Occult HBV infection status among chronic hepatitis C and hemodialysis patients in Northeastern Egypt : regional and national overview

Introduction: Occult hepatitis B infection (OBI) is considered to be one of the major risks for patients suffering from end-stage renal disease (ESRD) on regular hemodialysis (HD) and patients with chronic hepatitis C virus (HCV) infection. This study compared the prevalence of OBI among these two high-risk groups in the Suez Canal region, Northeastern Egypt, to obtain a better national overview of the magnitude of OBI in this region. Methods: Serum samples were collected from 165 HD patients and 210 chronic HCV-infected patients. Anti-HCV antibody, hepatitis B surface antigen (HBsAg), total hepatitis B core (anti-HBc) antibody, and hepatitis B surface antibody (anti-HBs) were detected by enzyme-linked immunosorbent assay (ELISA). HCV RNA was detected using a quantitative real-time RT-PCR assay, and HBV was detected using a nested PCR. Results: All patients were negative for HBsAg. A total of 49.1% and 25.2% of the patients in the HD and HCV groups, respectively, were anti-HBc-positive. In addition, more anti-HBs-positive patients were detected in the HD group compared to the HCV group (52.1% and 11.4%, respectively). Three cases were positive for HBV DNA in the HD group, while eighteen positive cases were detected in the HCV group. Both study groups showed signifi cant differences in serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) level as well as anti-HBc, anti-HBs and HBV-DNA positivity. Conclusions: OBI was more prevalent among chronic HCV patients than HD patients in the Suez Canal region, Egypt, with rates of 8.5% and 1.8%, respectively. However, more precise assessment of this infection requires regular patient follow-up using HBV DNA detection methods.

The continual existence of hepatitis B surface antigen (HBsAg) in blood has been established as a marker of overt hepatitis B virus (HBV) infection.Occult hepatitis B infection (OBI) is defi ned as the continuous existence of the HBV genome in liver tissues and/or serum in the absence of serum HBsAg.This phenomenon could result from the persistent presence of covalently closed circular deoxyribonucleic acid (cccDNA), which accumulates in the nuclei of hepatocytes and acts as a template for HBV transcription (1) .The risk of OBI transmission is decreased by the transfusion of blood components containing hepatitis B surface antibodies (anti-HBs) (2) (3) .
Although Africa is considered to be a highly endemic region for HBV, it is considered to have an intermediate prevalence (2-6%) in Egypt.This discrepancy is mostly due to the introduction of HBV vaccination into compulsory immunization programs in Egypt in 1991 and the availability of this vaccine to populations at high risk for HBV infection.However, a considerable incidence of OBI has been reported in Egypt (4) .
OBI may occur in serologically positive patients with markers of prior HBV infection (anti-HBs and/or hepatitis B core antibody (anti-HBc) as well as serologically negative patients without markers of previous exposure.Generally, approximately 20% of OBI patients are serologically negative, whereas 80% are serologically positive for one or more markers of prior infection (5) .OBI is primarily spread among hemodialysis (HD) patients, individuals under going frequent blood transfusions, and intravenous drug abusers (6) (7) (8) (9) (10) (11) (12) .Patients on chronic HD represent an important risk group for acquiring OBI, which may be attributed to the persistent exposure to blood-borne infections through the dialysis process and the need for frequent blood transfusions (13) .
Hepatitis C virus (HCV) infection suppresses not only HBV replication but also HBV surface protein expression

METHODS
(low or undetectable serum HBsAg) (14) (15) .Whereas some studies have failed to demonstrate any association between OBI and HCV (16) (17) , others have reported a high prevalence of OBI in chronically infected HCV patients (18) (19) (20) .Similarly, a close association between OBI infection and the existence of anti-HCV antibodies among HD patients was reported (21) .There is a broad range of clinical implications for OBI because it carries the potential for HBV transmission through blood transfusions, organ transplantations and HD.Moreover, OBI can result in fulminant hepatitis, poor responses to interferon/ribavirin (IFN/RBV) antiviral therapy, progression of hepatocellular carcinoma (HCC) and acute exacerbation of chronic hepatitis B (22) .
The prevalence of OBI in HD patients varies markedly from one locality to another worldwide, with a range of 0% to 36 % (13) .In Egypt, the prevalence was reported at 4% in Minia and Assuit (Upper Egypt) and reached 26.8% among patients in Alexandria (North Egypt) (23) (24) .Even with these recent data addressing the prevalence of OBI in patients on HD in Egypt, more comprehensive data may be needed to evaluate OBI among chronic HCV carriers and to determine whether the prevalence differs following HD, as both HCV carriers and chronic HD patients may serve as a potential source for the spread of OBI in the community.
Our study investigated the prevalence of occult hepatitis B in two groups of patients who were potentially immunocompromised and at higher risk for acquiring OBI (chronic HCV-infected patients and patients on regular HD due to end-stage renal disease (ESRD) using serological tests and nested polymerase chain reaction (PCR).A highly specifi c and sensitive nested PCR method was used to test for OBI.

