In silico evaluation of WHO-endorsed molecular methods to detect drug resistant tuberculosis

Universal drug susceptibility testing (DST) for tuberculosis is a major goal of the END TB strategy. PCR-based molecular diagnostic tests have been instrumental in increasing DST globally and several assays have now been endorsed by the World Health Organization (WHO) for use in the diagnosis of drug resistance. These endorsed assays, however, each interrogate a limited number of mutations associated with resistance, potentially limiting their sensitivity compared to sequencing-based methods. We applied an in silico method to compare the sensitivity and specificity of WHO-endorsed molecular based diagnostics to the mutation set identified by the WHO mutations catalogue using phenotypic DST as the reference. We found that, in silico, the mutation sets used by probe-based molecular diagnostic tests to identify rifampicin, isoniazid, pyrazinamide, levofloxacin, moxifloxacin, amikacin, capreomycin and kanamycin resistance produced similar sensitivities and specificities to the WHO mutation catalogue. PCR-based diagnostic tests were most sensitive for drugs where mechanisms of resistance are well established and localised to small genetic regions or a few prevalent mutations. Approaches using sequencing technologies can provide advantages for drugs where our knowledge of resistance is limited, or where complex resistance signatures exist

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

BACKGROUND: Rapid 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. METHODS: This 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. RESULTS: In 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). CONCLUSIONS: This 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 REGISTRATION: ClinicalTrials.gov (#NCT02170441). Registered 12 June 2014. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12879-016-1781-y) contains supplementary material, which is available to authorized users

Updating the approaches to define susceptibility and resistance to anti-tuberculosis agents: implications for diagnosis and treatment

11 páginas, 2 figuras, 1 tablaInappropriately high breakpoints have resulted in systematic false-susceptible AST results to anti-TB drugs. MIC, PK/PD and clinical outcome data should be combined when setting breakpoints to minimise the emergence and spread of antimicrobial resistance.I. Comas was supported by PID2019-104477RB-I00 from the Spanish Science Ministry and by ERC (CoG 101001038)Peer reviewe

Rapid Detection of Genetic Mutations Associated with Drug- resistant Mycobacterium tuberculosis

Background: Drug resistance continues to threaten recent global progress toward tuberculosis (TB) control. Rapid and accurate detection of resistance is imperative for successful treatment outcomes and reduction in transmission of resistant strains. Methods: Global frequency and distribution of mutations associated with isoniazid resistance were characterized through an exhaustive systematic literature review. The diagnostic performance of a newly redesigned line probe assay (LPA) among both Acid Fast Bacilli (AFB) smear positive and negative specimens was evaluated using cross-sectional data from a large multisite diagnostic study. Additionally, the association of clinical characteristics and valid test results (susceptible or resistant) was assessed using logistic regression. Individual probe results from two LPAs were compared to both sequencing data and phenotypic susceptibility results, and the relative contribution of absent wild type probe hybridization to the detection of phenotypic resistance was determined by comparing the areas under the receiver operating characteristic curves. Results: Globally 64% of isoniazid resistant specimens harbor the katG315 mutation and 19% harbor the inhA-15 mutation, together these two mutations are associated with 80% of isoniazid resistance. Sensitivity and specificity of the LPA for multidrug-resistant TB detection were 95.1% (95%CI 92.6, 96.9) and 99.1% (95%CI 97.1, 100.0) respectively, among both AFB smear positive and negative specimens. Sensitivity for isoniazid resistance was significantly lower among AFB smear negative specimens at 81.6% (95%CI 65.1, 91.7) compared to 94.9% (95%CI 92.4, 96.6) among AFB smear positive specimens. Additionally, AFB smear gradation was significantly associated with valid test results after controlling for HIV status, diabetic status, body mass index, and history of previous treatment. The inclusion of absent wild type probe hybridization in resistance classification for LPAs significantly improved detection of both rifampicin and flouroquinolone phenotypic resistance (P<0.001). Conclusions: Variation in mutation frequency justifies local monitoring of resistance associated mutations. The newly redesigned LPA performed well among both AFB smear positive and negative specimens, however, bacillary load influenced both the probability of obtaining an interpretable result and test sensitivity. The evaluation of individual LPA probe results demonstrated that current LPA resistance classification algorithms successfully differentiate between phenotypically susceptible and resistant specimen

Using Mycobacterium tuberculosis Single-Nucleotide Polymorphisms To Predict Fluoroquinolone Treatment Response.

