Pyrosequencing for rapid detection of Mycobacterium tuberculosis second-line drugs and ethambutol resistance

Lacoma, Alícia; Molina-Moya, Bárbara; Prat, Cristina; Pimkina, Edita; Díaz, Jéssica; Dudnyk, Andrii; García-Sierra, Nerea; Haba, L.; Maldonado, José; Samper, Sofía; Ruiz-Manzano, Juan; Ausina, Vicente; Domínguez, José

doi:10.1016/j.diagmicrobio.2015.07.004

Cited by 16 publications

(9 citation statements)

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“…As 93 % of all FQ-resistant strains have mutations in the gyrA gene region included in our assay [22], this result was lower than expected. However, our diagnostic sensitivity measures were in the range of those reported by Lacoma et al, who reported 40 % sensitivity for detection of OFX resistance and 70.8 % for the detection of MOX resistance for a PSQ assay including the same gyrA gene regions as our study, tested against strains from Spain and Lithuania [25]. Furthermore, only 14–15 phenotypically MOX- and OFX-resistant specimens were analyzed in Moldova, which may have led to a chance oversampling of specimens missing these common mutations.…”

Section: Discussionsupporting

confidence: 59%

Shedding light on the performance of a pyrosequencing assay for drug-resistant tuberculosis diagnosis

et al. 2016

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BackgroundRapid molecular diagnostics, with their ability to quickly identify genetic mutations associated with drug resistance in Mycobacterium tuberculosis clinical specimens, have great potential as tools to control multi- and extensively drug-resistant tuberculosis (M/XDR-TB). The Qiagen PyroMark Q96 ID system is a commercially available pyrosequencing (PSQ) platform that has been validated for rapid M/XDR-TB diagnosis. However, the details of the assay’s diagnostic and technical performance have yet to be thoroughly investigated in diverse clinical environments.MethodsThis study evaluates the diagnostic performance of the PSQ assay for 1128 clinical specimens from patients from three areas of high TB burden. We report on the diagnostic performance of the PSQ assay between the three sites and identify variables associated with poor PSQ technical performance.ResultsIn India, the sensitivity of the PSQ assay ranged from 89 to 98 % for the detection of phenotypic resistance to isoniazid, rifampicin, fluoroquinolones, and the injectables. In Moldova, assay sensitivity ranged from 7 to 94 %, and in South Africa, assay sensitivity ranged from 71 to 92 %. Specificity was high (94–100 %) across all sites. The addition of eis promoter sequencing information greatly improved the sensitivity of kanamycin resistance detection in Moldova (7 % to 79 %). Nearly all (89.4 %) sequencing reactions conducted on smear-positive, culture-positive specimens and most (70.8 %) reactions conducted on smear-negative, culture-positive specimens yielded valid PSQ reads. An investigation into the variables influencing sequencing failures indicated smear negativity, culture negativity, site (Moldova), and sequencing of the rpoB, gyrA, and rrs genes were highly associated with poor PSQ technical performance (adj. OR > 2.0).ConclusionsThis study has important implications for the global implementation of PSQ as a molecular TB diagnostic, as it demonstrates how regional factors may impact PSQ diagnostic performance, while underscoring potential gene targets for optimization to improve overall PSQ assay technical performance.Trial registrationClinicalTrials.gov (#NCT02170441). Registered 12 June 2014.Electronic supplementary materialThe online version of this article (doi:10.1186/s12879-016-1781-y) contains supplementary material, which is available to authorized users.

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Section: Discussionsupporting

confidence: 59%

Shedding light on the performance of a pyrosequencing assay for drug-resistant tuberculosis diagnosis

et al. 2016

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“…Molecular tests may show discordant results when compared to phenotypic DST based on critical concentration testing. 74,98,[105][106][107][108][109][110][111][112] The clinical consequences of the limitations in the accuracy of molecular resistance assays depend on the drug, but follow a general pattern. False-positive test results will lead to drug-susceptible TB being treated with one or more second-line drugs, i.e., treatment that is generally more toxic, less effective, prolonged and more expensive.…”

Section: Relationship Between the Results Of Genotypic And Phenotypicmentioning

confidence: 99%

Clinical implications of molecular drug resistance testing for <I>Mycobacterium tuberculosis</I>: a TBNET/RESIST-TB consensus statement

