Molecular technologies of mycobacterial research


  • Olga Shapovalova National University of Pharmacy, Ukraine
  • Svitlana Pozmogova NNC "IEKVM", Ukraine
  • Andriy Zavgorodniy NNC "IEKVM", Ukraine



Introduction. Despite significant successes in the fight against human and animal tuberculosis, many aspects of laboratory diagnostics, prevention and treatment of the disease require further improvement. Recently, complex technologies have been successfully implemented that effectively support the advantages of routine and innovative methods of molecular diagnostics and allow solving the urgent problems of detecting extrapulmonary forms of the disease, epidemiology and epizootology of tuberculosis, identifying isolates of non-tuberculous mycobacteria and forms of pathogens resistant to anti-tuberculosis drugs. The purpose of the work is to conduct a comparative analysis of technologies and methodological approaches used in modern laboratories specializing in molecular identification and differentiation of mycobacteria in human and veterinary medicine. We conducted a search for sources of literature covering the problems of diagnostics, treatment and prevention of tuberculosis in the PubMed, Medline, Web of Science, Google Scholar databases from 2001 to 2022. Full-text articles were selected for analysis, including the biological characteristics of mycobacteria, technologies of molecular diagnostic of human and animals tuberculosis and mycobacteriosis, issues of molecular epidemiology of mycobacterial infections, modern methods of detecting resistance of pathogens to antituberculosis drugs. The results of the literature data analysis show that common molecular genetic technologies allow detection MTBC and NTM in culture or specific genetic markers directly in the biomaterial: ITS 16S-23S рРНК, immunodominant antigen HSP65, specific proteins DnaJ, МРТ64, MPB64, IS (IS6110, IS901, IS900, IS1245 and other),  genes of resistance to antituberculosis drugs, mutation or deletion of the corresponding genes, etc. Most often, for this purpose, NAAT technologies are used in various modifications, MLPA, MOL-PCR, SNP- typing, DNA probe, PFGE- typing and other. WHO has introduced CB-NAAT, LPA and LAMP cartridge technology for widespread use, on which commercially available test systems from various manufacturers are based. The main molecular technologies used to determine the drug resistance of mycobacteria and the mechanisms of its occurrence are whole-genome sequencing of isolates and analysis of individual genes isolated from clinical samples. Currently, for each antituberculosis drug of the first and second line, as well as for most reserve drugs, genes, specific mutations in which lead to the development of drug resistance, have been identified. For the most studied M. tuberculosis, the list of molecular markers for detection of drug resistance includes a number of constitutive genes encoding the cellular targets of antituberculosis drugs. For this purpose, MLPA technologies with many drug resistance markers (rpoB, katG, inhA, embB) are widely used along with MTBC and NTM genotype detection. Conclusion. Today, molecular technologies are an integral element of almost all diagnostic and epidemiological/epizootological studies of mycobacterial infections. They expand our understanding of the biology of mycobacteria and serve as powerful tools for improving strategies and tactics for the treatment and prevention of diseases caused by these pathogens. The evolution of molecular technologies is in the direction of increasing their sensitivity, specificity, speed, simplicity and reducing the cost of analysis. Molecular technologies known and available today differ in reproducibility, reliability, production cost, degree of standardization, methods of interpreting results, availability of databases for interlaboratory comparative studies conducting. In each specific case, depending on the purpose and object of the study, taking into account the capabilities of the laboratory, the optimal technology for the identification and differentiation of mycobacteria isolated from biomaterial should be selected.

Author Biography

Olga Shapovalova, National University of Pharmacy

Доцент закладу вищої освіти кафедри мікробіології, вірусології та імунології  Національного фармацевтичного університеті, м. Харьків, Україна.


Adikaram Ch. P. Overview of Non Tuberculosis Mycobacterial Lung Diseases. Mycobacterium – Research and Development. 2018. Ch. 13. Р. 257-286.

Afanasiev M. V. Molecular typing of clinical strains of Mycobacterium tuberculosis: abstract dis. ... cand. biol. sciences: 03.00.04. M., SRI Research Institute of Physical and Chemical Medicine, 2008. 24 р.

Algorithm for laboratory diagnosis and treatment-monitoring of pulmonary tuberculosis and drug-resistant tuberculosis using state-of-the-art rapid molecular diagnostic technologies: expert opinion of the European Tuberculosis Laboratory Initiative core group members for the WHO European Region. World Health Organization. Regional Office for Europe. 2017. 29 р. URL:

Bagre A., Kumar Lariya N., Lal Kori M. Diagnosis of Tuberculosis: A core curriculum review IJRAR. 2019, V. 6. 1. Р.1193-1217.

