MTBDRplus Version Research Articles

Effect of sputum quality and role of Xpert® MTB/ RIF assay for detection of smear-negative pulmonary tuberculosis in same-day diagnosis strategy in Addis Ababa, Ethiopia.

BackgroundThere is limited information on the performance of the Xpert® MTB/RIF test for diagnosis of smear-negative pulmonary tuberculosis (SNPT) and rifampicin resistance (RR) in the same-day diagnosis approach. The effects of sputum quality and other factors affecting the Xpert performance are also under-investigated.ObjectiveThis study aimed to determine the performance of the Xpert® MTB/RIF test for detection of SNPT and RR in the same-day diagnosis strategy and the effect of sputum quality and other factors on its performance.MethodsA cross-sectional study was conducted from August 2017 to January 2018 across 16 health facilities in Addis Ababa, Ethiopia. Two spot sputum samples were collected from 418 presumptive SNPT patients, tested with Xpert® MTB/RIF, then compared to tuberculosis culture. Additionally, culture isolates were tested for RR by BACTEC MGIT™ 960 drug susceptibility testing (DST) and MTBDRplus version 2.ResultsThe Xpert® MTB/RIF test detected 24 (5.7%) SNPT cases, with a sensitivity of 92.3% (75.9% – 97.9%) and specificity of 99.2% (97.8% – 99.7%) compared with tuberculosis culture. Xpert® MTB/RIF also detected three (11.58%) RR strains with 100.0% concordance with BACTEC MGIT™ 960 DST and MTBDRplus results. Three blood-stained SNPT samples were positive by Xpert (30.0%), which was 6.9 times higher compared to salivary sputum (odds ratio: 6.9, 95% confidence interval: 1.36–34.96, p = 0.020).ConclusionThe performance of the Xpert® MTB/RIF to detect SNPT and RR in same-day diagnosis is high. However, SNPT positivity varies among sputum qualities, and good sample collection is necessary for better test performance.

Treatment With a Three-Drug Regimen for Pulmonary Tuberculosis Based on Rapid Molecular Detection of Isoniazid Resistance: A Noninferiority Randomized Trial (FAST-TB).

BackgroundThe rationale behind the use of ethambutol in the standard tuberculosis treatment is to prevent the emergence of resistance to rifampicin in case of primary resistance to isoniazid. We evaluated whether early detection of isoniazid resistance using molecular testing allows the use an ethambutol-free regimen.MethodsFAST-TB, a phase 4, French, multicenter, open-label, non-inferiority trial, compared 2 strategies: (1) polymerase chain reaction (PCR)-based detection of isoniazid and rifampicin resistance at baseline using Genotype MTBDRplus version 2.0 followed by ethambutol discontinuation if no resistance was detected (PCR arm) and (2) a standard 4-drug combination, pending phenotypic drug-susceptibility results (C arm). Adult patients with smear-positive pulmonary tuberculosis were enrolled. The primary endpoint was the proportion of patients with treatment success defined as bacteriological or clinical cure at the end of treatment. A non-inferiority margin of 10% was used.ResultsTwo hundred three patients were randomized, 104 in the PCR arm and 99 in the C arm: 26.6% were female, median age was 37 (interquartile range, 28–51) years, 72.4% were born in Africa, and 5.4% were infected with human immunodeficiency virus. Chest x-ray showed cavities in 64.5% of the cases. Overall, 169 patients met criteria of treatment success: 87 of 104 (83.7%) in the PCR arm and 82 of 99 (82.8%) in the C arm with a difference of +0.8% (90% confidence interval, −7.9 to 9.6), meeting the noninferiority criteria in the intention-to-treat population (P = .02).ConclusionsIn a setting with low prevalence of primary isoniazid resistance, a 3-drug combination with isoniazid, rifampicin, and pyrazinamide, based on rapid detection of isoniazid resistance using molecular testing, was noninferior to starting the recommended 4-drug regimen.

