 (39.1 %) and 75/251 (29.9 %) samples in the prospective and retrospective studies, respectively. LightMix® was found to be more sensitive than the ''in-house" nested PCR (91/297 (30.6 %) vs 48/297 (16.1 %)). Borrelia miyamotoi DNA was detected in 1/251 (0.4 %) and in 1/46 (2.2 %) heart specimens, in the retrospective and prospective parts of the study, respectively. We determined the prevalence of B. afzelii in rodents and report for the first time the presence of B. miyamotoi in Slovenia.

  12. Haematological changes in the blood of cultured Clarias gariepinus ...

    African Journals Online (AJOL)

    This study investigated the artifactual changes in the haematological values of Clarias gariepinus blood stored at room (32oC) and refrigerator (4oC) temperatures. Blood samples were collected from 12 apparently healthy fish weighing between 0.8 and 1kg. Samples were divided into two parts immediately after collection ...

  13. PCR-based pooling of dried blood spots for detection of malaria parasites: optimization and application to a cohort of Ugandan children.

    Science.gov (United States)

    Hsiang, Michelle S; Lin, Michael; Dokomajilar, Christian; Kemere, Jordan; Pilcher, Christopher D; Dorsey, Grant; Greenhouse, Bryan

    2010-10-01

    Sensitive, high-throughput methods to detect malaria parasites in low-transmission settings are needed. PCR-based pooling strategies may offer a solution. We first used laboratory-prepared samples to compare 2 DNA extraction and 4 PCR detection methods across a range of pool sizes and parasite densities. Pooled Chelex extraction of DNA, followed by nested PCR of cytochrome b, was the optimal strategy, allowing reliable detection of a single low-parasitemic sample (100 parasites/μl) in pool sizes up to 50. This PCR-based pooling strategy was then compared with microscopy using 891 dried blood spots from a cohort of 77 Ugandan children followed for 2 years in an urban setting of low endemicity. Among 419 febrile episodes, 35 cases of malaria were detected using the PCR-based pooling strategy and 40 cases using microscopy. All five cases of malaria not detected by PCR were from samples stored for >2 years with parasitemia of parasites using dried blood spots offers a sensitive and efficient approach for malaria surveillance in low-transmission settings, enabling improved detection of asymptomatic submicroscopic infections and dramatic savings in labor and costs.

  14. Clinical condition and comorbidity as determinants for blood culture positivity in patients with skin and soft-tissue infections

    NARCIS (Netherlands)

    van Daalen, F. V.; Kallen, M. C.; van den Bosch, C. M. A.; Hulscher, M. E. J. L.; Geerlings, S. E.; Prins, J. M.

    2017-01-01

    The utility of performing blood cultures in patients with a suspected skin infection is debated. We investigated the association between blood culture positivity rates and patients' clinical condition, including acute disease severity and comorbidity. We performed a retrospective study, including

  15. Evaluation of the Verigene® Blood Culture Nucleic Acid test for rapid identification of gram positive pathogens from positive blood cultures

    Directory of Open Access Journals (Sweden)

    Agnese Cellini

    2015-06-01

    Full Text Available Background. The rapid identification of the etiology and the evaluation of the antimicrobial susceptibility of the bacteria causing bacteremia is of outmost relevance to set up an adequate treatment of sepsis. In this study we evaluated the microarray based method, Verigene Gram-positive blood cultures (BC-GP nucleic acid test (Nanosphere Inc., Northbrook, IL, USA for the identification of Gram positive pathogens from positive blood cultures. The panel BC-GP is capable to identify 13 germs and 3 genes associated with antimicrobial resistance. Materials and Methods. In this study a total of 100 positive, non replicated and monomicrobic blood cultures have been evaluated. For testing on the Verigene platform using the BC-GP assay, 350 L of blood culture media from a positive the blood culture bottle.Results. A total of 100 positive blood cultures were tested by the Verigene BC-GP assay: out of these a total of 100 Gram-positive cocci were identified. The most frequent bacteria identified included staphylococci, streptococci and enterococci. Among staphylococci, Staphylococcus aureus accounted for 25% (15/60, with 38% of S. epidermidis 37% (23/60 and 37% (22/60 other CoNS. All the S. aureus isolates were correctly identified by BC-GP whereas in 2/45 cases (4% BC-GP misidentified CoNS. In the case of enterococci 7/10 were E. faecalis and 3 E. faecium, all of these were correctly identified.Conclusions. The overall agreement with the results obtained by standard procedure is quite elevated (88% and as a consequence the BC-GP panel could be used as a rapid diagnostic tool to give a faster response in the case of bacteremia associated with sepsis.

  16. Automated extraction of DNA from blood and PCR setup using a Tecan Freedom EVO liquid handler for forensic genetic STR typing of reference samples

    DEFF Research Database (Denmark)

    Stangegaard, Michael; Frøslev, Tobias G; Frank-Hansen, Rune

    2011-01-01

    17025 using the Qiagen MagAttract DNA Mini M48 kit (Qiagen GmbH, Hilden, Germany) from fresh whole blood and blood from deceased individuals. The workflow was simplified by returning the DNA extracts to the original tubes minimizing the risk of misplacing samples. The tubes that originally contained...... the samples were washed with MilliQ water before the return of the DNA extracts. The PCR was setup in 96-well microtiter plates. The methods were validated for the kits: AmpFlSTR Identifiler, SGM Plus and Yfiler (Applied Biosystems, Foster City, CA), GenePrint FFFL and PowerPlex Y (Promega, Madison, WI......). The automated protocols allowed for extraction and addition of PCR master mix of 96 samples within 3.5h. In conclusion, we demonstrated that (1) DNA extraction with magnetic beads and (2) PCR setup for accredited, forensic genetic short tandem repeat typing can be implemented on a simple automated liquid...

  17. Detection of Yersinia Enterocolitica Species in Pig Tonsils and Raw Pork Meat by the Real-Time Pcr and Culture Methods.

    Science.gov (United States)

    Stachelska, M A

    2017-09-26

    The aim of the present study was to establish a rapid and accurate real-time PCR method to detect pathogenic Yersinia enterocolitica in pork. Yersinia enterocolitica is considered to be a crucial zoonosis, which can provoke diseases both in humans and animals. The classical culture methods designated to detect Y. enterocolitica species in food matrices are often very time-consuming. The chromosomal locus _tag CH49_3099 gene, that appears in pathogenic Y. enterocolitica strains, was applied as DNA target for the 5' nuclease PCR protocol. The probe was labelled at the 5' end with the fluorescent reporter dye (FAM) and at the 3' end with the quencher dye (TAMRA). The real-time PCR cycling parameters included 41 cycles. A Ct value which reached a value higher than 40 constituted a negative result. The developed for the needs of this study qualitative real-time PCR method appeared to give very specific and reliable results. The detection rate of locus _tag CH49_3099 - positive Y. enterocolitica in 150 pig tonsils was 85 % and 32 % with PCR and culture methods, respectively. Both the Real-time PCR results and culture method results were obtained from material that was enriched during overnight incubation. The subject of the study were also raw pork meat samples. Among 80 samples examined, 7 ones were positive when real-time PCR was applied, and 6 ones were positive when classical culture method was applied. The application of molecular techniques based on the analysis of DNA sequences such as the Real-time PCR enables to detect this pathogenic bacteria very rapidly and with higher specificity, sensitivity and reliability in comparison to classical culture methods.

  18. Reproducibility of detection of tyrosinase and MART-1 transcripts in the peripheral blood of melanoma patients: a quality control study using real-time quantitative RT-PCR

    NARCIS (Netherlands)

    de Vries, T. J.; Fourkour, A.; Punt, C. J.; van de Locht, L. T.; Wobbes, T.; van den Bosch, S.; de Rooij, M. J.; Mensink, E. J.; Ruiter, D. J.; van Muijen, G. N.

    1999-01-01

    In recent years, large discrepancies were described in the success rate of the tyrosinase reverse transcription polymerase chain reaction (RT-PCR) for detecting melanoma cells in the peripheral blood of melanoma patients. We present a quality control study in which we analysed the reproducibility of

  19. Bayesian techniques for comparison of the test performance of PCR and culture for the identification of Campylobacter in enriched comminuted chicken samples.

    Science.gov (United States)

    Ebel, E D; Williams, M S; Golden, N J; Tankson, J

    2016-05-01

    Using Bayesian methods that do not require the definition of a gold standard, the diagnostic sensitivity and specificity of a real-time polymerase chain reaction (PCR) assay are compared to those of an enriched culture assay for detection of Campylobacter in enriched comminuted chicken samples. Food Safety and Inspection Service comminuted chicken samples were collected from production facilities across the United States. Enriched samples were examined using a commercial real-time PCR kit and plated for culture. Allowing for conditional dependence between these approaches and defining relatively uninformed prior distributions, the 'no gold standard' Bayesian methods generated estimates of the means (95% credible interval) of the posterior distributions for sensitivity and specificity of the PCR as 93% (79, 100%) and 95% (87, 100%) respectively. The estimated sensitivity implies a mean false negative frequency of 7%. The estimated means of the posterior distributions for sensitivity and specificity of the culture assay were 91% (76, 100%) and 96% (88, 100%) respectively. In this case, the mean false negative frequency is 9%. Graphical comparisons of the posterior distributions with their corresponding prior distributions suggested only subtle differences in the sensitivities of both tests, but the posterior distributions for specificities are substantially more certain than the prior distributions. The study suggests that the commercial real-time PCR assay is a more sensitive screening test that would provide timelier negative test results. The modest 1% reduction in specificity of this PCR assay, as compared to an enriched culture assay, is less of a concern for regulatory testing programs if a culture-based confirmatory assay is applied to all presumptive positive samples. The sensitivity and specificity of a PCR assay and a culture assay for Campylobacter in comminuted poultry produced in the United States were estimated. The PCR assay was shown to be an

  20. Polymerase chain reaction (PCR) identification of rodent blood meals confirms host sharing by flea vectors of plague.

    Science.gov (United States)

    Franklin, Heather A; Stapp, Paul; Cohen, Amybeth

    2010-12-01

    Elucidating feeding relationships between hosts and parasites remains a significant challenge in studies of the ecology of infectious diseases, especially those involving small or cryptic vectors. Black-tailed prairie dogs (Cynomys ludovicianus) are a species of conservation importance in the North American Great Plains whose populations are extirpated by plague, a flea-vectored, bacterial disease. Using polymerase chain reaction (PCR) assays, we determined that fleas (Oropsylla hirsuta) associated with prairie dogs feed upon northern grasshopper mice (Onychomys leucogaster), a rodent that has been implicated in the transmission and maintenance of plague in prairie-dog colonies. Our results definitively show that grasshopper mice not only share fleas with prairie dogs during plague epizootics, but also provide them with blood meals, offering a mechanism by which the pathogen, Yersinia pestis, may be transmitted between host species and maintained between epizootics. The lack of identifiable host DNA in a significant fraction of engorged Oropsylla hirsuta collected from animals (47%) and prairie-dog burrows (100%) suggests a rapid rate of digestion and feeding that may facilitate disease transmission during epizootics but also complicate efforts to detect feeding on alternative hosts. Combined with other analytical approaches, e.g., stable isotope analysis, molecular genetic techniques can provide novel insights into host-parasite feeding relationships and improve our understanding of the role of alternative hosts in the transmission and maintenance of disease. © 2010 The Society for Vector Ecology.

  1. Detection of Tilapia Lake Virus in Clinical Samples by Culturing and Nested Reverse Transcription-PCR

    OpenAIRE

    Kembou Tsofack, Japhette Esther; Zamostiano, Rachel; Watted, Salsabeel; Berkowitz, Asaf; Rosenbluth, Ezra; Mishra, Nischay; Briese, Thomas; Lipkin, W. Ian; Kabuusu, Richard M.; Ferguson, Hugh; del Pozo, Jorge; Eldar, Avi; Bacharach, Eran

    2017-01-01

    Tilapia are an important group of farmed fish that serve as a significant protein source worldwide. In recent years, substantial mortality of wild tilapia has been observed in the Sea of Galilee and in commercial ponds in Israel and Ecuador. We have identified the etiological agent of these mass die-offs as a novel orthomyxo-like virus and named it tilapia lake virus (TiLV). Here, we provide the conditions for efficient isolation, culturing, and quantification of the virus, including the use ...

  2. Comparison of bacterial culture and real-time PCR for the detection of Salmonella in grow-finish pigs in Western Canada using a Bayesian approach.

    Science.gov (United States)

    Wilkins, W; Waldner, C; Rajić, A; McFall, M; Muckle, A; Mainar-Jaime, R C

    2010-11-01

    The study objective was to evaluate the accuracy of a real-time polymerase chain reaction (RT-PCR) and a culture protocol used to detect Salmonella in the faeces of grow-finish pigs using a Bayesian approach. The RT-PCR was invA-gene-based assay, while the culture protocol included pre-enrichment in buffered peptone water, selective enrichment in tetrathionate and Rappaport-Vassiliadis broths, and isolation on semi-solid (modified semi-solid RV) or solid (XLT4, Rambach) agar plates. Bayesian analysis was performed using a two-test, two-population model with dependence between culture and RT-PCR and compared to a second model with conditional independence between these two tests. Two hundred and ninety three individual faecal and 294 pooled pen samples from grow-finish pig collected from 10 farms were tested and results were divided into two groups according to herd size (five herds 400 sows). In the dependence model, RT-PCR sensitivity (Se) and specificity (Sp) were estimated to be 90% (95% probability interval 74, 97) and 99% (98, 99), respectively. Culture Se was 92% (75, 99), while culture Sp was considered 100% as all culture-positive samples were confirmed by serotyping. In the conditional independence model, RT-PCR Se and Sp, and culture Se, were 96% (93, 98), 99% (98, 100) and 97% (94, 100), respectively. The dependence model resulted in posterior estimates of Se that were lower and with broader probability intervals than the independence model, indicating that when RT-PCR and culture are evaluated relative to each other, the correlation between these tests is an important source of bias and should be adjusted for during analysis. The RT-PCR evaluated in this study performed almost comparably to culture; given the cost savings associated with using this test and more timely results, the RT-PCR may be a useful alternative to culture for screening large numbers of samples, particularly when Salmonella prevalence is low. © 2010 Blackwell Verlag GmbH.

  3. Recent Progress in the Diagnosis of Pathogenic Candida Species in Blood Culture.

    Science.gov (United States)

    Phoompoung, Pakpoom; Chayakulkeeree, Methee

    2016-06-01

    Candidemia has become an emerging invasive fungal disease. Prompt treatment with appropriate antifungal agent is crucial to reduce the mortality of candidemia. The conventional blood culture method, which is considered the gold standard for candidemia diagnosis, has a low sensitivity and is time-consuming to perform. Recently, several novel advanced diagnostic methods that have a higher sensitivity and a shorter turnaround time than the conventional blood culture method have been developed for the early detection of Candida in blood samples or in blood culture broth. Most of these newer methods were developed using various molecular techniques, such as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, peptide nucleic acid fluorescence in situ hybridization, and a number of DNA-based techniques including in-house and commercial polymerase chain reactions. In this article, we review and summarize the novel molecular methods that have been recently used for the detection and identification of Candida organisms in blood specimens.

  4. Measurement of endotoxin levels in blood of hemodialysis Patients by 'Lal' test and comparision of its efficacy with blood culture

    Directory of Open Access Journals (Sweden)

    Gh Vazirzadeh

    2006-01-01

    Full Text Available Introduction: Presently, bacteremia is the principal cause of morbidity in patients undergoing hemodialysis. Gram-negative bacteria account for approximately 50 percent of documented infections. Endotoxins released during lysis of gram negative bacteremia result in inflammatory and defense response by the body and if not treated promptly result in septic shock and ultimately death of the patient. This study describes the detection of endotoxins in blood of patients with bacteremia due to gram - negative bacteria by LAL test. Method: Blood samples of 278 hemodialysis patients were analyzed in this study and pathogens were isolated from blood culture samples. Then, their antibiotic sensitivity was determined. In patients with positive blood culture, endotoxin levels were measured by LAL-test. Results: Frequency of bacteremia in patients was 13.6% . The prevalence of gram – negative bacteremia was 44.7%. E coli were the major pathogens, while staphylococcus aureus was the most common gram positive bacterium. Endotoxin was detected in 15 patients (3.8 ± 1.08 EU/ml . The sensitivity and specificity of endotoxins for gram – negative bacteremia were 88% and 95%, respectively. Conclusion: The results indicate that the LAL method is a fast, sensitive and simple method. There was no significant difference between the results of blood culture and LAL – test ( P > 0.05 .

  5. Anticoagulant Carryover May Influence Clot Formation in Direct Tube Coagulase Tests from Blood Cultures

    OpenAIRE

    Varettas, Kerry; Mukerjee, Chinmoy; Taylor, Peter C.

    2005-01-01

    The tube coagulase test (TCT) performed directly from positive blood culture bottles has been used to reduce the turnaround time for identifying Staphylococcus aureus. Most reports have shown the test to be specific but often lacking sufficient sensitivity to be useful. In a prospective study of blood culture bottles (BCB) signaling positive, with a Gram-stained smear showing gram-positive cocci resembling staphylococci, the sensitivity of the direct TCT was improved by diluting the BCB broth...

  6. The diagnostic utility of stabilized blood for detection of foot-and-mouth disease virus RNA by RT-qPCR

    DEFF Research Database (Denmark)

    S. Fontél, Kristina; Bøtner, Anette; Belsham, Graham

    In Europe, clinical signs indicative of foot-and-mouth disease (FMD), would immediately lead to collection of blood and relevant organ material for further laboratory examination for this vesicular disease virus. Today, the first line system for detection of virus in the sample material is real t...... time RT-PCR (RT-qPCR). The aim of this study was to investigate the diagnostic utility of stabilized blood for detection of FMDV RNA in this system.......In Europe, clinical signs indicative of foot-and-mouth disease (FMD), would immediately lead to collection of blood and relevant organ material for further laboratory examination for this vesicular disease virus. Today, the first line system for detection of virus in the sample material is real...

  7. The blood-brain barrier in vitro using primary culture

    DEFF Research Database (Denmark)

    Larsen, Annette Burkhart

    of the thesis involves the establishment and characterization of an in vitro BBB models based on primary cells isolated from the rat brain. Co-culture and triple culture models with astrocytes and pericytes were found to be the superior to mono cultured BCECs with respect to many important BBB characteristics...... obstacle for the treatment of central nervous system (CNS) diseases, as many potentially CNS active drugs are unable to reach their site of action within the brain. In vitro BBB models are, therefore, being developed to investigate the BBB permeability of a drug early in its development. The first part....... In the second part of the thesis, the ability of turning BCECs into protein factories is investigated using a non-viral gene carrier. Transfection and protein synthesis of BCECs cultured with confined BBB properties were found to be feasible without disrupting the BBB properties, although it was not possible...

  8. Tumor necrosis factor-alpha (TNF-alpha) concentrations from whole blood cultures correlate with isolated peripheral blood mononuclear cell cultures

    Science.gov (United States)

    Many cellular immune assays are impractical because they require labor-intensive isolation of cells from their natural environment. The objectives of this study were to determine the relationship between cell culture supernatant TNF-alpha from isolated peripheral blood mononuclear cells (PBMC) and w...

  9. Blood culture contamination in hospitalized pediatric patients: a single institution experience

    Science.gov (United States)

    Min, Hyewon; Park, Cheong Soo; Kim, Dong Soo

    2014-01-01

    Purpose Blood culture is the most important tool for detecting bacteremia in children with fever. However, blood culture contamination rates range from 0.6% to 6.0% in adults; rates for young children have been considered higher than these, although data are limited, especially in Korea. This study determined the contamination rate and risk factors in pediatric patients visiting the emergency room (ER) or being admitted to the ward. Methods We conducted a retrospective chart review of blood cultures obtained from children who visited Yonsei Severance Hospital, Korea between 2006 and 2010. Positive blood cultures were labeled as true bacteremia or contamination according to Centers for Disease Control and Prevention/National Healthcare Safety Network definitions for laboratory-confirmed bloodstream infection, after exclusion of cultures drawn from preexisting central lines only. Results Among 40,542 blood cultures, 610 were positive, of which 479 were contaminations and 131 were true bacteremia (overall contamination rate, 1.18%). The contamination rate in the ER was significantly higher than in the ward (1.32% vs. 0.66%, P6 years, respectively). Conclusion Overall, contamination rates were higher in younger children than in older children, given the difficulty of performing blood sampling in younger children. The contamination rates from the ER were higher than those from the ward, not accounted for only by overcrowding and lack of experience among personnel collecting samples. Further study to investigate other factors affecting contamination should be required. PMID:24868215

  10. Incorporation of real-time PCR into routine public health surveillance of culture negative bacterial meningitis in São Paulo, Brazil.

    Directory of Open Access Journals (Sweden)

    Claudio T Sacchi

    Full Text Available Real-time (RT-PCR increases diagnostic yield for bacterial meningitis and is ideal for incorporation into routine surveillance in a developing country. We validated a multiplex RT-PCR assay for Streptococcus pneumoniae, Neisseria meningitidis, and Haemophilus influenzae in Brazil. Risk factors for being culture-negative, RT-PCR positive were determined. The sensitivity of RT-PCR in cerebrospinal fluid (CSF was 100% (95% confidence limits, 96.0%-100% for N. meningitidis, 97.8% (85.5%-99.9% for S. pneumoniae, and 66.7% (9.4%-99.2% for H. influenzae. Specificity ranged from 98.9% to 100%. Addition of RT-PCR to routine microbiologic methods increased the yield for detection of S. pneumoniae, N. meningitidis, and H. influenzae cases by 52%, 85%, and 20%, respectively. The main risk factor for being culture negative and RT-PCR positive was presence of antibiotic in CSF (odds ratio 12.2, 95% CI 5.9-25.0. RT-PCR using CSF was highly sensitive and specific and substantially added to measures of meningitis disease burden when incorporated into routine public health surveillance in Brazil.

  11. Frequency and antibiotic resistance patterns of isolated bacteria from positive blood culture of hospitalized patients

    Directory of Open Access Journals (Sweden)

    Azadeh Vahedi

    2018-03-01

    Conclusion: The most prevalent bacterial isolate among the blood cultures of patients was Pseudomonas. The patients more than 50 years were more susceptible to blood stream infections. The most bacteria were isolated from the internal medicine department of hospital. The antibiotic resistance was also increasing especially in Acinetobacter, Staphylococcus coagulase negative, Escherichia coil and Klebsiella

  12. How to optimize the use of blood cultures for the diagnosis of bloodstream infections? A state-of-the art

    Directory of Open Access Journals (Sweden)

    Brigitte eLamy

    2016-05-01

    Full Text Available Bloodstream infection (BSI is a major cause of death in developed countries and the detection of microorganisms is essential in managing patients. Despite major progress has been made to improve identification of microorganisms, blood culture remains the gold standard and the first line tool for detecting BSIs. Consensus guidelines are available to ensure optimal BSI procedures, but blood culture practices often deviate from the recommendations. This review provides an update on clinical and technical issues related to blood collection and to blood culture performance, with a special focus on the blood sample strategy to optimize the sensitivity and specificity of blood cultures.

