About Bioline  All Journals  Testimonials  Membership  News

Medical Journal of The Islamic Republic of Iran
National Research Centre of Medical Sciences of I.R. IRAN
ISSN: 1016-1430
Vol. 19, Num. 2, 2005, pp. 147-151

Medical Journal of the Islamic Republic of Iran , Vol. 19, No. 2, August, 2005, pp. 147-151



From the Research Center of the Iranian Blood Transfusion Organization, Tehran, Iran.

*Scientific Member & Assistant Professor of IBTO, Iranian Blood Transfusion Organization, Sheikh Fazlollah Expressway, Shahid Hemmat, Tehran, Iran. Fax: 00 98 21 88601518, Tel: 88288581-3 rnegative

Code Number: mr05009


Background: Hepatitis B virus (HBV) infection in patients who lack detectable hepatitis B surface antigen (HBsAg) is called occult hepatitis B infection. Such infections have been frequently identified in patients with chronic hepatitis C liver disease, but their prevalence is not known.
Methods: 207 patients with chronic hepatitis C who were HCV-RNA and anti-HCV positive were studied for HBV-DNA by PCR, and for HBsAg and anti-HBc by ELISA. DNA was extracted by high pure nucleic acid kit (Roche-Germany). HBVDNA amplification was done with a set of primer directed to the pre-S region. HBsAg and anti-HBc were evaluated by a commercially available ELISA kit (Dade Behring).
Results: 23 of 207 patients with chronic hepatitis C liver disease (11.1%) were positive for HBV-DNA (co-infection). Among this group 17 patients (8.2%) were HBsAg negative (occult infection). 8 of 17 patients with occult infection (47%) were anti-HBc positive and 9 were anti-HBc negative (53%). No significant difference was found in epidemiological and biochemical parameters in patients with HCV alone in comparison with HCV co-infected with occult hepatitis B (p= 0.453 for ALT and p= 0.498 for AST).
Conclusion: Occult hepatitis B virus infections occur frequently in patients with chronic hepatitis C liver disease and may have clinical significance.

Keywords: Occult hepatitis B, Surface antigen, chronic hepatitis C, PCR.


Hepatitis B virus (HBV) and hepatitis C virus (HCV) infections are the most common causes of chronic liver disease in the world. Both viruses induce chronic hepatitis, which may progress to cirrhosis and eventually to hepatocellular carcinoma.1 It is estimated that there are 350 million HBV carriers and 170 million HCV carriers worldwide. HCV is an RNA virus of the Flaviviridae family and HBV is a DNA virus of the Hepadnaviridae family.2 HBV and HCV share modes of transmission, and their combined infection seems to be frequent, particularly in areas where the two viruses are endemic and among people at high risk for infection such as IV drug abusers, blood receivers like hemophiliacs and thalassemics, and by parenteral transmission.3,4 The diagnosis of HBV infection is usually based on the detection of hepatitis B surface antigen (HBsAg) in serum, and disappearance of this antigen indicates the clear ance of HBV. HCV infection is diagnosed by detection of anti-HCV and viral RNA in the serum.5 However, previous studies have shown that HBV DNA can be detected in patients with chronic liver disease who are negative for HBsAg but positive for antibodies to hepatitis B core antigen (anti-HBe).6-7 More recently, this so-called occult hepatitis B has frequently been identified in patients with chronic HCV infection. Patients with occult HBV and HCV co-infection usually have a delayed appearance, lower level, and shortened duration of HBsAg. Studies showed that HCV core protein is able to inhibit HBV replication, and for this suppressor effect the phosphorylated serines at position 99 and 116 seems to be essential. In addition, a high frequency of an eight-nucleotide deletion in HBV core promoter/distal X region was recently reported in patients with HCV and occult HBV infection, and this deletion mutation appears not to be present in patients with active HBV infection alone.8 Considerable data suggest that this occult infection is clinically relevant because it may be associated with severe forms of liver disease, poorly sensitive to interferon treatment and with a high risk of hepatocellular carcinoma development.9

However, whether the presence of such small amounts of HBS will lead to progressive disease has been questioned and indeed needs further confirmation from other areas of the world where HBV infection is rampant.

