Role of phenotypic methods for drug susceptibility testing of M. tuberculosis isolates in the era of MDR and XDR epidemics.

ABSTRACTForMycobacterium tuberculosis, phenotypic methods for drug susceptibility testing of second-line drugs are poorly standardized and technically challenging. The Sensititre MYCOTB MIC plate (MYCOTB) is a microtiter plate containing lyophilized antibiotics and configured for determination of MICs to first- and second-line antituberculosis drugs. To evaluate the performance of MYCOTB forM. tuberculosisdrug susceptibility testing using the Middlebrook 7H10 agar proportion method (APM) as the comparator, we conducted a two-site study using archivedM. tuberculosisisolates from Uganda and the Republic of Korea. Thawed isolates were subcultured, and dilutions were inoculated into MYCOTB wells and onto 7H10 agar. MYCOTB results were read at days 7, 10, 14, and 21; APM results were read at 21 days. A total of 222 isolates provided results on both platforms. By APM, 106/222 (47.7%) of isolates were resistant to at least isoniazid and rifampin. Agreement between MYCOTB and APM with respect to susceptibility or resistance was ≥92% for 7 of 12 drugs when a strict definition was used and ≥96% for 10 of 12 drugs when agreement was defined by allowing a ± one-well range of dilutions around the APM critical concentration. For ethambutol, agreement was 80% to 81%. For moxifloxacin, agreement was 83% to 85%; incorporating existing DNA sequencing information for discrepant analysis raised agreement to 91% to 96%. For MYCOTB, the median time to plate interpretation was 10 days and interreader agreement was ≥95% for all drugs. MYCOTB provided reliable results forM. tuberculosissusceptibility testing of first- and second-line drugs except ethambutol, and results were available sooner than those determined by APM.

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Drug susceptibility testing of Mycobacterium tuberculosis by two phenotypic methods

International Journal of Infectious Diseases ◽

10.1016/j.ijid.2012.05.968 ◽

2012 ◽

Vol 16 ◽

pp. e292

Author(s):

R. Saksena ◽

C.P. Baveja ◽

S. Kumar ◽

H.S. Hira ◽

A. Khanna

Keyword(s):

Mycobacterium Tuberculosis ◽

Susceptibility Testing ◽

Drug Susceptibility ◽

Drug Susceptibility Testing ◽

Phenotypic Methods

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Direct Susceptibility Testing of Mycobacterium tuberculosis for Pyrazinamide by Use of the Bactec MGIT 960 System

Journal of Clinical Microbiology ◽

10.1128/jcm.03162-15 ◽

2016 ◽

Vol 54 (5) ◽

pp. 1276-1281 ◽

Cited By ~ 10

Author(s):

Anne-Marie Demers ◽

Amour Venter ◽

Sven O. Friedrich ◽

Gabriel Rojas-Ponce ◽

Daniel Mapamba ◽

...

Keyword(s):

Susceptibility Testing ◽

Direct Method ◽

Drug Susceptibility ◽

Drug Susceptibility Testing ◽

New Drugs ◽

Indirect Methods ◽

Future Studies ◽

Patient Screening ◽

Phenotypic Methods ◽

Direct And Indirect Methods

Pyrazinamide (PZA) is a key antituberculosis drug, yet no rapid susceptibility test is commercially available. PZA drug susceptibility testing (DST) was performed directly on sputum samples from 327 patients and compared with the indirect method by using the Bactec MGIT 960 system in the context of patient screening for participation in a drug trial. Compared to standard indirect PZA DST, direct DST was successful in only 59% of cases, but results obtained were highly accurate and available faster. Agreement between the direct and indirect methods varied from 90 to 100% in each laboratory. The median times for obtaining PZA results from the time when the specimen was collected ranged from 11 to 16 days for the direct test and 18 to 95 days for the indirect test across laboratories. The direct method is accurate and reproducible across laboratories. It can be expected to accelerate results in >50% of cases, but it cannot replace indirect DST for PZA. Phenotypic methods remain the gold standard for DST in drug trials. If future studies can optimize the method to decrease the number of uninterpretable results, direct MGIT DST could be the new phenotypic DST standard for clinical trials, providing more rapid detection of resistance to new drugs in experimental regimens.

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Non-pncAGene-Mutated but Pyrazinamide-Resistant Mycobacterium tuberculosis: Why Is That?

