{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,7]],"date-time":"2026-09-07T20:50:17Z","timestamp":1788814217613,"version":"build-2803163510"},"reference-count":40,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2024,11,20]],"date-time":"2024-11-20T00:00:00Z","timestamp":1732060800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100011094","name":"Public Health Agency of Canada","doi-asserted-by":"publisher","id":[{"id":"10.13039\/100011094","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Microbiol."],"abstract":"<jats:sec><jats:title>Introduction<\/jats:title><jats:p>Chronic hepatitis D virus (HDV) is associated with rapid progression to severe liver disease. Co-infection with HDV and hepatitis B virus is likely underdiagnosed due to challenges in diagnostic test availability and standardization. With new HDV antiviral options, HDV RNA quantification is essential for understanding the patient response to treatment. To this end, a quantitative real-time reverse transcription PCR (qRT-PCR) assay utilizing synthetic RNA calibrators and a conversion factor to quantify HDV RNA in WHO international standard units (IU\/mL) was developed and validated.<\/jats:p><\/jats:sec><jats:sec><jats:title>Methods<\/jats:title><jats:p>qRT-PCR primers and probes were selected within the ribozyme region. Thermocycling conditions and reactions were optimized. Synthetic RNA transcripts were prepared as quantification standards and calibrators. Transcript dilutions (log<jats:sub>10<\/jats:sub> 8 to log<jats:sub>10<\/jats:sub> 1 copies\/\u03bcL) were calibrated against the WHO standard and a conversion factor calculated to convert copies\/\u03bcL to IU\/mL. Assay validation and evaluation was conducted, including use of specimens from 8 HDV genotypes and comparison to a commercial assay.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results<\/jats:title><jats:p>The assay lower limit of detection was determined by probit analysis to be 11\u2009IU\/mL (8.63\u201315.78 95% CI). Inter- and intra-assay coefficient of variation analysis showed 96.6% precision and 90.6% accuracy. A conversion factor of 16.5 was used to convert copies\/\u03bcL to IU\/mL. All 8 HDV genotypes were quantified by the assay and commercial assay comparison showed good agreement.<\/jats:p><\/jats:sec><jats:sec><jats:title>Discussion<\/jats:title><jats:p>The developed assay has clinical utility for the sensitive and specific quantitative monitoring of HDV RNA, appropriate for medium to high throughput laboratories.<\/jats:p><\/jats:sec>","DOI":"10.3389\/fmicb.2024.1472826","type":"journal-article","created":{"date-parts":[[2024,11,20]],"date-time":"2024-11-20T12:17:01Z","timestamp":1732105021000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":2,"title":["Laboratory development of an RNA quantitative RT-PCR assay reporting in international units for hepatitis D virus"],"prefix":"10.3389","volume":"15","author":[{"given":"Carla","family":"Osiowy","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jacqueline","family":"Day","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Emma R.","family":"Lee","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2024,11,20]]},"reference":[{"key":"ref1","doi-asserted-by":"publisher","first-page":"533","DOI":"10.1016\/j.jhep.2020.02.030","article-title":"Chronic hepatitis D and hepatocellular carcinoma: a systematic review and meta-analysis of observational studies","volume":"73","author":"Alfaiate","year":"2020","journal-title":"J. Hepatol."},{"key":"ref2","doi-asserted-by":"publisher","first-page":"831","DOI":"10.1111\/liv.15795","article-title":"Quantification of serum HDV RNA by Robogene 2.0 in HDV patients is significantly influenced by the extraction methods","volume":"44","author":"Anolli","year":"2024","journal-title":"Liver Int."