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Journal articles on the topic 'Microbiology assay'
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1
Dekker, John P. "Molecular Assay Validation Using Genomic Sequence Databases." Journal of Clinical Microbiology 54, no. 12 (October 12, 2016): 2854–56. http://dx.doi.org/10.1128/jcm.01797-16.
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Whole-genome sequence databases offer newin silicoapproaches for designing and validating PCR assays in the clinical microbiology laboratory. An article in this issue of theJournal of Clinical Microbiology(M. J. Jansen van Rensburg, C. Swift, A. J. Cody, C. Jenkins, and M. C. J. Maiden, J Clin Microbiol, 54:2882–2890, 2016,http://dx.doi.org/10.1128/JCM.01522-16) demonstrates the use of publicly available genomic sequence data to evaluate a PCR assay for distinguishingCampylobacterspecies.
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Ratnam, Samuel, Graham Tipples, Carol Head, Micheline Fauvel, Margaret Fearon, and Brian J. Ward. "Performance of Indirect Immunoglobulin M (IgM) Serology Tests and IgM Capture Assays for Laboratory Diagnosis of Measles." Journal of Clinical Microbiology 38, no. 1 (January 2000): 99–104. http://dx.doi.org/10.1128/jcm.38.1.99-104.2000.
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ABSTRACT As progress is made toward elimination of measles, the laboratory confirmation of measles becomes increasingly important. However, both false-positive and false-negative results can occur with the routinely used indirect measles immunoglobulin M (IgM) serology tests. The measles IgM capture assay is considered to be more specific, and therefore, its use is indicated for confirmatory testing, but its relative performance has not been fully assessed. Four commercial indirect measles IgM serology test kits (the Behring, Clark, Gull, and PanBio assays) and a commercial IgM capture assay (the Light Diagnostics assay) were evaluated for their abilities to detect measles virus-specific IgM antibody with a total of 308 serum samples from patients involved in a measles outbreak and with confirmed cases of measles and 454 samples from subjects without measles. The Centers for Disease Control and Prevention (CDC) IgM capture assay was also used in a part of the evaluation. Among the indirect assays, the overall sensitivities ranged from 82.8% (Clark assay) to 88.6% (Behring assay) and specificity ranged from 86.6% (PanBio assay) to 99.6% (Gull assay). These rates were 92.2 and 86.6%, respectively, for the Light Diagnostics capture assay and 87.0 and 94.8%, respectively, for the CDC capture assay. While the Light Diagnostics capture assay had the best detection rate (80%) with the acute-phase samples compared with those for the rest of the tests (CDC capture assay, 77%; Behring assay, 70%; Gull assay, 69%; PanBio assay, 58%; and Clark assay, 57%), all tests showed a significantly improved sensitivity in the range of 92% (Clark and PanBio assays) to 97% (Light Diagnostics and CDC capture assays) with the convalescent-phase samples, as expected. The best seropositivity rates (in the range of 92 to 100%) were observed with samples collected 6 to 14 days after the onset of symptoms. The Gull assay showed the highest positive predictive value (99.6%), followed by the Behring assay (97.8%) and the CDC capture assay (96.1%). Overall, the Gull and Behring assays were found to be as good as or better than the capture assays. In conclusion, laboratory diagnosis of measles based on IgM serology varies depending on the timing of specimen collection and the test used, and the case for the use of the IgM capture assay as the confirmatory test appears to be uncertain.
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Leiby, David A., Silvano Wendel, Deise T. Takaoka, Roberta M. Fachini, Lea C. Oliveira, and Melinda A. Tibbals. "Serologic Testing for Trypanosoma cruzi: Comparison of Radioimmunoprecipitation Assay with Commercially Available Indirect Immunofluorescence Assay, Indirect Hemagglutination Assay, and Enzyme-Linked Immunosorbent Assay Kits." Journal of Clinical Microbiology 38, no. 2 (2000): 639–42. http://dx.doi.org/10.1128/jcm.38.2.639-642.2000.
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The radioimmunoprecipitation assay (RIPA) has been used as a confirmatory test in several ongoing and published studies ofTrypanosoma cruzi in blood donors in the United States. Despite its use as a confirmatory test, few studies are available comparing RIPA to commercially available serologic test methods. Thus, we compared RIPA with two indirect hemagglutination assays (Biolab Diagnostica SA, São Paulo, Brazil; Hemagen Diagnostics, Inc., Waltham, Mass.) and four different enzyme-linked immunosorbent assays (Abbott Laboratories, Abbott Park, Ill.; Embrabio, São Paulo, Brazil; Organon Teknika, São Paulo, Brazil; and Gull Laboratories, Salt Lake City, Utah) using a panel of 220 serum specimens from Brazilian blood donors with a range of T. cruzi antibody titers as determined by indirect immunofluorescence assay (IFA). A titer of 1:20 was used as the baseline for seropositivity. All IFA-negative serum specimens (n = 19) were nonreactive on all tests. At a titer of 1:20 (n = 9), reactivity rates varied considerably among the tests, with only the RIPA and the Organon and Gull assays identifying reactive specimens. For specimens at a 1:40 titer (n = 35), most assays identified at least 32 of 35 (91%) specimens as reactive, but the Biolab assay only identified 24 (69%). At higher titers (1:80, n = 56; 1:160,n = 101) the assays were comparable, with the exception of the Biolab assay, demonstrating rates of agreement with IFA of ≥98%. Overall, when compared with several other test formats, RIPA demonstrated equivalent or superior rates of agreement with IFA-positive specimens across all titers examined. In particular, at titers of >1:40, the RIPA compared favorably with other test methods currently in use, supporting its application as a confirmatory test, particularly in a research setting.
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Forbes, Betty A. "Introducing a Molecular Test Into the Clinical Microbiology Laboratory: Development, Evaluation, and Validation." Archives of Pathology & Laboratory Medicine 127, no. 9 (September 1, 2003): 1106–11. http://dx.doi.org/10.5858/2003-127-1106-iamtit.
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Abstract Context.—In the mid-1980s, the polymerase chain reaction methodology for the amplification of minute amounts of target DNA was successfully developed and then introduced into clinical use; such technology has led to a revolution in diagnostic testing. Despite enormous advances in the detection of infectious agents by amplification methods, there are also limitations that must be addressed. Objective.—To highlight the pertinent steps and issues associated with the introduction of an amplification assay into a clinical microbiology laboratory as well as the subsequent ongoing activities following its introduction into routine laboratory use. Data Sources.—Data were obtained from literature searches from 1990 through September 2002 using the subject headings “polymerase chain reaction,” “molecular assays,” and “amplification” as well as publications of the National Committee for Clinical Laboratory Standards. Data Extraction and Synthesis.—Using the findings obtained from these studies and publications, the process of introducing a molecular assay into the clinical microbiology laboratory was broken down into 4 major components: (1) initial phase of assay development, (2) polymerase chain reaction assay verification in which analytic sensitivity and specificity is determined, (3) assay validation to determine clinical sensitivity and specificity, and (4) interpretation of results and ongoing, required activities. The approach, as well as the advantages and limitations involved in each step of the process, was highlighted and discussed within the context of the published literature. Conclusions.—The application of molecular testing methods in the clinical laboratory has dramatically improved our ability to diagnose infectious diseases. However, the clinical usefulness of molecular testing will only be maximized to its fullest benefit by appropriate and careful studies correlating clinical findings with assay results.
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Elbeik, Tarek, Edwin Charlebois, Patricia Nassos, James Kahn, Frederick M. Hecht, David Yajko, Valerie Ng, and Keith Hadley. "Quantitative and Cost Comparison of Ultrasensitive Human Immunodeficiency Virus Type 1 RNA Viral Load Assays: Bayer bDNA Quantiplex Versions 3.0 and 2.0 and Roche PCR Amplicor Monitor Version 1.5." Journal of Clinical Microbiology 38, no. 3 (2000): 1113–20. http://dx.doi.org/10.1128/jcm.38.3.1113-1120.2000.
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Quantification of human immunodeficiency virus type 1 (HIV-1) RNA as a measure of viral load has greatly improved the monitoring of therapies for infected individuals. With the significant reductions in viral load now observed in individuals treated with highly active anti-retroviral therapy (HAART), viral load assays have been adapted to achieve greater sensitivity. Two commercially available ultrasensitive assays, the Bayer Quantiplex HIV-1 bDNA version 3.0 (bDNA 3.0) assay and the Roche Amplicor HIV-1 Monitor Ultrasensitive version 1.5 (Amplicor 1.5) assay, are now being used to monitor HIV-1-infected individuals. Both of these ultrasensitive assays have a reported lower limit of 50 HIV-1 RNA copies/ml and were developed from corresponding older generation assays with lower limits of 400 to 500 copies/ml. However, the comparability of viral load data generated by these ultrasensitive assays and the relative costs of labor, disposables, and biohazardous wastes were not determined in most cases. In this study, we used matched clinical plasma samples to compare the quantification of the newer bDNA 3.0 assay with that of the older bDNA 2.0 assay and to compare the quantification and costs of the bDNA 3.0 assay and the Amplicor 1.5 assay. We found that quantification by the bDNA 3.0 assay was approximately twofold higher than that by the bDNA 2.0 assay and was highly correlated to that by the Amplicor 1.5 assay. Moreover, cost analysis based on labor, disposables, and biohazardous wastes showed significant savings with the bDNA 3.0 assay as compared to the costs of the Amplicor 1.5 assay.
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Mashock, Michael J., Matthew L. Faron, Blake W. Buchan, and Nathan A. Ledeboer. "Evaluation of Copan FecalSwab as Specimen Type for Use in Xpert C. difficile Assay." Journal of Clinical Microbiology 55, no. 10 (August 9, 2017): 3123–29. http://dx.doi.org/10.1128/jcm.00369-17.
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ABSTRACT Liquid-based microbiology (LBM) devices incorporating flocked swabs and preservation medium ease transport of specimens and improve specimen yield compared to traditional fiber wound swabs; however, the performance of LBM collection devices has not been evaluated in many molecular assays. It is unclear how the differences in matrix and specimen loading with an LBM device will affect test performance compared to traditional collection devices. The purpose of this study was to evaluate the performance of specimens collected in FecalSwab transport medium (Copan Diagnostics, Murrieta, CA) compared to unpreserved stool using the Cepheid Xpert C. difficile assay (Cepheid, Sunnyvale, CA). Results equivalent to unpreserved stool samples were obtained when 400 μl of FecalSwab-preserved stool was employed in the Xpert assay. The positive and negative percent agreement of specimens inoculated with FecalSwab medium ( n = 281) was 97.0% (95% confidence interval [CI], 90.9 to 96.4%) and 99.4% (95% CI, 96.4 to 99.9%), respectively, compared to reference results obtained using unpreserved stool. Throughout this study, only four discrepant results occurred when comparing preserved specimens to unpreserved stool specimens in the Xpert C. difficile PCR assay. Post discrepant analysis, using the BD MAX Cdiff assay, the specificity and sensitivity both increased to 100%. The high positive and negative percent agreements observed in this study suggest that stool preserved in FecalSwab media yields equivalent results to using unpreserved stool when tested on the Xpert C. difficile assay, allowing laboratories to adopt this liquid-based microbiology collection device.
