Prevalence of Occult Hepatitis B and Hepatitis C Virus Infections in Turkish Hemodialysis Patients

Abstract

Background and Objective. Hepatitis B virus (HBV) and hepatitis C virus (HCV) infections are important causes of morbidity and mortality in maintenance hemodialysis patients. Although their exact prevalence is not known, HBV and HCV viral infections and occult viral hepatitis are frequent in these patients. This study aimed to determine the prevalence of occult HBV and HCV infections in maintenance hemodialysis patients. Materials and Methods. One hundred and eighty-eight end-stage renal disease patients on maintenance hemodialysis (100 male, mean age 49±29 [16–80] years, and mean duration of hemodialysis 98±66 [12–228] months) were enrolled in this study. Serological markers for HBV and HCV were determined with immunoenzymatic assay (ELISA) by using commercial diagnostic kits (Access and BioRad, Beckman-Coulter). HCV-RNA (Cobas Amplicor HCV kit) and HBV-DNA (Artus GmbH HBV kit) were determined quantitatively by polymerase chain reaction. Results. Among the patients screened, 25 (13.3%) had HBV infection alone and 38 (20.2%) had HCV infection alone, while seven (3.7%) had dual infection of both viruses. Serological markers for occult hepatitis B and occult hepatitis C were positive in five (2.7%) and nine (4.8%) of the patients, respectively. Isolated anti-HBc was positive in 12 (6.4%) of all patients, three (7.9%) of the patients with anti-HCV and two (40%) of the patients with occult hepatitis B. Isolated anti-HBc positivity was more frequent in patients with occult hepatitis B than in those without (40% [2/5] vs. 5.5% [10/183], p=0.002). None of the patients with HCV had occult hepatitis B. Conclusions. Both occult and non-occult forms of HCV infection are more prevalent than HBV infection in hemodialysis patients. Especially the patients with isolated anti-HBc positivity should be tested for probable occult hepatitis B infection.

Keywords:

• hemodialysis
• occult hepatitis
• chronic renal failure

INTRODUCTION

Hepatitis B virus (HBV) and Hepatitis C virus (HCV) are transmitted primarily through the parenteral route, and nosocomial infections are not rare. HBV and HCV infections are the most common causes of liver disease in hemodialysis patients[1–4] and pose problems in the management of the patients in the renal dialysis units. Chronic renal failure (CRF) patients do not clear these viral infections efficiently, and several outbreaks of hepatitis have occurred in these settings.[5] HBV infection is less prevalent than HCV infection in hemodialysis units.[6] The introduction of HBV vaccination, isolation of HBV-positive patients, use of dedicated dialysis machines, and regular surveillance for HBV infection dramatically reduced the spread of HBV in this setting.[7] However, the relatively low acceptance of and response rates to the HBV vaccine among dialysis patients likely contributes to ongoing transmission, as does the need for vaccine boosts to maintain antibody to HBsAg (anti-HBs) at protective levels.[8] The prevalence of HCV infection among patients on hemodialysis is high and varies between countries (2–60%) and between dialysis units within a single country.[9] Dual infection with HBV and HCV leads to more aggressive liver disease.[10] There are very few reports on the prevalence of such dual infections in hemodialysis patients. Hepatitis B surface antigen (HBsAg) and HCV antibody (anti-HCV) prevalence were reported as 2.3% and 0.37%, respectively, in Turkish population.[11] While the risk of the transmission of these viruses has been considerably reduced in developed countries owing to increased screening procedures,[12–14] the problem is not properly addressed in developing countries like Turkey. However, an abnormally high prevalence of these two viral infections has not been documented in Turkish patients.

Maintenance hemodialysis patients, hemophiliacs, and thalassemics form a major risk group for HBV and HCV infections owing to the frequent use of blood products and hemodialysis apart from the multiple invasive medical procedures to which these patients are exposed. It is now well documented that the prevalence of HCV in maintenance hemodialysis patients averages between 10 and 40%.[15] As these viral infections have common modes of transmission, the presence of co-infection in patients is not rare and is relatively high in transfusion recipients.[15–17]

With the development of specific and sensitive polymerase chain reaction (PCR)-based testing for HBV-DNA and HCV-RNA, few studies within dialysis units have demonstrated occult HBV (HBsAg-negative patients with HBV viremia) and occult HCV (anti-HCV negative patients with HCV viremia) infections. The prevalence rates reported for occult HBV and HCV infections in these studies ranged from 0–50% and from 1–15%, respectively.[18],[19] However, data on the prevalence rates of these two viral infections in maintenance hemodialysis Turkish patients is lacking. Although exact prevalence rates are not known, occult HBV and HCV infections carry risks of disease transmission, acute exacerbation, and development of hepatocellular carcinoma.[20],[21] Due to a high risk of infection during dialysis, it is necessary to determine whether HBsAg and anti-HCV negative sera contain HBV-DNA and HCV-RNA to prevent ongoing transmission.[22],[23] The present study was conducted on 188 maintenance hemodialysis patients to determine the prevalence of occult HBV and HCV infections as the primary objective.