  13. Evaluation of MolYsis™ Complete5 DNA Extraction Method for Detecting Staphylococcus aureus DNA from Whole Blood in a Sepsis Model Using PCR/Pyrosequencing

    OpenAIRE

    McCann, Chase D.; Jordan, Jeanne A.

    2014-01-01

    Bacterial bloodstream infections (BSI) and ensuing sepsis are important causes of morbidity and mortality. Early diagnosis and rapid treatment with appropriate antibiotics are vital for improving outcome. Nucleic acid amplification of bacteria directly from whole blood has the potential of providing a faster means of diagnosing BSI than automated blood culture. However, effective DNA extraction of commonly low levels of bacterial target from whole blood is critical for this approach to be suc...

  14. Evaluation of PCR procedures for detecting and quantifying Leishmania donovani DNA in large numbers of dried human blood samples from a visceral leishmaniasis focus in Northern Ethiopia.

    Science.gov (United States)

    Abbasi, Ibrahim; Aramin, Samar; Hailu, Asrat; Shiferaw, Welelta; Kassahun, Aysheshm; Belay, Shewaye; Jaffe, Charles; Warburg, Alon

    2013-03-27

    Visceral Leishmaniasis (VL) is a disseminated protozoan infection caused by Leishmania donovani parasites which affects almost half a million persons annually. Most of these are from the Indian sub-continent, East Africa and Brazil. Our study was designed to elucidate the role of symptomatic and asymptomatic Leishmania donovani infected persons in the epidemiology of VL in Northern Ethiopia. The efficacy of quantitative real-time kinetoplast DNA/PCR (qRT-kDNA PCR) for detecting Leishmania donovani in dried-blood samples was assessed in volunteers living in an endemic focus. Of 4,757 samples, 680 (14.3%) were found positive for Leishmania k-DNA but most of those (69%) had less than 10 parasites/ml of blood. Samples were re-tested using identical protocols and only 59.3% of the samples with 10 parasite/ml or less were qRT-kDNA PCR positive the second time. Furthermore, 10.8% of the PCR negative samples were positive in the second test. Most samples with higher parasitemias remained positive upon re-examination (55/59 =93%). We also compared three different methods for DNA preparation. Phenol-chloroform was more efficient than sodium hydroxide or potassium acetate. DNA sequencing of ITS1 PCR products showed that 20/22 samples were Leishmania donovani while two had ITS1 sequences homologous to Leishmania major. Although qRT-kDNA PCR is a highly sensitive test, the dependability of low positives remains questionable. It is crucial to correlate between PCR parasitemia and infectivity to sand flies. While optimal sensitivity is achieved by targeting k-DNA, it is important to validate the causative species of VL by DNA sequencing.

  15. Blood meal identification and parasite detection in laboratory-fed and field-captured Lutzomyia longipalpis by PCR using FTA databasing paper

    Science.gov (United States)

    Sant’Anna, Mauricio R.V.; Jones, Nathaniel G.; Hindley, Jonathan A.; Mendes-Sousa, Antonio F.; Dillon, Rod J.; Cavalcante, Reginaldo R.; Alexander, Bruce; Bates, Paul A.

    2008-01-01

    The phlebotomine sand fly Lutzomyia longipalpis takes blood from a variety of wild and domestic animals and transmits Leishmania (Leishmania) infantum chagasi, etiological agent of American visceral leishmaniasis. Blood meal identification in sand flies has depended largely on serological methods but a new protocol described here uses filter-based technology to stabilise and store blood meal DNA, allowing subsequent PCR identification of blood meal sources, as well as parasite detection, in blood-fed sand flies. This technique revealed that 53.6% of field-collected sand flies captured in the back yards of houses in Teresina (Brazil) had fed on chickens. The potential applications of this technique in epidemiological studies and strategic planning for leishmaniasis control programmes are discussed. PMID:18606150

  16. Blood meal identification and parasite detection in laboratory-fed and field-captured Lutzomyia longipalpis by PCR using FTA databasing paper.

    Science.gov (United States)

    Sant'Anna, Mauricio R V; Jones, Nathaniel G; Hindley, Jonathan A; Mendes-Sousa, Antonio F; Dillon, Rod J; Cavalcante, Reginaldo R; Alexander, Bruce; Bates, Paul A

    2008-09-01

    The phlebotomine sand fly Lutzomyia longipalpis takes blood from a variety of wild and domestic animals and transmits Leishmania (Leishmania) infantum chagasi, etiological agent of American visceral leishmaniasis. Blood meal identification in sand flies has depended largely on serological methods but a new protocol described here uses filter-based technology to stabilise and store blood meal DNA, allowing subsequent PCR identification of blood meal sources, as well as parasite detection, in blood-fed sand flies. This technique revealed that 53.6% of field-collected sand flies captured in the back yards of houses in Teresina (Brazil) had fed on chickens. The potential applications of this technique in epidemiological studies and strategic planning for leishmaniasis control programmes are discussed.

  17. Blood-group-related carbohydrates are expressed in organotypic cultures of human skin and oral mucosa

    DEFF Research Database (Denmark)

    Grøn, B; Andersson, A; Dabelsteen, Erik

    1999-01-01

    the function of cell-surface carbohydrates, we established organotypic cultures of skin and buccal mucosa. In these cultures, keratinocytes are grown at the air-liquid interface on a supporting matrix consisting of homologous fibroblasts embedded in a collagen type I gel. We examined the expression of blood-group...

  18. The additional value of blood culture bottles in the diagnosis of endophthalmitis

    NARCIS (Netherlands)

    Tan, H. S.; Ghyczy-Carlborg, E. A. E.; Spanjaard, L.; de Smet, M. D.

    2011-01-01

    To assess the additional value of blood culture bottles (BCBs) in the diagnosis of endophthalmitis by comparing its culture yield with that of conventional media (CM). Retrospective consecutive case series. We included patients who were treated between January 2001 and January 2010 for clinically

  19. Effect of a training programme on blood culture contamination rate in critical care.

    Science.gov (United States)

    Sánchez-Sánchez, M M; Arias-Rivera, S; Fraile-Gamo, P; Jareño-Collado, R; López-Román, S; Vadillo-Obesso, P; García-González, S; Pulido-Martos, M T; Sánchez-Muñoz, E I; Cacho-Calvo, J; Martín-Pellicer, A; Panadero-Del Olmo, L; Frutos-Vivar, F

    2018-03-30

    Blood culture contamination can occur from extraction to processing; its rate should not exceed 3%. To evaluate the impact of a training programme on the rate of contaminated blood cultures after the implementation of sample extraction recommendations based on the best evidence. Prospective before-after study in a polyvalent intensive care unit with 18 beds. Two phases were established (January-June 2012, October 2012-October 2015) with a training period between them. Main recommendations: sterile technique, surgical mask, double skin disinfection (70° alcohol and 2% alcoholic chlorhexidine), 70° alcohol disinfection of culture flasks and injection of samples without changing needles. Including all blood cultures of patients with extraction request. demographic, severity, pathology, reason for admission, stay and results of blood cultures (negative, positive and contaminated). Basic descriptive statistics: mean (standard deviation), median (interquartile range) and percentage (95% confidence interval). Calculated contamination rates per 100 blood cultures extracted. Bivariate analysis between periods. Four hundred and eight patients were included. Eight hundred and forty-one blood cultures were taken, 33 of which were contaminated. In the demographic variables, severity, diagnosis and stay of patients with contaminated samples, no differences were observed from those with uncontaminated samples. Pre-training vs post-training contamination rates: 14 vs 5.6 per 100 blood cultures extracted (P=.00003). An evidence-based training programme reduced the contamination of samples. It is necessary to continue working on the planning of activities and care to improve the detection of pollutants and prevent contamination of samples. Copyright © 2018 Sociedad Española de Enfermería Intensiva y Unidades Coronarias (SEEIUC). Publicado por Elsevier España, S.L.U. All rights reserved.

  20. Relative sensitivity of conventional and real-time PCR assays for detection of SFG Rickettsia in blood and tissue samples from laboratory animals.

    Science.gov (United States)

    Zemtsova, Galina E; Montgomery, Merrill; Levin, Michael L

    2015-01-01

    Studies on the natural transmission cycles of zoonotic pathogens and the reservoir competence of vertebrate hosts require methods for reliable diagnosis of infection in wild and laboratory animals. Several PCR-based applications have been developed for detection of infections caused by Spotted Fever group Rickettsia spp. in a variety of animal tissues. These assays are being widely used by researchers, but they differ in their sensitivity and reliability. We compared the sensitivity of five previously published conventional PCR assays and one SYBR green-based real-time PCR assay for the detection of rickettsial DNA in blood and tissue samples from Rickettsia- infected laboratory animals (n = 87). The real-time PCR, which detected rickettsial DNA in 37.9% of samples, was the most sensitive. The next best were the semi-nested ompA assay and rpoB conventional PCR, which detected as positive 18.4% and 14.9% samples respectively. Conventional assays targeting ompB, gltA and hrtA genes have been the least sensitive. Therefore, we recommend the SYBR green-based real-time PCR as a tool for the detection of rickettsial DNA in animal samples due to its higher sensitivity when compared to more traditional assays.

  1. A field trial of a PCR-based Mansonella ozzardi diagnosis assay detects high-levels of submicroscopic M. ozzardi infections in both venous blood samples and FTA card dried blood spots.

    Science.gov (United States)

    Medeiros, Jansen Fernandes; Almeida, Tatiana Amaral Pires; Silva, Lucyane Bastos Tavares; Rubio, Jose Miguel; Crainey, James Lee; Pessoa, Felipe Arley Costa; Luz, Sergio Luiz Bessa

    2015-05-20

    Mansonella ozzardi is a poorly understood human filarial parasite with a broad distribution throughout Latin America. Most of what is known about its parasitism has come from epidemiological studies that have estimated parasite incidence using light microscopy. Light microscopy can, however, miss lighter, submicroscopic, infections. In this study we have compared M. ozzardi incidence estimates made using light microscopy, with estimates made using PCR. 214 DNA extracts made from Large Volume Venous Blood Samples (LVVBS) were taken from volunteers from two study sites in the Rio Solimões region: Codajás [n = 109] and Tefé [n = 105] and were subsequently assayed for M. ozzardi parasitism using a diagnostic PCR (Mo-dPCR). Peripheral finger-prick blood samples were taken from the same individuals and used for microscopic examination. Finger-prick blood, taken from individuals from Tefé, was also used for the creation of FTAcard dried blood spots (DBS) that were subsequently subjected to Mo-dPCR. Overall M. ozzardi incidence estimates made with LVVBS PCRs were 1.8 times higher than those made using microscopy (44.9% [96/214] compared with 24.3% [52/214]) and 1.5 times higher than the PCR estimates made from FTAcard DBS (48/105 versus 31/105). PCR-based detection of FTAcard DBS proved 1.3 times more sensitive at diagnosing infections from peripheral blood samples than light microscopy did: detecting 24/105 compared with 31/105. PCR of LVVBS reported the fewest number of false negatives, detecting: 44 of 52 (84.6%) individuals diagnosed by microscopy; 27 of 31 (87.1%) of those diagnosed positive from DBSs and 17 out of 18 (94.4%) of those diagnosed as positive by both alternative methodologies. In this study, Mo-dPCR of LVVBS was by far the most sensitive method of detecting M. ozzardi infections and detected submicroscopic infections. Mo-dPCR FTAcard DBS also provided a more sensitive test for M. ozzardi diagnosis than light microscopy based diagnosis did and

  2. A rapid, highly sensitive and culture-free detection of pathogens from whole blood by removal of white blood cells using immuno-magnetic beads.

    Science.gov (United States)

    Vutukuru, Manjula Ramya; Sharma, Divya Khandige; Ms, Ragavendar; Mitra, Nivedita

    2016-08-01

    Using anti-human CD45 antibody coated beads, we show a 98% reduction of WBCs from spiked blood samples in 1h, thereby enriching it for pathogens. This enrichment allowed the detection of blood using quantitative PCR; something not observed in unenriched samples. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. Reconstitution activity of hypoxic cultured human cord blood CD34-positive cells in NOG mice

    International Nuclear Information System (INIS)

    Shima, Haruko; Takubo, Keiyo; Iwasaki, Hiroko; Yoshihara, Hiroki; Gomei, Yumiko; Hosokawa, Kentaro; Arai, Fumio; Takahashi, Takao; Suda, Toshio

    2009-01-01

    Hematopoietic stem cells (HSCs) reside in hypoxic areas of the bone marrow. However, the role of hypoxia in the maintenance of HSCs has not been fully characterized. We performed xenotransplantation of human cord blood cells cultured in hypoxic or normoxic conditions into adult NOD/SCID/IL-2Rγ null (NOG) mice. Hypoxic culture (1% O 2 ) for 6 days efficiently supported the maintenance of HSCs, although cell proliferation was suppressed compared to the normoxic culture. In contrast, hypoxia did not affect in vitro colony-forming ability. Upregulation of a cell cycle inhibitor, p21, was observed in hypoxic culture. Immunohistochemical analysis of recipient bone marrow revealed that engrafted CD34 + CD38 - cord blood HSCs were hypoxic. Taken together, these results demonstrate the significance of hypoxia in the maintenance of quiescent human cord blood HSCs.

  4. A multiplex nested PCR for the detection and identification of Candida species in blood samples of critically ill paediatric patients

    OpenAIRE

    Taira, Cleison Ledesma; Okay, Thelma Suely; Delgado, Artur Figueiredo; Ceccon, Maria Esther Jurfest Rivero; Almeida, Margarete Teresa Gottardo de; Del Negro, Gilda Maria Barbaro

    2014-01-01

    Abstract Background Nosocomial candidaemia is associated with high mortality rates in critically ill paediatric patients; thus, the early detection and identification of the infectious agent is crucial for successful medical intervention. The PCR-based techniques have significantly increased the detection of Candida species in bloodstream infections. In this study, a multiplex nested PCR approach was developed for candidaemia detection i...

  5. Comparison of lysis-centrifugation with lysis-filtration and a conventional unvented bottle for blood cultures.

    OpenAIRE

    Gill, V J; Zierdt, C H; Wu, T C; Stock, F; Pizzo, P A; MacLowry, J D

    1984-01-01

    Evaluation of a commercially available lysis-centrifugation blood culture system (Isolator, DuPont Co., Wilmington, Del.) and a lysis-filtration blood culture system for 3,111 cultures showed that both methods had comparable recoveries (73 and 68%, respectively) of significant aerobic and facultatively anaerobic isolates. The unvented conventional blood culture bottle had a recovery rate of 59%. Although the lysis-centrifugation and lysis-filtration systems had comparable recoveries of pathog...

  6. Evaluation of quantitative PCR and culture methods for detection of house dust fungi and streptomycetes in relation to moisture damage of the house.

    Science.gov (United States)

    Lignell, U; Meklin, T; Rintala, H; Hyvärinen, A; Vepsäläinen, A; Pekkanen, J; Nevalainen, A

    2008-10-01

    Microbial concentrations in vacuumed house dust samples (n = 71) were analysed by culture and quantitative polymerase chain reaction (qPCR) methods and their association with extent of moisture damage in the house was studied. Microbial concentrations measured by qPCR correlated with concentrations obtained by culture method, but were orders of magnitude higher. qPCR also had better sensitivity. Concentrations of several microbes in house dust, determined with qPCR, were associated with the extent of moisture damage in the house. This association was strongest for Penicillium brevicompactum, one of the fungi detected in highest concentrations by qPCR. Furthermore, house dust concentrations of Wallemia sebi, Trichoderma viride, Cladosporium sphaerospermum, Eurotium amstelodami and the combined assay group for Penicillium spp., Aspergillus spp. and Paecilomyces variotii were significantly associated with the extent of the moisture damage. These species or assay groups could probably be used as indicators of moisture damage in the house. This finding indicates the benefits of the qPCR method, which is sensitive enough to reveal the differences in microbial concentrations of house dust between moisture-damaged and undamaged houses.

  7. [Detection of human parvovirus B19, human bocavirus and human parvovirus 4 infections in blood samples among 95 patients with liver disease in Nanjing by nested PCR].

    Science.gov (United States)

    Tong, Rui; Zhou, Wei-Min; Liu, Xi-Jun; Wang, Yue; Lou, Yong-Liang; Tan, Wen-Jie

    2013-04-01

    To analyze the infection of human parvovirus B19, human bocavirus (HBoV) and human parvovirus 4 (PARV4) in blood samples among patients with liver disease in Nanjing by molecular detection. Nested PCR assays were designed and validated to detect B19, HBoV and PARV4, respectively. The assays were used to screen three parvoviruses in blood samples from 95 patients with different liver disease in Nanjing. The parvovirus infection was analyzed statistically. The detection limits were 10 copies of genomic DNA equivalents per reaction for each assays and the good specificity were observed. The frequency of B19 and HBoV were 2/95 (2.1%) and 9/95 (9.5%) in blood samples respectively. No PARV4 was detected. HBoV was detected in 3/5 patients with drug-induced hepatitis. Both B19 and HBoV infection were detected in blood from patients with liver disease.

  8. Detection of Hepatitis B Virus (HBV) in Blood Serum By Means of PCR (Polymerase Chain Reaction) Technique

    International Nuclear Information System (INIS)

    Lina, M.; Dadang, S.; Suhadi, F.

    2002-01-01

    Research for detecting the presence of HBV DNA in serum with PCR technique by using two pairs of oligonucleotide primers, has been carried out. Ten serum consisted of 5 HBsAg positive serum, I HBsAg weak positive serum, 3 HBsAg negative serum, and I sampel with negative HBV DNA as a previous PCR product trom another laboratory, were used to purify and to extract the DNA of virus, the sample pretreatment was done with Boom method. The two pairs of primers used for the- PCR process, were PC1 and PC2 and P1 and P2. The amplification process by means of PC1 and PC2 primer was carried out with two treatments, l.a. and l.b treatments of 5 HBsAg positive serum samples, 3 were positive for HBV DNA by PCR test with l.a. treatment. The PCR test by means of either the same primer but different treaunent (l.b treatment) or different pair of primer (pI and P2 pimer), revealed the presence of HBV DNA in all of HBsAg serum mentioned above of HBsAg negative Seruln, I serum was positive for HBV DNA and it was an amplification product of PCR test by using PI and P2 primer. The amplification products of PCR processwith either l.b treatment or PI and P2 primer, showed the positive results for I HBV positive serum as a previous PCR product trom another laboratory. All of the PCR test in this research provided the negative HBV DNA result in the HBsAg weak positive serum. The DNA amplification process by means of PI and P2 primer was more sensitive compared with PC I and PC2 primer

  9. Evaluation of a low-cost procedure for sampling, long-term storage, and extraction of RNA from blood for qPCR analyses

    DEFF Research Database (Denmark)

    Mærkedahl, Rasmus Baadsgaard; Frøkiær, Hanne; Lauritzen, Lotte

    2015-01-01

    -extraction. We investigated the use of lysis buffer for long-term storage of blood samples for qPCR analysis. Methods: Blood was collected from 13 healthy adults and diluted in MagMAX lysis/binding solution or PAXgene Blood RNA tubes and stored at -20 °C for 0, 1, or 4 months before RNA extraction....../binding solution over time and between samples stored and extracted by the two systems. Conclusions: The MagMAX system can be used for storage of human blood for up to 4 months and is equivalent to the PAXgene system for RNA extraction. It furthermore, provides a means for significant cost reduction in large......Abstract Background: In large clinical trials, where RNA cannot be extracted immediately after sampling, preserving RNA in whole blood is a crucial initial step in obtaining robust qPCR data. The current golden standard for RNA preservation is costly and designed for time-consuming column-based RNA...

  10. Diversity of thermophilic bacteria in raw, pasteurized and selectively-cultured milk, as assessed by culturing, PCR-DGGE and pyrosequencing.

    Science.gov (United States)

    Delgado, Susana; Rachid, Caio T C C; Fernández, Elena; Rychlik, Tomasz; Alegría, Angel; Peixoto, Raquel S; Mayo, Baltasar

    2013-10-01

    Thermophilic lactic acid bacteria (LAB) species, such as Streptococcus thermophilus, Lactobacillus delbrueckii and Lactobacillus helveticus, enjoy worldwide economic importance as dairy starters. To assess the diversity of thermophilic bacteria in milk, milk samples were enriched in thermophilic organisms through a stepwise procedure which included pasteurization of milk at 63 °C for 30 min (PM samples) and pasteurization followed by incubation at 42 °C for 24 h (IPM samples). The microbial composition of these samples was analyzed by culture-dependent (at 42 °C) and culture-independent (PCR-DGGE and pyrosequencing of 16S rRNA gene amplicons) microbial techniques. The results were then compared to those obtained for their corresponding starting raw milk counterparts (RM samples). Twenty different species were scored by culturing among 352 isolates purified from the counting plates and identified by molecular methods. Mesophilic LAB species (Lactococcus lactis, Lactococcus garvieae) were dominant (87% of the isolates) among the RM samples. However, S. thermophilus and Lb. delbrueckii were found to be the dominant recoverable organisms in both PM and IPM samples. The DGGE profiles of RM and PM samples were found to be very similar; the most prominent bands belonging to Lactococcus, Leuconostoc and Streptococcus species. In contrast, just three DGGE bands were obtained for IPM samples, two of which were assigned to S. thermophilus. The pyrosequencing results scored 95 operational taxonomic units (OTUs) at 3% sequence divergence in an RM sample, while only 13 were encountered in two IPM samples. This technique identified Leuconostoc citreum as the dominant microorganism in the RM sample, while S. thermophilus constituted more than 98% of the reads in the IPM samples. The procedure followed in this study allowed to estimate the bacterial diversity in milk and afford a suitable strategy for the isolation of new thermophilic LAB strains, among which adequate

  11. Rapid detection of coliforms in drinking water of Arak city using multiplex PCR method in comparison with the standard method of culture (Most Probably Number).

