Prediction of drug resistance by Sanger sequencing of Mycobacterium tuberculosis complex strains isolated from multidrug resistant tuberculosis suspect patients in Ethiopia.

Autor: Mesfin EA; Ethiopian Public Health Institute, National Laboratory Capacity Building Directorate, Addis Ababa, Ethiopia., Merker M; Molecular and Experimental Mycobacteriology, Research Center Borstel, Sülfeld, Germany.; Evolution of the Resistome, Research Center Borstel, Sülfeld, Germany., Beyene D; Department of Microbial, Cellular and Molecular Biology, Addis Ababa University, Addis Ababa, Ethiopia., Tesfaye A; Addis Ababa City Administration Health Bureau Health Research and Laboratory Services, Addis Ababa, Ethiopia., Shuaib YA; Molecular and Experimental Mycobacteriology, Research Center Borstel, Sülfeld, Germany.; College of Veterinary Medicine, Sudan University of Science and Technology, Khartoum North, Sudan., Addise D; Ethiopian Public Health Institute, National Laboratory Capacity Building Directorate, Addis Ababa, Ethiopia., Tessema B; Department of Medical Microbiology, College of Medicine and Health Sciences, University of Gondar, Gondar, Ethiopia., Niemann S; Molecular and Experimental Mycobacteriology, Research Center Borstel, Sülfeld, Germany.; German Center for Infection Research, Partner Site Hamburg-Lübeck- Borstel-Riems, Hamburg, Germany.
Jazyk: angličtina
Zdroj: PloS one [PLoS One] 2022 Aug 05; Vol. 17 (8), pp. e0271508. Date of Electronic Publication: 2022 Aug 05 (Print Publication: 2022).
DOI: 10.1371/journal.pone.0271508
Abstrakt: Background: Ethiopia is one of the high multidrug-resistant tuberculosis (MDR-TB) burden countries. However, phenotypic drug susceptibility testing can take several weeks due to the slow growth of Mycobacterium tuberculosis complex (MTBC) strains. In this study, we assessed the performance of a Sanger sequencing approach to predict resistance against five anti-tuberculosis drugs and the pattern of resistance mediating mutations.
Methods: We enrolled 226 MTBC culture-positive MDR-TB suspects and collected sputum specimens and socio-demographic and TB related data from each suspect between June 2015 and December 2016 in Addis Ababa, Ethiopia. Phenotypic drug susceptibility testing (pDST) for rifampicin, isoniazid, pyrazinamide, ethambutol, and streptomycin using BACTEC MGIT 960 was compared with the results of a Sanger sequencing analysis of seven resistance determining regions in the genes rpoB, katG, fabG-inhA, pncA, embB, rpsL, and rrs.
Result: DNA isolation for Sanger sequencing was successfully extracted from 92.5% (209/226) of the MTBC positive cultures, and the remaining 7.5% (17/226) strains were excluded from the final analysis. Based on pDST results, drug resistance proportions were as follows: isoniazid: 109/209 (52.2%), streptomycin: 93/209 (44.5%), rifampicin: 88/209 (42.1%), ethambutol: 74/209 (35.4%), and pyrazinamide: 69/209 (33.0%). Resistance against isoniazid was mainly mediated by the mutation katG S315T (97/209, 46.4%) and resistance against rifampicin by rpoB S531L (58/209, 27.8%). The dominating resistance-conferring mutations for ethambutol, streptomycin, and pyrazinamide affected codon 306 in embB (48/209, 21.1%), codon 88 in rpsL (43/209, 20.6%), and codon 65 in pncA (19/209, 9.1%), respectively. We observed a high agreement between phenotypic and genotypic DST, such as 89.9% (at 95% confidence interval [CI], 84.2%-95.8%) for isoniazid, 95.5% (95% CI, 91.2%-99.8%) for rifampicin, 98.6% (95% CI, 95.9-100%) for ethambutol, 91.3% (95% CI, 84.6-98.1%) for pyrazinamide and 57.0% (95% CI, 46.9%-67.1%) for streptomycin.
Conclusion: We detected canonical mutations implicated in resistance to rifampicin, isoniazid, pyrazinamide, ethambutol, and streptomycin. High agreement with phenotypic DST results for all drugs renders Sanger sequencing promising to be performed as a complementary measure to routine phenotypic DST in Ethiopia. Sanger sequencing directly from sputum may accelerate accurate clinical decision-making in the future.
Competing Interests: The authors declare that they have no competing interests.
Databáze: MEDLINE
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