Uncertainty regarding etiology impedes implementation of treatment and prevention measures. We sought to determine the disease etiology in patients with acute nonAnonE hepatitis in Tunisia, the UK and Vietnam. MATERIALS & METHODS: A total of 119 serum specimens from cases of nonA-E hepatitis were studied. All specimens had previously tested negative by serological and NATbased assays for hepatitis viruses A, B, C and E, EBV, CMV, HSV and HPV. Sera from 60 healthy US blood donors were used as controls. Sequence-independent reverse transcription by modified SMART technology (Clontech), followed by amplification was used to insure that both DNA and RNA entities were retained. The amplified cDNAs were sheared, converted into individual random libraries and tested by Next generation sequencing (NGS) on Illumina. The serum metagenome of patients from cases with expressed symptoms of viral hepatitis and specimens from the healthy blood donors were analyzed after excluding human genetic information in silico. Individual metagenome testing was possible for 22 of the symptomatic cases and 28 of the control normal human sera. RESULTS: HBV (genotypes A, D and G) was identified in seven patients (30.4%) individually tested by NGS; HEV genotype 3 was identified in three patients and HCV genotype 1b was identified in four patients (21.7%). The limited amount of study serum prevented sufficient cDNA yield from specimens for the remainder of the patients. We were able to assemble and characterize a complete HBV genome in one case and large parts of the HCV genome in others. No evidence of hepatitis viruses was detected in any of the serum specimens from the healthy US blood donor controls. CONCLUSIONS: Sequence independent amplification and NGS enabled the identification of known hepatitis viruses in 50% of individually tested presumed nonA-E cases; hence the disease etiology could be ascribed to those agents. It is concluded that conventional serological and NAT-based assays may not always detect cryptic infections by known agents of viral hepatitis.
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