Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2022 May 19;14(1):52.
doi: 10.1186/s13073-022-01054-6.

Rapid molecular diagnostics of tuberculosis resistance by targeted stool sequencing

Doctor B Sibandze #  1   2   3   4 , Alexander Kay #  5   6 , Viola Dreyer #  7   8 , Welile Sikhondze #  4   9 , Qiniso Dlamini  2   3 , Andrew DiNardo  3 , Godwin Mtetwa  2   3 , Bhekumusa Lukhele  2   3 , Debrah Vambe  9 , Christoph Lange  3   8   10   11 , Muyalo Glenn Dlamini  1 , Tara Ness  3 , Rojelio Mejia  12 , Barbara Kalsdorf  8   10   11   13 , Jan Heyckendorf  8   11   14 , Martin Kuhns  15 , Florian P Maurer  8   15   16 , Sindisiwe Dlamini  1 , Gugu Maphalala  1 , Stefan Niemann #  7   8 , Anna Mandalakas #  17   18   19
Affiliations

Rapid molecular diagnostics of tuberculosis resistance by targeted stool sequencing

Doctor B Sibandze et al. Genome Med. .

Erratum in

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.

Keywords: Drug resistance; Feces; Tuberculosis.

PubMed Disclaimer

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Study flow chart demonstrating results classified in accordance with real-time polymerase chain reaction (qPCR) and concentrations in fg/μl (1 and 100 fg/uL of DNA equates to 62.65 CFU and 2316.13 CFU of H37Rv Mtb/50 mg of stool). Targeted sequencing quality was graded as quality (ND) not detected (red), quality – indicative of partial reads (yellow), and quality + , +  + , and +  +  + indicating complete reads with adequate depth of coverage (green). A Cohort 1 and (B) Cohort 2. *partial resistance report possible for one sample. Abbreviations: ND, not detected
Fig. 2
Fig. 2
Deeplex® Myc-TB resistotype and pDST resistance detection. Protionamide was tested as a class representative for the thioamides. Mutations associated with resistance are specified in the cells when present. A Cohort 1 and (B) Cohort 2. Abbreviations: pDST, phenotypic drug susceptibility; RMP, rifampicin; INH, isoniazid; PZA, pyrazinamide; EMB, ethambutol; SM, streptomycin; FQ, fluoroquinolones; KAN, kanamycin; AMI, amikacin; CAP, capreomycin; TA, thionamide; LNZ, linezolid; BDQ, bedaquiline; CFZ, clofazamine
See this image and copyright information in PMC

References

    1. Global Tuberculosis Report 2020. Geneva: World Health Organization; 2020. Licence: CC BY-NC-SA 3.0 IGO.
    1. Knight GM, McQuaid CF, Dodd PJ, Houben RMGJ. Global burden of latent multidrug-resistant tuberculosis: trends and estimates based on mathematical modelling. Lancet Infect Dis. 2019;19:903–912. doi: 10.1016/S1473-3099(19)30307-X. - DOI - PMC - PubMed
    1. Dodd PJ, Sismanidis C, Seddon JA. Global burden of drug-resistant tuberculosis in children: a mathematical modelling study. Lancet Infect Dis. 2016;16:1193–1201. doi: 10.1016/S1473-3099(16)30132-3. - DOI - PubMed
    1. Lange C, Dheda K, Chesov D, Mandalakas AM, Udwadia Z, Horsburgh CR., Jr Management of drug-resistant tuberculosis. Lancet. 2019;394:953–966. doi: 10.1016/S0140-6736(19)31882-3. - DOI - PMC - PubMed
    1. Lange C, Aarnoutse RE, Alffenaar JWC, et al. Management of patients with multidrug-resistant tuberculosis. Int J Tuberc Lung Dis. 2019;23:645–662. doi: 10.5588/ijtld.18.0622. - DOI - PubMed

Publication types