title: Development and assessment of a novel real-time PCR assay for quantitation of hepatitis D virus RNA to study viral kinetics in chronic hepatitis D.
abstract: Hepatitis delta virus (HDV) infection is a usually severe type of viral hepatitis associated with increased mortality and rapid evolution to cirrhosis. Currently, treatment is limited to extended interferon administration and measurement of HDV RNA blood levels is essential to judge the response. The aim of this study was to develop a highly sensitive and reproducible real-time reverse transcriptase-polymerase chain reaction (real-time RT-PCR) for the quantitation of circulating HDV RNA of all clades (1-8), and assess its usefulness in the follow-up of patients. The amplification was combined with molecular beacon technology using the LightCycler 2.0 system. The assay was specific and showed linearity over a wide range from 13 to 13 × 10(10) copies/mL. The 95% detection limit was 43.2 copies/mL. Intra-assay reproducibility, as expressed by the coefficient of variation, ranged from 1.84 to 18.61%, whereas the corresponding estimates for the inter-assay variability ranged from 0.57 to 10.18%. Finally, the dynamic profiles of six patients regarding virological (HDV RNA, HBV DNA), biochemical and serological data were constructed. We were able to observe that most patients who were treated with an interferon-based regime showed a significant reduction in delta viremia. In conclusion, our real-time RT-PCR for HDV RNA quantification combines high sensitivity and reproducibility in a high dynamic range, can provide important information for patient management and can be a useful tool for monitoring the response to antiviral therapies.
authors.affiliation: National Retrovirus Reference Center, Department of Hygiene, Epidemiology and Medical Statistics, Athens University Medical School, Athens, Greece. adakat@med.uoa.gr
authors.forename: A
authors.initials: A
authors.lastname: Katsoulidou
authors.forename: E
authors.initials: E
authors.lastname: Manesis
authors.forename: C
authors.initials: C
authors.lastname: Rokka
authors.forename: C
authors.initials: C
authors.lastname: Issaris
authors.forename: A
authors.initials: A
authors.lastname: Pagoni
authors.forename: V
authors.initials: V
authors.lastname: Sypsa
authors.forename: A
authors.initials: A
authors.lastname: Hatzakis
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D016903:Drug Monitoring / Q000379:methods; D005260:Female; D019701:Hepatitis D, Chronic / Q000821:virology*; D003698:Hepatitis Delta Virus / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D008297:Male; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D019562:Viral Load / Q000379:methods*; D055815:Young Adult
chemical_list: D012367:RNA, Viral