Whole genome sequencing of drug resistant Mycobacterium tuberculosis isolates from a high burden tuberculosis region of North West Pakistan

Abstract

Tuberculosis (TB), caused by Mycobacterium tuberculosis bacteria, is a leading infectious cause of mortality worldwide, including in Pakistan. Drug resistant M. tuberculosis is an emerging threat for TB control, making it important to detect the underlying genetic mutations, and thereby inform treatment decision making and prevent transmission. Whole genome sequencing has emerged as the new diagnostic to reliably predict drug resistance within a clinically relevant time frame, and its deployment will have the greatest impact on TB control in highly endemic regions. To evaluate the mutations leading to drug resistance and to assess for evidence of the transmission of resistant strains, 81 M. tuberculosis samples from Khyber Pakhtunkhwa province (North West Pakistan) were subjected to whole genome sequencing and standard drug susceptibility testing for eleven anti-TB drugs. We found the majority of M. tuberculosis isolates were the CAS/Delhi strain-type (lineage 3; n = 57; 70.4%) and multi-drug resistant (MDR; n = 62; 76.5%). The most frequent resistance mutations were observed in the katG and rpoB genes, conferring resistance to isoniazid and rifampicin respectively. Mutations were also observed in genes conferring resistance to other first and second-line drugs, including in pncA (pyrazinamide), embB (ethambutol), gyrA (fluoroquinolones), rrs (aminoglycosides), rpsL, rrs and giB (streptomycin) loci. Whilst the majority of mutations have been reported in global datasets, we describe unreported putative resistance markers in katG, ethA (ethionamide), gyrA and gyrB (fluoroquinolones), and pncA. Analysis of the mutations revealed that acquisition of rifampicin resistance often preceded isoniazid in our isolates. We also observed a high proportion (17.6%) of pre-MDR isolates with fluoroquinolone resistance markers, potentially due to unregulated anti-TB drug use. Our isolates were compared to previously sequenced strains from Pakistan in a combined phylogenetic tree analysis. The presence of lineage 2 was only observed in our isolates. Using a cut-off of less than ten genome-wide mutation differences between isolates, a transmission analysis revealed 18 M. tuberculosis isolates clustering within eight networks, thereby providing evidence of drug-resistant TB transmission in the Khyber Pakhtunkhwa province. Overall, we have demonstrated that drug-resistant TB isolates are circulating and transmitted in North West Pakistan. Further, we have shown the usefulness of whole genome sequencing as a diagnostic tool for characterizing M. tuberculosis isolates, which will assist future epidemiological studies and disease control activities in Pakistan.

<p>Detection of Resistance to Fluoroquinolones and Second-Line Injectable Drugs Among <em>Mycobacterium tuberculosis</em> by a Reverse Dot Blot Hybridization Assay</p>

“…In the present study, among 27 SLID resistant isolates, the most frequent mutation detected was rrs A1401G (40.7%), which was consistent with the reports from China, 4,6,49 Georgia America, 50 eSwatini, Uganda and Somalia 48 and Northwest Pakistan, 51 while different from the report from India which showed that the most prevalent mutation was eis C (−12)T (46.1%). 52 As for the SLID resistance, the RDBH assay showed 51.9% sensitivity and 100% specificity compared to the phenotypic DST.…”

Wild-Type MIC Distribution for Re-evaluating the Critical Concentration of Anti-TB Drugs and Pharmacodynamics Among Tuberculosis Patients From South India

“…Whole-genome sequencing has emerged as a new diagnostic tool that assists in clinical decision making in infectious diseases (Jabbar et al, 2019 ). The whole-genome sequencing of tuberculosis isolates provides genomic information that is responsible for causing resistance (Cohen et al, 2019 ).…”

Frequency of Codon 306 Mutations in embB Gene of Mycobacterium tuberculosis Resistant to Ethambutol: A Systematic Review and Meta-Analysis

“…[ 61 ] According to a study in Pakistan in 2019, the majority of M. tuberculosis isolates were CAS/Delhi strain-type and MDR (76.5%), and mutations with resistance were observed in kat G, rpo B, pnc A, embB , gyr A, rrs , rps L, and rrs genes. [ 62 ] Drug-resistant TB isolates and their genes circulate among the population and they are transmitted between different communities worldwide. Therefore, performing more epidemiological studies and designing appropriate disease control plans are of great importance.…”

“…Therefore, performing more epidemiological studies and designing appropriate disease control plans are of great importance. [ 2 62 ] In this study, embB codon 306 was investigated. Since other codons of this gene contribute to the development of EMB resistance as well, thus, it is advisable to research the rate of association of other codons with EMB resistance, in terms of systematic review and meta-analysis, to obtain comprehensive results.…”