{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,21]],"date-time":"2026-05-21T16:04:54Z","timestamp":1779379494669,"version":"3.53.1"},"reference-count":32,"publisher":"MDPI AG","issue":"5","license":[{"start":{"date-parts":[[2026,5,21]],"date-time":"2026-05-21T00:00:00Z","timestamp":1779321600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"ANLIS Competitive Funds 2019","award":["NRU-1904"],"award-info":[{"award-number":["NRU-1904"]}]},{"name":"Administraci\u00f3n Nacional de Laboratorios e Institutos de Salud (ANLIS), Argentina"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>Human herpesvirus 8 (HHV-8) is the etiologic agent of Kaposi\u2019s sarcoma (KS), primary effusion lymphoma (PEL), multicentric Castleman disease (MCD), and KS-associated immune reconstitution inflammatory syndrome (IRIS-KS). Quantifying HHV-8 DNA in whole blood is clinically relevant, yet laboratory practices remain heterogeneous. Here, we developed and validated an in-house quantitative PCR (qPCR) assay targeting ORF26, optimized for whole blood. Assay calibration used plasmid, BCBL-1 cell\u2013derived, and commercial HHV-8 DNA standards. Analytical validation was performed following the Clinical and Laboratory Standards Institute (CLSI) guidelines and the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines and showed a 95% limit of detection of 65.7 copies\/reaction, efficiencies of 90\u2013101% (R2 &gt; 0.99), and intra\/inter-assay coefficients of variation &lt; 6.5%. Strong correlations were observed among the three calibrators (R2 &gt; 0.97).Clinical validation against a composite reference yielded 100% sensitivity, specificity, PPV, and NPV. Viral loads (log10 copies\/mL) varied by clinical condition: classic KS and transplant-associated KS showed the lowest medians (2.30\u20132.23), MCD HIV\u2212 and PEL intermediate values (2.83\u20133.72), and epidemic KS, MCD HIV+, and IRIS-KS the highest (4.12, 4.86, and 5.03, respectively). Viremia &gt; 5 log10 copies\/mL was associated with uncontrolled E-KS, MCD HIV+, and IRIS-KS. Longitudinal follow-up revealed viral load decline paralleled clinical improvement. This validated assay provides a robust, affordable tool for HHV-8 quantification in whole blood and supports its integration into diagnostic workflows and patient monitoring.<\/jats:p>","DOI":"10.3390\/v18050578","type":"journal-article","created":{"date-parts":[[2026,5,21]],"date-time":"2026-05-21T15:02:27Z","timestamp":1779375747000},"page":"578","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Validated Quantification of HHV-8 DNA Using Inter-Convertible Plasmid and Cell-Derived Calibrators: Optimization of a Whole-Blood qPCR Assay"],"prefix":"10.3390","volume":"18","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-2971-2226","authenticated-orcid":false,"given":"Celeste Luj\u00e1n","family":"P\u00e9rez","sequence":"first","affiliation":[{"name":"Tissue Culture Service, Virology Department, INEI-ANLIS \u201cDr. Carlos G. Malbr\u00e1n\u201d (1282AAF), Buenos Aires C1282AAF, Argentina"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Carlos","family":"Ochoa Gamboa","sequence":"additional","affiliation":[{"name":"Tissue Culture Service, Virology Department, INEI-ANLIS \u201cDr. Carlos G. Malbr\u00e1n\u201d (1282AAF), Buenos Aires C1282AAF, Argentina"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"M\u00f3nica","family":"Tous","sequence":"additional","affiliation":[{"name":"Tissue Culture Service, Virology Department, INEI-ANLIS \u201cDr. Carlos G. Malbr\u00e1n\u201d (1282AAF), Buenos Aires C1282AAF, Argentina"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0006-1338-755X","authenticated-orcid":false,"given":"Juli\u00e1n","family":"Hazan","sequence":"additional","affiliation":[{"name":"Tissue Culture Service, Virology Department, INEI-ANLIS \u201cDr. Carlos G. Malbr\u00e1n\u201d (1282AAF), Buenos Aires C1282AAF, Argentina"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9037-9979","authenticated-orcid":false,"given":"Marcelo","family":"Rodr\u00edguez","sequence":"additional","affiliation":[{"name":"INEI-ANLIS \u201cDr. Carlos G. Malbr\u00e1n\u201d(1282AAF), Buenos Aires C1282AFF, Argentina"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Daniela","family":"Feliciotti","sequence":"additional","affiliation":[{"name":"Tissue Culture Service, Virology Department, INEI-ANLIS \u201cDr. Carlos G. Malbr\u00e1n\u201d (1282AAF), Buenos Aires C1282AAF, Argentina"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Luc\u00eda","family":"Irazu","sequence":"additional","affiliation":[{"name":"INEI-ANLIS \u201cDr. Carlos G. Malbr\u00e1n\u201d(1282AAF), Buenos Aires C1282AFF, Argentina"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Carlos","family":"Zala","sequence":"additional","affiliation":[{"name":"Department of Microbiology, School of Medicine, University of Buenos Aires, Buenos Aires C1121A6B, Argentina"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,5,21]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Poizot-Martin, I., Br\u00e9gigeon, S., Palich, R., Marcelin, A.-G., Valantin, M.-A., Solas, C., Veyri, M., Spano, J.-P., and Makinson, A. (2022). Immune Reconstitution Inflammatory Syndrome Associated Kaposi Sarcoma. Cancers, 14.","DOI":"10.3390\/cancers14040986"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"707","DOI":"10.1038\/nrc2888","article-title":"Kaposi\u2019s Sarcoma and Its Associated Herpesvirus","volume":"10","author":"Mesri","year":"2010","journal-title":"Nat. Rev. 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