Characterisation of Resistance Associated Variants (RAVs) to the newly approved anti-tuberculosis drugs Delamanid, Pretomanid, Bedaquiline, Clofazimine and Linezolid in isolates from a 2016/2017 Tanzanian Anti-Tuberculosis Drug Resistance Survey

Abstract

Background: Drug-resistant Tuberculosis (TB) undermines the effort to curb TB disease. It is associated with higher treatment costs, longer treatment periods and more adverse effects (Jeon, 2015). Recently, three new (Delamanid, Bedaquiline and Pretomanid) and two re-purposed drugs (Linezolid and Clofazimine) have been approved to treat drug-resistant TB. This study was set out to establish the prevalence of resistance-associated variants to the new and re-purposed anti-TB drugs using whole genome sequences derived from isolates from a 2016/2017 Tanzanian drug resistance survey. Methodology: Briefly, the genomic reads were visualized for quality using FastQC and MultiQC; followed by adapter sequence contamination screening and quality trimming using Trimmomatic. The reads were then aligned to the NC_000962.3 reference H37Rv genome, while variants and variant annotation was done using Freebaayes and Snpeff, respectively. Custom bash scripts were used to identify potential resistance-conferring mutations in the genes Rv0678, pepQ, and Rv1979c for both Bedaquiline and Clofazimine resistance while the atpE gene was also included for Bedaquline resistance. Genes fbiA, fbiB, fbiC, Rv2983, fgd1, and ddn were interrogated for potential resistance-conferring variants to Delamanid and Pretomanid, while for Linezolid resistance, the genes rplC, rrl, were analysed. The MultiAgEnt Stability pRedictiOn tool (MAESTRO) was used to infer changes in protein stability following point mutations. Results:144 samples were identified as having variants in either one or more of these genes. A total of 973 variants were identified in all genes, following filtering out of all complex variants. 5/192 samples (~3%) were identified to have variants in Rv0678 (p.Glu104Lys, p.Leu117Arg, p.Glu138Gly, p.Ser63Gly, p.Glu21* and p.Leu122Pro) that have been previously linked or found in Bedaquiline/Clofazimine resitant samples. One sample was found to have a pepQ p.E89* mutaton that has also been linked to resistance to both Bedaquiline and Clofazimine. The study also found six previously described mutations in genomic regions implicated in Delamanid resistance. 3 were in the ddn gene (Gly81Ser, p.Trp27fs,p.Tyr29*) and 1 in each of the genes fbiA (p.Cys287*), fbiC (p.Gly112Ala), fgd1(p.Gly314Glu), all of these have been implicated in Delamanid/Pretomanid resistance. Conclusions: This study shows that approximately 3% of the isolates habored Resistance Associated Variants to Bedaquiline/Clofazimine and Delamanid/Pretomanid, these patients did not have any documented use of any of the drugs, this raises concern of pre-existing natural resistance to these drugs.