Evaluation of genotype MTBDRsl assay to detect drug resistance associated with fluoroquinolones, aminoglycosides and ethambutol on clinical sediments

Abstract

Background: The emergence of resistant tuberculosis (TB) is a major setback to the global control of the disease as the treatment of such resistance is complex and expensive. Use of direct detection of mutations by molecular methods could facilitate rapid diagnosis of resistance to offset diagnostic delays. We evaluated the performance of the Genotype MTBDRsl (Hain Life Sciences) for the detection of second line resistant TB directly from stored smear positive sputum sediments.

Methodology/principal findings: The assay showed a diverse range of sensitivity and specificity, 91.26% [95% CI, 84-96] and 95.5% [95% CI, 87-99] for FQ (PPV ∼97% & NPV ∼ 87.67%), 56.19% [95%CI, 46-66] and 81% [95%CI, 66-91] for EMB (PPV ∼ 88.06% & NPV ∼ 43.21%) and 100% for SLD. Diagnostic accuracy for FQ, SLD and EMB was 94%, 100% and 63.51%, respectively. 1.17% (2/170) were heteroresistance strains, where the heteroresistance was linked to rrs gene. A varying rate of validity was observed 100% (170/170) for FQ, 94.11% (160/170) for EMB, 88.23% (150/170) for SLD.

Conclusions/significance: Genotype MTBDRsl is simple, rapid, economical assay that can be used to detect commonly known resistance associated with Fluoroquinolone, second line injectable drugs and ethambutol. The assay detects the targeted resistance in less time as compared to phenotypic DST. But due to low NPV to FQ (88%) and EMB (43.21%), the assay results must be interpreted in coordination with the phenotypic DST.

Figure 1. Representative DNA patterns obtained with GenoType MTBDRsl.

The positions of the oligonucleotides and control probes are given on the left. The targeted genes and specific probes lines are shown from top to bottom as follows: conjugate control (CC); amplification control (AC); M. tuberculosis complex-specific control (TUB); gyrA amplification control; gyrA wild-type probes WT1 to WT3 (85–90, 89–93 and 92–97); gyrA mutant probes MUT1, MUT2, MUT3A, MUT3B, MUT3C, and MUT3D for codons A90V, S91P, D94A, D94N, D94Y, D94G, and D94H, respectively; rrs amplification control; rrs wild-type probes WT1 (codons 1401 and 1402) and WT2 (codon 1484); rrs mutant probes MUT1 and MUT2, with A1401G and G1484T changes, respectively; embB amplification control; embB wild-type probe WT1, covering codon 306; and embB probes MUT1A and MUT1B for the mutations M306I and M306V, respectively. Lane 1, example of an fluoroquinolone, second line aminoglycoside and ethambutol susceptible; lane 2, fluoroquinolone resistance due to mut 3C, aminoglycosides susceptible and ethambutol resistance due to embB mutant M306V; lane 3, fluoroquinolone and aminoglycoside susceptible and ethambutol resistance with M306V.