    Science.gov (United States)

    Fatemeh, Dehghan; Reza, Zolfaghari Mohammad; Mohammad, Arjomandzadegan; Salomeh, Kalantari; Reza, Ahmari Gholam; Hossein, Sarmadian; Maryam, Sadrnia; Azam, Ahmadi; Mana, Shojapoor; Negin, Najarian; Reza, Kasravi Alii; Saeed, Falahat

    2014-05-01

    To analyse molecular detection of coliforms and shorten the time of PCR. Rapid detection of coliforms by amplification of lacZ and uidA genes in a multiplex PCR reaction was designed and performed in comparison with most probably number (MPN) method for 16 artificial and 101 field samples. The molecular method was also conducted on isolated coliforms from positive MPN samples; standard sample for verification of microbial method certificated reference material; isolated strains from certificated reference material and standard bacteria. The PCR and electrophoresis parameters were changed for reducing the operation time. Results of PCR for lacZ and uidA genes were similar in all of standard, operational and artificial samples and showed the 876 bp and 147 bp bands of lacZ and uidA genes by multiplex PCR. PCR results were confirmed by MPN culture method by sensitivity 86% (95% CI: 0.71-0.93). Also the total execution time, with a successful change of factors, was reduced to less than two and a half hour. Multiplex PCR method with shortened operation time was used for the simultaneous detection of total coliforms and Escherichia coli in distribution system of Arak city. It's recommended to be used at least as an initial screening test, and then the positive samples could be randomly tested by MPN.

  12. Comparison of automated BAX PCR and standard culture methods for detection of Listeria monocytogenes in blue Crabmeat (Callinectus sapidus) and blue crab processing plants.

    Science.gov (United States)

    Pagadala, Sivaranjani; Parveen, Salina; Schwarz, Jurgen G; Rippen, Thomas; Luchansky, John B

    2011-11-01

    This study compared the automated BAX PCR with the standard culture method (SCM) to detect Listeria monocytogenes in blue crab processing plants. Raw crabs, crabmeat, and environmental sponge samples were collected monthly from seven processing plants during the plant operating season, May through November 2006. For detection of L. monocytogenes in raw crabs and crabmeat, enrichment was performed in Listeria enrichment broth, whereas for environmental samples, demi-Fraser broth was used, and then plating on both Oxford agar and L. monocytogenes plating medium was done. Enriched samples were also analyzed by BAX PCR. A total of 960 samples were examined; 59 were positive by BAX PCR and 43 by SCM. Overall, there was no significant difference (P ≤ 0.05) between the methods for detecting the presence of L. monocytogenes in samples collected from crab processing plants. Twenty-two and 18 raw crab samples were positive for L. monocytogenes by SCM and BAX PCR, respectively. Twenty and 32 environmental samples were positive for L. monocytogenes by SCM and BAX PCR, respectively, whereas only one and nine finished products were positive. The sensitivities of BAX PCR for detecting L. monocytogenes in raw crabs, crabmeat, and environmental samples were 59.1, 100, and 60%, respectively. The results of this study indicate that BAX PCR is as sensitive as SCM for detecting L. monocytogenes in crabmeat, but more sensitive than SCM for detecting this bacterium in raw crabs and environmental samples.

  13. Anticoagulant carryover may influence clot formation in direct tube coagulase tests from blood cultures.

    Science.gov (United States)

    Varettas, Kerry; Mukerjee, Chinmoy; Taylor, Peter C

    2005-09-01

    The tube coagulase test (TCT) performed directly from positive blood culture bottles has been used to reduce the turnaround time for identifying Staphylococcus aureus. Most reports have shown the test to be specific but often lacking sufficient sensitivity to be useful. In a prospective study of blood culture bottles (BCB) signaling positive, with a Gram-stained smear showing gram-positive cocci resembling staphylococci, the sensitivity of the direct TCT was improved by diluting the BCB broth 1:10 in saline before inoculating 0.1 ml into 1.0 ml of 10% pooled human plasma. It was hypothesized that the improved sensitivity might be explained by reduced carryover of the anticoagulant sodium polyanetholesulfonate (SPS) used in blood culture media. By titrating the inoculum size and the concentration of SPS in an in vitro checkerboard assay, it was shown that concentrations of SPS >0.0008% prevented plasma coagulation. The 1:10 dilution of blood culture broth reduced the amount of residual SPS carried over to the TCT to a level (0.0005%) that did not impair plasma coagulation. The direct TCT inoculated with a 1:10 saline dilution of blood culture broth achieved 100% specificity and sensitivity within 4 h of inoculation without reducing the quality or quantity of coagulum.

  14. Effectiveness of a Novel Specimen Collection System in Reducing Blood Culture Contamination Rates.

    Science.gov (United States)

    Bell, Mary; Bogar, Catherine; Plante, Jessica; Rasmussen, Kristen; Winters, Sharon

    2018-04-20

    False-positive blood-culture results due to skin contamination of samples remain a persistent problem for health care providers. Our health system recognized that our rates of contamination across the 4 emergency department campuses were above the national average. A unique specimen collection system was implemented throughout the 4 emergency departments and became the mandatory way to collect adult blood cultures. The microbiology laboratory reported contamination rates weekly to manage potential problems; 7 months of data are presented here. There was an 82.8% reduction in false positives with the unique specimen collection system compared with the standard method (chi-squared test with Yates correction, 2-tailed, P = 0.0001). Based on the historical 3.52% rate of blood-culture contamination for our health facilities, 2.92 false positives were prevented for every 100 blood cultures drawn, resulting from adoption of the unique specimen collection system as the standard of care. This unique collection system can reduce the risk of blood culture contamination significantly and is designed to augment, rather than replace, the standard phlebotomy protocol already in use in most health care settings. Copyright © 2018 Elsevier Inc. All rights reserved.

  15. Philadelphia chromosome detection in chronic myeloid leukemia: Utility of phytohemagglutinin-stimulated peripheral blood culture

    Directory of Open Access Journals (Sweden)

    Man Updesh Singh Sachdeva

    2012-01-01

    Full Text Available Background: The conventional cytogenetic approach to demonstrate Philadelphia (Ph chromosome at times does not yield enough number of metaphases or are of suboptimal quality. Further, the rapid molecular tests have completely pushed this simple technique into disrepute. Aims: This study aimed to evaluate usefulness of phytohemagglutinin (PHA-stimulated peripheral blood culture for detection of Ph chromosome in chronic myeloid leukemia (CML patients. Materials and Methods: Fifty-six patients, including 11 newly diagnosed cases of CML and 45 patients of CML on imatinib therapy showing the presence of Ph chromosome in unstimulated samples, were included in the study. Cytogenetic analysis was done on unstimulated samples, i.e. bone marrow aspirate, 24- and 48-h peripheral blood culture, and compared with PHA-stimulated 72-h peripheral blood culture. Results: The preparations from PHA-stimulated peripheral blood culture samples in all 56 patients yielded high number of good-quality metaphases. All the 11 (100% newly diagnosed patients and 39/45 (87% of the patients on imatinib therapy showed the presence of Ph chromosome in PHA-stimulated samples. Addition of PHA-stimulated 72-h peripheral blood culture preparation can be of use for increasing the diagnostic yield in cases of CML with suboptimal results on conventional cytogenetics from bone marrow aspirate sample.

  16. Evaluation of the antimicrobial removal device when used with the BACTEC blood culture system

    International Nuclear Information System (INIS)

    Strand, C.L.

    1982-01-01

    A study to determine the value of the Antimicrobial Removal Device (ARD) used in conjunction with radiometric detection of bacteremia using three media was conducted. During a 12-month period, 622 duplicate ARD/BACTEC blood-culture sets were collected. There were 88 positive cultures that yielded 68 pathogenic isolates and 28 probable contaminant isolates. When all patients were considered, 31 pathogenic isolates were detected by both systems, 25 pathogenic isolates were detected faster or only by the BACTEC system, and 12 pathogenic isolates were detected faster or only when the ARD was employed. This difference is statistically significant (P less than 0.05). Thus, the standard BACTEC blood-culture system using three different media was superior to the same BACTEC system using ARD-processed blood specimens. When only patients receiving antimicrobial therapy were considered, there were more pathogenic isolates detected from unprocessed blood than from blood processed in the ARD; however, this difference was not statistically significant. In conclusion, there appears to be no advantage to using the ARD system in conjunction with the three-bottle BACTEC blood-culture system

  17. Detection of the BLV provirus from nasal secretion and saliva samples using BLV-CoCoMo-qPCR-2: Comparison with blood samples from the same cattle.

    Science.gov (United States)

    Yuan, Yuan; Kitamura-Muramatsu, Yuri; Saito, Susumu; Ishizaki, Hiroshi; Nakano, Miwa; Haga, Satoshi; Matoba, Kazuhiro; Ohno, Ayumu; Murakami, Hironobu; Takeshima, Shin-Nosuke; Aida, Yoko

    2015-12-02

    Bovine leukemia virus (BLV) induces enzootic bovine leukosis, which is the most common neoplastic disease in cattle. Sero-epidemiological studies show that BLV infection occurs worldwide. Direct contact between infected and uninfected cattle is thought to be one of the risk factors for BLV transmission. Contact transmission occurs via a mixture of natural sources, blood, and exudates. To confirm that BLV provirus is detectable in these samples, matched blood, nasal secretion, and saliva samples were collected from 50 cattle, and genomic DNA was extracted. BLV-CoCoMo-qPCR-2, an assay developed for the highly sensitive detection of BLV, was then used to measure the proviral load in blood (n=50), nasal secretions (n=48), and saliva (n=47) samples. The results showed that 35 blood samples, 14 nasal secretion samples, and 6 saliva samples were positive for the BLV provirus. Matched blood samples from cattle that were positive for the BLV provirus (either in nasal secretion or saliva samples) were also positive in their blood. The proviral load in the positive blood samples was >14,000 (copies/1×10(5) cells). Thus, even though the proviral load in the nasal secretion and saliva samples was much lower (blood, prolonged direct contact between infected and healthy cattle may be considered as a risk factor for BLV transmission. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. Improving molecular detection of Candida DNA in whole blood: comparison of seven fungal DNA extraction protocols using real-time PCR.

    Science.gov (United States)

    Metwally, L; Fairley, D J; Coyle, P V; Hay, R J; Hedderwick, S; McCloskey, B; O'Neill, H J; Webb, C H; Elbaz, W; McMullan, R

    2008-03-01

    The limitations of classical diagnostic methods for invasive Candida infections have led to the development of molecular techniques such as real-time PCR to improve diagnosis. However, the detection of low titres of Candida DNA in blood from patients with candidaemia requires the use of extraction methods that efficiently lyse yeast cells and recover small amounts of DNA suitable for amplification. In this study, a Candida-specific real-time PCR assay was used to detect Candida albicans DNA in inoculated whole blood specimens extracted using seven different extraction protocols. The yield and quality of total nucleic acids were estimated using UV absorbance, and specific recovery of C. albicans genomic DNA was estimated quantitatively in comparison with a reference (Qiagen kit/lyticase) method currently in use in our laboratory. The extraction protocols were also compared with respect to sensitivity, cost and time required for completion. The TaqMan PCR assay used to amplify the DNA extracts achieved high levels of specificity, sensitivity and reproducibility. Of the seven extraction protocols evaluated, only the MasterPure yeast DNA extraction reagent kit gave significantly higher total nucleic acid yields than the reference method, although nucleic acid purity was highest using either the reference or YeaStar genomic DNA kit methods. More importantly, the YeaStar method enabled C. albicans DNA to be detected with highest sensitivity over the entire range of copy numbers evaluated, and appears to be an optimal method for extracting Candida DNA from whole blood.

  19. Determining the prevalence of inv-positive and ail-positive Yersinia enterocolitica in pig tonsils using PCR and culture methods.

    Science.gov (United States)

    Stachelska, Milena Alicja

    2017-01-01

    Yersiniosis is believed to be the third most common intestinal zoonosis in the European Union, after campylobacteriosis and salmonellosis. Yersinia enterocolitica is the most common species responsible for human infections. Pigs are regarded as the biggest reservoir of pathogenic Y. enterocolitica strains, which are mainly isolated from pig tonsils. The aim of this paper is to examine the prevalence of inv-positive and ail-positive Y. enterocolitica in pigs which were slaughtered in a Polish abattoir. Real-time PCR and culture methods were used to assess the prevalence of patho- genic Y. enterocolitica strains in pig tonsils. Real-time PCR was applied to detect inv-positive and ail-positive Y. enterocolitica. Y. enterocolitica was also isolated by applying direct plating, unselective (tryptic soy broth) and selective (irgasan-ticarcillin-potassium chlorate bouillon) enrichment. A total of 180 pigs were studied, of which 85% and 32% respectively were found to be infected with inv-positive and ail-positive Y. enterocolitica. The 92 inv-positive and ail-positive isolates, from 57 culture- positive tonsils, underwent bio- and serotyping. The most common was bioserotype 4/O:3, which was found in 53 (93%) out of 57 culture-positive tonsils. Strains of bioserotypes 2/O:5, 2/O:9 and 2/O:5.27 occurred in significantly lower numbers. The prevalence of inv-positive and ail-positive Y. enterocolitica was found to be high in the ton- sils of slaughtered pigs, using real-time PCR. The real-time PCR method for the detection and identification of pathogenic Y. enterocolitica is sensitive and specific, which has been verified by specificity and sensitivity tests using the pure cultures. Serotypes were distinguished from each other using PCR serotyping. The PCR method was essential in forming our conclusions.

  20. Comparison of two commercial broadrange PCR and sequencing assays for identification of bacteria in culture-negative clinical samples

    DEFF Research Database (Denmark)

    Stavnsbjerg, Camilla; Frimodt-Moller, Niels; Moser, Claus Ernst

    2017-01-01

    in a real-time PCR and sequenced. Results 22 of 76 samples (28.9%) were positive for bacteria with the UMD SelectNA, which was significantly more (p = 0.0055) than the MicroSeq ID where 11 of 76 samples (14.5%) were positive. The UMD SelectNA assay identified more relevant bacterial pathogens than the Micro...... applied. The aim of the present study was to compare the current method used at our Department of Clinical Microbiology, based on the MicroSeq ID system (Applied Biosystems, USA) with the Universal Microbe Detection (UMD) SelectNA kit (Molzym, Germany). Methods 76 culture-negative samples were first...... processed with the MicroSeq ID analysis, where total DNA was extracted and the 16S gene amplified and sequenced with the MicroSeq ID system. Samples were subsequently processed with the UMD SelectNA analysis, where human DNA was removed during the DNA extraction procedure and the 16S gene amplified...

  1. Detection of Strongylus vulgaris in equine faecal samples by real-time PCR and larval culture - method comparison and occurrence assessment.

    Science.gov (United States)

    Kaspar, A; Pfister, K; Nielsen, M K; Silaghi, C; Fink, H; Scheuerle, M C

    2017-01-11

    Strongylus vulgaris has become a rare parasite in Germany during the past 50 years due to the practice of frequent prophylactic anthelmintic therapy. To date, the emerging development of resistance in Cyathostominae and Parascaris spp. to numerous equine anthelmintics has changed deworming management and the frequency of anthelmintic usage. In this regard, reliable detection of parasitic infections, especially of the highly pathogenic S. vulgaris is essential. In the current study, two diagnostic methods for the detection of infections with S. vulgaris were compared and information on the occurrence of this parasite in German horses was gained. For this purpose, faecal samples of 501 horses were screened for S. vulgaris with real-time PCR and an additional larval culture was performed in samples of 278 horses. A subset of 26 horses underwent multiple follow-up examinations with both methods in order to evaluate both the persistence of S. vulgaris infections and the reproducibility of each diagnostic method. The real-time PCR revealed S. vulgaris-DNA in ten of 501 investigated equine samples (1.9%). The larval culture demonstrated larvae of S. vulgaris in three of the 278 samples (1.1%). A direct comparison of the two methods was possible in 321 samples including 43 follow-up examinations with the result of 11 S. vulgaris-positive samples by real-time PCR and 4 S. vulgaris-positive samples by larval culture. The McNemar's test (p-value = 0.016) revealed a significant difference and the kappa values (0.525) showed a moderate agreement between real-time PCR and larval culture. The real-time PCR detected a significantly higher proportion of positives of S. vulgaris compared to larval culture and should thus be considered as a routine diagnostic method for the detection of S. vulgaris in equine samples.

  2. Aggregatibacter aphrophilus infective endocarditis confirmed by broad-range PCR diagnosis: A case report

    Directory of Open Access Journals (Sweden)

    Koji Hirano

    2017-01-01

    Conclusion: A rare disease, Aggregatibacter aphrophilus infective endocarditis was successfully treated with surgical repair and appropriate antibiotic therapy. To avoid misdiagnosis, br-PCR testing should be performed in patients with blood culture-negative endocarditis.

  3. The evaluation of histo-blood group ABO typing by flow cytometric and PCR-amplification of specific alleles analyses and their application in clinical laboratories.

    Science.gov (United States)

    Aki, Kensaku; Izumi, Azusa; Hosoi, Eiji

    2012-01-01

    ABO antigens are oligosaccharide antigens, and are widely distributed on red blood and tissue cells as well as in saliva and body fluid. Therefore, these antigens are important not only for blood transfusion, but also for tissue cell and organ transplantations. Also, blood, hair, and seminal fluid are important sources of evidence at crime scenes, and these antigens are some of the most important markers for personal identification in forensic investigations. Here, we describe the development and use of quantitative analysis of A, B, and H antigens on red blood cells by employing flow cytometric analysis and the ABO genotyping method based on PCR-amplification of specific alleles (PASA) within DNA, especially from blood and saliva. In this study, flow cytometric analysis could be used to compare the differences between the expression of A and/or B and H antigens on red blood cells with various phenotypes, and the PASA method was able to determine the genotype of the type cisA(2)B(3) pedigree using only DNA extracted from saliva. These analysis methods are simple and useful for judging the ABO blood group system and genotyping, and are used widely throughout research and clinical laboratories and forensic fields.

  4. Validity of direct identification and antibiotic susceptibility of microrganisms from bottles of blood culture

    OpenAIRE

    Carmela Mazzone; Maria Luisa Laterza; Lucio Tauro

    2009-01-01

    The blood culture is a very important laboratory test: if bacteremia or sepsis are suspected, the diagnosis of the pathogen and antibiotic therapy may be achieved making use of it. Identification and antibiotic susceptibility test carried out directly from the bottle may give important information in a shorter time. The introduction of the automatic instrumentation has improved the discovering of pathogens in the blood, however the elapsing time between the positive detection and the microbio...

  5. Hot topic: Bovine milk samples yielding negative or nonspecific results in bacterial culturing--the possible role of PCR-single strand conformation polymorphism in mastitis diagnosis.

    Science.gov (United States)

    Schwaiger, K; Wimmer, M; Huber-Schlenstedt, R; Fehlings, K; Hölzel, C S; Bauer, J

    2012-01-01

    A large proportion of mastitis milk samples yield negative or nonspecific results (i.e., no mastitis pathogen can be identified) in bacterial culturing. Therefore, the culture-independent PCR-single strand conformation polymorphism method was applied to the investigation of bovine mastitis milk samples. In addition to the known mastitis pathogens, the method was suitable for the detection of fastidious bacteria such as Mycoplasma spp., which are often missed by conventional culturing methods. The detection of Helcococcus ovis in 4 samples might indicate an involvement of this species in pathogenesis of bovine mastitis. In conclusion, PCR-single-strand conformation polymorphism is a promising tool for gaining new insights into the bacteriological etiology of mastitis. Copyright © 2012 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  6. Challenges in culture-negative cases of Madurella mycetomatis: A case report re-accentuating PCR as an essential diagnostic tool.

    Science.gov (United States)

    K, S; Das, S; Pandhi, D; Rai, G; Ansari, M A; Gupta, C; Haque, S; Dar, S A

    2017-12-01

    Identification of dematiaceous fungi responsible for black-grain mycetoma has remained cumbersome and time consuming for years leading to delayed diagnosis and thereby increased agony to patients. Moreover, difficult morphology of some of these fungi demanding enough expertise for species identification in addition to culture-negativity has often led to misdiagnosis and hence inapt treatment to the patients. We report the identification of Madurella mycetomatis from culture-negative black granules discharged from foot nodular lesions of a 27 years old male using PCR followed by sequencing of the internal transcribed spacer region. The patient's lesions were successfully treated using a combination of itraconazole (200mg) and terbinafine (250mg), confirming our diagnosis. Our case study proves the clinical value of PCR as the best, rapid and accurate diagnostic method for the identification of Madurella mycetomatis and related fungi, particularly in culture-negative cases. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  7. [Quality indicators for blood culture: three years of monitoring at a university hospital in Chile].

    Science.gov (United States)

    Guzmán, Ana María; Sánchez, Tomás; de la Barra, Ricardo

    2012-08-01

    Blood culture is considered the "gold standard" for the diagnosis of bacteremia, critical condition with high morbidity and mortality. Because of its importance, it is estimated that the blood culture is a critical test that requires close monitoring on the quality with which the process is performed. The objective of this work is to show the results of the monitoring carried out during the past three years, of 5 quality indicators of blood cultures in the laboratory of the Hospital Clínico de la Pontificia Universidad Católica de Chile, considering pre-analytical, analytical and post-analytical aspects. In the 3 years monitored the mean contamination was 0,7%, 46% of adult bottles had adequate volume, match between Gram stain with final identification was 99.4%, 100% of correct participations were achieved in surveys of external quality control and Gram staining notification before 1 hour was 88.7%. With regard to proposed aims, in 2011 the laboratory complies with all, except the percentage of bottles with appropriate volume of blood inoculated. This indicator is very low and should be corrected as soon as possible since it is known that it is an important condition for optimum performance of blood cultures.

  8. Validity of direct identification and antibiotic susceptibility of microrganisms from bottles of blood culture

    Directory of Open Access Journals (Sweden)

    Carmela Mazzone

    2009-12-01

    Full Text Available The blood culture is a very important laboratory test: if bacteremia or sepsis are suspected, the diagnosis of the pathogen and antibiotic therapy may be achieved making use of it. Identification and antibiotic susceptibility test carried out directly from the bottle may give important information in a shorter time. The introduction of the automatic instrumentation has improved the discovering of pathogens in the blood, however the elapsing time between the positive detection and the microbiological report is still along.The aim of our work was to verify the validity of the direct use of blood culture broth in which growth of microorganisms has been detected, which could reduce the response time of the bacteremia diagnosis. During the period February - July 2009, a total of 150 blood cultures were analysed:we compared the results obtained both by direct method and by reference method. 20 Gram positive microrganisms and 13 Gram negative microrganisms were respectively isolated and identified. The identification of Gram-negative and Gram-positive microrganisms showed an agreement of 100% between the direct and the reference method. For antibiotic susceptibility tests, among the Gram positive has reported 1.3% very major error, 2.9% major error and 1.4% minor error, while the Gram negative, respectivety 0.3%, 1.4%, 0%. The use of direct identification and susceptibility testing from positive blood cultures, can improve the response time and better efficiency in diagnostic procedures.

  9. Large-scale clinical comparison of the lysis-centrifugation and radiometric systems for blood culture

    International Nuclear Information System (INIS)

    Brannon, P.; Kiehn, T.E.

