{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,23]],"date-time":"2026-09-23T01:42:08Z","timestamp":1790127728379,"version":"4.0.1"},"reference-count":48,"publisher":"MDPI AG","issue":"6","license":[{"start":{"date-parts":[[2025,5,30]],"date-time":"2025-05-30T00:00:00Z","timestamp":1748563200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"EU funding within the MUR PNRR Extended Partnership Initiative on Emerging Infectious Diseases","award":["PE00000007"],"award-info":[{"award-number":["PE00000007"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>Wastewater-based epidemiology (WBE) offers valuable insight into viral circulation at the community level. In this study, we combined digital PCR (dPCR) with molecular typing to investigate the prevalence and diversity of human adenoviruses (HAdVs) in untreated wastewater samples collected throughout Italy. HAdV genomes were detected in over 93% of the 168 samples analyzed, with concentrations up to 4.5 \u00d7 106 genome copies per liter. For genotypic characterization, we used nested PCR followed by Sanger and Oxford Nanopore Technologies (ONTs) long-read sequencing. While Sanger sequencing identified three dominant genotypes (HAdV-A12, HAdV-B3, and HAdV-F41), ONT sequencing provided enhanced resolution, confirming all previously identified types and revealing seven additional genotypes: HAdV-B21, HAdV-C5, HAdV-D45, HAdV-D46, HAdV-D49, HAdV-D83, and HAdV-F40. This comprehensive approach highlights the added value of ONT long-read sequencing in uncovering the genetic complexity of adenoviruses in wastewater, particularly in detecting rare or low abundance types that conventional methods may miss. Our findings highlight the value of integrating quantitative and high-resolution molecular tools in WBE to improve surveillance and better understand the epidemiology of viral pathogens circulating in the human population.<\/jats:p>","DOI":"10.3390\/v17060791","type":"journal-article","created":{"date-parts":[[2025,5,30]],"date-time":"2025-05-30T09:22:41Z","timestamp":1748596961000},"page":"791","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":9,"title":["Wastewater-Based Surveillance of Human Adenoviruses in Italy: Quantification by Digital PCR and Molecular Typing via Nanopore Amplicon Sequencing"],"prefix":"10.3390","volume":"17","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-4187-4471","authenticated-orcid":false,"given":"Carolina","family":"Veneri","sequence":"first","affiliation":[{"name":"National Center for Water Safety (CeNSia), Istituto Superiore di Sanit\u00e0, 00161 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1998-7517","authenticated-orcid":false,"given":"G.","family":"Bonanno Ferraro","sequence":"additional","affiliation":[{"name":"National Center for Water Safety (CeNSia), Istituto Superiore di Sanit\u00e0, 00161 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0006-4522-1677","authenticated-orcid":false,"given":"D.","family":"Congiu","sequence":"additional","affiliation":[{"name":"National Center for Water Safety (CeNSia), Istituto Superiore di Sanit\u00e0, 00161 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"A.","family":"Franco","sequence":"additional","affiliation":[{"name":"National Center for Water Safety (CeNSia), Istituto Superiore di Sanit\u00e0, 00161 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7780-5355","authenticated-orcid":false,"given":"D.","family":"Brandtner","sequence":"additional","affiliation":[{"name":"Department of Infectious Disease, Istituto Superiore di Sanit\u00e0, 00161 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1685-9982","authenticated-orcid":false,"given":"P.","family":"Mancini","sequence":"additional","affiliation":[{"name":"National Center for Water Safety (CeNSia), Istituto Superiore di Sanit\u00e0, 00161 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5402-927X","authenticated-orcid":false,"given":"M.","family":"Iaconelli","sequence":"additional","affiliation":[{"name":"National Center for Water Safety (CeNSia), Istituto Superiore di Sanit\u00e0, 00161 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"name":"The SARI Network","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"L.","family":"Lucentini","sequence":"additional","affiliation":[{"name":"National Center for Water Safety (CeNSia), Istituto Superiore di Sanit\u00e0, 00161 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7605-0539","authenticated-orcid":false,"given":"E.","family":"Suffredini","sequence":"additional","affiliation":[{"name":"Department of Food Safety, Nutrition and Veterinary Public Health, Istituto Superiore di Sanit\u00e0, 00161 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2657-100X","authenticated-orcid":false,"given":"Giuseppina","family":"La Rosa","sequence":"additional","affiliation":[{"name":"National Center for Water Safety (CeNSia), Istituto Superiore di Sanit\u00e0, 00161 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,5,30]]},"reference":[{"key":"ref_1","first-page":"001721","article-title":"ICTV Virus Taxonomy Profile: Adenoviridae 2022","volume":"103","author":"Aoki","year":"2022","journal-title":"J. Gen. Virol."