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J Med Virol. 2014 Aug 27. doi: 10.1002/jmv.24057. [Epub ahead of print]

Mutations associated with occult hepatitis B in HIV-positive South Africans.

Powell EA1, Gededzha MP, Rentz M, Rakgole NJ, Selabe SG, Seleise TA, Mphahlele MJ, Blackard JT.

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Abstract

Occult hepatitis B is characterized by the absence of hepatitis B surface antigen (HBsAg) but the presence of HBV DNA. Because diagnosis of hepatitis B virus (HBV) typically includes HBsAg detection, occult HBV remains largely undiagnosed. Occult HBV is associated with increased risk of hepatocellular carcinoma, reactivation to chronic HBV during immune suppression, and transmission during blood transfusion and liver transplant. The mechanisms leading to occult HBV infection are unclear, although viral mutations are likely a significant factor. In this study, sera from 394 HIV-positive South Africans were tested for HBV DNA and HBsAg. For patients with detectable HBV DNA, the overlapping surface and polymerase open reading frames (ORFs) were sequenced. Occult-associated mutations-those mutations found exclusively in individuals with occult HBV infection but not in individuals with chronic HBV infection from the same cohort or GenBank references-were identified. Ninety patients (22.8%) had detectable HBV DNA. Of these, 37 had detectable HBsAg, while 53 lacked detectable surface antigen. The surface and polymerase ORFs were cloned successfully for 19 patients with chronic HBV and 30 patients with occult HBV. In total, 235 occult-associated mutations were identified. Ten occult-associated mutations were identified in more than one patient. Additionally, 15 amino acid positions had two distinct occult-associated mutations at the same residue. Occult-associated mutations were common and present in all regions of the surface and polymerase ORFs. Further study is underway to determine the effects of these mutations on viral replication and surface antigen expression in vitro. J. Med. Virol. 2014. © 2014 Wiley Periodicals, Inc.

© 2014 Wiley Periodicals, Inc.

Hepat Mon. 2014 May 3;14(5):e15275. doi: 10.5812/hepatmon.15275. eCollection 2014.

Potential mutations associated with occult hepatitis B virus status.

Besharat S1, Katoonizadeh A2, Moradi A3.

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Abstract

CONTEXT:

Occult hepatitis B virus (HBV) status (OHBS) is simply defined as the presence of HBV DNA in the liver (with or without detectable HBV DNA in the serum), in the absence of serum HBV surface antigen (HBsAg). Importance of OHBS is mostly clinical, related to its possible role in spreading through blood transfusion and liver transplantation; causing classic forms of HBV. Mechanisms underlying this entity are poorly defined. Several possibilities have been suggested, with major classification into two groups: defective host immune response and viral replication activity through mutations of HBV DNA sequence. Mutations are extensively investigated in all four overlapping open reading frames (ORFs) of HBV genome, to define their possible role in the pathogenesis of OHBS. Some of these mutations like S-escape mutants could not be detected by the routine available assays, making them difficult to diagnosis. Therefore, trying to detect this covert condition could be more helpful for defining better preventive and therapeutic strategies.

EVIDENCE ACQUISITION:

In the present study we provided an in-depth review of the most important new data available on different mutations in HBV genome of patients with OHBS, which may play a role in the pathogenesis of OHBS. The data were collected through reviewing the full-text articles, identified by the PubMed search, using the following keywords and their different combinations: occult hepatitis B, HBV genome, "a" determinant, HBV open reading frames, S mutations, X mutations, P mutations and C mutations.

RESULTS:

Variants within the major hydrophilic region (MHR) of the HBsAg, deletions in the pre-S1region, codon stop in the S open reading frames (ORF), sporadic non common mutations, some mutations affecting the posttranslational production of HBV proteins in the S ORF like deletion mutations, mutations in start codon and nucleotide changes in the X ORF, deletion and point mutations in P ORF and sometimes, nucleotide substitution in the C ORF are among the assumed mutations detected to have a role in OHBS appearance.

