Evaluation of the effectiveness of molecular genetic and bacteriological methods in determining heteroresistance Mycobacterium tuberculosis to rifampicin

We analyzed bacterial suspensions (mixtures) containing various proportions of resistant and susceptible strains to assess the effectiveness of molecular genetic tests and bacteriological methods for determination of the heteroresistant population of M. tuberculosis (MTB) to rifampicin (RIF). We established that the efficiency of determining the RIF-resistant MTB population in mixtures depends on the method of mutation detection used in the test system. The Genotype MTBDRplus, TB-TEST, and TB-BIOCHIP-1 tests showed the same results in detecting a resistant population with the Ser531Leu mutation. Genotype MTBDRplus more effectively detected His526Tyr, His526Asp, Asp516Val substitutions and less efficiently detected mutations determined by the absence of wild-type probes compared to TB-TEST and TB-BIOCHIP-1 tests.

The efficiency of bacteriological detection of the resistant MTB population depends on the terms of cultivation. The method of serial microdilutions in the Middlebrook 7H9 liquid medium is the most effective in detecting RIF-resistance in the heteroresistant MTB population with the MTB cultivation for 21 days in comparison with the automated system Bactec MGIT 960 and the method of absolute concentrations on the L-J solid medium with the MTB cultivation for 28 days. It was noted that the proportion of resistant MTB strains detectection by Bactec MGIT 960, the method of absolute concentrations on a solid L-J medium for 28 days and by the method of serial microdilutions for 14 days did not differ significantly and amounted to 45.1; 44.1 and 38.9 respectively

1. Определение лекарственной чувствительности микобактерий к противотуберкулезным препаратам / Приказ № 109 Минздрава России от 21 марта 2003 г. «О совершенствовании противотуберкулезных мероприятий в Российской Федерации». – М., 2003. − С. 186-198.

2. Andres S., Hillemann D., Rüsch-Gerdes S. et al. Occurrence of rpoB mutations in isoniazid-resistant but rifampin-susceptible Mycobacterium tuberculosis isolates from Germany // Antimicrob. Agents Chemother. – 2014. – Vol. 58. – P. 590-592.

3. Cambau E., Viveiros M., Machado D. et al. Revisiting susceptibility testing in MDR-TB by a standardized quantitative phenotypic assessment in a European multicentre study // J. Antimicrob. Chemother. – 2015. – Vol. 70. – P. 686-696.

4. Campbell E.A., Korzheva N., Mustaev A. et al. Structural mechanism for rifampicin inhibition of bacterial rna polymerase // Cell. – 2001. – Vol. 104. – N. 6. – P. 901-912.

5. Canetti G. Present aspects of bacterial resistance in tuberculosis // Am. Rev. Resp. Dis. – 1965. – Vol. 5. – P. 687-703.

6. Canetti G., Froman S., Grosset J. et al. Mycobacteria: Laboratory methods for testing drug sensitivity and resistance // Bull WHO. − 1963. – Vol. 29. – P. 565-578. 7. Cavusoglu C., Karaca-Derici Y., Bilgic A. In-vitro activity of rifabutin against rifampicin-resistant Mycobacterium tuberculosis isolates with known rpoB mutations // Clin. Microbiol. Infect. – 2004. - Vol. 10. – N. 7. – P. 662-665. doi: 10.1111/j.1469-0691.2004.00917.x.

7. Folkvardsen D.B., Thomsen V.O., Rigouts L. et al. Rifampin heteroresistance in Mycobacterium tuberculosis cultures as detected by phenotypic and genotypic drug susceptibility test methods // J. Clin. Microbiol. – 2013. – Vol. 51. – N. 12. – P. 4220-4222.

8. Hofmann-Thiel S., van Ingen J., Feldmann K. et al. Mechanisms of heteroresistance to isoniazid and rifampin of Mycobacterium tuberculosis in Tashkent, Uzbekistan // Eur. Respir. J. – 2009. – Vol. 33. – N. 2. – P. 368-374.

9. Jamieson F.B., Guthrie J.L., Neemuchwala A. et al. Profiling of rpoB mutations and MICs for rifampin and rifabutin in Mycobacterium tuberculosis // J. Clin. Microbiol. – 2014. – Vol. 52. – P. 2157-2162.

10. Jeong D.H., Kang Y.W., Kim J.Y. et al. Successful Treatment with a High-dose Rifampin-containing regimen for Pulmonary Tuberculosis with a Disputed rpoB Mutation // Intern. Med. – 2018. – Vol. 57(22). – P. 3281-3284.

