Next-generation sequencing of drug resistant Mycobacterium tuberculosis clinical isolates in low-incidence countries

Cover Page

Cite item


Drug resistant tuberculosis (TB), especially multidrug (MDR) and extensively drug-resistant (XDR) TB, is still a serious problem in global TB control. Slovenia and North Macedonia are low-incidence countries with TB incidence rates of 5.4 and 10.4 in 2017, respectively. In both countries, the percentage of drug resistant TB is very low with sporadic cases of MDR-TB. However, global burden of drug-resistant TB continues to increase imposing huge impact on public health systems and strongly stimulating the detection of gene variants related with drug resistance in TB. Next-generation sequencing (NGS) can provide comprehensive analysis of gene variants linked to drug resistance in Mycobacterium tuberculosis. Therefore, the aim of our study was to examine the feasibility of a full-length gene analysis for the drug resistance related genes (inhA, katG, rpoB, embB) using Ion Torrent technology and to compare the NGS results with those obtained from conventional phenotypic drug susceptibility testing (DST) in TB isolates. Between 1996 and 2017, we retrospectively selected 56 TB strains from our National mycobacterial culture collection. Of those, 33 TB isolates from Slovenian patients were isolated from various clinical samples and subjected to phenotypic DST testing in Laboratory for Mycobacteria (University Clinic Golnik, Slovenia). The remaining 23 TB isolates were isolated from Macedonian patients and sent to our laboratory for assistance in phenotypic DST testing. TB strains included were either mono-, poly- or multidrug resistant. For control purposes, we also randomly selected five TB strains susceptible to first-line anti-TB drugs. High concordance between genetic (Ion Torrent technology) and standard phenotypic DST testing for isoniazid, rifampicin and ethambutol was observed, with percent of agreement of 77%, 93.4% and 93.3%, sensitivities of 68.2%, 100% and 100%, and specificities of 100%, 80% and 88.2%, respectively. In conclusion, the genotypic DST using Ion Torrent semiconductor NGS successfully predicted drug resistance with significant shortening of time needed to obtain the resistance profiles from several weeks to just a few days.

About the authors

E. Sodja

University Clinic of Respiratory and Allergic Diseases Golnik

Author for correspondence.

Sodja Eva, PhD, Research Associate, National Reference Laboratory for Mycobacteria

Golnik 36, 4204 Golnik, Slovenia.

Phone: +386 4 2569 409. Fax: +386 4 2569 117.


N. Toplak

Omega d.o.o., Ljubljana

PhD, Field Application Specialist for Molecular Biology, Research Team of Omega d.o.o. Slovenia

S. Koren

Omega d.o.o., Ljubljana

PhD, Manager of Life Science Perkin Elmer and Field Application Specialist, Research Team of Omega d.o.o. Slovenia

M. Kovač

Omega d.o.o., Ljubljana

Manager of Thermo Fisher Scientific Sales Team and Research Team of Omega d.o.o. Slovenia