},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"La Rosa, G., and Suffredini, E. (2018). Adenovirus. Handbook of Foodborne Diseases, CRC Press.","DOI":"10.1201\/b22030-2"},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Kajon, A.E. (2024). Adenovirus infections: New insights for the clinical laboratory. J. Clin. Microbiol., 62.","DOI":"10.1128\/jcm.00836-22"},{"key":"ref_4","unstructured":"Centers for Disease Control (CDC) (1992). Outbreak of pharyngoconjunctival fever at a summer camp\u2014North Carolina, 1991. MMWR Morb. Mortal. Wkly. Rep., 41, 342\u2013344."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1759","DOI":"10.1086\/647987","article-title":"Health care transmission of a newly emergent adenovirus serotype in health care personnel at a military hospital in Texas, 2007","volume":"200","author":"Lessa","year":"2009","journal-title":"J. Infect. Dis."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"ofae192","DOI":"10.1093\/ofid\/ofae192","article-title":"Detection of a Human Adenovirus Outbreak, Including Some Critical Infections, Using Multipathogen Testing at a Large University, September 2022\u2013January 2023","volume":"11","author":"Montgomery","year":"2024","journal-title":"Open Forum Infect. Dis."},{"key":"ref_7","unstructured":"WHO (2025). Adenoviruses: Background Document for the WHO Guidelines for Drinking Water Quality and the WHO Guidelines on Sanitation and Health, World Health Organization."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"2548","DOI":"10.1016\/0043-1354(95)00070-2","article-title":"Survival of the enteric adenoviruses 40 and 41 in tap, sea, and waste water","volume":"29","author":"Enriquez","year":"1995","journal-title":"Water Res."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"7132","DOI":"10.1021\/es060892o","article-title":"Human Adenoviruses in Water: Occurrence and Health Implications: A Critical Review","volume":"40","author":"Jiang","year":"2006","journal-title":"Environ. Sci. Technol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"2504","DOI":"10.1080\/09603123.2023.2255559","article-title":"Exploring adenovirus in water environments: A systematic review and meta-analysis","volume":"34","author":"Takuissu","year":"2024","journal-title":"Int. J. Environ. Health Res."},{"key":"ref_11","first-page":"133","article-title":"Waterborne adenovirus","volume":"198","author":"Mena","year":"2009","journal-title":"Rev. Environ. Contam. Toxicol."},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Bonadonna, L., and La Rosa, G. (2019). A Review and Update on Waterborne Viral Diseases Associated with Swimming Pools. Int. J. Environ. Res. Public Health, 16.","DOI":"10.3390\/ijerph16020166"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"79","DOI":"10.1007\/s12560-016-9263-3","article-title":"One-year Surveillance of Human Enteric Viruses in Raw and Treated Wastewaters, Downstream River Waters, and Drinking Waters","volume":"9","author":"Iaconelli","year":"2017","journal-title":"Food Environ. Virol."},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"La Rosa, G., Brandtner, D., Mancini, P., Veneri, C., Ferraro, G.B., Bonadonna, L., Lucentini, L., and Suffredini, E. (2021). Key SARS-CoV-2 Mutations of Alpha, Gamma, and Eta Variants Detected in Urban Wastewaters in Italy by Long-Read Amplicon Sequencing Based on Nanopore Technology. Water, 13.","DOI":"10.3390\/w13182503"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"e00614","DOI":"10.1128\/mSystems.00614-20","article-title":"SARS-CoV-2 titers inwastewater are higher than expected from clinically confirmed cases","volume":"5","author":"Wu","year":"2020","journal-title":"mSystems"},{"key":"ref_16","first-page":"266","article-title":"Quantitative real-time PCR of enteric viruses in influent and effluent samples from wastewater treatment plants in Italy","volume":"46","author":"Pourshaban","year":"2010","journal-title":"Ann. Dell\u2019istituto Super. Sanita"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"1587","DOI":"10.1007\/s00705-005-0722-7","article-title":"Molecular typing of human adenoviruses by PCR and sequencing of a partial region of the hexon gene","volume":"151","author":"Lu","year":"2006","journal-title":"Arch. Virol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"262","DOI":"10.1007\/s12560-016-9246-4","article-title":"Quantitative PCR Detection and Characterisation of Human Adenovirus, Rotavirus and Hepatitis A Virus in Discharged Effluents of Two Wastewater Treatment Facilities in the Eastern Cape, South Africa","volume":"8","author":"Adefisoye","year":"2016","journal-title":"Food Environ. Virol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"98","DOI":"10.1186\/s12985-015-0327-z","article-title":"Incidence of human adenoviruses and Hepatitis A virus in the final effluent of selected wastewater treatment plants in Eastern Cape Province, South Africa","volume":"12","author":"Osuolale","year":"2015","journal-title":"Virol. J."