OBJECTIVES: Rapidly diagnosing drug-resistant TB is crucial for improving treatment and transmission control. WGS is becoming increasingly accessible and has added value to the diagnosis and treatment of TB. The aim of the study was to perform WGS to determine the rate of false-positive results of phenotypic drug susceptibility testing (pDST) and characterize the molecular mechanisms of resistance and transmission of mono- and polyresistant Mycobacterium (M.) tuberculosis. METHODS: WGS was performed on 53 monoresistant and 25 polyresistant M. tuberculosis isolates characterized by pDST. Sequencing data were bioinformatically processed to infer mutations encoding resistance and determine the origin of resistance and phylogenetic relationship between isolates studied. RESULTS: The data showed the variable sensitivity and specificity of WGS in comparison with pDST as the gold standard: isoniazid 92.7% and 92.3%; streptomycin 41.9% and 100.0%; pyrazinamide 15% and 94.8%; and ethambutol 75.0% and 98.6%, respectively. We found novel mutations encoding resistance to streptomycin (in gidB) and pyrazinamide (in kefB). Most isolates belonged to lineage 4 (80.1%) and the overall clustering rate was 11.5%. We observed lineage-specific gene variations encoding resistance to streptomycin and pyrazinamide. CONCLUSIONS: This study highlights the clinical potential of WGS in ruling out false-positive drug resistance following phenotypic or genetic drug testing, and recommend this technology together with the WHO catalogue in designing an optimal individualized treatment regimen and preventing the development of MDR TB. Our results suggest that resistance is primarily developed through spontaneous mutations or selective pressure.

Biomedical Centre Martin Jessenius Faculty of Medicine in Martin Comenius University Bratislava Slovakia

Department of Clinical Microbiology and Department of Pneumophthiology National Institute of Tuberculosis Lung Diseases and Thoracic Surgery Vyšné Hágy Slovakia

Department of Molecular Medicine Jessenius Faculty of Medicine in Martin Comenius University Bratislava Slovakia

Department of Pharmacology Jessenius Faculty of Medicine in Martin Comenius University Bratislava Slovakia

Emerging Bacterial Pathogens Unit Division of Immunology Transplantation and Infectious Diseases IRCCS San Raffaele Scientific Institute Milan Italy

Faculty of Health Catholic University Ružomberok Slovakia

International Reference Laboratory of Mycobacteriology Statens Serum Institut Copenhagen Denmark

National Reference Laboratory for Mycobacteria National Institute of Public Health Prague Czech Republic

Vita Salute San Raffaele University Milan Italy

Zobrazit více v PubMed

WHO . Global Tuberculosis Report 2021. 2021. https://www.who.int/publications/i/item/9789240037021.

WHO . Companion Handbook to the WHO Guidelines for the Programmatic Management of Drug-Resistant Tuberculosis. 2014. https://www.ncbi.nlm.nih.gov/books/NBK247420/. PubMed

Cohen KA, Abeel T, Manson McGuire Aet al. . Evolution of extensively drug-resistant tuberculosis over four decades: whole genome sequencing and dating analysis of mycobacterium tuberculosis isolates from KwaZulu-Natal. PLoS Med 2015; 12: e1001880. 10.1371/journal.pmed.1001880 PubMed DOI PMC

Köser CU, Cirillo DM, Miotto P. How to optimally combine genotypic and phenotypic drug susceptibility testing methods for pyrazinamide. Antimicrob Agents Chemother 2020; 64: e01003-20. 10.1128/AAC.01003-20 PubMed DOI PMC

Ngabonziza JCS, Decroo T, Migambi Pet al. . Prevalence and drivers of false-positive rifampicin-resistant Xpert MTB/RIF results: a prospective observational study in Rwanda. Lancet Microbe 2020; 1: e74–83. 10.1016/S2666-5247(20)30007-0 PubMed DOI

WHO . Technical Manual for Drug Susceptibility Testing of Medicines Used in the Treatment of tuberculosis. 2018. https://www.who.int/publications/i/item/9789241514842.

