Memórias do Instituto Oswaldo Cruz Fundação Oswaldo Cruz, Fiocruz ISSN: 1678-8060 EISSN: 1678-8060 Vol. 97, Num. 1, 2002, pp. 105-107

Mem Inst Oswaldo Cruz, Rio de 
  Janeiro, Vol. 97(1) 2002, pp. 105-107
Detection of Hepatitis B Virus 
  Antigens in Paraffin-embedded Liver Specimens from the Amazon Region, Brazil 
   
SRR Simonetti, HG Schatzmayr, OM Barth+, 
  JP Simonetti  
Departamento de Virologia, Instituto Oswaldo 
  Cruz-Fiocruz, Av. Brasil 4365, 21045-900 Rio de Janeiro, RJ, Brasil 

  +Corresponding author. Fax: +55-21-2270.6397. E-mail: barth@ioc.fiocruz.br 
  
This work received financial support from 
  the Conselho Nacional de Desenvolvimento Científico e Tecnológico 
  (CNPq), Brazil. 
Received 3 May 2001 

  Accepted 5 September 2001 
Code Number: oc02019 
Hepatic viscerotomy of paraffin-preserved 
  old specimens, collected in the period from 1934 to 1967, were analyzed by immunohistochemical 
  assays to detect hepatitis B, hepatitis D, dengue and yellow fever virus antigens. 
  The material belongs to the Yellow Fever Collection, Department of Pathology, 
  Instituto Oswaldo Cruz, Rio de Janeiro, Brazil and the cases were diagnosed 
  at that time according to clinical aspects and histopathological findings reporting 
  viral hepatitis, yellow fever, focal necrosis and hepatic atrophy. From the 
  79 specimens, 69 were collected at the Labrea Region and the other 10 in different 
  other localities in the Amazon Region. The five micra thick histological slices 
  were analyzed for the presence of hepatitis B surface antigen (HBsAg) and hepatitis 
  B core antigen (HBcAg) by immunoperoxidase technique. An immunofluorescence 
  assay was applied to the detection of hepatitis D, yellow fever and dengue virus 
  antigens. Nine (11.4%) histological samples were HBsAg reactive and 5 (6.3%) 
  were HBcAg reactive. The oldest reactive sample was from 1934. Viral antigens 
  related to the other pathologies were not detected in this study. Our results 
  confirm that the methodology described may be used to elucidate the aetiology 
  of hepatitis diseases even after a long time of conservation of the specimens. 
   
Key words: hepatitis B virus - human liver - 
  Amazon - Brazil  
According to the World Health Organization, hepatitis 
  B virus (HBV) carriers account for 5% of the world population. These potential 
  infection transmitter individuals can evolve from the asymptomatic condition 
  to a severe hepatic damage and even hepatocellular carcinoma. A similar HBV 
  epidemiological distribution is observed for hepatitis D virus (HDV) that shares 
  the obligatory association to HBV for its replication (Smedile et al. 1981). 
  Frequently related to HBV chronic carriers and less frequently to the acute 
  disease forms (Colombo et al. 1983, Rizzetto et al. 1983), HDV antigen was identified 
  by immunohistochemical assays in liver cell nuclei of patients with chronic 
  persistent or chronic active hepatitis, hepatitis B surface antigens (HBsAg-positive) 
  (Rizzetto et al. 1977). In South America HDV infection was first observed in 
  Venezuela as a severe and fulminant attack among Yucpa indians (Purcell & 
  Gerin 1983) and in Colombia where 60% of the individuals studied were simultaneously 
  HBV and HDV reactive (Ljunggren et al. 1984). In Brazil, the Amazon Region endemicity 
  for both viruses is well known (Figueiredo Mendes et al. 1984, Fonseca et al. 
  1986, Simonetti et al. 1986) showing high prevalence pattern compared to the 
  other parts of the world (Purcell & Gerin 1983, Nordenfelt et al. 1983, 
  Fonseca et al. 1988). This region is also endemic for yellow fever and it is 
  thought whether another human hepatitis viruses had been introduced in this 
  area by human serum present in vaccines against yellow fever, since there are 
  observations on the occurrence of icterus in England and in Brazil, following 
  vaccination against yellow fever in the 1930s and 1940s decades (Findlay & 
  Mac Callum 1937, 1938, Soper & Smith 1938, Fox et al. 1942). We included 
  the dengue virus antigen research in this study, once the same pathological 
  liver findings are observed in yellow fever and haemorrhagic dengue, such as 
  hepatomegaly, focal visceral haemorrhages, focal necrosis, sinusoidal acidophilic 
  bodies, Kupffer cell hypertrophy and portal tract mononuclear cell infiltration. 
   
