Molecular epidemiology of rifampicin-resistant Mycobacterium tuberculosis in Vietnam

Vietnam is a top 20 burden country for multi-drug resistant/rifampicin-resistant tuberculosis (MDR/RR-TB), with nearly 10,000 cases a year. With the emergence of new diagnostic assays for M. tuberculosis detection and resistance, along with new drugs for both treatment and prevention, we sought to better understand the molecular epidemiology of RR-TB in this high-burden setting, through the study of clinical trial isolates from the VQUIN MDR trial. We assembled a sample of cultured isolates, collected from patients with RR- M. tuberculosis detected with Xpert MTB/RIF across 10 provinces, enriching for isolates from outside of the two major cities, Hanoi and Ho Chi Minh City. We subjected these isolates to whole genome sequencing (WGS) and bioinformatic analysis, with a subset subjected to phenotypic drug susceptibility testing to evaluate phenotypic/genotypic concordance. New genome sequences were phylogenetically contextualised to publicly available M. tuberculosis genome sequences sampled in Vietnam from National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA). Of 252 isolates classified as RR-TB by Xpert MTB/RIF, 244 (97%) were confirmed to have rifampicin-resistance mutations by WGS. A high proportion of these (235/244, 96%) had mutations associated with resistance to at least one other first- or second-line antibiotic. Phenotypic drug susceptibility testing (pDST) for rifampicin, isoniazid, and levofloxacin was completed for a random sample of 77 isolates, demonstrating a high concordance between pDST and genomic-based resistance predictions (67/77, 87% RIF; 76/77, 99% INH; 73/77, 95% LFX). In a phenotypic/genotypic comparison of 59 isolates to new and repurposed antibiotics, no resistance was detected to pretomanid or linezolid, whereas high concordance was observed for bedaquiline (56/59, 95%). RIF-resistant strains were more likely to be L2.2.1, compared to RIF-susceptible M. tuberculosis strains in Vietnam, particularly in the major cities. The high prevalence of secondary drug-resistance beyond RIF and INH, along with the dominance of one major lineage across geographic regions, provides insights into the spread of MDR/RR-TB in Vietnam and reinforces the importance of prompt and broad detection of drug-resistance to inform the timely initiation of effective drug regimens.