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"10977","DOI":"10.1007\/s11356-018-1399-2","article-title":"Molecular detection and genotypic characterization of enteric adenoviruses in a hospital wastewater","volume":"25","author":"Ibrahim","year":"2018","journal-title":"Environ. Sci. Pollut. Res."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"287","DOI":"10.2166\/wh.2019.303","article-title":"Molecular detection of human adenovirus in urban wastewater in Egypt and among children suffering from acute gastroenteritis","volume":"17","author":"Elmahdy","year":"2019","journal-title":"J. Water Health"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"85","DOI":"10.1007\/s12560-009-9013-x","article-title":"Viral Removal by Wastewater Treatment: Monitoring of Indicators and Pathogens","volume":"1","author":"Carducci","year":"2009","journal-title":"Food Environ. Virol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"240","DOI":"10.1016\/j.watres.2017.05.039","article-title":"Molecular characterization of human adenoviruses in urban wastewaters using next generation and Sanger sequencing","volume":"121","author":"Iaconelli","year":"2017","journal-title":"Water Res."},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Pellegrinelli, L., Renteria, S.C.U., Ceriotti, F., Ammoni, E., Galli, C., Seiti, A., Castiglioni, S., Cereda, D., Binda, S., and Pariani, E. (2022). Wastewater Surveillance Captured an Increase in Adenovirus Circulation in Milan (Italy) during the First Quarter of 2022. Viruses, 14.","DOI":"10.3390\/v14112351"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"800","DOI":"10.1111\/j.1365-2672.2009.04477.x","article-title":"Evaluation of methodology for detection of human adenoviruses in wastewater, drinking water, stream water and recreational waters","volume":"108","author":"Dong","year":"2010","journal-title":"J. Appl. Microbiol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"715","DOI":"10.1128\/AEM.01316-09","article-title":"Quantitative Detection of Human Adenoviruses in Wastewater and Combined Sewer Overflows Influencing a Michigan River","volume":"76","author":"Fong","year":"2010","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"110","DOI":"10.2166\/wh.2012.123","article-title":"Assessment of burden of virus agents in an urban sewage treatment plant in Rio de Janeiro, Brazil","volume":"11","author":"Fumian","year":"2013","journal-title":"J. Water Health"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"720","DOI":"10.1007\/s10661-015-4917-4","article-title":"Human adenovirus spread, rainfalls, and the occurrence of gastroenteritis cases in a Brazilian basin","volume":"187","author":"Rodrigues","year":"2015","journal-title":"Environ. Monit. Assess."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"41","DOI":"10.1016\/j.watres.2017.09.063","article-title":"Viral diversity and abundance in polluted waters in Kampala, Uganda","volume":"127","author":"Nakyazze","year":"2017","journal-title":"Water Res."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"354","DOI":"10.1007\/s12560-017-9294-4","article-title":"DNA Heat Treatment for Improving qPCR Analysis of Human Adenovirus in Wastewater","volume":"9","author":"Rames","year":"2017","journal-title":"Food Environ. Virol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"112","DOI":"10.1007\/s12560-015-9194-4","article-title":"Human Adenovirus Diversity in Water Samples Using a Next-Generation Amplicon Sequencing Approach","volume":"7","author":"Ogorzaly","year":"2015","journal-title":"Food Environ. Virol."},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"Khales, P., Razizadeh, M.H., Ghorbani, S., Moattari, A., Sarvari, J., Saadati, H., Sayyahfar, S., Salavatiha, Z., Hasanabad, M.H., and Poortahmasebi, V. (2024). Human adenoviruses in children with gastroenteritis: A systematic review and meta-analysis. BMC Infect. Dis., 24.","DOI":"10.1186\/s12879-024-09386-x"},{"key":"ref_33","doi-asserted-by":"crossref","unstructured":"Nascimento, L.G.D., Fialho, A.M., Andrade, J.d.S.R.d., de Assis, R.M.S., and Fumian, T.M. (2022). Human enteric adenovirus F40\/41 as a major cause of acute gastroenteritis in children in Brazil, 2018 to 2020. Sci. Rep., 12.","DOI":"10.1038\/s41598-022-15413-1"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"751","DOI":"10.3201\/eid2904.221878","article-title":"Adeno-Associated Virus 2 and Human Adenovirus F41 in Wastewater during Outbreak of Severe Acute Hepatitis in Children, Ireland","volume":"29","author":"Martin","year":"2023","journal-title":"Emerg. Infect. Dis."