    1985-01-01

    The Isolator 10 lysis-centrifugation blood culture system (E. I. du Pont de Nemours and Co., Inc., Wilmington, Del.) was compared with the BACTEC radiometric method (Johnston Laboratories, Inc., Towson, Md.) with 6B and 7D broth media for the recovery of bacteria and yeasts. From 11,000 blood cultures, 1,174 clinically significant organisms were isolated. The Isolator system recovered significantly more total organisms, members of the family Enterobacteriaceae, Staphylococcus spp., and yeasts. The BACTEC system recovered significantly more Pseudomonas spp., Streptococcus spp., and anaerobes. Of the Isolator colony counts, 87% measured less than 11 CFU/ml of blood. Organisms, on an average, were detected the same day from each of the two culture systems. Only 13 of the 975 BACTEC isolates (0.01%) were recovered by subculture of growth-index-negative bottles, and 12 of the 13 were detected in another broth blood culture taken within 24 h. Contaminants were recovered from 4.8% of the Isolator 10 and 2.3% of the BACTEC cultures

  10. Blood culture procedures and diagnosis of Malassezia furfur bloodstream infections: Strength and weakness.

    Science.gov (United States)

    Iatta, Roberta; Battista, Michela; Miragliotta, Giuseppe; Boekhout, Teun; Otranto, Domenico; Cafarchia, Claudia

    2017-12-27

    The occurrence of Malassezia spp. bloodstream infections (BSIs) in neonatal intensive care unit was evaluated by using pediatric Isolator, BacT/Alert systems and central venous catheter (CVC) culture. The efficacy of BacT/Alert system in detecting Malassezia was assessed by conventional procedures, culturing 1 ml of bottle content before incubation and by studying the survival of Malassezia spp. strains in BacT/Alert bottles. Of the 492 neonates enrolled, blood was collected by pediatric Isolator (290 patients; group I) or by BacT/Alert bottles (202 patients; group II). The survival of Malassezia furfur and Malassezia pachydermatis in BacT/Alert bottles was evaluated by culturing the inoculum suspension (from 106 to 10 colony-forming units, cfu/ml) and assessing the cfu/ml for 15 days. In total, 15 Malassezia BSIs were detected, of which six (2.1%) from both blood and CVC culture in Dixon agar (DixA) in patients belong to group I (blood collected by paediatric Isolator tube) and nine (4.4%) only from CVC culture in DixA in patients of group II (blood collected by BacT/Alert bottle). Only one patient (0.5%) from group II scored positive for M. furfur also by culturing in DixA 1 ml blood content of BacT/Alert bottle before incubation in BacT/Alert system.M. furfur population size in BacT/Alert bottles decreased during the incubation time, whereas that of M. pachydermatis increased. The BacT/Alert system detected M. pachydermatis even at very low concentration (i.e., 10 cfu/ml) but not any positive blood culture for M. furfur. For a correct diagnosis of Malassezia furfur BSI, the blood should be culture in lipid-enriched fungal medium, and the BacT/Alert system implemented by adding lipid substrates to increase the method sensibility. Finally, CVC cultures on lipid-supplemented media may be proposed as a routine procedure to diagnose the Malassezia fungemia. © The Author 2017. Published by Oxford University Press on behalf of The International Society for Human and

  11. Is non-union of tibial shaft fractures due to nonculturable bacterial pathogens? A clinical investigation using PCR and culture techniques

    Directory of Open Access Journals (Sweden)

    Gille Justus

    2012-05-01

    Full Text Available Abstract Background Non-union continues to be one of the orthopedist’s greatest challenges. Despite effective culture methods, the detection of low-grade infection in patients with non-union following tibial fracture still presents a challenge. We investigated whether “aseptic” tibial non-union can be the result of an unrecognized infection. Methods A total of 23 patients with non-union following tibial shaft fractures without clinical signs of infection were investigated. Intraoperative biopsy samples obtained from the non-union site were examined by means of routine culture methods and by polymerase chain reaction (PCR for the detection of 16 S ribosomal RNA (rRNA. Control subjects included 12 patients with tibial shaft fractures. Results 23 patients (8 women and 15 men; mean age: 47.4 years were included into this study. Preoperative C-reactive protein levels (mean: 20.8 mg/l and WBC counts (mean: 8,359/μl in the study group were not significantly higher than in the control group. None of the samples of non-union routine cultures yielded microorganism growth. Bacterial isolates were found by conventional culturing methods in only 1 case of an open fracture from the control group. In this case, PCR yielded negative results. 16 S rRNA was detected in tissue specimens from 2 patients (8.7% with non-union. The analysis of these variable species-specific sequences enabled the identification of specific microorganisms (1x Methylobacterium species, 1x Staphylococcus species. Both PCR-positive patients were culture-negative. Conclusions The combination of microbiological culture and broad-range PCR seems to substantially add to the number of microbiological diagnoses obtained and may improve the clinican’s ability to tailor therapy to the individual patient’s needs.

  12. Applicability of integrated cell culture quantitative PCR (ICC-qPCR) for the detection of infectious adenovirus type 2 in UV disinfection studies

    Science.gov (United States)

    Human adenovirus is relatively resistant to UV radiation and has been used as a conservative testing microbe for evaluations of UV disinfection systems as components of water treatment processes. In this study, we attempted to validate the applicability of integrated cell culture...

  13. The Epidemiological And Susceptibility Study Of Inpatient Blood Cultures In Amir Alam Hospital 1998 - 2000

    OpenAIRE

    Karimi Shahidi M; Dabbagh Mohammady. A; Iravani B

    2002-01-01

    Sepsis is one of the most critical medical emergency situations. Treatment with anti microbial drugs should be initiated as soon as samples of blood and other relevant sites have been cultured. Available information about patterns of anti microbial Susceptibility among bacterial isolates from the community, the hospital, and the patient should be taken in to account. It is important, pending culture results, to initiate empirical anti microbial therapy."nMaterials and methods: In a descr...

  14. Blood Culture Contamination and the Type of Microorganisms in True and False Positive Results in Patients Admitted at Avicenna Qazvin

    OpenAIRE

    E Sajadi; S Asefzade; M Asefzade; F Manuchehri

    2010-01-01

    Introduction: Diagnosis of infection based on blood culture alone is not a suitable method, but it is important to understand the clinical diagnosis for interpreting blood cultures. The goal of this study was to determine blood culture contamination and the type of microorganisms in false and real positive cases in patients admitted at Avicenna Hospital. Methods: This cross sectional study was done on all patients in the emergency and internal medicine departments from April, 2008 to October,...

  15. A prospective study of two isothermal amplification assays compared with real-time PCR, CCNA and toxigenic culture for the diagnosis of Clostridium difficile infection.

    Science.gov (United States)

    Neuendorf, Martina; Guadarrama-Gonzalez, Raquel; Lamik, Birgit; MacKenzie, Colin R

    2016-02-12

    New molecular methods of detecting Clostridium difficile infection (CDI) provide the routine lab with a sensitive random access method to produce results that are available in a shorter time than traditional methods. In this prospective study a total of 989 stool specimens were tested over a period of 16 months in parallel using two isothermal amplification assays, AmpliVue® (Quidel) and Illumigene® (Meridian) and the results compared to those from toxigenic culture. In addition all specimens were tested using a cytotoxic cell neutralisation assay (CCNA) and three different Real-time PCR targeting a C. difficile-specific 16S rDNA sequence or the toxin genes tcdA, tcdB/tcdB027 or cdtB. AmpliVue® was positive in 242 (24.5%) and Illumigene® in 228 (23.1%) specimens. 167 (16.9%) specimens were positive in toxigenic culture. Real-time-tcdA and -tcdB PCR was positive in 211 (21.3%) specimens, Real-time-cdtB PCR was positive in 101 (10.2%) specimens and C. difficile-PCR (16S rDNA) in 267 (27.0%) specimens. The respective sensitivity, specificity, positive predictive value and negative predictive value compared to toxigenic culture were 91, 89, 62 and 98% for AmpliVue® and 91, 91, 67 and 98% for Illumigene®.

  16. Real-Time PCR in the early detection of invasive fungal infection in ...

    African Journals Online (AJOL)

    Blood samples were cultured for fungi and were analyzed with real-time PCR using universal fungal primers. For positive samples of fungal infection, aspergillus-specific primers were used for detection of aspergillus. Results: Seventeen patients (56.7%) proved to have IFI. Blood culture detected Candida in 2 patients only, ...

  17. Latent class analysis of the diagnostic characteristics of PCR and conventional bacteriological culture in diagnosing intramammary infections caused by Staphylococcus aureus in dairy cows at dry off

    Directory of Open Access Journals (Sweden)

    Cederlöf Sara Ellinor

    2012-11-01

    Full Text Available Abstract Background Staphylococcus aureus is one of the most common causes of intramammary infections in dairy cows at dry off. Reliable identification is important for disease management on herd level and for antimicrobial treatment of infected animals. Our objective was to evaluate the test characteristics of PathoProof ™ Mastitis PCR Assay and bacteriological culture (BC in diagnosing bovine intramammary infections caused by S. aureus at dry off at different PCR cycle threshold (Ct-value cut-offs. Methods Sterile quarter samples and non-sterile composite samples from 140 animals in seven herds were collected in connection with the dairy herd improvement (DHI milk recording. All quarter samples were analyzed using BC whereas all composite samples were analyzed with PathoProof ™ Mastitis PCR Assay. Latent class analysis was used to estimate test properties for PCR and BC in the absence of a perfect reference test. The population was divided into two geographically divided subpopulations and the Hui-Walter 2-test 2-populations model applied to estimate Se, Sp for the two tests, and prevalence for the two subpopulations. Results The Se for PCR increased with increasing Ct-value cut-off, accompanied by a small decrease in Sp. For BC the Se decreased and Sp increased with increasing Ct-value cut-off. Most optimal test estimates for the real-time PCR assay were at a Ct-value cut-off of 37; 0.93 [95% posterior probability interval (PPI 0.60-0.99] for Se and 0.95 [95% PPI 0.95-0.99] for Sp. At the same Ct-value cut-off, Se and Sp for BC were 0.83 [95% PPI 0.66-0.99] and 0.97 [95% PPI 0.91-0.99] respectively. Depending on the chosen PCR Ct-value cut-off, the prevalence in the subpopulations varied; the prevalence increased with increasing PCR Ct-value cut-offs. Conclusion Neither BC nor real-time PCR is a perfect test in detecting IMI in dairy cows at dry off. The changes in sensitivity and prevalence at different Ct-value cut-offs for both PCR and

  18. Latent class analysis of the diagnostic characteristics of PCR and conventional bacteriological culture in diagnosing intramammary infections caused by Staphylococcus aureus in dairy cows at dry off

    DEFF Research Database (Denmark)

    Cederlöf, Sara Ellinor; Toft, Nils; Aalbæk, Bent

    2012-01-01

    ABSTRACT: BACKGROUND: Staphylococcus aureus is one of the most common causes of intramammary infections in dairy cows at dry off. Reliable identification is important for disease management on herd level and for antimicrobial treatment of infected animals. Our objective was to evaluate the test...... characteristics of PathoProof TM Mastitis PCR Assay and bacteriological culture (BC) in diagnosing bovine intramammary infections caused by S. aureus at dry off at different PCR cycle threshold (Ct)-value cut-offs. METHODS: Sterile quarter samples and non-sterile composite samples from 140 animals in seven herds...

  19. New method using quantitative PCR to follow the tick blood meal and to assess the anti-feeding effect of topical acaricide against Rhipicephalus sanguineus on dogs.

    Science.gov (United States)

    Fourie, J J; Joubert, A; Labuschagné, M; Beugnet, F

    2014-05-01

    A 28-day study was conducted to assess the dynamic of blood feeding by Rhipicephalus sanguineus ticks on dogs treated or not with a novel topical combination of fipronil, amitraz and (S)-methoprene. Dogs were infested weekly through exposure to ticks in crates for 4h. Ticks were then counted in the crates at 2h and 4h post dog exposure. Ticks were also counted and removed from the dogs at 2h, 4h, 6h, 12h and 24h post tick exposure. The inhibition of blood feeding was assessed by both tick quantification and designing and performing a quantitative PCR (qPCR) to detect the canine hydroxymethylbilane synthase (HMBS) gene in ticks. The percentage of repellency sensu lato based on the ticks collected in crates at 2h varied from 4.7% at day 28 to 48.3% at day 7. The immediate mortality rate of the ticks expelled at 2h varied from 1.5% at day 21 to 31.7% at day 7. The efficacy calculation showed that the acaricidal combination started to kill ticks in as little as 2h. The average efficacy reached 90.0% at 12h post crate challenges and 100% at 24h post exposure in crates. The inclusion of an internal amplification control was used to ensure that no significant template-derived PCR inhibition (≤ 6.2%) affected the overall results. The reduction of blood feeding was significant at 4h (>80.0%) and >99.0% at 24h post tick exposure in the crate. The high repellency rate and the lethal efficacy of CERTIFECT(®) resulted in significantly fewer live attached ticks, consequently reducing blood intake and fluid exchanges. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

  20. Screening and DetectingSalmonellain Different Food Matrices in Southern Tunisia Using a Combined Enrichment/Real-Time PCR Method: Correlation with Conventional Culture Method.

    Science.gov (United States)

    Siala, Mariam; Barbana, Amina; Smaoui, Salma; Hachicha, Salma; Marouane, Chema; Kammoun, Sana; Gdoura, Radhouane; Messadi-Akrout, Férièle

    2017-01-01

    A combined enrichment/ newly developed invA TaqMan ® real-time PCR (qPCR) method as a screening assay to detect Salmonella spp. in 500 naturally food matrices is evaluated. DNA template for qPCR was extracted from an overnight pre-enriched sample in buffered peptone water using lysis-guanidine isothiocyanate method. Heterologous internal amplification control (IAC) was incorporated during qPCR assays and co-amplified with the invA gene of the target pathogen. InvA qPCR exhibited 100% specificity when testing 94 Salmonella strains (inclusivity) and 32 non- Salmonella strains (exclusivity). The qPCR showed a consistent detection of two copies of the invA gene/PCR reaction, a good intra- and inter-run reproducibility with a good PCR efficiency (89.6%). QPCR was sensitive and showed Salmonella detection at 8.5 × 10 0 CFU mL -1 of artificially spiked poultry meat -BWP solution in less than 40 cycles. When analyzing 500 different food matrices and comparing the results with the ISO 6579:2002 conventional culture method, the sensitivity and specificity were 100 and 76.6%, respectively. QPCR showed Salmonella spp. DNA in raw poultry meat 27/45 (60%), milk 31/93 (33.3%), raw red meat 5/13 (38.5%), and fish 11/46 (23.9%) samples. The prevalence of Salmonella spp. in cakes, dairy, cooked meals, charcuterie products using qPCR was 11/14 (26.8%), 5/22 (22.7%), 32/150 (21.3%), and 5/20 (25%), respectively, compared to 0% as demonstrated by culture. S. Anatum was the most common serovar found associated with red meat compared to S. kentucky isolated from fish and poultry meat. In conclusion, our study is the first to use a combined enrichment/ invA qPCR method as a screening assay to detect Salmonella DNA in different types of commercialized food in Southern Tunisia. QPCR results indicate that Salmonella contamination is common in milk and in other types of food samples.

  1. Screening and Detecting Salmonella in Different Food Matrices in Southern Tunisia Using a Combined Enrichment/Real-Time PCR Method: Correlation with Conventional Culture Method

    Directory of Open Access Journals (Sweden)

    Mariam Siala

    2017-12-01

    Full Text Available A combined enrichment/ newly developed invA TaqMan® real-time PCR (qPCR method as a screening assay to detect Salmonella spp. in 500 naturally food matrices is evaluated. DNA template for qPCR was extracted from an overnight pre-enriched sample in buffered peptone water using lysis–guanidine isothiocyanate method. Heterologous internal amplification control (IAC was incorporated during qPCR assays and co-amplified with the invA gene of the target pathogen. InvA qPCR exhibited 100% specificity when testing 94 Salmonella strains (inclusivity and 32 non-Salmonella strains (exclusivity. The qPCR showed a consistent detection of two copies of the invA gene/PCR reaction, a good intra- and inter-run reproducibility with a good PCR efficiency (89.6%. QPCR was sensitive and showed Salmonella detection at 8.5 × 100 CFU mL-1 of artificially spiked poultry meat -BWP solution in less than 40 cycles. When analyzing 500 different food matrices and comparing the results with the ISO 6579:2002 conventional culture method, the sensitivity and specificity were 100 and 76.6%, respectively. QPCR showed Salmonella spp. DNA in raw poultry meat 27/45 (60%, milk 31/93 (33.3%, raw red meat 5/13 (38.5%, and fish 11/46 (23.9% samples. The prevalence of Salmonella spp. in cakes, dairy, cooked meals, charcuterie products using qPCR was 11/14 (26.8%, 5/22 (22.7%, 32/150 (21.3%, and 5/20 (25%, respectively, compared to 0% as demonstrated by culture. S. Anatum was the most common serovar found associated with red meat compared to S. kentucky isolated from fish and poultry meat. In conclusion, our study is the first to use a combined enrichment/invA qPCR method as a screening assay to detect Salmonella DNA in different types of commercialized food in Southern Tunisia. QPCR results indicate that Salmonella contamination is common in milk and in other types of food samples.

  2. Measurement of Blood Coagulation Factor Synthesis in Cultures of Human Hepatocytes.

    Science.gov (United States)

    Heinz, Stefan; Braspenning, Joris

    2015-01-01

    An important function of the liver is the synthesis and secretion of blood coagulation factors. Within the liver, hepatocytes are involved in the synthesis of most blood coagulation factors, such as fibrinogen, prothrombin, factor V, VII, IX, X, XI, XII, as well as protein C and S, and antithrombin, whereas liver sinusoidal endothelial cells produce factor VIII and von Willebrand factor. Here, we describe methods for the detection and quantification of most blood coagulation factors in hepatocytes in vitro. Hepatocyte cultures indeed provide a valuable tool to study blood coagulation factors. In addition, the generation and expansion of hepatocytes or hepatocyte-like cells may be used in future for cell-based therapies of liver diseases, including blood coagulation factor deficiencies.

  3. HB&L System: rapid determination of antibiotic sensitivity of bacteria isolated from blood cultures.

    Directory of Open Access Journals (Sweden)

    Simone Barocci

    2010-03-01

    Full Text Available Introduction. Blood culture is an important method to detect microbial pathogens on blood, very useful for diagnosing bacterial infections. Unfortunately, classical diagnostic protocols cannot directly identify bacteria responsible for sepsis and accordingly their antimicrobial profiles. This problem causes a delay of almost two days in the availability of a specific antimicrobial profile. Objective. Among the main causes of death, sepsis have a relevant importance. For this reason it is important both to identify pathogens and to perform an antimicrobial susceptibility test in the shortest time as possible. For this purpose, the main aim of this study is the evaluation of the performances of an antimicrobial susceptibility determination directly performed on positive blood cultures. Materials and methods. This study has been performed on 70 positive blood cultures, during the period from January to July 2009. A number of 35 blood cultures were positive for Gram negative bacteria, and 35 were positive for Gram positive bacteria. From these positive blood cultures, after a short sample preparation, it has been possible to directly determine antimicrobial susceptibility profiles by using the HB&L (formerly URO-QUICK instrument. Results. The HB&L system results showed a very good correlation with both the classical disk diffusion method and VITEK 2 automatic system.The performances between the methods carried out in this study were equivalent. Conclusions. From data reported, thanks to the rapidity and simplicity of the method used, we can assert that the direct susceptibility test available with the HB&L system, is useful for a rapid and early choice of the antibiotic treatment.

  4. Molecular screening for Candida orthopsilosis and Candida metapsilosis among Danish Candida parapsilosis group blood culture isolates: proposal of a new RFLP profile for differentiation

    DEFF Research Database (Denmark)

    Mirhendi, Hossein; Bruun, Brita; Schønheyder, Henrik Carl

    2010-01-01

    Candida orthopsilosis and Candida metapsilosis are recently described species phenotypically indistinguishable from Candida parapsilosis . We evaluated phenotyping and molecular methods for the detection of these species among 79 unique blood culture isolates of the C. parapsilosis group obtained...... during the years 2004-2008. The isolates were screened by PCR amplification of the secondary alcohol dehydrogenase-encoding gene ( SADH) followed by digestion with the restriction enzyme Ban I, using C. parapsilosis ATCC 22019, C. orthopsilosis ATCC 96139 and C. metapsilosis ATCC 96144 as controls....... Isolates with RFLP patterns distinct from C. parapsilosis were characterized by sequence analysis of the ITS1-ITS2, 26S rRNA (D1/D2) and SADH regions. Restriction patterns for the 3 species with each of 610 restriction enzymes were predicted in silico using 12 available sequences. By PCR-RFLP of the SADH...

  5. Evaluation of conventional castaneda and lysis centrifugation blood culture techniques for diagnosis of human brucellosis.

    Science.gov (United States)

    Mantur, Basappa G; Mangalgi, Smita S

    2004-09-01

    We investigated the role of the lysis centrifugation blood culture technique over the conventional Castaneda technique for the diagnosis of human brucellosis. The lysis centrifugation technique has been found to be more sensitive in both acute (20% higher sensitivity; P centrifugation was in the mean detection time, which was only 2.4 days in acute and 2.7 days in chronic cases, with 103 out of 110 (93.6%) and 17 out of 20 (85%) cultures from acute and chronic brucellosis, respectively, detected before the conventional culture was positive. Our results confirmed the potential usefulness of the lysis technique in diagnosis and institution of appropriate antibiotic therapy.

  6. Culture-independent identification and quantification of Gallibacterium anatis (G. anatis) by real-time quantitative PCR

    DEFF Research Database (Denmark)

    Wang, Chong; Robles, Francisco; Ramirez, Saul

    2016-01-01

    -time quantitative PCR (qPCR) method allowing species-specific identification and quantification of G. anatis. A G. anatis specific DNA sequence was identified in the gyrase subunit B gene (gyrB) and used to design a TaqMan probe and corresponding primers. The specificity of the assay was tested on 52 bacterial...

  7. Comparison of BACTEC™ blood culture media for the detection of fungemia

    DEFF Research Database (Denmark)

    Datcu, Raluca; Boel, J; Jensen, I M

    2017-01-01

    with a positive blood culture with Candida species delivered to the Department of Clinical Microbiology, Herlev and Gentofte Hospital, Denmark in the 8-year period 2006 through 2014. The patients had at least one BACTEC™ aerobic and one Mycosis bottle sampled at the same time and at least one of the bottles...

  8. Discrepancy between growth of Coccidioides immitis in bacterial blood culture media and a radiometric growth index

    International Nuclear Information System (INIS)

    Ampel, N.M.; Wieden, M.A.