Taking advantage of the fact that HBV and HCV infections are common in Iran, we determined the prevalence of occult HBV infection in patients with HCV-related chronic liver disease and studied the possible influence of occult HBV infection on the clinical outcomes of infected patients.


Selection of cases

Serum and plasma samples from 207 referral patients to the molecular diagnostic lab of the Iranian Blood Transfusion Organization plus 130 cases as control were obtained by vacutainer system.

All of the patients had a history of positive HCVPCR and HCV-Elisa. Both patient and case control samples were subject to HBV-DNA, HBsAg, HBcAb, HBcAg, HBeAb, HBsAb, ALT, and AST tests.

None of them were under treatment. 173 of the patients were male and 34 female with age between 8-79 years old.

Serological markers

Tests for HBsAg, anti-HBs, antiHBc and anti-HCV were done with commercially available kits (Avecina, Russia, Ortho, Dada Behring, Biorad, USA).

Nucleic acid extraction

Plasma samples were frozen at -70°C before starting the nucleic acid extraction procedure. RNA was extracted preserved at -70°C.


Serum was assayed for HCV-RNA by reverse-transcription-PCR with random hexamer and specific primer for 5´ untranslated region.

The sensitivity of the assay was estimated by vqc panel ( less than 380 copy/mL).

HBV-DNA was analyzed by 2 set primer for pre S conserved region.

We used hot start method to decrease non-specificity and increase both sensitivity and specificity of the reaction.

All precautions for avoiding PCR contamination were followed strongly. Each positive result was confirmed by a second independent PCR.

PCR products for both HCV and HBV were electrophoresed on 1.8 % agarose. A 150 bp and 240 bp corresponded to positive PCR reaction for HCV and HBV genome.

Serum ALT and AST level

ALT and AST activity were estimated by Pars Azmun kit ( Tehran- Iran ) with a Hitachi auto-analyzer.


HBV-DNA evaluation in HCV-RNA positive patients

Of 207 patients with chronic hepatitis C (confirmed by Elisa-Riba and PCR) 23 patients (11.1%) were positive for HBV-DNA PCR for pre S region ( co-infection of HBV and HCV).

Among co-infected patients 9.7 % were male and 1.4% female. Also the majority of patients were between 20-40 years old (47.8%). Table I characterizes the prevalence and distribution of co-infected patients. In addition 130 HBsAg, HBcAb, and HBsAb negative referral patients were also subject to HBV-DNA analysis.

All cases were HBV-DNA negative according to vqc (viral quality control–Netherlands), and the sensitivity of our PCR and RT-PCR assay results was 300 and 380 geg/mL (Fig. 1).

Occult versus co-infected

In order to further clarify the nature of HBV-DNA positivity among the HCV-RNA population, we evaluated HBsAg status in this group. As Table I shows by modified Sacchi Chomzynski method (Trisol-LS, invitrogen-USA). Briefly 250 µL plasma plus 750 µL Trisol were mixed and 2 phase separate and RNA were precipitated with cold ethanol. Pellets were dissolved in 20 µL DEPC treated water. DNA was extracted by high pure viral nucleic acid kit (Roche, Germany) according to the manufacturer’s procedure. Both DNA and RNA were among this group 8.2% (17) of the 11% (23) who were HBV-DNA+ (HCV-RNA+) were HBsAg negative.

For further characterization of HBsAg negatives we determined total anti-HBc for this group. Prevalence of anti-HBc positivity in Table II is shown. 53% (9/17) of HBsAg-, HBV-DNA+> , HCV-RNA+ patients have negative HBcAb and in contrast 47% (8/17) have positive HBcAb. According to our criteria the group with HBsAg (-), antiHBc(-) HBV-DNA (+), HCV-RNA (+) were suitable candidates for the occult HBV form (Fig. 2).