Barbova A. I., Zhurylo O. A., Trofimova P. S., Myronchenko S. V. Comparative results of drug resistance determining by molecular-genetic and phenotypic research methods. Ukrainian Pulmonology Journal. 2018, № 1 (S). P. 6-8.

Bergval I. L., Vijzelaar R. N. C. P., Dalla Costa E. R., Schuitema A. R. J., Oskam L., Kritski A. L., Klatser P. R., Anthony R. M. Development of Multiplex Assay for Rapid Characterization of Mycobacterium tuberculosis. J. Clin. Microbiol. 2008. 46. 2. P. 689–699.

Catalogue of mutations in Mycobacterium tuberculosis complex and their association with drug resistance. Geneva: World Health Organization; 2021. Licence: CC BY-NC-SA 3.0 IGO.

Cherednyk Yu. A., Anoprienko O. V., Feshchenko Yu. I. Molecular and genetic typing of clinical isolates of Mycobacterium tuberculosis in Ukraine. Ukrainian Pulmonology Journal. 2005. 4. P. 66 - 68.

Clarridge J. E., III. Impact of 16S rRNA Gene Sequence Analysis for Identification of Bacteria on Clinical Microbiology and Infectious Diseases. Clinical microbiology reviews. 2004. Vol. 17, No. 4. P. 840–862. doi: 10.1128/CMR.17.4.840–862.2004.

Core Curriculum on Tuberculosis: What the Clinician Should Know. Sixth Edition 2013. Chapter 4. Diagnosis of Tuberculosis Disease. URL:

Dicks K.V., Stout J.E. Molecular Diagnostics for Mycobacterium tuberculosis Infection. Annu. Rev. Med. 2019. 70. Р. 77-90.

Ergeshov A.E., Chernousova L.N., Andreevskaya S.N. New Technologies for the Diagnosis of Drug-Resistant Tuberculosis. Annals of the Russian Academy of Medical Sciences. 2019. 74 (6). Р. 413–422. doi: 10.15690/vramn1163.

Faksri K., Xia E., Tan J.H., Teo Y.Y., Ong R.T. In silico region of difference (RD) analysis of Mycobacterium tuberculosis complex from sequence reads using RD-Analyzer. BMC Genomics. 2016. 17 (1). Р. Article number: 847. doi: 10.1186/s12864-016-3213-1.

Feshchenko Y. I. Up-to-date tendencies in tuberculosis research. Ukrainian Pulmonology Journal. 2019. № 1. P. 8–24.

Fujiwara M., Kawasaki М., Hariguchi N., Liu Y., Matsumoto M. Mechanisms of resistance to delamanid, a drug for Mycobacterium tuberculosis. Tuberculosis. 2018. V. 108. P. 186-194.

Gils T., Lynen L., de Jong B.C., Van Deun A., Decroo T. Pretomanid for tuberculosis: a systematic review. Clin Microbiol Infect. 2022. 28 (1). Р. 31-42. doi: 10.1016/j.cmi.2021.08.007. Epub 2021 Aug 14.

Global tuberculosis report 2019. Geneva: World Health Organization. 2019. 297 р. URL:

Islam M.M., Hameed H.M.A., Mugweru J., Chhotaray C., Wang C., Tan Y., Liu J., Li X., Tan S., Ojima I., Yew W.W., Nuermberger E., Lamichhane G., Zhang T. Drug resistance mechanisms and novel drug targets for tuberculosis therapy. J Genet Genomics. 2017. 44 (1). Р. 21-37. doi: 10.1016/j.jgg.2016.10.002.

Islam M.M., Tan Y., Hameed H.M.A., Liu Z., Chhotaray C., Liu Y., Lu Z., Cai X., Tang Y., Gao Y., Surineni G., Li X., Tan S., Guo L., Cai X., Yew W.W., Liu J., Zhong N., Zhang T. Detection of novel mutations associated with independent resistance and cross-resistance to isoniazid and prothionamide in Mycobacterium tuberculosis clinical isolates. Clinical Microbiology and Infection 2019. 25. P. 1041.e1-1041e7.

Jagielski T., Minias A., van Ingen J., Rastogi N., Brzostek A., Z˙aczek A., Dziadek J. Methodological and clinical aspects of the molecular epidemiology of Mycobacterium tuberculosis and other mycobacteria. Clin Microbiol Rev. 2016. 29. P. 239-290. doi:10.1128/CMR.00055-15.

