Nanopore sequencing panel for saliva-based host pharmacogenomic screening in anti-tubercular therapy

Abstract Rationale: Host genotypes can predict subtherapeutic anti-tubercular drug exposures and treatment-associated toxicities. Screening for these variants could enable personalized dosing, but scalable assays for second-line drugs are lacking. Objectives: We developed a nanopore sequencing panel to detect host variants affecting anti-tuberculosis drug troughs and toxicities, and evaluated its performance as a saliva-based screening tool. Methods: We designed a 16-plex panel targeting 23 variants (21 clinically validated, 2 predicted actionable) relevant to linezolid, bedaquiline, clofazimine, moxifloxacin, and ethambutol exposure. We first sequenced 50 Coriell DNA (1000 Genomes Project) to benchmark accuracy against Illumina, then sequenced saliva from 202 individuals treated for drug-resistant tuberculosis in India using MinION Mk1C (R10.4). Plasma trough concentrations and toxicity frequencies were stratified by genotype. Data were analyzed using in-house pipelines. Measurements and Main Results: The panel showed high coverage in saliva (median 3,609X). Several suggestive genotype-phenotype trends reached nominal significance in distinct subsets. Among patients on high-dose moxifloxacin (800mg daily), UGT1A1 rs3755319 A>C was associated with higher troughs in heterozygotes (6/14, p<0.01) and homozygous alternates (4/14, p<0.05). Among patients with linezolid-associated toxicity dose-reduced to 300mg, ABCB1 rs2032582 A>C homozygous alternates (7/98) had significantly lower Cmin versus wild-type (p<0.05) and heterozygotes (p<0.01); neither association held at standard dosing. Linezolid toxicity was more frequent among ABCB1 rs1128503 A>G heterozygotes versus homozygous reference (58.3% vs. 29.1%), and UGT1A1 rs4148323 G>A heterozygotes showed higher moxifloxacin toxicity rates than wild-type (42.9% vs. 14.3%). Conclusions: Portable, saliva-based sequencing reliably detects pharmacogenetic variants and could inform pre-treatment screening for drug exposure or toxicity.