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Keiser, Patrick T., Manu Anantpadma, Hilary Staples, Ricardo Carrion, and Robert A. Davey. "Automation of Infectious Focus Assay for Determination of Filovirus Titers and Direct Comparison to Plaque and TCID50 Assays." Microorganisms 9, no. 1 (January 12, 2021): 156. http://dx.doi.org/10.3390/microorganisms9010156.
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Ongoing efforts to develop effective therapies against filoviruses rely, to different extents, on quantifying the amount of viable virus in samples by plaque, TCID50, and focus assays. Unfortunately, these techniques have inherent variance, and laboratory-specific preferences make direct comparison of data difficult. Additionally, human errors such as operator errors and subjective bias can further compound the differences in outcomes. To overcome these biases, we developed a computer-based automated image-processing method for a focus assay based on the open-source CellProfiler software platform, which enables high-throughput screening of many treatment samples at one time. We compared virus titers calculated using this platform to plaque and TCID50 assays using common stocks of virus for 3 major Filovirus species, Zaire ebolavirus, Sudan ebolavirus, and Marburg marburgvirus with each assay performed by multiple operators on multiple days. We show that plaque assays give comparable findings that differ by less than 3-fold. Focus-forming unit (FFU) and TCID50 assays differ by 10-fold or less from the plaque assays due a higher (FFU) and lower (TCID50) sensitivity. However, reproducibility and accuracy of each assay differs significantly with Neutral Red Agarose Overlay plaque assays and TCID50 with the lowest reproducibility due to subjective analysis and operator error. Both crystal violet methylcellulose overlay plaque assay and focus assays perform best for accuracy and the focus assay performs best for speed and throughput.
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Theel, Elitza S., Marc Roger Couturier, Laura Filkins, Elizabeth Palavecino, Stephanie Mitchell, Sheldon Campbell, Michael Pentella, et al. "Application, Verification, and Implementation of SARS-CoV-2 Serologic Assays with Emergency Use Authorization." Journal of Clinical Microbiology 59, no. 1 (October 5, 2020): e02148-20. http://dx.doi.org/10.1128/jcm.02148-20.
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ABSTRACTInterest continues to grow regarding the role of serologic assays for the detection of prior infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The U.S. Food and Drug Administration (FDA) has granted emergency use authorization (EUA) status to many SARS-CoV-2 serologic assays. In this document, expert recommendations from clinical microbiologist members of the American Society for Microbiology (ASM) concerning detailed verification strategies for SARS-CoV-2 serologic assays with FDA EUA are provided, as are insights into assay limitations and reporting considerations for laboratories. Assessments concerning single-antibody and multiantibody isotype detection assays, which may provide either differentiated or nondifferentiated (i.e., total antibody) antibody class results, are addressed. Additional considerations prior to assay implementation are also discussed, including biosafety, quality control, and proficiency testing strategies. As the landscape of SARS-CoV-2 serologic testing is rapidly changing, this document provides updated guidance for laboratorians on application of these assays.
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Jansen van Rensburg, Melissa J., Craig Swift, Alison J. Cody, Claire Jenkins, and Martin C. J. Maiden. "Exploiting Bacterial Whole-Genome Sequencing Data for Evaluation of Diagnostic Assays: Campylobacter Species Identification as a Case Study." Journal of Clinical Microbiology 54, no. 12 (October 12, 2016): 2882–90. http://dx.doi.org/10.1128/jcm.01522-16.
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The application of whole-genome sequencing (WGS) to problems in clinical microbiology has had a major impact on the field. Clinical laboratories are now using WGS for pathogen identification, antimicrobial susceptibility testing, and epidemiological typing. WGS data also represent a valuable resource for the development and evaluation of molecular diagnostic assays, which continue to play an important role in clinical microbiology. To demonstrate this application of WGS, this study used publicly available genomic data to evaluate a duplex real-time PCR (RT-PCR) assay that targetsmapAandceuEfor the detection ofCampylobacter jejuniandCampylobacter coli, leading global causes of bacterial gastroenteritis.In silicoanalyses ofmapAandceuEprimer and probe sequences from 1,713 genetically diverseC. jejuniandC. coligenomes, supported by RT-PCR testing, indicated that the assay was robust, with 1,707 (99.7%) isolates correctly identified. The high specificity of themapA-ceuEassay was the result of interspecies diversity and intraspecies conservation of the target genes inC. jejuniandC. coli. Rare instances of a lack of specificity amongC. coliisolates were due to introgression inmapAor sequence diversity inceuE. The results of this study illustrate how WGS can be exploited to evaluate molecular diagnostic assays by using publicly available data, online databases, and open-source software.
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Wylie, John L., Stephen Moses, Ryan Babcock, Ann Jolly, Sandra Giercke, and Greg Hammond. "Comparative Evaluation of Chlamydiazyme, PACE 2, and AMP-CT Assays for Detection of Chlamydia trachomatis in Endocervical Specimens." Journal of Clinical Microbiology 36, no. 12 (1998): 3488–91. http://dx.doi.org/10.1128/jcm.36.12.3488-3491.1998.
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We conducted a comparative evaluation of the Chlamydiazyme (Abbott Laboratories), PACE 2 (Gen-Probe), and AMP-CT (Gen-Probe) assays for the detection of Chlamydia trachomatis in endocervical samples. Specimens from 787 females were included in the study. The sensitivities of the PACE 2 and Chlamydiazyme assays in comparison to the results of the AMP-CT assay were 79.3 and 63.4%, respectively. The specificities of the Chlamydiazyme and PACE 2 assays were 100%. All of the positive specimens detected in this study were positive by the AMP-CT assay. On the basis of the final results of the comparison, the prevalence of C. trachomatis in the population was 10.4%. Retesting of specimens whose results were in the intermediate zone by the PACE 2 assay by a probe competition assay identified some additional true-positive specimens. Amplification assay testing of such specimens did not significantly increase the yield. The majority of specimens which tested positive by the AMP-CT assay only were not in the intermediate zone by the PACE 2 assay. We were unable to identify demographic or clinical factors which could predict those individuals who tested positive by amplified tests but not by nonamplified tests. The Gen-Probe PACE 2 assay proved to be superior to the Chlamydiazyme assay for the screening and diagnosis of C. trachomatisinfections in female endocervical specimens.
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Ho, Stephen K. N., Tak Mao Chan, Ignatius K. P. Cheng, and Kar Neng Lai. "Comparison of the Second-Generation Digene Hybrid Capture Assay with the Branched-DNA Assay for Measurement of Hepatitis B Virus DNA in Serum." Journal of Clinical Microbiology 37, no. 8 (1999): 2461–65. http://dx.doi.org/10.1128/jcm.37.8.2461-2465.1999.
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The optimal hepatitis B virus (HBV) DNA quantitative assay for clinical use remains to be determined. We examined the sensitivity, linearity, and variability of a novel second-generation antibody capture solution hybridization assay, the Digene Hybrid Capture II assay (HCII), and compared it with another widely used solution hybridization assay, the branched-DNA (bDNA) assay (Quantiplex; Chiron Corp.). Our results showed similar and satisfactory assay linearity values, as well as interassay and intra-assay variability values, for both HCII and bDNA assays across different ranges of HBV DNA. Ninety-one percent of 102 serum samples from hepatitis B surface antigen-positive patients showed concordant results with the two assays. The HCII assay was more sensitive than the bDNA assay by 1 dilution, with the lowest reading being 0.9 pg/ml (3.8 pg/ml by bDNA assay). The HBV DNA seropositivity rates for the 102 samples were 58, 67, and 97% by bDNA, HCII, and nested PCR, respectively. While the relationship between results obtained with the bDNA assay and those with the HCII assay was nonlinear, with the bDNA assay yielding values 2.83 ± 0.92-fold higher than those of the HCII assay, especially at high HBV DNA levels, a linear relationship was observed between the two sets of data after logarithmic conversion. The formula for interassay conversion of results was derived as follows: HBV DNA by HCII (picograms per milliliter) = 3.19 × [HBV DNA by bDNA (megaequivalents per milliliter)]0.866. The HCII assay was technically less complex and required a shorter assay time (4 h) than the bDNA assay (24 h). We conclude that the HCII assay compares favorably with the bDNA assay and offers the additional advantages of increased sensitivity and shorter assay time. The increased sensitivity should be particularly useful in monitoring the efficacy of antiviral therapies and detecting the emergence of drug-resistant HBV mutants.
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Gomes-Solecki, Maria J. C., John J. Dunn, Benjamin J. Luft, Jonathan Castillo, Daniel E. Dykhuizen, Xiaohua Yang, John D. Glass, and Raymond J. Dattwyler. "Recombinant Chimeric Borrelia Proteins for Diagnosis of Lyme Disease." Journal of Clinical Microbiology 38, no. 7 (2000): 2530–35. http://dx.doi.org/10.1128/jcm.38.7.2530-2535.2000.
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Current serologic Lyme disease tests use whole borrelia cells as the source of antigen. These assays are difficult to standardize and to optimize for sensitivity and specificity. To help solve these problems, we constructed a library of recombinant chimeric proteins composed of portions of key antigens of Borrelia burgdorferi. These proteins were then used to develop an enzyme-linked immunosorbent assay. We compared our assay with the most sensitive of three whole-cell borrelia assays. We found that the recombinant assay could detect antibodies significantly better from early Lyme disease sera (P < 0.05), and had the same sensitivity for late Lyme disease sera, as the most sensitive whole-cell borrelia assay. On potentially cross-reactive sera, the recombinant assay was more specific, but not significantly so, than the best whole-cell borrelia assay. Optimization of the recombinant assay offers the potential for a significant improvement in both sensitivity and specificity.
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Allen, Marchelle I., Josee Gauthier, Manon DesLauriers, Eric J. Bourne, Kevin M. Carrick, Fausto Baldanti, Lisa L. Ross, Michael W. Lutz, and Lynn D. Condreay. "Two Sensitive PCR-Based Methods for Detection of Hepatitis B Virus Variants Associated with Reduced Susceptibility to Lamivudine." Journal of Clinical Microbiology 37, no. 10 (1999): 3338–47. http://dx.doi.org/10.1128/jcm.37.10.3338-3347.1999.
