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Keywords:

  • Hemodialysis (HD);
  • Isolated hepatitis B core antibody (anti-HBc);
  • Occult hepatitis B virus (HBV) infection

Abstract

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSION
  7. Acknowledgments
  8. REFERENCES

Occult hepatitis B virus (HBV) infection is characterized by presence of HBV infection with undetectable hepatitis B surface antigen (HBsAg). Occult HBV infection harbors potential risk of HBV transmission through hemodialysis (HD). The aim of this study was to assess the occult HBV infection in hemodialysis patients with isolated hepatitis B core antibody (anti-HBc). A total of 289 HD patients from five dialysis units in Tehran, Iran, were included in this study. Hepatitis B surface antigen (HBsAg), Hepatitis B surface antibody (anti-HBs), anti-HBc, Hepatitis C antibody (anti-HCV), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were tested in all subjects. The presence of HBV-DNA was determined quantitatively in plasma samples of HD patients with isolated anti-HBc (HBsAg negative, anti-HBs negative and anti-HBc positive) by real-time PCR using the artus HBV RG PCR kit on the Rotor-Gene 3000 real-time thermal cycler. Of 289 patients enrolled in this study, 18 subjects (6.2%, 95% confidence interval (CI), 3.5%–8.9%) had isolated anti-HBc. HBV-DNA was detectable in 9 of 18 patients (50%, 95% CI, 27%–73%) who had isolated anti-HBc. Plasma HBV-DNA load was less than 50 IU/ml in all of these patients. Our study showed that detection of isolated anti-HBc could reflect unrecognized occult HBV infection in HD patients. The majority of these infections are associated with low viral loads.

Hepatitis B virus (HBV) is the most important cause of transmitted infections by the parenteral route in patients on maintenance hemodialysis (HD).The prevalence of HBV infection in HD patients varies markedly from country to country(1). In a study by Eslamifar et al. (2) 6.49% of Iranian HD patients had HBV infection. However improvement of the people's knowledge about HBV transmission risk factors, national vaccination program and vaccination of high risk groups justifies the decreased HBV incidence in the general population and also in those who started hemodialysis (3). Now Iran is in the low endemic area of HBV infection (3). A nationwide study in Iran showed that the prevalence of Hepatitis B surface antigen (HBsAg) ranges from 1.7% to over 5% in different provinces (4).

Occult HBV infection is characterized by the presence of HBV infection with undetectable HBsAg. Serum HBV level is usually less than 104 copies/mL in these patients. Diagnosis of occult HBV infection requires a sensitive HBV-DNA PCR assay (5). A number of explanations for the persistence of HBV-DNA in HBsAg negative samples have been proposed, including integration of HBV-DNA into the host's chromosomes (6), genetic variations in the S gene (7,8), and the presence of immune complexes in which HBsAg may be hidden (9,10). Occult hepatitis B may also be due to the window period following acute HBV infection, poor laboratory detection of HBsAg due to low level of HBs antigenemia, underlying HCV co-infection, immunosuppression, or other host factors (5,11).

The clinical implications of occult HBV infection involve different clinical aspects. First of all, occult HBV infection harbors potential risk of HBV transmission through blood transfusion, hemodialysis, and organ transplantation. Second, it may serve as the cause of cryptogenic liver disease; contribute to acute exacerbation of chronic hepatitis B, or even fulminant hepatitis. Third, it is associated with development of hepatocellular carcinoma. Fourth, it may affect disease progression and treatment response of chronic hepatitis C (5).

Occult HBV infection is most frequently seen in patients with hepatitis B core antibody (anti-HBc) as the only HBV serological marker (12). It is estimated that in individuals with markers of HBV infection, 10–20% present with a positive anti-HBc as the only HBV maker (isolated anti-HBc) (11). In one study on 4930 subjects in Iran, 5.13% were only positive for anti-HBc, without any detectable HBsAg (4).

To date, few studies have documented the prevalence of occult HBV infection in renal dialysis patients. In three small studies of 33, 5, and 67 HBsAg-negative dialysis patients, 50%, 40%, and 0%, respectively, were HBV-DNA positive (13–15). Of note, the majority of HBV-DNA positive individuals in these studies had serological evidence of previous HBV infection (HBV seropositive), but as many as 39% were HBV seronegative (16).

The epidemiology and clinical significance of occult HBV infection remains controversial with only limited information about its prevalence in Iranian patients on long-term hemodialysis. The aim of this study was to assess the occult HBV infection in hemodialysis patients with isolated anti-HBc (HBsAg negative, anti-HBs negative and anti-HBc positive).

PATIENTS AND METHODS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSION
  7. Acknowledgments
  8. REFERENCES

In this cross-sectional study 289 patients on chronic HD were recruited from five dialysis units in Tehran, Iran. A questionnaire was used to collect data such as age, sex and length of time on dialysis. This project was approved by the Pasteur Institute of Iran ethics committee and informed consent was obtained from patients prior to their enrollment.