According to Table II, males between 20-40 years old are the predominant group of occult HBV.

As shown in Table III, no significant difference was found in epidemiological parameters in the two groups (HCV alone in comparison with HCV and occult hepatitis B co-infection). In the two groups, transfusion and IV drug use was the most frequent epidemiological characteristic.


HBV infection is diagnosed when circulating HBsAg is detected.10 However, a unique persistent infection known as occult HBV infection, which is characterized by positivity for HBV-DNA in serum by using nested PCR assays, has been identified in HBsAg-negative pa-

Table I. Prevalence of co-infection and occult hepatitis.

Variable % No. of HCV-RNA (+) studies No. (%) with HBV-DNA (+) Co-infection No. (%) with HBs-Ag (-) Occult hepatitis
Sex (Total)
Male Female
Age (years)




                    HBV- DNA (+)

A= % ————————————

                  Total no. of patients( 207 )

                         HBS-Ag (-)

B= ————————————

            Total no. of patients ( 207 )

Table II. Prevalence of Anti-HBc in occult hepatitis.

Variable Anti-HBc(+) Anti-HBc(-)
Sex (Total) 8(47%)a 9(53%)b
Male 7(41.1%) 7(41.2%)
Femal 1(5.8%) 2(11.7%)
Age (years)
8-20 1(12.5%) 3(33.3%)
20-40 5(62.5%) 4(44.4%)
40-60 2(25%) 2(22.2%)
60-80 0(0.0) 0(0.0)

                    HBV- DNA (+)                                    HBS-Ag (-)                      

A= % ————————————      B= ————————————

            Total no. of patients ( 207 )                   Total no. of patients( 207 )                       

 tients with or without serological markers of previous HBV infection ( anti-HBs or anti-HBc).11,12,13,14 Several recent studies have indicated that this occult HBV infection can be found in patients with chronic HCV infection at various frequencies.15,16,17,18 The high prevalence of occult HBV infection in such patients has been suggested to have clinical implications in the pathogenesis of HCV-induced chronic liver disease.

The reason for disappearance of HBsAg is not well known, but in subjects co-infected with HCV, it seems that the HCV core protein can inhibit HBV replication15

HBV viral level in serum is usually less than 104 copies/mL in patients with occult infection which is significantly lower than in those with HBsAg positive infection.3 Thus we need a sensitive PCR technique for detection of this form of infection.

Table III. Epidemiological characteristics of the two groups.

Transfusion 70(38%) 9(52.9%)
IV Drug use 48(26%) 5(29.4%)
Thalassemia 18(9.7%) 4(23.5%)
Hemophilia 25(13.5%) 3(17.6%)
Tattoo 16(8.6%) 3(17.6%)
Transfusion 7(3.8%) 2(11.7%)
+ IV Drug use


This study indicates a high rate of hepatic HBV-DNA turn-over in patient with HBsAg-negative chronic liver disease. Indeed when DNA-PCR results were analyzed, we found that 11.1 percent of patients with HBsAg-negative chronic liver disease had been detected by HBVDNA .

Previous studies have shown that patients co-infected with occult hepatitis B and HCV suffer from a more severe disease. Studies have shown that patients with HCV and occult HBV had a significantly higher incidence of cirrhosis than those with HCV infection alone.16 Recent studies demonstrate that patients with occult hepatitis B and HCV infection have a poorer response to α-interferon compared to those with chronic hepatitis C alone.16

We investigated HBV infection in HBsAg negative patients with chronic hepatitis C. It seems the frequency of the occult form of HBV is significant in chronic hepatitis C. The prevalence of occult HBV infection was particularly high among patients with Anti-HBc antibodies. And we found that occult HBV infection co-infected with HCV was particularly high among patients at high risk for two viruses like multi-transfusion patients and IV drug abusers.


