Direct detection of resistance to fluoroquinolones/SLIDs in sputum specimen by GenoType MTBDRsl v.2.0 assay A study from Eastern Uttar Pradesh, India

Abstract Background According to World Health Organization (WHO), drug-resistant tuberculosis (DR-TB) is a major contributor to antimicrobial resistance globally and continues to be a public health threat. Annually, about half a million people fall ill with DR-TB globally. The gradual increase in resistance to fluoroquinolones (FQs) and second-line injectable drugs (SLIDs), poses a serious threat to effective TB control and adequate patient management. Therefore, WHO suggests the use of GenoType MTBDRsl v.2.0 assay for detection of multiple mutations associated with FQs and SLIDs. Hence, the study was conducted to determine the prevalence of resistance to FQs and SLIDs by comparing direct GenoType MTBDRsl v.2.0 assay with phenotypic drug susceptibility testing (DST). Methods The study was conducted on 1320 smear positive sputum samples from a total of 2536 RR-TB, confirmed by GeneXpert MTB/RIF. The smear positive specimens were decontaminated, and DNA extraction was performed. Furthermore, the extracted DNA was used for GenoType MTBDRsl v.2.0 assay. While 20% of the decontaminated specimens were inoculated in Mycobacterium growth indicator tube (MGIT) for drug susceptibility testing (DST). Results Out of 1320 smear positive sputum samples, 1178 were identified as Mycobacterium tuberculosis complex (MTBC) and remaining were negative by GenoType MTBDRsl v.2.0 assay. Of the 1178 MTBC positive, 26.6% were sensitive to both FQs and SLIDs, whereas 57.3% were only FQs resistant and 15.9% were resistant to both FQs and SLIDs. Further DST of 225 isolates by liquid culture showed that 17% were sensitive to both FQs and SLIDs, 61.3% were only FQs resistant and 21.3% were resistant to both. The specificity for FQs and SLIDs was 92.31% and 100% whereas sensitivity was 100% respectively by GenoType MTBDRsl v.2.0 assay in direct sputum samples. Conclusions Our study clearly suggests that GenoType MTBDRsl v.2.0 assay is a reliable test for the rapid detection of resistance to second-line drugs after confirmation by GeneXpert MTB/RIF assay for RR-TB. Though, high rate FQ (ofloxacin) resistance was seen in our setting, moxifloxacin could be used as treatment option owing to very low resistance.

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Validation of Bedaquiline Phenotypic Drug Susceptibility Testing Methods and Breakpoints: a Multilaboratory, Multicountry Study

Journal of Clinical Microbiology ◽

10.1128/jcm.01677-19 ◽

2020 ◽

Vol 58 (4) ◽

Cited By ~ 5

Author(s):

Koné Kaniga ◽

Akio Aono ◽

Emanuele Borroni ◽

Daniela Maria Cirillo ◽

Christel Desmaretz ◽

...

Keyword(s):

