Comparison of Xpert MTB/RIF Assay and GenoType MTBDRplus DNA Probes for Detection of Mutations Associated with Rifampicin Resistance in Mycobacterium tuberculosis.

BACKGROUND:GeneXpert MTB/RIF (Xpert) and Genotype MTBDRplus (DRplus) are two World Health Organization (WHO) endorsed probe based molecular drug susceptibility testing (DST) methods for rapid diagnosis of drug resistant tuberculosis. Both methods target the same 81 bp Rifampicin Resistance Determining Region (RRDR) of bacterial RNA polymerase β subunit (rpoB) for detection of Rifampicin (RIF) resistance associated mutations using DNA probes. So there is a correspondence of the probes of each other and expected similarity of probe binding. METHODS:We analyzed 92 sputum specimens by Xpert, DRplus and LJ proportion method (LJ-DST). We compared molecular DSTs with gold standard LJ-DST. We wanted to see the agreement level of two molecular methods for detection of RIF resistance associated mutations. The 81bp RRDR region of rpoB gene of discrepant cases between the two molecular methods was sequenced by Sanger sequencing. RESULTS:The agreement of Xpert and DRplus with LJ-DST for detection of RIF susceptibility was found to be 93.5% and 92.4%, respectively. We also found 92.4% overall agreement of two molecular methods for the detection of RIF susceptibility. A total of 84 out of 92 samples (91.3%) had agreement on the molecular locus of RRDR mutation by DRplus and Xpert. Sanger sequencing of 81bp RRDR revealed that Xpert probes detected seven of eight discrepant cases correctly and DRplus was erroneous in all the eight cases. CONCLUSION:Although the overall concordance with LJ-DST was similar for both Xpert and DRplus assay, Xpert demonstrated more accuracy in the detection of RIF susceptibility for discrepant isolates compared with DRplus. This observation would be helpful for the improvement of probe based detection of drug resistance associated mutations especially rpoB mutation in M. tuberculosis

Comparison of Xpert and DR<i>plus</i> assay for detection of RIF susceptibility, n = 92.

<p>Comparison of Xpert and DR<i>plus</i> assay for detection of RIF susceptibility, n = 92.</p

Comparison of molecular and phenotypic DST methods for detection of RIF susceptibility, n = 92.

<p>Comparison of molecular and phenotypic DST methods for detection of RIF susceptibility, n = 92.</p

Data from: Performance of TaqMan Array Card to detect TB drug resistance on direct specimens

Culture based phenotypic drug susceptibility testing (DST) for Mycobacterium tuberculosis (TB) is time consuming therefore rapid genotypic methods are increasingly being utilized. We previously developed and evaluated on TB isolates a rapid genotypic TaqMan array card (TAC) that detects mutations in several resistance-associated genes using dozens of primer pairs, probes, and high resolution melt analysis, with >96% accuracy versus Sanger sequencing. In this study we examined the performance of TAC on sputum, comparing results between 71 paired sputum and multi drug resistant TB isolates. We also adapted the TAC to include wild-type probes and broadened coverage for rpoB and gyrA mutations. TAC was 89% successful at detecting wild-type or mutations within inhA, katG, rpoB, eis, gyrA, rplC, and pncA on smear positive sputa and 33% successful on smear negative sputa. The overall accuracy of these detections as compared to the TAC results of the paired isolate was 95% ± 7 (average sensitivity 98% ± 3; specificity 92% ± 14). Accuracy of sputum TAC results versus phenotypic DST for isoniazid, rifampin, amikacin/kanamycin, ofloxacin/moxifloxacin, and pyrazinamide was 87% ± 11. This was similar to that of the paired isolate TAC results (accuracy 90% ± 12) and inaccuracies primarily reflected intrinsic genotypic-phenotypic discordance. The TAC is a rapid, modular, comprehensive, and accurate TB DST for the major first and second line TB drugs on smear positive sputum

Drug susceptibility result of 8 discrepant cases between two molecular DST methods.

<p>Drug susceptibility result of 8 discrepant cases between two molecular DST methods.</p

Aligned sequences of discrepant cases (n = 8) and associated mutations.

