{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,11]],"date-time":"2026-10-11T14:42:04Z","timestamp":1791729724630,"version":"4.3.4"},"reference-count":26,"publisher":"American Society for Microbiology","issue":"9","license":[{"start":{"date-parts":[[2025,9,10]],"date-time":"2025-09-10T00:00:00Z","timestamp":1757462400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2025,9,10]],"date-time":"2025-09-10T00:00:00Z","timestamp":1757462400000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["J Clin Microbiol"],"published-print":{"date-parts":[[2025,9,10]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:sec>\n                    <jats:title\/>\n                    <jats:p>\n                      Human papillomavirus (HPV) is comprised of &gt;200 genotypes and has an ~8 kb, circular, double-stranded DNA genome. Transmission of HPV occurs through skin-to-skin contact and infection of squamous epithelial cells of cutaneous and mucosal surfaces. HPV genotypes are categorized as low- or high-risk (hrHPV) based on oncogenic potential. There are approximately 14 types of hrHPV that can cause several types of cancer, including HPV-associated oropharyngeal squamous cell carcinoma (HPV(+)OPSCC). Detection of HPV(+)OPSCC is traditionally accomplished using p16 immunohistochemistry (IHC) and HPV-specific testing, either DNA or RNA\n                      <jats:italic toggle=\"yes\">in situ<\/jats:italic>\n                      hybridization (ISH) staining or DNA-based PCR of suspected tumor biopsy tissue. More recently, platelet-poor plasma (PPP) samples from patients with HPV(+)OPSCC have proven useful for detection and quantitation of fragments of HPV circulating tumor DNA (ctDNA). ctDNA has been shown to be useful in determining treatment response and monitoring for disease recurrence. In this study, a novel droplet digital PCR assay (ddPCR) was developed and validated for the detection and quantitation of ctDNA from 5 hrHPV genotypes in PPP. Analytical sensitivity ranged from 7.71 to 19.45 fragments of HPV ctDNA per milliliter of PPP across five hrHPV genotypes. In patients with confirmed primary or recurrent HPV(+)OPSCC or HPV(-)OPSCC, testing of corresponding PPP samples (\n                      <jats:italic toggle=\"yes\">n<\/jats:italic>\n                      = 32) by ddPCR demonstrated 90.63% (29\/32) overall agreement with p16\/HPV-ISH biopsy results. Compared with reference ddPCR assays performed at outside laboratories, our ddPCR assay yielded 90% (9\/10) overall agreement. This assay may provide clinicians with a tool for monitoring HPV ctDNA prior to, during, and after treatment of an HPV-associated cancer.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>IMPORTANCE<\/jats:title>\n                    <jats:p>At least 14 genotypes of human papillomavirus (HPV) have been identified to have high oncogenic potential. While molecular diagnostic testing for HPV is widely available for liquid cytologic cervical samples, testing is limited for other sample types, including liquid biopsy samples, such as platelet-poor plasma (PPP). With the rising incidence of HPV-associated oropharyngeal squamous cell carcinoma (HPV(+)OPSCC), laboratory testing is an essential part of patient diagnosis, management, and surveillance. Here, we summarize the development and analytical performance validation of a multiplexed, droplet digital PCR (ddPCR) assay for the detection and quantitation of HPV circulating tumor DNA (ctDNA) in PPP. This assay may provide clinicians with a tool to address minimal residual disease for patients with an HPV-associated cancer.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1128\/jcm.00585-25","type":"journal-article","created":{"date-parts":[[2025,8,19]],"date-time":"2025-08-19T13:00:07Z","timestamp":1755608407000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":7,"title":["Detection, quantitation, and genotyping of human papillomavirus circulating tumor DNA by droplet digital PCR"],"prefix":"10.1128","volume":"63","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-9300-4451","authenticated-orcid":true,"given":"Emily C.","family":"Fernholz","sequence":"first","affiliation":[{"name":"Division of Clinical Microbiology, Department of Laboratory Medicine and Pathology, Mayo Clinic","place":["Rochester, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"David M.","family":"Routman","sequence":"additional","affiliation":[{"name":"Department of Radiation Oncology, Mayo Clinic","place":["Rochester, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kathryn