New Subgenotyping and Consensus Real-Time Reverse Transcription-PCR Assays for Hepatitis A Outbreak Surveillance

Abstract

Laboratory surveillance plays an important role in the detection and control of hepatitis A outbreaks and requires the application of rapid and accurate molecular diagnostic tools for hepatitis A virus (HAV) RNA detection, subgenotype identification, and sequence-based genotyping. We describe the development and validation of a triplex real-time, reverse transcription-PCR (triplex rRT-PCR) assay for the identification and discrimination of HAV subgenotypes IA, IB, and IIIA and a singleplex rRT-PCR assay designed to detect all HAV genotypes infecting humans. Overall, the accuracy, sensitivity, and specificity of the new assays were >97% for serum and plasma specimens collected during unrelated outbreaks of HAV in California and Michigan compared to a nested RT-PCR genotyping assay and the ISO 15216-1 rRT-PCR method for HAV detection. The new assays will permit the rapid detection of HAV RNA and discrimination among subgenotypes IA, IB, and IIIA in serum and plasma specimens, which will strengthen public health surveillance efforts for HAV outbreak detection and response.

Keywords: genotyping; hepatitis A virus; outbreak; public health; reverse transcription-PCR.

FIG 1

Phylogenetic relationship of 50 unique HAV VP1-P2B sequences detected in serum and plasma specimens. HAV reference sequences are indicated by GenBank accession numbers. Sequence alignments were performed with ClustalW, and the dendrogram was generated using the neighbor-joining algorithm and the maximum composite likelihood model. Evolutionary distance is indicated by the scale at the bottom of the dendrogram. Evolutionary branches corresponding to genotype lineage are labeled. The Michigan and California HAV outbreak strains are indicated with the brackets to the right of the dendrogram.