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Rapid molecular diagnostics of resistance tuberculosis by targeted stool sequencing

V Dreyer(1,5) D B Sibandze(2,3,4,13) A Kay(3,4) W Sikhondze(6,13) Q Dlamini(3,4) A DiNardo(4) G Mtetwa(3,4) B Lukhele(3,4) D Vambe(6) C Lange(4,5,7,8) M G Dlamini(2) R Mejia(9) B Kalsdorf(5,7,8,10) J Heyckendorf(7,8) M Kuhns(11) F P Maurer(5,11,12) S Dlamini(2) G Maphalala(2) S Niemann(1,5) A Mandalakas(4) T Ness(4)

1:Research Center Borstel, Leibniz Lung Center; 2:National Tuberculosis Reference Laboratory, Eswatini National Health Services Laboratory, Ministry of Health, Eswatini; 3:Baylor College of Medicine Children’s Foundation-Eswatini, Mbabane, Eswatini; 4:Global Tuberculosis Program, Baylor College of Medicine, Texas, USA; 5:German Center for Infection Research (DZIF), Partner Site Hamburg-Lübeck-Borstel-Riems, Borstel, Germany; 6:Eswatini National Tuberculosis Program, Ministry of Health, Mbabane, Eswatini; 7:Clinical Infectious Diseases, Research Center Borstel, Borstel, Germany; 8:Respiratory Medicine & International Health, University of Lübeck, Lübeck, Germany; 9:The National School of Tropical Medicine, Baylor College of Medicine, Houston, Texas, USA; 10:Cluster Precision Medicine in Inflammation, University of Kiel, Kiel, Germany; 11:National and WHO Supranational Reference Center for Mycobacteria, Research Center Borstel, Borstel, Germany; 12:Institute of Medical Microbiology, Virology and Hygiene, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; 13:Department of Global Health, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands


A stool specimen is important to diagnose Mycobacterium tuberculosis in populations who struggle to provide sputum, such as children or people living with HIV. However, colonies of M tuberculosis complex strains from stool culture performs poorly on drug resistance testing and provides limited utility.



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 with tuberculosis from a prospective cohort in Eswatini, and an independent German validation cohort.



Analysis of the Eswatini cohort included stool specimens from 56 unique participants with and 10 participants without M tuberculosis complex DNA detected by real-time quantitative PCR. 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. 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/ml), 1 to 100 fg/ml, and <1 fg/ml, respectively (p = 0.0004).  The German cohort confirmed these results and demonstrated a high concordance between stool tNGS and sputum phenotypic drug susceptibility results (k = 0.84). 

tNGS is an important diagnostic tool to identify drug resistance from stool specimen provided by tuberculosis patients who struggle to provide respiratory specimens. This discovery affords the opportunity to obtain critical diagnostic information from all tuberculosis patients.

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