Patients
This study included two groups.The fi rst group included 165 patients with ESRD who had been undergoing regular HD for more than 6 months and were HCV-negative (negative for anti-HCV antibody).The second group included 210 patients with chronic HCV infection.The patients were selected from two HD centers in the Suez Canal region, Northeastern Egypt.HCV diagnosis was based on the detection of serum anti-HCV antibodies and confi rmed by the detection of serum ribonucleic acid (HCV/RNA).Patients with acute or chronic HBV infection (as determined by positive HBsAg), other causes of liver diseases (i.e., autoimmune hepatitis and continued alcohol abuse), or currently being treated with IFN and/or ribavirin were excluded.The medical history was collected for the entire study population, including HBV vaccination history and clinical and biochemical assessments.The study was conducted in accordance with the ethical standards of the Declaration of Helsinki.The study was approved by the research ethical committee of the Faculty of Medicine, Suez Canal University, and informed written consent was obtained from all patients included in this study.Samples Blood samples were collected from all patients.The serum was separated and stored at -80°C prior to analysis.Both alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were measured in all samples using a colorimetric method.All patients were screened for human immunodefi ciency virus (HIV) 1 and 2 with commercial kits (AxSYM HIV 1/2gO, Abbott Diagnostics, Wiesbaden, Germany).

Hepatitis C virus testing
A third-generation enzyme-linked immunosorbent assay (ELISA; HCV 3.0 ELISA Ortho, Raritan, NJ, USA) was used to test for anti-HCV antibodies according to the manufacturer's instructions.HCV RNA was detected using a quantitative real time reverse transcription polymerase chain reaction (RT-PCR) assay with a detection limit of 33.6IU/ml (Artus HCV RG RT-PCR, Qiagen, Hilden, Germany).
Nested PCR amplification of HBV region S: DNA was extracted from 200µl of serum using the QIAamp Min Elute Virus Spin Kit (QIAGEN, Inc., Hilden, Germany) and re-suspended in 100µl of a storage buffer provided by the kit manufacturer.
The second PCR (nucleotide positions 414-989) used the primers (SB1) 5'-TGCTGCTATGCCTCATCTTC-3' and (HS4R) 5'-CATACTTTCCAATCAATAGG-3'.The PCR was performed in a 25µl reaction mixture volume containing 2µl of DNA template (from the fi rst PCR product), 25µM of each primer, 12.5µl of One Taq® Quick-Load® 2× Master Mix (New England Bio labs), and PCR-grade water.The cycling conditions were the same as those described forthe fi rst PCR.
Positive and negative controls were run along with the clinical samples.DNA from anHBsAg-positive patient with a high viral load (under treatment) was used as a positive DNA control, while DNA from a healthy blood donor was used as a negative reference.The PCRs were performed using an Eppendorf Master Cycler nexus PCR thermal cycler.Proper precautions were taken to avoid cross contamination.Gel electrophoresis: The resulting PCR amplicons were electrophoresed in 1.5% agarose gels in 1× Tris-borate-ethylene diaminetetraacetic acid (EDTA) buffer and stained with ethidium bromide.The images were captured using the Syngene G: Box documentation system (Syngene, UK).

Statistical analysis
Statistical Package for Social Science (SPSS) version 16 was used for data processing and analysis.The results are expressed in ranges, percentages and mean (SD).For comparisons between the study groups, the Student's t-test was employed for quantitative variables, and the Chi-square test was used for categorical (qualitative) variables.P values less than 5% were considered to be statistically signifi cant.

