{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,9]],"date-time":"2026-03-09T01:47:36Z","timestamp":1773020856396,"version":"3.50.1"},"reference-count":28,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2022,1,15]],"date-time":"2022-01-15T00:00:00Z","timestamp":1642204800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>Hepatitis A virus (HAV) is an emerging public health concern and there is an urgent need for ways to rapidly identify cases so that outbreaks can be managed effectively. Conventional testing for HAV relies on anti-HAV IgM seropositivity. However, studies estimate that 10\u201330% of patients may not be diagnosed by serology. Molecular assays that can directly detect viral nucleic acids have the potential to improve diagnosis, which is key to prevent the spread of infections. In this study, we developed a real-time PCR (RT-PCR) assay to detect HAV RNA for the identification of acute HAV infection. Primers were designed to target the conserved 5\u2032-untranslated region (5\u2032-UTR) of HAV, and the assay was optimized on both the Qiagen Rotor-Gene and the BD MAX. We successfully detected HAV from patient serum and stool samples with moderate differences in sensitivity and specificity depending on the platform used. Our results highlight the clinical utility of using a molecular assay to detect HAV from various specimen types that can be implemented in hospitals to assist with diagnostics, treatment and prevention.<\/jats:p>","DOI":"10.3390\/v14010159","type":"journal-article","created":{"date-parts":[[2022,1,16]],"date-time":"2022-01-16T20:46:06Z","timestamp":1642365966000},"page":"159","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":15,"title":["Development and Evaluation of a Molecular Hepatitis A Virus Assay for Serum and Stool Specimens"],"prefix":"10.3390","volume":"14","author":[{"given":"Robert A.","family":"Kozak","sequence":"first","affiliation":[{"name":"Sunnybrook Research Institute, Sunnybrook Health Sciences Centre, Toronto, ON M4N 3M5, Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Candace","family":"Rutherford","sequence":"additional","affiliation":[{"name":"St. Joseph\u2019s Healthcare, Hamilton, ON L8N 4A6, Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6717-8856","authenticated-orcid":false,"given":"Melissa","family":"Richard-Greenblatt","sequence":"additional","affiliation":[{"name":"St. Joseph\u2019s Healthcare, Hamilton, ON L8N 4A6, Canada"},{"name":"Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"N. Y. Elizabeth","family":"Chau","sequence":"additional","affiliation":[{"name":"Sunnybrook Research Institute, Sunnybrook Health Sciences Centre, Toronto, ON M4N 3M5, Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ana","family":"Cabrera","sequence":"additional","affiliation":[{"name":"Pathology and Laboratory Medicine, London Health Sciences Centre, London, ON N6A 5W9, Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1409-9908","authenticated-orcid":false,"given":"Mia","family":"Biondi","sequence":"additional","affiliation":[{"name":"Toronto Centre for Liver Disease, University Health Network, Toronto, ON M6H 3M1, Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0003-7553-4795","authenticated-orcid":false,"given":"Jamie","family":"Borlang","sequence":"additional","affiliation":[{"name":"National Microbiology Laboratory, Winnipeg, MB R3E 3PG, Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jaqueline","family":"Day","sequence":"additional","affiliation":[{"name":"National Microbiology Laboratory, Winnipeg, MB R3E 3PG, Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5429-7220","authenticated-orcid":false,"given":"Carla","family":"Osiowy","sequence":"additional","affiliation":[{"name":"National Microbiology Laboratory, Winnipeg, MB R3E 3PG, Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6359-5003","authenticated-orcid":false,"given":"Sumathi","family":"Ramachandran","sequence":"additional","affiliation":[{"name":"Division of Viral Hepatitis, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Nancy","family":"Mayer","sequence":"additional","affiliation":[{"name":"Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Laurel","family":"Glaser","sequence":"additional","affiliation":[{"name":"Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Marek","family":"Smieja","sequence":"additional","affiliation":[{"name":"St. Joseph\u2019s