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Two novel assays, a restriction fragment length polymorphism (RFLP) assay and an assay based on the 5′-nuclease activity of TaqDNA polymerase, were developed for screening viral variants in lamivudine-treated patients’ sera containing <1,000 copies of the hepatitis B virus (HBV) genome per ml. Both assays were designed to detect single-nucleotide changes within the HBV DNA polymerase gene that are associated with lamivudine resistance in vitro and have been used to screen a number of patients’ sera for variant virus. Results obtained with these assays and standard sequencing technology were compared with regard to throughput, ability to detect individual virus species present at low concentrations, and ability to detect, distinguish, and quantitate wild-type (wt) and HBV tyrosine methionine552 aspartate aspartate motif variants in mixed viral populations. Unlike DNA sequencing, both assays are amenable to high-throughput screening and were shown to be able to quantitatively detect variant virus in the presence of a background of wt virus. As with DNA sequencing, both new assays incorporate a PCR amplification step and are able to detect the relatively low amounts of virus found in lamivudine-treated patients’ sera. However, these assays are far less labor intensive than the DNA-sequencing techniques presently in use. Overall, the RFLP assay was more sensitive than DNA sequencing in detecting and determining the ratios of wt to variant virus. Furthermore, the RFLP assay and 5′-nuclease assay were equally sensitive in the detection of mixed viral species, but the RFLP assay was superior to the 5′-nuclease assay in the quantitation of mixed viral species. These assays should prove useful for further understanding of virological response to therapy and disease progression.
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Chan, Henry L. Y., Nancy W. Y. Leung, Tracy C. M. Lau, May L. Wong, and Joseph J. Y. Sung. "Comparison of Three Different Sensitive Assays for Hepatitis B Virus DNA in Monitoring of Responses to Antiviral Therapy." Journal of Clinical Microbiology 38, no. 9 (2000): 3205–8. http://dx.doi.org/10.1128/jcm.38.9.3205-3208.2000.
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The aim of our study was to compare the performances of two new hepatitis B virus (HBV) DNA assays, a cross-linking assay (NAXCOR) and a hybrid-capture amplification assay (Digene), versus the widely used branched-DNA (bDNA) assay (Chiron) in the monitoring of HBV DNA levels during antiviral treatment. Serial serum samples from 12 chronically HBV infected patients undergoing a phase II trial of an antiviral drug, 2′,3′-dideoxy-5-fluoro-3′-thiacytidine (FTC), were studied. A total of 96 serum samples were tested for HBV DNA using the cross-linking, hybrid-capture amplification, and bDNA assays. In the comparison of the cross-linking and bDNA assays, concordant results were found in 77 (80.3%) samples, no significant difference was found between the median log10 HBV DNA levels (6.66 versus 7.17 meq/ml), and the results of the two assays were closely correlated (r = 0.95). In the comparison of the hybrid-capture amplification and bDNA assays, concordant results were found in 79 (82.3%) samples, no significant difference was found between the median log10 HBV DNA levels (6.98 versus 6.99 meq/ml), and the results of the two assays were closely correlated (r = 0.99). Six (6.3%) samples by the cross-linking assay and 10 (10.4%) samples by the bDNA assay required retesting because of unacceptably high within-run coefficients of variance. No sample required retesting in the hybrid-capture amplification assay according to the internal validation. In conclusion, the cross-linking and hybrid-capture amplification assays were as sensitive as the bDNA assay for HBV DNA detection and can be recommended for monitoring of HBV DNA levels during antiviral treatment.
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de Andrade, Fabrício Havy Dantas, Rayanne Sales de Araújo Batista, Taynara Batista Lins, Felipe Hugo Alencar Fernandes, Deysiane Oliveira Brandão, Rui Oliveira Macedo, Fábio Santos de Souza, and Almir Gonçalves Wanderley. "Thermal characterization and microbiology assay of Annona muricata L. leaves." Journal of Thermal Analysis and Calorimetry 138, no. 5 (February 25, 2019): 3737–45. http://dx.doi.org/10.1007/s10973-019-08050-w.
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Vernon, Suzanne D., Elizabeth R. Unger, and Deon Williams. "Comparison of Human Papillomavirus Detection and Typing by Cycle Sequencing, Line Blotting, and Hybrid Capture." Journal of Clinical Microbiology 38, no. 2 (2000): 651–55. http://dx.doi.org/10.1128/jcm.38.2.651-655.2000.
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We compared the results of human papillomavirus (HPV) detection and typing from 781 cervical samples assayed by three methods: L1 consensus PCR followed by cycle sequencing, L1 consensus PCR with biotinylated primers followed by hybridization to a line blot, and Hybrid Capture assay. Both PCR assays used L1 consensus PCR with primers MY09 and MY11. We evaluated the amplification efficiencies of both PCR assays and also compared the specific HPV types detected by each method. The samples positive by the Hybrid Capture assay were compared to the specific types detected by the PCR-based assays. The concordance between the two PCR assays in producing an HPV amplicon visible by gel electrophoresis or in detecting any HPV type was moderate: kappa values were 0.61 (95% confidence interval [CI] = 0.56 to 0.67) and 0.51 (95% CI = 0.46 to 0.58), respectively. The McNemar test for correlated proportions indicated that biotinylated PCR was less likely to produce a band (P = 0.001) and to detect an HPV type (P = 0.001) than the other PCR assay. In comparing the Hybrid Capture assay results with the HPV types detected by the PCR-based assays, we found that positivity by the Hybrid Capture assay for a number of samples may be due to cross-hybridization with HPV types not included in the Hybrid Capture assay probe cocktails.
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Basavanna, Uma, Tim Muruvanda, Eric W. Brown, and Shashi K. Sharma. "Development of a Cell-Based Functional Assay for the Detection ofClostridium botulinumNeurotoxin Types A and E." International Journal of Microbiology 2013 (2013): 1–7. http://dx.doi.org/10.1155/2013/593219.
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The standard procedure for definitive detection of BoNT-producingClostridiais a culture method combined with neurotoxin detection using a standard mouse bioassay (MBA). The mouse bioassay is highly sensitive and specific, but it is expensive and time-consuming, and there are ethical concerns due to use of laboratory animals. Cell-based assays provide an alternative to the MBA in screening for BoNT-producingClostridia. Here, we describe a cell-based assay utilizing a fluorescence reporter construct expressed in a neuronal cell model to study toxin activityin situ. Our data indicates that the assay can detect as little as 100 pM BoNT/A activity within living cells, and the assay is currently being evaluated for the analysis of BoNT in food matrices. Among availablein vitroassays, we believe that cell-based assays are widely applicable in high-throughput screenings and have the potential to at least reduce and refine animal assays if not replace it.
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Sam, Soya S., Jaclynn R. Kurpewski, Susan Cu-Uvin, and Angela M. Caliendo. "Evaluation of Performance Characteristics of the Aptima HIV-1 Quant Dx Assay for Detection and Quantitation of Human Immunodeficiency Virus Type 1 in Plasma and Cervicovaginal Lavage Samples." Journal of Clinical Microbiology 54, no. 4 (February 3, 2016): 1036–41. http://dx.doi.org/10.1128/jcm.03289-15.
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Quantification of HIV-1 RNA has become the standard of care in the clinical management of HIV-1-infected individuals. The objective of this study was to evaluate performance characteristics and relative workflow of the Aptima HIV-1 Quant Dx assay in comparison with the Abbott RealTime HIV-1 assay using plasma and cervicovaginal lavage (CVL) specimens. Assay performance was evaluated by using an AcroMetrix HIV-1 panel, AcroMetrix positive controls, Qnostics and SeraCare HIV-1 evaluation panels, 208 clinical plasma samples, and 205 matched CVL specimens on the Panther and m2000 platforms. The Aptima assay demonstrated good linearity over the quantification range tested (2 to 5 log10copies/ml), and there was strong linear correlation between the assays (R2= 0.99), with a comparable coefficient of variance of <5.5%. For the plasma samples, Deming regression analyses and Bland-Altman plots showed excellent agreement between the assays, with an interassay concordance of 91.35% (kappa = 0.75; 95% confidence interval [CI], 0.65 to 0.85), and on average, the viral loads determined by the Aptima assay were 0.21 log10copies/ml higher than those determined by the RealTime assay. The assays differed in their sensitivity for quantifying HIV-1 RNA loads in CVL samples, with the Aptima and RealTime assays detecting 30% and 20%, respectively. Aptima had fewer invalid results, and on average, the viral loads in CVL samples quantified by the Aptima assay were 0.072 log10copies/ml higher than those of the RealTime assay. Our results demonstrate that the Aptima assay is sensitive and accurate in quantifying viral loads in both plasma and CVL specimens and that the fully automated Panther system has all the necessary features suitable for clinical laboratories demanding high-throughput sample processing.
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Nkengasong, John N., Mirielle Kalou, Chantal Maurice, Celestin Bile, Marie-Yolande Borget, Stéphania Koblavi, Emmanuel Boateng, et al. "Comparison of NucliSens and Amplicor Monitor Assays for Quantification of Human Immunodeficiency Virus Type 1 (HIV-1) RNA in Plasma of Persons with HIV-1 Subtype A Infection in Abidjan, Côte d’Ivoire." Journal of Clinical Microbiology 36, no. 9 (1998): 2495–98. http://dx.doi.org/10.1128/jcm.36.9.2495-2498.1998.
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We compared the sensitivity and accuracy of the NucliSens assay and those of both the standard and modified (addition of a new primer set, primer mix 1, supplied by Roche) Amplicor HIV Monitor assays to quantify human immunodeficiency virus type 1 (HIV-1) RNA in persons infected with HIV-1 subtype A in Abidjan, Côte d’Ivoire. Seventy-one plasma samples from HIV-1-seropositive persons at different stages of HIV infection and 15 samples from HIV antibody-negative persons were analyzed. The HIV-1 genetic subtype was determined either by DNA sequencing or by a restriction fragment length polymorphism assay. Of the 71 samples, 70 (98%) were subtype A and 1 was subtype G. Of the 70 subtype A samples, the proportion of RNA-positive plasma samples and mean HIV-1 RNA levels were significantly higher by the modified HIV Monitor assay (n = 67 [96%]; mean RNA levels, 5.2 log10 HIV-1 RNA copies/ml) than the NucliSens assay (n = 56 [80%]; 4.3 log10 HIV-1 RNA copies/ml) or the standard HIV Monitor assay (n = 44 [63%]; mean RNA levels, 3.8 log10 HIV-1 RNA copies/ml) (all P values were <0.05). The HIV-1 RNA levels by the modified HIV Monitor assay correlated significantly with those by the NucliSens assay (r = 0.76; P< 0.001) and the standard HIV Monitor assay (r = 0.57; P < 0.001), as did the RNA levels by the NucliSens and the standard HIV Monitor assays (r = 0.60; P < 0.001). Lower CD4 cell counts were significantly correlated with higher HIV-1 RNA levels by all three assays (r = −0.47 for the NucliSens assay, −0.45 for the standard HIV Monitor assay, and −0.62 for the modified HIV Monitor assay). These results indicate that the modified HIV Monitor assay has the highest sensitivity and efficiency at quantifying the levels of RNA in persons infected with HIV-1 subtype A and thus constitutes a valuable tool for the monitoring of RNA levels in areas of Africa were HIV-1 subtype A is predominant.