General characteristics of the studied populations
The current study included two groups.The fi rst group included 165 patients with ESRD who received regular HD.These patients ranged from 12 to 76 years in age with a mean (SD) of 44.35 years (13.40 years); additionally, 53.9% of the patients were male and 46.1% were female.The second group included 210 patients with chronic HCV infection.These patients ranged from17 to 56 years with a mean (SD) of 40.61 years (9.09 years); additionally, 67.2% of the patients were male and 32.8% were female.There were signifi cant differences in age and gender between the study groups.
The ALT values ranged from 5 to 90 with a mean (SD) of 20.17 (11.89) in the HD group and from 5 to 246 with a mean (SD) of 53.43 (33.67) in the chronic HCV group.The AST means (SD) were 21.93 (10.98) and 51.07 (30.01) in the HD and HCV patients, respectively.Both the ALT and AST levels were signifi cantly different between the HD and chronic HCV groups (Table 1).

Hepatitis B serological status
All the patients were negative for HBsAg.Positive cases for total anti-HBc were signifi cantly higher in the HD group compared to the HCV group (49.1% and 25.2%, respectively).Moreover, the number of positive cases for anti-HBs was higher in the HD group compared to the HCV group (52.1% and 11.4%, respectively).The results revealed signifi cant differences in anti-HBc and anti-HBs seropositivity in both groups, with p values < 0.01 for both markers (Table 1).
In both study populations, males were more common than females among the anti-HBc-positive cases.Table 2 shows that serum ALT and AST levels were signifi cantly higher in the HCV group, where as the HD group showed more positive anti-HBs cases than the HCV group (63% and 18.9%, respectively).

Nested PCR results
All the DNA samples extracted from patients in the current study were subjected to nested PCR targeting the S region of the hepatitis B genome.The resulting amplifi ed product was approximately 681bp in size, as shown in Figure 1.Highly signifi cant variation was observed in positive cases between the two groups (p value <0.01);only 3 cases were positive for HBV DNA in the HD group, while 18 cases were positive in the HCV group (Table 1).The OBI prevalence was 1.8% and 8.5% in the HD and HCV groups, respectively.The characterization of HBV DNA-positive cases in both study groups is shown in Table 3.All three cases in the HD group were negative for anti-HBs, and only two cases were positive for anti-HBc; in contrast, more than half (55.5%) of the cases in the HCV group were positive for anti-HBs, and all cases were positive for anti-HBc.There were no signifi cant differences in age, gender, ALT and AST levels, and anti-HBc and anti-HBs positivity between the HBV DNA-positive cases from both study groups.

