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.
Highlights
Tuberculosis (TB), caused by Mycobacterium tuberculosis bacteria, is a global public health problem responsible for 10 million new cases and 1.6 million deaths worldwide in 20171
Sputum smear microscopy is used as a primary screening test for the diagnosis of TB at local clinics, while GeneXpert MTB/RIF assays are employed for the rapid detection of rifampicin resistant TB at the district level[3]
Beyond multi-drug resistant (MDR)-TB, the study found that only 43% of pyrazinamide could be explained by pncA SNPs, fluoroquinolone resistance was mostly explained by gyrA (91–94 codon) mutations, and resistance to aminoglycoside injectables was associated with rrs mutations
Summary
Tuberculosis (TB), caused by Mycobacterium tuberculosis bacteria, is a global public health problem responsible for 10 million new cases and 1.6 million deaths worldwide in 20171. One study characterized drug resistance mutations across 42 XDR-TB isolates from the Aga Khan University (Karachi) strain bank (years 2004–2009), which were sourced from 4 provinces (Sindh (21), Punjab (16), Khyber Pakhtunkhwa (4), Baluchistan (1))[19]. These isolates were predominantly CAS lineage 3 strains[19], in keeping with previous genotyping-based studies[20]. We performed WGS on 81 drug resistant M. tuberculosis from the Khyber Pakhtunkhwa province, which is endemic for TB across its tribal and migrant populations, but where public health surveillance systems are not strong.
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