Prevalence and extent of heteroresistance by next generation sequencing of multidrug-resistant tuberculosis

Abstract

Amplicon-based Next Generation Sequencing (NGS) is an emerging method for Mycobacterium tuberculosis drug susceptibility testing (DST) but has not been well described. We examined 158 clinical multidrug-resistant M. tuberculosis isolates via NGS of 11 resistance-associated gene regions covering 3519 nucleotides. Across these gene regions, complete resistance or heteroresistance (defined as 1%-99% mutation) was present in at least one isolate in 6.3% of loci. The number of isolates with heteroresistance was highest for gyrA codon 94, rpoB codons 526 and 531, and embB codons 306, 372 and 406 (range 11–26% of isolates exhibited heteroresistance). 57% of MDR strains had heteroresistance of one or more recognized resistance-associated mutation. Heteroresistant loci generally exhibited high or low degrees of mutation (>90% or <10%). The deep sensitivity of NGS for detecting low level pncA heteroresistance appeared to improve genotypic-phenotypic PZA susceptibility correlations over that of Sanger. NGS demonstrates that heteroresistance in TB in the regions of key genes is common and will need to be bioinformatically managed. The clinical significance of such heteroresistance is unclear, and further study of pncA should be pursued.

Prevalence and Characterization of Heterogeneous Variable-Number Tandem-Repeat Clusters Comprising Drug-Susceptible and/or Variable Resistant Mycobacterium tuberculosis Complex Isolates in the Netherlands from 2004 to 2016

“…Other explanations for the occurrence of heterogeneous clusters can be discussed, for example, the existence of heteroresistance within M. tuberculosis isolates, i.e., the presence of drug-susceptible and drug-resistant bacteria in the same isolate. Recently, a study investigating the prevalence of heteroresistance by WGS showed that for around 50% of the isolates tested, heteroresistance was present in at least one resistance-associated genomic locus (24). Moreover, the existence of resistant minority subpopulations in M. tuberculosis was described previously (25).…”

A Systematic Evaluation of High-Throughput Sequencing Approaches to Identify Low-Frequency Single Nucleotide Variants in Viral Populations

“…However, this study showed that for RNA Medium and DNA High inputs, additional technical replicates allowed for SNVs to be called at lower percentage frequencies ( Table 2 ) whist maintaining maximum accuracy. In previous studies, where a percentage frequency cut-off has been used to identify SNVs, no consideration for input material or PCR amplification was applied to reduce processed-introduced errors being called as real SNVs [ 8 , 37 , 38 ]. This study highlights the need for a tailored approach to frequency thresholds depending on template input concentration.…”

How well do routine molecular diagnostics detect rifampicin heteroresistance inMycobacterium tuberculosis?

“…In the case of classic Xpert, previous studies report LOD values ranging from 65 to 100% (14, 15), for Ultra, the first validation study conducted by the manufacturer presented LODs only for mutations L430P, H445N (20-40%), and S450L (5-10%) (16), whereas LODs of Genoscholar NTM+MDRTB II have not been reported yet. High coverage depths achieved through pre-selected amplified genes allow targeted deep sequencing to capture and quantify minority resistant variants of Mycobacterium tuberculosis mutants and detect RIF heteroresistance with high sensitivity (17, 18). As an example of such an approach, Deeplex®-MycTB (Genoscreen, France; Deeplex) employs ultra-deep sequencing of a single, 24-plexed amplicon mix to detect drug resistance-associated mutations in M. tuberculosis complex (MTBC) strains, in addition to mycobacterial species identification and MTBC strain genotyping, with a 24-48 turnaround time starting from smear positive clinical samples or primary cultures.…”