Establishment of a novel quantitative hepatitis D virus (HDV) RNA assay using the Cobas TaqMan platform to study HDV RNA kinetics.
Determination of hepatitis D virus (HDV) viremia represents the "gold standard" for the diagnosis of HDV infection. Hepatitis B virus (HBV)-HDV coinfection frequently leads to end-stage liver disease and hepatocellular carcinoma. No commercial assay for HDV RNA quantification that includes automated nucleic acid extraction is available, and in-house PCR tests are not well standardized. However, knowledge of HDV RNA levels may give important information for patient management and could be a useful tool for monitoring the response to antiviral therapies. One platform that is widely used for HBV DNA or HCV RNA quantification is the Cobas Ampliprep/TaqMan system. Using the utility channel of this platform, we established a novel protocol for TaqMan-based HDV RNA quantification after automatic extraction of RNA by the Ampliprep system. The assay was specific and showed linearity over a wide range from 3 x 10(2) to 10(7) copies/ml. Reproducibility was demonstrated by determination of the interrun and intrarun variabilities, which were similar to those achieved with the commercially available Cobas TaqMan assays for HCV RNA and HBV DNA. HDV RNA levels were stable in whole blood (n = 4), plasma (n = 3), and serum (n = 3) samples at room temperature for up to 6 days. Importantly, HDV RNA viremia showed only minor fluctuations, with the log(10) coefficient of variation being between 1.3 and 11.2% for hepatitis delta patients studied every 2 weeks for up to 3 months (n = 6), while a rapid viral decline was observed early during treatment with pegylated alfa-2a interferon (n = 6). In conclusion, this novel automated HDV RNA assay is a useful tool for monitoring HDV-infected patients both before and during antiviral therapy.
Mederacke
Ingmar
I
Department of Gastroenterology, Hepatology, and Endocrinology, Hannover Medical School, Carl-Neuberg-Str. 1, Hannover D-30625, Germany.
Bremer
Birgit
B
Heidrich
Benjamin
B
Kirschner
Janina
J
Deterding
Katja
K
Bock
Thomas
T
Wursthorn
Karsten
K
Manns
Michael P
MP
Wedemeyer
Heiner
H
D001331:Automation / Q000379:methods; D001769:Blood / Q000821:virology; D003699:Hepatitis D / Q000175:diagnosis* / Q000821:virology; D003698:Hepatitis Delta Virus / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D012367:RNA, Viral / Q000097:blood*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013048:Specimen Handling / Q000379:methods; D019562:Viral Load / Q000379:methods*; D014766:Viremia / Q000175:diagnosis / Q000821:virology
D012367:RNA, Viral