P031

1:ECDC Fellowship Programme, Public Health Microbiology path (EUPHEM), European Centre for Disease Prevention and Control (ECDC); 2:Infectious Diseases Department, National Institute of Health Doctor Ricardo Jorge; 3:Genomics and Bioinformatics Unit, Infectious Diseases Department, National Institute of Health Doctor Ricardo Jorge; 4:National Reference Laboratory for Mycobacteria, Infectious Diseases Department, National Institute of Health Doctor Ricardo Jorge; 5:Animal and Veterinary Research Center (CECAV), Faculty of Veterinary Medicine, Lusófona University - Lisbon University Centre

The emergence of drug-resistant Mycobacterium tuberculosis strains poses a challenge to TB-control and global eradication efforts. Most methods for drug susceptibility testing (DST) are culture-dependent and time consuming, possibly delaying optimal treatment. This study aimed to develop an extensive targeted next-generation sequencing (tNGS) approach for rapid genotypic DST directly from clinical samples. We designed a tNGS panel comprising 30 amplicons targeting 19 genomic regions associated with resistance to 20 antibiotics. This method was applied to 71 smear-positive (0–3+) pulmonary TB clinical samples collected at the Portuguese National Reference Laboratory. DNA was extracted and amplified using multiplex PCRs, followed by sequencing on Oxford Nanopore Technologies MinION platform. Sequencing data were analysed using the online version of TB-Profiler and the tNGS results compared to phenotypic DST and whole genome sequencing (WGS) data from corresponding isolates. The tNGS demonstrated high concordance with both phenotypic and WGS-based DST across different sample types and smear positivity levels. For first-line drugs, tNGS showed 88% categorical agreement (CA) with pDST, increasing to 97% when excluding undetermined results. Compared to WGS across all analysed antibiotics, tNGS achieved 92% CA, increasing to >99% when excluding undetermined results. Validation of the tNGS panel showed 90% (1,895/2,076) of amplicons reaching >10x depth of coverage at all analysed positions and 43 (61%) samples with all amplicons above this threshold. Non-specific amplification of contaminant bacterial DNA was minimal. In conclusion, this method enables comprehensive resistance prediction directly from clinical samples and may aid in TB diagnostics and resistance monitoring.