Susceptibility Testing ◽

Improve Patient Care ◽

Drug Susceptibility ◽

Drug Susceptibility Testing ◽

Error Rates ◽

World Health ◽

Health Concern ◽

Content Type ◽

Indicator Tube ◽

Health Organization

ABSTRACT Drug-resistant tuberculosis persists as a major public health concern. Alongside efficacious treatments, validated and standardized drug susceptibility testing (DST) is required to improve patient care. This multicountry, multilaboratory external quality assessment (EQA) study aimed to validate the sensitivity, specificity, and reproducibility of provisional bedaquiline MIC breakpoints and World Health Organization interim critical concentrations (CCs) for categorizing clinical Mycobacterium tuberculosis isolates as susceptible/resistant to the drug. Three methods were used: Middlebrook 7H11 agar proportion (AP) assay, broth microdilution (BMD) assay, and mycobacterial growth indicator tube (MGIT) assay. Each of the five laboratories tested the 40-isolate (20 unique isolates, duplicated) EQA panel at three time points. The study validated the sensitivity and specificity of a bedaquiline MIC susceptibility breakpoint of 0.12 μg/ml for the BMD method and WHO interim CCs of 1 μg/ml for MGIT and 0.25 μg/ml for the 7H11 AP methods. Categorical agreements between observed and expected results and sensitivities/specificities for correctly identifying an isolate as susceptible/resistant were highest at the 0.25, 0.12, and 1 μg/ml bedaquiline concentrations for the AP method, BMD (frozen or dry plates), and MGIT960, respectively. At these concentrations, the very major error rates for erroneously categorizing an isolate as susceptible when it was resistant were the lowest and within CLSI guidelines. The most highly reproducible bedaquiline DST methods were MGIT960 and BMD using dry plates. These findings validate the use of standardized DST methodologies and interpretative criteria to facilitate routine phenotypic bedaquiline DST and to monitor the emergence of bedaquiline resistance.

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Testing Susceptibility of M. tuberculosis to Second Line Anti-Tuberculosis Drugs Using the XDR Test in Clinical Trials and International Professional Testing Cycles

Tuberculosis and lung diseases ◽

10.21292/2075-1230-2021-99-8-13-20 ◽

2021 ◽

Vol 99 (8) ◽

pp. 13-20

Author(s):

L. V. Domotenko ◽

T. P. Morozova ◽

M. V. Khramov ◽

А. P. Shepelin

Keyword(s):

Clinical Trials ◽

Susceptibility Testing ◽

Drug Susceptibility ◽

Drug Susceptibility Testing ◽

World Health ◽

Clinical Samples ◽

Second Line ◽

Tuberculosis Patients ◽

Testing Susceptibility ◽

Health Organization

The objective of the study: to evaluate the commercial XDR test for susceptibility testing of M. tuberculosis to second line anti-tuberculosis drugs in clinical trials and as part of annual professional testing cycles coordinated by the World Health Organization (WHO).Subjects and Methods. Cultures of M. tuberculosis (n = 90) freshly isolated on egg media from clinical samples collected in tuberculosis patients were tested using the Bactec MGIT 960 system and the XDR test under identical conditions. Well-studied strains of M. tuberculosis (n = 216) obtained from the WHO supranational laboratories were repeatedly cultured on Middlebrook 7H10 medium before the study. The drug susceptibility of the cultures was assessed using the XDR test by the nitrate reductase method.Results. A high concurrence (96.7-100%) of the results was shown when testing susceptibility of 90 M. tuberculosis isolates to kanamycin, amikacin, capreomycin and ofloxacin using the XDR test and the Bactec MGIT 960 system with comparable test periods. The use of the XDR test for drug susceptibility testing of 216 M. tuberculosis strains in eleven annual professional testing cycles coordinated by the WHO supranational laboratories provided the results consistent with the consensus one for kanamycin, capreomycin, ofloxacin and amikacin in 98.6, 99.4, 99.4, and 99.0% of cases, respectively. For moxifloxacin and levofloxacin additionally incorporated to the XDR test, completely identical results were obtained.

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TB Elimination Requires Discovery and Development of Transformational Agents

Applied Sciences ◽

10.3390/app10072605 ◽

2020 ◽

Vol 10 (7) ◽

pp. 2605 ◽

Cited By ~ 1

Author(s):

Christian Lienhardt ◽

Mario C. Raviglione

Keyword(s):