DISCUSSION
HBV infection is a major health concern due to its widespread prevalence, with 350,000 million infected individuals worldwide (26) .HBV infection leads to major complications, including fulminant hepatitis, chronic liver disease and HCC, which carry the signifi cant burden of high health costs.OBI is a variant of HBV infection that occurs when patients show no HBsAg in the serum but persistent HBV DNA in the liver tissue with or without detection of HBV genome in the serum.Patients with ESRD on HD and chronic HCV-infected patients are highly susceptible to acquiring HBV infection.Our study was designed to measure the of this infection in these two groups of patients in a large district in our country.The current study showed marked variations in patients' ages, because we included variable age groups (from children to elderly patients with a range of 12 to 76 years in the HD group and from 17 to 56 years in the HCV group).This approach enabled a more accurate assessment of OBI in our patients, as these samples were representative of the age groups in the general population.
Our results showed signifi cantly different ALT and AST levels between the study groups.The levels were more elevated in the HCV group, which may be attributed to hepatic dysfunction due to chronic hepatitis.
Anti-HBc constitutes the fi rst antibody response to HBV infection, and its detection in the serum may denote acute or chronic infection.Patients remain anti-HBc-positive after recovery, and this antibody therefore serves as the only serologic marker for HBV during the window period of infection.As a result, this marker is considered one of the most valuable serological markers for the diagnosis of OBI (27) (28) (29) and has been proposed as a surrogate screening marker for OBI diagnosis when viral DNA detection methods cannot be applied (26) .In contrast, the anti-HBs antibody appears late in infection after the disappearance of HBsAg and has a protective neutralizing effect.Anti-HBs is the only HBV serologic marker that can be detected after vaccination against this virus.
In the current study, the rates of detection of both anti-HBs and anti-HBc differed signifi cantly between the study populations, and both markers were detected at higher levels in the HD group compared to the HCV group.This fi nding can be explained either by a true difference in the infection rate between groups or by immune unresponsiveness or a weak immune response to HBV antigens as a result of the suppression of replication and expression of this virus in HCV co-infected patients (14) (15) .Moreover, the positivity for both an ti-HBc and anti-HBs could be explained by either recovery from previous acute or chronic HBV infection (26) or OBI seropositivity in which HBsAg cannot be detected in the serum because it is cleared to an undetectable level (30) .Additionally, the increased number of anti-HBs-positive patients in the HD group may be a result of vaccination against HBV, which is routinely performed in HD patients in our region.As shown in Table 2, a considerable number of the anti-HBc-positive patients were negative for both anti-HBs and HBV DNA; this result may be due to either recovery from previous HBV infection or true occult HBV infection without detectable DNA in the serum.Thus, the rate of DNA detection may increase if liver biopsies are examined.
The detection of HBV DNA is considered the most defi nitive diagnostic tool for the detection of occult hepatitis infection.In our study, we used nested PCR because this method is simple and accurate, easy to interpret, can detect even very low amounts of viral DNA and has been previously applied on a wide scale for the detection, genotyping and phylogenetic analysis of HBV (31) (32) (33) (34) .Specifi cally, we targeted the S region of the HBV genome in patient serum samples; this region was selected as a target for amplifi cation by PCR because it was found to be more sensitive for the detection of HBV DNA in serum (26) .
Our nested PCR demonstrated that the prevalence of OBI was 1.8% and 8.5% in the HD and HCV groups, respectively; the difference in these prevalence rates was statistically signifi cant.Marked variation was found by comparing these rates with OBI prevalence rates reported in other related national or international studies (23) (24) (35) (36) (37) (38) (39) .This variation may be attributed to differences in the sample size of the study populations, demographics, immunologic status of the studied patients, endemicity of HBV infection, levels of viral DNA in the blood, sampling conditions and the types of diagnostic tool(s) used for diagnosis (40) .This comparison also revealed that the prevalence of OBI in our target HD patients was relatively low, possibly due to the application of safety precautions during the dialysis process in these units and the implementation of the routine vaccination program.Additionally, the low rate can be attributed to the use of serum samples to detect HBV DNA in the current study.The likelihood of detecting DNA is much lower in serum samples than in liver tissue, although the use of liver biopsies for the detection of OBI is diffi cult, especially in HD patients.Moreover, the higher prevalence rate of OBI in the chronic HCV group can be explained by the occurrence of coinfection with HBV, as both of these viruses share common routes of infection (41) .
Two of the three HBV DNA-positive patients in the HD group were seropositive for anti-HBc alone, while the third patient was seronegative.In contrast, all HBV DNA-positive cases in the HCV group were positive for anti-HBc, and more than half were positive for anti-HBs.This result is in accordance with the fi nding that OBI is more prevalent in seropositive patients who are positive for anti-HBc and/or anti-HBs (23) (29) (37) (42) (43) .The occurrence of occult infection despite the presence of neutralizing an ti-HBs can result from mutations affecting the S region of the HBV genome (39) .However, the absence of all HBV serological markers (even anti-HBc) in some HBV DNApositive patients strengthens the proposal that the detection of viral DNA is the gold standard for the diagnosis of OBI.Therefore, the presence of seronegative cases should be given consideration during the management of HD and HCV patients.
From the current study, we can conclude that OBI occurs in HD and chronic hepatitis C patients in our area at rates of 1.8% and 8.5%, respectively.Despite these relatively low rates, this infection is considered to be a real health problem in these debilitated patients and necessitates regular follow-up for the early detection of OBI using DNA detection methods.

TABLE 1 -Demographic and laboratory data of the study group populations.
HCV: hepatitis C virus; SD: standard deviation; n: number; ALT: alanine aminotransferase; AST: aspartate aminotransferase; IU/L: international unit per liter; anti-HBc: hepatitis B core antibody; anti-HBs: hepatitis B surface antibody; HBV DNA: hepatitis B virus deoxyribonucleic acid.