Hepatitis D (delta) virus genotyping: Relationship with the geographical origin and the clinical course of the disease - Antigen; Total and IgM antibodies; Molecular diagnosis (RT-PCR); Genotyping HDV-1 to HDV-8 (RT-PCR and sequencing)

 

Information 20-01-2018.

 

The hepatitis delta virus (HDV) is responsible for the most severe form of acute and chronic viral hepatitis. Infection with HDV is one of the important etiologies of fulminant hepatitis and may aggravate the clinical course of chronic infection with hepatitis B virus (HBV), leading to cirrhosis and liver failure.

The HDV is unique in animal virology, constituting the only representative of the family Deltaviridae, genus Deltavirus. It is a small defective RNA virus and an obligate satellite of HBV, which requires surface antigens (HBsAg) for its own assembly, propagation and transmission of infectious virions. In addition, this virus parasitizes the transcriptional machinery of the host, sequestering cellular RNA polymerases to replicate its RNA genome, which is replicated by a rolling circle mechanism with the help of the ribozymes contained in the RNA itself. The HDV virion is 36 nm in diameter and is composed of an outer envelope with the HBV surface glycoproteins (HBsAg) and host lipids, an internal nucleocapsid formed by hepatitis delta antigen (HDAg) molecules, and a chain of single-stranded circular RNA of negative polarity. Its genome is approximately 1.7 kb in length, constituting the smallest genome of the known human RNA viruses. The anti-genome of HDV contains a single open reading frame that encodes the two isoforms of hepatitis delta antigen, the small antigen (S-HDAg), responsible for the start of replication, and the large one (L-HDAg), required for viral assembly.

Like hepatitis B, hepatitis D is transmitted parenterally, by contact with blood or other body fluids of infected people. Infection with HDV can occur as a coinfection, simultaneously with HBV, or as a superinfection, in a patient who was already a chronic carrier of HBV. The clinical evolution of these two infections is different. While patients with HBV/HDV co-infection usually evolve to cure, superinfection is associated with a more severe form of liver disease, since it almost always evolves into chronicity and, on occasion, can cause severe cases of liver failure or worsening of chronic hepatitis B that the patient had previously had, leading to a rapid progression to cirrhosis and increasing the risk of hepatocellular carcinoma.

Currently there is no effective antiviral treatment against hepatitis D. The virus does not have its own replicative function, which represents a major obstacle to being attacked by antiviral drugs. However, infection with HDV can be prevented by vaccination against hepatitis B. In the last 20 years, vaccination against HBV has decreased the circulation of HDV in industrialized countries; However, hepatitis D is returning to Western Europe through immigration from endemic areas. Worldwide, there are approximately 250 million HBV carriers, of whom between 15 and 20 million have serologic evidence of exposure to HDV. HDV is ubiquitous and is present in six of the seven continents. However, the prevalence of infection varies according to the geographical area. Traditionally, regions with high rates of endemicity are central and northern Africa, the Amazon basin, eastern Europe and the Mediterranean, the Middle East and parts of Asia.

HDV genotypes: geographical distribution and clinical importance

Although clinical studies in all continents have shown that infection with HDV aggravates the clinical course of the underlying infection with HBV, in Greece, Samoa and the Far East, infection with HDV has also been associated with benign clinical conditions or with normal hepatic function. These data suggest that the expression of the disease may vary, probably due to infection with the different genotypes of HDV.

The genome of HDV is characterized by its variability, which reaches up to 40% of divergence in the complete RNA sequence. This diversity can be generated by the selection of viral variants under the immunological pressure of the host during chronic infection and/or by possible events in the editing of new genomes that can occur in different sites. In addition, it has been suggested that the intragenotypic or intergenotypic recombination events, as well as the low efficiency of correction of the DNA polymerase II of the host that amplifies the HDV RNA, may contribute to this variability.

The genetic diversity of the HDV makes that it is currently classified in eight phylogenetically distinct genotypes that have different geographical distributions: HDV-1, HDV-2, HDV-3, HDV-4, HDV-5, HDV-6, HDV-7, and HDV-8. With the exception of HDV-1, which is geographically widespread, the other genotypes are closely related to specific geographic areas. HDV-1 is ubiquitous, it is mainly isolated from the US, Europe and the Middle East, but it has also been isolated in Russia, Africa, Asia and Brazil. The genotypes HDV-2 and HDV-4 are found in Japan and Taiwan, and HDV-2 is also found in Russia. The HDV-3 is found exclusively in the North of South America, being common in the Amazon region. Finally, genotypes HDV-5, HDV-6, HDV-7 and HDV-8 have been isolated in Africa. Recently, the genotype HDV-8 has also been isolated in Brazil.

In addition to its linkage with certain geographical areas, as already mentioned above, the different genotypes seem to be associated with different clinical courses. Thus, the genotype HDV-1 has been associated with a broad spectrum of pathogenicity, while the genotypes HDV-2 and HDV-4 are considered to cause milder forms of liver disease. In contrast, HDV-3 is the most divergent genotype and apparently related to the more aggressive nature of delta hepatitis, being responsible for outbreaks of severe fulminant hepatitis. At present, the association of genotypes of HDV-5 to -8 with the severity of liver disease is unknown.

Tests carried out in IVAMI:

  • Molecular detection of HDV RNA by RT-PCR.
  • Genotyping of HDV by amplification (RT-PCR) and subsequent sequencing of the coding region of the major antigen L-HDAg.

Recommended sample:

  • Plasma or serum (1 mL) separated from blood extracted with EDTA.
  • Total blood extracted with EDTA (5 mL).

Conservation and shipment of the sample:

  • Refrigerated (preferred) for less than 2 days, or frozen if the storage period is longer.
  • In the case of sending whole blood extracted with EDTA, the sample must be kept refrigerated, not frozen, and arrive at our laboratory in a period not exceeding 24 hours.

Delivery of results:

  • Molecular detection of HDV RNA (RT-PCR): 24 hours.
  • Genotyping of HDV by amplification (RT-PCR) and subsequent sequencing of the coding region of the major antigen L-HDAg: 48 to 72 hours

Cost of the test:

  • Genotyping of HDV by amplification (RT-PCR) and subsequent sequencing of the coding region of the major antigen L-HDAg: Consult ivami@ivami.com