Clinical utility of target amplicon sequencing test for rapid diagnosis of drug-resistant Mycobacterium tuberculosis from respiratory specimens

Abstract

An in-house-developed target amplicon sequencing by next-generation sequencing technology (TB-NGS) enables simultaneous detection of resistance-related mutations in Mycobacterium tuberculosis (MTB) against 8 anti-tuberculosis drug classes. In this multi-center study, we investigated the clinical utility of incorporating TB-NGS for rapid drug-resistant MTB detection in high endemic regions in southeast China. From January 2018 to November 2019, 4,047 respiratory specimens were available from patients suffering lower respiratory tract infections in Hong Kong and Guangzhou, among which 501 were TB-positive as detected by in-house IS6110-qPCR assay with diagnostic sensitivity and specificity of 97.9 and 99.2%, respectively. Preliminary resistance screening by GenoType MTBDRplus and MTBDRsl identified 25 drug-resistant specimens including 10 multidrug-resistant TB. TB-NGS was performed using MiSeq on all drug-resistant specimens alongside 67 pan-susceptible specimens, and demonstrated 100% concordance to phenotypic drug susceptibility test. All phenotypically resistant specimens with dominating resistance-related mutations exhibited a mutation frequency of over 60%. Three quasispecies were identified with mutation frequency of less than 35% among phenotypically susceptible specimens. They were well distinguished from phenotypically resistant cases and thus would not complicate TB-NGS results interpretations. This is the first large-scale study that explored the use of laboratory-developed NGS platforms for rapid TB diagnosis. By incorporating TB-NGS with our proposed diagnostic algorithm, the workflow would provide a user-friendly, cost-effective routine diagnostic solution for complicated TB cases with an average turnaround time of 6 working days. This is critical for timely management of drug resistant TB patients and expediting public health control on the emergence of drug-resistant TB.

Direct detection of drug-resistant Mycobacterium tuberculosis using targeted next generation sequencing

“…tNGS assays require careful selection of targets and high confidence resistance mutations ( 50 ). Our assay targets full-length loci associated with resistance to first- and second-line MTBC antimicrobials and is consistent with other targeted assays ( 31–38 ), with some variation in number and size of loci included. In contrast to molecular beacon and line probe assays which focus just on hot spot regions, the assay described in this study examines full-length genes and promoter regions of many targets to allow for detection of rare and atypical resistance mutations.…”

“…Targeted NGS (tNGS) assays can further reduce the time required for comprehensive DR profiling by amplifying numerous loci directly from clinical specimens. Several tNGS assays for DR profiling have been described in the literature, including laboratory-developed assays ( 31–35 ) and the commercially available Genoscreen Deeplex ( 36 , 37 ) and Ion AmpliSeq ( 38 ). These assays vary in a number of ways including the selection and size of targets, how multiplexed the PCR reactions are, and the sequencing platforms employed, which include Illumina ( 31 , 35 , 36 ), Ion Torrent ( 32 , 34 , 38 ), and Oxford Nanopore Technologies ( 33 , 39 , 40 ).…”

Exploring Diagnostic Methods for Drug-Resistant Tuberculosis: A Comprehensive Overview

“…Here are the four most common challenges/limitations that we found by reviewing the literature. First, the large majority of the analyzed studies considered the old definition of XDR and did not consider the pre-XDR definition, with few exceptions [13,43,50]; thus, it is impossible to translate the findings in the context of the new pre-and XDR definitions. Also, we found few studies that also analysed isolates resistant to LZD or BDQ, for example [17,97], both in terms of resistance mutations (via WGS) or pDST.…”

“…These minor variants, with a frequency < 35%, were differentiated from dominant resistance mutations typically seen at frequencies > 60%. While minor variants do not pose challenges to initial resistance determination, it would be important to further study them for potential progression to full resistance [50].…”