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Occult hepatitis B virus infection in HIV-infected Lebanese patients with isolated antibodies to hepatitis B core antigen
S Ramia, J Mokhbat, F Ramlawi and M El-Zaatari Int J STD AIDS 2008 19: 197 DOI: 10.1258/ijsa.2007.007200 The online version of this article can be found at: http://std.sagepub.com/content/19/3/197

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ORIGINAL RESEARCH ARTICLE

Occult hepatitis B virus infection in HIV-infected Lebanese patients with isolated antibodies to hepatitis B core antigen
S Ramia
PhD FRCPath*,

J Mokhbat

MD,

F Ramlawi

MSc*

and M El-Zaatari

MD*

*Department of Medical Laboratory Technology, Faculty of Health Sciences, American University of Beirut, Beirut; Department of Medicine, Faculty of Medicine, Lebanese University; Department of Laboratory Medicine, Hammoud Hospital, Saida, Lebanon

Summary: The presence of hepatitis B virus (HBV) serological markers have been investigated in 101 Lebanese patients (69 men, 32 women; mean age 32.7 + 1.7 years) infected with human immunodeciency virus type 1 (HIV-1). Seven patients (6.9%) were HBsAg carriers compared with 54 patients (53.5%) who had no evidence of exposure to HBV infection. Twenty-four patients (23.8%) had anti-HBc alone as a serological marker compared with four patients who were positive for anti-HBs alone and 12 patients (11.9%) who were anti-HBc and anti-HBs-positive. Occult HBV infection ( presence of HBV DNA in the absence of HBsAg) is found to be relatively high (28.7%) in HIV-infected Lebanese patients and the overwhelming majority (83.3%) of those who were positive for anti-HBc alone had a detectable HBV DNA in their serum. However, none of our HIV-positive patients with occult HBV infection had abnormal alanine aminotrasferase level, which also raises the question as to whether occult HBV plays a role in the aetiology of liver disease in HIV-infected patients. Further, studies on the association between HBV DNA levels and markers of liver function in addition to data on liver biopsy would help in answering this question. Keywords: human immunodeciency virus, hepatitis B virus, occult hepatitis B, Lebanon

INTRODUCTION
Hepatitis B virus (HBV) remains a major global health problem with more than 350 million people affected worldwide.1 Infection with virus could range from fulminant hepatitis, acute infection, chronic infection to asymptomatic carriage.2,3 The status of HBV infection can be determined using serological proles: for instance, the simultaneous presence of hepatitis B surface antigen (HBsAg) and antibodies to the hepatitis B core antigen (anti-HBc) reveals current infection whereas the presence of anti-HBc and antibodies to HBsAg (anti-HBs) usually indicates a past, resolved infection. Recently, more attention has been given to a serological pattern dened as the presence of anti-HBc alone4 but, its signicance remains unclear. Anti-HBc alone have been reported in 7.2 9.7% among the Chinese5 and in 10 20% of individuals with HBV markers in areas with low HBV endemicity, such as Europe and the United States.4 Lack of detection of HBsAg in individuals with anti-HBc alone could be attributed to several factors, such as the presence of mutations in the pre-s/s genome region of HBV or low level of HBV DNA.6,7 The recent availability of polymerase chain reaction (PCR) assay for the detection of HBV DNA has shown that HBV DNA could be detected in patients who are negative for HbsAg, but positive for anti-HBc.8,9 This so-called occult HBV infection ( presence of HBV DNA in the absence of HBsAg)10,11 have been recently reported and with the high
Correspondence to: Dr Sami Ramia Email: sramia@aub.edu.lb

frequencies in intravenous drug users,12 in patients with hepatitis C virus (HCV) infection13,14 and in patients with human immunodeciency virus (HIV) infection.15,16 Recently, we have shown that occult HBV infection is relatively high (12 44%) among the HCV-infected Lebanese patients and that the prevalence increased with the severity of liver disease.17 In this study, we aim to describe the frequency and signicance of anti-HBc alone and HBV DNA in HIV-infected Lebanese patients.

MATERIALS AND METHODS Patients and serum samples

A total of 101 HIV-infected Lebanese patients (69 men, 32 women; age range 25 60 years with mean age of 32.7 + 1.7 years) were involved in the study. These were from different regions of Lebanon and the majority acquired the infection by the sexual route (92 out of 101 patients) whereas the rest (nine out of 101 patients) acquired the infection via intravenous drug use (IDU). All patients were anti-HIV-1 positive by conventional serological tests and conrmed by Western blot hybridization technique.

Serological tests
Blood samples were collected and re-tested at our Laboratory for HBsAg, anti-HBc and anti-HBs using the Axsym assays (Abbott Laboratories, North Chicago, IL, USA). All samples

DOI: 10.1258/ijsa.2007.007200. International Journal of STD & AIDS 2008; 19: 197 199
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International Journal of STD & AIDS

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that were found to be positive for anti-HBc alone were further re-tested for anti-HBc by another commercially available ELISA (DiaSorin srl Saluggia, Italy) with a sensitivity of 100% and specicity of 99.5% and only samples positive by both assays were included in the study. All anti-HBc alone positive samples were tested for HBV DNA. The concentration of alanine aminotrasferase (ALT) was determined in all anti-HBc alone samples by an autoanalyser (Hitachi 912), and considered abnormally high if  60 IU/mL.

Diagnostics, Raritan, NJ, USA) used in our study has a reported sensitivity of 400 copies/mL. ALT levels on all our anti-HBc alone positive samples were within normal (  60 Iu/mL), but unfortunately no liver biopsy was performed on our patients.

