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

2017 ◽  
Vol 11 (6) ◽  
pp. e0005506 ◽  
Author(s):  
Sergio Araujo ◽  
Luiz Ricardo Goulart ◽  
Richard W. Truman ◽  
Isabela Maria B. Goulart ◽  
Varalakshmi Vissa ◽  
...  
Keyword(s):  
High Resolution ◽  
Susceptibility Testing ◽  
Mycobacterium Leprae ◽  
Drug Susceptibility ◽  
Drug Susceptibility Testing ◽  
High Resolution Melt ◽  
Clinical Specimens ◽  
High Resolution Melt Analysis ◽  
Leprosy Patients

scholarly journals Integrated Microfluidic Card with TaqMan Probes and High-Resolution Melt Analysis To Detect Tuberculosis Drug Resistance Mutations across 10 Genes

mBio ◽  
2015 ◽  
Vol 6 (2) ◽  
Author(s):  
Suporn Pholwat ◽  
Jie Liu ◽  
Suzanne Stroup ◽  
Jean Gratz ◽  
Sayera Banu ◽  
...  
Keyword(s):  
High Resolution ◽  
Susceptibility Testing ◽  
Drug Susceptibility ◽  
Multidrug Resistant ◽  
Drug Susceptibility Testing ◽  
Resistance Mutations ◽  
High Resolution Melt ◽  
Content Type ◽  
High Resolution Melt Analysis ◽  
Taqman Array

ABSTRACT Genotypic methods for drug susceptibility testing of Mycobacterium tuberculosis are desirable to speed the diagnosis and proper therapy of tuberculosis (TB). However, the numbers of genes and polymorphisms implicated in resistance have proliferated, challenging diagnostic design. We developed a microfluidic TaqMan array card (TAC) that utilizes both sequence-specific probes and high-resolution melt analysis (HRM), providing two layers of detection of mutations. Twenty-seven primer pairs and 40 probes were designed to interrogate 3,200 base pairs of critical regions of the inhA, katG, rpoB, embB, rpsL, rrs, eis, gyrA, gyrB, and pncA genes. The method was evaluated on 230 clinical M. tuberculosis isolates from around the world, and it yielded 96.1% accuracy (2,431/2,530) in comparison to that of Sanger sequencing and 87% accuracy in comparison to that of the slow culture-based susceptibility testing. This TAC-HRM method integrates assays for 10 genes to yield fast, comprehensive, and accurate drug susceptibility results for the 9 major antibiotics used to treat TB and could be deployed to improve treatment outcomes. IMPORTANCE Multidrug-resistant tuberculosis threatens global tuberculosis control efforts. Optimal therapy utilizes susceptibility test results to guide individualized treatment regimens; however, the susceptibility testing methods in use are technically difficult and slow. We developed an integrated TaqMan array card method with high-resolution melt analysis that interrogates 10 genes to yield a fast, comprehensive, and accurate drug susceptibility result for the 9 major antituberculosis antibiotics.

scholarly journals Evaluation of the Abbott RealTi m e MTB and RealTi m e MTB INH/RIF Assays for Direct Detection of Mycobacterium tuberculosis Complex and Resistance Markers in Respiratory and Extrapulmonary Specimens

2016 ◽  
Vol 54 (12) ◽  
pp. 3022-3027 ◽  
Author(s):  
Sabine Hofmann-Thiel ◽  
Nikolay Molodtsov ◽  
Uladzimir Antonenka ◽  
Harald Hoffmann
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Susceptibility Testing ◽  
Drug Susceptibility ◽  
Drug Susceptibility Testing ◽  
Nucleic Acid Amplification ◽  
Content Type ◽  
Clinical Specimens ◽  
Different Types ◽  
Resistance Markers ◽  
Smear Negative

The Abbott RealTi m e MTB (RT MTB) assay is a new automated nucleic acid amplification test for the detection of Mycobacterium tuberculosis complex (MTBC) in clinical specimens. In combination with the RealTi m e MTB INH/RIF (RT MTB INH/RIF) resistance assay, which can be applied to RT MTB-positive specimens as an add-on assay, the tests also indicate the genetic markers of resistance to isoniazid (INH) and rifampin (RIF). We aimed to evaluate the diagnostic sensitivity and specificity of RT MTB using different types of respiratory and extrapulmonary specimens and to compare performance characteristics directly with those of the FluoroType MTB assay. The resistance results obtained by RT MTB INH/RIF were compared to those from the GenoType MTBDR plus and from phenotypic drug susceptibility testing. A total of 715 clinical specimens were analyzed. Compared to culture, the overall sensitivity of RT MTB was 92.1%; the sensitivity rates for smear-positive and smear-negative samples were 100% and 76.2%, respectively. The sensitivities of smear-negative specimens were almost identical for respiratory (76.3%) and extrapulmonary (76%) specimens. Specificity rates were 100% and 95.8% for culture-negative specimens and those that grew nontuberculous mycobacteria, respectively. RT MTB INH/RIF was applied to 233 RT MTB-positive samples and identified resistance markers in 7.7% of samples. Agreement with phenotypic and genotypic drug susceptibility testing was 99.5%. In conclusion, RT MTB and RT MTB INH/RIF allow for the rapid and accurate diagnosis of tuberculosis (TB) in different types of specimens and reliably indicate resistance markers. The strengths of this system are the comparably high sensitivity with paucibacillary specimens, its ability to detect INH and RIF resistance, and its high-throughput capacities.