Meehan CJ, Goig GA, Kohl TAet al. . Whole genome sequencing of Mycobacterium tuberculosis: current standards and open issues. Nat Rev Microbiol 2019; 17: 533–45. 10.1038/s41579-019-0214-5 PubMed DOI

Tagliani E, Anthony R, Kohl TAet al. . Use of a whole genome sequencing-based approach for Mycobacterium tuberculosis surveillance in Europe in 2017–2019: an ECDC pilot study. Eur Res J 2021; 57: 2002272. 10.1183/13993003.02272-2020 PubMed DOI PMC

Kohl TA, Utpatel C, Schleusener Vet al. . MTBseq: a comprehensive pipeline for whole genome sequence analysis of Mycobacterium tuberculosis complex isolates. PeerJ 2018; 2018: e5895. 10.7717/peerj.5895 PubMed DOI PMC

Walker TM, Fowler PW, Knaggs Jet al. . The 2021 WHO catalogue of Mycobacterium tuberculosis complex mutations associated with drug resistance: a genotypic analysis. Lancet Microbe 2022; 3: e265–73. 10.1016/S2666-5247(21)00301-3 PubMed DOI PMC

Stamatakis A. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 2014; 30: 1312–3. 10.1093/bioinformatics/btu033 PubMed DOI PMC

Letunic I, Bork P. Interactive tree of life (iTOL) v5: an online tool for phylogenetic tree display and annotation. Nucleic Acids Res 2021; 49: W293–6. 10.1093/nar/gkab301 PubMed DOI PMC

Walker TM, Ip CLC, Harrell RHet al. . Whole-genome sequencing to delineate Mycobacterium tuberculosis outbreaks: a retrospective observational study. Lancet Infect Dis 2013; 13: 137–46. 10.1016/S1473-3099(12)70277-3 PubMed DOI PMC

Zhou Z, Alikhan NF, Sergeant MJet al. . GrapeTree: visualization of core genomic relationships among 100,000 bacterial pathogens. Genome Res 2018; 28: 1395–404. 10.1101/gr.232397.117 PubMed DOI PMC

Galili T. dendextend: an R package for visualizing, adjusting and comparing trees of hierarchical clustering. Bioinformatics 2015; 31: 3718–20. 10.1093/bioinformatics/btv428 PubMed DOI PMC

Coll F, McNerney R, Guerra-Assunção JAet al. . A robust SNP barcode for typing Mycobacterium tuberculosis complex strains. Nat Commun 2014; 5: 4812. 10.1038/ncomms5812 PubMed DOI PMC

Miotto P, Zhang Y, Cirillo DMet al. . Drug resistance mechanisms and drug susceptibility testing for tuberculosis. Respirology 2018; 23: 1098–113. 10.1111/resp.13393 PubMed DOI

Gegia M, Cohen T, Kalandadze Iet al. . Outcomes among tuberculosis patients with isoniazid resistance in Georgia, 2007–2009. Int J Tuberc Lung Dis 2012; 16: 812–6. 10.5588/ijtld.11.0637 PubMed DOI PMC

Dean AS, Zignol M, Cabibbe AMet al. . Prevalence and genetic profiles of isoniazid resistance in tuberculosis patients: a multicountry analysis of cross-sectional data. PLoS Med 2020; 17: e1003008. 10.1371/journal.pmed.1003008 PubMed DOI PMC

Binkhamis KM, Bahatheg MA, Altahan FAet al. . Prevalence and outcome of isoniazid-monoresistant tuberculosis at a university hospital in Saudi Arabia. Saudi Med J 2021; 42: 636–42. 10.15537/smj.2021.42.6.20200832 PubMed DOI PMC

Liu Z, Zhang M, Wang Jet al. . Longitudinal analysis of prevalence and risk factors of rifampicin-resistant tuberculosis in Zhejiang, China. Biomed Res Int 2020; 2020: 3159482. 10.1155/2020/3159482 PubMed DOI PMC

André E, Goeminne L, Colmant Aet al. . Novel rapid PCR for the detection of Ile491Phe rpoB mutation of Mycobacterium tuberculosis, a rifampicin-resistance-conferring mutation undetected by commercial assays. Clin Microbiol Infect 2017; 23: 267.e5–e7. 10.1016/j.cmi.2016.12.009 PubMed DOI

Rigouts L, Gumusboga M, De Rijk WBet al. . Rifampin resistance missed in automated liquid culture system for Mycobacterium tuberculosis isolates with specific rpoB mutations. J Clin Microbiol 2013; 51: 2641. 10.1128/JCM.02741-12 PubMed DOI PMC

Wang L, Yang J, Chen Let al. . Whole-genome sequencing of Mycobacterium tuberculosis for prediction of drug resistance. Epidemiol Infect 2022; 150: e22. 10.1017/S095026882100279X PubMed DOI PMC

Jajou R, Van Der Laan T, De Zwaan Ret al. . WGS more accurately predicts susceptibility of Mycobacterium tuberculosis to first-line drugs than phenotypic testing. J Antimicrob Chemother 2019; 74: 2605–16. 10.1093/jac/dkz215 PubMed DOI