M.","family":"Van Abel","sequence":"additional","affiliation":[{"name":"Department of Otolaryngology-Head and Neck Surgery, Mayo Clinic","place":["Rochester, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Eric J.","family":"Moore","sequence":"additional","affiliation":[{"name":"Department of Otolaryngology-Head and Neck Surgery, Mayo Clinic","place":["Rochester, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Daniel J.","family":"Ma","sequence":"additional","affiliation":[{"name":"Department of Radiation Oncology, Mayo Clinic","place":["Rochester, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Danielle E.","family":"Hunter","sequence":"additional","affiliation":[{"name":"Department of Otolaryngology-Head and Neck Surgery, Mayo Clinic","place":["Rochester, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kathleen R.","family":"Bartemes","sequence":"additional","affiliation":[{"name":"Department of Otolaryngology-Head and Neck Surgery, Mayo Clinic","place":["Rochester, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"suffix":"Jr.","given":"James S.","family":"Lewis","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/03jp40720","id-type":"ROR","asserted-by":"publisher"}],"name":"Division of Anatomic Pathology, Department of Laboratory Medicine and Pathology, Mayo Clinic Arizona","place":["Scottsdale, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Erik B.","family":"Wendlandt","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/009jvpf03","id-type":"ROR","asserted-by":"publisher"}],"name":"Integrated DNA Technologies","place":["Coralville, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2537-7165","authenticated-orcid":true,"given":"Matthew J.","family":"Binnicker","sequence":"additional","affiliation":[{"name":"Division of Clinical Microbiology, Department of Laboratory Medicine and Pathology, Mayo Clinic","place":["Rochester, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","reference":[{"key":"e_1_3_2_2_2","doi-asserted-by":"publisher","DOI":"10.1097\/OLQ.0000000000001356"},{"key":"e_1_3_2_3_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1416074111"},{"key":"e_1_3_2_4_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.jasc.2019.06.001"},{"key":"e_1_3_2_5_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.hoc.2021.05.004"},{"key":"e_1_3_2_6_2","volume-title":"Oropharyngeal squamous cell carcinoma","author":"Jamal Z","year":"2023","unstructured":"Jamal Z, Anjum F. 2023. Oropharyngeal squamous cell carcinoma. StatPearls, Treasure Island (FL). https:\/\/www.ncbi.nlm.nih.gov\/books\/NBK563268\/."},{"key":"e_1_3_2_7_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.oraloncology.2022.105776"},{"key":"e_1_3_2_8_2","doi-asserted-by":"publisher","DOI":"10.1002\/hed.24442"},{"key":"e_1_3_2_9_2","doi-asserted-by":"publisher","DOI":"10.1002\/ijc.27739"},{"key":"e_1_3_2_10_2","doi-asserted-by":"publisher","DOI":"10.1007\/s12105-021-01318-4"},{"key":"e_1_3_2_11_2","doi-asserted-by":"publisher","DOI":"10.1002\/cncr.24309"},{"key":"e_1_3_2_12_2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0250530"},{"key":"e_1_3_2_13_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.jcv.2011.03.003"},{"key":"e_1_3_2_14_2","doi-asserted-by":"publisher","DOI":"10.1101\/gr.241141.118"},{"key":"e_1_3_2_15_2","doi-asserted-by":"publisher","DOI":"10.1093\/jnci\/djv403"},{"key":"e_1_3_2_16_2","doi-asserted-by":"publisher","DOI":"10.1002\/cjp2.111"},{"key":"e_1_3_2_17_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.oraloncology.2023.106436"},{"key":"e_1_3_2_18_2","doi-asserted-by":"publisher","DOI":"10.1200\/JCO.2015.61.6995"},{"key":"e_1_3_2_19_2","doi-asserted-by":"publisher","DOI":"10.1373\/clinchem.2018.293712"},{"key":"e_1_3_2_20_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.jviromet.2021.114193"},{"key":"e_1_3_2_21_2","first-page":"S49","article-title":"Limit of blank, limit of detection and limit of quantitation","volume":"29","author":"Armbruster DA","year":"2008","unstructured":"Armbruster DA, Pry T. 2008. Limit of blank, limit of detection and limit of quantitation. Clin Biochem Rev 29:S49\u2013S52.","journal-title":"Clin Biochem Rev"},{"key":"e_1_3_2_22_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.virol.2013.07.018"},{"key":"e_1_3_2_23_2","doi-asserted-by":"publisher","DOI":"10.3390\/v16060904"},{"key":"e_1_3_2_24_2","doi-asserted-by":"publisher","DOI":"10.1186\/s12985-023-02188-8"},{"key":"e_1_3_2_25_2","unstructured":"Droplet digital PCR applications guide. 2025 Bulletin 6407. Version B. 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