Rapid molecular diagnostics of tuberculosis resistance by targeted stool sequencing

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

doi:10.1186/s13073-022-01054-6

Rapid molecular diagnostics of tuberculosis resistance by targeted stool sequencing

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

National Tuberculosis Reference Laboratory, Ministry of Health, Mbabane, Eswatini
Baylor College of Medicine Children's Foundation-Eswatini
Divisions of Clinical Infectious Diseases, Research Center Borstel, German Center for Infection Research (DZIF), Clinical Tuberculosis Unit, Borstel
Department of Sciences, Amsterdam University College, VU Amsterdam/University of Amsterdam, 1012 WX, Amsterdam, The Netherlands
Baylor College of Medicine
Eswatini National Tuberculosis Program
National Tuberculosis Reference Laboratory
German Center for Infection Research (DZIF), Germany

Research output: Contribution to journal › Article › Academic › peer-review

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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.

Original language	English
Article number	52
Number of pages	11
Journal	Genome medicine
Volume	14
DOIs	https://doi.org/10.1186/s13073-022-01054-6
Publication status	Published - 19 May 2022

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

SDG 3 Good Health and Well-being

Keywords

Antitubercular Agents/pharmacology
Child
DNA
Humans
Mycobacterium tuberculosis/genetics
Pathology, Molecular
Prospective Studies
Real-Time Polymerase Chain Reaction
Sensitivity and Specificity
Tuberculosis/diagnosis

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10.1186/s13073-022-01054-6

Rapid-molecular-diagnostics-of-tuberculosis-resistance-by-targeted-stool-sequencing.pdfFinal published version, 1.18 MBLicence: Taverne

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Sibandze, D. B., Kay, A., Dreyer, V., Sikhondze, W., Dlamini, Q., DiNardo, A., Mtetwa, G., Lukhele, B., Vambe, D., Lange, C., Glenn Dlamini, M., Ness, T., Mejia, R., Kalsdorf, B., Heyckendorf, J., Kuhns, M., Maurer, F. P., Dlamini, S., Maphalala, G., ... Mandalakas, A. (2022). Rapid molecular diagnostics of tuberculosis resistance by targeted stool sequencing. Genome medicine, 14, Article 52. https://doi.org/10.1186/s13073-022-01054-6

@article{6f9c8726b649408893d945f25dab1197,

title = "Rapid molecular diagnostics of tuberculosis resistance by targeted stool sequencing",

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{\textregistered} 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.",

keywords = "Antitubercular Agents/pharmacology, Child, DNA, Humans, Mycobacterium tuberculosis/genetics, Pathology, Molecular, Prospective Studies, Real-Time Polymerase Chain Reaction, Sensitivity and Specificity, Tuberculosis/diagnosis",

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

note = "{\textcopyright} 2022. The Author(s).",

year = "2022",

month = may,

day = "19",

doi = "10.1186/s13073-022-01054-6",

language = "English",

volume = "14",

journal = "Genome medicine",

issn = "1756-994X",

publisher = "BioMed Central Ltd.",

}

Sibandze, DB, Kay, A, Dreyer, V, Sikhondze, W, Dlamini, Q, DiNardo, A, Mtetwa, G, Lukhele, B, Vambe, D, Lange, C, Glenn Dlamini, M, Ness, T, Mejia, R, Kalsdorf, B, Heyckendorf, J, Kuhns, M, Maurer, FP, Dlamini, S, Maphalala, G, Niemann, S & Mandalakas, A 2022, 'Rapid molecular diagnostics of tuberculosis resistance by targeted stool sequencing', Genome medicine, vol. 14, 52. https://doi.org/10.1186/s13073-022-01054-6

TY - JOUR

T1 - Rapid molecular diagnostics of tuberculosis resistance by targeted stool sequencing

AU - Sibandze, Doctor B

AU - Kay, Alexander

AU - Dreyer, Viola

AU - Sikhondze, Welile

AU - Dlamini, Qiniso

AU - DiNardo, Andrew

AU - Mtetwa, Godwin

AU - Lukhele, Bhekumusa

AU - Vambe, Debrah

AU - Lange, Christoph

AU - Glenn Dlamini, Muyalo

AU - Ness, Tara

AU - Mejia, Rojelio

AU - Kalsdorf, Barbara

AU - Heyckendorf, Jan

AU - Kuhns, Martin

AU - Maurer, Florian P

AU - Dlamini, Sindisiwe

AU - Maphalala, Gugu

AU - Niemann, Stefan

AU - Mandalakas, Anna

PY - 2022/5/19

Y1 - 2022/5/19

N2 - 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.

AB - 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.

KW - Antitubercular Agents/pharmacology

KW - Child

KW - DNA

KW - Humans

KW - Mycobacterium tuberculosis/genetics

KW - Pathology, Molecular

KW - Prospective Studies

KW - Real-Time Polymerase Chain Reaction

KW - Sensitivity and Specificity

KW - Tuberculosis/diagnosis

U2 - 10.1186/s13073-022-01054-6

DO - 10.1186/s13073-022-01054-6

M3 - Article

SN - 1756-994X

VL - 14

JO - Genome medicine

JF - Genome medicine

M1 - 52

ER -

Rapid molecular diagnostics of tuberculosis resistance by targeted stool sequencing

Abstract

UN SDGs

Keywords

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