},{"key":"ref3","doi-asserted-by":"publisher","first-page":"133","DOI":"10.1056\/NEJMoa2314134","article-title":"Bulevirtide combined with pegylated interferon for chronic hepatitis D","volume":"391","author":"Asselah","year":"2024","journal-title":"N. Engl. J. Med."},{"key":"ref4","doi-asserted-by":"publisher","first-page":"734","DOI":"10.1016\/j.cgh.2013.01.025","article-title":"Commercial real-time reverse transcriptase PCR assays can underestimate or fail to quantify hepatitis delta virus viremia","volume":"11","author":"Brichler","year":"2013","journal-title":"Clin. Gastroenterol. Hepatol."},{"key":"ref5","doi-asserted-by":"publisher","first-page":"1694","DOI":"10.1128\/JCM.03521-13","article-title":"Serological and molecular diagnosis of hepatitis delta virus infection: results of a French national quality control study","volume":"52","author":"Brichler","year":"2014","journal-title":"J. Clin. Microbiol."},{"key":"ref6","doi-asserted-by":"publisher","first-page":"1749","DOI":"10.3390\/v14081749","article-title":"A review of HDV infection","volume":"14","author":"Caviglia","year":"2022","journal-title":"Viruses"},{"key":"ref7","doi-asserted-by":"publisher","first-page":"512","DOI":"10.1136\/gutjnl-2018-316601","article-title":"Prevalence and burden of hepatitis D virus infection in the global population: a systematic review and meta-analysis","volume":"68","author":"Chen","year":"2019","journal-title":"Gut"},{"key":"ref8","year":"2017"},{"key":"ref9","doi-asserted-by":"publisher","first-page":"156","DOI":"10.3138\/canlivj.2018-0008","article-title":"Management of hepatitis B virus infection: 2018 guidelines from the Canadian Association for the Study of liver disease and association of medical microbiology and infectious disease Canada","volume":"1","author":"Coffin","year":"2018","journal-title":"Can. Liver. J."},{"key":"ref10","doi-asserted-by":"publisher","first-page":"100686","DOI":"10.1016\/j.jhepr.2023.100686","article-title":"Treating hepatitis D with bulevirtide - real-world experience from 114 patients","volume":"5","author":"Dietz-Fricke","year":"2023","journal-title":"JHEP Rep."},{"key":"ref11","doi-asserted-by":"publisher","first-page":"e0003521","DOI":"10.1128\/CMR.00035-21","article-title":"The standardization and control of serology and nucleic acid testing for infectious diseases","volume":"34","author":"Dimech","year":"2021","journal-title":"Clin. Microbiol. Rev."},{"key":"ref12","doi-asserted-by":"publisher","first-page":"1970","DOI":"10.1515\/cclm-2018-0351","article-title":"Comparison of four methods of establishing control limits for monitoring quality controls in infectious disease serology testing","volume":"56","author":"Dimech","year":"2018","journal-title":"Clin. Chem. Lab. Med."},{"key":"ref13","doi-asserted-by":"publisher","first-page":"329","DOI":"10.1515\/cclm-2014-0546","article-title":"Determination of quality control limits for serological infectious disease testing using historical data","volume":"53","author":"Dimech","year":"2015","journal-title":"Clin. Chem. Lab. Med."},{"key":"ref14","doi-asserted-by":"publisher","first-page":"433","DOI":"10.1016\/j.jhep.2023.05.001","article-title":"EASL clinical practice guidelines on hepatitis delta virus","volume":"79","year":"2023","journal-title":"J. Hepatol."},{"key":"ref15","doi-asserted-by":"publisher","first-page":"1129","DOI":"10.1097\/HEP.0000000000000642","article-title":"Association of hepatitis delta virus with liver morbidity and mortality: a systematic literature review and meta-analysis","volume":"79","author":"Gish","year":"2024","journal-title":"Hepatology"},{"key":"ref16","doi-asserted-by":"publisher","first-page":"323654","DOI":"10.1155\/2010\/323654","article-title":"Evolution and diversity of the human hepatitis D virus genome","volume":"2010","author":"Huang","year":"2010","journal-title":"Adv. Bioinforma."