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

Abstract

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.

Resistance Rates of Mycobacterium tuberculosis Complex Isolates to Primary Anti-tuberculosis Drugs: A 5-Year Retrospective Study

“…TB mortality has surpassed that of acquired immunodeficiency syndrome (AIDS), making Mycobacterium tuberculosis the leading infectious agent that threatens human well-being (16). TB control strategies have been massively hindered by the emergence and transmission of antituberculosisresistant strains, specifically rifampicin-resistant and MDR-TB isolates despite the striking declines in the prevalence of TB in recent years (17). The estimated annual incidence of RR-TB in new cases was 3.4% (18).…”

Mycobacterium tuberculosis Lineage Distribution Using Whole-Genome Sequencing and Bedaquiline, Clofazimine, and Linezolid Phenotypic Profiles among Rifampicin-Resistant Isolates from West Java, Indonesia

“…Te analysis information is then compiled into a fle. Mykrobe v0.10 and TB-Profler v4.4.1 were used to predict the lineage classifcation, and only Mykrobe was used to predict the type of drug resistance among the isolates [24][25][26][27]. Descriptive statistics were used to describe the results of this study.…”

Direct detection of drug-resistant Mycobacterium tuberculosis using targeted next generation sequencing

“…Sequencing-based methods provide greater resolution of these loci. Assays developed for the detection of DR include pyrosequencing ( 19–22 ) and Sanger sequencing ( 23 ) of individual targets; however, these methods are typically limited to single-plex reactions analyzing limited sections of DR determining loci. NGS assays offer more comprehensive DR profiles by identifying novel and high confidence DR-associated mutations throughout the genome ( 24 , 25 ).…”