Ausceptibility testing and resistance mechanisms to second-line agents against Mycobacterium tuberculosis in Pretoria, South Africa

Abstract

Multidrug-resistant (MDR) and extensively drug-resistant tuberculosis (XDR-TB) is a serious problem in South Africa. Standardised and accurate drug susceptibility testing for first-line and second-line anti-TB drugs are critical elements in battling the MDR- and XDR-TB epidemic. Furthermore, understanding of the population structure and transmission patterns of circulating drug-resistant Mycobacterium tuberculosis (M. tuberculosis) strains is of utmost importance. Previous studies have shown that different genotypes of M. tuberculosis strains predominate in different geographical regions and strain to strain variations may have important consequences when it comes to transmissibility, diagnostics and management. Despite the high prevalence of MDR- and XDR-TB in South Africa, data with regard to the population structure of drug-resistant strains is lacking in many regions. Therefore, the aim of the study was to evaluate phenotypic and genotypic methods for susceptibility testing of firstline and second-line anti-TB drugs and molecular characterisation of drug-resistant strains from high-burden TB areas in South Africa. Consecutive MDR-TB isolates from four provinces were included in this study. The results of routine drug susceptibility testing of ethambutol, streptomycin, kanamycin and ofloxacin using the BACTEC MGIT 960 system in MDR-TB isolates was compared to the agar proportion method. The GenoType® MTBDRsl assay was evaluated for the detection of resistance against ethambutol, capreomycin, kanamycin and ofloxacin. Finally, the study investigated the population structure and transmission patterns of drug-resistant M. tuberculosis isolates using spoligotyping and MIRU-VNTR typing. Additionally, the possible association of genotypes with drug-resistance patterns and demographic information were explored. The sensitivity and specificity of the BACTEC MGIT 960 system using the agar proportion method as a gold standard was 18.8% and 96.5% for ethambutol, 95.2% and 37.4% for streptomycin, 54.6% and 91.8% for kanamycin and 100% and 89% for ofloxacin, respectively. The GenoType® MTBDRsl assay gave comparable results for ofloxacin; however, a low performance was found for the detection of resistance to capreomycin, kanamycin and ethambutol. In order to resolve the discrepancies between the two methods, DNA sequencing was done for the target genes (gyrA, rrs, embB) from the discrepant results as well as two additional genes (gyrB, tlyA) that were not included in the assay. The DNA sequencing identified mutations in the gyrA, gyrB, rrs and tlyA genes that were not detected by the GenoType® MTBDRsl assay. The prevalence of XDR-TB and pre-XDR TB was 7.1% and 9.5%, respectively. A highdiversity of M. tuberculosis strains were found in this study, with the Beijing and EAI1_SOM families being predominant. No association was found between genotypes and specific drugresistance or demographic information. The sensitivity of the BACTEC MGIT 960 system for ofloxacin and streptomycin was excellent; however, the sensitivity was low for ethambutol and kanamycin. The GenoType® MTBDRsl assay was promising for the detection of ofloxacin; however, the sensitivity of the assay needs to be improved for capreomycin, kanamycin and ethambutol. The high-level of diversity and the geographical distribution of the drug-resistant M. tuberculosis isolates in this study suggested that the transmission of drug-resistant TB in these study settings is not caused by clonal spread of a specific M. tuberculosis strain.