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Arduino, M. J., L. A. Bland, S. M. Aguero, L. Carson, M. Ridgeway, and M. S. Favero. "Comparison of microbiologic assay methods for hemodialysis fluids." Journal of Clinical Microbiology 29, no. 3 (1991): 592–94. http://dx.doi.org/10.1128/jcm.29.3.592-594.1991.
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Rubin, Lorry G., and Atqia Rizvi. "PCR-based assays for detection of Streptococcus pneumoniae serotypes 3, 14, 19F and 23F in respiratory specimens." Journal of Medical Microbiology 53, no. 7 (July 1, 2004): 595–602. http://dx.doi.org/10.1099/jmm.0.45550-0.
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Current culture-based assays are insensitive for detection of simultaneous respiratory tract colonization by more than one pneumococcal serotype. Separate single-tube, nested PCR-based assays have been developed to detect Streptococcus pneumoniae serotypes 3, 14, 19F and 23F by amplifying unique DNA sequences in the capsular polysaccharide gene cluster of each serotype. Pairs of 27–32-base outer primers and 20–21-base inner primers and a 20–22-base probe were designed to amplify and detect a 200–221-base sequence by dot blotting using the labelled probe. Sensitivity of the assays was 0.01–10 fg using chromosomal DNA and ⩽ 1 viable cell using DNA extracted from exponential-phase bacteria. Each serotype-specific assay detected chromosomal DNA from all of five to ten clinical isolates of the homologous type and did not detect DNA sequences from any of 190–204 strains from 51–52 different serotypes or 28 non-pneumococcal bacterial strains. Sixteen throat swabs from children that had been cultured for S. pneumoniae were tested in PCR assays following DNA extraction. All of six that grew S. pneumoniae serotype 3, 14, 19F or 23F were positive in the PCR assay for the homologous serotype (and in a PCR assay for sequences in lytA, present in all pneumococci) and were negative in assays for other serotypes. Of eight culture-negative specimens in children not receiving antimicrobials, three were positive for both the lytA assay and an assay for one of the four serotypes, suggesting true positive results; in three others all five PCR assays were negative and, in the remaining two, the lytA assay was positive but each of the four assays for individual serotypes was negative, suggesting either false-positive results or presence of DNA sequences from an S. pneumoniae serotype other than 3, 14, 19F or 23F. These preliminary clinical data suggest that these PCR-based assays are sensitive and specific for detection of individual serotypes of pneumococci and may be used with respiratory tract specimens.
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Eltringham, I. J., S. M. Wilson, and F. A. Drobniewski. "Evaluation of a Bacteriophage-Based Assay (Phage Amplified Biologically Assay) as a Rapid Screen for Resistance to Isoniazid, Ethambutol, Streptomycin, Pyrazinamide, and Ciprofloxacin among Clinical Isolates of Mycobacterium tuberculosis." Journal of Clinical Microbiology 37, no. 11 (1999): 3528–32. http://dx.doi.org/10.1128/jcm.37.11.3528-3532.1999.
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Rapid molecular assays for the detection of mutations associated with rifampin resistance in Mycobacterium tuberculosis are commercially available. However, they are complex and expensive and have predictive values of 90 to 95%. Molecular assays for other drugs are less predictive of resistance. Ideally, assays based on phenotypic markers should be used for susceptibility testing, but these can take weeks to complete. We previously described a rapid phenotypic assay, the phage amplified biologically (PhaB) assay, for the rapid determination of rifampin and isoniazid susceptibility in clinical isolates of M. tuberculosis. In this study, we extended the assay to the study of ethambutol, pyrazinamide, streptomycin, and ciprofloxacin. After the optimization of antibiotic concentrations and incubation conditions, the assay was applied to each drug for a total of 157 isolates. The correlations between the results of the PhaB assay and the resistance ratio method were 94% for isoniazid, 96% for streptomycin, 100% for ciprofloxacin, 88% for ethambutol, and 87% for pyrazinamide. For ciprofloxacin, ethambutol, and pyrazinamide, significantly better correlations were found when a 90% reduction in plaque count was used as the cutoff. Turnaround times for the PhaB assay were 2 to 3 days, compared with 10 days for the resistance ratio method. We believe that this low-cost assay may have widespread applicability for the rapid screening of drug resistance in M. tuberculosis isolates, especially in developing countries.
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Schmid, D. Scott, Denise R. Brown, Rosane Nisenbaum, Rae Lyn Burke, D’Anna Alexander, Rhoda Ashley, Philip E. Pellett, and William C. Reeves. "Limits in Reliability of Glycoprotein G-Based Type-Specific Serologic Assays for Herpes Simplex Virus Types 1 and 2." Journal of Clinical Microbiology 37, no. 2 (1999): 376–79. http://dx.doi.org/10.1128/jcm.37.2.376-379.1999.
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Type-specific serologic assays for herpes simplex virus (HSV) types 1 and 2 based on glycoprotein G-1 (gG-1) (HSV-1) and gG-2 (HSV-2) discriminate between antibodies against HSV-1 and HSV-2. We previously developed a Western blot assay using gG-1 and gG-2 expressed in baculovirus, performed extensive validation studies, and determined that it was both sensitive and specific for type-specific detection of HSV antibody. Here we report that, among a cohort of Thai military recruits, the serostatus of some individuals changed from positive to negative over time (6.6% among those ever positive for HSV-1, and 14.9% among those ever positive for HSV-2). We tested a subset of these specimens in three other gG-based assays: an enzyme-linked immunosorbent assay, an immunoblot strip assay, and a Western blot assay. Positive-to-negative shifts occurred in every assay; the frequency of the shifts ranged from 6.1% to 21.2% of the specimen sets tested. There was only limited agreement among the assays concerning which individuals lost reactivity. This inaccuracy, exhibited by all of the assay protocols, was not predicted by validation studies employing specimens from cross-sectional studies and was most pronounced in HSV-2 testing. This argues for the inclusion of serial blood specimens in serologic assay validation procedures.
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Eis-Hübinger, Anna Maria, Martin Däumer, Bertfried Matz, and Karl Eduard Schneweis. "Evaluation of Three Glycoprotein G2-Based Enzyme Immunoassays for Detection of Antibodies to Herpes Simplex Virus Type 2 in Human Sera." Journal of Clinical Microbiology 37, no. 5 (1999): 1242–46. http://dx.doi.org/10.1128/jcm.37.5.1242-1246.1999.
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Three new glycoprotein G-based enzyme immunoassays (ETI-HSVK-G 2, Sorin Diagnostics Biomedica [assay A]; HSV Type 2 Specific IgG ELISA, Gull Laboratories, Inc. [assay B]; Cobas Core HSV-2 IgG EIA, Roche [assay C]) for the detection of herpes simplex virus (HSV) type 2 (HSV-2)-specific antibodies were evaluated. By testing sera from 25 individuals with culture-proven HSV-2 infection, the assays showed a sensitivity of 96%. The specificities, evaluated with sera from 70 HSV antibody-negative children, 75 HSV antibody-positive children, and 69 HSV antibody-negative adults, were 100% for assay A, 96.2% for assay B, and 97.8% for assay C, respectively. Discrepant results by any of the three assays, i.e., reactivity of a specimen in only one or two assays, occurred with similar frequencies for HSV-seronegative individuals as well as HSV-seropositive children and adults. For sera with discrepant results, the positive reactivity was mostly low. Thus, for determination of the prevalence of HSV-2 antibodies, only concordantly positive results were considered. On the basis of the results obtained with sera from 41 adults with culture-proven HSV-1 infection and from 173 HSV-antibody-positive pregnant women, the HSV-2 seroprevalence was 9.8%. The results show that the new glycoprotein G2-based enzyme immunoassays are useful tools for the detection of type-specific HSV-2 antibodies. However, if only one assay is performed, careful interpretation of the results is indicated, especially if the exhibited reactivity is low, and for determination of the definitive HSV-2 serostatus, confirmatory assays may still be necessary.
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Eberhardt, Kirsten Alexandra, Felix Dewald, Eva Heger, Lutz Gieselmann, Kanika Vanshylla, Maike Wirtz, Franziska Kleipass, et al. "Evaluation of a New Spike (S)-Protein-Based Commercial Immunoassay for the Detection of Anti-SARS-CoV-2 IgG." Microorganisms 9, no. 4 (March 31, 2021): 733. http://dx.doi.org/10.3390/microorganisms9040733.
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Background: The investigation of the antibody response to SARS-CoV-2 represents a key aspect in facing the COVID-19 pandemic. In the present study, we compared the new Immundiagnostik IDK® anti-SARS-CoV-2 S1 IgG assay with four widely-used commercial serological assays for the detection of antibodies targeting S (spike) and NC (nucleocapsid) proteins. Methods: Serum samples were taken from an unbiased group of convalescent patients and from a negative control group. Sample were simultaneously analyzed by the new Immundiagnostik IDK® anti-SARS-CoV-2 S1 IgG assay, by the DiaSorin LIAISON® SARS-CoV-2 S1/S2 IgG assay, and by the Euroimmun anti-SARS-CoV-2 S1 IgG ELISA. Antibodies binding NC were detected by the Abbott SARS-CoV-2 IgG assay and by the pan-immunoglobulin immunoassay Roche Elecsys® anti-SARS-CoV-2. Moreover, we investigated samples of a group of COVID-19 convalescent subjects that were primarily tested S1 IgG non-reactive. Samples were also tested by live virus and pseudovirus neutralization tests. Results: Overall, the IDK® anti-SARS-CoV-2 S1 IgG assay showed the highest sensitivity among the evaluated spike (S) protein-based assays. Additionally, the Immundiagnostik assay correlated well with serum-neutralizing activity. Conclusions: The novel IDK® anti-SARS-CoV-2 S1 IgG assay showed high sensitivity and specificity, representing a valid option for use in the routine diagnostic.
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Teoh, Boon-Teong, Sing-Sin Sam, Kim-Kee Tan, Mohammed Bashar Danlami, Meng-Hooi Shu, Jefree Johari, Poh-Sim Hooi, et al. "Early Detection of Dengue Virus by Use of Reverse Transcription-Recombinase Polymerase Amplification." Journal of Clinical Microbiology 53, no. 3 (January 7, 2015): 830–37. http://dx.doi.org/10.1128/jcm.02648-14.