    1988-01-01

    Spherules of Coccidioides immitis grew readily after inoculation in vented trypticase soy broth, biphasic brain heart infusion media, and aerobic tryptic soy broth bottles used in a radiometric system (BACTEC). However, visible growth was not accompanied by a significant radiometric growth index. Growth of C. immitis can be visually detected in routine bacterial blood culture media while the radiometric growth index remains negative

  9. Isolation of Mycobacterium chelonei with the lysis-centrifugation blood culture technique.

    OpenAIRE

    Fojtasek, M F; Kelly, M T

    1982-01-01

    Mycobacterium chelonei was isolated from a patient by the lysis-centrifugation and the conventional two-bottle blood culture methods. The lysis-centrifugation method was significantly more sensitive and rapid than the conventional method in detecting and isolating this organism; quantitations done by this method were useful for monitoring response to therapy.

  10. Comparison of the lysis-centrifugation and agitated biphasic blood culture systems for detection of fungemia.

    Science.gov (United States)

    Murray, P R

    1991-01-01

    Although the detection of fungemia has been improved by the use of vented or biphasic blood culture bottles, the best recovery and earliest detection have been reported in the Isolator lysis-centrifugation system. It was recently demonstrated that improved detection of both bacteria and fungi was accomplished by mechanically agitating blood culture bottles for the first 24 h of incubation. In this study the detection of fungemia by use of the Isolator system was compared with that of an agitated biphasic system. A total of 182 fungi were isolated from blood specimens inoculated into both culture systems. No difference in the overall recovery of fungi or individual species of yeasts was observed between the two systems. However, all seven isolates of Histoplasma capsulatum were recovered in the Isolator system only. The time required to detect fungemia with each of the two systems was also compared. No statistically significant difference was observed. From the data collected during this 18-month study, it can be concluded that the overall recovery and time of detection of yeasts are equivalent in the lysis-centrifugation system and the agitated biphasic blood culture system. The lysis-centrifugation system is still superior for the detection of filamentous fungi such as H. capsulatum. PMID:1993772

  11. Antimicrobial Susceptibility and Microorganisms Isolated from Blood Cultures of Hospitalized Patients in Intensive Care Units

    Directory of Open Access Journals (Sweden)

    Emine Küçükateş

    2016-06-01

    Full Text Available Aim: The aim of this study was to evaluate microorganism growth in blood cultures of hospitalized patients in our intensive care units and to determine appropriate antimicrobial agents for treatment. Methods: We retrospectively investigated the blood cultures obtained from the patients hospitalized in the Coronary and Surgical Intensive Care Units at the Institute of Cardiology, İstanbul University, between July 2013 and December 2014. All microorganisms were identified using the conventional methods. Results: A total of 1034 blood cultures were obtained from 324 patients. Microbial growth was detected in 174 (16.8% blood cultures of 68 patients. Among all microbial growth, 113 (58.55% were gram-positive bacteria, 69 (35.75% were gram-negative rods and 11 (5.7% were fungi. Staphylococcus aureus was the most frequent microorganism (48; 24.87%, followed by coagulasenegative Staphylococci (35; 18,13%, Enterococcus spp. (30; 15,54%, Stenotrophomonas maltophilia, Escherichia coli, and Pseudomonas spp. 60.4% of Staphylococcus aureus were methicillin-resistant and 65.7% of coagulase-negative Staphylococci were also methicillin-resistant. All Staphylococci and Enterococci were not resistant to vancomycin, teicoplanin and tigecycline. All the gram-negative rods were susceptible to colistin and tigecycline, followed by imipenem (71.6% and meropenem (70.7%. Conclusions: We assume that infection control measures must be increased due to high antibiotic resistance and besides, antibiotic policies should be improved.

  12. Organ donation registration among current blood donors in The Netherlands. Personal, cultural and network determinants

    NARCIS (Netherlands)

    Merz, E.M.; van den Hurk, Katja; De Kort, Wim L.A.M.

    2017-01-01

    Introduction: In the Netherlands, there is a constant shortage in donor organs, resulting in long waiting lists. The decision to register as organ donor is associated with several demographic, cultural, and personal factors. Previous research on attitudes and motivations toward blood and organ

  13. Bacteriological Profile of Blood Culture Positive Sepsis in Newborn at BPKIHS, Dharan Nepal

    Directory of Open Access Journals (Sweden)

    Piush Kanodia

    2017-03-01

    Full Text Available Background & Objectives: Neonatal infections currently cause about 1.6 million deaths annually in developing countries. Sepsis and meningitis is responsible for most of these deaths. This study was undertaken to determine the bacteriological profiles and antibiotic sensitivity patterns of isolates from blood cultures of neonates admitted in a tertiary care hospital in Eastern Nepal.Materials & Methods: A retrospective study was conducted at pediatric department from January, 2014 to December 2014. Total 1009 newborns blood sample with suspected and clinical sepsis were cultured by using standard microbiological technique and antibiotic sensitivity patterns were studied. Results: The positive blood culture was 32.4% (327/1009. Gram positive bacteria were more common 231(71% than gram negative bacteria 96(29%. Staphylococcus aureus 174 (53.2% and acinetobacter 46(14.1% were the commonest isolates in blood culture. Most of the organisms showed sensitivity with aminoglycosides (gentamicin and amikacin and third generation cephalosporins.Conclusion: Staphylococcus aureus, Acinetobacter and Klebsiella species remain the principal organisms causing neonatal sepsis and antibiotics like amino glycosides should be first choice of drugs.

  14. Sodium polyanethole sulfonate as an inhibitor of activation of complement function in blood culture systems

    DEFF Research Database (Denmark)

    Palarasah, Yaseelan; Skjoedt, Mikkel-Ole; Vitved, Lars

    2010-01-01

    Sodium polyanethole sulfonate (SPS; trade name, Liquoid) is a constituent in culture media used to grow bacteria from blood samples from patients suspected of bacteremia. SPS prevents the killing of bacteria by innate cellular and humoral factors. We analyzed the effect of SPS on the three...

  15. Mortality and prognostic factors of patients who have blood cultures performed in the emergency department

    DEFF Research Database (Denmark)

    Prier Lindvig, Katrine; Nielsen, Stig Lønberg; Henriksen, Daniel P

    2016-01-01

    BACKGROUND: Early identification and treatment of patients with severe infection improve their prognosis. The aims of this study were to describe the 30-day mortality and to identify prognostic factors among blood-cultured patients in a medical emergency department (MED). PATIENTS AND METHODS: Th...

  16. Draft Genome Sequence of "Terrisporobacter othiniensis" Isolated from a Blood Culture from a Human Patient

    DEFF Research Database (Denmark)

    Lund, Lars Christian; Sydenham, Thomas Vognbjerg; Høgh, Silje Vermedal

    2015-01-01

    "Terrisporobacter othiniensis" (proposed species) was isolated from a blood culture. Genomic DNA was sequenced using a MiSeq benchtop sequencer (Illumina) and assembled using the SPAdes genome assembler. This resulted in a draft genome sequence comprising 3,980,019 bp in 167 contigs containing 3...

  17. Predicting bacteremia based on nurse-assessed food consumption at the time of blood culture.

    Science.gov (United States)

    Komatsu, Takayuki; Onda, Toshihito; Murayama, Go; Yamanouchi, Masashi; Inukai, Minori; Sakai, Ai; Kikuta, Masumi; Branch, Joel; Aoki, Makoto; Tierney, Lawrence M; Inoue, Kenji

    2012-01-01

    Bacteremia and its complications are important causes of morbidity and mortality in hospitalized patients. However, the yield of blood cultures is relatively low, with many false-positive results from bacterial contamination. We investigated the relationship between patient food consumption and the presence of bacteremia. This was an observational analysis of a cohort of 1179 patients who underwent blood culture analysis between January 2005 and December 2009. Patients with anorexia-inducing conditions, such as gastrointestinal illness and malignant disease treated with chemotherapy, were excluded. Food consumption was rated by nurses as the percentage of food consumed during the meal preceding the blood culture. Groupings were as follows: low consumption (50% to 80%). Low consumption was observed in 39.8% of patients, moderate in 17.8%, and high in 41.6%. The average body temperature was 38.1 ± 1.1°C. Bacteremia was present in 18.5%, 3.9%, and 1.4% of patients in the low, moderate, and high food consumption groups, respectively. The negative predictive value was 98.3%, suggesting that bacteremia is very unlikely in the setting of good food intake. Bacteremia is an unlikely occurrence in hospitalized patients who maintain adequate food consumption at the time of blood culture. Copyright © 2012 Society of Hospital Medicine.

  18. Analytical performance of a multiplex Real-Time PCR assay using TaqMan probes for quantification of Trypanosoma cruzi satellite DNA in blood samples.

    Directory of Open Access Journals (Sweden)

    Tomas Duffy

    Full Text Available BACKGROUND: The analytical validation of sensitive, accurate and standardized Real-Time PCR methods for Trypanosoma cruzi quantification is crucial to provide a reliable laboratory tool for diagnosis of recent infections as well as for monitoring treatment efficacy. METHODS/PRINCIPAL FINDINGS: We have standardized and validated a multiplex Real-Time quantitative PCR assay (qPCR based on TaqMan technology, aiming to quantify T. cruzi satellite DNA as well as an internal amplification control (IAC in a single-tube reaction. IAC amplification allows rule out false negative PCR results due to inhibitory substances or loss of DNA during sample processing. The assay has a limit of detection (LOD of 0.70 parasite equivalents/mL and a limit of quantification (LOQ of 1.53 parasite equivalents/mL starting from non-boiled Guanidine EDTA blood spiked with T. cruzi CL-Brener stock. The method was evaluated with blood samples collected from Chagas disease patients experiencing different clinical stages and epidemiological scenarios: 1- Sixteen Venezuelan patients from an outbreak of oral transmission, 2- Sixty three Bolivian patients suffering chronic Chagas disease, 3- Thirty four Argentinean cases with chronic Chagas disease, 4- Twenty seven newborns to seropositive mothers, 5- A seronegative receptor who got infected after transplantation with a cadaveric kidney explanted from an infected subject. CONCLUSIONS/SIGNIFICANCE: The performing parameters of this assay encourage its application to early assessment of T. cruzi infection in cases in which serological methods are not informative, such as recent infections by oral contamination or congenital transmission or after transplantation with organs from seropositive donors, as well as for monitoring Chagas disease patients under etiological treatment.

  19. Quantitative Microbiological Study of Human Carious Dentine by Culture and Real-Time PCR: Association of Anaerobes with Histopathological Changes in Chronic Pulpitis

    Science.gov (United States)

    Martin, F. Elizabeth; Nadkarni, Mangala A.; Jacques, Nicholas A.; Hunter, Neil

    2002-01-01

    The bacteria found in carious dentine were correlated with the tissue response of the dental pulps of 65 teeth extracted from patients with advanced caries and pulpitis. Standardized homogenates of carious dentine were plated onto selective and nonselective media under anaerobic and microaerophilic conditions. In addition, real-time PCR was used to quantify the recovery of anaerobic bacteria. Primers and fluorogenic probes were designed to detect the total anaerobic microbial load, the genera Prevotella and Fusobacterium, and the species Prevotella melaninogenica, Porphyromonas endodontalis, Porphyromonas gingivalis, and Micromonas (formerly Peptostreptococcus) micros. The pulpal pathology was categorized according to the cellular response and degenerative changes. Analysis of cultured bacteria showed a predominance of gram-positive microorganisms, particularly lactobacilli. Gram-negative bacteria were also present in significant numbers with Prevotella spp., the most numerous anaerobic group cultured. Real-time PCR analysis indicated a greater microbial load than that determined by colony counting. The total number of anaerobes detected was 41-fold greater by real-time PCR than by colony counting, while the numbers of Prevotella and Fusobacterium spp. detected were 82- and 2.4-fold greater by real-time PCR than by colony counting, respectively. Real-time PCR also identified M. micros, P. endodontalis, and P. gingivalis in 71, 60, and 52% of carious samples, respectively. Correlation matrices of the real-time PCR data revealed significant positive associations between M. micros and P. endodontalis detection and inflammatory degeneration of pulpal tissues. These anaerobes have been strongly implicated in endodontic infections that occur as sequelae to carious pulpitis. Accordingly, the data suggest that the presence of high levels of these bacteria in carious lesions may be indicative of irreversible pulpal pathology. PMID:11980945

  20. Development of an internal amplification control system for a real-time PCR assay for detection of Neisseria meningitidis in CSF and EDTA blood.

    Science.gov (United States)

    McIver, Christopher J; Bell, Sydney M; Er, Noel

    2014-06-01

    The aim of this study was to assemble and assess a non-competitive internal amplification control (IAC) system targeting the Escherichia coli alanine racemase (alr) gene to include in a real-time polymerase chain reaction (PCR) assay for Neisseria meningitidis. Primers and hybridisation probes specific for the IAC were designed and assessed for specificity. Amplification efficiency and limit of detection for the assembled assay was extrapolated using standard curves constructed with serial dilutions of N. meningitidis in saline, pooled cerebrospinal fluid (CSF) and EDTA blood. The 95% confidence limits (CI) were calculated for IAC crossing-points recorded for assays for N. meningitidis ctrA in saline (negative blank), and N. meningitides-negative samples of CSF and EDTA blood. These limits served as a reference range against which the IAC crossing-points recorded for prospective assays are compared to detect sample inhibition. This system was used in testing consecutive EDTA blood samples from two cases of meningococcal disease. The IAC system is specific for Escherichia coli and Shigella species. The amplification efficiency of the assembled assay for N. meningitidis and ability to detect low target DNA levels was not compromised with the inclusion of the IAC system. The IAC crossing-points varied in clinical samples of CSF and EDTA blood. The elucidated reference range for EDTA blood was used to detect sample inhibition in one of the two clinical cases investigated.The IAC system monitors the performance of all processes in the assembled assay for N. meningitidis. Measuring IAC crossing-points serves as an indicator of sample stability and inhibitory properties when testing single or multiple samples from the same patient. Specificity for E. coli and Shigella species enables inclusion in assays of different targets within the same laboratory. Reporting PCR assay results in the context of the IAC crossing-points and reference ranges validates against sample

  1. Assessing the performance of multiplexed tandem PCR for the diagnosis of pathogenic genotypes of Theileria orientalis using pooled blood samples from cattle.

    Science.gov (United States)

    Gebrekidan, Hagos; Gasser, Robin B; Stevenson, Mark A; McGrath, Sean; Jabbar, Abdul

    2017-02-01

    Oriental theileriosis caused by multiple genotypes of Theileria orientalis is an important tick-borne disease of bovines. Here, we assessed the performance of an established multiplexed tandem PCR (MT-PCR) for the diagnosis of the two recognized, pathogenic genotypes (chitose and ikeda) of T. orientalis in cattle using pooled blood samples. We used a total of 265 cattle blood samples, which were divided into two groups according to previous MT-PCR results for individual samples. Samples in group 1 (n = 155) were from a herd with a relatively high prevalence of T. orientalis infection; and those in group 2 (n = 110) were from four herds with a low prevalence. For group 1, 31 and 15 batches of five- and ten-pooled samples (selected at random), respectively, were formed. For group 2, 22 and 11 batches of five- and ten-pooled samples (selected at random), respectively, were formed. DNAs from individual pooled samples in each batch and group were then tested by MT-PCR. For group 1, the apparent prevalences estimated using the 31 batches of five-pooled samples (97%) and 15 batches of ten-pooled samples (100%) were significantly higher compared with individual samples (75%). For group 2, higher apparent prevalences (9% and 36%) were also recorded for the 22 and 11 batches of pooled samples, respectively, compared with individual samples (7%). Overall, the average infection intensity recorded for the genotypes of chitose and ikeda were considerably lower in pooled compared with individual samples. The diagnostic specificities of MT-PCR were estimated at 95% and 94%, respectively, when batches of five- and ten-pooled samples were tested, and 94% for individual samples. The diagnostic sensitivity of this assay was estimated at 98% same for all individual, five- and ten-pooled samples. This study shows that screening batches of five- and ten-pooled blood samples from cattle herds are similar to those obtained for individual samples, and, importantly, that the reduced cost

  2. Comparison of bacterial culture and qPCR testing of rectal and pen floor samples as diagnostic approaches to detect enterotoxic Escherichia coli in nursery pigs

    DEFF Research Database (Denmark)

    Weber, N. R.; Nielsen, J. P.; Hjulsager, Charlotte Kristiane

    2017-01-01

    Enterotoxigenic E. coli (ETEC) are a major cause of diarrhoea in weaned pigs. The objective of this study was to evaluate the agreement at pen level among three different diagnostic approaches for the detection of ETEC in groups of nursery pigs with diarrhoea. The diagnostic approaches used were......: bacterial culturing of faecal samples from three pigs (per pen) with clinical diarrhoea and subsequent testing for virulence genes in E. coli isolates; bacterial culturing of pen floor samples and subsequent testing for virulence genes in E. coli isolates; qPCR testing of pen floor samples in order...... to determine the quantity of F18 and F4 genes. The study was carried out in three Danish pig herds and included 31 pens with a pen-level diarrhoea prevalence of > 25%, as well as samples from 93 diarrhoeic nursery pigs from these pens. All E. coli isolates were analysed by PCR and classified as ETEC when genes...

  3. Analysis of mtDNT 4977bp deletion induced by ionizing radiation in human peripheral blood nucleated cells using real-time PCR

    International Nuclear Information System (INIS)

    Fan Tianli; Wang Ping; Han Lin; Liu Yulong; Liu Yumin

    2010-01-01

    To detect mitochondrial DNA(mtDNA) 4977bp deletion(triangle open mtDNA 4977 ) in human peripheral blood nucleated cells exposed to ionizing radiation in vitro by using real-time PCR, and explore possibility of the index as biodosimetry for estimating biological dose in radiation accident,six healthy individuals' peripheral blood was collected,and the blood samples were irradiated with 0,1,2,3,4 and 5 Gy 60 Co gamma-ray. The triangle open mtDNA 4977 and total mtDNA copy number(mtDNA total ) in the mtDNA samples were detected, and then the deletion rates were calculated. The results showed that the mtDNA total and triangle open mtDNA 4977 copy number, and the deletion rates of mtDNA 4977bp in the mtDNA samples from 6 healthy individuals' blood exposed to 1-5 Gy radiation were higher than that with the samples exposed to 0 Gy radiation(p 0.05). The results indicated that ionizing radiation can induce accumulation of the triangle open mtDNA 4977 and increase of mtDNA total copy number in human peripheral blood nucleated cells,but both the mtDNA 4977bp deletion and exposure dose(0-5 Gy) were not obviously correlated. (authors)

  4. Real-time PCR-based genotyping from whole blood using Taq DNA polymerase and a buffer supplemented with 1,2-propanediol and trehalose

    Czech Academy of Sciences Publication Activity Database

    Utekal, Pavol; Kocanda, Lukáš; Matoušek, P.; Wagner, P.; Bugajev, Viktor; Dráber, Petr

    2015-01-01

    Roč. 416, January (2015), s. 178-182 ISSN 0022-1759 R&D Projects: GA ČR(CZ) GBP302/12/G101; GA MPO FR-TI3/067; GA ČR(CZ) GA14-09807S; GA ČR(CZ) GA14-00703S Institutional support: RVO:68378050 Keywords : 1,2-Propanediol * real-time PCR * SYBR Green I * Taq DNA polymerase * trehalose * Unseparated blood Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.858, year: 2015

  5. BIOCHEMICAL AND MOLECULAR CHARACTERISTICS OF LISTERIA MONOCYTOGENES ISOLATES FROM A PROSTHETIC MITRAL HEART VALVE-BEARING PATIENT´S BLOOD CULTURES

    Directory of Open Access Journals (Sweden)

    Nilma Cintra Lea

    2013-09-01

    Full Text Available Background: In Brazil, listeriosis is not a notifiable disease; thus, the incidence of Brazilian cases remains unknown. Listeria monocytogenes is not always included in automated systems, and its detection depends on the high skill level of microbiology laboratory professionals. This paper describes the characteristics of L. monocytogenes isolates fortuitously obtained from an endocarditis case in Recife, PE, Brazil. Methods: Six bacterial isolates obtained from six blood cultures from a 28-year-old male bearing a prosthetic mitral heart valve were analyzed by PCR using primers specific of L. monocytogenes to confirm a presumptive identification, determine the serotype and presence of the virulence genes (inlA, inlB, inlC, inlJ, hly, plcA, actA, prfA in an attempt to determine the Listeria genotype by PCR-ribotyping. Results: The samples were identified as L. monocytogenes 4b. All investigated virulence genes were amplified by PCR, and the identity of the amplified segments was confirmed by sequencing. A deletion of 105 base pairs was detected in the actA gene. All of the samples generated the same PCR-ribotype pattern, clustered into a single ribotype, and were considered a single strain. Conclusion: L. monocytogenes infection should be considered in endocarditis differential diagnoses, especially among high-risk groups, due to its high pathogenicity and the environmental ubiquity.

  6. Biochemical and molecular characteristics of Listeria monocytogenes isolates from a prosthetic mitral heart valve-bearing patient´s blood cultures

    Directory of Open Access Journals (Sweden)

    Nilma Cintra Leal

    2013-09-01

    Full Text Available Background: In Brazil, listeriosis is not a notifiable disease; thus, the incidence of Brazilian cases remains unknown. Listeria monocytogenes is not always included in automated systems, and its detection depends on the high skill level of microbiology laboratory professionals. This paper describes the characteristics of L. monocytogenes isolates fortuitously obtained from an endocarditis case in Recife, PE, Brazil. Methods: Six bacterial isolates obtained from six blood cultures from a 28-year-old male bearing a prosthetic mitral heart valve were analyzed by PCR using primers specific of L. monocytogenes to confirm a presumptive identification, determine the serotype and presence of the virulence genes (inlA, inlB, inlC, inlJ, hly, plcA, actA, prfA in an attempt to determine the Listeria genotype by PCR-ribotyping. Results: The samples were identified as L. monocytogenes 4b. All investigated virulence genes were amplified by PCR, and the identity of the amplified segments was confirmed by sequencing. A deletion of 105 base pairs was detected in the actA gene. All of the samples generated the same PCR-ribotype pattern, clustered into a single ribotype, and were considered a single strain. Conclusion: L. monocytogenes infection should be considered in endocarditis differential diagnoses, especially among high-risk groups, due to its high pathogenicity and the environmental ubiquity.

  7. Correlation of Culture Positivity, PCR Positivity, and Burden of Borrelia burgdorferi Sensu Lato in Skin Samples of Erythema Migrans Patients with Clinical Findings.

