Minor Contribution of inhA-15 Mutations to the Rapid Detection of Isoniazid Resistance in Mycobacterium Tuberculosis Isolates

Dear Editor, In the present work, the necessity for the use of inhA-15 testing in detecting isoniazid resistance was challenged for the first time. We found that the molecular detection of inhA-15 C to T alterations in the fast detection of isoniazid-resistant tuberculosis (TB) is not necessary and should not be recommended for routine work. In this descriptive study, out of 98 clinical isolates of Mycobacterium tuberculosis at The Tuberculosis Research Center in the Iranian city of Arak, 64 isolates that had already been determined to be phenotypically resistant or susceptible to isoniazid were analyzed for mutations in the regulatory region of inhA-15 with 2 allele-specific polymerase chain reaction (AS-PCR) and sequence methods. The H37Rv strain was used as a negative control. The mutations in inhA-15 were determined by using 3 primers, namely TB92, TB93, and Rmut, via AS-PCR. The primer sequences were as: TB92: 3-CCTCGCTGCCCAGAAAGGGA-5, TB93: 3-ATCCCCCGGTTTCCTCCGGT-5, Rmut: 5-AGTCACCCCGACAACCTATTA-5. The detection of mutations in codon 315 of the katG gene was performed in accordance with a previous study.2 In order to prove whether or not the mutations occurred in the inhA-15 point and to confirm the methodology of AS-PCR, we employed the sequence method (the gold standard of molecular methods). Therefore, 248 bp bands containing the promoter region of inhA-15 were amplified with primers TB92 and TB93 by PCR. The PCR products were purified and sequenced using an ABI apparatus. The strains harboring mutation in inhA-15 generated a band at 174 bp. Primers TB92 and TB93 created 248 bp bands in all the strains. Molecular Evolutionary Genetics Analysis (MEGA) software determined that TB92 bound to the nucleotide -169 upstream of the inhA gene and to the inside of the fabG1 gene (or mabA) and that it was a forward primer in PCR. TB93 bound to the nucleotide +79 of the inhA gene, and it was a reverse primer making a 248 bp band with TB92. The Rmut primer is a reverse primer that binds to the nucleotide +5 to -15 inhA promoter region in mutant strains and creates a 174 bp band with TB92. This primer is designed for mutant strains, and strains without mutations in this region do not show this band. Figure 1 depicts a view of the situation of the primers and AS-PCR-resulted products in a strain harboring mutation at inhA-15. The AS-PCR product was electrophoresed on 1.5% agarose gel. In this context, the mutant strains in inhA-15 showed 2 bands of 248 bp and 174 bp, and the strains with no mutations in this region were only 248 bp band, where there was 98% phenotypic compliance. As a result, of the 42 strains resistant to isoniazid, 5 strains had mutations in inhA-15. Also, of the 22 strains susceptible to isoniazid, 1 strain (901H) had a mutation in that area. In other words, this strain was phenotypically susceptible to isoniazid, and while there was no mutation in katG315 (which is perfectly logical), both AS-PCR and sequence methods showed mutations in inhA-15.