P54

False-positive cultures caused by laboratory cross-contamination of Mycobacterium tuberculosis are well-documented. Such contamination events are often confirmed with molecular techniques. In contrast, laboratory cross-contamination of non-tuberculous mycobacteria (NTM) is much less documented. Here we describe the investigation of two suspected Mycobacterium avium cross-contamination cases in an ISO15189 accredited reference laboratory using whole genome sequencing (WGS). Cross-contamination was considered in two patients, each with a single positive-culture and with no clinical suspicion of NTM lung disease. Further investigation was performed by reviewing all laboratory processes and WGS of retrospective M. avium isolates that were identified in the laboratory in 2022. WGS of M. avium isolates (n=65) was performed using the Illumina MiSeq and the species identification was confirmed using NTM-Profiler. WGS analysis was performed using MTBseq and Snippy, and de novo assembly was also performed on the suspected source isolate and used as a reference genome for confirmation. Two cases of M. avium cross-contamination were identified and confirmed by WGS. WGS results suggested cross-contamination from a strongly positive M. avium culture (3+ AFB smear and time to positivity 3 days) that occurred on two separate occasions, which may have been caused by aerosol generation during laboratory testing. There were no further cases of M. avium cross-contamination detected. There was no impact on patient care and corrective actions were implemented in the laboratory. This study highlights the added value of WGS for NTM identification and relatedness analysis, and the possibility of cross-contamination in a setting where NTM infections are on the rise.