    Science.gov (United States)

    Stupica, Daša; Lusa, Lara; Maraspin, Vera; Bogovič, Petra; Vidmar, Darja; O'Rourke, Maria; Traweger, Andreas; Livey, Ian; Strle, Franc

    2015-01-01

    Limited data are available regarding the relationship of Borrelia burden in skin of patients with erythema migrans (EM) and the disease course and post-treatment outcome. We studied 121 adult patients with EM in whom skin biopsy specimens were cultured and analyzed by quantitative PCR for the presence of Borreliae. Evaluation of clinical and microbiological findings were conducted at the baseline visit, and 14 days, 2, 6, and 12 months after treatment with either amoxicillin or cefuroxime axetil. In 94/121 (77.7%) patients Borrelia was detected in skin samples by PCR testing and 65/118 (55.1%) patients had positive skin culture result (96.8% B. afzelii, 3.2% B. garinii). Borrelia culture and PCR results correlated significantly with the presence of central clearing and EM size, while Borrelia burden correlated significantly with central clearing, EM size, and presence of newly developed or worsened symptoms since EM onset, with no other known medical explanation (new or increased symptoms, NOIS). In addition, the logistic regression model for repeated measurements adjusted for time from inclusion, indicated higher Borrelia burden was a risk factor for incomplete response (defined as NOIS and/or persistence of EM beyond 14 days and/or occurrence of new objective signs of Lyme borreliosis). The estimated association between PCR positivity and unfavorable outcome was large but not statistically significant, while no corresponding relationship was observed for culture positivity. Higher Borrelia burden in EM skin samples was associated with more frequent central clearing and larger EM lesions at presentation, and with a higher chance of incomplete response.

  8. Correlation of Culture Positivity, PCR Positivity, and Burden of Borrelia burgdorferi Sensu Lato in Skin Samples of Erythema Migrans Patients with Clinical Findings.

    Directory of Open Access Journals (Sweden)

    Daša Stupica

    Full Text Available Limited data are available regarding the relationship of Borrelia burden in skin of patients with erythema migrans (EM and the disease course and post-treatment outcome.We studied 121 adult patients with EM in whom skin biopsy specimens were cultured and analyzed by quantitative PCR for the presence of Borreliae. Evaluation of clinical and microbiological findings were conducted at the baseline visit, and 14 days, 2, 6, and 12 months after treatment with either amoxicillin or cefuroxime axetil.In 94/121 (77.7% patients Borrelia was detected in skin samples by PCR testing and 65/118 (55.1% patients had positive skin culture result (96.8% B. afzelii, 3.2% B. garinii. Borrelia culture and PCR results correlated significantly with the presence of central clearing and EM size, while Borrelia burden correlated significantly with central clearing, EM size, and presence of newly developed or worsened symptoms since EM onset, with no other known medical explanation (new or increased symptoms, NOIS. In addition, the logistic regression model for repeated measurements adjusted for time from inclusion, indicated higher Borrelia burden was a risk factor for incomplete response (defined as NOIS and/or persistence of EM beyond 14 days and/or occurrence of new objective signs of Lyme borreliosis. The estimated association between PCR positivity and unfavorable outcome was large but not statistically significant, while no corresponding relationship was observed for culture positivity.Higher Borrelia burden in EM skin samples was associated with more frequent central clearing and larger EM lesions at presentation, and with a higher chance of incomplete response.

  9. ELIME assay vs Real-Time PCR and conventional culture method for an effective detection of Salmonella in fresh leafy green vegetables.

    Science.gov (United States)

    Fabiani, L; Pucci, E; Delibato, E; Volpe, G; Piermarini, S; De Medici, D; Capuano, F; Palleschi, G

    2017-05-01

    The detection of Salmonella according to EC regulation is still primarily based on traditional microbiological culture methods that may take several days to be completed. The purpose of this work is to demonstrate the applicability of an Enzyme-Linked-Immuno-Magnetic-Electrochemical (ELIME) assay, recently developed by our research group for the detection of salmonella in irrigation water, in fresh (raw and ready-to-eat) leafy green vegetables by comparison with Real-Time PCR (RTi-PCR) and ISO culture methods. Since vegetables represent a more complex matrix than irrigation water, preliminary experiments were carried out on two leafy green vegetables that resulted negative for salmonella by the ISO method. 25g of these samples were experimentally inoculated with 1-10 CFU of S. Napoli or S. Thompson and pre-enriched for 20h in two different broths. At this time aliquots were taken, concentrated at different levels by centrifugation, and analyzed by ELIME and RTi-PCR. Once selected the best culture medium for salmonella growth, and the optimal concentration factor suitable to reduce the sample matrix effect, enhancing the out-put signal, several raw and ready-to-eat leafy green vegetables were artificially inoculated and pre-enriched. Aliquots were then taken at different incubation times and analyzed with both techniques. Results obtained showed that 20 and 8h of pre-enrichment were required to allow the target salmonella (1-10 CFU/25g) to multiply until reaching a detectable concentration by ELIME and RTi-PCR assays, respectively. A confirmation with the ISO culture method was carried out. Based on the available literature, this is the first report of the application of an ELISA based method for the detection of Salmonella in vegetables. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Development of real-time PCR array for simultaneous detection of eight human blood-borne viral pathogens.

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    Natalia Pripuzova

    Full Text Available BACKGROUND: Real-time PCR array for rapid detection of multiple viral pathogens should be highly useful in cases where the sample volume and the time of testing are limited, i.e. in the eligibility testing of tissue and organ donors. FINDINGS: We developed a real-time PCR array capable of simultaneously detecting eight human viral pathogens: human immunodeficiency virus types 1 and 2 (HIV-1 and -2, hepatitis B virus (HBV, hepatitis C virus (HCV, human T-cell leukemia virus-1 and -2 (HTLV-1 and -2, vaccinia virus (VACV and West Nile virus (WNV. One hundred twenty (120 primers were designed using a combination of bioinformatics approaches, and, after experimental testing, 24 primer sets targeting eight viral pathogens were selected to set up the array with SYBR Green chemistry. The specificity and sensitivity of the virus-specific primer sets selected for the array were evaluated using analytical panels with known amounts of viruses spiked into human plasma. The array detected: 10 genome equivalents (geq/ml of HIV-2 and HCV, 50 geq of HIV-1 (subtype B, HBV (genotype A and WNV. It detected 100-1,000 geq/ml of plasma of HIV-1 subtypes (A - G, group N and CRF (AE and AG isolates. Further evaluation with a panel consisting of 28 HIV-1 and HIV-2 clinical isolates revealed no cross-reactivity of HIV-1 or HIV-2 specific primers with another type of HIV. All 28 viral isolates were identified with specific primer sets targeting the most conserved genome areas. The PCR array correctly identified viral infections in a panel of 17 previously quantified clinical plasma samples positive for HIV-1, HCV or HBV at as low as several geq per PCR reaction. CONCLUSIONS: The viral array described here demonstrated adequate performance in the testing of donors' clinical samples. Further improvement in its sensitivity for the broad spectrum of HIV-1 subtypes is under development.

  11. Growth of human T lymphocyte colonies from whole blood: culture requirements and applications

    International Nuclear Information System (INIS)

    Knox, S.J.; Wilson, F.D.; Greenberg, B.R.; Shifrine, M.

    1982-01-01

    Growth of human lymphocyte colonies from whole blood following stimulation with PHA, Con A, or PPD is described. Individual colony cells were identified as T lymphocytes on the basis of surface marker and enzyme cytochemical characterizations. Colony formation increased as a power function over a wide range of cell concentrations above a critical minimal concentration. The whole blood culture system eliminates possible selective effects of lymphocyte colony techniques utilizing gradient-enriched lymphocyte fractions and more closely approximates the in vivo milieu. The whole blood colony method is more sensitive for the detection of low-level radiation effects on lymphocytes than widely used tests that measure 3 H-thymidine incorporation. In preliminary studies, researchers used the whole blood method to determine the relative radiosensitivity of lymphocytes from humans with various hematopoietic disorders, and observed abnormalities in mitogen responsiveness and colony formation in some of the patient groups. This method has wide application for studies in cellular and clinical immunology

  12. Use of Tissue Culture Techniques for Producing Virus-Free Plant in Garlic and Their Identification through Real-Time PCR

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    Hatıra Taşkın

    2013-01-01

    Full Text Available This study was performed for comparison of meristem culture technique with shoot tip culture technique for obtaining virus-free plant, comparison of micropropagation success of two different nutrient media, and determination of effectiveness of real-time PCR assay for the detection of viruses. Two different garlic species (Allium sativum and Allium tuncelianum and two different nutrient media were used in this experiment. Results showed that Medium 2 was more successful compared to Medium 1 for both A. tuncelianum and A. sativum (Kastamonu garlic clone. In vitro plants obtained via meristem and shoot tip cultures were tested for determination of onion yellow dwarf virus (OYDV and leek yellow stripe virus (LYSV through real-time PCR assay. In garlic plants propagated via meristem culture, we could not detect any virus. OYDV and LYSV viruses were detected in plants obtained via shoot tip culture. OYDV virus was observed in amount of 80% and 73% of tested plants for A. tuncelianum and A. sativum, respectively. LYSV virus was found in amount of 67% of tested plants of A. tuncelianum and in amount of 87% of tested plants of A. sativum in this study.

  13. Use of tissue culture techniques for producing virus-free plant in garlic and their identification through real-time PCR.

    Science.gov (United States)

    Taşkın, Hatıra; Baktemur, Gökhan; Kurul, Mehmet; Büyükalaca, Saadet

    2013-01-01

    This study was performed for comparison of meristem culture technique with shoot tip culture technique for obtaining virus-free plant, comparison of micropropagation success of two different nutrient media, and determination of effectiveness of real-time PCR assay for the detection of viruses. Two different garlic species (Allium sativum and Allium tuncelianum) and two different nutrient media were used in this experiment. Results showed that Medium 2 was more successful compared to Medium 1 for both A. tuncelianum and A. sativum (Kastamonu garlic clone). In vitro plants obtained via meristem and shoot tip cultures were tested for determination of onion yellow dwarf virus (OYDV) and leek yellow stripe virus (LYSV) through real-time PCR assay. In garlic plants propagated via meristem culture, we could not detect any virus. OYDV and LYSV viruses were detected in plants obtained via shoot tip culture. OYDV virus was observed in amount of 80% and 73% of tested plants for A. tuncelianum and A. sativum, respectively. LYSV virus was found in amount of 67% of tested plants of A. tuncelianum and in amount of 87% of tested plants of A. sativum in this study.

  14. Multicenter Evaluation of the Portrait Staph ID/R Blood Culture Panel for Rapid Identification of Staphylococci and Detection of themecAGene.

    Science.gov (United States)

    Denys, Gerald A; Collazo-Velez, Vanessa; Young, Stephen; Daly, Judy A; Couturier, Marc Roger; Faron, Matthew L; Buchan, Blake W; Ledeboer, Nathan

    2017-04-01

    Bloodstream infections are a leading cause of morbidity and mortality in the United States and are associated with increased health care costs. We evaluated the Portrait Staph ID/R blood culture panel (BCP) multiplex PCR assay (Great Basin Scientific, Salt Lake City, UT) for the rapid and simultaneous identification (ID) of Staphylococcus aureus , Staphylococcus lugdunensis , and Staphylococcus species to the genus level and the detection of the mecA gene directly from a positive blood culture bottle. A total of 765 Bactec bottles demonstrating Gram-positive cocci in singles or clusters were tested during the prospective trial at 3 clinical sites. The Portrait Staph ID/R BCP results were compared with results from conventional biochemical and cefoxitin disk methods performed at an independent laboratory. Discordant ID and mecA results were resolved by rpoB gene sequencing and mecA gene sequencing, respectively. A total of 658 Staphylococcus species isolates ( S. aureus , 211 isolates; S. lugdunensis , 3 isolates; and Staphylococcus spp., 444 isolates) were recovered from monomicrobial and 33 polymicrobial blood cultures. After discrepant analysis, the overall ratios of Portrait Staph ID/R BCP positive percent agreement and negative percent agreement were 99.4%/99.9% for Staphylococcus ID and 99.7%/99.2% for mecA detection. Copyright © 2017 American Society for Microbiology.

  15. Staph ID/R: a rapid method for determining staphylococcus species identity and detecting the mecA gene directly from positive blood culture.

    Science.gov (United States)

    Pasko, Chris; Hicke, Brian; Dunn, John; Jaeckel, Heidi; Nieuwlandt, Dan; Weed, Diane; Woodruff, Evelyn; Zheng, Xiaotian; Jenison, Robert

    2012-03-01

    Rapid diagnosis of staphylococcal bacteremia directs appropriate antimicrobial therapy, leading to improved patient outcome. We describe herein a rapid test (Staph ID/R, combines a rapid isothermal nucleic acid amplification method, helicase-dependent amplification (HDA), with a chip-based array that produces unambiguous visible results. The analytic sensitivity was 1 CFU per reaction for the mecA gene and was 1 to 250 CFU per reaction depending on the staphylococcal species present in the positive blood culture. Staph ID/R has excellent specificity as well, with no cross-reactivity observed. We validated the performance of Staph ID/R by testing 104 frozen clinical positive blood cultures and comparing the results with rpoB gene or 16S rRNA gene sequencing for species identity determinations and mecA gene PCR to confirm mecA gene results. Staph ID/R agreed with mecA gene PCR for all samples and agreed with rpoB/16S rRNA gene sequencing in all cases except for one sample that contained a mixture of two staphylococcal species, one of which Staph ID/R correctly identified, for an overall agreement of 99.0% (P Staph ID/R could potentially be used to positively affect patient management for Staphylococcus-mediated bacteremia.

  16. Primary quantitative analysis of the mtDNA4977bp deletion induced by lonizing radiation in human peripheral blood u-sing real-time PCR

    International Nuclear Information System (INIS)

    Duan Zhikai; Liu Jiangong; Guo Wanlong; Zhang Shuxian

    2011-01-01

    Objective: To observe the influence of mtDNA4977bp deletion induced by different dose of γ ray in human peripheral blood in order to explore the feasibility of mtDNA4977bp deletion as biodosimeter. Methods: Human peripheral blood samples were collected from three healthy donors and irradiated by γ ray, MtDNA4977bp deletion was detected by real-time PCR. Results: It indicated that that from the range of 0 ∼ 8 Gy, the relationship between mtDNA4977bp deletion and irradiation dose represents certain curvilinear correlation (Y=1.2693+1.0660X+0.0198X 2 ). Conclusion: We find that γ ray has influence on the mtDNA4977bp deletion, so it may be an important biodosmeter in future. (authors)

  17. Profile of GenMark's ePlex® blood culture identification fungal pathogen panel.

    Science.gov (United States)

    Maubon, Danièle; Dard, Céline; Garnaud, Cécile; Cornet, Muriel

    2018-02-01

    Fungemia presents high morbi-mortality and thus rapid microbiological diagnosis may contribute to appropriate patient management. In the last decade, kits based on molecular technologies have become available and health care institutes are increasingly facing critical investment choices. Although all these tools aim to achieve rapid fungal detection and species identification, they display different inherent characteristics. Areas covered: Considering technologies allowing detection and identification of fungal species in a sepsis context, the market proposes either tests on positive blood culture or tests on patient's whole blood. In this review, the authors describe and compare the ePlex® Blood Culture Identification Fungal Pathogen (BCID-FP) test, a fully automated one-step single-use cartridge assay that has been designed to detect identify frequent or rare but emerging, fungal species, from positive blood culture. A comparison with the competing kits is provided. Expert commentaries: The ePlex BCID-FP test provides a diversified and rather relevant panel. Its easy-to-use cartridges allow flexible use around the clock. Nevertheless, prospective clinical studies assessing the time-to-result benefit on antifungal stewardship and on hospital length of stay are not available yet. New tools aim to benefit clinicians and patients, but they should be accompanied by supervision of result interpretation and adaptation of antifungal stewardship.

  18. Capacity and Utilization of Blood Culture in Two Referral Hospitals in Indonesia and Thailand.

    Science.gov (United States)

    Teerawattanasook, Nittaya; Tauran, Patricia M; Teparrukkul, Prapit; Wuthiekanun, Vanaporn; Dance, David A B; Arif, Mansyur; Limmathurotsakul, Direk

    2017-10-01

    It is generally recommended that sepsis patients should have at least two blood cultures obtained before antimicrobial therapy. From 1995 to 2015, the number of blood cultures taken each year in a 1,100-bed public referral hospital in Ubon Ratchathani northeast Thailand rose from 5,235 to 56,719, whereas the number received in an 840-bed referral public hospital in South Sulawesi, Indonesia, in 2015 was 2,779. The proportion of patients sampled for blood cultures out of all inpatients in South Sulawesi in 2015 (9%; 2,779/30,593) was lower than that in Ubon Ratchathani in 2003 (13%; 8,707/66,515), at a time when health expenditure per capita in the two countries was comparable. Under-use of bacterial cultures may lead to an underestimate and underreporting of the incidence of antimicrobial-resistant infections. Raising capacity and utilization of clinical microbiology laboratories in developing countries, at least at sentinel hospitals, to monitor the antimicrobial resistance situation should be prioritized.

  19. Optimal turnaround time for direct identification of microorganisms by mass spectrometry in blood culture.

    Science.gov (United States)

    Randazzo, Adrien; Simon, Marc; Goffinet, Pierre; Classen, Jean-François; Hougardy, Nicolas; Pierre, Pascal; Kinzinger, Philippe; Mauel, Etienne; Goffinet, Jean-Sébastien

    2016-11-01

    During the past few years, several studies describing direct identification of bacteria from blood culture using mass spectrometry have been published. These methods cannot, however, be easily integrated into a common laboratory workflow because of the high hands-on time they require. In this paper, we propose a new method of identification with a short hands-on time and a turnaround time shorter than 15min. Positive blood bottles were homogenised and 600μL of blood were transferred to an Eppendorf tube where 600μL of lysis buffer were added. After homogenisation, a centrifugation step of 4min at 10,500g was performed and the supernatant was discarded. The pellet was then washed and loaded in quadruplicate into wells of a Vitek® MS-DS plate. Each well was covered with a saturated matrix solution and a MALDI-TOF mass spectrometry analysis was performed. Species were identified using the software Myla 3.2.0-2. We analysed 266 positive blood culture bottles. A microorganism grew in 261 cultures, while five bottles remained sterile after 48h of incubation in subculture. Our method reaches a probability of detection at the species level of 77.8% (203/261) with a positive predictive value of 99.5% (202/203). We developed a new method for the identification of microorganisms using mass spectrometry, directly performed from a positive blood culture. This method has short hands-on time and turnaround time and can easily take place in the workflow of a laboratory, with comparable results in performance with other methods reported in the literature. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. Rapid detection of positive blood cultures with the BACTEC NR-660 does not require first-day subculturing.

    OpenAIRE

    Levi, M H; Gialanella, P; Motyl, M R; McKitrick, J C

    1988-01-01

    An analysis of blood culture data was performed to determine whether subculturing within the first 24 h of incubation decreased the time to detection of positive blood cultures when compared with the routine use of the BACTEC NR-660 system (Johnston Laboratories, Inc., Towson, Md.). During a 9-month period (June 1985 to February 1986), 17,913 blood cultures were received in our laboratory, of which 1,463 (8.2%) became positive. Of the positive cultures, 97% were detected with equal or greater...

  1. Simple Sample Preparation Method for Direct Microbial Identification and Susceptibility Testing From Positive Blood Cultures

    Directory of Open Access Journals (Sweden)

    Hong-wei Pan

    2018-03-01

    Full Text Available Rapid identification and determination of the antibiotic susceptibility profiles of the infectious agents in patients with bloodstream infections are critical steps in choosing an effective targeted antibiotic for treatment. However, there has been minimal effort focused on developing combined methods for the simultaneous direct identification and antibiotic susceptibility determination of bacteria in positive blood cultures. In this study, we constructed a lysis-centrifugation-wash procedure to prepare a bacterial pellet from positive blood cultures, which can be used directly for identification by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS and antibiotic susceptibility testing by the Vitek 2 system. The method was evaluated using a total of 129 clinical bacteria-positive blood cultures. The whole sample preparation process could be completed in <15 min. The correct rate of direct MALDI-TOF MS identification was 96.49% for gram-negative bacteria and 97.22% for gram-positive bacteria. Vitek 2 antimicrobial susceptibility testing of gram-negative bacteria showed an agreement rate of antimicrobial categories of 96.89% with a minor error, major error, and very major error rate of 2.63, 0.24, and 0.24%, respectively. Category agreement of antimicrobials against gram-positive bacteria was 92.81%, with a minor error, major error, and very major error rate of 4.51, 1.22, and 1.46%, respectively. These results indicated that our direct antibiotic susceptibility analysis method worked well compared to the conventional culture-dependent laboratory method. Overall, this fast, easy, and accurate method can facilitate the direct identification and antibiotic susceptibility testing of bacteria in positive blood cultures.

  2. Identification by culture, PCR, and immunohistochemistry of mycoplasmas and their molecular typing in sheep and lamb lungs with pneumonia in Eastern Turkey.

    Science.gov (United States)

    Kılıc, Ayşe; Kalender, Hakan; Eroksuz, Hatice; Muz, Adile; Tasdemir, Bülent

    2013-10-01

    This study used cultures, polymerase chain reaction (PCR), and immunoperoxidase to examine samples from 216 lungs from sheep and lambs with macroscopic pneumonia lesions for the presence of Mycoplasma species. DNA was extracted from lung tissue samples and broth cultures with the help of a DNA extraction kit and replicated using genus-specific and species-specific primers for mycoplasma. The lung samples were examined by the immunoperoxidase method using hyperimmune Mycoplasma ovipneumoniae serum. The randomly amplified polymorphic DNA (RAPD) test was used for the molecular typing of M. ovipneumoniae isolates. Mycoplasma was isolated in the cultures of 80 (37.03 %) of a total of 216 lung samples. Genus-specific mycoplasma DNA was identified by PCR in 96 (44.44 %) samples in broth cultures and 36 (16.66 %) directly in the lung tissue. Of these 96 cases in which genus-specific identification was made, 57 (59.37 %) were positive for reaction with species-specific primers for M. ovipneumoniae and 31 (32.29 %) for Mycoplasma arginini. The DNA of neither of the latter two species could be identified in the remaining eight samples (8.33 %) where mycoplasma had been identified. As for the immunoperoxidase method, it identified M. ovipneumoniae in 61 of 216 lung samples (28 %). Positive staining was concentrated in the bronchial epithelium cell cytoplasm and cell surface. RAPD analysis resulted in 15 different profiles. Our results suggest that PCR methods could be successfully used in the diagnosis of mycoplasma infections as an alternative to culture method and identifying this agent at the species level.