},{"key":"ref17","doi-asserted-by":"publisher","first-page":"1826","DOI":"10.1002\/hep.29574","article-title":"Genetic diversity and worldwide distribution of the deltavirus genus: a study of 2,152 clinical strains","volume":"66","author":"Le Gal","year":"","journal-title":"Hepatology"},{"key":"ref18","doi-asserted-by":"publisher","first-page":"1483","DOI":"10.1002\/hep.28772","article-title":"First international external quality assessment for hepatitis delta virus RNA quantification in plasma","volume":"64","author":"Le Gal","year":"2016","journal-title":"Hepatology"},{"key":"ref19","doi-asserted-by":"publisher","first-page":"431","DOI":"10.1128\/JCM.02027-16","article-title":"Performance characteristics of a new consensus commercial kit for hepatitis D virus RNA viral load quantification","volume":"55","author":"Le Gal","year":"","journal-title":"J. Clin. Microbiol."},{"key":"ref20","doi-asserted-by":"publisher","first-page":"2363","DOI":"10.1128\/JCM.43.5.2363-2369.2005","article-title":"Quantification of hepatitis delta virus RNA in serum by consensus real-time PCR indicates different patterns of virological response to interferon therapy in chronically infected patients","volume":"43","author":"Le Gal","year":"2005","journal-title":"J. Clin. Microbiol."},{"key":"ref21","doi-asserted-by":"publisher","first-page":"1912","DOI":"10.3390\/v13101912","article-title":"Hepatitis D review: challenges for the resource-poor setting","volume":"13","author":"Lee","year":"2021","journal-title":"Viruses"},{"key":"ref22","doi-asserted-by":"publisher","first-page":"3334","DOI":"10.1128\/jcm.00940-14","article-title":"Relevance of a full-length genomic RNA standard and a thermal-shock step for optimal hepatitis delta virus quantification","volume":"52","author":"Maria","year":"2020","journal-title":"J. Clin. Microbiol."},{"key":"ref23","doi-asserted-by":"publisher","first-page":"1677","DOI":"10.1093\/infdis\/jiz633","article-title":"Estimating the global prevalence, disease progression, and clinical outcome of hepatitis delta virus infection","volume":"221","author":"Miao","year":"2020","journal-title":"J. Infect. Dis."},{"key":"ref24","doi-asserted-by":"publisher","first-page":"100461","DOI":"10.1016\/j.jhepr.2022.100461","article-title":"Molecular epidemiology and clinical characteristics of hepatitis D virus infection in Canada","volume":"4","author":"Osiowy","year":"2022","journal-title":"JHEP Rep."},{"key":"ref25","year":"2024"},{"key":"ref26","doi-asserted-by":"publisher","first-page":"100356","DOI":"10.1016\/j.jhepr.2021.100356","article-title":"Clinical establishment of a laboratory developed quantitative HDV PCR assay on the cobas6800 high-throughput system","volume":"3","author":"Pfl\u00fcger","year":"2021","journal-title":"JHEP Rep."},{"key":"ref27","doi-asserted-by":"publisher","first-page":"232","DOI":"10.1016\/j.jhep.2023.10.043","article-title":"Adjusted estimate of the prevalence of hepatitis delta virus in 25 countries and territories","volume":"80","year":"2024","journal-title":"J. Hepatol."},{"key":"ref28","doi-asserted-by":"publisher","first-page":"576","DOI":"10.1016\/j.jhep.2023.02.041","article-title":"Hepatitis D double reflex testing of all hepatitis B carriers in low-HBV- and high-HBV\/HDV-prevalence countries","volume":"79","author":"Razavi","year":"2023","journal-title":"J. Hepatol."},{"key":"ref29","doi-asserted-by":"publisher","first-page":"1200","DOI":"10.1016\/j.jhep.2021.01.014","article-title":"The changing context of hepatitis D","volume":"74","author":"Rizzetto","year":"2021","journal-title":"J. Hepatol."