Diagnostic Accuracy and Reproducibility of WHO-Endorsed Phenotypic Drug Susceptibility Testing Methods for First-Line and Second-Line Antituberculosis Drugs

Our objective was to establish reference MIC quality control (QC) ranges for drug susceptibility testing of antimycobacterials, including first-line agents, second-line injectables, fluoroquinolones, and World Health Organization category 5 drugs for multidrug-resistant tuberculosis using a 7H9 broth microdilution MIC method. A tier-2 reproducibility study was conducted in eight participating laboratories using Clinical Laboratory and Standards Institute (CLSI) guidelines. Three lots of custom-made frozen 96-well polystyrene microtiter plates were used and prepared with 2× prediluted drugs in 7H9 broth-oleic acid albumin dextrose catalase. The QC reference strain wasMycobacterium tuberculosisH37Rv. MIC frequency, mode, and geometric mean were calculated for each drug. QC ranges were derived based on predefined, strict CLSI criteria. Any data lying outside CLSI criteria resulted in exclusion of the entire laboratory data set. Data from one laboratory were excluded due to higher MIC values than other laboratories. QC ranges were established for 11 drugs: isoniazid (0.03 to 0.12 μg/ml), rifampin (0.03 to 0.25 μg/ml), ethambutol (0.25 to 2 μg/ml), levofloxacin (0.12 to 1 μg/ml), moxifloxacin (0.06 to 0.5 μg/ml), ofloxacin (0.25 to 2 μg/ml), amikacin (0.25 to 2 μg/ml), kanamycin (0.25 to 2 μg/ml), capreomycin (0.5 to 4 μg/ml), linezolid (0.25 to 2 μg/ml), and clofazimine (0.03 to 0.25 μg/ml). QC ranges could not be established for nicotinamide (pyrazinamide surrogate), prothionamide, or ethionamide, which were assay nonperformers. Using strict CLSI criteria, QC ranges against theM. tuberculosisH37Rv reference strain were established for the majority of commonly used antituberculosis drugs, with a convenient 7H9 broth microdilution MIC method suitable for use in resource-limited settings.

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Clinical evaluation of the Xpert MTB/XDR assay for rapid detection of isoniazid, fluoroquinolone, ethionamide and second-line drug resistance: A cross-sectional multicentre diagnostic accuracy study

10.1101/2021.05.06.21256505 ◽

2021 ◽

Author(s):

Adam Penn-Nicholson ◽

Sophia B Georghiou ◽

Nelly Ciobanu ◽

Mubin Kazi ◽

Manpreet Bhalla ◽

...

Keyword(s):

Drug Resistance ◽

Diagnostic Accuracy ◽

Susceptibility Testing ◽

Drug Susceptibility ◽

Drug Susceptibility Testing ◽

Foreign Affairs ◽

Second Line ◽

Reference Standard ◽

Composite Reference Standard ◽

Resistance Detection

Background The WHO End TB Strategy requires universal drug susceptibility testing and treatment of all people with tuberculosis. However, available second-line diagnostic tools are cumbersome and require sophisticated laboratory infrastructure, and ultimately less than half of those with drug-resistant tuberculosis receive appropriate treatment. Xpert MTB/XDR was developed to help overcome these limitations. Methods We assessed the diagnostic accuracy of sputum-based Xpert MTB/XDR for isoniazid, fluoroquinolone, ethionamide and second-line injectable resistance detection in adults with an Xpert MTB/RIF or Ultra Mycobacterium tuberculosis-positive result against a composite reference standard of phenotypic drug-susceptibility testing and whole genome sequencing (NCT03728725). Participants with pulmonary tuberculosis symptoms and ≥1 risk factor for drug resistance were consecutively enrolled between four clinical sites in India, Moldova and South Africa. Findings Between 31 July 2019 and 21 March 2020, we enrolled 710 patients, of which 611 (86.1%) had results from index and composite reference standard tests and were included in analysis. The sensitivity of Xpert MTB/XDR was 94% for isoniazid, 95% for fluoroquinolones, 54% for ethionamide, 73% for amikacin, 86% for kanamycin, and 61% for capreomycin resistance detection. Specificity was 98-100% for all drugs. Performance was equivalent to line-probe assays. The non-determinate rate of Xpert MTB/XDR was 2.96%. Interpretation This first prospective, multicentre clinical study of the Xpert MTB/XDR assay demonstrated high diagnostic test accuracy, meeting target product profile criteria for a next-generation drug susceptibility test. Funding German Federal Ministry of Education and Research through KfW, Dutch Ministry of Foreign Affairs, and Australian Department of Foreign Affairs and Trade.