To observe the simultaneous HBV and HDV antigen 
  frequency and to detect yellow fever and dengue viral antigens in histological 
  sections we studied 79 hepatic samples by immunohistochemical assays. Hepatic 
  viscerotomy samples belong to the Yellow Fever Collection (Department of Pathology, 
  Instituto Oswaldo Cruz, Brazil) created in the 1930s decade from an agreement 
  between Brazilian Government and the Rockefeller Foundation International Division 
  to study yellow fever in Brazil. 
MATERIALS AND 
  METHODS 
Material - Seventy-nine paraffin-embedded 
  hepatic samples collected from 1934 to 1967 were analyzed. Sixty-nine samples 
  were from the Labrea Region, State of Amazonas and ten random samples were from 
  other different localities in the same state. The cases were diagnosed at that 
  time as viral hepatitis, yellow fever, focal necrosis or hepatic atrophy according 
  to clinical aspects and histopathological findings.  
Methods - Four or five micra thick paraffin-embedded 
  histological sections were prepared for hepatitis B, hepatitis D, yellow fever 
  and dengue virus antigen detection by immunoperoxidase or immunofluorescence 
  techniques. HBsAg and hepatitis B core antigens (HBcAg) were detected by the 
  immunoperoxi-dase assay (Immuno Tag S - Immunon-Lipshaw Corporation). Paraffin 
  sections were deparaffinated and treated with 90% and 85% ethyl alcohol solutions 
  followed by 10% ammonium hydroxide solution. To block endogenous peroxidase, 
  sections were incubated with 3 to 10% hydrogen peroxidase solution for 5 min. 
  Sections were then incubated with peroxidase/anti-peroxidase conjugate, stained 
  with hematoxylin solution (1 min) and treated with 0.2% ammonium hydroxide solution. 
  The reaction colour was developed after 3-amino-9-ethylcarbazole substrate addition. 
  Positive HBsAg and HBcAg controls were included for every batch. HDAg was detected 
  by the immunofluorescence assay as follows: 5 µm thick paraffin-embedded 
  sections were deparaffinated, rehydrated and digested with 0.1% protease solution 
  (type XXVII Sigma) in Tris-HCl 0.5M pH 7.4 for 2 h at 37ºC or 0.05% trypsin 
  (Difco) in phosphate buffered saline pH 7.4 for 10 min at 37ºC. Digested 
  sections were incubated at room temperature for 30 min with FITC-anti HD conjugate 
  diluted 1:100 in PBS pH 7.4. Yellow fever and dengue virus antigens were tested 
  by immunofluorescence (Huang et al. 1976, Walker & Cain 1978, Schatzmayr 
  et al. 1984, Barth et al. 1988): 4 µm thick paraffin sections were deparaffinated, 
  rehydrated and digested with 0.1% trypsin with 0.1% CaCl2 solution 
  in PBS pH 7.8 for 2 h at 37ºC. Digested sections were incubated at 37ºC 
  for 30 min with FITC-conjugated yellow fever positive simian serum diluted at 
  1:20 in PBS pH 7.2. Flavivirus hyperimmune ascitic fluid obtained from mouse 
  was used to detect dengue virus antigen. After washing sections were mounted 
  with buffered glycerol and examined at the UV microscope. Four controls were 
  used for yellow fever virus detection: not infected Vero cells, yellow fever 
  virus (17D strain)-infected Vero cells, not infected liver cells and HBV-infected 
  liver cells. Aedes albopictus C6/36 infected cell line was included as 
  positive control for dengue virus detection. 
RESULTS 
  
