Evaluation of the GenoType MTBDRplus assay for rifampin and isoniazid susceptibility testing of Mycobacterium tuberculosis strains and clinical specimens

Abstract

The new GenoType MTBDRplus assay (Hain Lifescience GmbH, Nehren, Germany) was tested with 125 clinical isolates and directly with 72 smear-positive sputum specimens for its ability to detect rifampin (RMP) and isoniazid (INH) resistance in Mycobacterium tuberculosis complex (MTBC) strains. In total, 106 RMP(r)/INH(r), 10 RMP(s)/INH(r), and 80 RMP(s)/INH(s) MTBC strains were comparatively analyzed with the new and the old MTBDR assays. Besides the detection of mutations within the 81-bp hot spot region of rpoB and katG codon 315, the GenoType MTBDRplus assay is designed to detect mutations in the regulatory region of inhA. The applicability of the new assay directly to specimens was shown, since 71 of 72 results for smear-positive sputa and all 125 results for clinical isolates were interpretable and no discrepancies compared with the results of real-time PCR or DNA sequencing were obtained. In comparison to conventional drug susceptibility testing, both assays were able to identify RMP resistance correctly in 74 of 75 strains (98.7%) and 30 of 31 specimens (96.8%). The misidentification of RMP resistance was obtained for two strains containing rpoB P533L mutations. Compared to the old MTBDR assay, the new GenoType MTBDRplus assay enhanced the rate of detection of INH resistance from 66 (88.0%) to 69 (92.0%) among the 75 INH-resistant strains and 36 (87.8%) to 37 (90.2%) among the 41 specimens containing INH-resistant strains. Thus, the new GenoType MTBDRplus assay represents a reliable and upgraded tool for the detection of INH and RMP resistance in strains or directly from smear-positive specimens.

FIG. 1.

Representative patterns of a pansusceptible strain (rows 1) and an MDR strain (rows 2) which had an inhA C15T mutation in the regulatory region of inhA obtained by the new MTBDRplus assay (B) but not the MTBDR assay (A). The positions of the oligonucleotides and the marker lines are given. The targeted genes and specificity are shown from left to right, as follows: for the MTBDR assay (A), conjugate control (CC); amplification control (UC); M. tuberculosis complex-specific control (Tub); rpoB amplification control; rpoB wild-type probes WT1 to WT5 located in the hot spot region WT 1 to WT 5, respectively; rpoB mutant probes (probes MUT1, MUT2A, MUT2B, and MUT3) with mutations in codons rpoB D516V, H526Y, H526D, and S531L, respectively; katG amplification control; katG codon 315 wild-type probe (WT); and katG codon 315 mutation probes (T1 and T2, respectively) with AGC-to-ACC (S315T1) and AGC-to-ACA (S315T2) exchanges, respectively. The MTBDR plus assay (A) has three additional rpoB wild-type probes (resulting in probes WT1 to WT8) and targets the regulatory region of the inhA gene with the inhA amplification control (WT 1 to WT 8, respectively); inhA gene wild-type probes WT1, which spans the region from positions −9 to −22, and WT, which spans the region from positions −1 to −12; and inhA mutant probes MUT1, MUT2, MUT3A, and MUT3B with mutations −15C/T, −16A/G, −8T/C, and −8T/A, respectively (MUT 1, MUT 2A, MUT 2B, and MUT 3, respectively). Pansusceptible isolate 1 was positive with the wild-type rpoB and katG probes of the MTBDR assay and, additionally, with the inhA wild-type probes of the MTBDRplus assay. MDR isolate 2 showed RMP resistance in both assays (for the MTBDR assay, the omission of rpoB WT 5 and positivity for rpoB Mut 3; for the MTBDRplus assay, the omission of rpoB WT 8 and positivity for rpoB Mut 3). Concerning INH resistance, isolate 2 had to be interpreted as INH susceptible (katG wild-type probe) by the MTBDR assay, whereas the MTBDRplus assay indicated INH resistance (omission of the inhA WT1 probe and positivity for the inhA MUT1 probe). Lanes M, colored markers.