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

Area of Science:

• Microbiology
• Genetics
• Infectious Diseases

Background:

• Tuberculosis (TB) treatment relies on antibiotic combinations, but efficacy is limited against multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains.
• Conventional phenotypic drug susceptibility testing for Mycobacterium tuberculosis complex (MTBC) is time-consuming, delaying appropriate patient management.

Purpose of the Study:

• To develop and validate a multiplexed, targeted next-generation sequencing (tNGS) assay for rapid prediction of MTBC drug resistance (DR).
• To enable direct testing on clinical respiratory specimens, reducing turnaround time compared to culture-based methods.

Main Methods:

• Designed a multiplex PCR assay to amplify 13 full-length genes and promoter regions associated with MTBC drug resistance.
• Utilized Oxford Nanopore Technologies for long-read amplicon sequencing and an in-house bioinformatics pipeline for real-time mutation identification.
• Validated the tNGS assay's sensitivity, specificity, reproducibility, and accuracy on 72 primary specimens and 55 MTBC-positive cultures, comparing results with whole genome sequencing (WGS).

Main Results:

• Achieved complete or partial susceptibility profiles from 82% of smear-positive primary specimens.
• Demonstrated 100% concordance between tNGS-identified resistance mutations and WGS results.
• The tNGS assay offers a 2-3 day turnaround time, significantly faster than culture-derived WGS, and can process mixed mycobacterial cultures.

Conclusions:

• The tNGS assay reliably predicts MTBC drug resistance directly from clinical specimens and cultures.
• This rapid diagnostic tool provides critical information for timely and appropriate treatment of patients with drug-resistant tuberculosis.
• The assay is suitable for clinical implementation, improving patient outcomes by accelerating treatment decisions.