Evaluation of the Genotype MTBDR assay for rapid detection of rifampin and isoniazid resistance in Mycobacterium tuberculosis isolates

Abstract

A novel PCR-based reverse hybridization method Genotype MTBDR assay (Hain Lifescience GmbH, Nehren, Germany) was evaluated for rapid detection of rifampin (RIF) and isoniazid (INH) resistance in Turkish Mycobacterium tuberculosis isolates. The Genotype MTBDR assay is designed to detect mutations within the 81-bp hotspot region of rpoB and mutations at katG codon 315. A total of 41 RIF-resistant M. tuberculosis isolates with rpoB mutations that were previously tested by the INNO-LiPA Rif.TB kit and also characterized by DNA sequencing were included in the study. Thirty-seven of these isolates were also resistant to INH. RIF resistance was correctly identified in 39 of 41 isolates (95.1%) with the Genotype MTBDR assay probes specific for these mutations. One isolate with a Gln-490-His mutation and another one with a CGG insertion between codons 514 and 515 were identified as RIF sensitive by the Genotype MTBDR assay. While the INNO-LiPA Rif.TB kit was able to determine the CGG insertion between codons 514 and 515, the Gln-490-His mutation outside the 81-bp hotspot region was not detected by the INNO-LiPA Rif.TB kit. These isolates had MICs of >or=32 microg/ml for RIF. The Genotype MTBDR assay also correctly identified 27 of 37 INH-resistant isolates (73%) with mutations in katG codon 315. In conclusion, the Genotype MTBDR assay may be useful for the rapid diagnosis of the most common mutations found in multidrug-resistant M. tuberculosis strains. However, the test results should always be confirmed with phenotypic methods.

FIG. 1.

Locations of Genotype MTBDR and INNO-LiPA Rif.TB probes within the 81-bp hotspot cluster of the rpoB gene.

FIG. 2.

Representative results for patterns of the isolate which had a CGG insertion between codons 514 and 515 obtained with the Genotype MTBDR assay and INNO-LiPA Rif.TB assay. The positions of the oligonucleotides and the marker lines are given. The specificity and targeted genes of the lines are shown from top to bottom as follows. The Genotype MTBDR assay (lane 1): conjugate control; amplification control (23S rRNA); M. tuberculosis complex-specific control (23S rRNA); control for rpoB amplification; rpoB wild-type probes located in the 81-bp hotspot region 5 to 9; rpoB mutant (Mut) probes with mutations in codons 516, 526, and 531; control for katG amplification; katG codon 315 wild-type probe; katG codon 315 mutation probes (sequences in parentheses). The INNO-LiPA Rif.TB assay (lane 2): conjugate control; M. tuberculosis complex-specific control; rpoB wild-type probes located in the 81-bp hotspot region 3 to 7; rpoB mutant probes with mutations in codons 516, 526, and 531. In lane 1, the isolate was positive with the control probes, wild-type rpoB probes, and katG S315T2 (ACA) probe and evaluated as RIF sensitive and INH resistant by the Genotype MTBDR assay. In lane 2, the isolate was positive with the control probes and wild-type rpoB probes S3, S4, and S5 but negative with the wild-type rpoB probes S1 and S2 and evaluated as RIF resistant by INNO-LiPA Rif.TB assay.