DISCUSSION
Recurrent reports in the literature focus on the increasing detection of occult HBV infection in HIV-positive individuals and in particular in those with anti-HBc-alone serological prole.15,20 24 The failure to detect any other HBV serological marker, particularly anti-HBs, suggests that these patients are most likely to be chronic HBV carriers rather than recovering from recent HBV infection. This is probably true as follow-up on some of our patients (9 patients) failed to detect anti-HBs even after a period of 11 months. Failure to detect HBsAg in the anti-HBc alone patients could be due to the low level of HbsAg, which cannot be detected by current enzyme linked immunosorbant assays or possibly due to HBV variants with mutations or deletions.25 Point mutations in the S-gene associated with HBsAg- negative HBV infection have been reported.25,26 HBV-DNA in the absence of HBsAg (Occult HBV infection) was detected in the serum of 28.7% of our HIV-infected patients. It should be mentioned, however, that HBV-DNA is more often detected in Liver than in serum.23 The relatively high frequency of HBV infection in our HIV-positive patients is somehow surprising as Lebanon is a country of lowendemicity of HBV ( , 1%).27 The low prevalence of HCV infection in our patients (9%) is possibly due to the fact that the overwhelming majority (91%) of the HIV-positive patients that we investigated acquired HIV via the sexual route compared with only 9% who acquired the infection via intravenous drug uses. In fact, three out of the nine HIV-positive patients who were anti-HCV-positive acquired HIV via IDU conrming the efcient transmission of HCV by this route.28 Occult HBV infection have been reported in a wide range of clinical manifestations ranging from a symptomatic carriage to hepatocellular carcinoma.29,30 Of interest is the nding that none of our HIV-positive patients with detectable HBV-DNA had abnormal level of ALT although normal ALT does not exclude signicant liver disease. Unfortunately, the extent of liver damage could not be assessed since no liver biopsy was done on our patients. Implementation of highly active antiretroviral therapy (HAART) for treatment of HIV infection has lead to a decrease in HIV-related morbidity and mortality.31 In a recent study, the impact of chronic HBV infection or the presence of isolated antibodies to HBV core antigen on survival in the area of HAART was investigated.32 It was shown that HIV-positive patients with isolated anti-HBc did not have a lower rate of survival or a higher rate of liver disease than in patients who had resolved HBV infection.32 Further studies are needed to conrm this conclusion. Currently administered initial ART regimens usually include at least one drug active against HBV, such as lamivudine, tenofovir, or entricitabine. Sometimes, both lamivudine or entribitabine are combined with tenofovir as a nucleoside backbone of most initial regimens. This could certainly affect HBV replication and activity and may play a role in altering the course of HBV disease in a favourable way. A study to evaluate the role of ART on future course of HBV HIV coinfection, particularly in the subgroup of anti-HBc-alone, is

HBV DNA detection

HBV DNA detection was performed as described previously.18 Briey, DNA extraction was performed using the QIAamp DNA Blood Mini kit (Qiagen, Chatsworth, CA, USA) according to the manufacturers instructions. PCR was used for the detection of HBV DNA with primers P7 and P8 as described by Lindh et al. 19 whereas HBV genotyping was investigated by restriction fragment length polymorphism analysis of the fragment of the HBV genome, which was amplied by PCR as described by Lindh et al. 19

Hepatitis B viral load

Samples with the detectable HBV DNA by PCR were further tested for HBV viral load using the Amplicor HBV Monitor test (Roche, Switzerland). All manufacturers instructions were followed. The detection limit of the assay was 400 genome copies/mL.

RESULTS
A summary of HBV DNA in 101 HIV-positive patients in different HBV serogroups is shown in Table 1. Only seven patients (6.9%) were HBsAg carriers whereas 54 patients (53.5%) had no evidence of exposure to HBV. Among those who were exposed to HBV 24 patients (51.1%) were positive for anti-HBc alone compared with four patients (8.5%) who were anti-HBs alone positive. The majority of those who were positive for anti-HBc alone were also positive for HBV DNA (83.3%) (Table 1). HBV DNA could be detected in only 16.7% of HIV-positive patients who were both anti-HBc and anti-HBs-positive. The Amplicor HBV monitor test (Roche,

Table 1 Summary of HBV DNA in HIV-positive Lebanese patients in different HBV serogroups HbsAg (94 patients), no. (%) HBsAg (7 patients) No. (%) HBV DNA 1 HBV DNA 2 Total 7 (100%) 0 7 Anti HBc Anti HBc 2 anti HBs anti HBs 2 (12 (54 patients) patients) 0 54 54 2 (16.7%) 10 12 Anti HBc 2 anti HBs (4 patients) 2 (16.7%) 10 12 Anti HBc anti HBs 2 (24 patients) 20 (83.3%) 4 24

HBV hepatitis B virus; HIV human immunodeciency virus; HBsAg hepatitis B surface antigen; anti-HBc hepatits B core antigen; anti-HBs antibodies to HBsAg

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currently ongoing on our patients. Another conclusion may be drawn is that initial testing of HIV-infected individuals for HBV necessitates only testing for anti-HBc. However, it is important to delineate the need for other tests such as PCR according to potential future courses of illness in each subgroup.

ACKNOWLEDGEMENTS

This work was supported partially by a grant from the University of Research Board (Grant No 688501) of the American University of Beirut and partially by Abbott Diagnostics (Wiesbaden, Germany). Special thanks go for Ms Dina Martinos at Rizk Clinic and for Mrs M Abul Naja for her secretarial help.

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