Domínguez

Boettger

Cirillo

et al. 2016

int j tuberc lung dis

120

105

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“… Kontos 2012 Conference abstract. Lacoma 2015 Not a diagnostic accuracy study. Lemus 2011 Conference abstract.…”

Section: Characteristics Of Excluded Studies [Ordered By Study Id]mentioning

confidence: 99%

GenoType^® MTBDRsl assay for resistance to second-line anti-tuberculosis drugs

Theron

Peter

Richardson

et al. 2016

Cochrane Database of Systematic Reviews

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Background Genotype® MTBDR sl (MTBDR sl ) is a rapid DNA‐based test for detecting specific mutations associated with resistance to fluoroquinolones and second‐line injectable drugs (SLIDs) in Mycobacterium tuberculosis complex. MTBDR sl version 2.0 (released in 2015) identifies the mutations detected by version 1.0, as well as additional mutations. The test may be performed on a culture isolate or a patient specimen, which eliminates delays associated with culture. Version 1.0 requires a smear‐positive specimen, while version 2.0 may use a smear‐positive or ‐negative specimen. We performed this updated review as part of a World Health Organization process to develop updated guidelines for using MTBDR sl . Objectives To assess and compare the diagnostic accuracy of MTBDR sl for: 1. fluoroquinolone resistance, 2. SLID resistance, and 3. extensively drug‐resistant tuberculosis, indirectly on a M. tuberculosis isolate grown from culture or directly on a patient specimen. Participants were people with rifampicin‐resistant or multidrug‐resistant tuberculosis. The role of MTBDR sl would be as the initial test, replacing culture‐based drug susceptibility testing (DST), for detecting second‐line drug resistance. Search methods We searched the following databases without language restrictions up to 21 September 2015: the Cochrane Infectious Diseases Group Specialized Register; MEDLINE; Embase OVID; Science Citation Index Expanded, Conference Proceedings Citation Index‐Science, and BIOSIS Previews (all three from Web of Science); LILACS; and SCOPUS; registers for ongoing trials; and ProQuest Dissertations & Theses A&I. We reviewed references from included studies and contacted specialists in the field. Selection criteria We included cross‐sectional and case‐control studies that determined MTBDR sl accuracy against a defined reference standard (culture‐based DST, genetic sequencing, or both). Data collection and analysis Two review authors independently extracted data and assessed quality using the Quality Assessment of Diagnostic Accuracy Studies (QUADAS‐2) tool. We synthesized data for versions 1.0 and 2.0 separately. We estimated MTBDR sl sensitivity and specificity for fluoroquinolone resistance, SLID resistance, and extensively drug‐resistant tuberculosis when the test was performed indirectly or directly (smear‐positive specimen for version 1.0, smear‐positive or ‐negative specimen for version 2.0). We explored the influence on accuracy estimates of individual drugs within a drug class and of different reference standards. We performed most analyses using a bivariate random‐effects model with culture‐based DST as reference ...

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Pyrosequencing for rapid detection of Mycobacterium tuberculosis second-line drugs and ethambutol resistance

Cited by 16 publications

References 40 publications

Shedding light on the performance of a pyrosequencing assay for drug-resistant tuberculosis diagnosis

Shedding light on the performance of a pyrosequencing assay for drug-resistant tuberculosis diagnosis

Clinical implications of molecular drug resistance testing for <I>Mycobacterium tuberculosis</I>: a TBNET/RESIST-TB consensus statement

GenoType^® MTBDRsl assay for resistance to second-line anti-tuberculosis drugs

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Pyrosequencing for rapid detection of Mycobacterium tuberculosis second-line drugs and ethambutol resistance

Cited by 16 publications

References 40 publications

Shedding light on the performance of a pyrosequencing assay for drug-resistant tuberculosis diagnosis

Shedding light on the performance of a pyrosequencing assay for drug-resistant tuberculosis diagnosis

Clinical implications of molecular drug resistance testing for <I>Mycobacterium tuberculosis</I>: a TBNET/RESIST-TB consensus statement

GenoType® MTBDRsl assay for resistance to second-line anti-tuberculosis drugs

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Product

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GenoType^® MTBDRsl assay for resistance to second-line anti-tuberculosis drugs