Healthcare, Hamilton, ON L8N 4A6, Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2022,1,15]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"227","DOI":"10.1016\/j.meegid.2013.10.023","article-title":"Hepatitis A virus: Host interactions, molecular epidemiology and evolution","volume":"21","author":"Vaughan","year":"2014","journal-title":"Infect. Genet. Evol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"S15","DOI":"10.1093\/infdis\/171.Supplement_1.S15","article-title":"Clinical manifestations of hepatitis A: Recent experience in a community teaching hospital","volume":"171","author":"Tong","year":"1995","journal-title":"J. Infect. Dis."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"413","DOI":"10.15585\/mmwr.mm6818a2","article-title":"Increase in Hepatitis A Virus Infections\u2014United States, 2013\u20132018","volume":"68","author":"Foster","year":"2019","journal-title":"MMWR Morb. Mortal. Wkly. Rep."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"a031716","DOI":"10.1101\/cshperspect.a031716","article-title":"Globalization and the Changing Epidemiology of Hepatitis A Virus","volume":"8","author":"Jacobsen","year":"2018","journal-title":"Cold Spring Harb. Perspect. Med."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"68","DOI":"10.4254\/wjh.v4.i3.68","article-title":"Hepatitis A: Epidemiology and prevention in developing countries","volume":"4","author":"Franco","year":"2012","journal-title":"World J. Hepatol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"1208","DOI":"10.15585\/mmwr.mm6743a3","article-title":"Hepatitis A Virus Outbreaks Associated with Drug Use and Homelessness - California, Kentucky, Michigan, and Utah, 2017","volume":"67","author":"Foster","year":"2018","journal-title":"MMWR Morb. Mortal. Wkly. Rep."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"3","DOI":"10.1016\/j.ijid.2007.04.012","article-title":"The underestimated risk of hepatitis A and hepatitis B: Benefits of an accelerated vaccination schedule","volume":"12","author":"Keystone","year":"2008","journal-title":"Int. J. Infect. Dis."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"8536","DOI":"10.1016\/j.vaccine.2007.10.001","article-title":"Cost-utility of universal hepatitis A vaccination in Canada","volume":"25","author":"Bauch","year":"2007","journal-title":"Vaccine"},{"key":"ref_9","unstructured":"Public Health Ontario (2020, September 04). Infectious Disease Trends in Ontario. Available online: https:\/\/www.publichealthontario.ca\/-\/media\/documents\/i\/2019\/idto-technical-notes.pdf?la=en."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"e86","DOI":"10.1086\/497073","article-title":"Detection of immunoglobulin M antibody to hepatitis A virus in Alaska residents without other evidence of hepatitis","volume":"41","author":"Castrodale","year":"2005","journal-title":"Clin. Infect. Dis."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"56","DOI":"10.3350\/kjhep.2012.18.1.56","article-title":"Optimal time for repeating the IgM anti-hepatitis A virus antibody test in acute hepatitis A patients with a negative initial test","volume":"18","author":"Hyun","year":"2012","journal-title":"Korean J. Hepatol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"46","DOI":"10.1016\/j.jviromet.2018.01.014","article-title":"Improved TaqMan real-time assays for detecting hepatitis A virus","volume":"254","author":"Chou","year":"2018","journal-title":"J. Virol. Methods"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"254","DOI":"10.1016\/S1386-6532(03)00165-3","article-title":"Detection of hepatitis A virus RNA in serum during the window period of infection","volume":"29","author":"Villar","year":"2004","journal-title":"J. Clin. Virol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"98","DOI":"10.1016\/j.jcv.2011.06.011","article-title":"Molecular epidemiology of hepatitis A virus infections in Catalonia, Spain, 2005\u20132009: Circulation of newly emerging strains","volume":"52","author":"Costafreda","year":"2011","journal-title":"J. Clin. Virol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"50","DOI":"10.1128\/jvi.61.1.50-59.1987","article-title":"Complete nucleotide sequence of wild-type hepatitis A virus: Comparison with different strains of hepatitis A virus and other picornaviruses","volume":"61","author":"Cohen","year":"1987","journal-title":"J. Virol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"63","DOI":"10.1128\/CMR.19.1.63-79.2006","article-title":"Diagnosis of hepatitis a virus infection: A molecular approach","volume":"19","author":"Nainan","year":"2006","journal-title":"Clin. Microbiol. Rev."