MATERIALS AND METHODS

The present study was conducted on maintenance hemodialysis patients at the Department of Nephrology, Ministry of Health Ankara Etlik Specialization Hospital, Ankara, Turkey. A total of 188 patients (102 male) with a mean age of 49±2 (16–80) years and a mean hemodialysis period of 61±4 (12–228) months were included in the study. None of the patients had cirrhosis.

Blood samples from all patients were obtained during periodic (monthly) follow-ups, and they were centrifuged at 2500 rpm for 5 min for the separation of serum samples, which were then stored at −80ºC until the study. HBsAg, Hepatitis B early antigen (HBeAg), anti-HBe, antibody to hepatitis B core antigen (anti-HBc), and anti-HCV in these serum samples were determined using commercial diagnostic kits of ELISA (Access and BioRad, Beckman-Coulter, California, USA) according to the manufacturer's protocol.

HBV-DNA was measured by a PCR technique (Artus GmbH HBV RG PCR kit, Hamburg, Germany) with a lower detection limit of 100 copies/mL HBV-DNA. PCR was performed in 100 μl of serum samples, and DNA dissolved in 50 μl buffers.[24] Positive and negative controls were used for each group during DNA extraction and PCR steps. HCV-RNA was also determined by a PCR technique (Cobas Amplicor HCV Monitor^™ Test, version 2.0 kit, Roche Diagnostic Systems, California, USA) with a lower detection limit of 600 IU/mL. Extractions were added on a working master mix. The COBAS Amplicor analyzer automatically performed reverse transcription, amplification, and detection. In order to avoid contamination, a maximum physical separation between the pre- and post-amplification steps (separate rooms, separate pipettes and other laboratory material, separate laboratory coats and gloves) was done. All the protocols for avoiding contamination were applied as described previously.[25] Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were also measured in all blood samples. Normal levels of AST and ALT were considered as 8–33 U/L and 5–40 U/L, respectively. HBV-DNA or HCV-RNA positivity by PCR was accepted as marker of viremia.

Written informed consent was obtained from each patient included in the study, and the Local Ethics Committee approved the study protocol.

Statistical Analysis

Statistical analyses were performed using SPSS 11.0 version for Windows (SPSS Inc., Chicago, Illinois, USA). Comparisons of groups for parametric data were done by χ² test, and for non-parametric data by Wilcoxon test. AST and ALT levels were compared between groups with multivariate variance analysis. Statistical significance was assumed to be less than 0.05.

RESULTS

Table 1 summarizes the results of screening 188 patients for seromarkers of HBV and HCV. It can be readily observed that the prevalence of active HCV infection was higher than active HBV infection, defined as the presence of HCV-RNA or HBV-DNA in the serum, respectively (30 [16.0%] vs. 9 [4.8%]), although the difference was statistically not significant (p=0.6). Out of 188 patients, 25 (13.3%) were positive for HBsAg (21 [84%] patients HBV-DNA-negative and four [16%] patients HBV-DNA-positive) and 5 (2.7%) had occult hepatitis B (HBsAg-negative and HBV-DNA-positive). Anti-HBc positivity was 84 (44.7%) and “isolated anti-HBc” positivity was 12 (6.4%) in all patients. Isolated anti-HBc positivity was more prevalent in patients with occult hepatitis B compared to those without (40% vs. 5.5%, p=0.002). Of all patients, 38 (20.2%) were positive for anti-HCV (17 [44.7%] patients HCV-RNA-negative and 21 [55.3%] patients HCV-RNA-positive) and nine (4.8%) had occult hepatitis C (anti-HCV-negative and HCV-RNA-positive). Isolated anti-HBc was positive in three (7.9%) of the patients with anti-HCV. Seven (3.7%) patients were positive for both HBsAg and anti-HCV indicating dual infection. None of the patients with HCV had occult hepatitis B.

