Multiple strain infections in TB are now recognized as common occurrences and identifying patients with multiple MTB strains is critical in clinical practice, public health and molecular epidemiology. This is because not only does it provide insight into the disease patterns but also aids in the management and control of TB. This study revealed that, one out of every sixteen PTB patients (6.4%) was infected with multiple strains of MTB. This prevalence is almost similar to the 7.1% reported in Kampala, Uganda 11 but much lower than the 11% observed in Mubende, Uganda 21. It is probable that the disparities in estimations are due to discrepancies in sensitivities of the genotyping techniques used to differentiate between MTB strains. While there are diverse genotyping approaches employed in the identification of mixed infections, the degree of sensitivity of each method varies. While the Mubende and Kampala investigations used 15 loci MIRU-VNTR typing, our study used 24 loci MIRU-VNTR typing with a single target conventional PCR. This method has been demonstrated to be very sensitive and discriminative, rendering it the gold standard in the diagnosis of multiple strain infections 31,32. Other significant discrepancies can be seen in the laboratory methods utilized. As with any other approach used to identify multiple strain infections, detection of an underlying strain can only be established when there are sufficient DNA copies of that strain in the sample being studied. Many studies, including the Kampala and Mubende studies, use culture to increase the mycobacterial population 11,21,33. However, this can drastically change the clonal composition thus changing the frequency with which multiple strains are detected 9,31,34. In our study, we utilized DNA isolated directly from processed sputum samples. Our findings are also much higher than the 2.8% 12 reported in Malawi and 3.2% in Zambia 35 but lower than the 9.6% 36 and 10% 37 reported in Botswana. Differences between the study settings may partly account for this difference whereby the annual risk of TB infection in Malawi is approximately 1% 3,38 while in Botswana its 3% 39.
Our study also revealed that unlike the relapse patients, who were not infected with multiple strains, all (100%) of the patients in our study with multiple strain infections were newly diagnosed cases. This is consistent with the findings of the Mubende study 21, which observed that the majority (87.5%) of patients with multiple strains were the newly diagnosed cases. This might reflect a high level of transmission and heterogeneity of strains in this category of patients (Cohen et al., 2012). This hypothesis is supported by the proportion of newly diagnosed cases that exhibited the multiple strain infection phenomena, an attribute that is reported to indicate high transmission rates 40–42. Furthermore, a third (9.7%) of the patients with multiple strain infections were also HIV-infected. This finding is consistent with other studies, in which nearly all multiple strain TB infected people were HIV positive 11,21. This appears to support the notion of the link between multiple strain TB infection and HIV/TB co-infection 16,21,36,43,44. Given the high prevalence of HIV and HIV/TB co-infection in this region 45, it is plausible to suggest that HIV-induced immune deficiency exposes patients to the risk of concurrent infections. HIV removes the security of being reinfected as one battles an ongoing infection there by creating a scenario where one can be infected even before they clear an ongoing infection (Elizabeth Glaser Foundation, 2015).
In conclusion, patients experiencing their initial episodes of the disease and those co-infected with HIV are more susceptible to being infected with multiple strains of M. tuberculosis. This points to a critical component of disease dynamics that is most likely being overlooked at the clinical level, emphasizing the need to further study the potential high risk of exposure to this category of patients at the community level.