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Molecular drug susceptibility testing against the first-line (rifampin and isoniazid) and second-line (ciprofloxacin–amikacin and kanamycin) treatment in different subtypes of Mycobacterium simiae

International Journal of Mycobacteriology ◽

10.1016/j.ijmyco.2016.11.006 ◽

2016 ◽

Vol 5 ◽

pp. S215

Author(s):

Parissa Farnia ◽

Donya Malekshahian ◽

Payam Tabarsi ◽

Shima Seif ◽

Ali Akbar Velayati

Keyword(s):

Susceptibility Testing ◽

Drug Susceptibility ◽

Drug Susceptibility Testing ◽

Second Line ◽

First Line ◽

Mycobacterium Simiae

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Molecular Detection of Mutations Associated with First- and Second-Line Drug Resistance Compared with Conventional Drug Susceptibility Testing of Mycobacterium tuberculosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01550-10 ◽

2011 ◽

Vol 55 (5) ◽

pp. 2032-2041 ◽

Cited By ~ 236

Author(s):

Patricia J. Campbell ◽

Glenn P. Morlock ◽

R. David Sikes ◽

Tracy L. Dalton ◽

Beverly Metchock ◽

...

Keyword(s):

Drug Resistance ◽

Mycobacterium Tuberculosis ◽

Dna Sequencing ◽

Susceptibility Testing ◽

Drug Susceptibility ◽

Drug Susceptibility Testing ◽

Second Line ◽

First Line ◽

Content Type ◽

Line Drug

ABSTRACTThe emergence of multi- and extensively drug-resistant tuberculosis is a significant impediment to the control of this disease because treatment becomes more complex and costly. Reliable and timely drug susceptibility testing is critical to ensure that patients receive effective treatment and become noninfectious. Molecular methods can provide accurate and rapid drug susceptibility results. We used DNA sequencing to detect resistance to the first-line antituberculosis drugs isoniazid (INH), rifampin (RIF), pyrazinamide (PZA), and ethambutol (EMB) and the second-line drugs amikacin (AMK), capreomycin (CAP), kanamycin (KAN), ciprofloxacin (CIP), and ofloxacin (OFX). Nine loci were sequenced:rpoB(for resistance to RIF),katGandinhA(INH),pncA(PZA),embB(EMB),gyrA(CIP and OFX), andrrs,eis, andtlyA(KAN, AMK, and CAP). A total of 314 clinicalMycobacterium tuberculosiscomplex isolates representing a variety of antibiotic resistance patterns, genotypes, and geographical origins were analyzed. The molecular data were compared to the phenotypic data and the accuracy values were calculated. Sensitivity and specificity values for the first-line drug loci were 97.1% and 93.6% forrpoB, 85.4% and 100% forkatG, 16.5% and 100% forinhA, 90.6% and 100% forkatGandinhAtogether, 84.6% and 85.8% forpncA, and 78.6% and 93.1% forembB. The values for the second-line drugs were also calculated. The size and scope of this study, in numbers of loci and isolates examined, and the phenotypic diversity of those isolates support the use of DNA sequencing to detect drug resistance in theM. tuberculosiscomplex. Further, the results can be used to design diagnostic tests utilizing other mutation detection technologies.

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Resistance to Second-Line Antituberculosis Drugs and Delay in Drug Susceptibility Testing among Multidrug-Resistant Tuberculosis Patients in Shanghai

BioMed Research International ◽

10.1155/2016/2628913 ◽

2016 ◽

Vol 2016 ◽

pp. 1-8 ◽

Cited By ~ 2

Author(s):

Yong Chen ◽

Zhengan Yuan ◽

Xin Shen ◽

Jie Wu ◽

Zheyuan Wu ◽

...

Keyword(s):

Susceptibility Testing ◽

Drug Susceptibility ◽

Multidrug Resistant ◽

Drug Susceptibility Testing ◽

Second Line ◽

Total Delay ◽

Antituberculosis Drugs ◽

Resistant Tuberculosis ◽

Multidrug Resistant Tuberculosis ◽

Mdr Tb

Introduction. Second-line antituberculosis drugs (SLDs) are used for treating multidrug-resistant tuberculosis (MDR-TB). Prolonged delays before confirming MDR-TB with drug susceptibility testing (DST) could result in transmission of drug-resistant strains and inappropriate use of SLDs, thereby increasing the risk of resistance to SLDs. This study investigated the diagnostic delay in DST and prevalence of baseline SLD resistance in Shanghai and described the distribution of SLD resistance with varied delays to DST.Methods. All registered patients from 2011 to 2013 in Shanghai were enrolled. Susceptibility to ofloxacin, amikacin, kanamycin, and capreomycin was tested. Total delay in DST completion was measured from the onset of symptoms to reporting DST results.Results. Resistance to SLDs was tested in 217 of the 276 MDR-TB strains, with 118 (54.4%) being resistant to at least one of the four SLDs. The median total delay in DST was 136 days. Patients with delay longer than median days were roughly twice more likely to have resistance to at least one SLD (OR 2.22, 95% CI 1.19–4.11).Conclusions. During prolonged delay in DST, primary and acquired resistance to SLDs might occur more frequently. Rapid diagnosis of MDR-TB, improved nosocomial infection controls, and regulated treatment are imperative to prevent SLD resistance.