Table 1 Prevalence of HBV and HCV infections

Screening test (n=188)	Positivity n (%)
HBsAg	25 (13.3)
HBV-DNA	4 (16.0)
Anti-HBc	84 (44.7)
“Isolated anti-HBc”	12 (6.4)
HBV-DNA	2 (16.7)
Occult hepatitis B	5 (2.7)
“Isolated anti-HBc”	2 (40)
Anti-HCV	38 (20.2)
HCV-RNA	21 (55.3)
“Isolated anti-HBc”	3 (7.9)
Occult hepatitis C	9 (4.8)
Dual infection	7 (3.7)

Table 2 shows clinical features of the patients with and without occult hepatitis B and occult hepatitis C. Age, gender, and dialysis period were not significantly different among patients with and without occult hepatitis B and occult hepatitis C.

Table 2 Clinical features of the patients depending on occult HBV and occult HCV infections

	Occult HBV-positive (n=5)	Occult HBV-negative (n=183)	p	Occult HCV-Positive (n=9)	Occult HCV-Negative (n=179)	p
Age (years)	44.0±22.8	49.3±13.6	NS	50.2±12.2	49.1±14.0	NS
Gender (F:M)	2:3	84:99	NS	2:7	84:95	NS
Dialysis period (months)	34±22.8	62±49.2	NS	74±43.2	61±48.5	NS
Anti-HCV	0	38 (20.8)	NS
Isolated anti-HBc	2 (40)	10 (5.5)	0.002	1(11.1)	11 (6.1)	NS

Data are given as mean ± SD or n (%).

NS: not significant, F: female, M: male.

Table 3 summarizes the results of aminotransferase measurements. AST and ALT levels were similar in patients with and without HBV viremia; ALT levels, however, were higher in patients with non-occult HCV viremia than with occult HCV viremia and without HCV viremia (p = 0.004).

Table 3 Aminotransferase levels in maintenance hemodialysis patients

	HBV viremia-negative (n=179)	HBV viremia-positive (nonoccult) (n=4)	HBV viremia-positive (occult) (n=5)	p	HCV viremia-negative (n=158)	HCV viremia-positive (nonoccult) (n=21)	HCV viremia-positive (occult) (n=9)	p
AST (mean, IU/L)	16.7±16.8	12.0±3.2	12.0±1.4	NS	15.8±16.8	22.8±16.3	12.9±3.9	NS
ALT (mean, IU/L)	17.0±16.4	13.5±5.1	12.8±1.9	NS	15.4±12.9	27.6±29.8	16.3±6.2	0.004

AST: aspartate aminotransferase, ALT: alanine aminotransferase, NS: not significant.

DISCUSSION

The present study reports on the prevalence of hepatitis B and C in 188 hemodialysis patients in Ankara, Turkey. Twenty-five (13.3%) patients had HBV infection alone and 38 (20.2%) patients had HCV infection alone, while seven (3.7%) patients had dual infection of both viruses. Frequencies of occult hepatitis B and occult hepatitis C were 2.7% and 4.8%, respectively. Isolated anti-HBc was positive in 12 (6.4%) of all patients, three (7.9%) of the patients with anti-HCV, and two (40%) of the patients with occult hepatitis B. None of the patients with HCV had occult hepatitis B. AST and ALT levels were similar in patients with and without HBV viremia. ALT levels were higher in patients with non-occult HCV viremia than with occult HCV viremia and without HCV viremia. None of the patients had cirrhosis.

Hemodialysis patients are an important risk group for HBV and HCV infections, and these infections are the most common causes of liver disease in hemodialysis patients.[1–3] HBV prevalence in maintenance hemodialysis patients varies among studies and geographical areas. It has been reported to be as low as 0.8–17.0%.[26–34] In the present study, HBV was positive in 13.3% of hemodialysis patients. Hepatitis C prevalence, in the largest studies in various countries, was reported between 5.9% and 41.0%.[26],[29–31],[34–43] However, the prevalence rates of 46% in India,[31] 75% in Moldavia,[32] 49% in Turkey,[44] and 68% in Saudi Arabia[45] were also reported. A prevalence rate of 20.2% was observed for HCV in the present study's patients, which was similar to the majority of reports in the literature[20],[41],[42] but lower than the rates from different countries.[31],[43–45] The possible explanation for the lower HCV prevalence in this study than the above-mentioned reports may be related to the regular screening of HCV markers and isolation of dialysis devices.

Hemodialysis patients are at high risk for hepatitis viral infections due to the high number of blood transfusions, prolonged vascular access, and potential for exposure to infected patients and contaminated equipment.[46] The prevalence of HBV and HCV infections among blood donors from Turkey was reported to be 2.3% and 0.37%, respectively.[11] In the current hemodialysis patient group, blood transfusion history was positive in two patients with HBV, ten patients with HCV, and all of patients with dual infection.

