Analytical and clinical validation of a multiplex PCR assay for detection of Neisseria gonorrhoeae and Chlamydia trachomatis including simultaneous LGV serotyping on an automated high-throughput PCR system

ABSTRACTFor effective infection control measures for Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG), a reliable tool for screening and diagnosis is essential. Here, we aimed to establish and validate a multiplex PCR assay on an automated system using a dual-target approach for the detection of CT/NG and differentiation between lymphogranuloma venereum (LGV) and non-LGV from genital and extra-genital specimens. Published primer/probe sets (CT: pmpH, cryptic plasmid; NG: porA, opa) were modified for the cobas 5800/6800/8800. Standards quantified by digital PCR were used to determine linearity and lower limit of detection (LLoD; eSwab, urine). For clinical validation, prospective samples (n = 319) were compared with a CE-marked in vitro diagnostics (CE-IVD) assay. LLoDs ranged from 21.8 to 244 digital copies (dcp)/mL and 10.8 to 277 dcp/mL in swab and urine, respectively. A simple linear regression analysis yielded slopes ranging from −4.338 to −2.834 and Pearson correlation coefficients from 0.956 to 0.994. Inter- and intra-run variability was 128 million and >82 million people, respectively, were newly infected in 2020. For effective infection control measures, a reliable tool for sensitive diagnosis and screening of CT/NG is essential. We established a multiplex PCR assay for the detection of CT/NG and simultaneous discrimination between lymphogranuloma venereum (LGV) and non-LGV strains, which has been validated for genital and extra-genital specimens on a fully automated system. To increase assay sensitivity, a dual-target approach has been chosen for both pathogens. This strategy reduces false-positive results in oropharyngeal swabs due to the detection of commensal N. species that may harbor NG DNA fragments targeted in the PCR due to horizontal gene transmission following previous infection. In sum, the established assay provides a powerful tool for use as either a screening/diagnostic or a typing/confirmatory assay.