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A method for the rapid diagnosis of early dengue virus (DENV) infection is highly needed. Here, a prototype reverse transcription-recombinase polymerase amplification (RT-RPA) assay was developed. The assay detected DENV RNA in <20 min without the need for thermocycling amplification. The assay enabled the detection of as few as 10 copies of DENV RNA. The designed RT-RPA primers andexoprobe detected the DENV genome of at least 12 genotypes of DENV circulating globally without cross-reacting with other arboviruses. We assessed the diagnostic performance of the RT-RPA assay for the detection of DENV RNA in 203 serum samples of patients with clinically suspected dengue. The sera were simultaneously tested for DENV using a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay, quantitative RT-PCR (qRT-PCR), and IgM- and IgG-capture enzyme-linked immunosorbent assays (ELISA). Acute DENV infection was confirmed in 130 samples and 61 of the samples (46.9%) were classified as viremic with qRT-PCR. The RT-RPA assay showed good concordance (κ of ≥0.723) with the RT-LAMP and qRT-PCR assays in detecting the dengue viremic samples. When used in combination with ELISA, both the RT-RPA and RT-LAMP assays increased the detection of acute DENV infection to ≥95.7% (≥45/47) in samples obtained within 5 days of illness. The results from the study suggest that the RT-RPA assay is the most rapid molecular diagnostic tool available for the detection of DENV. Hence, it is possible to use the RT-RPA assay in a laboratory to complement routine serology testing for dengue.
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Yek, Christina, Marta Massanella, Tashi Peling, Kristen Lednovich, Sangeetha V. Nair, Andrew Worlock, Milenka Vargas, et al. "Evaluation of the Aptima HIV-1 Quant Dx Assay for HIV-1 RNA Quantitation in Different Biological Specimen Types." Journal of Clinical Microbiology 55, no. 8 (June 7, 2017): 2544–53. http://dx.doi.org/10.1128/jcm.00425-17.
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ABSTRACTThe search for a cure for HIV infection has highlighted the need for increasingly sensitive and precise assays to measure viral burden in various tissues and body fluids. We describe the application of a standardized assay for HIV-1 RNA in multiple specimen types. The fully automated Aptima HIV-1 Quant Dx assay (Aptima assay) is FDA cleared for blood plasma HIV-1 RNA quantitation. In this study, the Aptima assay was applied for the quantitation of HIV RNA in peripheral blood mononuclear cells (PBMCs;n= 72), seminal plasma (n= 20), cerebrospinal fluid (CSF;n= 36), dried blood spots (DBS;n= 104), and dried plasma spots (DPS;n= 104). The Aptima assay was equivalent to or better than commercial assays or validated in-house assays for the quantitation of HIV RNA in CSF and seminal plasma. For PBMC specimens, the sensitivity of the Aptima assay in the detection of HIV RNA decayed as background uninfected PBMC counts increased; proteinase K treatment demonstrated some benefit in restoring signal at higher levels of background PBMCs. Finally, the Aptima assay yielded 100% detection rates of DBS in participants with plasma HIV RNA levels of ≥35 copies/ml and 100% detection rates of DPS in participants with plasma HIV RNA levels of ≥394 copies/ml. The Aptima assay can be applied to a variety of specimens from HIV-infected subjects to measure HIV RNA for studies of viral persistence and cure strategies. It can also detect HIV in dried blood and plasma specimens, which may be of benefit in resource-limited settings.
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Le Roy, Chloé, Sabine Pereyre, Nadège Hénin, and Cécile Bébéar. "French Prospective Clinical Evaluation of the Aptima Mycoplasma genitalium CE-IVD Assay and Macrolide Resistance Detection Using Three Distinct Assays." Journal of Clinical Microbiology 55, no. 11 (August 9, 2017): 3194–200. http://dx.doi.org/10.1128/jcm.00579-17.
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ABSTRACTThe aim of this study was to evaluate the clinical performance of the AptimaMycoplasma genitaliumtranscription-mediated amplification (MG-TMA) CE-marked forin vitrodiagnosis (CE-IVD) assay for the detection ofMycoplasma genitaliumin male and female clinical samples in comparison with the in-house real-time PCR (in-house PCR) assay routinely used in our laboratory. A total of 1,431 clinical specimens obtained from 1,235 patients were prospectively collected at the Bacteriology Department of Bordeaux University Hospital (France). Additional research-use-only AptimaM. genitaliumtranscription-mediated amplification (TMA) assays, Alt1-TMA and Alt2-TMA, were performed on discordant specimens to determineM. genitaliuminfection status. All confirmedM. genitalium-positive specimens were tested for macrolide resistance using three assays: the in-house 23S rRNA FRET PCR assay, the SpeeDx ResistancePlus MG assay and the nested reverse transcription-PCR (RT-PCR) sequencing assay. The comparison of the MG-TMA assay with the in-house PCR results showed a moderate correlation (kappa value, 0.69). The MG-TMA assay had higher clinical sensitivity compared to that of the in-house PCR assay (100% versus 59.74%, respectively) and similar specificity (99.10% versus 100%, respectively) forM. genitaliumdetection. In this study, the prevalence ofM. genitaliuminfection was 5.90% (72/1,220 patients). The nested RT-PCR sequencing assay was the most sensitive but the most laborious assay for detecting macrolide-resistance-associated mutations. The prevalence of resistance was 8.33% (6/72). To our knowledge, this is the first clinical evaluation of the MG-TMA CE-IVD assay. The MG-TMA assay performed on the automated Panther system is a very sensitive and specific method for the detection ofM. genitaliumin clinical specimens.
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Laperche, Syria, Silvia Sauleda, Maria Piron, Annelies Mühlbacher, Harald Schennach, Volkmar Schottstedt, Lucinda Queirós, et al. "Evaluation of Sensitivity and Specificity Performance of Elecsys HTLV-I/II Assay in a Multicenter Study in Europe and Japan." Journal of Clinical Microbiology 55, no. 7 (May 3, 2017): 2180–87. http://dx.doi.org/10.1128/jcm.00169-17.
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ABSTRACTScreening of blood for human T-cell lymphotropic virus type 1 and type 2 (HTLV-1 and -2, respectively) is important to diagnose and prevent infection and ensure the safety of blood supplies. The Elecsys HTLV-I/II assay is a newly developed, electrochemiluminescence screening assay for the detection of HTLV-1/2 infection. The sensitivity and specificity of the Elecsys HTLV-I/II assay were determined using well-characterized HTLV-1/2-positive serum and plasma samples and routine diagnostic and blood donor samples expected to be HTLV negative, respectively. These results were compared with those for at least one of the following CE-marked assays at seven independent laboratories and the Roche Diagnostics facility in Penzberg, Germany: Abbott Architect rHTLV-I/II, Ortho Avioq HTLV-I/II Microelisa system, Abbott Prism HTLV-I/HTLV-II, and DiaSorin Murex HTLV I+II. Fujirebio INNO-LIA HTLV-I/II Score was used as a confirmatory assay. The Elecsys HTLV-I/II, Abbott Architect rHTLV-I/II, and Abbott Prism HTLV-I/HTLV-II assays detected all HTLV-1/2-positive samples (sensitivity, 100%). Sensitivity for Ortho Avioq HTLV-I/II was 98.63%. The Elecsys HTLV-I/II assay had a specificity of 99.95% in blood donor samples, which was comparable to results for the other assays (range, 99.91 to 100%). In routine diagnostic samples, the specificity of the Elecsys HTLV-I/II assay was 99.83%, compared with 99.70% for Abbott Architect rHTLV-I/II. Specificity for the Elecsys HTLV-I/II assay in potentially cross-reactive samples was 100%, compared with 99.0% for Ortho Avioq HTLV-I/II and 99.2% for DiaSorin Murex HTLV I+II. The Elecsys HTLV-I/II assay has the sensitivity and specificity to support its use as a routine screening assay for detecting HTLV infection.
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Pas, Suzan D., Edwin Fries, Robert A. De Man, Albert D. M. E. Osterhaus, and Hubert G. M. Niesters. "Development of a Quantitative Real-Time Detection Assay for Hepatitis B Virus DNA and Comparison with Two Commercial Assays." Journal of Clinical Microbiology 38, no. 8 (2000): 2897–901. http://dx.doi.org/10.1128/jcm.38.8.2897-2901.2000.
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A highly reproducible and sensitive real-time detection assay based on TaqMan technology was developed for the detection of hepatitis B virus (HBV) DNA and compared with two commercially available assays. The assay was validated with the Viral Quality Control panel, which also includes EUROHEP HBV DNA standards. This real-time PCR detection system had a dynamic range of 373 to 1010 genome copies per ml and showed an excellent correlation with both the commercial HBV Digene Hybrid Capture II microplate assay (Digene Diagnostics) and the HBV MONITOR assay (Roche Diagnostics). To demonstrate its clinical utility, four chronically HBV-infected patients treated with lamuvidine were monitored using the three different assays. From the results we concluded that this assay is an excellent alternative for monitoring of HBV-infected patients in routine diagnostics and clinical practice, enabling the analysis of a large dynamic range of HBV DNA in a single, undiluted sample.
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Lai, Vicky C. H., Richard Guan, Michael L. Wood, Su Kong Lo, Man-Fung Yuen, and Ching-Lung Lai. "Nucleic Acid-Based Cross-Linking Assay for Detection and Quantification of Hepatitis B Virus DNA." Journal of Clinical Microbiology 37, no. 1 (1999): 161–64. http://dx.doi.org/10.1128/jcm.37.1.161-164.1999.
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A nucleic acid photo-cross-linking technology was used to develop a direct assay for the quantification of hepatitis B virus (HBV) DNA levels in serum. Cross-linker-modified DNA probes complementary to the viral genomes of the major HBV subtypes were synthesized and used in an assay that could be completed in less than 6 h. The quantification range of the assay, as determined by testing serial dilutions of Eurohep HBV reference standards and cloned HBV DNA, was 5 × 105 to 3 × 109 molecules of HBV DNA/ml of serum. Within-run and between-run coefficients of variation (CVs) for the assay were 4.3 and 4.0%, respectively. The assay was used to determine HBV DNA levels in 302 serum samples, and the results were compared to those obtained after testing the same samples with the Chiron branched-DNA (bDNA) assay for HBV DNA. Of the samples tested, 218 were positive for HBV DNA by both assays and 72 gave results below the cutoff for both assays. Of the remaining 12 samples, 10 were positive for HBV DNA by the cross-linking assay only; the 2 other samples were positive by the bDNA assay only. Twenty-eight samples had to be retested by the bDNA assay (CV, >20% between the results obtained from the testing of each sample in duplicate), whereas only three samples required retesting by the cross-linking assay. The correlation between the HBV DNA levels, as measured by the two tests, was very high (r = 0.902; P = 0.01). We conclude that the cross-linking assay is a sensitive and reproducible method for the detection and quantification of HBV DNA levels in serum.
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van der Giessen, M., T. H. The, and W. J. van Son. "Cytomegalovirus antigenemia assay." Journal of Clinical Microbiology 29, no. 12 (1991): 2909–10. http://dx.doi.org/10.1128/jcm.29.12.2909-2910.1991.