Clinical phenotypic fluoroquinolone susceptibility testing of Mycobacterium tuberculosis is currently based on M. tuberculosis growth at a single critical concentration, which provides limited information for a nuanced clinical response. We propose using specific resistance-conferring M. tuberculosis mutations in gyrA together with population pharmacokinetic and pharmacodynamic modeling as a novel tool to better inform fluoroquinolone treatment decisions. We sequenced the gyrA resistance-determining region of 138 clinical M. tuberculosis isolates collected from India, Moldova, Philippines, and South Africa and then determined each strain's MIC against ofloxacin, moxifloxacin, levofloxacin, and gatifloxacin. Strains with specific gyrA single-nucleotide polymorphisms (SNPs) were grouped into high or low drug-specific resistance categories based on their empirically measured MICs. Published population pharmacokinetic models were then used to explore the pharmacokinetics and pharmacodynamics of each fluoroquinolone relative to the empirical MIC distribution for each resistance category to make predictions about the likelihood of patients achieving defined therapeutic targets. In patients infected with M. tuberculosis isolates containing SNPs associated with a fluoroquinolone-specific low-level increase in MIC, models suggest increased fluoroquinolone dosing improved the probability of achieving therapeutic targets for gatifloxacin and moxifloxacin but not for levofloxacin and ofloxacin. In contrast, among patients with isolates harboring SNPs associated with a high-level increase in MIC, increased dosing of levofloxacin, moxifloxacin, gatifloxacin, or ofloxacin did not meaningfully improve the probability of therapeutic target attainment. We demonstrated that quantifiable fluoroquinolone drug resistance phenotypes could be predicted from rapidly detectable gyrA SNPs and used to support dosing decisions based on the likelihood of patients reaching therapeutic targets. Our findings provide further supporting evidence for the moxifloxacin clinical breakpoint recently established by the World Health Organization

Impact of Fluoroquinolone Use on Mortality Among a Cohort of Patients With Suspected Drug-Resistant Tuberculosis.

BackgroundPrevious retrospective and in vitro studies suggest that use of later-generation fluoroquinolones may reduce mortality risk and improve treatment outcomes for drug-resistant tuberculosis (TB) patients, including individuals resistant to a fluoroquinolone. Meta-analysis results are mixed and few studies have examined this relationship prospectively.MethodsAs part of a comparative diagnostic study, we conducted a prospective cohort study with 834 Mycobacterium tuberculosis-infected patients from selected hospitals and clinics with high prevalence of drug-resistant TB in India, Moldova, and South Africa. We used Cox proportional hazards regression models to assess the association between later-generation fluoroquinolone (moxifloxacin or levofloxacin) use and patient mortality, adjusting for risk factors typically associated with poor treatment outcomes.ResultsAfter adjusting for phenotypic resistance profile, low body mass index (<18.5 kg/m2), human immunodeficiency virus status, and study site, participants treated with a later-generation fluoroquinolone had half the risk of mortality compared with participants either not treated with any fluoroquinolone or treated only with an earlier-generation fluoroquinolone (adjusted hazard ratio, 0.46 [95% confidence interval, .26-.80]) during follow-up.ConclusionsUse of later-generation fluoroquinolones significantly reduced patient mortality risk in our cohort, suggesting that removal of a later-generation fluoroquinolone from a treatment regimen because of demonstrated resistance to an earlier-generation fluoroquinolone might increase mortality risk. Further studies should evaluate the effectiveness of later-generation fluoroquinolones among patients with and without resistance to early-generation fluoroquinolones.Clinical trials registrationNCT02170441