Blood samples were collected from all patients and plasmas were stored at –80°C. All samples were tested for hepatitis B surface antigen (HBsAg), hepatitis B surface antibody (anti-HBs), hepatitis C antibody (anti-HCV) and anti-HBc by enzyme-linked immunosorbent assay (ELISA). The commercial enzyme immunoassay kits used were as follows: HBsAg and anti-HBs (Hepanosticka Biomerieux, Boxtel, The Netherlands), anti-HBc (Dia.Pro Diagnostic BioProbes, Milan, Italy), anti-HCV (Biorad, Segrate, Italy). Recombinant immunoblot assay (RIBA Innogenetics, Ghent, Belgium) was employed to confirm anti-HCV reactivity. Liver enzymes [alanine aminotransferase (ALT) and aspartate aminotransferase (AST)] were also determined in all of the patients.

Human immunodeficiency virus antibody (anti-HIV) was determined by ELISA (MP Biomedicals, Illkirch, France); with positive tests confirmed by Western blot assay (Diaplus, San Francisco, CA, USA). All assay protocols, cutoffs and result interpretations were carried out according to the manufacturers' instructions.

HBV-DNA was extracted using High Pure Viral Nucleic Acid kit (Roche Diagnostics GmbH, Mannheim, Germany) following the manufacturer's instructions. HBV-DNA was determined quantitatively by real-time PCR using the artus HBV RG PCR kit (QIAGEN, Hamburg, Germany) on the Rotor-Gene 3000 real-time thermal cycler (Corbett Research, Sydney, Australia). The analytical detection limit of the kit is 20 IU/mL according to the user manual. Results were presented as IU/mL. HBV-DNA below 50 IU/mL is reported as less than 50 IU/ml.

Statistical analysis

The Mann-Whitney U, Chi-square and t2-tests were used with the SPSS 16 Package program for statistical analysis (Chicago, IL, USA). Data are presented as mean ± SD or, when indicated, as an absolute number and percentage. The 95% confidence interval (95% CI) was calculated.

RESULTS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSION
  7. Acknowledgments
  8. REFERENCES

A total of 289 HD patients with mean age 55 ± 16 (range: 15–89) years were enrolled in the study. 60% of patients were male and 40% were female. The duration of HD was 5.2 ± 5.1 years. All of them had past history of blood transfusion but none of them had organ transplantation. The mean ALT and AST levels were 16.4 ± 9.4 and 16.9 ± 9.8 IU/L, respectively. HBsAg, anti-HBs, anti-HCV and anti-HIV were found in 2.8%, 77.5%, 3.1% and 0.34% of patients, respectively.

Of the 289 patients, 18 subjects (6.2%, 95% CI, 3.5%–8.9%) had isolated anti-HBc. The characteristics of these patients are summarized in Table 1. Patients with isolated anti-HBc were found in only 2 dialysis units. There was not any significant difference between patients with and without isolated anti-HBc regarding age, sex, length of time on dialysis, ALT and AST levels (Table 2).

Table 1. Characteristics of hemodialysis patients with isolated hepatitis B core antibody (anti-HBc)
CharacteristicValue
  1. Data are indicated as mean (range) and number (%); ALT, alanine aminotransferase; AST, aspartate aminotransferase; HCV, hepatitis C virus.

Sex 
 Male11 (61.1%)
 Female7 (38.9%)
Age (years)59.6 ± 15 (37–85)
Duration of dialysis (years)3.5 ± 4.4
ALT (IU/L)21.1 ± 15.2 (8–66)
AST (IU/L)21.1 ± 14.5 (8–65)
Anti-HCV (+)1 (5.55%)
Table 2. Comparison of isolated anti-HBc positive and negative hemodialysis patients
CharacteristicIsolated anti-HBc positive patients (n = 18)Isolated anti-HBc negative patients (n = 271)P value
  1. Data are indicated as mean (range) and number (%); ALT, alanine aminotransferase; AST, aspartate aminotransferase; anti-HBc, hepatitis B core antibody; NS, not significant.

Sex (M/F)11/7162/109NS
Age (years)59.6 ± 1554.7 ± 16NS
Duration of dialysis(years)3.5 ± 4.45.3 ± 5.2NS
ALT (IU/L)21.1 ± 15.216 ± 8.8NS
AST (IU/L)21.1 ± 14.516.6 ± 9.4NS

HBV-DNA was detectable in 9 out of 18 patients (50%, 95% CI, 27%–73%) who had isolated anti-HBc. One dialysis unit showed 5 cases of occult HBV infection from 6 patients with isolated anti-HBc and the other one had 4 subjects of occult HBV infection from 12 isolated anti-HBc patients. As low-level contamination of some samples might be a problem, we tested 11 voluntary blood donors with isolated anti-HBc by real-time PCR using the same kit and HBV-DNA was not detected in any of them.

Five (55.5%) of HBV-DNA positive HD patients were male and 4 (44.5%) were female. Their mean age was 66.3 ± 14.3 years. The duration of HD in them was 2.6 ± 1.2 years. The mean ALT and AST levels were 22.9 ± 18.1and 21.3 ± 17.6 IU/L, respectively. In all of them plasma HBV-DNA load was less than 50 IU/mL. Only one of them was anti-HCV positive. None of them were HIV infected. There was no significant difference between HBV-DNA positive and negative patients regarding age, sex, ALT and AST levels or length of time on dialysis (Table 3).