<p>Aligned sequences of discrepant cases (n = 8) and associated mutations.</p

Comparison of Xpert MTB/RIF Assay and GenoType MTBDR<i>plus</i> DNA Probes for Detection of Mutations Associated with Rifampicin Resistance in <i>Mycobacterium tuberculosis</i>

<div><p>Background</p><p>GeneXpert MTB/RIF (Xpert) and Genotype MTBDR<i>plus</i> (DR<i>plus</i>) are two World Health Organization (WHO) endorsed probe based molecular drug susceptibility testing (DST) methods for rapid diagnosis of drug resistant tuberculosis. Both methods target the same 81 bp Rifampicin Resistance Determining Region (RRDR) of bacterial RNA polymerase β subunit (<i>rpoB</i>) for detection of Rifampicin (RIF) resistance associated mutations using DNA probes. So there is a correspondence of the probes of each other and expected similarity of probe binding.</p><p>Methods</p><p>We analyzed 92 sputum specimens by Xpert, DR<i>plus</i> and LJ proportion method (LJ-DST). We compared molecular DSTs with gold standard LJ-DST. We wanted to see the agreement level of two molecular methods for detection of RIF resistance associated mutations. The 81bp RRDR region of <i>rpoB</i> gene of discrepant cases between the two molecular methods was sequenced by Sanger sequencing.</p><p>Results</p><p>The agreement of Xpert and DR<i>plus</i> with LJ-DST for detection of RIF susceptibility was found to be 93.5% and 92.4%, respectively. We also found 92.4% overall agreement of two molecular methods for the detection of RIF susceptibility. A total of 84 out of 92 samples (91.3%) had agreement on the molecular locus of RRDR mutation by DR<i>plus</i> and Xpert. Sanger sequencing of 81bp RRDR revealed that Xpert probes detected seven of eight discrepant cases correctly and DR<i>plus</i> was erroneous in all the eight cases.</p><p>Conclusion</p><p>Although the overall concordance with LJ-DST was similar for both Xpert and DR<i>plus</i> assay, Xpert demonstrated more accuracy in the detection of RIF susceptibility for discrepant isolates compared with DR<i>plus</i>. This observation would be helpful for the improvement of probe based detection of drug resistance associated mutations especially <i>rpoB</i> mutation in <i>M</i>. <i>tuberculosis</i>.</p></div

Prevalence and incidence of tuberculosis infection among healthcare workers in chest diseases hospitals, Bangladesh: Putting infection control into context.

BackgroundHealthcare workers (HCWs) are at increased risk of tuberculosis infection (TBI). We estimated the prevalence and incidence of TBI and risk factors among HCWs in Bangladeshi hospitals to target TB infection prevention and control (IPC) interventions.MethodsDuring 2013-2016, we conducted a longitudinal study among HCWs in four chest disease hospitals. At baseline, we administered a questionnaire on sociodemographic and occupational factors for TB, tuberculin skin tests (TST) in all hospitals, and QuantiFERON ®-TB Gold in-Tube (QFT-GIT) tests in one hospital. We assessed factors associated with baseline TST positivity (induration ≥10mm), TST conversion (induration increase ≥10mm from baseline), baseline QFT-GIT positivity (interferon-gamma ≥0.35 IU/mL), and QFT-GIT conversion (interferon-gamma ResultsOf the 758 HCWs invited, 732 (97%) consented to participate and 731 completed the one-step TST, 40% had a positive TST result, and 48% had a positive QFT-GIT result. In multivariable models, HCWs years of service 11-20 years had 2.1 (95% CI: 1.5-3.0) times higher odds of being TST-positive and 1.6 (95% CI 1.1-2.5) times higher odds of QFT-GIT-positivity at baseline compared with those working ≤10 years. HCWs working 11-20 years in pulmonary TB ward had 2.0 (95% CI: 1.4-2.9) times higher odds of TST positivity, and those >20 years had 2.5 (95% CI: 1.3-4.9) times higher odds of QFT-GIT-positivity at baseline compared with those working ConclusionsPrevalent TST and QFT-GIT positivity was associated with an increased number of years working as a healthcare worker and in pulmonary TB wards. The incidence of TBI among HCWs suggests ongoing TB exposure in these facilities and an urgent need for improved TB IPC in chest disease hospitals in Bangladesh