  3. Self-sustained circadian rhythm in cultured human mononuclear cells isolated from peripheral blood.

    Science.gov (United States)

    Ebisawa, Takashi; Numazawa, Kahori; Shimada, Hiroko; Izutsu, Hiroyuki; Sasaki, Tsukasa; Kato, Nobumasa; Tokunaga, Katsushi; Mori, Akio; Honma, Ken-ichi; Honma, Sato; Shibata, Shigenobu

    2010-02-01

    Disturbed circadian rhythmicity is associated with human diseases such as sleep and mood disorders. However, study of human endogenous circadian rhythm is laborious and time-consuming, which hampers the elucidation of diseases. It has been reported that peripheral tissues exhibit circadian rhythmicity as the suprachiasmatic nucleus-the center of the biological clock. We tried to study human circadian rhythm using cultured peripheral blood mononuclear cells (PBMCs) obtained from a single collection of venous blood. Activated human PBMCs showed self-sustained circadian rhythm of clock gene expression, which indicates that they are useful for investigating human endogenous circadian rhythm.

  4. Association of different types of milk feeding with blood culture positive neonatal sepsis

    International Nuclear Information System (INIS)

    Anwar, M.; Waheed, K.A.I.; Rehman, A.

    2014-01-01

    To ascertain and compare microbial growth pattern in blood culture of septic neonates who were either totally breast or formula fed. Study Design: Cross sectional study. Place and Duration of Study: The Children's Hospital Lahore, Pakistan from Feb 2012 to Dec 2012. Methodology: All clinically septic neonates, who were either exclusively breast fed or formula fed, were enrolled in the study. They were divided into two groups and studied for the type of organisms grown on blood culture. Group-A were breast fed and group-B were formula fed. Neonates who were blood culture negative or had growth of multiple organisms or had incomplete data or who died / left against medical advice before completing the required data or babies receiving milk feeding from multiple sources or no feeding at all were excluded. BACTEC technique was used for obtaining bacterial growth. SPSS version 19 was used for statistical analysis. Results: A total of 380 clinically septic neonates were enrolled. Each group consisted of 190 subjects. Incidence of culture positive sepsis in breast fed and in formula fed was 6.7% and 15.7% respectively (p-value = 0.0001). Overall, gram-negative organisms constituted the majority (16.1%). Thirty seven percent cultures grew coagulase negative Staphylococcus (CoNS) followed by Klebsiella spp (23.4%). In group A, gram-negative and gram-positive organisms were equally distributed whilst in group-B, gram-negative organisms were three times more frequent than gram-positive organisms. Predominant pattern of organisms was also different in the two groups. In group-A, CoNS was predominant while in group-B, Klebsiella spp. was most frequent. Conclusion: Culture positive sepsis is more than two times greater in formula fed babies and is caused predominantly by gram-negative organisms whilst in breast fed babies, CoNS is the commonest organism. (author)

  5. Towards cultural materialism in the medical humanities: the case of blood rejuvenation

    Science.gov (United States)

    2018-01-01

    This paper argues for an approach within the medical humanities that draws on the theoretical legacy of cultural materialism as a framework for reading cultural practices and their relationship to the social and economic order. It revisits the origins and development of cultural materialism in cultural studies and literary studies between the 1970s and 1990s and considers how, with adaptation, this methodology might facilitate ideological criticism focused on material formations of health, disease and the human body. I outline three key characteristics of a medicocultural materialist approach along these lines: (a) interdisciplinary work on a broad range of medical and cultural sources, including those drawn from ‘popular’ forms of culture; (b) the combination of historicist analysis with scrutiny of present-day contexts; (c) analyses that engage with political economy perspectives and/or the work of medical sociology in this area. The subsequent sections of the paper employ a medicocultural materialist approach to examine conjectural understandings of, and empirical investigations into, the capacity of transfused human blood to rejuvenate the ageing body. I trace textual faultlines that expose the structures of power which inform the movement of blood between bodies in ‘medical gothic’ fictions from the 19th-century fin de siècle, including Mary Elizabeth Braddon's ‘Good Lady Ducayne’ (1896) and Bram Stoker's Dracula (1897). I conclude with a critique of biomedical innovations in blood rejuvenation in the era of medical neoliberalism, before considering the potential applications of medicocultural materialism to other topics within the field of the medical humanities. PMID:28495908

  6. Towards cultural materialism in the medical humanities: the case of blood rejuvenation.

    Science.gov (United States)

    Oakley, Catherine

    2018-03-01

    This paper argues for an approach within the medical humanities that draws on the theoretical legacy of cultural materialism as a framework for reading cultural practices and their relationship to the social and economic order. It revisits the origins and development of cultural materialism in cultural studies and literary studies between the 1970s and 1990s and considers how, with adaptation, this methodology might facilitate ideological criticism focused on material formations of health, disease and the human body. I outline three key characteristics of a medicocultural materialist approach along these lines: (a) interdisciplinary work on a broad range of medical and cultural sources, including those drawn from 'popular' forms of culture; (b) the combination of historicist analysis with scrutiny of present-day contexts; (c) analyses that engage with political economy perspectives and/or the work of medical sociology in this area. The subsequent sections of the paper employ a medicocultural materialist approach to examine conjectural understandings of, and empirical investigations into, the capacity of transfused human blood to rejuvenate the ageing body. I trace textual faultlines that expose the structures of power which inform the movement of blood between bodies in 'medical gothic' fictions from the 19th-century fin de siècle, including Mary Elizabeth Braddon's 'Good Lady Ducayne' (1896) and Bram Stoker's Dracula (1897). I conclude with a critique of biomedical innovations in blood rejuvenation in the era of medical neoliberalism, before considering the potential applications of medicocultural materialism to other topics within the field of the medical humanities. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

  7. A comparison of antigen dipstick assays with polymerase chain reaction (PCR) technique and blood film examination in the rapid diagnosis of malaria.

    Science.gov (United States)

    Lee, M A; Aw, L T; Singh, M

    1999-07-01

    Early diagnosis of malaria for military personnel training in the field is crucial in providing proper treatment for the infected and in taking appropriate preventive measures for the non-infected. Present preliminary diagnosis of malaria in the field depends on the clinical symptoms of the patients and there is a need for rapid diagnosis of malaria in the field. The presence of drug-resistant strains of the Plasmodium species in the region also increases the urgency of finding a quick and sensitive way of identifying the different strains. This study evaluated current methods available for the diagnosis of Plasmodium falciparum and Plasmodium vivax. The dipstick assays, the ParaSight F test and the OptiMAL malaria rapid test were compared with the methods of microscopic examination of blood film and polymerase chain reaction (PCR). On comparison to the blood film and PCR methods, the ParaSight F test has specificity of 98.6% and sensitivity of 91% for P. falciparum detection. The OptiMAL malaria rapid test has a specificity of 100% and 98.6% and sensitivity of 92.8% and 92.6% for P. vivax and P. falciparum detection respectively. We conclude that both tests are suitable for use for rapid malaria diagnosis in the field but the OptiMAL rapid malaria test, which can detect both vivax and falciparum malaria, would be more useful.

  8. Squamous cell carcinoma antigen 1 and 2 expression in cultured normal peripheral blood mononuclear cells and in vulvar squamous cell carcinoma

    Science.gov (United States)

    Kowalewska, Magdalena; Brzoska-Wojtowicz, Edyta; Radziszewski, Jakub; Ptaszynski, Konrad; Rys, Janusz; Kaminska, Janina; Nowak, Radoslawa

    2010-01-01

    Squamous cell carcinoma antigen (SCCA) is expressed in normal squamous cell epithelia and in squamous cell carcinomas (SCC). Two nearly identical genes encode the inhibitory serpins SCCA1 (SERPINB3) and SCCA2 (SERPINB4). Serum levels of SCCA are elevated in patients with benign skin diseases and in patients with SCC. SCCA, used for the monitoring of SCC patients, presents no satisfactory diagnostic specificity. As we have shown previously, the reverse transcription polymerase chain reaction (RT-PCR)-based SCCA messenger RNA (mRNA) testing aimed at detecting disseminated cancer cells may be hampered by the false-positive results due to SCCA expression in activated peripheral blood mononuclear cells (PBMC). The aim of this study was to assess the expression of SCCA at mRNA and protein levels in cultured normal PBMC, compared to that in vulvar SCC (VSCC) samples. High SCCA concentrations were found in vulvar tumours and in metastatic lymph nodes, while negative inguinal lymph nodes from the same patients often presented significantly less SCCA. In normal activated PBMC, the level of SCCA protein was the lowest. At the mRNA level SCCA was detectable in normal PBMC even in cultures with no mitogen stimulation, but only by the nested RT-PCR, contrary to VSCC samples found to be SCCA positive already in one-step PCR. Both SCCA1 and SCCA2 transcripts were present in cultured PBMC; SCCA1 was expressed at a higher level than SCCA2. In conclusion, both SCCA forms are detectable in normal PBMC cultured in vitro. SCCA expression level in normal PBMC is much lower than in the squamous epithelium-derived cells. In VSCC, in addition to tumour itself, metastatic lymph nodes seem also to be a potential source of serum SCCA. PMID:20589490

  9. Use of high throughput qPCR screening to rapidly clone low frequency tumour specific T-cells from peripheral blood for adoptive immunotherapy

    Directory of Open Access Journals (Sweden)

    Serrano Oscar K

    2008-10-01

    Full Text Available Abstract Background The adoptive transfer of autologous tumor reactive lymphocytes can mediate significant tumor regression in some patients with refractory metastatic cancer. However, a significant obstacle for this promising therapy has been the availability of highly efficient methods to rapidly isolate and expand a variety of potentially rare tumor reactive lymphocytes from the natural repertoire of cancer patients. Methods We developed a novel in vitro T cell cloning methodology using high throughput quantitative RT-PCR (qPCR assay as a rapid functional screen to detect and facilitate the limiting dilution cloning of a variety of low frequency T cells from bulk PBMC. In preclinical studies, this strategy was applied to the isolation and expansion of gp100 specific CD8+ T cell clones from the peripheral blood of melanoma patients. Results In optimization studies, the qPCR assay could detect the reactivity of 1 antigen specific T cell in 100,000 background cells. When applied to short term sensitized PBMC microcultures, this assay could detect T cell reactivity against a variety of known melanoma tumor epitopes. This screening was combined with early limiting dilution cloning to rapidly isolate gp100154–162 reactive CD8+ T cell clones. These clones were highly avid against peptide pulsed targets and melanoma tumor lines. They had an effector memory phenotype and showed significant proliferative capacity to reach cell numbers appropriate for adoptive transfer trials (~1010 cells. Conclusion This report describes a novel high efficiency strategy to clone tumor reactive T cells from peripheral blood for use in adoptive immunotherapy.

  10. The San Luigi Gonzaga Hospital experience: improving blood and urine culture preanalytical quality by shared protocols

    Directory of Open Access Journals (Sweden)

    Angela Samiolo

    2017-07-01

    Full Text Available Background and aims: Reduction in the number of blood culture and urine culture contamination samples. Materials and methods: We have designed a partly retrospective and partly prospective observational study. On one hand, we have been striving for the creation, dissemination and promotion of shared operational rules in all departments/hospital services to improve the quality of the levy; on the other hand, we analysed data. We considered blood cultures and urine cultures analysed in the laboratory from March to August 2015, and from March to August 2016. The data were processed with R and the incidence of contaminated samples was calculated by dividing the number of blood cultures/urine cultures contaminated by the total. The results of 2015 and 2016 were compared by χ2. To highlight the possible differences between departments and identify those at higher risk of contamination, the data of each year were stratified dividing departments into five groups: Medicine, Surgery, Critical Area, Specialties and ER. To assess the strength of the association, a risk analysis was carried out using the risk ratio (RR. The RR was calculated by dividing the contamination rates of 2015 by the those of 2016. The value of α was set at 0.05. Results: After implementation of the shared protocols, blood culture contamination was substantially reduced (−56.8%, P=1.783e-05, confirmed by an RR of 2.2 (95%CI: 1.54±3.27. The evidence is strengthened by the finding of a lower number of isolates belonging to the group of possible contaminants (−32.7%, P=2.042e-07 and confirmed by an RR of 1.5 (95%CI: 1.27±1.73. Urine culture data analysis showed no change in the incidence of contamination between 2015 and 2016 (P=0.8808, as confirmed by a non-informational RR (95%CI: 0.62±1:46. Even the analysis of the individual areas showed no change in the two semesters, as confirmed by the risk analysis that does not show any association between outcome and group

  11. Utility of Acridine Orange staining for detection of bacteria from positive blood cultures.

    Science.gov (United States)

    Neeraja, M; Lakshmi, V; Padmasri, C; Padmaja, K

    2017-08-01

    The diagnostic performance of AO stain was evaluated for the detection of bacteria and or fungi from positive blood cultures. The sensitivity of Gram stain (GS) was 98.26% while Acridine Orange (AO) stain proved to be more sensitive (100%) with a Positive and Negative Predictive Value of 100% each. The specificity of both the stains was 100%. Overall agreement between the two stains was 98.23% (688/700). The organisms that were missed by GS and positive by AO were Candida species (Sutton, 2006) and Gram negative bacilli (GNB) (Sutton, 2006). Sensitivity of GS was 82.35% and AO was 100% among mixed cultures. Immediate reporting of the results of AO stain would have a significant impact on clinical management of patients with serious blood stream infections. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Impact of reporting gram stain results from blood culture bottles on the selection of antimicrobial agents.

    Science.gov (United States)

    Uehara, Yuki; Yagoshi, Michiko; Tanimichi, Yumiko; Yamada, Hiroko; Shimoguchi, Kazuo; Yamamoto, Sachiyo; Yanai, Mitsuru; Kumasaka, Kazunari

    2009-07-01

    We assessed the usefulness of reporting direct blood Gram stain results compared with the results of positive blood cultures in 482 episodes and monitored impact on selection of antimicrobial treatment. We found that the reporting groups "Staphylococcus spp," "Pseudomonas spp and related organisms," and "yeasts" identified in this way matched perfectly with later culture identification. When the report indicated Staphylococcus spp or Pseudomonas spp and related organisms, physicians started or changed antimicrobials suitable for these bacteria more frequently than when "other streptococci" and "family Enterobacteriaceae" were reported (P Gram stain results that definitively identify Staphylococcus spp, Pseudomonas spp and related organisms, and yeasts reliably can be rapidly provided by clinical laboratories; this information has a significant impact on early selection of effective antimicrobials. Further investigation is needed to assess the clinical impact of reporting Gram stain results in bacteremia.

  13. Evaluation of substrates for radiometric detection of bacteria in blood cultures

    International Nuclear Information System (INIS)

    Bopp, H.; Ellner, P.D.

    1988-01-01

    Various 14 C-labeled substrates were evaluated for their potential use in blood culture media. These uniformly labeled compounds were added to hypertonic and anaerobic formulations of modified Columbia broth and compared with analogous BACTEC media with the BACTEC 460. Different bacterial species gave significant growth indices when 2.0 microCi of labeled glucose, glutamic acid, aspartic acid, arginine, or formate was used alone or in combinations in the experimental media. The combination of glucose, glutamic acid, and sodium formate was selected, and simulated blood cultures with representative aerobic, facultative, and anaerobic bacteria and a yeast were compared with BACTEC vials. Under these conditions, the experimental media often became positive several hours earlier than the BACTEC vials and usually produced higher growth indices

  14. Rapid identification and quantification of Campylobacter coli and Campylobacter jejuni by real-time PCR in pure cultures and in complex samples

    Directory of Open Access Journals (Sweden)

    Denis Martine

    2011-05-01

    Full Text Available Abstract Background Campylobacter spp., especially Campylobacter jejuni (C. jejuni and Campylobacter coli (C. coli, are recognized as the leading human foodborne pathogens in developed countries. Livestock animals carrying Campylobacter pose an important risk for human contamination. Pigs are known to be frequently colonized with Campylobacter, especially C. coli, and to excrete high numbers of this pathogen in their faeces. Molecular tools, notably real-time PCR, provide an effective, rapid, and sensitive alternative to culture-based methods for the detection of C. coli and C. jejuni in various substrates. In order to serve as a diagnostic tool supporting Campylobacter epidemiology, we developed a quantitative real-time PCR method for species-specific detection and quantification of C. coli and C. jejuni directly in faecal, feed, and environmental samples. Results With a sensitivity of 10 genome copies and a linear range of seven to eight orders of magnitude, the C. coli and C. jejuni real-time PCR assays allowed a precise quantification of purified DNA from C. coli and C. jejuni. The assays were highly specific and showed a 6-log-linear dynamic range of quantification with a quantitative detection limit of approximately 2.5 × 102 CFU/g of faeces, 1.3 × 102 CFU/g of feed, and 1.0 × 103 CFU/m2 for the environmental samples. Compared to the results obtained by culture, both C. coli and C. jejuni real-time PCR assays exhibited a specificity of 96.2% with a kappa of 0.94 and 0.89 respectively. For faecal samples of experimentally infected pigs, the coefficients of correlation between the C. coli or C. jejuni real-time PCR assay and culture enumeration were R2 = 0.90 and R2 = 0.93 respectively. Conclusion The C. coli and C. jejuni real-time quantitative PCR assays developed in this study provide a method capable of directly detecting and quantifying C. coli and C. jejuni in faeces, feed, and environmental samples. These assays represent a new

  15. Rapid identification and quantification of Campylobacter coli and Campylobacter jejuni by real-time PCR in pure cultures and in complex samples.

    Science.gov (United States)

    Leblanc-Maridor, Mily; Beaudeau, François; Seegers, Henri; Denis, Martine; Belloc, Catherine

    2011-05-22

    Campylobacter spp., especially Campylobacter jejuni (C. jejuni) and Campylobacter coli (C. coli), are recognized as the leading human foodborne pathogens in developed countries. Livestock animals carrying Campylobacter pose an important risk for human contamination. Pigs are known to be frequently colonized with Campylobacter, especially C. coli, and to excrete high numbers of this pathogen in their faeces. Molecular tools, notably real-time PCR, provide an effective, rapid, and sensitive alternative to culture-based methods for the detection of C. coli and C. jejuni in various substrates. In order to serve as a diagnostic tool supporting Campylobacter epidemiology, we developed a quantitative real-time PCR method for species-specific detection and quantification of C. coli and C. jejuni directly in faecal, feed, and environmental samples. With a sensitivity of 10 genome copies and a linear range of seven to eight orders of magnitude, the C. coli and C. jejuni real-time PCR assays allowed a precise quantification of purified DNA from C. coli and C. jejuni. The assays were highly specific and showed a 6-log-linear dynamic range of quantification with a quantitative detection limit of approximately 2.5 × 10² CFU/g of faeces, 1.3 × 10² CFU/g of feed, and 1.0 × 10³ CFU/m² for the environmental samples. Compared to the results obtained by culture, both C. coli and C. jejuni real-time PCR assays exhibited a specificity of 96.2% with a kappa of 0.94 and 0.89 respectively. For faecal samples of experimentally infected pigs, the coefficients of correlation between the C. coli or C. jejuni real-time PCR assay and culture enumeration were R² = 0.90 and R² = 0.93 respectively. The C. coli and C. jejuni real-time quantitative PCR assays developed in this study provide a method capable of directly detecting and quantifying C. coli and C. jejuni in faeces, feed, and environmental samples. These assays represent a new diagnostic tool for studying the epidemiology of

  16. Comparison of latex agglutination and immunofluorescence for direct Lancefield grouping of streptococci from blood cultures.

    OpenAIRE

    Shlaes, D M; Toossi, Z; Patel, A

    1984-01-01

    Simulated positive blood cultures with 84 known stock strains of streptococci were used to comparatively evaluate the direct identification of these organisms by fluorescein-tagged antibody staining (immunofluorescence [IF]) and latex agglutination (LA). IF was not evaluated for Lancefield group D strains (a total of 81 strains tested) and had 89% sensitivity and 91% specificity. IF was least sensitive for the identification of Lancefield group F, in which three of seven strains showed no flu...

  17. Lack of requirement for blind subcultures of BACTEC blood culture media

    International Nuclear Information System (INIS)

    McLaughlin, J.C.; Evers, J.L.; Officer, J.L.

    1981-01-01

    To determine the need for blind subculturing of BACTEC (Johnston Laboratories, Cockeysville, Md.) blood culture media, we compared results of radiometric readings, visual inspection, and blind subculturing for nearly 7,500 blood specimens. Visual inspection and radiometric testing were performed on day 1 through 7, and blind subcultures were made on day 3. In the first phase of the study, 402 of 3,896 aerobic bottles were positive by radiometric testing (growth index, greater than 25), visual inspection, or subculturing. Only six bottles were radiometrically negative but subculture positive on day 3. The second phase of the study was designed to determine if aerobic bottles eventually became radiometrically positive in those cases in which they were radiometrically negative but subculture positive on day 3. Two bottles were subculture positive but never gave a growth index of greater than or equal to 25 by day 7. One yielded Staphylococcus epidermidis, and one yielded viridans, Streptococcus sp. A total of 35 anaerobic organisms were isolated from 3,896 blood specimens. All of these anaerobes were detected by both radiometric testing and subculturing. We examined a total of 14,972 blood culture bottles. Twenty-nine bottles considered negative by visual inspection or radiometric readings were found to be positive by subculturing. Fifteen of these were shown, by chart review, to contain contaminants. Organisms in the other negative bottles would not have gone undetected because companion bottles from the same patients were radiometrically or visually positive. We concluded that it is necessary to perform blind subcultures of BACTEC 7B and 8B blood culture bottles

  18. Lack of requirement for blind subcultures of BACTEC blood culture media

    Energy Technology Data Exchange (ETDEWEB)

    McLaughlin, J.C.; Evers, J.L.; Officer, J.L.

    1981-11-01

    To determine the need for blind subculturing of BACTEC (Johnston Laboratories, Cockeysville, Md.) blood culture media, we compared results of radiometric readings, visual inspection, and blind subculturing for nearly 7,500 blood specimens. Visual inspection and radiometric testing were performed on day 1 through 7, and blind subcultures were made on day 3. In the first phase of the study, 402 of 3,896 aerobic bottles were positive by radiometric testing (growth index, greater than 25), visual inspection, or subculturing. Only six bottles were radiometrically negative but subculture positive on day 3. The second phase of the study was designed to determine if aerobic bottles eventually became radiometrically positive in those cases in which they were radiometrically negative but subculture positive on day 3. Two bottles were subculture positive but never gave a growth index of greater than or equal to 25 by day 7. One yielded Staphylococcus epidermidis, and one yielded viridans, Streptococcus sp. A total of 35 anaerobic organisms were isolated from 3,896 blood specimens. All of these anaerobes were detected by both radiometric testing and subculturing. We examined a total of 14,972 blood culture bottles. Twenty-nine bottles considered negative by visual inspection or radiometric readings were found to be positive by subculturing. Fifteen of these were shown, by chart review, to contain contaminants. Organisms in the other negative bottles would not have gone undetected because companion bottles from the same patients were radiometrically or visually positive. We concluded that it is necessary to perform blind subcultures of BACTEC 7B and 8B blood culture bottles.

