Molecular epidemiology of multi-drug resistant Mycobacterium tuberculosis strains in Swaziland

Background: The tuberculosis (TB) epidemic remains a major global health problem, noting that the Swaziland government had declared the disease a national emergency. Hence the study was aimed to determine the prevalence of TB in Swaziland and further to ascertain whether the circulating susceptible M. tuberculosis strain later develops to MDR-TB on the same patient or whether patients acquire a completely new multi-drug resistant strain. Methods: Participants were recruited from four TB testing facilities (n=560), which are regional TB referral facilities in Swaziland. Willing participants who had been selected using a systematic random sampling method, and who met the inclusion criteria, were included in this quantitative descriptive cohort study upon signing an informed consent form (n=103). Sputum samples collected from these participants (at baseline and at follow-up) were tested for the presence of Mycobacterium tuberculosis using the GeneXpert® MTB/RIF assay (Cephied, USA). When found to be positive, the samples were then cultured on the BACTEC™ MGIT™ 960 Mycobacteria Culture System (Becton Dickinson, USA), which was also utilised for drug sensitivity testing and thereafter DNA was extracted for spoligotyping, using a GenoLyse (Hain Life Science, Germany). The study results were analysed using the Statistical Package for the Social Sciences (SSPS) version 25, Epi Info (version 3.5.1, 2008) and STATA 13.0 software packages. Frequencies of multiple-drug resistant-TB (MDR-TB) amongst all the genotype families’ categories based on spoligotyping were compared among the participants from the four regions using a Fisher Exact and Chi-square statistical methods, where associations with a pvalue of < 0.05 were considered statistically significant. The spoligotyping findings were entered into an MS Excel spreadsheet as a binary code signifying either a positive or negative hybridization outcome. The spoligotyping results were then entered into SITVIT2 database (Pasteur Institute of Guadeloupe) to generate spoligotype families. Results: The prevalence of TB infection in this study population was 26.41% (n=103/390) and MDR-TB prevalence was 33.01% (n=34/103). Notably, the TB infection was high among males (64%; n=103) and among the young adults of both genders (18-35 years, with a mean age of 34.4 years). Most of the strains at both baseline and follow-up (58.74%) were susceptible to all of these anti-TB drugs, followed by those strains classified as MDR-TB, with 28.64% and polyresistant and RIF mono-resistant strains being 8.25% and 2.91%% respectively, although with no statistical significance (p=0.786). The Beijing sub-lineage (lineage 2) was the predominant sub-lineage in 28.82% isolates (n=49/170), followed by lineage 4 (Euro American) the S and T1 sub-lineages (of lineage 4) in 20.0% and 11.76% isolates, respectively. The emerging U sublineage was isolated as well with n=1 (2.2%) p=0.001. We then compared sub-lineage changes of baseline and follow-up specimens, together with DST patterns. A total of 33 (53.23%) participants with a pattern of ‘changed sub-lineage and the same DST pattern’ followed by n=16 (25.81%) participants showing ‘changed sub-lineage with changed DST pattern’. The least represented categories were of seven (11.29%) participants with ‘same sub-lineage with the same DST pattern’ category as well as the ‘same sub-lineage with changed drug sensitivity pattern’ category with n=6 (9.68%). The Beijing genotype was significantly detected in the group with the ‘changed sub-lineage and the same DST pattern’ (p ≤0.001). Conclusion: Since high prevalence of TB infection was observed in this current study, mainly among men and young adults, hence mass TB screening and testing campaigns in all health centres and workplace wellness centres are recommended, to reduce the high TB prevalence. A key finding of this study was n=24/103 (23.30%) participants who were susceptible to all first line anti-TB drugs at baseline developed MDR-TB at follow-up. A significant proportion of those developed MDR-TB were infected with the Beijing sub-lineage, which is linked with MDR-TB outbreaks in many regions. Notably, Swaziland has a high M. tuberculosis lineage diversity, with eighteen sub-lineages noted. A significant proportion of the TB infected participants had the Beijing sub-lineage, which is linked with MDR-TB outbreaks in many regions. In addition, the emerging U sub-lineage, also linked to MDR-TB, was noted. Since different strains are reported to uniquely respond to treatment, therefore, it is hence recommended that the various genotypes of M. tuberculosis strains circulating in Swaziland be investigated and monitored, so as to improve on the TB treatment outcomes, control and prevention programs and detect timeously the drug resistant TB strains in Swaziland