Introduction

Approximately 30% of the world’s population, or about 2 billion persons, have serologic evidence of current or past HBV infection (Perz, et al, 2006). Of these, an estimated 361 million persons have chronic HBV infection. The WHO estimates that around 4.3 million persons are infected with HBV and around 600 000 persons die each year due to HBV-related chronic liver disease (WHO, 2008)

The worldwide prevalence of carriage of hepatitis B virus (HBV) ranges from 0.1 to 20 percent. This wide range is largely related to differences in age at the time of infection, a factor that is inversely related to the risk of chronic infection. Perinatal infection is the predominant mode of transmission in high-prevalence areas, and horizontal transmission during early childhood is the most common form in areas with an intermediate prevalence, whereas sexual contact and injection-drug use in adults are the main routes of spread in low-prevalence areas (Lok, 2002).

Hepatitis B virus infection is ende­mic in the Kingdom of Saudi Arabia with a marked variation in prevalence between different regions (Al-Faleh, 1988). Following the integration of hepatitis B vaccination into the extended program of immunization, the pre­valence of HBsAg among children declined from 6.7% to 0.15%. (Madani, 2005,  Al-Hazmi, 1989; Al-Faleh et al 1992).

The outcome of HBV infection is mostly determined by the immune response of the host. The most complete immune response is associated with the most severe liver injury and the greatest likelihood of viral clearance, whereas an immature immune response leads to chronic hepatitis (Viola, et al, 1981).

The natural history of chronic HBV infection is characterized by four distinct phases (Villeneuve 2005). These phases are dependent on a complex interaction between host, viral and environmental factors, and particularly the age at infection. The first stage is characterized by high viral loads and immune tolerance followed by an immune competence (clearance) phase during which hepatocyte necrosis occur. When the immune response decreases the number of infected cells, a third stage begins with low viral replication, referred to as the inactive carrier state. In the fourth stage, Immune escape phase, patients become negative for HBeAg and positive for anti-HBs, and HBV DNA is usually no longer detectable in serum, although still present in liver tissue. This phase is characterized by negative HBeAg, positive anti-HBe and detectable viral load (HBV DNA > 2000 IU/mL).

Another group of patients who is HBeAg-negative may follow seroconversion from HBeAg to anti-HBe antibodies during the immune reactive phase and represents a later phase in the natural history of CHB. Patients can reach this phase from the immune control state (5–10%), ( Liaw et al, 1983)  or can progress directly from HBeAg-positive chronic hepatitis to HBeAg-negative chronic hepatitis (10-30%) (Zacharakis et al, 2005).

HBeAg-negative chronic HBV infection is reported in all parts of the world, but is more common in Asian and Mediterranean countries (Papatheodoridis and Hadziyannis, 2001). It occurs due to the selection of a mutant HBV, which does not produce HBeAg but is still able to replicate.

Data on pattern of CHB in Saudi Arabia are still scarce. We investigated the patterns of chronic hepatitis B virus (HBV) related disease in a cohort of HBsAg  positive patients, in the eastern region of the Kingdom of Saudi Arabia, by collecting demographic, clinical and laboratory data.

Materials and Methods

A cross-sectional study was conducted with HBsAg positive patients recruited from king Fahad military Medical Complex, Dhahran, Saudi Arabia during the year 2009. Ninety-six patients were recruited in this study.

Serum samples of patients with HBsAg were tested for, HBeAg, anti-HBc immunoglobulin G (IgG), IgM, anti-HBs, and anti-HBe, using Axym (Abbott Laboratories, North Chicago, Illinois, USA).

Biochemical tests for liver disease including alanine aminotransferase [ALT], and aspartate aminotransferase [AST], Bilirubin, GGT, AFB, ALP, and serum albumin were detected using Dimension RxL (Dade Behring, USA). The upper normal limit of for ALT of 41 U/L was considered. DNA quantification of HBV was performed by real-time TaqMan PCR assay at Bioscientia laboratory, Germany, with lower limit of detection of 12 IU/ml and an upper limit is 17 X 10⁷ IU/ml.