},{"key":"ref30","doi-asserted-by":"publisher","first-page":"2126","DOI":"10.1128\/JCM.06829-11","article-title":"Standardized one-step real-time reverse transcription-PCR assay for universal detection and quantification of hepatitis delta virus from clinical samples in the presence of a heterologous internal-control RNA","volume":"50","author":"Scholtes","year":"2012","journal-title":"J. Clin. Microbiol."},{"key":"ref31","doi-asserted-by":"publisher","first-page":"55","DOI":"10.1016\/j.jviromet.2012.05.008","article-title":"Development and validation of an efficient in-house real-time reverse transcription polymerase chain reaction assay for the quantitative detection of serum hepatitis delta virus RNA in a diverse South London population","volume":"184","author":"Shang","year":"2012","journal-title":"J. Virol. Methods"},{"key":"ref32","doi-asserted-by":"publisher","first-page":"104932","DOI":"10.1016\/j.jcv.2021.104932","article-title":"Reliable quantification of plasma HDV RNA is of paramount importance for treatment monitoring: a European multicenter study","volume":"142","author":"Stelzl","year":"2021","journal-title":"J. Clin. Virol."},{"key":"ref33","doi-asserted-by":"publisher","first-page":"523","DOI":"10.1016\/j.jhep.2020.04.008","article-title":"The global prevalence of hepatitis D virus infection: systematic review and meta-analysis","volume":"73","author":"Stockdale","year":"2020","journal-title":"J. Hepatol."},{"key":"ref34","doi-asserted-by":"publisher","first-page":"947","DOI":"10.1016\/j.jhep.2022.04.014","article-title":"Feasibility of hepatitis B elimination in high-income countries with ongoing immigration","volume":"77","author":"Tian","year":"2022","journal-title":"J. Hepatol."},{"key":"ref35","doi-asserted-by":"publisher","first-page":"937","DOI":"10.1016\/j.cld.2023.05.008","article-title":"State of the art: test all for anti-hepatitis D virus and reflex to hepatitis D virus RNA polymerase chain reaction quantification","volume":"27","author":"Umukoro","year":"2023","journal-title":"Clin. Liver Dis."},{"key":"ref36","doi-asserted-by":"publisher","DOI":"10.1097\/HEP.0000000000000584","article-title":"HDV RNA assays: performance characteristics, clinical utility, and challenges","author":"Wedemeyer","year":"2023","journal-title":"Hepatology"},{"key":"ref37","doi-asserted-by":"publisher","first-page":"324","DOI":"10.1111\/jvh.13939","article-title":"An updated assessment of hepatitis delta prevalence among adults in Canada: a meta-analysis","volume":"31","author":"Wong","year":"2024","journal-title":"J. Viral Hepat."},{"key":"ref38","volume-title":"Global health sector strategy on viral hepatitis 2016-2021. Towards ending viral hepatitis","year":"2016"},{"key":"ref39","doi-asserted-by":"publisher","first-page":"2442","DOI":"10.1111\/liv.16006","article-title":"Long-term outcome of hepatitis delta in different regions world-wide: results of the hepatitis delta international network","volume":"44","author":"Wranke","year":"2024","journal-title":"Liver Int."},{"key":"ref40","doi-asserted-by":"publisher","first-page":"1551","DOI":"10.1002\/hep.32259","article-title":"A phase 2 dose-finding study of lonafarnib and ritonavir with or without interferon alpha for chronic delta hepatitis","volume":"75","author":"Yurdaydin","year":"2022","journal-title":"Hepatology"}],"container-title":["Frontiers in Microbiology"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fmicb.2024.1472826\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2024,11,20]],"date-time":"2024-11-20T12:17:06Z","timestamp":1732105026000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fmicb.2024.1472826\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,11,20]]},"references-count":40,"alternative-id":["10.3389\/fmicb.2024.1472826"],"URL":"https:\/\/doi.org\/10.3389\/fmicb.2024.1472826","relation":{},"ISSN":["1664-302X"],"issn-type":[{"value":"1664-302X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2024,11,20]]},"article-number":"1472826"}}