Kadura S., King N., Nakhoul M., Zhu H., Theron G., Köser C.U., Farhat M.J. Systematic review of mutations associated with resistance to the new and repurposed Mycobacterium tuberculosis drugs bedaquiline, clofazimine, linezolid, delamanid and pretomanid. Antimicrob Chemother. 2020. 75 (8). Р. 2031-2043. doi: 10.1093/jac/dkaa136.

Kim H., Kim S.-H., Shim T.-S., Kim Mi-na, Bai G.-H. Differentiation of Mycobacterium species by analysis of the heat-shock protein 65 gene (hsp65). International Journal of Systematic and Evolutionary Microbiology 2005. 55. P. 1649-1656. doi 10.1099/ijs.0.63553-0.

Leao S.C., Martin A., Mejia G.I., Palomino J.C., Robledo, J., da Silva Telles, M.A., Portaels F. Practical handbook for the phenotypic and genotypic identification of mycobacteria. 2004. 164 рр.

Liashenko O.O. Genotyping methods in phthisiology. Tuberculosis Lung Diseases HIV Infection. 2015. № 1 (20). P. 98 - 103.

Liu W., Li B., Chu H., Zhang Z., L. Luo, W. Ma, S. Yang, Q. Guo. Rapid Detection of Mutations in erm(41) and RRL Associated With Clarithromycin Resistance in Mycobacterium Abscessus Complex by Denaturing Gradient Gel Electrophoresis J Microbiol. Methods 2017. Vol. 143. P. 87-93. doi: 10.1016/j.mimet.2017.10.010.

Manual of Commercial Methods in Clinical Microbiology. Allan L. Truant, editor-in-chief. 2nd edition. Hoboken, New Jersey : John Wiley & Sons, Inc., 2016. Ch. 14 Mycobacteria. Р. 273 - 283.

Mase A., Yamaguchi F., Funaki T., Yamazaki Y., Shikama Y., Fukuchi K. PCR amplification of the erm(41) gene can be used to predict the sensitivity of Mycobacterium abscessus complex strains to clarithromycin. Experimental and Therapeutic Medicine 2020. 19. Р. 945-955. DOI: 10.3892/etm.2019.8289.

Meeting report of the WHO expert consultation on the definition of extensively drug-resistant tuberculosis, 27-29 October 2020. Geneva: World Health Organization; 2021. URL:

Mehta P., Dahiya B., Sharma S., Singh N., Dharra R., Thakur Z., Mehta N., Gupta K.B., Gupta M.C., Chaudhary D. Immuno-PCR, a new technique for the serodiagnosis of tuberculosis. J Microbiol Methods. 2017. 139. P. 218-229.

Mokrousov I.V. Some features of genome structure and evolution of Mycobacterium tuberculosis Infekc. immun. 2011. Vol. 1. 3. P. 211–220.

Neonakis I. K., Gitti Z., Krambovitis E., Spandidos D. A. Molecular diagnostic tools in mycobacteriology. Journal of Microbiological Methods 2008. 75. P. 1–11.

Nguyen T.V.A., Anthony R.M., Cao T.T.H., Bañuls A.L., Nguyen V.A.T., Vu D.H., Nguyen N.V., Alffenaar J.C. Delamanid Resistance: Update and Clinical Management. Clin Infect Dis. 2020. 71 (12). Р. 3252-3259. doi: 10.1093/cid/ciaa755.

On the approval of the Instructions for Microbiological Diagnosis of Tuberculosis Order of the Ministry of Health from 27.06.2019 № 1462. URL:

Parveen S., Arya D. Recent Advances in Diagnosis of Tuberculosis: A Review. Int. J. Life. Sci. Scienti. Res. 2018. 4 (1). P 1557-1562. doi:10.21276/ijlssr.2018.4.1.8.

Policy guidance on drug-susceptibility testing (DST) of second-line antituberculosis drugs. World Health Organization. Geneva. 2008.

Practical Manual on Tuberculosis laboratory strengthening, 2022 update. Geneva: World Health Organization; 2022. URL:

Rueda J., Realpe T., Mejia G.I., Zapata E., Rozo J.C., Ferro B.E., Robledo J. Genotypic Analysis of Genes Associated with Independent Resistance and Cross-Resistance to Isoniazid and Ethionamide in Mycobacterium tuberculosis Clinical Isolates. Antimicrob. Agents Chemother. 2015. 59(12). P. 7805-7810. doi: 10.1128/AAC.01028-15.

Sevаstyanovа E. V., Chernousovа L. N. Modern algorithms of microbiological diagnostics of Tuberculosis. Tuberculosis and Lung Diseases. 2018. Vol. 96. 7. P. 11-17. doi: 10.21292/2075-1230-2018-96-7-11-17.