The study was presented and approved by ethical committee of the KFMMC.

All statistical analyses were carried out using SPSS v17.0 software (Chicago, Illinois). A value of P < 0.05 was considered statistically significant.

Results

A total of 96 patients with HBsAg were included in the study with a mean age (± SD) of 34.1 + 11.1 years. There were 58 (60.4%) males and 38 (39.6%) females.

Out of 96 HBsAg carriers 79.2% were HBeAg-negative/anti-HBe positive, and 12.5% were HBeAg-positive/anti-HBe negative, and 8.3% were HBeAg-positive/anti-HBe positive (Table 1).

The average ages of those who are HBeAg-negative/anti-HBe positive, HBeAg-positive/anti-HBe negative, and HBeAg-positive/anti-HBe positive are 35.1 + 11.6,   31.5 + 6.8 and 27.5 + 6.3, respectively. Patients with HBeAg-positive were younger.

Table 1 also shows the serum DNA various levels among the various e antigen/antibody patterns. Serum HBV DNA was detected in 69 (71.9 %) patients whereas 27 (28.1%) had undetectable DNA level (<12 IU/ml).

Out of the 96 cases studied, 50.0% showed a low level of 13 – 2000 IU/ml., 12.5% with a moderate level of 2001-20000 IU/ml, and 9.4% with a high level of >20000. DNA was detected in all HBeAg-positive/anti-HBe negative group and 72.4% of HBeAg-negative ones.

Patients who are negative for e antigen constitute 92.6% of those with an undetected DNA (<12) and only 55.6% of the individuals with high DNA level (>20000).

Table 1

According to their ALT levels, patients with HBeAg and anti-HBe were divided into further groups (table 2).

Table 2

Normal ALT levels were observed in 42 (43.8%) of the total cases, while 29 were 1-1.5X upper limit of normal (ULN), 21 were >1.5-2X ULN, and 8 were >2 ULN.

There was no difference in the distribution of DNA levels among sex groups (P=0.15).

DNA levels are higher among younger age groups. 10.9% (n=46) of the age group 17-30 years showed a DNA level >2000 compared to 7.1% (n=28) and 4.5 (n=22) of the age groups of 31-40 and 41-66 years, respectively. On the other hand, 50% of the age group 41-66 years have shown an undetectable DNA level compared to 21.7% and 25% of the age groups 17-30 years and 31-40 years, respectively. This distribution of DNA levels among age groups was statistically significant (P=0.034).

Discussion

96 patients were studied with various stages of chronic HBV infection. Out of these 25 patients were classified as HBeAg negative/anti-HBe positive inactive carriers. All of the 25 individuals have shown an undetectable HBV DNA and normal ALT level. These patients can be classified at the immune control phase and are at low risk of developing advanced liver disease and its related complications (Manno et al, 2004).

Among HBeAg-negative/anti-HBe positive patients, 11 cases showed a DNA level of >2000-20000 IU/ml and an elevated ALT level.

The average ages of HBeAg-positive/anti-HBe positive group is less than HBeAg-positive/anti-HBe negative ones, which is in turn less than HBeAg-negative/anti-HBe positive ones. Although the difference in age groups among different HB e antigen pattern was not statistically significant (P=0.1), patients with HBeAg in their serum were younger than those with HBeAb which may indicate an earlier stage in the natural history of the disease. The HBV replication level is known to fluctuate over a broad range during the course of its chronic infection. In accordance with other studies, serum HBV concentration was significantly higher in HBeAg-positive patients than in anti-HBe-positive patients (e.g. Fiordalisi et al 1990).

The trend of increasing prevalence of HBeAb with age may also indicate a considerable horizontal transmission still present in this age range.