Rapid Detection ◽

Susceptibility Testing ◽

Drug Susceptibility ◽

Drug Susceptibility Testing ◽

New Drugs ◽

World Health ◽

Drug Resistant ◽

The World ◽

Health Organization ◽

Combination Regimens

The World Health Organization (WHO) End Tuberculosis (TB) Strategy has set ambitious targets to reduce 2015 TB incidence and deaths by 80% and 90%, respectively, by the year 2030. Given the current rate of TB incidence decline (about 2% per year annually), reaching these targets will require new transformational tools and innovative ways to deliver them. In addition to improved tests for early and rapid detection of TB and universal drug-susceptibility testing, as well as novel vaccines for improved prevention, better, safer, shorter and more efficacious treatments for all forms of TB are needed. Only a handful of new drugs are currently in phase II or III clinical trials, and a few combination regimens are being tested, mainly for drug-resistant TB. In this article, capitalising on an increasingly rich medicine pipeline and taking advantage of new methodological designs with great potential, the main areas where progress is needed for a transformational improvement of treatment of all forms of TB are described.

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Epidemiological cutoff values for a 96-well broth microdilution plate for high-throughput research antibiotic susceptibility testing of M. tuberculosis

10.1101/2021.02.24.21252386 ◽

2021 ◽

Author(s):

◽

Philip W Fowler

Keyword(s):

Genetic Variants ◽

Susceptibility Testing ◽

Drug Susceptibility ◽

Drug Susceptibility Testing ◽

Broth Microdilution ◽

Wild Type ◽

Genetic Sequencing ◽

Indicator Tube ◽

Cutoff Values ◽

Type Population

AbstractDrug susceptibility testing of M. tuberculosis is rooted in a binary susceptible/resistant paradigm. There are considerable advantages in measuring the minimum inhibitory concentrations (MICs) of a panel of drugs for an isolate, including quantifying the magnitude of effect conferred by genetic variants and being able to identify isolates with elevated MICs that can still be treated with standard therapy. It is necessary, however, to measure the epidemiological cutoff values (ECOFF/ECVs) to permit comparison with qualitative data. Here we present ECOFF/ECVs for 13 anti-TB compounds, including bedaquiline and delamanid, derived from 20,637 clinical isolates collected by 14 laboratories based in 11 countries on five continents. Each isolate was incubated for 14 days on a dry 96-well broth microdilution plate and then read. Resistance to the majority of the drugs due to prior exposure is expected and the MIC distributions for many of the compounds are complex and therefore a phenotypically wild-type population could not be defined. Since a majority of samples also underwent genetic sequencing, we defined a genotypically wild-type population and measured the MIC of the 99th percentile by direct measurement and via fitting a Gaussian using interval regression. The proposed ECOFF/ECV values were then validated by comparing to the MIC distributions of high-confidence genetic variants that confer resistance and to qualitative drug susceptibility tests obtained via Mycobacterial Growth Indicator Tube and the Microscopic-Observation Drug-Susceptibility assay.

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Quantitative measurement of antibiotic resistance in Mycobacterium tuberculosis reveals genetic determinants of resistance and susceptibility in a target gene approach

10.1101/2021.09.14.460353 ◽

2021 ◽

Author(s):

◽

Joshua J Carter

Keyword(s):

Mycobacterium Tuberculosis ◽

Quantitative Measurement ◽

Target Gene ◽

Microtiter Plate ◽

Drug Susceptibility ◽

Drug Susceptibility Testing ◽

World Health ◽

Genetic Determinants ◽

Microtiter Plate Assay ◽

Health Organization

AbstractThe World Health Organization goal of universal drug susceptibility testing for patients with tuberculosis is most likely to be achieved through molecular diagnostics; however, to date these have focused largely on first-line drugs, and always on predicting binary susceptibilities. Here, we used whole genome sequencing and a quantitative microtiter plate assay to relate genomic mutations to minimum inhibitory concentration in 15,211 Mycobacterium tuberculosis patient isolates from 27 countries across five continents.This work identifies 449 unique MIC-elevating genetic determinants across thirteen drugs, as well as 91 mutations resulting in hypersensitivity for eleven drugs. Our results provide a guide for further implementation of personalized medicine for the treatment of tuberculosis using genetics-based diagnostics and can serve as a training set for novel approaches to predict drug resistance.

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