Language
English
Publication Date
11-17-2025
Journal
Microbiology Spectrum
DOI
10.1128/spectrum.02335-25
PMID
41247050
Abstract
Alaska has the highest incidence of tuberculosis (TB) in the United States, with 8% mortality while undergoing TB treatment. With a quarter of TB cases lacking sputum culture to enable drug resistance testing, FreezeTB aimed to develop tools tailored to meet the challenges in Alaska while being translatable to other settings. We designed a rapid and cost-effective laboratory workflow and software to identify drug-resistant mutations in Mycobacterium tuberculosis using targeted next-generation sequencing (tNGS). FreezeTB, a Fast, Reliable, Economical Evaluation tool to Zap Endemic Tuberculosis, amplifies 22 gene loci associated with resistance to 16 anti-tuberculosis drugs. M. tuberculosis isolates from Alaska (2011-2024) were blinded and underwent analysis with FreezeTB, then compared with phenotypic drug susceptibility testing (pDST) and whole genome sequencing (WGS). Compared with WGS (n = 79), FreezeTB provided the same mutations in 96% (n = 76/79) of samples with 100% lineage agreement (n = 79/79). Compared with pDST using Cohen's kappa, FreezeTB had almost perfect agreement for rifampin (RIF, 0.90; n = 97/98) and ethambutol (EMB, 1.00; n = 98/98), strong agreement for isoniazid (INH, 0.80; n = 88/98), moderate agreement for ethionamide (ETO, 0.78; n = 95/98), weak agreement for streptomycin (STR, 0.56; n = 95/98), and no agreement for pyrazinamide (PZA, 0.20; n = 91/98). Using a portable nanopore sequencer, each sample cost under $30 for sequencing, which included a flow cell, a barcoding kit, a flow cell wash kit, a polymerase, and primers. FreezeTB provides a portable, 1-day laboratory and bioinformatic workflow for sequencing M. tuberculosis. Freeze TB can accurately determine mycobacterial lineage and resistance for RIF, INH, ETH, and ETO at a low cost.IMPORTANCEGlobally, tuberculosis is the leading infectious cause of mortality with 10.8 million new cases and 1.25 million deaths occurring in 2024. Targeted next-generation sequencing (tNGS) is a rapid, cost-effective method for identifying mutations in the Mycobacterium tuberculosis genome associated with drug resistance. FreezeTB was created to provide low-cost, portable sequencing and tNGS analysis for drug resistance. FreezeTB selectively amplifies and sequences 24 targets of the M. tuberculosis genome that cover regions the WHO has designated as containing mutations conferring drug resistance, as well as provides species confirmation and rapid lineage determination. Freeze TB is a laboratory workflow and free, open-source (OS) low dependency bioinformatic software that can be downloaded onto a computer with no further need for internet access. Using M. tuberculosis isolates from Alaska, FreezeTB provided the same mutations in 96% (n = 76/79) of samples with 100% lineage agreement (n = 79/79) compared with whole genome sequencing.
Keywords
AMR, Alaska, DR-TB, MDR-TB, Mycobacterium tuberculosis, TB, nanopore, sequencing, spoligotyping, tNGS, targeted, tuberculosis
Published Open-Access
yes
Recommended Citation
Inman, Bryce; Butler, Jeremy; George-Nichol, Soren; et al., "Application of FreezeTB, a Targeted Nanopore Sequencing Assay, for Identification of Drug Resistance and Lineages Among Pulmonary Tuberculosis Cases in Alaska" (2025). Faculty and Staff Publications. 4692.
https://digitalcommons.library.tmc.edu/baylor_docs/4692