Development of a reverse transcription quantitative real-time PCR-based system for rapid detection and quantitation of hepatitis delta virus in the western Amazon region of Brazil

Abstract

The hepatitis delta virus (HDV) is a pathogen that causes a severe and rapidly progressive disease of hepatocytes. The measurement of viral load in the peripheral blood of patients with HDV infections is important for diagnosis, treatment monitoring, and support for follow-up studies of viral replication during the course of the disease. This study reports the development of an assay capable of detecting and quantifying the abundance of HDV particles in serum samples, based on reverse-transcription quantitative PCR (RT-qPCR). Two standards for calibration were produced for determining the viral load of HDV: a cDNA cloned into a linear plasmid and a transcribed RNA. For validating this assay, 140 clinical samples of sera were used, comprising 100 samples from patients who tested positive for anti-HDV and hepatitis B virus surface antigen (HBsAg) by ELISA; 30 samples from blood donors; 5 samples monoinfected with hepatitis B virus (HBV); and 5 samples monoinfected with hepatitis C virus (HCV). The HDV RT-qPCR assay performed better when calibrated using the standard based on HDV cDNA cloned into a linear plasmid, yielding an efficiency of 99.8% and a specificity of 100% in the in vitro assays. This study represents the first HDV RT-qPCR assay developed with clinical samples from Brazil and offers great potential for new clinical efficacy studies of antiviral therapeutics for use in patients with hepatitis delta in the western Amazon region.

First international external quality assessment for hepatitis delta virus RNA quantification in plasma

“…Very interestingly, L27, which failed to quantify 10 of the 18 positive samples, used a forward primer exhibiting only one mismatch, and, inversely, lab 2, which detected all samples correctly, used forward primer exhibiting three mismatches. Similarly, S19 (HDV-1Afr), which was not detected by eight laboratories (labs 4,5,9,13,15,20,26,and 27;Figs. 4 and 5), presented one to six mismatches within the corresponding forward primer and probe sequences.…”

“…It is based on TaqMan real-time reverse-transcriptase polymerase chain reaction (RT-PCR) technology and capable of quantifying RNA load for most, if not all, known strains of HDV. (19) Several other in-house and commercial assays have been developed in specialized laboratories using realtime RT-PCR technologies, (19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29) but they performed poorly compared to the FNRL-HDV assay in recent studies. (30,31) The high genetic variability of HDV is a key factor in the discrepancies observed for these tests, which, furthermore, suffer from a lack of standardization.…”

Characterization of the Genotypic Profile of Hepatitis Delta Virus: Isolation of HDV Genotype-1 in the Western Amazon Region of Brazil

“…Serum samples from 54 patients with HBV/HDV infection (with detectable HDV RNA and HB s Ag) [40] were obtained from the Research Center for Tropical Medicine of Rondônia - CEPEM. A control group of 30 donors who were negative for HIV-1, HIV-2, HB s Ag, anti-HBc, and anti-HCV (hepatitis C virus) was used.…”

Treatment of hepatitis delta virus genotype 3 infection with peg-interferon and entecavir

“…26 To monitor HDV, the quantification method described by Botelho-Souza et al was used (sensitivity of 75 copies/ml of viral RNA). 27 Some of the samples were sent at random to the Genome Center in São Paulo for external control and to validate the results.…”