Prevalence and resistance gene mutations of multi-drug resistant and extensively drug resistant mycobacterium tuberculosis in the Eastern Cape

The emergence and spread of multi-drug resistant (MDR-TB) and extensively drugresistant tuberculosis (XDR-TB) are a major medical and public problem threatening the global health. The objectives of this study were to (i) determine the prevalence of MDR-TB and XDR-TB in the Eastern Cape; (ii) analyze patterns of gene mutations in MDR-TB and (iii) identify gene mutations associated with resistance to second line injectable drugs in XDR-TB isolates. A total of 1520 routine sputum specimens sequentially received within a period of 12 months i.e. February 2012 to February 2013 from all MDR-TB and XDR-TB patients treated by Hospitals and clinics in the Eastern Cape were included in this study, of which 1004 had interpretable results. Samples were analyzed with the Genotype MTBDRplus VER 2.0 assay kit (Hain Lifescience) for detection of resistance to Rifampicin and Isoniazid while solid and liquid culture drug susceptibility tests were used for ethambutol, streptomycin, ethionamide, ofloxacin, capreomycin and amikacin. PCR and sequence analysis of short regions of target genes gyrA, (encode subunit of DNA topoisomerase gyrase), rrs (16S rRNA) and tlyA (encodes a 2’-O-methyltransferase) were performed on 20 XDR-TB isolates. MTBDRplus kit results and drug susceptibility tests identified 462 MDR-TB, 284 pre-XDR and 258 XDR-TB isolates from 267 clinics and 25 hospitals in the Eastern Cape. There was a high frequency of resistance to streptomycin, ethionamide, amikacin, ofloxacin and capreomycin. Mutation patterns indicated differences between the health districts as well as differences between the facilities within the health districts. The most common mutation patterns observed were: (i) ΔWT3, ΔWT4, MUT1 [D516V+del515] (rpoB), ΔWT, MUT1 [S315T1] (katG), ΔWT1 [C15T] (inhA) [39 MDR, 204 XDR-TB and 214 pre XDR-TB isolates], (ii) ΔWT8, MUT3 [L533P+S531L] (rpoB), ΔWT, MUT1 [S315T1] [145 MDR, 18 pre-XDR and 3 XDR-TB solates] and (iii) ΔWT3, WT4 [D516Y+del515] (rpoB), ΔWT, MUT1 [S315T1] (katG) [75 MDR, 1 pre-XDR and 7 XDR-TB isolates]. Mutations in inhA promoter regions were strongly associated with XDR-TB isolates. Two thirds (66.6 percent (669/1004) of the isolates had inhA mutations present with 25.4 percent (170/669) found among the MDR isolates, 39.2 percent (262/669) among the pre-XDR isolates and 35.4 percent (237/669) among the XDR-TB isolates, which implies that these resistant isolates are being spread by transmission within the community and circulating in the province. There was good correlation between XDR-TB drug susceptibility test results and sequence analyses of the gyrA and rrs genes. The majority of XDR-TB isolates contained mutations at positions C269T (6/20) and 1401G (18/20) in gyrA and rrs genes respectively. Sequence analysis of short regions of gyrA and rrs genes may be useful for detection of fluoroquinolone and amikacin/ kanamycin resistance in XDR-TB isolates but the tlyA gene is not a sensitive genetic marker for capreomycin resistance. This study highlighted the urgent need for the development of rapid diagnostics for XDR-TB and raised serious concerns regarding ineffective patientmanagement resulting in ongoing transmission of extremely resistant strains of XDRTB in the Eastern Cape suggesting that the Eastern Cape could be fast becoming the epicenter for the development of Totally Drug-resistant Tuberculosis (TDR-TB) in South Afric