S. Truden

University Clinic of Respiratory and Allergic Diseases Golnik

MSc, Analytics, National Reference Laboratory for Mycobacteria Slovenia

M. Žolni-Dovč

University Clinic of Respiratory and Allergic Diseases Golnik

PhD, Head of National Reference Laboratory for Mycobacteria Slovenia


  1. Cegielski J.P., Kurbatova E., van der Walt M., Brand J., Ershova J., Tupasi T., Caoili J.C., Dalton T., Contreras C., Yagui M., Bayona J., Kvasnovsky C., Leimane V., Kuksa L., Chen M.P., Via L.E., Hwang S.H., Wolfgang M., Volchenkov G.V., Somova T., Smith S.E., Akksilp S., Wattanaamornkiet W., Kim H.J., Kim C.K., Kazennyy B.Y., Khorosheva T., Kliiman K., Viiklepp P., Jou R., Huang A.S., Vasilyeva I.A., Demikhova O.V.; Global PETTS Investigators, Lancaster J., Odendaal R., Diem L., Perez T.C., Gler T., Tan K., Bonilla C., Jave O., Asencios L., Yale G., Suarez C., Walker A.T., Norvaisha I., Skenders G., Sture I., Riekstina V., Cirule A., Sigman E., Cho S.N., Cai Y., Eum S., Lee J., Park S., Jeon D., Shamputa I.C., Metchock B., Kuznetsova T., Akksilp R., Sitti W., Inyapong J., Kiryanova E.V., Degtyareva I., Nemtsova E.S., Levina K., Danilovits M., Kummik T., Lei Y.C., Huang W.L., Erokhin V.V., Chernousova L.N., Andreevskaya S.N., Larionova E.E., Smirnova T.G. Multidrug-resistant tuberculosis treatment outcomes in relation to treatment and initial versus acquired second-line drug resistance. Clin. Infect. Dis., 2016, vol. 62, no. 4, pp. 418–430.
  2. European Centre for Disease Prevention and Control. Molecular typing for surveillance of multidrug-resistant tuberculosis in the EU/EEA – January 2016. Stockholm: ECDC, 2016.
  3. European Centre for Disease Prevention and Control/WHO Regional Office for Europe. Tuberculosis surveillance and monitoring in Europe 2018. 2016 data. 206 p. URL:
  4. Global tuberculosis report 2017. Geneva: World Health Organization, 2017. 295 p. URL:
  5. Hazbón M.H., Bobadilla del Valle M., Guerrero M.I., Varma-Basil M., Filliol I., Cavatore M., Colangeli R., Safi H., Billman-Jacobe H., Lavender C., Fyfe J., García-García L., Davidow A., Brimacombe M., León C.I., Porras T., Bose M., Chaves F., Eisenach K.D., Sifuentes-Osornio J., Ponce de León A., Cave M.D., Alland D. Role of embB codon 306 mutations in Mycobacterium tuberculosis revisited: a novel association with broad drug resistance and IS6110 clustering rather than ethambutol resistance. Antimicrob. Agents Chemother., 2005, vol. 49, no. 9, pp. 3794–3802. doi: 10.1128/AAC.49.9.3794-3802.2005
  6. Hazbón M.H., Brimacombe M., Bobadilla del Valle M., Cavatore M., Guerrero M.I., Varma-Basil M., Billman-Jacobe H., Lavender C., Fyfe J., García-García L., León C.I., Bose M., Chaves F., Murray M., Eisenach K.D., Sifuentes-Osornio J., Cave M.D., Ponce de León A., Alland D. Population genetics study of isoniazid resistance mutations and evolution of multidrug-resistant Mycobacterium tuberculosis. Antimicrob. Agents. Chemother., 2006, vol. 50, no. 8, pp. 2640–2649. doi: 10.1128/AAC.00112-06
  7. Kim S.Y., Park Y.J., Kim W.I., Lee S.H., Ludgerus Chang C., Kang S.J., Kang C.S. Molecular analysis of isoniazid resistance in Mycobacterium tuberculosis isolates recovered from South Korea. Diagn. Microbiol. Infect. Dis., 2003, vol. 47, no. 3, pp. 497–502.
  8. Mokrousov I., Otten T., Vyshnevskiy B., Narvskaya O. Detection of embB306 mutations in ethambutol-susceptible clinical isolates of Mycobacterium tuberculosis from Northwestern Russia: implications for genotypic resistance testing. J. Clin. Microbiol., 2002, vol. 40, no. 10, pp. 3810–3813.
  9. Park J., Jang W., Kim M., Kim Y., Shin S.Y., Park K., Kim M.S., Shin S.J. Molecular drug resistance profiles of Mycobacterium tuberculosis from sputum specimens using ion semiconductor sequencing. Microbiol. Methods, 2018, vol. 145, pp. 1–6. doi: 10.1016/j.mimet.2017
  10. Park J., Shin S.Y., Kim K., Park K., Shin S., Ihm C. Determining Genotypic Drug Resistance by Ion Semiconductor Sequencing With the Ion AmpliSeq™ TB Panel in Multidrug-Resistant Mycobacterium tuberculosis Isolates. Ann. Lab. Med., 2018, vol. 38, no. 4, pp. 316–323. doi: 10.3343/alm.2018.38.4.316
  11. Sandgren A., Strong M., Muthukrishnan P., Weiner B.K., Church G.M., Murray M.B. Tuberculosis drug resistance mutation database. PLoS Med., 2009, vol. 6, no. 2: e2. doi: 10.1371/journal.pmed.1000002
  12. Seifert M., Catanzaro D., Catanzaro A., Rodwell T.C. Genetic mutations associated with isoniazid resistance in Mycobacterium tuberculosis: a systematic review. PLoS One, 2015, vol. 10, no. 3: e0119628. doi: 10.1371/journal.pone.0119628
  13. Somerville W., Thibert L., Schwartzman K., Behr M. A. Extraction of Mycobacterium tuberculosis DNA: a question of containment. J. Clin. Microbiol., 2005, vol. 43, pp. 2996–2997. doi: 10.1128/JCM.43.6.2996-2997.2005
  14. Unissa A.N., Subbian S., Hanna L.E., Selvakumar N. Overview on mechanisms of isoniazid action and resistance in Mycobacterium tuberculosis. Infect. Genet. Evol., 2016, vol. 45, pp. 474–492. doi: 10.1016/j.meegid.2016.09.004
  15. Zaw M.T., Emran N.A., Lin Z.J. Mutations inside rifampicin-resistance determining region of rpoB gene associated with rifampicin-resistance in Mycobacterium tuberculosis. Infect. Public. Health., 2018, vol. 11, no. 5, pp. 605–610. doi: 10.1016/j.jiph.2018.04.005
  16. Zignol M., Cabibbe A.M., Dean A.S., Glaziou P., Alikhanova N., Ama C., Andres S., Barbova A., Borbe-Reyes A., Chin D.P., Cirillo D.M., Colvin C., Dadu A., Dreyer A., Driesen M., Gilpin C., Hasan R., Hasan Z., Hoffner S., Hussain A., Ismail N., Kamal S.M.M., Khanzada F.M., Kimerling M., Kohl T.A., Mansjö M., Miotto P., Mukadi Y.D., Mvusi L., Niemann S., Omar S.V., Rigouts L., Schito M., Sela I., Seyfaddinova M., Skenders G., Skrahina A., Tahseen S., Wells W.A., Zhurilo A., Weyer K., Floyd K., Raviglione M.C. Genetic sequencing for surveillance of drug resistance in tuberculosis in highly endemic countries: a multi-country population-based surveillance study. Lancet. Infect. Dis., 2018, vol. 18, no. 6, pp. 675–683. doi: 10.1016/S1473-3099(18)30073-2

Supplementary files

There are no supplementary files to display.

Copyright (c) 2020 Sodja E., Toplak N., Koren S., Kovač M., Truden S., Žolni-Dovč M.

Creative Commons License
This work is licensed under a Creative Commons Attribution 4.0 International License.

СМИ зарегистрировано Федеральной службой по надзору в сфере связи, информационных технологий и массовых коммуникаций (Роскомнадзор).
Регистрационный номер и дата принятия решения о регистрации СМИ: серия ПИ № ФС 77 - 64788 от 02.02.2016.

This website uses cookies

You consent to our cookies if you continue to use our website.

About Cookies