A-304 A RT-PCR Assay for Detection and Quantification of Hepatitis D Virus (HDV)
Authors: Silva J D , Zauli D A 
Published: September 2023

Abstract

Background
Hepatitis D virus (HDV), also called Delta Hepatitis, is associated with the presence of the Hepatitis B virus (HBV) and is responsible for the infection and inflammation of liver cells. It is estimated that 15–20 million people worldwide are affected by HDV. In Brazil, Hepatitis D has high prevalence rates in the Amazon Basin. HDV is an enveloped virus with a 36 nm average diameter. The HDV virion involves a negative-sense, single-stranded circular RNA molecule associated with an inner-core delta antigen surrounded by envelope glycoproteins. HDV-infected individuals may not show signs or symptoms of the disease. When present, the most common are: tiredness, dizziness, nausea and/or vomiting, fever, abdominal pain, and yellow skin/eyes. Chronic Hepatitis D is considered the most severe form of chronic viral hepatitis, with more rapid progression to cirrhosis and an increased risk for decompensation, hepatocellular carcinoma (HCC), and death. Molecularly, determining the concentration of virus circulating in the blood of HDV patients is extremely important for diagnosis. The Quantitative Polymerase Chain Reaction (qPCR) is a molecular technique capable of detecting the viral genome with high sensitivity and specificity, ensuring the safety of the result. Therefore, this study aims to describe the analytical validation of a one-step RT-qPCR assay for HDV detection.

Methods
Assay performance was evaluated using commercial quantified positive control to HDV (1000, 100, 10, 1, and 0.5 IU/µL), and the parameters of analysis included: standardization assays, efficiency, and detection limit. For specificity evaluation, positive samples for Epstein-Barr Virus (EBV), Herpes Simplex Virus (HSV), Cytomegalovirus (CMV), Hepatitis B Virus (HBV), and Hepatitis C Virus (HCV) were used. RNA isolation was performed with the MagNA Pure 24 Total NA Isolation Kit at the automated platform MagNA Pure 24 System (Roche Diagnostics, Basel, Switzerland), according to the manufacturer's instructions. Altona RealStar HDV RT-PCR Kit 1.0 (Altona Diagnostics, Hamburg, Germany) was used to directly amplify RNA samples on the qPCR instrument, according to the manufacturer's instructions.

Results
The standardization assays indicated good qPCR amplification curves and high fluorescence levels between the controls evaluated. The assay demonstrated an average efficiency of 95%, with a coefficient of determination (r2) of 0.99. The detection limit of the test was 1 IU/µL for the HDV target (95% confidence interval). No cross-reactions with closely related viruses were observed (15 HDV-negative human plasma samples), indicating 100% specificity. The experiments performed to evaluate the precision demonstrated optimal repeatability and reproducibility.

Conclusion
The HDV diagnostic is required to ensure sensitive, specific and accurate identification of the viral agent and thus prompt surveillance actions such as epidemiological monitoring and control measures of infection. The RT-qPCR assay described here provides a reliable, highly specific, and sensitive method for the detection and quantification of HDV. Nevertheless, the assay needs to be evaluated in conjunction with a clinical and epidemiological context.