11. Kamela C.S.Ng., Supply Ph., Cobelens F.G.J. et al. How well do routine molecular diagnostics detect rifampin heteroresistance in Mycobacterium tuberculosis? // J. Clin Microbiol. – 2019. – Vol. 57. - N. 11: e00717-19. doi: 10.1128/JCM.00717-19.

12. Kao C.Y., Lee A.Y., Huang A.H. et al. Heteroresistance of Helicobacter pylori from the same patient prior to antibiotic treatment. // Infect. Genet. Evol. – 2014. – Vol. 23. – P.196-202.

13. Kumar P., Balooni V., Kumar B. et al. High degree of multi-drug resistance and hetero-resistance in pulmonary TB patients from Punjab state of India // Tuberculosis (Edinb). – 2014. – Vol. 94. – N. 1. – P. 73-80.

14. Mekonnen D., Admassu A., Mulu W. et al. Multidrug-resistant and heteroresistant Mycobacterium tuberculosis and associated gene mutations in Ethiopia. // Intern. J. Infect. Dis. – 2015. – Vol. 39. – P. 34–38.

15. Ocheretina O., Escuyer V. E., Mabou M.M. et al. Correlation between genotypic and phenotypic testing for resistance to rifampin in Mycobacterium tuberculosis clinical isolates in Haiti: investigation of cases with discrepant susceptibility results // PloS One. – 2014. – Vol. 9. – N. 3. – e9056.

16. Post F.A., Willcox P.A., Mathema B. et al. Genetic polymorphism in Mycobacterium tuberculosis isolates from patients with chronic multidrug-resistant tuberculosis // J. Infect. Dis. – 2004. – Vol. 190. – N. 1. – P. 99-106.

17. Reference protocol for MIC determination of anti-tuberculous agents against isolates of the Mycobacterium tuberculosis complex in Middlebrook 7H9 broth. EUCAST. Version 6.1. 4th of July, 2019. [Электронный ресурс] URL: https://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/Mycobacteria/Methods_in_AMST/Technical_protocol_AMST_MIC_reference_method_190719.pdf (Дата обращения 20.09.2022).

18. Rigouts L., Gumusboga M., Bram de Rijk W. et al. Rifampin resistance missed in automated liquid culture system for Mycobacterium tuberculosis isolates with specific rpoB mutations // J. Clinical Microbiol. – 2013. – Vol. 51. – N. 8. – P. 2641-2645.

19. Siddiqi S. H., Rusch-Gerdes S., Alexander H. et al. MGIT Procedure Manual for BACTECТМMGITТМ 960 TB System (Also applicable for Manual MGIT) – 2006. [Электронный ресурс] URL: https://www.finddx.org/wp-content/uploads/2016/02/mgit_manual_nov2006.pdf (Дата обращения 20.09.2022).

20. Springer B., Calligaris-Maibach R., Ritter C. et al. Tuberculosis drug resistance in an area of low endemicity in 2004 to 2006: semiquantitative drug susceptibility testing and genotyping // J. Clinical Microbiol. – 2008. – Vol. 46. – N. 12. – P. 4064-4067.

21. Telenti A., Imboden P., Marchesi F. et al. Detection of rifampicin resistance mutations in Mycobacterium tuberculosis // Lancet. – 1993. – Vol. 341. – P. 647-651.

22. Van Deun A., Barrera L., Bastian I. et al. Mycobacterium tuberculosis strains with highly discordant rifampin susceptibility test results // J. Clinical Microbiol. – 2009. – Vol. 47. – N. 11. – P. 3501-3506.

23. Van Deun A., Decroo T., Piubello A. et al. Principles for constructing a tuberculosis treatment regimen: the role and definition of core and companion drugs // Int. J. Tuberc. Lung Dis. – 2018. – Vol. 22. – P. 239-245.

24. Van Ingen J., Aarnoutse R., de Vries G. et al. Low-level rifampicin-resistant Mycobacterium tuberculosis strains raise a new therapeutic challenge // Int. J. Tuberc. Lung Dis. – 2011. – Vol. 15. – N. 7. – P. 990-992.

25. Williamson D.A., Roberts S.A., Bower J.E. et al. Clinical failures associated with rpoB mutations in phenotypically occult multidrug-resistant Mycobacterium tuberculosis // Int. J. Tuberc. Lung Dis. – 2012. – Vol. 16. – N. 2. – P. 216-220.

26. Zhang Z., Wang Y., Pang Yu. et al. Comparison of different drug susceptibility test methods to detect rifampin heteroresistance in Mycobacterium tuberculosis // Antimicrobial Agents and Chemotherapy. – 2014. – Vol. 58. – N. 9. – P. 5632–5635.