Phenotypic and genotypic characterization of isoniazid resistance mutations in Mycobacterium tuberculosis isolates from new and previously treated patients in the Tshwane region

Abstract

Background: Isoniazid (INH) is one of the most potent anti-tuberculosis (TB) drugs and a key component of the multi-drug regimen for the treatment of TB. Tuberculosis cases with initial INH resistance are more at risk of poor clinical outcomes than those susceptible to INH among cases receiving standard first-line chemotherapy. Drug resistance arises mainly from spontaneous mutations in the Mycobacterium tuberculosis (M. tuberculosis) genome. Resistance to INH is more complex mainly because it may involve mutations in one or more of several genes, such as katG, inhA, ndh, kasA and ahpC. However, resistance in the katG and inhA genes is responsible for approximately 65% of all instances of INH resistance. Continued usage of INH has been compromised by the increasing prevalence of INH resistance among M. tuberculosis strains. Furthermore, accurate molecular diagnosis of drug resistance depends on knowledge of mechanisms conferring resistance to anti-TB drugs. Early detection of INH resistance is essential to allow proper initial drug therapy and a decrease in MDR-TB.

Different M. tuberculosis lineages have been reported to be associated with different levels of pathogenicity, are thought to drive resistance and to play a role in virulence characteristics and transmission. Seven M. tuberculosis lineages have been identified, are geographically distributed in diverse but specific regions and have been associated with specific resistance-conferring mutations in the M. tuberculosis genome. The East Asian lineage (Beijing) has been shown to be the foremost virulent and most dominant among the known lineages. It is the predominant lineage in Far East Asia and causes more than 60% of TB cases in this region. The lineage has a worldwide distribution and is also dominant in some parts of South Africa. The aim of this study was to characterize INH resistance mutations in M. tuberculosis isolates from new and previously treated patients and determine the association of mutations with TB lineages in the Tshwane region, South Africa. Methods: A total of 150 M. tuberculosis resistant isolates were obtained (2016 to 2019) from the National Health Laboratory Service, Tshwane Academic Division. The study population included: 118/150 (79%) new cases and 32/150 (21%) previously treated cases. Of the new cases, 47/118 (40%) were females and 71/118 (60%) were males. Of the previously treated cases, 11/32 (34%) were females and 21/32 (67%) were males. Genomic DNA was extracted using the GenoLyse® kit (Hain Lifescience, Germany). Characterization of INH resistance-conferring mutations was

done using GenoType MTBDRplus (Hain Lifescience, Germany). Thereafter, screening of XDR- TB was done using GenoType MTBDRsl (Hain Lifescience, Germany). Genotyping was

performed using spoligotyping, a PCR-based method. Association of M. tuberculosis lineages and INH mutations was evaluated. Results: Overall results showed that 117/150 (78%) were both RIF and INH resistant and 33/150 (22%) were RIF mono-resistant. Of the 33 pre-MDR-TB isolates, 12/33 (36%) were phenotypically discrepant in RIF and INH resistance. Of these, 10/12 (83%) were new and 2/12 (17%) were previously treated TB cases. Mutation S315T of the katG gene was found in 75/118 (64%) of new TB cases and 13/32 (41%) in previously treated cases. The sensitivity and specificity of LPA for detecting INH resistance was between 29% to 71% and 84% to 100% respectively. Screening of MDR-TB isolates for XDR-TB showed that 139/150 were sensitive to second-line injectable drugs (SLIDs) and 11/150 were resistant. Only 90/150 were susceptible to fluoroquinolones (FLQs) and 60/150 were resistant. Spoligotyping identified 9 major distinct TB families including East African Indian (EAI) (14%), Latin American and Mediterranean (LAM)

(13%), Beijing (27%), T-family (13%), S-family (7%), H-family (1%), X-family (7%), Manu2- family (4%) and CAS-family (1%). Remaining isolates 13% of the isolates were orphans. Mutation S315T of the katG gene in new TB cases was mostly associated with Beijing (18/118 or 15%) and with T1-family (9/118 or 8%). In previously treated cases, mutation S315T was associated with Beijing 5/32 (16%) and with X2 (3/32 or 9%). Furthermore, mutation C-15T in new TB cases was mostly associated with Beijing (9/118 or 8%). However, the was no significant association between INH resistance-conferring mutations and M. tuberculosis lineage in new and previously treated cases. Conclusion: This study showed that male gender is more prominently associated with resistance to INH within the Tshwane region. This shows that male gender is prone to disease exposure than females. The line probe assay MTBDRplus 2.0 is commonly used as a rapid diagnostic for drug resistance to first-line drugs, including RIF and INH resistance. However, in comparison with phenotypic methods, around 8% of isolates produced discrepant results, i.e. INH resistance missed by the LPA. This indicates that a more discriminatory technique should be used to further analyse INH susceptible isolates. A high genetic diversity of M. tuberculosis lineages was observed in both new and previously treated cases. Furthermore, M. tuberculosis lineages associated with S315T mutation within the Tshwane region appears to be spreading rapidly compared to previous studies done in the region.