Detection of mutations associated with isoniazid and rifampin resistance in Mycobacterium tuberculosis isolates from Samara Region, Russian Federation

Abstract

High incidence rates of isoniazid-, rifampin-, and multiple-drug-resistant tuberculosis have been reported in countries of the former Soviet Union (FSU). Genotypic (unlike phenotypic) drug resistance assays do not require viable cultures but require accurate knowledge of both the target gene and the mutations associated with resistance. For these assays to be clinically useful, they must be able to detect the range of mutations seen in isolates from the population of tuberculosis patients to which they are applied. Two novel macroarrays were applied to detect mutations associated with rifampin (rpoB) and isoniazid (katG and inhA) resistance. In a sample of 233 isolates from patients in Samara, central Russia, 46.5% of isolates possessed mutations in both the rpoB and the katG (or inhA) genes. Combined results from the macroarrays demonstrated concordance in 95.4 and 90.4% of phenotypically defined rifampin- and isoniazid-resistant isolates, respectively. The contribution of different mutations to resistance was comparable to that reported previously for non-FSU countries, with 90% of rifampin-resistant isolates and 93% of isoniazid resistant isolates due to rpoB531 and katG315 mutations, respectively. The percentage of phenotypically resistant rifampin isolates with no mutations in the rpoB codons 509 to 536 was 4.2%, which was similar to previous reports. Novel macroarrays offer a rapid, accurate, and relatively cheap system for the identification of rifampin-, isoniazid-, and multiple-drug-resistant Mycobacterium tuberculosis isolates.

FIG. 1.

Macroarray design. (Panel 1) MDRTB screening array and examples of arrays hybridized with labeled PCR products. (A and B) Duplicate hybridized arrays showing WT rpoB, katG, and inhA genotypes, indicative of a rifampin- and isoniazid-susceptible isolate. Rifampin-sensitive probes, such as all rpoB probes (MRURP3, -6, -9, -12, -17, and -22), show hybridization and so have the WT phenotype; isoniazid-sensitive probes, such as K315WTC, show binding, indicating the WT K315GC genotype; and TOMIWT shows hybridization, indicating a WT inhA locus. (C and D) Duplicate arrays showing mutant rpoB (arrow labeled 1) and katG (arrow labeled 2) genotypes indicative of an MDRTB isolate. Rifampin-resistant probes, such as MRURP22, show absence of binding (indicating a mutation in codon 531); isoniazid-resistant probes, marked by an absence of binding to K315WTC and corresponding hybridization with K315GC, indicate the KG315 AGC→ACC genotype; hybridization to TOMIWT and an absence of binding to TOMIMUT1 indicate a WT inhA locus. (Panel 2) Diagram of the rpoB array in which numbers 1 to 27 represent WT rpoB probes MRURP1 to -27, respectively. A, orientation spot; B, color development control.