Table 3. Comparison of HBV-DNA positive and negative hemodialysis patients with isolated anti-HBc
CharacteristicHBV-DNA(+) (n = 9)HBV-DNA(−) (n = 9)P value
  1. Data are indicated as mean (range) and number (%); ALT, alanine aminotransferase; AST, aspartate aminotransferase; HCV, hepatitis C virus; anti-HBc, hepatitis B core antibody; NS, not significant

Sex (M/F)5/46/3NS
Age (years)66.3 ± 14.352.9 ± 13.1NS
Duration of dialysis(years)2.6 ± 1.24.5 ± 6.3NS
ALT (IU/L)22.9 ± 18.119.7 ± 12.6NS
AST (IU/L)21.3 ± 17.620.9 ± 11.6NS
Anti-HCV (+)1 (11.1%)0NS

DISCUSSION

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSION
  7. Acknowledgments
  8. REFERENCES

In this study the rate of occult HBV infection in Iranian HD patients with isolated anti-HBc was determined. The HBV-DNA was detectable in 50% of these patients. Our survey showed that occult HBV infection was common in HD patients with isolated anti-HBc regardless of age, sex, aminotransferases levels and length of time on dialysis.

Occult HBV infection can occur in different clinical scenarios. Typically, seroclearance of HBsAg is followed by development of anti-HBs with coexisting anti-HBc. If anti-HBs remain undetectable, anti-HBc serves as the only marker indicating past HBV infection (17,18). The latter may be seen in three different situations: (i) a resolving acute HBV infection after HBsAg disappeared, but anti-HBs remains undetectable (window period) (ii) a past HBV infection, especially an infection which occurred a long time ago and anti-HBs has fallen to an undetectable level (iii) a true occult HBV infection with low level of HBV replication (6,11,17–21).

The prevalence of occult HBV infection in renal dialysis patients ranges between 0 and 58% in published reports (15,16,22–25). Interpretation is complicated by important differences in the composition of the study populations and the level of sensitivity of the HBV-DNA assay.

In the studies of Cabrerizo et al. (13,23), the rate of occult HBV in HD patients was 57.6% but the frequency of serum HBV markers was 51.5%.In the series of Dueymes et al. (22) the HBV-DNA rate was 13.9% with an anti-HBc seropositivity of 64.7%.

Fabrizi et al. (24) investigated a large cohort (585) of Italian chronic dialysis patients. They found isolated anti-HBc in 20.8% of HD patients. Occult HBV was absent in their study group. Minuk et al. (16) reported that the prevalence of occult HBV in adult hemodialysis patients in a North American urban center was approximately 4–5 times higher than standard HBsAg testing. The majority of these infections are associated with low viral loads and a high prevalence of the sG145R mutant. These discrepancies in the rate of occult HBV infection in dialysis patients may reflect the diverse prevalence of HBV infection in different countries and within different dialysis units. Other possible explanations include sensitivity of molecular biology techniques, size and virological features of the patient groups. In our study the patients were selected on the basis of positive anti-HBc result. This selection criterion would favor the enrollment of more high-risk patients.

Liver enzyme abnormalities are uncommon in patients with occult HBV infection (25). We also did not find any relationship between biochemical liver tests and occult HBV infection in our study population. Serum transaminases values tend to be attenuated in dialysis patients (16) and regardless of whether they are dialysis dependent (26,27) or not (28) this hampers the recognition of liver damage on the basis of liver biochemical tests.

The failure of demographic and biochemical findings to suggest the presence of occult HBV is not unexpected. Detectable HBV-DNA in serum was not predicted by demographic, clinical or biochemical parameters (29) and occult infections have been described in both high and low prevalence regions and various ethnic populations with no age or gender predilections (6) as in our study.

Some studies showed associations between HCV and occult HBV infection (24,30), but because only one of our patients with occult HBV was co-infected with HCV, a conclusion can not be reached regarding the association of occult HBV infection and HCV. This may be due to the overall low prevalence of HCV infection in our study cohort.

Some studies found that time on dialysis was significantly greater in anti-HBc positive than negative HD patients (24). Other scholars found that hemodialysis duration was not significantly different in patients with and without occult HBV infection (31,32). Our study also showed that there was no association between occult HBV infection and length of time on dialysis.

CONCLUSION

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSION
  7. Acknowledgments
  8. REFERENCES

Our study showed that detection of isolated anti-HBc could reflect unrecognized occult HBV infection in HD patients. The majority of these infections are associated with low viral loads. Vaccination of susceptible patients and staff, avoidance of dialyzer reuse, use of dedicated dialysis rooms, machines, and staff for infected patients and isolation of the diagnosed patients in a specific ward have all been advocated as means of limiting HBV transmission within dialysis units.

Acknowledgments

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSION
  7. Acknowledgments
  8. REFERENCES

Acknowledgments:  The authors are grateful to the Pasteur Institute of Iran for financial support of this study.

REFERENCES

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSION
  7. Acknowledgments
  8. REFERENCES