For many years, HBeAg / anti-HBe status was the only widely available means of assessing viral replication in chronic HBsAg carriers. Earlier studies revealed that the presence of HbeAg may be taken as a surrogate marker for HBV DNA in hepatitis B virus carriers with raised serum ALT in the absence of HBV PCR testing (Badur, et al 2001, Hussein et al 2004). In the present study, 75% (n=8) of the individuals with a high viremia (>20000 IU/ml) and an elevated ALT level were positive for e antigen.

Out of 6 HBeAg-positive/anti-HBe negative patients, 4 patients were with elevated ALT level and the other 2 patients were with normal ALT level. All of the 6 HBeAg-positive/anti-HBe negative cases have a detectable HBV DNA irrespective of their ALT level.

HBeAg-positive patients with elevated ALT level have high level of HBV DNA. There is a marked difference in the level of HBV DNA in HBeAg-positive patients with raised and normal ALT level  in agreement with other studies, for example (Hussein et al 2004).

In this study, the ratio normal ALT levels to elevated ALT levels in anti-HBe positive patients is approximately 1:2.

The presence of HBV DNA in some patients who demonstrate anti-HBe or the absence of HBV DNA in some patients with positive HBeAg implied a complicated picture of the disease pathogenesis.

41 of the patients with detectable serum HBV DNA and anti-HBe antibodies had abnormal ALT levels which may explain why some patients developed chronic liver disease despite sustained HBeAg seroconversion.  8 of them have demonstrated a higher DNA level of >2000IU/ml. Our finding therefore supported the observation of other studies. The liver disease in anti-HBe-positive patients is associated either with mutants  which are now know to be common at least in Mediterranean countries or decrease in the amount of HBeAg secreted from infected hepatocytes.

Traditionally sero-conversion of HBeAg to anti-HBe coincides with the decrease or normalization of serum ALT concentration and a very low level of HBV replication (Alagiozian-Angelova et al, 1998). But some studies have concluded that presence or absence of HBeAg/anti-HBe may not necessarily reflect the serum HBV DNA concentration, particularly in persistent infection and thus absence of HBeAg and presence of anti-HBe poorly correlates with complete loss of HBV DNA from the serum (Luo, 1992)

In 28.7% (23 out of 80) of anti-HBe positive patients had normal ALT levels and 65.2% of them had detectable HBV DNA. This finding was in agreement with a recent study (Ljunggren,et al 2004) and provided further evidence that normal ALT levels, without DNA investigations, cannot be used to assess the infectivity of anti-HBe-positive patients.

The threshold HBV DNA level that is associated with chronic liver disease is not known and may be dependent on host factors such as immune response, viral factors such as HBV genotype and mutations in the core promoter and precore regions, and environmental factors such as alcohol consumption.

Further evaluation of HBeAg/ anti-HBe assays by HBV DNA has been recommended in assessment of possible infectivity and chronic liver disease in the HBsAg positive patients.

Similar to some previous studies (Ljunggrenet al 1993, Loriot et al, 1992), we found that the presence of HBV DNA is 100% in all HBeAg-positive/anti-HBe negative patients and in 67% of HbeAg negative/anti-HBe positive cases and this later group may be infected with precore mutant strain which is common in Asia and Mediterranean (Tong et al 1990, Fiordalisi et al, 1990).

Detection of high level of HBV DNA in 11 patients who are HBeAg-negative/anti-HBe positive in this study might indicate the presence of precore mutant virus which needs further investigations.

Several studies indicated that the presence of HBV DNA has been associated with raised ALT level even in presence of anti-HBe Kaneko, et al 1990, Baker et al 1991).

Such a picture has been reflected in this study. About 33 patients (78.6%) of anti-HBe positive patients with raised ALT had detectable level of HBV DNA (11 of them have shown high DNA level), compared with 52.97% with normal ALT and none of them has shown high DNA level.

Some of HBV infected patients presented with unexpected serological patterns. The simultaneous determination of both HbAg and HbAb may be the result of either breakage of immune complexes during experiment and determination of antigens and antibodies separately or less commonly a new infection by a different strain of HBV (Masuyama et al, 1993).

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