Variations in rifampicin and isoniazid resistance associated genetic mutations among drug naïve and recurrence cases of pulmonary tuberculosis

BackgroundThe resistance to first-line drugs can increase the risk of treatment failure and development of resistance to other anti-TB drugs. In TB endemic settings, a considerable rate of recurrence cases exhibited each, year which adds significant burden to the prevalence of disease worldwide. MethodsA total of 562 sputum samples were collected from presumptive positive clinical cases of MDR tuberculosis. Treatment history and demographic data of the patients were obtained after informed consent. Xpert MTB/RIF assay was performed for simultaneous detection of MTB and rifampicin resistance. The mutation patterns of isoniazid and rifampicin were observed after multiplex PCR and reverse hybridization by Genotype® MTBDRplus version 2.0 assay. ResultsA total of 73 of 97 cases (75.2%) of treatment failure were found positive for MDR-TB, whereas 79.6% newly diagnosed and 72.9% default cases were MDR in our isolates. The mutation of rpoB S531L was slightly higher in new treatment cases (89.3%) as compared to the default (80.4%) and failure cases (84.8%), whereas rpoB D516V mutation was more prevalent in default cases (19.6%) with complete absence of rpoB 526 mutation, which was observed in the other two types of cases. The mutation pattern of katG resistance differed among drug naïve and recurrence cases. The resistance in newly diagnosed cases was mostly conferred by katG 315 (49.1%) whereas in default (70.8%) and failure cases (63.3%) isoniazid resistance was commonly associated with katG S315T1 mutation. Mutations in inhA promoter region occurred at nucleotide position −8 and −15. In new cases the rate of mutation of C-15T was 3.7% and T-8A was 1.5% while in treatment failure cases the frequency for C-15T and T-8C was 2.5 and 3.8% respectively. However, no inhA promoter region mediated mutations were detected in default treatment cases. ConclusionRetreated cases are at more risk of developing hot spot mutations. An unusual difference in mutation pattern was determined in naïve and recurrence cases. Some mutations were exclusively associated with the retreatment of 35anti-TB drugs which suggest the increased risk of resistance with poor treatment outcome.

Study of the prevalence of Multidrug-Resistant Pulmonary Tuberculosis (MDR-TB) in Western Rajasthan using line probe assay.

Objective:To determine the prevalence of MDR-TB and find out the incidence of drug resistance using molecular diagnostic method. Line probe assay (LPA) is based on the principle of multiplex PCR is used to detect MTB (Mycobacterium tuberculosis) complex as well as its sensitivity to rifampicin and isoniazid.Method:This is a hospital-based prospective observational study. The sputum of MDR-TB suspected patients was subjected to Ziehl-Neelsen staining and smear positive samples were analyzed by LPA. Decontamination and digestion of the samples was done using the NALC- NaOH method (as defined in RNTCP guidelines). DNA extraction was done from the decontaminated samples using Geno Lyse kit. After DNA extraction, detection of MTB complex and rifampicin and/or INH resistance was done with the help of line probe assay (LPA) using GenoType ® MTBDRplus version 2.0.Results:Out of the 156 smear-positive sputum samples, 140 samples had LPA valid results. The most common age group of positive TB samples in this study was 30-40 years (26.42%). Twenty-five samples (17.85%) were found to be rifampicin resistant and 22 (15.71%) samples were found resistant to isoniazid. Sixteen patients (11.42%) were detected MDR. Nine patients (6.42%) were monoresistant to rifampicin and six patients (4.28%) were monoresistant to isoniazid. “Sputum positive retreatment cases” had the highest detection rate for MDR TB.Conclusion:Line probe assay is an economical and time saving method for the detection of MDR-TB and serves as a lifesaving tool for early diagnosis and treatment. This calls for a widespread national use of this assay. The detection of around 10% ZN-positive patients, who were not showing MTB complex in LPA may be a hidden iceberg for non-tubercular mycobacteria.

Variable ability of rapid tests to detect Mycobacterium tuberculosis rpoB mutations conferring phenotypically occult rifampicin resistance