Occult hepatitis B and occult hepatitis C in hemodialysis patients have been rarely reported. Minuk et al.[27] reported occult hepatitis B positivity as 3.8% in 239 adult hemodialysis patients. Besisik et al.[21] reported occult hepatitis B positivity as 36.4% in 33 hemodialysis patients with HCV. Bozdayi et al.[39] found that occult hepatitis C was positive in 3.2% of 94 hemodialysis patients. Schroter et al.[40] detected HCV infection in 12 (5.0%) cases among 238 sero-negative hemodialysis patients. Occult hepatitis B and occult hepatitis C were detected in five (2.7%) and in nine (4.8%) of our patients, respectively.

Anti-HBc may be the sole marker of resolved HBV infection, as anti-HBs, which is neutralizing and so appears after the clearance of HBsAg, may disappear from serum many years after the resolution of HBV infection. The clinical significance of “isolated anti-HBc” in serum (i.e., anti-HBc-positive status in HBsAg-negative patients) is still unclear in dialysis patients as well as in non-uremic populations. Some reports suggest that anti-HBc-positive status implies a potentially infectious state. Depending on the anti-HBc test used and the prevalence of true positive results, a certain proportion of anti-HBc-positive patients may be false positive.[47] Carpenter et al.[20] showed that the prevalence of anti-HBc is higher among anti-HCV-positive individuals. Besisik et al.[21] could find “isolated anti-HBc” positivity in none of the patients with anti-HCV, but this study found “isolated anti-HBc” in three (7.9%) patients with anti-HCV. Besisik et al.[21] and Haushofer et al.[48] could not find any difference in regard to anti-HBc seropositivity between patients with and without occult HBV infection. However, the present study showed that anti-HBc positivity was 84 (44.7%) and “isolated anti-HBc” positivity was 12 (6.4%) in all patients. Isolated anti-HBc positivity was more prevalent in patients with occult hepatitis B compared to those without (40% vs. 5.5%).

Dual infection with HBV and HCV, though rare, occurs more frequently in certain risk groups. The risk is greater among the CRF patients due to the frequent exposure to blood from transfusions and extracorporeal circulation during hemodialysis. Dual infection with HBV and HCV leads to more aggressive liver disease.[10] A significant risk of cirrhosis development and decompensation of liver function is observed in HBV- and HCV-infected hemodialysis patients.[26] However, no signs of cirrhosis had been observed in any of the present study's patients. Studies on the prevalence of HBV and HCV co-infection in hemodialysis units are rare. Prevalence of HBV and HCV co-infection in non-uremic patients was reported by several authors and ranged between 3–56%.[10],[26],[49],[50] Kara et al.[51] reported dual infection in 0.04% of hemodialysis patients. The present study found the prevalence of dual infection as 3.7% in hemodialysis patients.

The suppression of HBV replication in patients co-infected with HCV to the level of carrier status has been documented. It is believed that in such co-infected patients, HCV appears to be the cause of active viral hepatitis. Occult HBV infection was reported in patients with chronic hepatitis C liver disease;[20] However, its prevalence and clinical significance are unknown in chronic hepatitis C patients on hemodialysis. In the current study, none of the patients with HCV had occult hepatitis B.

It was previously shown that HBsAg-positive dialysis patients with active viral replication shows the greatest liver damage,[52] and detectable HBV-DNA in serum is a strong and independent predictor of raised aminotransferase activity among dialysis patients.[53] Previously, serum AST and ALT levels were found significantly higher in viremic patients than in individuals with no detectable HCV-RNA in serum, and an association between HCV viremia, anti-HCV positivity, and ALT activity was shown in logistic regression model.[54] On the other hand, in a recent study, it was reported that elevated liver enzymes are infrequent in patients with HCV infection undergoing chronic hemodialysis, suggesting that the ALT is a poor predictor of hepatocellular damage in this population.[55] In the current study, AST and ALT levels were similar in patients with and without HBV viremia. However, ALT levels were higher in patients with non-occult HCV viremia than with occult HCV viremia and without HCV viremia.

In conclusion, both occult and non-occult forms of HCV infection are more prevalent than HBV infection in hemodialysis patients. Dual infection is less prevalent than either infection. Especially the patients with “isolated anti-HBc” positivity should be tested for probable occult hepatitis B infection. Serum aminotransferase levels are not useful as a screening method for occult HBV and occult HCV infections on maintenance hemodialysis patients. Therefore, screening strategies for viral infections should be revised, including the addition of molecular diagnostic methods for these special sub-groups of hemodialysis patients.