Application of Next Generation Sequencing for Diagnosis and Clinical Management of Drug-Resistant Tuberculosis: Updates on Recent Developments in the Field

Abstract

The World Health Organization's End TB Strategy prioritizes universal access to an early diagnosis and comprehensive drug susceptibility testing (DST) for all individuals with tuberculosis (TB) as a key component of integrated, patient-centered TB care. Next generation whole genome sequencing (WGS) and its associated technology has demonstrated exceptional potential for reliable and comprehensive resistance prediction for Mycobacterium tuberculosis isolates, allowing for accurate clinical decisions. This review presents a descriptive analysis of research describing the potential of WGS to accelerate delivery of individualized care, recent advances in sputum-based WGS technology and the role of targeted sequencing for resistance detection. We provide an update on recent research describing the mechanisms of resistance to new and repurposed drugs and the dynamics of mixed infections and its potential implication on TB diagnosis and treatment. Whilst the studies reviewed here have greatly improved our understanding of recent advances in this arena, it highlights significant challenges that remain. The wide-spread introduction of new drugs in the absence of standardized DST has led to rapid emergence of drug resistance. This review highlights apparent gaps in our knowledge of the mechanisms contributing to resistance for these new drugs and challenges that limit the clinical utility of next generation sequencing techniques. It is recommended that a combination of genotypic and phenotypic techniques is warranted to monitor treatment response, curb emerging resistance and further dissemination of drug resistance.

Keywords: drug-resistance; mixed infection; next-generation sequencing; resistance mechanisms; targeted sequencing; tuberculosis; whole genome sequencing.

FIGURE 1

Illustration of (A) the current diagnostic pipeline of drug resistance in TB, (B) workflow of WGS from culture, and (C) proposed workflow of targeted NGS directly from sputum. TB, tuberculosis; WGS, whole genome sequencing; MTB, Mycobacterium tuberculosis; MGIT, mycobacterial growth indicator tube; DST, drug susceptibility testing; LPA, line probe assay; RIF, rifampicin; INH, isoniazid; FQ, fluoroquinolones; DNA, deoxyribonucleic acid; NGS, next generation sequencing; PCR, polymerase chain reaction.

FIGURE 2

Mycobacterium tuberculosis cell depicting the known resistance mechanisms against BDQ, i.e., atpE and Rv0678. The mechanism of action of pepQ, a cytoplasmic peptidase protein, remains to be elucidated. BDQ targets the c subunit of the ATP synthase complex to inhibit ATP production and respiration of mycobacteria. Mutations such as A28 and A63 in atpE prevent BDQ binding to the complex, rendering the drug ineffective. The Rv0678 protein represses mmpS5 and mmpL5 genes to prevent transcription and expression of the MmpS5-MmpL5 efflux pump. A mutated and non-functional Rv0678 repressor cannot bind to the promoter/operator region thereby allowing over-expression of efflux pumps to expel more BDQ out of the mycobacterial cell.