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Myers, L. E., L. J. McQuay, and F. B. Hollinger. "Dilution assay statistics." Journal of Clinical Microbiology 32, no. 3 (1994): 732–39. http://dx.doi.org/10.1128/jcm.32.3.732-739.1994.
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Erice, Alejo, Donald Brambilla, James Bremer, J. Brooks Jackson, Robert Kokka, Belinda Yen-Lieberman, and Robert W. Coombs. "Performance Characteristics of the QUANTIPLEX HIV-1 RNA 3.0 Assay for Detection and Quantitation of Human Immunodeficiency Virus Type 1 RNA in Plasma." Journal of Clinical Microbiology 38, no. 8 (2000): 2837–45. http://dx.doi.org/10.1128/jcm.38.8.2837-2845.2000.
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The QUANTIPLEX HIV-1 RNA assay, version 3.0 (a branched DNA, version 3.0, assay [bDNA 3.0 assay]), was evaluated by analyzing spiked and clinical plasma samples and was compared with the AMPLICOR HIV-1 MONITOR Ultrasensitive (ultrasensitive reverse transcription-PCR [US-RT-PCR]) method. A panel of spiked plasma samples that contained 0 to 750,000 copies of human immunodeficiency virus type 1 (HIV-1) RNA per ml was tested four times in each of four laboratories (1,344 assays). Negative results (<50 copies/ml) were obtained in 30 of 32 (94%) assays with seronegative samples, 66 of 128 (52%) assays with HIV-1 RNA at 50 copies/ml, and 5 of 128 (4%) assays with HIV-1 RNA at 100 copies/ml. The assay was linear from 100 to 500,000 copies/ml. The within-run standard deviation (SD) of the log10 estimated HIV-1 RNA concentration was 0.08 at 1,000 to 500,000 copies/ml, increased below 1,000 copies/ml, and was 0.17 at 100 copies/ml. Between-run reproducibility at 100 to 500 copies/ml was <0.10 log10 in most comparisons. Interlaboratory differences across runs were ≤0.10 log10 at all concentrations examined. A subset of the panel (25 to 500 copies/ml) was also analyzed by the US-RT-PCR assay. The within-run SD varied inversely with the log10 HIV-1 RNA concentration but was higher than the SD for the bDNA 3.0 assay at all concentrations. Log-log regression analysis indicated that the two methods produced very similar estimates at 100 to 500 copies/ml. In parallel testing of clinical specimens with low HIV-1 RNA levels, 80 plasma samples with <50 copies/ml by the US-RT-PCR assay had <50 copies/ml when they were retested by the bDNA 3.0 assay. In contrast, 11 of 78 (14%) plasma samples with <50 copies/ml by the bDNA 3.0 assay had ≥50 copies/ml when they were retested by the US-RT-PCR assay (median, 86 copies/ml; range, 50 to 217 copies/ml). Estimation of bDNA 3.0 values of <50 copies/ml by extending the standard curve of the assay showed that these samples with discrepant results had higher HIV-1 RNA levels than the samples with concordant results (median, 34 versus 17 copies/ml;P = 0.0051 by the Wilcoxon two-sample test). The excellent reproducibility, broad linear range, and good sensitivity of the bDNA 3.0 assay make it a very attractive method for quantitation of HIV-1 RNA levels in plasma.
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Saez-Alquézar, Amadeo, Ester C. Sabino, Nanci Salles, Dalton F. Chamone, Frank Hulstaert, Hans Pottel, Erik Stoops, and Maan Zrein. "Serological Confirmation of Chagas' Disease by a Recombinant and Peptide Antigen Line Immunoassay: INNO-LIA Chagas." Journal of Clinical Microbiology 38, no. 2 (2000): 851–54. http://dx.doi.org/10.1128/jcm.38.2.851-854.2000.
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Although screening for Trypanosoma cruzi antibodies is mandatory in most South American countries, current tests are insensitive and have poor specificity. A recently optimized line immunoassay (the INNO-LIA Chagas assay) for the serological confirmation of Chagas' disease was evaluated at a large blood bank in São Paulo, Brazil. Sera from blood donors who reacted in at least one of three serological screening assays (n = 1,604) and who returned for a follow-up were retested, and the donors were interviewed to assess their epidemiological risk. The results obtained by the confirmatory assay evaluated in this study were compared to those obtained by the three different screening assays. Upon consideration of the consensus results obtained by the three different screening assays as a “gold standard,” the INNO-LIA Chagas assay showed a sensitivity of 99.4% (95% confidence interval [CI], 98.3 to 99.9) and a specificity of 98.1% (95% CI, 96.6 to 99.0) for positive (n = 503) and negative (n = 577) sera. The INNO-LIA Chagas assay confirmed the results for significantly larger numbers of positive samples of at-risk individuals independent of the number of positive screening tests (P = 0.017, Mantel-Haenszel test). In conclusion, the INNO-LIA Chagas assay reliably confirmed the presence of antibodies toT. cruzi and can be implemented as a confirmatory assay for Chagas' disease serology.
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Huh, Hee Jae, Ji-Youn Kim, Hyeon Jeong Kwon, Sun Ae Yun, Myoung-Keun Lee, Nam Yong Lee, Jong-Won Kim, and Chang-Seok Ki. "Performance Evaluation of Allplex Respiratory Panels 1, 2, and 3 for Detection of Respiratory Viruses and Influenza A Virus Subtypes." Journal of Clinical Microbiology 55, no. 2 (November 30, 2016): 479–84. http://dx.doi.org/10.1128/jcm.02045-16.
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ABSTRACTThe Allplex respiratory panels 1, 2, and 3 (Allplex) comprise a one-step real-time reverse transcription-PCR assay for the detection of respiratory viruses (RVs) and influenza A subtypes based on multiple detection temperature (MuDT) technology. The performance of the Allplex assay was compared with those of the AdvanSure RV real-time PCR kit (AdvanSure) and the PowerChek pandemic H1N1/H3N2/H5N1 real-time PCR kit (PowerChek) using 417 clinical respiratory specimens. In comparison with the AdvanSure assay for RV detection by each virus, the ranges of positive percent agreement, negative percent agreement, and kappa values with the Allplex assay were 82.8 to 100%, 95.5 to 100%, and 0.85 to 1.00, respectively. For influenza A virus (INF A) subtyping, the kappa values between the Allplex and PowerChek assays were 0.67 and 1.00 for the INF A H1N1-pdm09 and H3 subtypes, respectively. Uniplex PCR and sequencing for samples with discrepant results demonstrated that the majority of results were concordant with those from the Allplex assay. When testing 24 samples, the turnaround and hands-on time required to perform the Allplex assay were 4 h 15 min and 15 min, respectively. In conclusion, the Allplex assay produced results comparable to those from the AdvanSure and PowerChek assays.
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Santos, Nathally C. de S., Regiane B. de L. Scodro, Dayane CB Leal, Silvia MT do Prado, Daniela F. Micheletti, Eloísa G. Sampiron, Giovana F. Costacurta, et al. "Determination of minimum bactericidal concentration, in single or combination drugs, against Mycobacterium tuberculosis." Future Microbiology 15, no. 2 (January 2020): 107–14. http://dx.doi.org/10.2217/fmb-2019-0050.
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Aim: To evaluate an assay to detect minimum bactericidal concentration (MBC) in Mycobacterium tuberculosis, using as single model rifampicin, isoniazid, levofloxacin (LVX) and linezolid (LNZ) and in combination. Material & methods: MBCs were carried out directly from resazurin microtiter assay plate and 3D checkerboard in M. tuberculosis H37Rv and five resistant clinical isolates. Results: The proposed MBC assay showed similar values to those determined by MGIT™, used as control. LVX and LNZ's MBC values were close to their MIC values. LNZ or LVX combined with isoniazid and rifampicin showed MBC value reduced in 63.7% of the assays. Conclusion: The proposed assay to determine MBCs of drugs can be applied to the study of new compounds with anti- M. tuberculosis activity to detect their bactericidal effect and also in laboratory routine for clinical dose adjustment of drugs according to the patient's profile.
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Kakumanu, Madhavi L., Loganathan Ponnusamy, Haley T. Sutton, Steven R. Meshnick, William L. Nicholson, and Charles S. Apperson. "Development and Validation of an Improved PCR Method Using the 23S-5S Intergenic Spacer for Detection of Rickettsiae in Dermacentor variabilis Ticks and Tissue Samples from Humans and Laboratory Animals." Journal of Clinical Microbiology 54, no. 4 (January 27, 2016): 972–79. http://dx.doi.org/10.1128/jcm.02605-15.
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A novel nested PCR assay was developed to detectRickettsiaspp. in ticks and tissue samples from humans and laboratory animals. Primers were designed for the nested run to amplify a variable region of the 23S-5S intergenic spacer (IGS) ofRickettsiaspp. The newly designed primers were evaluated using genomic DNA from 11Rickettsiaspecies belonging to the spotted fever, typhus, and ancestral groups and, in parallel, compared to otherRickettsia-specific PCR targets (ompA,gltA, and the 17-kDa protein gene). The new 23S-5S IGS nested PCR assay amplified all 11Rickettsiaspp., but the assays employing other PCR targets did not. The novel nested assay was sensitive enough to detect one copy of a cloned 23S-5S IGS fragment from “CandidatusRickettsia amblyommii.” Subsequently, the detection efficiency of the 23S-5S IGS nested assay was compared to those of the other three assays using genomic DNA extracted from 40 adultDermacentor variabilisticks. The nested 23S-5S IGS assay detectedRickettsiaDNA in 45% of the ticks, while the amplification rates of the other three assays ranged between 5 and 20%. The novel PCR assay was validated using clinical samples from humans and laboratory animals that were known to be infected with pathogenic species ofRickettsia. The nested 23S-5S IGS PCR assay was coupled with reverse line blot hybridization with species-specific probes for high-throughput detection and simultaneous identification of the species ofRickettsiain the ticks. “CandidatusRickettsia amblyommii,”R. montanensis,R. felis, andR. belliiwere frequently identified species, along with some potentially novelRickettsiastrains that were closely related toR. belliiandR. conorii.
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Beebe, Deborah P., Patrick J. Gaffney, and D. M. V. van Schie. "Potency assays for anistreplase: Comparison of the fibrin plate assay and a 96-well plate assay." Biologicals 20, no. 2 (June 1992): 129–33. http://dx.doi.org/10.1016/s1045-1056(05)80060-0.
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RYAN, GINA, SHERRY ROOF, LAURIE POST, and MARTIN WIEDMANN. "Evaluation of Rapid Molecular Detection Assays for Salmonella in Challenging Food Matrices at Low Inoculation Levels and Using Difficult-to-Detect Strains." Journal of Food Protection 78, no. 9 (September 1, 2015): 1632–41. http://dx.doi.org/10.4315/0362-028x.jfp-15-098.