We compared the ability of commercial and non-commercial, phenotypic and genotypic rapid drug susceptibility tests (DSTs) to detect rifampicin resistance (RR)-conferring ‘disputed’ mutations frequently missed by Mycobacterium Growth Indicator Tube (MGIT), namely L430P, D435Y, L452P, and I491F. Strains with mutation S450L served as positive control while wild-types were used as negative control. Of the 38 mutant strains, 5.7% were classified as RR by MGIT, 16.2% by Trek Sensititre MYCOTB MIC plate, 19.4% by resazurin microtiter plate assay (REMA), 50.0% by nitrate reductase assay (NRA), and 62.2% by microscopic observation direct susceptibility testing (MODS). Reducing MGIT rifampicin concentration to 0.5 µg/ml, and/or increasing incubation time, enhanced detection of disputed mutations from 5.7% to at least 65.7%, particularly for mutation I491F (from 0.0 to 75.0%). Compared with MGIT at standard pre-set time with 0.25 µg/ml ECOFF as breakpoint, we found a statistically significant increase in the ability of MGIT to resolve disputed mutants and WT strains at extended incubation period of 15 and 21 days, with 0.5 µg/ml and 1 µg/ml ECOFF respectively. MODS detected 75.0% of the I491F strains and NRA 62.5%, while it was predictably missed by all molecular assays. Xpert MTB/RIF, Xpert Ultra, and GenoscholarTB-NTM + MDRTB detected all mutations within the 81 bp RR determining region. Only GenoType MTBDRplus version 2 missed mutation L430P in 2 of 11 strains. Phenotypic and genotypic DSTs varied greatly in detecting occult rifampicin resistance. None of these methods detected all disputed mutations without misclassifying wild-type strains.

High prevalence of multi drug resistant tuberculosis in people living with HIV in Western India

BackgroundMost studies assessing drug resistant tuberculosis (DRTB) in human immunodeficiency virus (HIV) co-infected patients in India have used conventional culture- based systems to diagnose DRTB that have a longer turnaround time leading to risk of amplification of resistance to an empirical regimen. We determined the prevalence of DRTB amongst people living with HIV (PLHIV) using the line probe assay and determined risk factors associated with the presence of multi drug resistant tuberculosis (MDRTB).MethodsA Cross-sectional study was undertaken at Poona Hospital and Research Center (PHRC) and the Institute of Infectious Diseases, two tertiary level private care centers in Pune, India. Consenting PLHIV with confirmed Pulmonary TB (PTB) and/or extra-pulmonary TB (EPTB) diagnosed based on detection of Mycobacterium TB by line probe assay (Geno Type MTBDRplus version 2) on clinical specimens were included. Those with documented past history of DRTB were excluded. Resistance against anti-TB drugs was determined by the same assay. The prevalence of any form of drug resistant TB (DRTB), MDRTB, Rifampicin resistant TB (RRTB) and Isoniazid (INH) mono-resistant TB were determined as the proportion of these amongst all included PLHIV-TB. A multivariate analysis was conducted to determine risk factors that were statistically associated with MDRTB, DRTB, RRTB and INH mono-resistant TB.ResultsTwo hundred PLHIV were recruited. The prevalence (95% CI) of MDRTB, INH mono- resistance and RR resistance was 12.5% (7.9–17.1%), 9% (6.9–11.2%) and 2.5% (1.4–3.6%), respectively. The prevalence (95% CI) of MDRTB among new and relapsed patients was 8.8% (6.5–11.1%) and 23.1% (17.2–28.9%), respectively. Tuberculosis relapse was the only factor significantly associated with MDRTB, DRTB and INH mono-resistant TB.ConclusionWe document a high prevalence of drug resistance to anti-TB drugs including MDRTB among PLHIV in our setting using Geno Type MTBDRplus directly on clinical specimens. This validates the WHO recommendation of performing routine rapid molecular resistance testing prior to initiating anti-TB treatment among all PLHIV with presumptive TB. Using rapid molecular testing especially Geno Type MTBDRplus (that detects resistance to INH and Rifampicin simultaneously) reduces the turn-around time helping in optimizing treatment.

Diagnostic Performance of the GenoType MTBDRplus and MTBDRsl Assays to Identify Tuberculosis Drug Resistance in Eastern China.