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"vead023","DOI":"10.1093\/ve\/vead023","article-title":"Genomic epidemiology of human adenovirus F40 and F41 in coastal Kenya: A retrospective hospital-based surveillance study (2013\u20132022)","volume":"9","author":"Lambisia","year":"2023","journal-title":"Virus Evol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"292","DOI":"10.7883\/yoken.JJID.2024.035","article-title":"Prevalence of Human Adenovirus Type 3 Associated with Pharyngoconjunctival Fever in Children in Osaka, Japan during and after the COVID-19 Pandemic","volume":"77","author":"Koyama","year":"2024","journal-title":"Jpn. J. Infect. Dis."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"e70139","DOI":"10.1002\/jmv.70139","article-title":"An Outbreak of Human Adenovirus Infection Among Children Post COVID-19 Pandemic in Southern China","volume":"96","author":"Wang","year":"2024","journal-title":"J. Med. Virol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"1097","DOI":"10.1016\/j.jiac.2024.07.017","article-title":"Epidemiological impact of human adenovirus as causative agent of respiratory infections: An Italian multicentre retrospective study, 2022\u20132023","volume":"30","author":"Giardina","year":"2024","journal-title":"J. Infect. Chemother."},{"key":"ref_39","first-page":"277","article-title":"Investigation of an outbreak of adenovirus type 3 infection in a boys\u2019 boarding school","volume":"93","author":"Payne","year":"1984","journal-title":"Epidemiol. Infect."},{"key":"ref_40","first-page":"9","article-title":"A primary school outbreak of pharyngoconjunctival fever caused by adenovirus type 3","volume":"25","author":"Harley","year":"2001","journal-title":"Commun. Dis. Intell."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"200225","DOI":"10.1016\/j.tvr.2021.200225","article-title":"Almost famous: Human adenoviruses (and what they have taught us about cancer)","volume":"12","author":"Tessier","year":"2021","journal-title":"Tumour Virus Res."},{"key":"ref_42","doi-asserted-by":"crossref","unstructured":"Kim, J.-S., Lee, S.K., Ko, D.-H., Hyun, J., Kim, H.-S., Song, W., and Kim, H.S. (2017). Associations of Adenovirus Genotypes in Korean Acute Gastroenteritis Patients with Respiratory Symptoms and Intussusception. BioMed Res. Int., 2017.","DOI":"10.1155\/2017\/1602054"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"403","DOI":"10.1590\/0074-02760160030","article-title":"A non-enteric adenovirus A12 gastroenteritis outbreak in Rio de Janeiro, Brazil","volume":"111","author":"Portes","year":"2016","journal-title":"Mem. Do Inst. Oswaldo Cruz"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"490","DOI":"10.1016\/j.jinf.2014.06.015","article-title":"A human adenovirus species B subtype 21a associated with severe pneumonia","volume":"69","author":"Hage","year":"2014","journal-title":"J. Infect."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"99","DOI":"10.1016\/j.jcrc.2019.02.019","article-title":"Severe acute respiratory distress syndrome (ARDS) induced by human adenovirus B21: Report on 2 cases and literature review","volume":"51","author":"Pfortmueller","year":"2019","journal-title":"J. Crit. Care"},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"25","DOI":"10.1186\/1743-422X-11-25","article-title":"High prevalence and diversity of species D adenoviruses (HAdV-D) in human populations of four Sub-Saharan countries","volume":"11","author":"Pauly","year":"2014","journal-title":"Virol. J."},{"key":"ref_47","doi-asserted-by":"crossref","unstructured":"Kurskaya, O.G., Prokopyeva, E.A., Dubovitskiy, N.A., Solomatina, M.V., Sobolev, I.A., Derko, A.A., Nokhova, A.R., Anoshina, A.V., Leonova, N.V., and Simkina, O.A. (2024). Genetic Diversity of the Human Adenovirus C Isolated from Hospitalized Children in Russia (2019\u20132022). Viruses, 16.","DOI":"10.3390\/v16030386"},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"2417","DOI":"10.1128\/JVI.02392-08","article-title":"Latent Species C Adenoviruses in Human Tonsil Tissues","volume":"83","author":"Garnett","year":"2009","journal-title":"J. Virol."}],"container-title":["Viruses"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4915\/17\/6\/791\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,9]],"date-time":"2025-10-09T17:43:59Z","timestamp":1760031839000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4915\/17\/6\/791"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,5,30]]},"references-count":48,"journal-issue":{"issue":"6","published-online":{"date-parts":[[2025,6]]}},"alternative-id":["v17060791"],"URL":"https:\/\/doi.org\/10.3390\/v17060791","relation":{},"ISSN":["1999-4915"],"issn-type":[{"value":"1999-4915","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,5,30]]}}}