P069

1:Department of Clinical Microbiology and Infectious Diseases, Gregorio Marañón General University Hospital, Madrid, Spain; 2:Gregorio Marañón Health Research Institute (IiSGM), Madrid, Spain; 3:CIBER of Respiratory Diseases (CIBERES), Madrid, Spain; 4:Doctoral School, Autonomous University of Madrid, Madrid, Spain; 5:Department of Preventive Medicine, Public Health and Epidemiological Surveillance, Poniente University Hospital, Almería, Spain; 6:Microbiology service, Torrecárdenas Hospital Complex, Almería, Spain; 7:Department of Medicine, Complutense University of Madrid, Madrid, Spain; 8:Hospital Basurto, Bilbao, Spain; 9:National Center of Microbiology, Madrid, Spain; 10:Pediatric Infectious Diseases Department, Gregorio Marañón University Hospital, Madrid, Spain; 11:Biomedical Research Network Center for Infectious Diseases (CIBERINFEC), Spain; 12:CIEMAT, Extremadura Research Centre for Advanced Technologies (CETA-CIEMAT), Trujillo, Spain; 13:Faculty of infectious and tropical diseases, London School of Hygiene & Tropical Medicine, London, UK

Whole genome sequencing (WGS) enables accurate monitoring of tuberculosis transmission. Traditionally, genomic analysis lies on high-throughput Illumina sequencing, which needs to accumulate a high number of isolates per run, leading to delays in identifying new clustered cases. To address these limitations in contexts where epidemiological intervention is linked to genomic analysis, we leveraged the flexibility of nanopore sequencing to analyze 33 consecutive primary cultures on a case-by-case basis. All sequences achieved exploitable genome coverage (>90% at >20X), enabling identification of those associated to existing clusters, within the critical 21-day window when contact tracing remained ongoing. To further accelerate the availability of genomic results we applied the same strategy to sputa from 45 consecutive cases. Following human DNA partial depletion, 24% of the samples yielded exploitable coverage, and an additional 13% showed suboptimal performance but still provided partial information (from 27% at 10X to 49% at 20X). As an additional approach to accelerate the identification of new clustered cases when facing alarming transmission events, we also assessed the usefulness of fast-tracking potentially related cases by targeted sequencing. We applied this strategy in an interterritorial cluster (involving Madrid and Almería), associated with a super-spreader case/event. Sixteen key-marker SNPs of the strain involved were used to design a targeted amplicon-based sequencing approach. This allowed the rapid analysis of 96 cases from populations epidemiologically related to the involved cases, excluding further expansion of the aforementioned cluster, without the need for complete WGS of the cases.

Acknowledgments : COST-Action-AdvanceTB(CA21164), Computing facilities at CETA-CIEMAT with ERDF-funds.