Background:The WHO recently has recommended the GenoType MTBDRplus version 1.0 and MTBDRsl version 1.0 assays for widespread use in countries endemic with drug-resistant tuberculosis. Despite this, these assays have rarely been evaluated in China, where the burden of drug-resistant tuberculosis is among the highest globally.Methods:Mycobacterium tuberculosis clinical isolates were obtained between January 2008 and December 2008. Isolates were tested for drug resistance against rifampicin (RFP) and isoniazid (INH) using the GenoType MTBDRplus assay and drug resistance against ethambutol (EMB), ofloxacin (OFX), and kanamycin (KM) using the Genotype MTBDRsl assay. These results were compared with conventional drug-susceptibility testing (DST).Results:Readable results were obtained from 235 strains by GenoType MTBDRplus assay. Compared to DST, the sensitivity of GenoType MTBDRplus assay to detect RFP, INH, and multidrug resistance was 97.7%, 69.9%, and 69.8%, respectively, whereas the specificity for detecting RFP, INH, and multidrug resistance was 66.7%, 69.2%, and 76.8%, respectively. The sensitivity and specificity of the GenoType MTBDRsl assay were 90.9% and 95.2% for OFX, 77.8% and 99.5% for KM, 63.7% and 86.4% for EMB, respectively. Mutations in codon S531L of the rpoB gene and codon S315T1 of KatG gene were dominated in multidrug-resistant tuberculosis (MDR-TB) strains.Conclusions:In combination with DST, application of the GenoType MTBDRplus and MTBDRsl assays may be a useful supplementary tool to allow a rapid and safe diagnosis of multidrug resistance and extensively drug-resistant tuberculosis.

Analytical and clinical performance characteristics of the Abbott RealTime MTB RIF/INH Resistance, an assay for the detection of rifampicin and isoniazid resistant Mycobacterium tuberculosis in pulmonary specimens

Clinical management of drug-resistant tuberculosis patients continues to present significant challenges to global health. To tackle these challenges, the Abbott RealTime MTB RIF/INH Resistance assay was developed to accelerate the diagnosis of rifampicin and/or isoniazid resistant tuberculosis to within a day. This article summarizes the performance of the Abbott RealTime MTB RIF/INH Resistance assay; including reliability, analytical sensitivity, and clinical sensitivity/specificity as compared to Cepheid GeneXpert MTB/RIF version 1.0 and Hain MTBDRplus version 2.0. The limit of detection (LOD) of the Abbott RealTime MTB RIF/INH Resistance assay was determined to be 32 colony forming units/milliliter (cfu/mL) using the Mycobacterium tuberculosis (MTB) strain H37Rv cell line. For rifampicin resistance detection, the Abbott RealTime MTB RIF/INH Resistance assay demonstrated statistically equivalent clinical sensitivity and specificity as compared to Cepheid GeneXpert MTB/RIF. For isoniazid resistance detection, the assay demonstrated statistically equivalent clinical sensitivity and specificity as compared to Hain MTBDRplus. The performance data presented herein demonstrate that the Abbott RealTime MTB RIF/INH Resistance assay is a sensitive, robust, and reliable test for realtime simultaneous detection of first line anti-tuberculosis antibiotics rifampicin and isoniazid in patient specimens.

Evaluation &validation of a highly multiplexed LATE-PCR single-tube assay for M(X)DR-TB

Background: In 2006, the World Health Organization (WHO) and the Stop TB partnership called for strengthening of diagnostic services and highlighted the need for the development of rapid diagnostics to fight the tuberculosis pandemic. In 2011, WHO estimated that approximately 630,000 (5.3%) of the 12 million TB cases had multiple drug resistant (MDR)-TB, while more than 80 countries have reported cases of extremely drug-resistant (XDR)TB. Only a small fraction of reported cases (<20%) were correctly diagnosed and even fewer were treated according to WHO standards. In response the WHO endorsed the Genotype® MTBDRplus (version 1.0) line probe assay (LPA) in 2008 and theXpert®MTB/RIF assay in 2010. But these tests only provide evidence for resistance to isoniazid and/or rifampicin. There continues to be a critical need for a more comprehensive convenient diagnostic technology. The highly multiplexed LATE-PCR assay for M(X)DR-TB described here was designed to meet that need. Our goal was to firmly establish that a highly multiplexed Linear-After-the-Exponential (LATE) PCR single closed-tube assay can simultaneously detect and distinguish multiple mutations in multiple gene targets that are known to confer resistance to isoniazid, rifampicin, ethambutol, ofloxacin, amikacin, kanamycin and capreomycin. Methods & Materials: In this initial study, DNA from clinical isolates with different rpoB, katG, embB, inhA promoter, gyrB, gyrA and rrs genotypes were selected from a DNA bank housed at Stellenbosch University. Each DNA samples was amplified and the singled-strandedDNAproductswere scanned formutations at endpoint using the same mixture of Lights-On/Lights-Off Probes. The resulting fluorescent signatures were compared to that of H37Rv, a pan-susceptible “wildtype” strain. Results: Each clinical isolate harbouring a uniquemutation had its own, highly reproducible fluorescent signature distinct from that of H37Rv, as well as all other isolates with differentmutations. Conclusion: This study achieved the intended transfer of the Brandeis University technology to Stellenbosch University. This study also demonstrates that this single tube multiplexed assay can simultaneously distinguish the different mutations that confer resistance to rifampicin, isoniazid, ethambutol, fluoroquinolones, aminoglycosides and ethionamide in less than three hours. (Supported by NIH Grant R01 A1099532)

