{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,12,17]],"date-time":"2025-12-17T18:15:04Z","timestamp":1765995304812,"version":"3.41.2"},"reference-count":43,"publisher":"American Society for Microbiology","issue":"3","license":[{"start":{"date-parts":[[2024,3,5]],"date-time":"2024-03-05T00:00:00Z","timestamp":1709596800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2024,3,5]],"date-time":"2024-03-05T00:00:00Z","timestamp":1709596800000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"funder":[{"DOI":"10.13039\/100009139","name":"Deutsches Zentrum f\u00fcr Infektionsforschung","doi-asserted-by":"crossref","award":["TTU 08.815"],"award-info":[{"award-number":["TTU 08.815"]}],"id":[{"id":"10.13039\/100009139","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["Microbiol Spectr"],"published-print":{"date-parts":[[2024,3,5]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n          <jats:sec>\n            <jats:title\/>\n            <jats:p>\n              For effective infection control measures for\n              <jats:italic>Chlamydia trachomatis<\/jats:italic>\n              (\n              <jats:italic>CT<\/jats:italic>\n              ) and\n              <jats:italic>Neisseria gonorrhoeae<\/jats:italic>\n              (\n              <jats:italic>NG<\/jats:italic>\n              ), 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\n              <jats:italic>CT\/NG<\/jats:italic>\n              and differentiation between lymphogranuloma venereum (LGV) and non-LGV from genital and extra-genital specimens. Published primer\/probe sets (\n              <jats:italic>CT<\/jats:italic>\n              : pmpH, cryptic plasmid;\n              <jats:italic>NG<\/jats:italic>\n              : 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              <jats:italic>n<\/jats:italic>\n              = 319) were compared with a CE-marked\n              <jats:italic>in vitro<\/jats:italic>\n              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 \u22124.338 to \u22122.834 and Pearson correlation coefficients from 0.956 to 0.994. Inter- and intra-run variability was &lt;0.5 and &lt;1 cycle threshold (ct), respectively. No cross-reactivity was observed (\n              <jats:italic>n<\/jats:italic>\n              = 42). Clinical validation showed a sensitivity of 94.74% (95% confidence interval (CI): 87.23%\u201397.93%) and 95.51% (95% CI: 89.01%\u201398.24%), a specificity of 99.59% (95% CI: 97.71%\u201399.98%) and 99.57% (95% CI: 97.58%\u201399.98%), positive predictive values of 89.91% (estimated prevalence: 3.7%; 95% CI: 80.91%\u201395.6%) and 88.61% (estimated prevalence: 3.4%; 95% CI: 80.18%\u201394.34%), and negative predictive values of 99.81% (95% CI: 98.14%\u2013100%) and 99.85% (95% CI: 98.14%\u2013100%) for the detection of\n              <jats:italic>CT<\/jats:italic>\n              and\n              <jats:italic>NG<\/jats:italic>\n              , respectively. In conclusion, we established a dual-target, internally controlled PCR on an automated system for the detectiwon of\n              <jats:italic>CT\/NG<\/jats:italic>\n              from genital and extra-genital specimens. Depending on local regulations, the assay can be used as a screening or a confirmatory\/typing assay.\n            <\/jats:p>\n            <jats:sec>\n              <jats:title>IMPORTANCE<\/jats:title>\n              <jats:p>\n                <jats:italic>Chlamydia trachomatis<\/jats:italic>\n                (\n                <jats:italic>CT<\/jats:italic>\n                ) and\n                <jats:italic>Neisseria gonorrhoeae<\/jats:italic>\n                (\n                <jats:italic>NG<\/jats:italic>\n                ) represent a major global health burden, with the World Health Organization estimating that &gt;128 million and &gt;82 million people, respectively, were newly infected in 2020. For effective infection control measures, a reliable tool for sensitive diagnosis and screening of\n                <jats:italic>CT\/NG<\/jats:italic>\n                is essential. We established a multiplex PCR assay for the detection of\n                <jats:italic>CT\/NG<\/jats:italic>\n                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                <jats:italic>N<\/jats:italic>\n                . species that may harbor\n                <jats:italic>NG<\/jats:italic>\n                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.\n              <\/jats:p>\n            <\/jats:sec>\n          <\/jats:sec>","DOI":"10.1128\/spectrum.02756-23","type":"journal-article","created":{"date-parts":[[2024,2,12]],"date-time":"2024-02-12T14:09:57Z","timestamp":1707746997000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":3,"title":["Analytical and clinical validation of a multiplex PCR assay for detection of\n            <i>Neisseria gonorrhoeae<\/i>\n            and\n            <i>Chlamydia trachomatis<\/i>\n            including simultaneous LGV serotyping on an automated high-throughput PCR system"],"prefix":"10.1128","volume":"12","author":[{"ORCID":"https:\/\/orcid.org\/0009-0009-1649-5312","authenticated-orcid":false,"given":"Lisa Sophie","family":"Pfl\u00fcger","sequence":"first","affiliation":[{"name":"Center for Diagnostics, Institute of Medical Microbiology, Virology and Hygiene, University Medical Center Hamburg-Eppendorf (UKE), Hamburg, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4001-7192","authenticated-orcid":false,"given":"Dominik","family":"N\u00f6rz","sequence":"additional","affiliation":[{"name":"Center for Diagnostics, Institute of Medical Microbiology, Virology and Hygiene, University Medical Center Hamburg-Eppendorf (UKE), Hamburg, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Moritz","family":"Grunwald","sequence":"additional","affiliation":[{"name":"Center for Diagnostics, Institute of Medical Microbiology, Virology and Hygiene, University Medical Center Hamburg-Eppendorf (UKE), Hamburg, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7489-6557","authenticated-orcid":false,"given":"Susanne","family":"Pfefferle","sequence":"additional","affiliation":[{"name":"Center for Diagnostics, Institute of Medical Microbiology, Virology and Hygiene, University Medical Center Hamburg-Eppendorf (UKE), Hamburg, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Katja","family":"Giersch","sequence":"additional","affiliation":[{"name":"Center for Diagnostics, Institute of Medical Microbiology, Virology and Hygiene, University Medical Center Hamburg-Eppendorf (UKE), Hamburg, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Martin","family":"Christner","sequence":"additional","affiliation":[{"name":"Center for Diagnostics, Institute of Medical Microbiology, Virology and Hygiene, University Medical Center Hamburg-Eppendorf (UKE), Hamburg, 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