Molecular characterization of Mycobacterium tuberculosis complex from Kampala, Uganda

Uganda is one of the countries with the highest burden of tuberculosis (TB) in Sub-Saharan Africa, ranked 16 among the 22 high-burden countries in the world. Poor peri-urban areas of developing countries with inadequate living conditions and a high prevalence of HIV infection have been implicated most in the increase of TB. Different species, strain families and lineages of the Mycobacterium tuberculosis complex (MTC) are now known to have differences in virulence, clinical presentation as well as transmission potential. This study determined the predominant species as well as strain lineages that cause TB in Rubaga division, Kampala; analyzed TB transmission in HIV-seropositive and HIV-seronegative TB patients and the prevalence of resistance to key anti-tuberculosis drugs. Furthermore, the study characterized cattle-derived isolates of M. bovis from slaughter-cattle at a peri-urban city abattoir so as to set up a database of M. bovis strains for comparison with infections in humans in future studies. To achieve this, 386 consecutive newly presenting sputum smear positive patients resident in and attending TB clinics in Rubaga division were enrolled. Infecting species for 344 cultures were determined by a solely PCR-based typing panel that determined presence or absence of regions of difference (RDs) in the MTC; strain types and families were determined by spoligotyping, and dynamics of TB spread in HIV co-infected vis-a-vis HIV-seronegative TB patients by standard IS6110-RFLP fingerprinting methodology. All but one of the 344 isolates in the study were M. tuberculosis, the other being M. bovis. Spoligotyping revealed predominance of the T2 family, which was in turn predominated by a previously described “Uganda genotype” group of strains. Further characterization of 139 Uganda genotype strains revealed an internal deletion in the RD724 locus, a polymorphism that defines one of the major sub-lineages of M. tuberculosis commonly seen in the central African human host population. Resistance to isoniazid was found in 8.1% of 344 strains, while all 15 (4.4%) strains resistant to rifampicin were also multi-drug resistant. IS6110-RFLP analysis of isolates from 80 HIVseropositive and 103 HIV-seronegative patients revealed no difference in the level of diversity of DNA fingerprints observed in the two sero-groups (P = 0.615), patients aged <40 years (P = 0.100), and sex (P = 0.715). However, 54% (99/183) of the patients shared fingerprints (average cluster size of 2.9), suggesting a high transmission rate in this community. There was no association between any strain types in the sample with either drug resistance or HIV serostatus of the patients. Eleven M. bovis and six non tuberculous mycobacteria were isolated from tissue samples of 87 carcasses. Worryingly, six carcasses showing obvious and multiple sites of infection were not condemned as unfit for human consumption, creating a potential for spread of M. bovis in the food chain and to humans through consumption of contaminated meat, a very important public health concern in a resource-poor high disease-burden setting. The study has shown that M. tuberculosis is the predominate species of the MTC in Kampala, and the spoligotype-specific and RD724-deleted “Uganda genotype” the predominant strain type. The TB epidemic in Kampala is localized, mainly caused by the closely knit T2 spoligotype family of strains, and strain types common in neighboring countries were minimal. Additionally, strain types were neither associated with drug resistance, nor HIV sero-status. The study further showed evidence of a high rate of recent transmission of TB in Rubaga with a high average cluster size, but infection with an isolate with a fingerprint found to be part of a cluster was not associated with any demographic or clinical characteristic, including HIV sero-