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Delayed and Unreported Drug-Susceptibility Testing Results in the US National Tuberculosis Surveillance System, 1993-2014

Public Health Reports ◽

10.1177/0033354917708989 ◽

2017 ◽

Vol 132 (4) ◽

pp. 480-487

Author(s):

Jefferson Michael Jones ◽

Lori R. Armstrong

Keyword(s):

Surveillance System ◽

Susceptibility Testing ◽

Drug Susceptibility ◽

Drug Susceptibility Testing ◽

Drug Resistant ◽

First Line ◽

Drug Resistant Tuberculosis ◽

Antituberculosis Drugs ◽

Resistant Tuberculosis ◽

National Tuberculosis

Objectives: Drug-susceptibility testing (DST) of Mycobacterium tuberculosis is necessary for identifying drug-resistant tuberculosis, administering effective treatment regimens, and preventing the spread of drug-resistant tuberculosis. DST is recommended for all culture-confirmed cases of tuberculosis. We examined trends in delayed and unreported DST results in the Centers for Disease Control and Prevention’s National Tuberculosis Surveillance System. Methods: We analyzed culture-confirmed tuberculosis cases reported to the National Tuberculosis Surveillance System during 1993-2014 for annual trends in initial DST reporting for first-line antituberculosis drugs and trends in on-time, delayed, and unreported results. We defined on-time reporting as DST results received during the same calendar year in which the patient’s case was reported or ≤4 months after the calendar year ended and delayed reporting as DST results received after the calendar year. We compared cases with on-time, delayed, and unreported DST results by patient and tuberculosis program characteristics. Results: The proportion of cases with reported results for all first-line antituberculosis drugs increased during 1993-2011. Reporting of pyrazinamide results was lower than reporting of other drugs. However, during 2000-2012, of 134 787 tuberculosis cases reported to the National Tuberculosis Surveillance System, reporting was on time for 125 855 (93.4%) cases, delayed for 5332 (4.0%) cases, and unreported for 3600 (2.7%) cases. Conclusions: Despite increases in the proportion of cases with on-time DST results, delayed and unreported results persisted. Carefully assessing causes for delayed and unreported DST results should lead to more timely reporting of drug-resistant tuberculosis.

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Overcoming the Challenges of Pyrazinamide Susceptibility Testing in Clinical Mycobacterium tuberculosis isolates

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02617-20 ◽

2021 ◽

Author(s):

Simone Mok ◽

Emma Roycroft ◽

Peter R Flanagan ◽

Lorraine Montgomery ◽

Emanuele Borroni ◽

...

Keyword(s):

Sanger Sequencing ◽

Susceptibility Testing ◽

Critical Concentration ◽

Drug Susceptibility ◽

Rapid Identification ◽

Drug Susceptibility Testing ◽

Whole Genome ◽

Testing Methods ◽

First Line ◽

Phenotypic Susceptibility

Pyrazinamide (PZA) is one of the first-line agents used for the treatment of tuberculosis. However, current phenotypic PZA susceptibility testing in the BACTEC MGIT 960 system is unreliable and false resistance is well documented. Rapid identification of resistance-associated mutations can confirm the phenotypic result. This study aimed to investigate the use of genotypic methods in combination with phenotypic susceptibility testing for confirmation of PZA resistant M. tuberculosis isolates. Sanger sequencing and/or whole genome sequencing were performed to detect mutations in pncA, rpsA, panD and clpC1. Isolates were screened for heteroresistance, and PZA susceptibility testing was performed in the BACTEC MGIT 960 system using a reduced inoculum to investigate false resistance. Overall, 40 phenotypically PZA resistant isolates were identified. Of these, PZA resistance was confirmed in 22/40 (55%) isolates by detecting mutations in pncA, rpsA and panD genes. 16/40 (40%) isolates were found to be susceptible using the reduced inoculum method (i.e. false resistance). No mutations were detected in two PZA resistant isolates. False resistance was observed in isolates with MICs close to the critical concentration. In particular, EAI strains (lineage 1) appeared to have an elevated MIC that is close to the critical concentration. While this study illustrates the complexity and challenges associated with PZA susceptibility testing of M. tuberculosis, we conclude that a combination of genotypic and phenotypic drug susceptibility testing methods is required for accurate detection of PZA resistance.

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