Rapid Molecular Diagnostics of Tuberculosis Resistance by Targeted Stool Sequencing

Authors

Doctor B Sibandze
Alexander Kay
Viola Dreyer
Welile Sikhondze
Qiniso Dlamini
Andrew DiNardo
Godwin Mtetwa
Bhekumusa Lukhele
Debrah Vambe
Christoph Lange
Muyalo Glenn Dlamini
Tara Ness
Rojelio Mejia
Barbara Kalsdorf
Jan Heyckendorf
Martin Kuhns
Florian P Maurer
Sindisiwe Dlamini
Gugu Maphalala
Stefan Niemann
Anna Mandalakas

Publication Date

5-19-2022

Journal

Genome Medicine

DOI

10.1186/s13073-022-01054-6

PMID

35585607

PMCID

PMC9118838

PubMedCentral® Posted Date

5-19-2022

PubMedCentral® Full Text Version

Post-print

Published Open-Access

yes

Keywords

Antitubercular Agents, Child, DNA, Humans, Mycobacterium tuberculosis, Pathology, Molecular, Prospective Studies, Real-Time Polymerase Chain Reaction, Sensitivity and Specificity, Tuberculosis, Tuberculosis, Drug resistance, Feces

Abstract

BACKGROUND: Stool is an important diagnostic specimen for tuberculosis in populations who struggle to provide sputum, such as children or people living with HIV. However, the culture of Mycobacterium tuberculosis (M. tuberculosis) complex strains from stool perform poorly. This limits the opportunity for phenotypic drug resistance testing with this specimen. Therefore, reliable molecular methods are urgently needed for comprehensive drug resistance testing on stool specimens.

METHODS: We evaluated the performance of targeted next-generation sequencing (tNGS, Deeplex® Myc-TB) for the detection of mutations associated with M. tuberculosis complex drug resistance on DNA isolated from stool specimens provided by participants from a prospective cohort of patients treated for tuberculosis in Eswatini (n = 66; 56 with and 10 participants without M. tuberculosis complex DNA detected in stool by real-time quantitative PCR), and an independent German validation cohort of participants with culture-confirmed tuberculosis (n = 21).

RESULTS: The tNGS assay detected M. tuberculosis complex DNA in 38 of 56 (68%) samples; for 28 of 38 (74%) samples, a full M. tuberculosis complex drug resistance prediction report was obtained. There was a high degree of concordance with sputum phenotypic drug susceptibility results (κ = 0.82). The ability to predict resistance was concentration-dependent and successful in 7/10 (70%), 18/25 (72%), and 3/21 (14%) of samples with stool PCR concentration thresholds of > 100 femtogram per microliter (fg/μl), 1 to 100 fg/μl, and < 1 fg/μl, respectively (p = 0.0004). The German cohort confirmed these results and demonstrated a similarly high concordance between stool tNGS and sputum phenotypic drug susceptibility results (κ = 0.84).

CONCLUSIONS: tNGS can identify drug resistance from stool provided by tuberculosis patients. This affords the opportunity to obtain critical diagnostic information for tuberculosis patients who struggle to provide respiratory specimens.