Phelan JE, O’Sullivan DM, Machado Det al. . Integrating informatics tools and portable sequencing technology for rapid detection of resistance to anti-tuberculous drugs. Genome Med 2019; 11: 41. 10.1186/s13073-019-0650-x PubMed DOI PMC

Rocha DMGC, Magalhães C, Cá Bet al. . Heterogeneous streptomycin resistance level among Mycobacterium tuberculosis strains from the same transmission cluster. Front Microbiol 2021; 12: 1380. 10.3389/fmicb.2021.659545 PubMed DOI PMC

Glasauer S, Altmann D, Hauer Bet al. . First-line tuberculosis drug resistance patterns and associated risk factors in Germany, 2008–2017. PLoS One 2019; 14: e0217597. 10.1371/journal.pone.0217597 PubMed DOI PMC

Chernyaeva E, Rotkevich M, Krasheninnikova Ket al. . Whole-genome analysis of Mycobacterium tuberculosis from patients with tuberculous spondylitis, Russia. Emerg Infect Dis 2018; 24: 579–83. 10.3201/eid2403.170151 PubMed DOI PMC

Jnawali HN, Yoo H, Ryoo Set al. . Molecular genetics of Mycobacterium tuberculosis resistant to aminoglycosides and cyclic peptide capreomycin antibiotics in Korea. World J Microbiol Biotechnol 2013; 29: 975–82. 10.1007/s11274-013-1256-x PubMed DOI

Rahman SMM, Rahman A, Nasrin Ret al. . Molecular epidemiology and genetic diversity of multidrug-resistant Mycobacterium tuberculosis isolates in Bangladesh. Microbiol Spectr 2022; 10: e0184821. 10.1128/spectrum.01848-21 PubMed DOI PMC

Salfinger M, Heifets LB. Determination of pyrazinamide MICs for Mycobacterium tuberculosis at different pHs by the radiometric method. Antimicrob Agents Chemother 1988; 32: 1002–4. 10.1128/AAC.32.7.1002 PubMed DOI PMC

Farhat MR, Freschi L, Calderon Ret al. . GWAS for quantitative resistance phenotypes in Mycobacterium tuberculosis reveals resistance genes and regulatory regions. Nat Commun 2019; 10: 2128. 10.1038/s41467-019-10110-6 PubMed DOI PMC

Chan RCY, Hui M, Chan EWCet al. . Genetic and phenotypic characterization of drug-resistant Mycobacterium tuberculosis isolates in Hong Kong. J Antimicrob Chemother 2007; 59: 866–73. 10.1093/jac/dkm054 PubMed DOI PMC

Budzik JM, Jarlsberg LG, Higashi Jet al. . Pyrazinamide resistance, Mycobacterium tuberculosis lineage and treatment outcomes in San Francisco, California. PLoS One 2014; 9: e95645. 10.1371/journal.pone.0095645 PubMed DOI PMC

Bouzouita I, Cabibbe AM, Trovato Aet al. . Is sequencing better than phenotypic tests for the detection of pyrazinamide resistance? Int J Tuberc Lung Dis 2018; 22: 661–6. 10.5588/ijtld.17.0715 PubMed DOI

Lam C, Martinez E, Crighton Tet al. . Value of routine whole genome sequencing for Mycobacterium tuberculosis drug resistance detection. Int J Infect Dis 2021; 113: S48–54. 10.1016/j.ijid.2021.03.033 PubMed DOI

Cancino-Muñoz I, Moreno-Molina M, Furió Vet al. . Cryptic resistance mutations associated with misdiagnoses of multidrug-resistant tuberculosis. J Infect Dis 2019; 220: 316–20. 10.1093/infdis/jiz104 PubMed DOI PMC

Hakamata M, Takihara H, Iwamoto Tet al. . Higher genome mutation rates of Beijing lineage of Mycobacterium tuberculosis during human infection. Sci Rep 2020; 10: 17997. 10.1038/s41598-020-75028-2 PubMed DOI PMC

Gagneux S. Ecology and evolution of Mycobacterium tuberculosis. Nat Rev Microbiol 2018; 16: 202–13. 10.1038/nrmicro.2018.8 PubMed DOI

Takiff HE, Feo O. Clinical value of whole-genome sequencing of Mycobacterium tuberculosis. Lancet Infect Dis 2015; 15: 1077–90. 10.1016/S1473-3099(15)00071-7 PubMed DOI

Shi J, Zheng D, Zhu Yet al. . Role of MIRU-VNTR and spoligotyping in assessing the genetic diversity of Mycobacterium tuberculosis in Henan Province, China. BMC Infect Dis 2018; 18: 447. 10.1186/s12879-018-3351-y PubMed DOI PMC