Heteroresistance of Mycobacterium tuberculosis in the Sputum Detected by Droplet Digital PCR

SIMPLE SUMMARY: Tuberculosis caused by Mycobacterium tuberculosis (MTB) is a major public health concern globally, and efforts to control and eliminate the disease continue to be a priority. Heteroresistance in MTB describes a bacterial population where distinct clones or subpopulations exhibit varying levels of antibiotic susceptibility. In this study, we employed Droplet Digital PCRs to investigate the heteroresistance of MTB in sputum samples in new TB cases. We targeted mutations in the katG and rpoB genes, which are associated with isoniazid and rifampicin resistance, respectively. This study found that in a total of 79 new TB cases, drug-resistant and drug-susceptible TB were detected in 11.4% and 88.6%, respectively. The prevalence of INH mono-resistant, RIF mono-resistant, and MDR-TB among total cases was 1.3%, 6.3%, and 3.8%, respectively. Heteroresistance in katG, rpoB, and both genes were found in 2.5%, 5%, and 2.5% of total cases, respectively. Our findings suggest that the detected mutations may have occurred spontaneously, as the patients had not yet received anti-TB treatment. The ddPCR’s ability to detect both mutant and wild-type strains in a population makes it a valuable tool for early diagnosis and management of drug-resistant TB. Improved methods such as ddPCR for detecting heteroresistance in MTB are crucial for global TB control and elimination. ABSTRACT: Heteroresistance in MTB refers to the presence of distinct subpopulations of bacteria with varying levels of antibiotic susceptibility within a population. Multidrug-resistant and rifampicin-resistant TB are serious global health concerns. In this study, we aimed to determine the prevalence of heteroresistance in MTB from sputum samples of new TB cases using Droplet Digital PCR mutation detection assays for katG and rpoB genes, which are commonly associated with resistance to isoniazid and rifampicin, respectively. We found that out of 79 samples, 9 (11.4%) exhibited mutations in katG and rpoB genes. INH mono-resistant TB, RIF mono-resistant TB, and MDR-TB samples constituted 1.3%, 6.3%, and 3.8% of new TB cases, respectively. Heteroresistance in katG, rpoB, and both genes were found in 2.5%, 5%, and 2.5% of total cases, respectively. Our results suggest that these mutations may have arisen spontaneously, as the patients had not yet received anti-TB drugs. ddPCR is a valuable tool for the early detection and management of DR-TB, as it can detect both mutant and wild-type strains in a population, enabling the detection of heteroresistance and MDR-TB. Overall, our findings highlight the importance of early detection and management of DR-TB for effective TB control (in katG, rpoB, and katG/rpoB).