Sharma N., Sharma V., Singh P.R., Jawed B., Babu V., Kandpal J., Nautiyal S.C., Singh R.K. Tuberculosis and Molecular Diagnosis. WebmedCentral Biotechnology 2013. 4 (2): WMC003992.

Skrypnik A.V., Stegniy B.T., Skrypnik V.G., Zavgorodny A.I. Molecular genetic in-house methods for detection and species differentiation of mycobacteria used in veterinary phthisiology. Works of the Federal Center for Animal Health. 2008. 6. Р. 171-185

Soini H., Musser J. M. Molecular Diagnosis of Mycobacteria. Clinical Chemistry 2001. 47. 5. P. 809-814.

Somily A.M., Habib H.A., Sarwar M.S., Al-Beeshi N.Z., Alohali R.M., Shakoor Z.A. Performance of the BD ProbeTec ET direct detection assay for the analysis of Mycobacterium tuberculosis in respiratory and non-respiratory clinical specimens. J Taibah Univ Med Sci. 2016. 12(4). Р. 364-368. doi: 10.1016/j.jtumed.2016.09.005.

Surkova L. K., Slizen V. V., Zalutskaya O. M. Correlation between molecular-genetic characteristics of Mycobacterium tuberculosis and prevalence, manifestation and outcome of disease. Proceedings of the National Academy of Sciences of Belarus, mеdical series. 2016. 4. P. 114–125.

The use of molecular line probe assay for the detection of resistance to second-line anti-tuberculosis drugs. Expert group meeting report Geneva. 2013 World Health Organization 2013 WHO/HTM/TB/2013.01. 52 p.

Williams K. J., Ling C. L., Jenkins C., Gillespie S. H., McHugh T. D. A paradigm for the molecular identification of Mycobacterium species in a routine diagnostic laboratory. Journal of Medical Microbiology 2007, 56. P. 598-602. doi 10.1099/jmm.0.46855-0.

Yamada-Noda M., Ohkusua K., Hatac H., Monir Shaha M., Nhunga P.H., Suna X.S., Hayashia M., Ezakia T. Mycobacterium species identification – A new approach via dnaJ gene sequencing. Systematic and Applied Microbiology 2007. 30. P. 453-462. doi:10.1016/j.syapm.2007.06.003.

Yong Y.K., Tan H.Y., Saeidi A., Wong W.F., Vignesh R., Velu V., Eri R., Larsson M., Shankar E.M. Immune Biomarkers for Diagnosis and Treatment Monitoring of Tuberculosis: Current Developments and Future Prospects. 2019. Front. Microbiol. 10. Article 2789. doi: 10.3389/fmicb.2019.02789.

Zhao L, Sun Q, Liu H, Wu X, Xiao T, Zhao X, Li G, Jiang Y, Zeng C, Wan K. 2015. Analysis of embCAB mutations associated with ethambutol resistance in multidrug-resistant Mycobacterium tuberculosis isolates from China. Antimicrob Agents Chemother. 2015. 59 (4). Р. 2045–2050. doi:10.1128/AAC.04933-14.

Zhou L., Ma C., Xiao T., Li M., Liu H., Zhao X., Wan K., Wang R. A New Single Gene Differential Biomarker for Mycobacterium tuberculosis Complex and Non-tuberculosis Mycobacteria. Front. Microbiol. 2019. 10. Article 1887. doi: 10.3389/fmicb.2019.01887.

Zhuang Z.G., Zhang J.A, Luo H.L., Liu G.B., Lu Y.B., Ge N.H., Zheng B.Y., Li R.X., Chen C., Wang X, Liu Y.Q., Liu F.H., Zhou Y., Cai X.Z., Chen Z.W., Xu J.F. The circular RNA of peripheral blood mononuclear cells: Hsa_circ_0005836 as a new diagnostic biomarker and therapeutic target of active pulmonary tuberculosis. Mol Immunol. 2017. 90. Р. 264-272. doi: 10.1016/j.molimm.2017.08.008.

Zimina V.N., Degtyaryova S.Yu., Beloborodova E.N., Kulabukhova E.I., Rusakova L.I., Fesenko O.V. A current state of mycobacterioses. CMAC. 2017. 19.4. P. 276-282.



How to Cite

Shapovalova, O., Pozmogova , S., & Zavgorodniy, A. (2022). Molecular technologies of mycobacterial research. Annals of Mechnikov’s Institute, (1), 9–20.