  1. Ke-Qin KE: Occult hepatitis B virus infection and its clinical implication. J Viral Hepatitis 9: 243-257, 2002.
  2. Squadrito G, Orlando ME, Pollicini T, Raffa G, Restuccia T, et al: Virological profiles in patients with chronic hepatitis C and overt or occult HBV infection. Am J Gastroenterol 97(6): 1518-1523, 2002.
  3. Cacciol I, Pollicino T, Squadrito G, et al: Occult hepatitis B virus infection in patients with chronic hepatitis C liver disease. New Eng J Med 341(1): 22-26, 1999.
  4. Sato S, Shigetoshi F, Tanaka M, et al: Co-infection of hepatitis C virus in patients with chronic hepatitis B infection. J Hepatol 21: 159-166, 1994.
  5. Brechot C, Thiers V, Kremsdorf D, et al: Persistent hepatitis B virus infection in subjects without hepatitis B surface antigen: clinically significant or purely occult. Hepatology Jul 34(1): 194-203. 2001.
  6. Shintani Y, Yotsuyanagi H, Moriya K, et al: The significance of hepatitis B virus DNA detected in hepatocellular carcinoma of patients with hepatitis C. Cancer 88: 24782486, 2000.
  7. Shih CM, Miyamura T, et al: Suppression of hepatitis B virus expression and replication by hepatitis C virus core protein in HuH-7 cells. J Virol 67: 5823-5832. 1993.
  8. Chan HL,T Sang SW, Leung NW, Tse CH, Hui Y, Tam JS, Chan JJ: Occult HBV infection in cryptogenic liver cirrhosis in areas with high prevalence of HBV infection. Am J Gastroenterol May 97(5): 1211-5, 2002.
  9. Kubo S, Tamori A,Ohba K,Shuto T,Yamamoto T, Tanaka H, Nishiguchi S, Wakasa K: Previous or occult hepatitis B virus infection in hepatitis C virus associated hepatocellular carcinoma without fibrosis. Dig Dis Sci Nov 46(1): 240814, 2001.
  10. Shiota G, Oyama K, Udagawa A, et al: Occult hepatitis B virus infection in HBsAg negative hepatocellular carcinoma in a Japanese population: involvement of HBx and p 53. J Med Virol Oct 62(6): 151-8, 2000.
  11. Tamori A, Shuhei NI, Kubo S, et al: Possible contribution to hepatocarcinogenesis of x-transcript of hepatitis B virus in Japanese patients with hepatitis C virus. Hepatology 29: 1429-1434, 1999.
  12. Cabreizo M, Bartolome J, Caramelo C, et al: Analysis of hepatitis B virus DNA in serum and peripheral blood mononuclear cells from hepatitis B surface antigen negative cases. Hepatology 32: 116-123, 2000.
  13. Alberti A, Pontsso P, Chemello L, et al: The interaction between hepatitis B virus and hepatitis C virus in acute and chronic liver disease. J Hepatol 22(suppl): 38-41, 1995.
  14. Zarski JP, Bohn B, Bastie A, et al: Characteristics of patients with dual infection by hepatitis B and C viruses. J Hepatol 28: 27-33, 1998.
  15. Sato S, Shigetoshi F, Tanaka M, et al: Co-infection of hepatitis C virus in patients with chronic hepatitis B infection. J Hepatol 21: 159-6, 1994.
  16. Ohkawa K, Hayashi N, Yuki N, et al: Long term follow-up of hepatitis B virus and hepatitis C virus replication levels in chronic hepatitis patients co-infected with both viruses. J Med Virol 46: 258-64, 1995.

Copyright 2005 -Medical Journal of the Islamic Republic of Iran

The following images related to this document are available:

Photo images

[mr05009f2.jpg] [mr05009f1.jpg]
Home Faq Resources Email Bioline
© Bioline International, 1989 - 2024, Site last up-dated on 01-Sep-2022.
Site created and maintained by the Reference Center on Environmental Information, CRIA, Brazil
System hosted by the Google Cloud Platform, GCP, Brazil