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"65","DOI":"10.1016\/j.jviromet.2005.07.004","article-title":"A broadly reactive one-step real-time RT-PCR assay for rapid and sensitive detection of hepatitis E virus","volume":"131","author":"Jothikumar","year":"2006","journal-title":"J. Virol. Methods"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"1647","DOI":"10.1093\/bioinformatics\/bts199","article-title":"Geneious Basic: An integrated and extendable desktop software platform for the organization and analysis of sequence data","volume":"28","author":"Kearse","year":"2012","journal-title":"Bioinformatics"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"12","DOI":"10.1086\/315701","article-title":"Duration of viremia in hepatitis A virus infection","volume":"182","author":"Bower","year":"2000","journal-title":"J. Infect. Dis."},{"key":"ref_20","unstructured":"Centers for Disease Control and Prevention (2020, September 04). Widespread Person-to-Person Outbreaks of Hepatitis A across the United States, Available online: https:\/\/www.cdc.gov\/hepatitis\/outbreaks\/2017March-HepatitisA.htm."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"14","DOI":"10.1093\/cid\/ciz788","article-title":"Homelessness and Hepatitis A\u2014San Diego County, 2016\u20132018","volume":"71","author":"Peak","year":"2019","journal-title":"Clin. Infect. Dis."},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Coudray-Meunier, C., Fraisse, A., Mokhtari, C., Martin-Latil, S., Roque-Afonso, A.M., and Perelle, S. (2014). Hepatitis A virus subgenotyping based on RT-qPCR assays. BMC Microbiol., 14.","DOI":"10.1186\/s12866-014-0296-1"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1594","DOI":"10.3201\/eid2508.181489","article-title":"Molecular Genotyping of Hepatitis A Virus, California, USA, 2017\u20132018","volume":"25","author":"Probert","year":"2019","journal-title":"Emerg. Infect. Dis."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"e00500-19","DOI":"10.1128\/JCM.00500-19","article-title":"New Subgenotyping and Consensus Real-Time Reverse Transcription-PCR Assays for Hepatitis A Outbreak Surveillance","volume":"57","author":"Probert","year":"2019","journal-title":"J. Clin. Microbiol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1111\/j.1472-765X.2007.02140.x","article-title":"Hepatitis A virus detection in food: Current and future prospects","volume":"45","author":"Sanchez","year":"2007","journal-title":"Lett. Appl. Microbiol."},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Lee, H.W., Chang, D.-Y., Moon, H.J., Chang, H.Y., Shin, E.-C., Lee, J.S., Kim, K.-A., and Kim, H.J. (2015). Clinical Factors and Viral Load Influencing Severity of Acute Hepatitis, A. PLoS ONE, 10.","DOI":"10.1371\/journal.pone.0130728"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"3587","DOI":"10.1007\/s00705-017-3538-3","article-title":"The serological markers of acute infection with hepatitis A, B, C, D, E and G viruses revisited","volume":"162","author":"Ponde","year":"2017","journal-title":"Arch. Virol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"887","DOI":"10.1086\/500260","article-title":"Detection of immunoglobulin M antibody to hepatitis A virus in patients without acute hepatitis A: The usefulness of specific immunoglobulin G avidity","volume":"42","author":"Mackiewicz","year":"2006","journal-title":"Clin. Infect. Dis."}],"container-title":["Viruses"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4915\/14\/1\/159\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,13]],"date-time":"2025-10-13T13:27:30Z","timestamp":1760362050000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4915\/14\/1\/159"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,1,15]]},"references-count":28,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2022,1]]}},"alternative-id":["v14010159"],"URL":"https:\/\/doi.org\/10.3390\/v14010159","relation":{},"ISSN":["1999-4915"],"issn-type":[{"value":"1999-4915","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,1,15]]}}}