Nine (11.4%) of 79 hepatic samples were HBsAg-reactive 
  and 5 (6.3%) were HBcAg-reactive by immunoperoxi-dase assay (2 of them showed 
  reactivity for both viral markers). HDAg was detected in none of these reactive 
  samples. HBsAg and HBcAg were more frequently seen in samples from people at 
  lower ages since in both groups examined (male and female) we had a higher number 
  of samples from persons under 21 years old. Two samples showing simultaneous 
  HBsAg and HBcAg reactivity were from boys agged 2 and 6 years. Chronological 
  distribution of viral markers detected in this study showed that old preserved-paraffin 
  liver samples are still suitable for viral markers detection by immunoperoxidase 
  assay. The results are shown in the Table. 
   
An exclusive cytoplasmic HBsAg localization and 
  an exclusive nuclear HBcAg localization were observed by immunoperoxidase assay. 
  The results are shown in Figs 1-4.  

Yellow fever and dengue virus antigens were seen 
  in none of the 79 hepatic samples by immunofluorescence assay.
DISCUSSION 
  
The Labrea Region, State of Amazonas, shows a 
  high HBV endemical level (Figueiredo Mendes et al. 1984, Fonseca et al. 1988); 
  this was an important point in sample selection and could have influence on 
  the uneven age range distribution observed. Hepatitis B virus antigen detection 
  in the liver tissue samples by immunohistochemical assays showed a cytoplasmic 
  HBsAg and a nuclear HBcAg localization.  
Only the HBsAg-reactive (9 of 79) and HBcAg-reactive 
  (5 of 79) samples were analyzed for the presence of HDAg. None of them showed 
  reactivity. The possible explanations for this are (1) HDV is mainly found in 
  young adults and 71.3% of people analyzed in our study were children under 14 
  years old (Fonseca et al. 1988); (2) the immunofluorescence method had low sensitivity 
  that could not detect HDV antigen in low level concentration samples; and (3) 
  the more recent HDV introduction in this region, which would be subsequent to 
  the analysis period of the samples (Fonseca et al. 1986, 1988, Simonetti et 
  al. 1986).  
The retrospective analysis evidenced HBsAg preservation 
  in old histological samples. The oldest one, from 1934, was from a 9 year old 
  boy from Labrea Region, Amazonas, with a negative yellow fever diagnosis.  

All the 79 samples were analyzed for dengue and 
  yellow fever virus antigen detection. The results were negative. This could 
  be due to the low sensitivity of the method, as discussed for HDV (Fonseca et 
  al. 1988). Alternatively, other viruses causing acute liver inflamation and 
  necrosis, determining similar hepatic lesions to those described, or non-viral 
  fatal infections such as malaria and leptospirosis, could be considered aetiological 
  agents.  
Our results confirm that the methodology described 
  may be used to elucidate the aetiology of hepatitis diseases even after long 
  time of conservation of the specimens. Retrospective studies of viral infection 
  of the liver (Schatzmayr et al. 1984) were used in order to acquire a better 
  knowledge of the natural history of these diseases and to may compare the data 
  obtained with the actual distribution of hepatitis in the Amazon region.
ACKNOWLEDGMENTS  

To Ms Itália B Kerr, Department of Pathology, 
  Instituto Oswaldo Cruz, for delivery of specimens and related data. To Ms Ângela 
  T Pinhão, Department of Virology, Instituto Oswaldo Cruz and to Dr Carlos 
  Alberto Basílio, Pathology Service, Gafrée Guinle University Hospital, 
  Rio de Janeiro, for technical assistance.  
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  Instituto Oswaldo Cruz - Fiocruz

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