The impact of expanded testing for multidrug resistant tuberculosis using genotype [correction of geontype] MTBDRplus in South Africa: an observational cohort study.

IntroductionGlobally, multidrug resistant tuberculosis (MDR-TB) remains underdiagnosed. The Genotype MTBDRplus®, a rapid drug susceptibility testing (DST) assay used to detect resistance to isoniazid and rifampicin in the diagnosis of MDR-TB, has good diagnostic accuracy, but its impact on patient outcomes in routine practice is unproven. We assessed the clinical impact of routine DST using MTBDRplus in a single health district in South Africa.MethodsData were collected on all adult pulmonary TB patients registered at 25 public health clinics in the periods before and after introduction of an expanded DST algorithm using MTBDRplus version 1.0.ResultsWe collected data on 1176 TB patients before implementation and 1177 patients afterwards. In the before period, measured MDR-TB prevalence among new cases was 0.7% (95% CI1.4–3.1%), and among retreatment cases 6.2% (95% CI:3.5–8.8%), versus 3.7% (95% CI:2.4–5.0, p<0.01) and 6.6% (95% CI:3.8–9.4%, p = 0.83) respectively after MTBDRplus introduction. The median times from sputum collection to MDR treatment in the before and after periods were 78 days (IQR:52–93) and 62 days (IQR:32–86, p = 0.05), respectively. Among MDR-TB cases, 27% (95%CI:10–44) in the before period converted sputum cultures to negative by 8 months following treatment initiation, while 52% (95%CI:38–66) converted in the intervention period (p = 0.04).ConclusionsThe expanded use of MTBDRplus DST resulted in a substantial increase in the proportion of new cases identified as MDR-TB; though time to MDR treatment was reduced, it was still over two months. Culture conversion for MDR-TB patients improved after introduction of MTBDRplus. This work illustrates the mixture of successes and challenges resulting from increased access to rapid DST in a setting with a high TB burden.

The Diagnostic Performance of the GenoType MTBDR plus Version 2 Line Probe Assay Is Equivalent to That of the Xpert MTB/RIF Assay

Molecular diagnostics for Mycobacterium tuberculosis have recently been endorsed by the World Health Organization. The Xpert MTB/RIF assay was endorsed for use on patient material, regardless of smear gradation, while the GenoType MTBDRplus (version 1) has been limited for use on smear-positive patient material. In this study, we evaluated the diagnostic performance of the Xpert MTB/RIF and GenoType MTBDRplus (version 2) assays on smear-positive and smear-negative patient specimens submitted to a high-throughput diagnostic laboratory. A total of 282 consecutive specimens were subjected to the two new molecular assays, and their performance characteristics were assessed relative to the routine diagnostic standard. Both assays showed similar diagnostic performance characteristics. The sensitivities of the GenoType MTBDRplus (v2.0) and Xpert MTB/RIF assays for the detection of culture-positive M. tuberculosis were 73.1% and 71.2%, respectively, while the specificities of both assays were 100%. Both assays were able to diagnose the presence of M. tuberculosis in 57 to 58% of smear-negative cases, suggesting that the performance characteristics were dependent on bacillary load. The detection of M. tuberculosis in culture-negative specimens confirmed that molecular assays should not be used for treatment monitoring. The sensitivity and specificity for rifampin resistance detection were 100% in both assays; however, the GenoType MTBDRplus (v2.0) assay provided additional information on isoniazid susceptibility. The GenoType MTBDRplus (v2.0) assay will complement the Xpert MTB/RIF screening assay by validating rifampin susceptibility and providing information on isoniazid susceptibility. In addition, the GenoType MTBDRplus (v2.0) assay will provide pharmacogenetic information that may be critical in guiding appropriate treatment.