  19. Reducing time to identification of aerobic bacteria and fastidious micro-organisms in positive blood cultures.

    Science.gov (United States)

    Intra, J; Sala, M R; Falbo, R; Cappellini, F; Brambilla, P

    2016-12-01

    Rapid and early identification of micro-organisms in blood has a key role in the diagnosis of a febrile patient, in particular, in guiding the clinician to define the correct antibiotic therapy. This study presents a simple and very fast method with high performances for identifying bacteria by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) after only 4 h of incubation. We used early bacterial growth on PolyViteX chocolate agar plates inoculated with five drops of blood-broth medium deposited in the same point and spread with a sterile loop, followed by a direct transfer procedure on MALDI-TOF MS target slides without additional modification. Ninety-nine percentage of aerobic bacteria were correctly identified from 600 monomicrobial-positive blood cultures. This procedure allowed obtaining the correct identification of fastidious pathogens, such as Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae that need complex nutritional and environmental requirements in order to grow. Compared to the traditional pathogen identification from blood cultures that takes over 24 h, the reliability of results, rapid performance and suitability of this protocol allowed a more rapid administration of optimal antimicrobial treatment in the patients. Bloodstream infections are serious conditions with a high mortality and morbidity rate. Rapid identification of pathogens and appropriate antimicrobial therapy have a key role for successful patient outcome. In this work, we developed a rapid, simplified, accurate, and efficient method, reaching 99 % identification of aerobic bacteria from monomicrobial-positive blood cultures by using early growth on enriched medium, direct transfer to target plate without additional procedures, matrix-assisted laser desorption ionization-time of flight mass spectrometry and SARAMIS database. The application of this protocol allows to anticipate appropriate antibiotic therapy.

  20. Comparing the Accuracy Rate of Two Different Universal Primers in Enteric Pathogen Diagnosis From Blood

    Directory of Open Access Journals (Sweden)

    Esmaeil Soleimani

    2014-02-01

    Full Text Available Background: Detecting enteric bacteria in blood by culture is a slow assay with low accuracy rate. PCR might be a suitable alternative assay but as several species can cause bacteremia, it is necessary to use universal primers. Objectives: In this study we evaluated and compared two pairs of universal primers in detecting four enteric bacteria in blood, which are common causes of bacteremia in human. Materials and Methods: Standard strains of E. faecalis, S. typhi, E. coli, and S. Aeruginosa, were used in this study. A serially diluted bacterial suspension of all strains was made for inoculation to four sets of defibrinated sheep blood which were used to prepare blood specimens with different bacterial contents for performing routine assay and PCR. PCR was performed using two different universal primers designed from two ribosomal genes, 16sr RNA and 23sr RNA. Results: PCR with 16sr RNA universal primer showed more accuracy rate than both blood culture and PCR with 23sr RNA universal primer. Mean time for performing PCR assay and blood culture was eight and 48 hours, respectively. Conclusions: Both PCR with 16sr RNA and 23sr RNA universal primers have more accuracy rate than blood culture and are faster in detection of bacteremia. PCR with 16sr RNA universal primer is more accurate than both PCR with 16sr RNA universal primer and blood culture for diagnosis of bacteremia.

  1. New method for rapid Susceptibility Testing on blood culture with HB&L system: preliminary data

    Directory of Open Access Journals (Sweden)

    Vincenzo Rondinelli

    2010-12-01

    Full Text Available Blood culture, although represents the gold standard in detecting the ethiological agent of sepsis, is rather rarely required in relation to the real diagnostic importance. The result of this test depends in fact on many factors (sample volume, time of collection, accuracy, antibiotic therapy, contamination, number of drawings, drawing site, interpretation difficulties, etc. that are often considered by many clinicians so limited as to doubt about their actual value. The disadvantages are therefore represented by the lack of standardization but also by the low sensitivity and above all by the technical times too long for the clinical needs. Blood culture begins with the drawing of samples from the “septic” patient followed incubation of the bottles in automatic thermostated systems. In case of positive result (36 hours, the culture is Gram stained and streaked on solid media in order to obtain isolated colonies for the identification and the susceptibility testing (48 hours from positive result. The long time required for pathogen identification and susceptibility testing involves empirical broad spectrum antibiotic therapy that can promote the increase of bacterial resistance but also patient management costs. A clinically useful report should be available on short notice in order to guide the clinician to choose the most appropriate antibiotic. The microbiologist has therefore the hard work of reviewing the organization and the management of the procedures.We have therefore started to consider the possibility of treating the blood as an biological liquid in order to quickly determine the susceptibility of bacteria to antibiotics.

  2. Long-term molecular epidemiology of Staphylococcus epidermidis blood culture isolates from patients with hematological malignancies.

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    Erik Ahlstrand

    Full Text Available Staphylococcus epidermidis is an important cause of bloodstream infections in patients with hematological malignancies. Knowledge of the long-term epidemiology of these infections is limited. We surveyed all S. epidermidis blood culture isolates from patients treated for hematological malignancies at the University Hospital of Örebro, Sweden from 1980 to 2009. A total of 373 S. epidermidis isolates were identified and multilocus sequence typing, staphylococcal chromosome cassette mec (SCCmec typing and standard antibiotic susceptibility testing were employed to characterize these isolates. The majority of the isolates 361/373 (97% belonged to clonal complex 2, and the 373 isolates were divided into 45 sequence types (STs; Simpson's Diversity Index was 0.56. The most prevalent STs were ST2 (243/373, 65% and ST215 (28/373, 8%. Ninety three percent (226/243 of the ST2 isolates displayed either SCCmec type III or IV. ST2 and 215 were isolated during the entire study period, and together these STs caused temporal peaks in the number of positive blood cultures of S. epidermidis. Methicillin resistance was detected in 213/273 (78% of all isolates. In the two predominating STs, ST2 and ST215, methicillin resistance was detected in 256/271 isolates (95%, compared with 34/100 (34% in other STs (p<0.001. In conclusion, in this long-term study of patients with hematological malignancies, we demonstrate a predominance of methicillin-resistant ST2 among S. epidermidis blood culture isolates.

  3. Comparison of QIAsymphony automated and QIAamp manual DNA extraction systems for measuring Epstein-Barr virus DNA load in whole blood using real-time PCR.

    Science.gov (United States)

    Laus, Stella; Kingsley, Lawrence A; Green, Michael; Wadowsky, Robert M

    2011-11-01

    Automated and manual extraction systems have been used with real-time PCR for quantification of Epstein-Barr virus [human herpesvirus 4 (HHV-4)] DNA in whole blood, but few studies have evaluated relative performances. In the present study, the automated QIAsymphony and manual QIAamp extraction systems (Qiagen, Valencia, CA) were assessed using paired aliquots derived from clinical whole-blood specimens and an in-house, real-time PCR assay. The detection limits using the QIAsymphony and QIAamp systems were similar (270 and 560 copies/mL, respectively). For samples estimated as having ≥10,000 copies/mL, the intrarun and interrun variations were significantly lower using QIAsymphony (10.0% and 6.8%, respectively), compared with QIAamp (18.6% and 15.2%, respectively); for samples having ≤1000 copies/mL, the two variations ranged from 27.9% to 43.9% and were not significantly different between the two systems. Among 68 paired clinical samples, 48 pairs yielded viral loads ≥1000 copies/mL under both extraction systems. Although the logarithmic linear correlation from these positive samples was high (r(2) = 0.957), the values obtained using QIAsymphony were on average 0.2 log copies/mL higher than those obtained using QIAamp. Thus, the QIAsymphony and QIAamp systems provide similar EBV DNA load values in whole blood. Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

  4. Evaluation of DNA extraction methods and their clinical application for direct detection of causative bacteria in continuous ambulatory peritoneal dialysis culture fluids from patients with peritonitis by using broad-range PCR.

    Science.gov (United States)

    Kim, Si Hyun; Jeong, Haeng Soon; Kim, Yeong Hoon; Song, Sae Am; Lee, Ja Young; Oh, Seung Hwan; Kim, Hye Ran; Lee, Jeong Nyeo; Kho, Weon-Gyu; Shin, Jeong Hwan

    2012-03-01

    The aims of this study were to compare several DNA extraction methods and 16S rDNA primers and to evaluate the clinical utility of broad-range PCR in continuous ambulatory peritoneal dialysis (CAPD) culture fluids. Six type strains were used as model organisms in dilutions from 10(8) to 10(0) colony-forming units (CFU)/mL for the evaluation of 5 DNA extraction methods and 5 PCR primer pairs. Broad-range PCR was applied to 100 CAPD culture fluids, and the results were compared with conventional culture results. There were some differences between the various DNA extraction methods and primer sets with regard to the detection limits. The InstaGene Matrix (Bio-Rad Laboratories, USA) and Exgene Clinic SV kits (GeneAll Biotechnology Co. Ltd, Korea) seem to have higher sensitivities than the others. The results of broad-range PCR were concordant with the results from culture in 97% of all cases (97/100). Two culture-positive cases that were broad-range PCR-negative were identified as Candida albicans, and 1 PCR-positive but culture-negative sample was identified as Bacillus circulans by sequencing. Two samples among 54 broad-range PCR-positive products could not be sequenced. There were differences in the analytical sensitivity of various DNA extraction methods and primers for broad-range PCR. The broad-range PCR assay can be used to detect bacterial pathogens in CAPD culture fluid as a supplement to culture methods.

  5. Comparison of latex agglutination and immunofluorescence for direct Lancefield grouping of streptococci from blood cultures.

    Science.gov (United States)

    Shlaes, D M; Toossi, Z; Patel, A

    1984-08-01

    Simulated positive blood cultures with 84 known stock strains of streptococci were used to comparatively evaluate the direct identification of these organisms by fluorescein-tagged antibody staining (immunofluorescence [IF]) and latex agglutination (LA). IF was not evaluated for Lancefield group D strains (a total of 81 strains tested) and had 89% sensitivity and 91% specificity. IF was least sensitive for the identification of Lancefield group F, in which three of seven strains showed no fluorescence with the group F reagent. Since LA was more convenient and revealed comparable sensitivities and specificities on 84 simulated cultures, we tested this procedure using an additional 29 fresh positive clinical blood cultures, for a total of 113 cultures tested by this technique. Of 11 Streptococcus pneumoniae strains, 9 reacted with the LA group C reagent, a problem not observed with IF. However, all these strains were identified by a rapid modified bile solubility test. Of the 12 Streptococcus faecalis strains, 4 were falsely negative with the group D reagent, but all were correctly identified by a rapid litmus milk reduction test. Of 12 group A strains, 1 was not detected. Of all 113 strains tested by LA, eliminating S. faecalis and S. pneumoniae, the sensitivity and specificity were 97 and 98%, respectively. LA was simple and reliable in the rapid identification of streptococci from blood cultures and appeared to be preferable to IF. When LA is used, the group D reagent should not be used, and all samples reacting with the group C reagent should be tested by a modified rapid bile solubility test to exclude S. pneumoniae.

  6. Sensitivity, specificity and predictive value of blood cultures from cattle clinically suspected of bacterial endocarditis

    DEFF Research Database (Denmark)

    Houe, Hans; Eriksen, L.; Jungersen, Gregers

    1993-01-01

    This study investigated the number of blood culture-positive cattle among 215 animals clinically suspected of having bacterial endocarditis. For animals that were necropsied, the sensitivity, specificity and predictive value of the diagnosis of endocarditis were calculated on the basis...... of the isolation of the causative bacteria from blood. Furthermore, it was investigated whether the glutaraldehyde coagulation time, total leucocyte count, per cent neutrophil granulocytes, pulse rate and duration of disease could help to discriminate endocarditis from other diseases. Among 138 animals necropsied...... the sensitivity, specificity and predictive value of blood cultivation were 70.7 per cent, 93.8 per cent and 89.1 per cent, respectively. None of the other measurements could be used to discriminate between endocarditis and non-endocarditis cases....

  7. Development of the nested polymerase chain reaction (PCR) for detection of hepatitis C virus RNA in blood derivatives. Final report for the period 15 December 1994 - 15 December 1995

    International Nuclear Information System (INIS)

    Pavelic, J.

    1996-07-01

    Testing for the presence of hepatitis C virus (HCV) in blood derivatives used in clinical medicine is important to ensure the safety of such preparations. A reliable and reproducible method is described for the isolation of HCV RNA, subsequent reverse transcription and nested polymerase chain reaction (PCR) from blood derivatives. Of 17 batches of blood derivatives (14 negative for anti-HCV and 3 of unknown anti-HCV status) five were found to be positive in the nested PCR. (author). 4 refs, 3 figs, 1 tab

  8. A bovine mammary endothelial/epithelial cell culture model of the blood/milk barrier.

    Science.gov (United States)

    Guidry, A J; O'Brien, C N; Douglass, L W

    1998-04-01

    The complex nature of the mammary gland has hampered in-depth studies of the relationship of the circulatory system to cells lining the teat ducts and alveoli of the gland. This study reports an in vitro model of endothelial and epithelial cells separated by a subcellular matrix that simulates the blood milk barrier of the bovine mammary gland. Dual chamber culture dishes with a porous membrane separating the upper and lower chamber were used. Endothelial and epithelial cells were cultured on opposite sides of the porous membrane. A collagen and fibroblast subcellular matrix, separating the 2 cell layers, simulated the in vivo interstitial tissue. Changes in surface binding of anti-bodies to polymorphonuclear neutrophils (PMN) following their migration from the upper to the lower chamber simulated the passage of PMN from blood to milk. Changes in the binding of antibodies to PMN agreed with results observed following the migration of PMN from blood to milk in vivo. This gives credence to the model's potential value for studies where more direct observation of the blood/milk barrier is required. The model will be further tested for its usefulness as an assay for determining: 1) antibiotic diffusion from milk to blood and from blood to milk, 2) cytotoxicity of prophylactic and therapeutic mammary infusion products, 3) factors affecting bacterial adhesion and penetration of mammary epithelial tissue, 4) effectiveness of antibodies present in lacteal secretions in preventing bacterial adhesion, and 5) the feasibility of gene constructs to induce synthesis and secretion of mastitis-preventing compounds and prophylactic and therapeutic compounds for treatment of human disorders.

  9. The production of collagenase by adherent mononuclear cells cultured from human peripheral blood.

    Science.gov (United States)

    Louie, J S; Weiss, J; Ryhänen, L; Nies, K M; Rantala-Ryhänen, S; Uitto, J

    1984-12-01

    Mononuclear cells were isolated from human peripheral blood by Ficoll-Hypaque centrifugation, and the cells adherent to plastic substrata were cultured in serum-free media supplemented with lactalbumin hydrolysate. These cell cultures, which consisted predominantly of monocyte-macrophages as judged by nonspecific esterase staining, accumulated collagenase in the medium. This collagenase resembled other vertebrate collagenases in that it cleaved native triple-helical type I collagen at a locus 3/4-length away from the amino-terminal end of the molecule. The collagenase activity was inhibited by Na2EDTA, dithiothreitol, and fetal calf serum, while the addition of Ca++ or N-ethylmaleimide enhanced the enzyme activity. The accumulation of collagenase in the culture media was markedly enhanced by the incubation of cells with concanavalin A or phorbol myristic acetate. In the presence of cycloheximide, the levels of collagenase activity were markedly reduced, suggesting that active protein synthesis was required to express the enzyme activity. In additional experiments, monocytes were further purified by counterflow centrifugation-elutriation. The collagenase production was markedly increased in cultures enriched in monocyte-macrophages and devoid of polymorphonuclear leukocytes. The accumulation of collagenase in monocyte cultures incubated for 48 hours in the presence of concanavalin A or phorbol myristic acetate was of the same order of magnitude as in parallel cultures containing the same number of polymorphonuclear leukocytes purified by Ficoll-Hypaque centrifugation and Plasmagel sedimentation.(ABSTRACT TRUNCATED AT 250 WORDS)

  10. The Epidemiological And Susceptibility Study Of Inpatient Blood Cultures In Amir Alam Hospital 1998 - 2000

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    Karimi Shahidi M

    2002-07-01

    Full Text Available Sepsis is one of the most critical medical emergency situations. Treatment with anti microbial drugs should be initiated as soon as samples of blood and other relevant sites have been cultured. Available information about patterns of anti microbial Susceptibility among bacterial isolates from the community, the hospital, and the patient should be taken in to account. It is important, pending culture results, to initiate empirical anti microbial therapy."nMaterials and methods: In a descriptive study during 3 years (1377-1379, microbial and anti microbial susceptibility patterns evaluated in Amir alam clinical laboratory on 2000 specimen of blood culture received from 765 hospitalized patients at Amir Alam hospital wards."nResults: 113 specimens from 77 patient (10 percent were positive for microbial growth. Enterobacter, S. aureus, S.epidermidis, Pneumococci, Ecoli, and Pseudomonas were the most common isolated etiologic agents(80 percent . The most common organism was Entenobacter in 1377, S.aureus in 1378 and pseudomonas in 1379 There were significant change in patlern of organisms, increase resistance to some important available antibiotics and change in antibiotic susceptibility pattern during three years (disc diffusion method."nConclusions: According to Results of this study due to change in pattern of organism and their antibiotic susceptibility, dynamic microbiological study provide important data for Ordering empirical and culture oriented treatment of patients with bacteremia, Sepsis, anti microbial Chemotherapy, anti microbial susceptibility empirical anti microbial therapy, microbial pattern.

  11. Microarray-based identification and RT-PCR test screening for epithelial-specific mRNAs in peripheral blood of patients with colon cancer

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    Coppola Domenico

    2006-10-01

    Full Text Available Abstract Background The efficacy of screening for colorectal cancer using a simple blood-based assay for the detection of tumor cells disseminated in the circulation at an early stage of the disease is gaining positive feedback from several lines of research. This method seems able to reduce colorectal cancer mortality and may replace colonoscopy as the most effective means of detecting colonic lesions. Methods In this work, we present a new microarray-based high-throughput screening method to identifying candidate marker mRNAs for the early detection of epithelial cells diluted in peripheral blood cells. This method includes 1. direct comparison of different samples of colonic mucosa and of blood cells to identify consistent epithelial-specific mRNAs from among 20,000 cDNA assayed by microarray slides; 2. identification of candidate marker mRNAs by data analysis, which allowed selection of only 10 putative differentially expressed genes; 3. Selection of some of the most suitable mRNAs (TMEM69, RANBP3 and PRSS22 that were assayed in blood samples from normal subjects and patients with colon cancer as possible markers for the presence of epithelial cells in the blood, using reverse transcription – polymerase chain reaction (RT-PCR. Results Our present results seem to provide an indication, for the first time obtained by genome-scale screening, that a suitable and consistent colon epithelium mRNA marker may be difficult to identify. Conclusion The design of new approaches to identify such markers is warranted.

  12. Bacteriologic profile and antibiogram of blood culture isolates from a children's hospital in Kabul.

    Science.gov (United States)

    Tariq, Tariq Mahmud

    2014-06-01

    To identify the bacterial pathogens causing paediatric septicaemia in Kabul and to determine their antibiogram to improve empirical antibiotic therapy. Cross-sectional study. Microbiology Laboratory of FMIC, Kabul, Afghanistan, from January 2010 to June 2012. Blood cultures from suspected cases of sepsis were processed in BD (Becton Dickinson, USA) for culture BACTEC™ 9240 Blood Culture System. Positive growths were examined and isolates were identified by conventional biochemical tests. Bacteria were identified to the species level using various Analytical Profile Index (API) identification strips. Antibiotic susceptibility testing was performed by modified Kirby-Bauer disk diffusion method. Drug resistant strains were studied for extended spectrum beta lactamase (ESBL) production by combination disk method and for methicillin resistant Staphylococcus aureus (MRSA) by Cefoxitin disk diffusion method. Out of a total 3360 blood cultures received from in-patients, 410 yielded monomicrobial growth; hence the frequency of positive blood culture was 12.2%. Out of a total 410 isolates, 212 (51.71%) were gram-negative bacilli and 184 (44.88%) were gram-positive cocci. In addition, 14 (3.41%) Candida species were also isolated. The frequently isolated species of gram-negative bacteria belonged to Enterobacteriaceae and included 66 Klebsiella (16.1%), 42 Enterobacter (10.2%), 35 Escherichia (E.) coli (8.5%) and 16 Serratia (3.9%) species. In addition, 21 (5.12%) Pseudomonas species were also isolated. Correspondingly, amongst gram-positive cocci, the most frequently isolated species were 108 coagulase-negative Staphylococci (26.34%) followed by 49 Staphylococcus aureus (11.95%) and 21 Streptococcus species (5.12%). Among gram-negative isolates, those that produced ESBL i.e., 110 out of 212 (51.9%) were found to be multidrug-resistant and showed high resistance to commonly used antibiotics namely Ampicillin, Gentamicin, 3rd generation Cephalosporins, Fluoroquinolones and

  13. Immunophenotype of hematopoietic stem cells from placental/umbilical cord blood after culture

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    P. Pranke

    2005-12-01

    Full Text Available Identification and enumeration of human hematopoietic stem cells remain problematic, since in vitro and in vivo stem cell assays have different outcomes. We determined if the altered expression of adhesion molecules during stem cell expansion could be a reason for the discrepancy. CD34+CD38- and CD34+CD38+ cells from umbilical cord blood were analyzed before and after culture with thrombopoietin (TPO, FLT-3 ligand (FL and kit ligand (KL; or stem cell factor in different combinations: TPO + FL + KL, TPO + FL and TPO, at concentrations of 50 ng/mL each. Cells were immunophenotyped by four-color fluorescence using antibodies against CD11c, CD31, CD49e, CD61, CD62L, CD117, and HLA-DR. Low-density cord blood contained 1.4 ± 0.9% CD34+ cells, 2.6 ± 2.1% of which were CD38-negative. CD34+ cells were isolated using immuno-magnetic beads and cultured for up to 7 days. The TPO + FL + KL combination presented the best condition for maintenance of stem cells. The total cell number increased 4.3 ± 1.8-fold, but the number of viable CD34+ cells decreased by 46 ± 25%. On the other hand, the fraction of CD34+CD38- cells became 52.0 ± 29% of all CD34+ cells. The absolute number of CD34+CD38- cells was expanded on average 15 ± 12-fold when CD34+ cells were cultured with TPO + FL + KL for 7 days. The expression of CD62L, HLA-DR and CD117 was modulated after culture, particularly with TPO + FL + KL, explaining differences between the adhesion and engraftment of primary and cultured candidate stem cells. We conclude that culture of CD34+ cells with TPO + FL + KL results in a significant increase in the number of candidate stem cells with the CD34+CD38- phenotype.

  14. Detection of poliovirus type 2 in oysters by using cell cul