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

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.

Drug-resistance patterns of Mycobacterium tuberculosis strains and associated risk factors among multi drug-resistant tuberculosis suspected patients from Ethiopia.

BACKGROUND: Multidrug drug-resistant tuberculosis (MDR-TB) is a major health problem and seriously threatens TB control and prevention efforts globally. Ethiopia is among the 30th highest TB burden countries for MDR-TB with 14% prevalence among previously treated cases. The focus of this study was on determining drug resistance patterns of Mycobacterium tuberculosis among MDR-TB suspected cases and associated risk factors. METHODS: A cross-sectional study was conducted in Addis Ababa from June 2015 to December 2016. Sputum samples and socio-demographic data were collected from 358 MDR-TB suspected cases. Samples were analyzed using Ziehl-Neelsen technique, GeneXpert MTB/RIF assay, and culture using Lowenstein-Jensen and Mycobacterial growth indicator tube. Data were analyzed using SPSS version 23. RESULTS: A total of 226 the study participants were culture positive for Mycobacterium tuberculosis, among them, 133 (58.8%) participants were males. Moreover, 162 (71.7%) had been previously treated for tuberculosis, while 128 (56.6%) were TB/HIV co-infected. A majority [122 (54%)] of the isolates were resistant to any first-line anti-TB drugs. Among the resistant isolates, 110 (48.7%) were determined to be resistant to isoniazid, 94 (41.6%) to streptomycin, 89 (39.4%) to rifampicin, 72 (31.9%) to ethambutol, and 70 (30.9%) to pyrazinamide. The prevalence of MDR-TB was 89 (39.4%), of which 52/89 (58.4%) isolates were resistance to all five first-line drugs. Risk factors such as TB/HIV co-infection (AOR = 5.59, p = 0.00), cigarette smoking (AOR = 3.52, p = 0.045), alcohol drinking (AOR = 5.14, p = 0.001) hospital admission (AOR = 3.49, p = 0.005) and visiting (AOR = 3.34, p = 0.044) were significantly associated with MDR-TB. CONCLUSIONS: The prevalence of MDR-TB in the study population was of a significantly high level among previously treated patients and age group of 25-34. TB/HIV coinfection, smoking of cigarette, alcohol drinking, hospital admission and health facility visiting were identified as risk factors for developing MDR-TB. Therefore, effective strategies should be designed considering the identified risk factors for control of MDR-TB.