Whole genome sequencing of Mycobacterium tuberculosis and assessment of treatment outcome in tb patients from Vhembe District

Abstract

BACKGROUND Tuberculosis (TB) remains one of the most pressing global health challenges, contributing significantly to morbidity and mortality worldwide. The burden is particularly severe in regions with high HIV prevalence, where immunocompromised individuals are more susceptible to TB infection. South Africa, and specifically Limpopo Province, continues to face a dual epidemic of TB and HIV, with drug-resistant TB (DR-TB) posing a growing threat to public health. The emergence of multidrug-resistant TB (MDR-TB), pre-extensively drug-resistant TB (pre-XDR-TB), and extensively drug-resistant TB (XDR-TB) complicates treatment regimens, increases costs, and reduces treatment success rates. Despite national efforts to implement WHO-endorsed diagnostics such as Xpert MTB/RIF and Genotype MTBDRplus, limitations remain. These assays primarily detect mutations within the rifampicin resistance-determining region (RRDR) of the rpoB gene, but mutations outside this region may go undetected, leading to underestimation of resistance prevalence. AIM To investigate the genomic characteristics of Mycobacterium tuberculosis using whole-genome sequencing and their association with treatment outcomes among TB patients in the Vhembe District. METHODOLOGY This study employed a cross-sectional design incorporating both qualitative and quantitative components to comprehensively investigate tuberculosis (TB) within the Vhembe District of South Africa. Ethical approval was granted by the University of Venda Research Ethics Committee (SMNS/20/MBY/13/2104), with additional authorisation obtained from the Limpopo Department of Health (LP_2021_11_001). Institutional permission was secured from participating healthcare facilities prior to data collection. All participants received detailed information regarding the study objectives and procedures, and written informed consent was obtained in accordance with established ethical standards for research involving human participants. The study population comprised adults aged 18 years and older who were diagnosed with TB and actively receiving treatment, either as inpatients or outpatients. A total of 70 sputum samples were collected using sterile containers pre-filled with 4% NaOH. Specimens were transported to the University of Venda TB laboratory under temperature-controlled conditions to preserve sample integrity. DNA extraction and Real-time PCR [Anyplex™ and Allplex™ (Seegene, Korea)] were performed according to the manufacturer's protocols to determine the MTB type in sputum samples. Further analysis was performed using various sequencing techniques to identify mutations. RESULTS: The study enrolled more males (55%) and detected MTB as the predominant pathogen within the cohort, with a measurable prevalence of DR-TB [(4/100) 4%]. Resistance-associated mutations were detected in key loci, including rpoB, katG, and inhA, consistent with rifampicin and isoniazid resistance as determined by the Seegene assays. A highlight of an INH-R mono-resistant strain was identified, underscoring the importance of regular monitoring of TB epidemics. Importantly, genomic analyses revealed additional variants beyond regions routinely targeted by conventional diagnostics, highlighting the limitations of mutation-restricted assays. Notably, none of the DR-TB cases reported prior TB treatment, suggesting primary transmission of resistant strains rather than resistance acquired through therapeutic mismanagement. Shotgun metagenomics further revealed the presence of non-tuberculous mycobacteria (NTM) and other respiratory pathogens, underscoring the polymicrobial complexity of respiratory disease and the potential for diagnostic misclassification. The treatment outcome was more favourable [cure (46.34%) and treatment completion (18.29%)], with favourable outcomes outweighing unfavourable outcomes, and some factors were statistically significant (p < 0.05). Collectively, these findings indicate that, while favourable outcomes were achieved in the majority of cases, the presence of ongoing treatment and a small proportion of treatment failures highlight the continued need for effective monitoring and management strategies to improve treatment success rates within the study population. CONCLUSION The study highlights the importance of improving diagnostic techniques in low-income settings to prevent underdiagnosis and misdiagnosis, as NTM and INH-R mono-resistant strains were detected. Additionally, the study demonstrates the benefits of combining high-throughput genotyping, metagenomics, and structural modelling in TB research. This also highlights the effectiveness of molecular techniques in reducing turnaround time, preventing misdiagnosis, and detecting polymicrobial infections that may compromise TB treatment outcomes. By integrating molecular epidemiology and functional prediction, the results provide new insights into the dynamics of resistance transmission, diagnostic gaps, and targeted treatment approaches. The integration of genetic and statistical modelling provides a strong framework for enhancing DR-TB surveillance, improving treatment plans, and expanding individualized TB care in high-burden settings.