scholarly journals Pyrazinamide susceptibility testing of Mycobacterium tuberculosis by high resolution melt analysis

Tuberculosis ◽  
2014 ◽  
Vol 94 (1) ◽  
pp. 20-25 ◽  
Author(s):  
Suporn Pholwat ◽  
Suzanne Stroup ◽  
Jean Gratz ◽  
Varittha Trangan ◽  
Suporn Foongladda ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
High Resolution ◽  
Susceptibility Testing ◽  
High Resolution Melt ◽  
High Resolution Melt Analysis

scholarly journals High-resolution melt curve analysis for rapid detection of rifampicin resistance in mycobacterium tuberculosis: A single-center study in Iran

2019 ◽  
Author(s):  
Samaneh Arefzadeh ◽  
Mohammad Javad Nasiri ◽  
Taher Azimi ◽  
Zahra Nikpor ◽  
Hossein Dabiri ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
High Resolution ◽  
Diagnostic Accuracy ◽  
Predictive Value ◽  
Drug Susceptibility ◽  
Drug Susceptibility Testing ◽  
Clinical Samples ◽  
High Resolution Melt ◽  
Cross Sectional ◽  
Risk Of Death

AbstractIntroductionTuberculosis (TB) remains a leading cause of death worldwide, especially in developing countries. Early detection of resistance is extremely important to reduce the risk of death. This study was aimed to compare the diagnostic accuracy of high resolution melting (HRM) analysis in comparison with Xpert MTB/RIF as well as conventional drug susceptibility testing (DST) for the detection of rifampicin (RIF) resistance in Mycobacterium tuberculosis in Iran.Materials and methodsA comparative cross-sectional study was carried out from April 2017 to September 2018. A total of 80 culture-positive clinical samples selected during the study period were analyzed for detection of RIF-resistant TB by conventional DST, Xpert MTB/RIF, and sequencing. Sensitivity and specificity of the HRM calculated according to DST as our gold standard test in this study.ResultsThe overall sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of HRM assay were found to be 100%, 89.33%, 38.46%, and 100% respectively.ConclusionsThe analysis has demonstrated that the diagnostic accuracy of HRM tests is insufficient to replace Xpert MTB/RIF and conventional DST. HRM test may be used in combination with culture due to the advantage of the time to result. Further work to improve molecular tests would benefit from standardized reference standards and the methodology.

scholarly journals Improved determination of Neisseria gonorrhoeae gyrase A genotype results in clinical specimens

2019 ◽  
Author(s):  
Lao-Tzu Allan-Blitz ◽  
Olivia L Ellis ◽  
Rachel Wee ◽  
Annie Truong ◽  
Samantha M Ebeyan ◽  
...  
Keyword(s):  
High Resolution ◽  
Neisseria Gonorrhoeae ◽  
Drug Resistant ◽  
High Resolution Melt ◽  
Clinical Specimens ◽  
High Resolution Melt Analysis ◽  
Gyrase A ◽  
Rapid Molecular Assays ◽  
Sydney Australia

Abstract Background The emergence of drug-resistant Neisseria gonorrhoeae has prompted the development of rapid molecular assays designed to determine antimicrobial susceptibility. One common assay uses high-resolution melt analysis to target codon 91 of the gyrase A gene (gyrA) to predict N. gonorrhoeae susceptibility to ciprofloxacin. Methods We extracted DNA from remnant clinical specimens that had previously tested positive for N. gonorrhoeae using the Aptima Combo 2 for CT/NG assay (Hologic, San Diego, CA, USA). We selected DNA extracts from specimens with indeterminate, WT and mutant gyrA genotype results from a previous study using high-resolution melt analysis to detect the gyrA codon 91 mutation. We re-tested those specimens using the recently CE-marked ResistancePlus GC (beta) assay (SpeeDx, Sydney, Australia). Results Of 86 specimens with indeterminate gyrA genotypes on high-resolution melt analysis, the ResistancePlus GC (beta) assay (SpeeDx) identified 30 (35%) WT, 22 (26%) mutant and 34 (40%) indeterminate gyrA genotypes. Conclusions The ResistancePlus GC (beta) assay showed improved N. gonorrhoeae gyrA genotype determination compared with a prior gyrA genotypic high-resolution melt assay.

Accuracy of molecular drug susceptibility testing amongst tuberculosis patients in Karakalpakstan, Uzbekistan

2021 ◽  
Author(s):  
Horacio Gil ◽  
Hasmik Margaryan ◽  
Ismailov Azamat ◽  
Bekturdieva Ziba ◽  
Halmuratov Bayram ◽  
...  
Keyword(s):  
Susceptibility Testing ◽  
Drug Susceptibility ◽  
Drug Susceptibility Testing ◽  
Tuberculosis Patients
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