CONCLUSIONS:

Studies mostly lacked a control group and the whole-length HBV sequencing was scant with conflicting results, suggesting that OHBS is often a result of multiple mechanisms. Additional studies on full-length HBV genomes from occult and non-occult HBV cases may shed more light on the interplay between different mechanisms involved in the pathogenesis of OHBS.

PLoS One. 2013;8(1):e54486. doi: 10.1371/journal.pone.0054486. Epub 2013 Jan 18.

Naturally occurring mutations in large surface genes related to occult infection of hepatitis B virus genotype C.

Kim H1, Lee SA, Kim DW, Lee SH, Kim BJ.

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Abstract

Molecular mechanisms related to occult hepatitis B virus (HBV) infection, particularly those based on genotype C infection, have rarely been determined thus far in the ongoing efforts to determine infection mechanisms. Therefore, we aim to elucidate the mutation patterns in the surface open reading frame (S ORF) underlying occult infections of HBV genotype C in the present study. Nested PCRs were applied to 624 HBV surface antigen (HBsAg) negative Korean subjects. Cloning and sequencing of the S ORF gene was applied to 41 occult cases and 40 control chronic carriers. Forty-one (6.6%) of the 624 Korean adults with HBsAg-negative serostatus were found to be positive for DNA according to nested PCR tests. Mutation frequencies in the three regions labeled here as preS1, preS2, and S were significantly higher in the occult subjects compared to the carriers in all cases. A total of two types of deletions, preS1 deletions in the start codon and preS2 deletions as well as nine types of point mutations were significantly implicated in the occult infection cases. Mutations within the "a" determinant region in HBsAg were found more frequently in the occult subjects than in the carriers. Mutations leading to premature termination of S ORF were found in 16 occult subjects (39.0%) but only in one subject from among the carriers (2.5%). In conclusion, our data suggest that preS deletions, the premature termination of S ORF, and "a" determinant mutations are associated with occult infections of HBV genotype C among a HBsAg-negative population. The novel mutation patterns related to occult infection introduced in the present study can help to broaden our understanding of HBV occult infections.

Can J Gastroenterol Hepatol. 2014 Jun;28(6):315-8.

Screening, detecting and enhancing the yield of previously undiagnosed hepatitis B and C in patients with acute medical admissions to hospital: a pilot project undertaken at the Vancouver General Hospital.

Kapeluto J, Kadatz M, Wormsbecker A, Sidhu K, Yoshida EM.

Abstract

BACKGROUND:

Hepatitis B virus (HBV) and hepatitis C virus (HCV) represent an increasing health burden and morbidity in Canada. Viral hepatitis, specifically HCV, has high prevalence among persons born between 1945 and 1965, with 45% to 85% of infected adults asymptomatic and unaware of their infection. Screening has been shown to be cost effective in the detection and treatment of viral hepatitis.

OBJECTIVE:

To quantify incidence and identify undocumented HBV and HCV infection in hospitalized patients at a single centre with secondary analysis of risk factors as part of a quality improvement initiative.

METHODS:

A one-time antibody test was conducted in patients admitted to the acute medicine and gastroenterology services.

RESULTS:

Over a 12-week period, hospital screening for HBV and HCV was performed in 37.3% of 995 admitted patients. There was identification of 15 previously undiagnosed cases of HCV (4%) and 36 undocumented cases of occult (ie, antihepatitis B core antigen seropositive) or active (ie, hepatitis B surface antigen seropositive) HBV (9.7%). Among patients with positive screens, 60% of seropositive HCV patients had no identifiable risk factors.

CONCLUSIONS:

The prevalence of HBV and HCV infection among hospitalized patients in Vancouver was higher than that of the general population. Risk factors for contraction are often not identified. These results can be used as part of an ongoing discussion regarding a 'seek and treat' approach to the detection and treatment of chronic blood-borne viral illnesses.

Can J Gastroenterol Hepatol. 2014 Jun;28(6):315-8.

Screening, detecting and enhancing the yield of previously undiagnosed hepatitis B and C in patients with acute medical admissions to hospital: a pilot project undertaken at the Vancouver General Hospital.

