Infection by the hepatitis delta virus (HDV), a satellite of the hepatitis B virus (HBV), increases viral liver disease severity. Its diagnosis is thus vital for HBV-infected patients. HDV-RNA load (HDVL) should be assessed and monitored in plasma using real-time reverse-transcriptase polymerase chain reaction assays. Taking advantage of the recently-developed World Health Organization (WHO) HDV international standard (WHO-HDV-IS), the first international external quality control for HDVL quantification was performed. Two panels of samples were sent to 28 laboratories in 17 countries worldwide. Panel A comprised 20 clinical samples of various genotypes (1, 2, and 5-8) and viral loads, including two negative controls. Panel B, composed of dilutions of the WHO-HDV-IS, allowed the conversion of results from copies/mL into IU/mL for HDVL standardization and interlaboratory comparisons. Comprehensive analysis revealed a very high heterogeneity of assay characteristics, including their technical steps and technologies. Thirteen labs (46.3%) properly quantified all 18 positive samples; 16 (57.1%) failed to detect one to up to 10 samples, and several others underestimated (>3 log IU/mL) HDVL of African genotype strains (1 and 5-8). Discrepancies were mainly attributed to either primers or probe mismatches related to the high genetic variability of HDV and, possibly, to the complex secondary structure of the target genomic RNA. The labs were grouped in four clusters by the statistical analysis of their performances. The best clusters comprised the 17 labs that obtained the expected HDVL values, including five that otherwise failed to quantify one or two samples.
The results of this international quality-control study underline the urgent need to improve methods used to monitor HDV viremia and will be instrumental in achieving that goal. (Hepatology 2016;64:1483-1494).