Birgit Bremer1, Olympia E Anastasiou2,3, Sandra Ciesek3, Heiner Wedemeyer1,2. 1. Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany. 2. Department of Gastroenterology and Hepatology, University Hospital of Essen, Essen, Germany. 3. Institute of Virology, University Hospital of Essen, University of Duisburg-Essen, Essen, Germany.
Abstract
BACKGROUND: HDV infection is a cause of severe liver disease. Diagnosis and monitoring of HDV RNA are important to patient management. Since 2012, a WHO standard for HDV RNA quantification has been available; however, the impact of RNA extraction methods on HDV viral load quantification has never been evaluated. METHODS: The aim of this study was to compare four commonly used automated nucleic acid (NA) extraction methods (AmpliPrep, MagNA Pure, QIAcube QBK and QIAcube VRK) with a manual RNA extraction method (Instant Virus RNA/DNA kit) and evaluate the possible effect of each method on HDV RNA yield with subsequent amplification with the Robogene HDV assay. Serum samples from HDV-positive patients taken before treatment with pegylated interferon-α2a and at treatment weeks 12 and 48 were studied. RESULTS: The automated extraction methods MagNA Pure, Ampliprep and QIAcube VRK extraction led to about 10-fold lower HDV RNA values compared with the manual method of NA extraction, while the difference was smaller with QIAcube QBK (about 6-fold lower). The median viral load was 10,665 IU/ml for the manual method, 445 IU/ml for AmpliPrep, 3,209 IU/ml for MagNA Pure, 2,060 IU/ml for QIAcube QBK and 3,568 IU/ml for QIAcube VRK. Use of MagNA Pure led to misclassification of two on-treatment samples with low viral load as being false negative. CONCLUSIONS: The NA extraction method had a significant impact on the measured HDV viral loads determined by the commonly used Robogene assay, with the manual RNA method yielding consistently higher values of viral load. ClinicalTrials.gov Identifier: NCT00932971.
BACKGROUND:HDV infection is a cause of severe liver disease. Diagnosis and monitoring of HDV RNA are important to patient management. Since 2012, a WHO standard for HDV RNA quantification has been available; however, the impact of RNA extraction methods on HDV viral load quantification has never been evaluated. METHODS: The aim of this study was to compare four commonly used automated nucleic acid (NA) extraction methods (AmpliPrep, MagNA Pure, QIAcube QBK and QIAcube VRK) with a manual RNA extraction method (Instant Virus RNA/DNA kit) and evaluate the possible effect of each method on HDV RNA yield with subsequent amplification with the Robogene HDV assay. Serum samples from HDV-positive patients taken before treatment with pegylated interferon-α2a and at treatment weeks 12 and 48 were studied. RESULTS: The automated extraction methods MagNA Pure, Ampliprep and QIAcube VRK extraction led to about 10-fold lower HDV RNA values compared with the manual method of NA extraction, while the difference was smaller with QIAcube QBK (about 6-fold lower). The median viral load was 10,665 IU/ml for the manual method, 445 IU/ml for AmpliPrep, 3,209 IU/ml for MagNA Pure, 2,060 IU/ml for QIAcube QBK and 3,568 IU/ml for QIAcube VRK. Use of MagNA Pure led to misclassification of two on-treatment samples with low viral load as being false negative. CONCLUSIONS: The NA extraction method had a significant impact on the measured HDV viral loads determined by the commonly used Robogene assay, with the manual RNA method yielding consistently higher values of viral load. ClinicalTrials.gov Identifier: NCT00932971.