{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,19]],"date-time":"2026-05-19T05:12:45Z","timestamp":1779167565535,"version":"3.51.4"},"reference-count":30,"publisher":"MDPI AG","issue":"10","license":[{"start":{"date-parts":[[2026,5,16]],"date-time":"2026-05-16T00:00:00Z","timestamp":1778889600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100003196","name":"Italian Ministry of Health","doi-asserted-by":"crossref","id":[{"id":"10.13039\/501100003196","id-type":"DOI","asserted-by":"crossref"}]},{"name":"European Reference Laboratory for Public Health"},{"name":"EU4Health Programme","award":["101194810"],"award-info":[{"award-number":["101194810"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Differentiation and detection of viruses causing vesicular\/mucocutaneous lesions are essential for patient management. This study evaluated the analytical and clinical performance of the Novaplex\u2122 HSV-1&amp;2\/VZV\/MPXV multiplex real-time PCR assay (Novaplex), designed for the simultaneous detection of monkeypox virus (MPXV), herpes virus types 1 and 2 (HSV), and varicella-zoster virus (VZV). It was compared with Singleplex PCRs as reference methods. Analytical sensitivity was assessed only for MPXV using viral stocks of clades Ia, Ib, and IIb, while clinical performances for HSV, VZV and MPXV were evaluated on 93 residual clinical samples. Novaplex discriminated clade II from clade I viruses and demonstrated low limits of detection for MPXV (clade Ia: 2.9 Log TCID50\/mL; clade Ib: 1.9 Log TCID50\/mL; clade IIb: 1.4 Log TCID50\/mL). Clinically, the assay showed high overall sensitivity (97.1%) and specificity (100%), with almost perfect agreement with Singleplex PCR (\u03ba = 0.947). Stratified results by viruses: HSV showed \u03ba = 1.000 and 100% of sensitivity, MPXV showed a sensitivity of 97.8% with \u03ba = 0.969, and VZV showed a \u03ba = 0.914 with a sensitivity of 87.5%. Specificity was 100% for all three viruses. Novaplex offers a robust, efficient diagnostic approach for simultaneous detection of MPXV, HSV-1\/2, and VZV, supporting timely clinical decision making and enhanced outbreak preparedness.<\/jats:p>","DOI":"10.3390\/ijms27104477","type":"journal-article","created":{"date-parts":[[2026,5,18]],"date-time":"2026-05-18T14:40:31Z","timestamp":1779115231000},"page":"4477","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Evaluation of a Novel Multiplex PCR Assay for Vesicular Viruses"],"prefix":"10.3390","volume":"27","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-0955-2074","authenticated-orcid":false,"given":"Giuseppe","family":"Sberna","sequence":"first","affiliation":[{"name":"Laboratory of Virology and Laboratories of Biosafety, National Institute for Infectious Diseases Lazzaro Spallanzani\u2014IRCCS, 00149 Rome, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5022-5388","authenticated-orcid":false,"given":"Maria Beatrice","family":"Valli","sequence":"additional","affiliation":[{"name":"Laboratory of Virology and Laboratories of Biosafety, National Institute for Infectious Diseases Lazzaro Spallanzani\u2014IRCCS, 00149 Rome, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0640-8667","authenticated-orcid":false,"given":"Francesca","family":"Colavita","sequence":"additional","affiliation":[{"name":"Laboratory of Virology and Laboratories of Biosafety, National Institute for Infectious Diseases Lazzaro Spallanzani\u2014IRCCS, 00149 Rome, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Fabiano","family":"Brillo","sequence":"additional","affiliation":[{"name":"Laboratory of Virology and Laboratories of Biosafety, National Institute for Infectious Diseases Lazzaro Spallanzani\u2014IRCCS, 00149 Rome, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Gabriella","family":"Rozera","sequence":"additional","affiliation":[{"name":"Laboratory of Virology and Laboratories of Biosafety, National Institute for Infectious Diseases Lazzaro Spallanzani\u2014IRCCS, 00149 Rome, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7833-9539","authenticated-orcid":false,"given":"Claudia","family":"Minosse","sequence":"additional","affiliation":[{"name":"Laboratory of Virology and Laboratories of Biosafety, National Institute for Infectious Diseases Lazzaro Spallanzani\u2014IRCCS, 00149 Rome, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7489-5271","authenticated-orcid":false,"given":"Fabrizio","family":"Maggi","sequence":"additional","affiliation":[{"name":"Laboratory of Virology and Laboratories of Biosafety, National Institute for Infectious Diseases Lazzaro Spallanzani\u2014IRCCS, 00149 Rome, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Eleonora","family":"Lalle","sequence":"additional","affiliation":[{"name":"Laboratory of Virology and Laboratories of Biosafety, National Institute for Infectious Diseases Lazzaro Spallanzani\u2014IRCCS, 00149 Rome, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2026,5,16]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"97","DOI":"10.1016\/j.jviromet.2005.05.020","article-title":"Rapid, differential diagnosis of orthopox- and herpesviruses based upon real-time PCR product melting temperature and restriction enzyme analysis of amplicons","volume":"129","author":"Carletti","year":"2005","journal-title":"J. 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