NovelkatGmutations causing isoniazid resistance in clinicalM. tuberculosisisolates

Abstract

We report the discovery and confirmation of 23 novel mutations with previously undocumented role in isoniazid (INH) drug resistance, in catalase-peroxidase (katG) gene of Mycobacterium tuberculosis (Mtb) isolates. With these mutations, a synonymous mutation in fabG1g609a, and two canonical mutations, we were able to explain 98% of the phenotypic resistance observed in 366 clinical Mtb isolates collected from four high tuberculosis (TB)-burden countries: India, Moldova, Philippines, and South Africa. We conducted overlapping targeted and whole-genome sequencing for variant discovery in all clinical isolates with a variety of INH-resistant phenotypes. Our analysis showed that just two canonical mutations (katG 315AGC-ACC and inhA promoter-15C-T) identified 89.5% of resistance phenotypes in our collection. Inclusion of the 23 novel mutations reported here, and the previously documented point mutation in fabG1, increased the sensitivity of these mutations as markers of INH resistance to 98%. Only six (2%) of the 332 resistant isolates in our collection did not harbor one or more of these mutations. The third most prevalent substitution, at inhA promoter position -8, present in 39 resistant isolates, was of no diagnostic significance since it always co-occurred with katG 315. 79% of our isolates harboring novel mutations belong to genetic group 1 indicating a higher tendency for this group to go down an uncommon evolutionary path and evade molecular diagnostics. The results of this study contribute to our understanding of the mechanisms of INH resistance in Mtb isolates that lack the canonical mutations and could improve the sensitivity of next generation molecular diagnostics.

Detection of <I>kat</I>G and <I>inh</I>A mutations to guide isoniazid and ethionamide use for drug-resistant tuberculosis

“…As well as providing an accurate diagnosis for MDR-TB, LPAs also provide information about the level of INH resistance, depending on the mutation detected. 16 Katiyar et al compared high doses of INH (16–18 mg/kg), a normal dose of INH and placebo in a trial of 123 MDR-TB subjects. They found that high doses of INH led to rapid culture negativity of sputum and increased the likelihood of sputum being negative by month 6 of treatment compared with the other two groups.…”

Frequency and Distribution of Tuberculosis Resistance-Associated Mutations between Mumbai, Moldova, and Eastern Cape

“…Additionally, although the mutations that we identified provide a larger picture of the genetic basis of phenotypic antituberculosis drug resistance, they do not represent a complete genetic profile of the DR-TB specimens evaluated in this study. The inclusion of other gene regions, such as novel katG and fabG1 mutations recently associated with INH resistance (32), may further increase the sensitivity of rapid molecular diagnostic assays for DR-TB detection. Additional sequencing studies investigating other genes and gene regions, such as tlyA and gidB mutations and their association with injectable resistance (12), will be necessary to identify the genetic basis of drug resistance for the 4 to 16% of genetically wild-type, drugresistant specimens in this study.…”

Validation of novelMycobacterium tuberculosisisoniazid resistance mutations not detectable by common molecular tests

“…The current model of exclusively using molecular diagnostics to target INH resistance mutations inside of “hotspots” may ultimately select for clinical strains that have non-canonical INH resistance mutations by depleting those with common ones from the host population. This scenario was already hypothesized by Torres and colleagues, who found >20 clinical katG mutations outside codon S315 by WGS of and used site-directed mutagenesis to show that several could independently confer INH resistance to Mycobacterium smegmatis (43). There is evidence to suggest that the rise of non-canonical INH resistance mutations is already happening in India, where there is a high burden of drug-resistant TB (65).…”

“…Likewise, N138S, W300R, S315G, S315N, W328R were also expected to increase the INH MIC since mutations at all of those residues are associated with decreased INH-NADH adduct (active drug) formation in biochemical studies of KatG enzyme (40). Notably, the W161Q, W161R, E402stop, L415P, and A480del mutations were not reported in the literature and A110V has only been reported in the presence of other confounding mutations (43, 64).…”