Deep whole-genome sequencing reveals no evidence for heteroresistance influencing treatment outcomes among drug-susceptible tuberculosis patients

Abstract

The purpose of this study was to investigate the minimum level of heteroresistance that predicts poor tuberculosis treatment outcomes. This retrospective study enrolled 45 new tuberculosis patients with varied treatment outcomes and 16 drug-susceptible retreatment cases. Pretreatment isolates from these 61 patients were whole genome sequenced to detect heteroresistance. Heteroresistance was not found in isolates from any of the new patients, but was detected in isolates from retreatment patients who were nevertheless cured. The results of our small series of patients suggest that heteroresistance <1%, the threshold used to define resistance with the phenotypic proportion method, is not associated with poor treatment outcomes.

Introduction

Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), is among the 10 most frequent causes of death worldwide, with an estimated 10 million new cases and 1.4 million deaths in 2019 [1]. Although approximately 85% of patients with drug-susceptible TB are cured with the standard four-drug treatment, a subset of patients fail treatment. Perhaps treatment failures can be reduced if the host and bacterial factors associated with poor treatment outcomes can be identified and effectively countered. Previous studies have focused mainly on host attributes such as patient adherence [2], co-infection with the human immunodeficiency virus (HIV) [3] and TB treatment history [4], but the impact of bacterial factors on TB treatment outcomes is less well known. The most important bacterial risk factor for TB treatment failure is the presence or emergence of drug-resistance [5]. MTB isolates that are classified as susceptible by clinical breakpoints but have higher subminimal inhibitory concentrations (sub-MICs) are associated with treatment failure, and higher sub-MICs can predict relapse with a sensitivity of 75% [6].

Heteroresistance, defined as the coexistence of both drug-susceptible and drug-resistant bacteria within the same patient [7], has gained increasing attention in studies of drug resistance evolution. Heteroresistance occurs in two situations [7]: a mixed infection, with two or more distinct MTB strains; or the presence of different subpopulations within the host caused by microevolution of a single strain. Heteroresistance is thought to reduce the likelihood of treatment success, presumably because the resistant subpopulations can survive and grow during antimicrobial therapy. However, studies to document a correlation between heteroresistance and treatment outcomes have produced inconsistent results [[8], [9], [10]], perhaps due to differences in the definition of heteroresistance or the degree of heterogeneity in the patients studied. Deep whole-genome sequencing (WGS) of MTB isolates, which is increasing employed to study drug resistance, can detect very small heteroresistant populations of bacilli. In this report we studied the extent of heteroresistance in patients with phenotypic drug-susceptible TB, both new TB and retreatment cases, to determine whether these very small heteroresistant populations will affect treatment outcome.