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Assays for detection of foodborne pathogens are generally initially evaluated for performance in validation studies carried out according to guidelines provided by validation schemes (e.g., AOAC International or the International Organization for Standardization). End users often perform additional validation studies to evaluate the performance of assays in specific matrices (e.g., specific foods or raw material streams of interest) and with specific pathogen strains. However, these types of end-user validations are typically not well defined. This study was conducted to evaluate a secondary end user validation of four AOAC-validated commercial rapid detection assays (an isothermal nucleic acid amplification, an immunoassay, and two PCR-based assays) for their ability to detect Salmonella in two challenging matrices (dry pet food and dark chocolate). Inclusivity was evaluated with 68 diverse Salmonella strains at low population levels representing the limit of detection (LOD) for each assay. One assay detected all strains at the LOD, two assays detected multiple strains only at 10 times the LOD, and the fourth assay failed to detect two strains (Salmonella bongori and S. enterica subsp. houtenae) even at 1,000 times the LOD; this assay was not further evaluated. The three remaining assays were subsequently evaluated for their ability to detect five selected Salmonella strains in food samples contaminated at fractional levels. Unpaired comparisons revealed no significant difference between the results for each given assay and the results obtained with the reference assay. However, analysis of paired culture-confirmed results revealed assay false-negative rates of 4 to 26% for dry pet food and 12 to 16% for dark chocolate. Overall, our data indicate that rapid assays may have high false-negative rates when performance is evaluated under challenging conditions, including low-moisture matrices, strains that are difficult to detect, injured cells, and low inoculum levels.
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Vincart, Benoit, Ricardo De Mendonça, Sylvianne Rottiers, Françoise Vermeulen, Marc J. Struelens, and Olivier Denis. "A specific real-time PCR assay for the detection of Bordetella pertussis." Journal of Medical Microbiology 56, no. 7 (July 1, 2007): 918–20. http://dx.doi.org/10.1099/jmm.0.46947-0.
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A novel real-time PCR (RT-PCR) assay was developed for detection of Bordetella pertussis in respiratory specimens by targeting the pertactin gene. In vitro evaluation with reference strains and quality control samples showed analytical sensitivity equivalent to and specificity superior to those of PCR assays which target the IS481 element. The pertactin-based RT-PCR assay offers better discrimination between B. pertussis and other Bordetella species than previously described assays.
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Tong, C. Y. William, Luis E. Cuevas, Helen Williams, and Ali Bakran. "Comparison of Two Commercial Methods for Measurement of Cytomegalovirus Load in Blood Samples after Renal Transplantation." Journal of Clinical Microbiology 38, no. 3 (2000): 1209–13. http://dx.doi.org/10.1128/jcm.38.3.1209-1213.2000.
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A cohort of 77 renal transplant recipients was prospectively studied for comparison of two commercially available cytomegalovirus (CMV) load assays, i.e., the COBAS AMPLICOR CMV Monitor test (Amplicor), using plasma samples, and the Murex Hybrid Capture System (HCS), using whole blood. The manufacturer of the HCS assay changed the version of the test from 1.0 (HCS-1) to 2.0 (HCS-2) after the first 37 patients had been tested. Despite the differences in principle and type of specimen used, the Amplicor and HCS assays gave comparable results. The regression line correlating the HCS-1 assay to the Amplicor assay was similar to that correlating the HCS-2 assay to the Amplicor assay. The HCS results could be converted to Amplicor-equivalent units by using linear-regression equations [log10 HCS-1 result = 0.49 (log10 Amplicor result) + 2.58, and log10 HCS-2 result = 0.61 (log10 Amplicor result) + 2.18]. The HCS-2 assay appeared to have the lowest detection limit, followed by the Amplicor assay and then the HCS-1 assay. When a sliding scale of cutoff values in Amplicor-equivalent units (>1,000, >2,500, >6,000, >16,000, >40,000, and >100,000 copies/ml) was applied to diagnose CMV disease, similar patterns of sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were observed with the Amplicor and HCS assays. A cutoff value of >40,000 copies/ml has a low sensitivity (Amplicor, 29.4%; HCS, 41.2%) but is specific (Amplicor, 96.7%; HCS, 93.3%) and can be used for the differential diagnosis of CMV disease (PPV, 71.4% [Amplicor] or 63.6% [HCS]; NPV, 82.9% [Amplicor] or 84.8% [HCS]). A lower cutoff value of >1,000 copies/ml improves the sensitivity (Amplicor, 76.5%; HCS, 82.4%) and has a high NPV (Amplicor, 91.8%; HCS, 94.2%) but, due to the low PPV (Amplicor, 46.2%; HCS, 56%), is useful only for exclusion of CMV disease.
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Best, Susan J., Anthony P. Gust, Elizabeth I. M. Johnson, Catherine H. McGavin, and Elizabeth M. Dax. "Quality of Human Immunodeficiency Virus Viral Load Testing in Australia." Journal of Clinical Microbiology 38, no. 11 (2000): 4015–20. http://dx.doi.org/10.1128/jcm.38.11.4015-4020.2000.
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This study determined the proficiencies of laboratories measuring human immunodeficiency virus type 1 (HIV-1) viral loads and the accuracies of two assays used for HIV-1 viral load measurement in Australia and investigated the variability of the new versions of these assays. Quality assessment program panels containing (i) dilutions of HIV-1 subtype B, (ii) replicates of identical samples of HIV-1 subtype B, and (iii) samples of subtype E and B were tested by laboratories. Total variability (within and between laboratories) was tested with quality control samples. The coefficients of variation (CVs) for the Roche AMPLICOR HIV-1 MONITOR version (v) 1.0 and Chiron Quantiplex bDNA 2.0 assays ranged from 53 to 87% and 22 to 31%, respectively. The widespread occurrence of invalid runs with the AMPLICOR HIV-1 MONITOR 1.0 assay was identified. The CVs of the new versions of the assays were 82 to 86% for the AMPLICOR HIV-1 MONITOR v 1.5 assay and 16 to 23% for the Quantiplex bDNA 3.0 assay. For virus dilution samples, all but 5 of 19 laboratories obtained results within 2 standard deviations of the mean. The Quantiplex bDNA 2.0 assay reported values lower than those reported by the AMPLICOR HIV-1 MONITOR version 1.0 assay for samples containing HIV-1 subtype B, whereas the reverse was true for subtype E. Identification and resolution of the problem of invalid runs markedly improved the quality of HIV-1 viral load testing. The variability observed between laboratories and between assays, even the most recent versions, dictates that monitoring of viral load in an individual should always be by the same laboratory and by the same assay. Results for an individual which differ by less than 0.5 log10 HIV-1 RNA copy number/ml should not be considered clinically significant.
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Chae, Hansong, Seung Jung Han, Su-Young Kim, Chang-Seok Ki, Hee Jae Huh, Dongeun Yong, Won-Jung Koh, and Sung Jae Shin. "Development of a One-Step Multiplex PCR Assay for Differential Detection of Major Mycobacterium Species." Journal of Clinical Microbiology 55, no. 9 (June 28, 2017): 2736–51. http://dx.doi.org/10.1128/jcm.00549-17.
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ABSTRACTThe prevalence of tuberculosis continues to be high, and nontuberculous mycobacterial (NTM) infection has also emerged worldwide. Moreover, differential and accurate identification of mycobacteria to the species or subspecies level is an unmet clinical need. Here, we developed a one-step multiplex PCR assay using whole-genome analysis and bioinformatics to identify novel molecular targets. The aims of this assay were to (i) discriminate between theMycobacterium tuberculosiscomplex (MTBC) and NTM usingrv0577or RD750, (ii) differentiateM. tuberculosis(M. tuberculosis) from MTBC using RD9, (iii) selectively identify the widespreadM. tuberculosisBeijing genotype by targetingmtbk_20680, and (iv) simultaneously detect five clinically important NTM (M. avium,M. intracellulare,M. abscessus,M. massiliense, andM. kansasii) by targeting IS1311, DT1,mass_3210, andmkan_rs12360. An initial evaluation of the multiplex PCR assay using reference strains demonstrated 100% specificity for the targetedMycobacteriumspecies. Analytical sensitivity ranged from 1 to 10 pg for extracted DNA and was 103and 104CFU for pure cultures and nonhomogenized artificial sputum cultures, respectively, of the targeted species. The accuracy of the multiplex PCR assay was further evaluated using 55 reference strains and 94 mycobacterial clinical isolates. Spoligotyping, multilocus sequence analysis, and a commercial real-time PCR assay were employed as standard assays to evaluate the multiplex PCR assay with clinicalM. tuberculosisand NTM isolates. The PCR assay displayed 100% identification agreement with the standard assays. Our multiplex PCR assay is a simple, convenient, and reliable technique for differential identification of MTBC,M. tuberculosis,M. tuberculosisBeijing genotype, and major NTM species.
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Favresse, Julien, Christine Eucher, Marc Elsen, Constant Gillot, Sandrine Van Eeckhoudt, Jean-Michel Dogné, and Jonathan Douxfils. "Persistence of Anti-SARS-CoV-2 Antibodies Depends on the Analytical Kit: A Report for Up to 10 Months after Infection." Microorganisms 9, no. 3 (March 8, 2021): 556. http://dx.doi.org/10.3390/microorganisms9030556.
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Several studies have described the long-term kinetics of anti-SARS-CoV-2 antibodies but long-term follow-up data, i.e., >6 months, are still sparse. Additionally, the literature is inconsistent regarding the waning effect of the serological response. The aim of this study was to explore the temporal dynamic changes of the immune response after SARS-CoV-2 infection in hospitalized and non-hospitalized symptomatic patients over a period of 10 months. Six different analytical kits for SARS-CoV-2 antibody detection were used. Positivity rates, inter-assay agreement and kinetic models were determined. A high inter-individual and an inter-methodology variability was observed. Assays targeting total antibodies presented higher positivity rates and reached the highest positivity rates sooner compared with assays directed against IgG. The inter-assay agreement was also higher between these assays. The stratification by disease severity showed a much-elevated serological response in hospitalized versus non-hospitalized patients in all assays. In this 10-month follow-up study, serological assays showed a clinically significant difference to detect past SARS-CoV-2 infection with total antibody assays presenting the highest positivity rates. The waning effect reported in several studies should be interpreted with caution because it could depend on the assay considered.
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Schlaberg, Robert, Michael J. Mitchell, Edward W. Taggart, and Rosemary C. She. "Verification of Performance Specifications for a US Food and Drug Administration–Approved Molecular Microbiology Test: Clostridium difficile Cytotoxin B Using the Becton, Dickinson and Company GeneOhm Cdiff Assay." Archives of Pathology & Laboratory Medicine 136, no. 1 (January 1, 2012): 20–25. http://dx.doi.org/10.5858/arpa.2011-0138-oa.
