Detection of Mycoplasma pneumoniae and Chlamydia pneumoniae using a laboratory-developed high-throughput test for the Panther Fusion system.

Mycoplasma pneumoniae and Chlamydia pneumoniae are key agents of atypical pneumonia. During a 2023 M. pneumoniae outbreak in Denmark, a high-throughput laboratory-developed test (LDT) was created for the Hologic Panther Fusion system using Open Access software. This multiplex real-time PCR assay employs TaqMan® probes targeting the CARDS tx-gene (M. pneumoniae) and argR gene (C. pneumoniae). Validation included external quality controls, defined bacterial concentrations, and frozen patient samples. Sensitivity, specificity, precision, and robustness were assessed, with 95 % limits of detection at 111 and 878 copies/mL for M. pneumoniae and C. pneumoniae, respectively. Specificity was 100 % against 30 related pathogens. Over the course of 2024 we used the assay routinely analysing 56,773 samples finding a positive prevalence of 18.9 % for M. pneumoniae and 1.5 % for C. pneumoniae. The assay demonstrated high sensitivity and specificity, offering rapid sample processing.