Detection of drug resistant Mycobacterium tuberculosis by high-throughput sequencing of DNA isolated from acid fast bacilli smears

Abstract

BackgroundDrug susceptibility testing for Mycobacterium tuberculosis (MTB) is difficult to perform in resource-limited settings where Acid Fast Bacilli (AFB) smears are commonly used for disease diagnosis and monitoring. We developed a simple method for extraction of MTB DNA from AFB smears for sequencing-based detection of mutations associated with resistance to all first and several second-line anti-tuberculosis drugs. MethodsWe isolated MTB DNA by boiling smear content in a Chelex solution, followed by column purification. We sequenced PCR-amplified segments of the rpoB, katG, embB, gyrA, gyrB, rpsL, and rrs genes, the inhA, eis, and pncA promoters and the entire pncA gene.

Pyrazinamide-resistant Tuberculosis Obscured From Common Targeted Molecular Diagnostics

“…After elucidating that the majority PZA-R subpopulation ( pncA del-264:158 ) with PZA-S subpopulations ( pncA WT / pncA Ser65Ser ) accounted for the discrepant DST results, we investigated whether existing primer schemes and emerging primer-based amplification methods ( Banu et al, 2017 ; Hameed et al, 2020 ; Willby et al, 2017 ; Jouet et al, 2021 ; Kahbazi et al, 2018 ; Tam et al, 2019 ; Li et al, 2021 ; Liu et al, 2023 ; Mansjo et al, 2017 ; Rowneki et al, 2020 ; Sengstake et al, 2017 ; Tan et al, 2014 ) would capture the resistance-conferring pncA del-264:158 . None of the pncA amplification schemes had primers spanning the deletion boundaries of this majority PZA-R subpopulation ( Fig.…”

Experimental confirmation that an uncommon, yet clinically relevant mutation (G878A) in the rrs gene of Mycobacterium tuberculosis confers resistance to streptomycin

“…Then, to identify the genetic basis for resistance towards each of these antibiotics, PCR products corresponding to the genes that are most frequently associated with mutations conferring resistance towards these compounds were amplified and sequenced. This included products corresponding to the 81bp rifampicin resistance determining region (RRDR) of rpoB, the katG, inhA and inhA promoter sequences (associated with INH resistance), as well as the rpsL, rrs and gidB genes, mutations in which are most frequently responsible for resistance towards STR (10, 22). In this manner we identified that the strain possessed the S450W RpoB mutation that is frequently linked to RIF resistance, and the −15C/T inhA promoter mutation consistent with the low-level of INH resistance observed for strain #57001.…”

“…PCR was carried out according to standard protocols except for when amplifying a portion of the KAN resistance cassette where 5% DMSO was also included in the reaction mixtures. Primers used in this study for PCR and sequencing are shown in Supplementary Table S1, a number of which are based on those reported by Rowneki et al (22). Sanger sequencing of PCR products was carried out at the Centre d’expertise et de services Génome Québec (Montreal).…”

High Throughput Sequencing for Clinical Tuberculosis: An Overview

“…Although it does not provide information on the entirety of the genome, careful primer design to investigate specific areas leads to greater depth of coverage for less resources. Several research studies have designed "in-house" multiplex polymerase chain reaction (PCR) panels for use on culture isolates [13,14] and clinical specimens such as sputum or bronchial aspirates [15][16][17]. The Deeplex ® Myc-TB assay (Genoscreen, Lille, France) is the most well-described commercially available targeted sequencing assay in the peer-reviewed literature.…”