Next-generation Sequencing to Diagnose Cryptic Hep B and E
By Dean L. Winslow, MD, FACP, FIDSA
Dr. Winslow is Professor of Medicine, Division of General Medical Disciplines, Division of Infectious Diseases and Geographic Medicine, Stanford University School of Medicine.
Dr. Winslow reports no financial relationships relevant to this field of study.
SYNOPSIS: Sera from patients with hepatitis of unknown etiology (HUE) were studied using next-generation sequencing (NGS). Sequences from hepatitis B virus were detected in 7 patients and hepatitis E virus in 2 patients.
SOURCE: Ganova-Raeva L, et al. Cryptic hepatitis B and E in patients with hepatitis of unknown etiology. J Infect Dis 2015;212:1962-1969.
Serum specimens were collected from 32 patients from both the United Kingdom and Vietnam. None of the patients had received hepatotoxic drugs and none had laboratory evidence of autoimmune hepatitis. All patients’ sera tested negative by serologic tests for hepatitis A, B, C, E, CMV, and EBV, as well as by polymerase chain reaction (PCR) for HBV DNA, HCV RNA, and HEV RNA. No patients were immunocompromised or were infected with HIV. Twenty-six U.S. blood donors were used as controls.
After total nucleic acid extraction, the samples were reverse transcribed using random hexamer primers, then the cDNA product was amplified, purified, and finally used to generate shotgun libraries for sequencing.
Extraction of total nucleic acid from 12 patients with acute hepatitis and 26 controls yielded a sufficient amount of DNA for sequencing. Total nucleic acids from 26 acute hepatitis patient sera with insufficient nucleic acid quantity for sequencing were pooled and then used for NGS.
Sequences from HBV were detected in seven individuals with HUE and from the pooled library. HEV was detected in two individuals with HUE and from the pooled library. Both HEV patients were co-infected with HBV. HBV sequences were from genotypes A, D, and G. The HEV sequences were genotype 3. No known hepatitis viruses were detected in the tested normal sera.
COMMENTARY
Up to 30% of cases of presumed acute viral hepatitis have no known etiology. This study shows that NGS-based detection of HBV and HEV is more sensitive than commercial antibody and molecular assays, and that cryptic infection with these viruses is likely responsible for a high proportion of HUE. This still leaves about 40% of cases of HUE in this study in which no viral etiology was determined, suggesting that either HBV or HEV was present at levels of viremia below the threshold of detection using NGS or that yet-undiscovered pathogens were responsible.
NGS is emerging as a powerful tool to identify rare or unexpected pathogens. Fremond et al recently applied NGS to identify an astrovirus as the cause of unexplained progressive encephalitis in a child with X-linked agammaglobulinemia,1 and Wilson et al used NGS to diagnose neuroleptospirosis in a child with severe combined immunodeficiency.2
REFERENCES
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Fremond M-L, et al. Next-generation sequencing for diagnosis and tailored therapy: A case report of astrovirus-associated progressive encephalitis.
J Ped Infect Dis Soc 2015;4:e53-e57. - Wilson MR, et al. Actionable diagnosis of neuroleptospirosis by next-generation sequencing. N Engl J Med 2014;370:2408-2417.
ABSTRACT & COMMENTARY: Sera from patients with hepatitis of unknown etiology were studied using next-generation sequencing.
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