Commercial Real-Time Reverse Transcriptase PCR Assays Can Underestimate or Fail to Quantify Hepatitis Delta Virus Viremia

Abstract

Hepatitis delta virus (HDV) infection causes fulminant hepatitis and increases the severity of chronic hepatitis B virus infection, leading to cirrhosis, liver failure, or hepatocellular carcinoma. There are 8 HDV genotypes (genotypes 1-8). We previously developed a TaqMan real-time reverse transcriptase (RT)-PCR method that is able to quantify viral load of all HDV genotypes (linear from 2 to 8 log(10) copies/mL). We compared its results with those from 3 commercial real-time RT-PCR assays: the Lightmix HDV kit (designed to quantify HDV genotype 1 [HDV-1]), and the RoboGene and the DiaPro HDV RNA quantification kits (designed to quantify all genotypes). We selected RNA from 128 clinical samples of all HDV genotypes except HDV-4, with various HDV viral load values. We also analyzed 5 samples, collected over time, from each of 6 patients infected with strains of different genotypes. Quantification results from the commercial kits for HDV-1 from European or Asian samples were consistent with those from our method, however, they underestimated (0.5-1 log(10) with Lightmix and DiaPro) and did not detect (1 and 4 samples with Lightmix and DiaPro, respectively) HDV-1 African samples. Moreover, the commercial kits greatly underestimated HDV viral load of almost all non-genotype-1 strains (about 2-3 log(10)), and even did not detect HDV-7 or HDV-8 RNA in several samples with high concentrations of virus. Commercial kits accurately quantify HDV-1 in samples from European and Asian patients. However, they can dramatically underestimate or fail to quantify HDV viral load from samples from African patients infected with strains of genotypes 1 and 5 to 8.