Journal of Clinical Microbiology ◽

10.1128/jcm.02532-16 ◽

2017 ◽

Vol 55 (6) ◽

pp. 1920-1927 ◽

Cited By ~ 20

Author(s):

Jim Werngren ◽

Erik Alm ◽

Mikael Mansjö

Keyword(s):

Susceptibility Testing ◽

Gene Mutations ◽

Drug Susceptibility ◽

Multidrug Resistant ◽

Drug Susceptibility Testing ◽

Molecular Techniques ◽

Content Type ◽

Resistant Strains ◽

Clear Majority

ABSTRACTPyrazinamide (PZA) is a key component for the effective treatment of drug-susceptible and PZA-susceptible multidrug-resistant (MDRPZA-S) tuberculosis (TB).pncAgene mutations are usually detected in a clear majority (>90%) of PZA-resistant strains but obviously not in all. Rapid and reliable PZA drug susceptibility testing (DST) is critical whenever PZA is to be used in a treatment regimen, not least for the treatment of MDRPZA-STB. In this study, we selected 26 PZA-resistant isolates reported to carry a wild-typepncAgene. To confirm resistance, susceptibility testing was repeated using 100 mg/liter and 200 mg/liter PZA for all the 26 isolates and Sanger sequencing was repeated on the 18 isolates that remained PZA resistant. Apart from the eight isolates initially misclassified as PZA resistant, the retests identified three factors responsible for the phenotype-genotype discrepancy:panDorrpsAmutations identified by whole-genome sequencing (WGS) (n= 7), heteroresistance (n= 8), and mixed populations withMycobacterium avium(n= 3). Additionally, we performed WGS on 400 PZA-susceptible isolates and 15 consecutive MDRPZA-Rclinical isolates. Of the 400 PZA-susceptible isolates, only 1 harbored a nonsynonymouspncAmutation (Thr87Met), whereas a nonsynonymousrpsAmutation was found in 17 isolates. None of these isolates carried a nonsynonymouspanDmutation, while all 15 of the MDRPZA-Risolates harbored a nonsynonymouspncAmutation. Our findings indicate that it is necessary to consider the occurrence ofpanDmutations in PZA-resistant isolates, as well as heteroresistance, for the development and evaluation of new molecular techniques to ensure high-quality DST performance. The identification of nonsynonymousrpsAmutations in both PZA-susceptible and PZA-resistant isolates also implies that further studies are needed in order to determine the role ofrpsAin PZA resistance.

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Comparative Performance of Genomic Methods for the Detection of Pyrazinamide Resistance and Heteroresistance in Mycobacterium tuberculosis

Journal of Clinical Microbiology ◽

10.1128/jcm.01907-21 ◽

2021 ◽

Author(s):

Michael G. Whitfield ◽

David M. Engelthaler ◽

Christopher Allender ◽

Megan Folkerts ◽

Tim H. Heupink ◽

...

Keyword(s):

Diagnostic Accuracy ◽

Susceptibility Testing ◽

High Sensitivity ◽

Drug Susceptibility ◽

Drug Susceptibility Testing ◽

Comparative Performance ◽

Treatment Regimens ◽

Resistant Tuberculosis ◽

Targeted Deep Sequencing ◽

Phenotypic Methods

Background: Pyrazinamide is an important component of both drug-susceptible and drug-resistant tuberculosis treatment regimens. Although approximately 50% of rifampicin resistant isolates are also resistant to pyrazinamide, pyrazinamide susceptibility testing is not routinely performed due to the challenging nature of the assay. We investigated the diagnostic accuracy of genotypic and phenotypic methods, and explored the occurrence of pyrazinamide heteroresistance. Methods: We assessed pyrazinamide susceptibility among 358 individuals enrolled in the South African EXIT-RIF cohort using Sanger and targeted deep sequencing (TDS) of the pncA gene, whole genome sequencing (WGS), and phenotypic drug-susceptibility testing. We calculated the diagnostic accuracy of the different methods, and investigated the prevalence and clinical impact of pncA heteroresistance. True pyrazinamide susceptibility status was assigned to each isolate using the Koser classification and expert rules. Results: We observed 100% agreement across genotypic methods for detection of pncA fixed mutations, only TDS confidently identified three isolates (0.8%) with minor variants. For the 355 (99.2%) isolates that could be assigned true pyrazinamide status with confidence, phenotypic DST had a sensitivity of 96.5% (95% CI: 93.8-99.3%) and specificity of 100% (95% CI: 100-100%); both Sanger sequencing and WGS had a sensitivity of 97.1% (95% CI: 94.6-99.6%) and specificity of 97.8% (95% CI: 95.7-99.9%); and TDS, sensitivity of 98.8% (95% CI: 97.2-100%) and specificity of 97.8% (95% CI: 95.7-99.9%). Conclusions: We demonstrate high sensitivity and specificity for pyrazinamide susceptibility testing among all assessed genotypic methods. The prevalence of pyrazinamide heteroresistance in Mtb isolates was lower than that identified for other first-line drugs.

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