Kapeluto J, Kadatz M, Wormsbecker A, Sidhu K, Yoshida EM.

Abstract

BACKGROUND:

Hepatitis B virus (HBV) and hepatitis C virus (HCV) represent an increasing health burden and morbidity in Canada. Viral hepatitis, specifically HCV, has high prevalence among persons born between 1945 and 1965, with 45% to 85% of infected adults asymptomatic and unaware of their infection. Screening has been shown to be cost effective in the detection and treatment of viral hepatitis.

OBJECTIVE:

To quantify incidence and identify undocumented HBV and HCV infection in hospitalized patients at a single centre with secondary analysis of risk factors as part of a quality improvement initiative.

METHODS:

A one-time antibody test was conducted in patients admitted to the acute medicine and gastroenterology services.

RESULTS:

Over a 12-week period, hospital screening for HBV and HCV was performed in 37.3% of 995 admitted patients. There was identification of 15 previously undiagnosed cases of HCV (4%) and 36 undocumented cases of occult (ie, antihepatitis B core antigen seropositive) or active (ie, hepatitis B surface antigen seropositive) HBV (9.7%). Among patients with positive screens, 60% of seropositive HCV patients had no identifiable risk factors.

CONCLUSIONS:

The prevalence of HBV and HCV infection among hospitalized patients in Vancouver was higher than that of the general population. Risk factors for contraction are often not identified. These results can be used as part of an ongoing discussion regarding a 'seek and treat' approach to the detection and treatment of chronic blood-borne viral illnesses.

Virol J. 2014 Jan 28;11:16. doi: 10.1186/1743-422X-11-16.

Development of cost-effective real-time PCR test: to detect a wide range of HBV DNA concentrations in the western Amazon region of Brazil.

dos Santos Ade O1, Souza LF, Borzacov LM, Villalobos-Salcedo JM, Vieira DS.

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Abstract

BACKGROUND:

Currently there is a significant risk of infection with hepatitis B virus (HBV) during blood transfusion in high epidemic area. This is due to the pre-seroconversion window period, immunovariant viral strains and the presence of occult HBV infection (OBI). The aim of this study was to develop an in-house real-time PCR-based method, which was both ultra-sensitive and efficient offering an alternative method for nucleic acid testing (NAT).

METHODS:

A precore fragment with 109 bp was cloned and serial diluted to standard curve construction. The calibration of the HBV-DNA values was performed against OptiQuant® HBV-DNA Quantification Panel, Acrometrix Europe B.V.).

RESULTS:

From our in-house plasmid we prepared serial dilutions ranging from 2 × 10³-2 × 10⁹ copies/ml. The threshold was adjusted automatically during analysis and the data collected were analyzed by linear regression (r² = 0.99). The limit of detection for the assay with pHBVRO standards was 2000/ml in a total reaction volume of 30 μl. We found a strong correlation between the two methods (r² = 0.9965 and p 

The Best way to detect HBV is the detection of HBV DNA by PCR and atleast three regions. In the HBsAg negative cases antiHBc antibody should be confirmed first.

I agree with Dr. Rajesh Panigrahi

First antiHBc antibody positive then HBV DNA by PCR detected in sera; also in liver including the countries where it is possible to do it.

Thank you all.

The occult state of HBV infection could arise due to some mutations in the viral genome (Envelope gene promoter or 'a' determinant of the envelope gene (V. Chaudhuri et al., 2004). Further, no definitive correlation has been found between Occult HBV infection and core antigen levels. Therefore, the most definitive test for Occult infection would be viral DNA sequencing and its analysis.

The gold standard method to detect HBV-DNA in HBsAg negative individuals is amplification by PCR of at least 2-3 different regions around the HBV genome  to distinguish between  the presence of viral particles and defective viruses, possibly in liver tissue. If liver tissue is not available, HBV DNA should be searched for in sequential serum samples , since it is well known  the alternance of HBV-DNA detectability and undetectability during occult HBV infection. So, I suggest to explore such an infection in different serum plasma from the same patient, especially in immune compromised individuals.