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Context.—US Food and Drug Administration (FDA)–approved diagnostic tests based on molecular genetic technologies are becoming available for an increasing number of microbial pathogens. Advances in technology and lower costs have moved molecular diagnostic tests formerly performed for research purposes only into much wider use in clinical microbiology laboratories. Objective.—To provide an example of laboratory studies performed to verify the performance of an FDA-approved assay for the detection of Clostridium difficile cytotoxin B compared with the manufacturer's performance standards. Design.—We describe the process and protocols used by a laboratory for verification of an FDA-approved assay, assess data from the verification studies, and implement the assay after verification. Results.—Performance data from the verification studies conducted by the laboratory were consistent with the manufacturer's performance standards and the assay was implemented into the laboratory's test menu. Conclusion.—Verification studies are required for FDA-approved diagnostic assays prior to use in patient care. Laboratories should develop a standardized approach to verification studies that can be adapted and applied to different types of assays. We describe the verification of an FDA-approved real-time polymerase chain reaction assay for the detection of a toxin gene in a bacterial pathogen.
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Wattanamano, Pornthep, John L. Clayton, Jeffrey J. Kopicko, Patricia Kissinger, Steven Elliot, Christine Jarrott, Setlur Rangan, and Mark A. Beilke. "Comparison of Three Assays for Cytomegalovirus Detection in AIDS Patients at Risk for Retinitis." Journal of Clinical Microbiology 38, no. 2 (2000): 727–32. http://dx.doi.org/10.1128/jcm.38.2.727-732.2000.
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The purpose of this study was to determine the sensitivity and specificity of three different methods of cytomegalovirus (CMV) detection for AIDS patients at risk for CMV retinitis. Patients with CD4+ counts of <100/μl and negative baseline screening eye examinations were tested for CMV infection by (i) pp65 antigenemia expression in leukocytes, (ii) the Digene Hybrid Capture CMV DNA System, and (iii) the Roche Amplicor Qualitative PCR Test. The incidence of CMV retinitis in our study of 296 patients at the Medical Center of Louisiana—New Orleans HIV Outpatient Clinic was 7.2 per 100 person-years (a total of 20 episodes in 18 patients from April 1997 to February 1999). Receiver operating characteristic curves were calculated for each assay to determine optimal cutoff points which maximized the sensitivity and specificity of each assay. The sensitivities of the assays compared to the eye examinations were 80% for the pp65 antigenemia assay (cutoff, >0 cell per 1.5 × 105 leukocytes), 85% for the Digene assay (cutoff, 1,400 genome copies/ml of whole blood), and 60% for the Amplicor assay. The specificities of the assays were 84, 84, and 87%, respectively. The Digene assay with a cutoff of ≥1,400 genome copies/ml gave optimal sensitivity and specificity and was found to have predictive values equal to those of the more technically cumbersome antigenemia assay.
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Pankhurst, Louise, Louissa Macfarlane-Smith, James Buchanan, Luke Anson, Kerrie Davies, Lily O’Connor, Helen Ashwin, et al. "Can rapid integrated polymerase chain reaction-based diagnostics for gastrointestinal pathogens improve routine hospital infection control practice? A diagnostic study." Health Technology Assessment 18, no. 53 (August 2014): 1–167. http://dx.doi.org/10.3310/hta18530.
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BackgroundEvery year approximately 5000–9000 patients are admitted to a hospital with diarrhoea, which in up to 90% of cases has a non-infectious cause. As a result, single rooms are ‘blocked’ by patients with non-infectious diarrhoea, while patients with infectious diarrhoea are still in open bays because of a lack of free side rooms. A rapid test for differentiating infectious from non-infectious diarrhoea could be very beneficial for patients.ObjectiveTo evaluate MassCode multiplex polymerase chain reaction (PCR) for the simultaneous diagnosis of multiple enteropathogens directly from stool, in terms of sensitivity/specificity to detect four common important enteropathogens:Clostridium difficile,Campylobacterspp.,Salmonellaspp. and norovirus.DesignA retrospective study of fixed numbers of samples positive forC. difficile(n = 200),Campylobacterspp. (n = 200),Salmonellaspp. (n = 100) and norovirus (n = 200) plus samples negative for all these pathogens (n = 300). Samples were sourced from NHS microbiology laboratories in Oxford and Leeds where initial diagnostic testing was performed according to Public Health England methodology. Researchers carrying out MassCode assays were blind to this information. A questionnaire survey, examining current practice for infection control teams and microbiology laboratories managing infectious diarrhoea, was also carried out.SettingMassCode assays were carried out at Oxford University Hospitals NHS Trust. Further multiplex assays, carried out using Luminex, were run on the same set of samples at Leeds Teaching Hospitals NHS Trust. The questionnaire was completed by various NHS trusts.Main outcome measuresSensitivity and specificity to detectC. difficile,Campylobacterspp.,Salmonellaspp., and norovirus.ResultsNucleic acids were extracted from 948 clinical samples using an optimised protocol (200Campylobacterspp., 199C. difficile, 60S. enterica, 199 norovirus and 295 negative samples; some samples contained more than one pathogen). Using the MassCode assay, sensitivities for each organism compared with standard microbiological testing ranged from 43% to 94% and specificities from 95% to 98%, with particularly poor performance forS. enterica. Relatively large numbers of unexpected positives not confirmed with quantitative PCR were also observed, particularly forS. enterica,Giardia lambliaandCryptosporidiumspp. As the results indicated thatS. entericadetection might provide generic challenges to other multiplex assays for gastrointestinal pathogens, the Luminex xTag®gastrointestinal assay was also run blinded on the same extracts (937/948 remaining) and on re-extracted samples (839/948 with sufficient material). ForCampylobacterspp.,C. difficileand norovirus, high sensitivities (> 92%) and specificities (> 96%) were observed. ForS. enterica, on the original MassCode/Oxford extracts, Luminex sensitivity compared with standard microbiological testing was 84% [95% confidence interval (CI) 73% to 93%], but this dropped to 46% on a fresh extract, very similar to MassCode, with a corresponding increase in specificity from 92% to 99%. Overall agreement on the per-sample diagnosis compared with combined microbiology plus PCR for the main four/all pathogens was 85.6%/64.7%, 87.0%/82.9% and 89.8%/86.8% for the MassCode assay, Luminex assay/MassCode extract and Luminex assay/fresh extract, respectively. Luminex assay results from fresh extracts implied that 5% of samples did not represent infectious diarrhoea, even though enteropathogens were genuinely present. Managing infectious diarrhoea was a significant burden for infection control teams (taking 21% of their time) and better diagnostics were identified as having major potential benefits for patients.ConclusionsOverall, the Luminex xTag gastrointestinal panel showed similar or superior sensitivity and specificity to the MassCode assay. However, on fresh extracts, this test had low sensitivity to detect a key enteric pathogen,S. enterica; making it an unrealistic option for most microbiology laboratories. Extraction efficiency appears to be a major obstacle for nucleic acid-based tests for this organism, and possibly the whole Enterobacteriaceae family. To improve workflows in service microbiology laboratories, to reduce workload for infection control practitioners, and to improve outcomes for NHS patients, further research on deoxyribonucleic acid-based multiplex gastrointestinal diagnostics is urgently needed.FundingThe Health Technology Assessment programme of the National Institute for Health Research.
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Gade, Lalitha, Dale E. Grgurich, Thomas M. Kerkering, Mary E. Brandt, and Anastasia P. Litvintseva. "Utility of Real-Time PCR for Detection of Exserohilum rostratum in Body and Tissue Fluids during the Multistate Outbreak of Fungal Meningitis and Other Infections." Journal of Clinical Microbiology 53, no. 2 (December 17, 2014): 618–25. http://dx.doi.org/10.1128/jcm.02443-14.
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Exserohilum rostratumwas the major cause of the multistate outbreak of fungal meningitis linked to contaminated injections of methylprednisolone acetate produced by the New England Compounding Center. Previously, we developed a fungal DNA extraction procedure and broad-range andE. rostratum-specific PCR assays and confirmed the presence of fungal DNA in 28% of the case patients. Here, we report the development and validation of a TaqMan real-time PCR assay for the detection ofE. rostratumin body fluids, which we used to confirm infections in 57 additional case patients, bringing the total number of case patients with PCR results positive forE. rostratumto 171 (37% of the 461 case patients with available specimens). Compared to fungal culture and the previous PCR assays, this real-time PCR assay was more sensitive. Of the 139 identical specimens from case patients tested by all three methods, 19 (14%) were positive by culture, 41 (29%) were positive by the conventional PCR assay, and 65 (47%) were positive by the real-time PCR assay. We also compared the utility of the real-time PCR assay with that of the previously described beta-d-glucan (BDG) detection assay for monitoring response to treatment in case patients with serially collected CSF. Only the incident CSF specimens from most of the case patients were positive by real-time PCR, while most of the subsequently collected specimens were negative, confirming our previous observations that the BDG assay was more appropriate than the real-time PCR assay for monitoring the response to treatment. Our results also demonstrate that the real-time PCR assay is extremely susceptible to contamination and its results should be used only in conjunction with clinical and epidemiological data.
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LiPuma, John J., Betty Jo Dulaney, Jennifer D. McMenamin, Paul W. Whitby, Terrence L. Stull, Tom Coenye, and Peter Vandamme. "Development of rRNA-Based PCR Assays for Identification of Burkholderia cepacia Complex Isolates Recovered from Cystic Fibrosis Patients." Journal of Clinical Microbiology 37, no. 10 (1999): 3167–70. http://dx.doi.org/10.1128/jcm.37.10.3167-3170.1999.
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PCR assays targeting rRNA genes were developed to identify species (genomovars) within the Burkholderia cepacia complex. Each assay was tested with 177 bacterial isolates that also underwent taxonomic analysis by whole-cell protein profile. These isolates were from clinical and environmental sources and included 107 B. cepacia complex strains, 23 Burkholderia gladiolistrains, 20 Ralstonia pickettii strains, 10Pseudomonas aeruginosa strains, 8 Stenotrophomonas maltophilia strains, and 9 isolates belonging to nine other species. The sensitivity and specificity of the 16S rRNA-based assay for Burkholderia multivorans (genomovar II) were 100 and 99%, respectively; for Burkholderia vietnamiensis(genomovar V), sensitivity and specificity were 87 and 92%, respectively. An assay based on 16S and 23S rRNA gene analysis ofB. cepacia ATCC 25416 (genomovar I) was useful in identifying genomovars I, III, and IV as a group (sensitivity, 100%, and specificity, 99%). Another assay, designed to be specific at the genus level, identified all but one of the Burkholderia andRalstonia isolates tested (sensitivity, 99%, and specificity, 96%). The combined use of these assays offers a significant improvement over previously published PCR assays forB. cepacia.