Rapid Identification of Drug-Resistant Tuberculosis Genes Using Direct PCR Amplification and Oxford Nanopore Technology Sequencing

Abstract

Mycobacterium tuberculosis antimicrobial resistance has been continually reported and is a major public health issue worldwide. Rapid prediction of drug resistance is important for selecting appropriate antibiotic treatments, which significantly increases cure rates. Gene sequencing technology has proven to be a powerful strategy for identifying relevant drug resistance information. This study established a sequencing method and bioinformatics pipeline for resistance gene analysis using an Oxford Nanopore Technologies sequencer. The pipeline was validated by Sanger sequencing and exhibited 100% concordance with the identified variants. Turnaround time for the nanopore sequencing workflow was approximately 12 h, facilitating drug resistance prediction several weeks earlier than that of traditional phenotype drug susceptibility testing. This study produced a customized gene panel assay for rapid bacterial identification via nanopore sequencing, which improves the timeliness of tuberculosis diagnoses and provides a reliable method that may have clinical application.

Diagnostic Accuracy of Nanopore Sequencing for DetectingMycobacterium tuberculosisand Drug-Resistant Strains: A Systematic Review and Meta-Analysis

“…To facilitate our analysis, studies were classified into two categories: those focusing on the detection of MTB (Table 1) and those focusing on the detection of DR-MTB (Table 2). Among the 32 articles, 13 articles related to MTB detection only [18][19][20][21][22][23][24][25][26][27][28][29][30] ,15 articles focused on DR-MTB detection only [31][32][33][34][35][36][37][38][39][40][41][42][43][44][45] , while 4 articles examined both [46][47][48][49] .…”

“…Among the 16 DR-MTB studies, two studies followed STARD recommendations 50 ; one study stated the use of consecutive sampling 47 and one study mentioned use of random sampling 41 .…”

Application of Nanopore Sequencing in the Diagnosis and Treatment of Pulmonary Infections

“…Zhao et al . [ 91 ] performed nanopore sequencing and Sanger sequencing of sputum specimens from patients with TB to identify drug resistance. The results showed 100% identity between the two methods of identification of drug-resistance genes, with a sequencing time of 3 h, data analysis of 1 h, and turnaround of less than 12 h using nanopore sequencing, and several days using Sanger sequencing, demonstrating the potential of nanopore sequencing for rapid assessment of drug susceptibility prior to starting treatment, enabling appropriate antibiotic selection.…”

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.…”