Detection of Hepatitis Virus B And C in Archival Autopsy Specimens of Liver Cirrhosis

3Department of Internal Medicine, Kojin Hospital, Nagoya, Aichi, Japan 4Department of Pathology & Microbiology, Faculty of Medicine, Saga University, Saga, Japan 5Department of Anatomic Pathology, Pathological Sciences, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan 6Histopathology Core Facility, Niigata University Faculty of Medicine, Niigata, Japan 7Division of Molecular and Diagnostic Pathology, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan 8Department of Pathology, Niigata Medical Center, Niigata, Japan

Faculty of Medicine, Niigata, were chosen. Hepatitis B Virus (HBV) was demonstrated by immunostaining for HBs antigen, while Hepatitis C Virus (HCV) was detected by nested Reverse Transcription-Polymerase Chain Reaction (RT-PCR) using total Ribonucleic Acid (RNA) extracted from fixed tissues or paraffin sections. The present report gives the direct evidence for hepatitis virus detection in archival specimens: Infection of HBV and HCV was proven even in 110-year-old formalin-fixed liver samples.

Target Archival Samples
A total of ten specimens of advanced liver cirrhosis displayed in Kyushu University Museum, Fukuoka, were sampled from long-fixed tissues. The autopsy was done in the period from 1907 to 1930 (the fixation periods reached 86-109 years). The specimens were chosen by gross appearance of cirrhosis, so that paraffin sections prepared from all the ten lesions microscopically showed liver cirrhosis of common type (Table 1). Another series included twentytwo paraffin blocks of liver specimens autopsied in the period from 1940 to 1957 and kept at room temperature in the Department of Pathology, Niigata University Faculty of Medicine, Niigata, including chronic active hepatitis and liver cirrhosis ( Table 2). The specimens were chosen by three pathologists, RO, YA and MN, according to the autopsy diagnosis on the autopsy note. Hematoxylin and Eosin (HE) and Azan stains were performed to confirm histological features.  were employed as the secondary reagent. Diaminobenzidine color reaction and nuclear counterstaining with Mayer's hematoxylin followed. The specificity of immunostaining was confirmed both by using known positive and negative control liver specimens and by comparing with immunostaining with a polyclonal anti-HBs antibody (Agilent Technologies, Hachioji, Japan).

Patients' Summary
Cases K1-K10 were autopsied at Kyushu University in 1907 through 1930. The male to female ratio was nine-to-one, and the age ranged from 30 to 59 years (mean 43.1, median 41.5). All the livers were cirrhotic, and three lesions were associated with Hepatocellular Carcinoma (HCC). Brief comments on histologic features are described in Table 1.
HCC was associated in four cirrhotic lesions. The male to female ratio was fourteen-to-eight, and the age ranged from 14 to 65 years (mean 40.4, median 39). The age of case N13 was unknown. Brief comments on histological features are described in Table 2.

Status of Hepatitis Virus Infection
Among ten cirrhotic lesions of the Kyushu University series, HBs antigen was immunohistochemically detected in eight (80%).
HCV genome was detected in two liver lesions (cases K2 and K4), and both belonged to the genotype 1b (Table 1 and Figure 1). Of   By RT-PCR analysis, GAPDH signals were consistently demonstrated in all the RNA samples.

Discussion
We demonstrated the usefulness and value of archival liver specimens for detecting HBV and HCV. Of surprising note is that HBs antigen and HCV genome tolerated prolonged storage of paraffin sections, as well as long fixation in formalin. Reportedly, HBs antigen and HBV genome were detectable after 60 days storage of blood at room temperature [8]. It is nearly impossible for us to obtain archival serum or blood samples without keeping in freezers. The pathology tissues can thus be incarnated as historical As shown in our archival specimens, genotype 1 is known to be most common among HCV prevailing in the world [9]. Co-infection of both HBV and HCV, common in highly endemic areas and among high-risk individuals such as injection drug users and hemodialysis patients, accelerates progression to cirrhosis and HCC [10,11].
Infrequently, cases of fulminant hepatitis C have been recorded so far [12,13]. Pathobiology of viral hepatitis in the era before 1957 significantly differed from the present status.
In 1963, Blumberg et al. discovered Australian (Au) antigen or HBs antigen from the blood of Australian aborigine [14]. HCV was identified by Choo et al. much later in 1989 [15]. At present, natural history of infection of HBV and HCV has been detailed [16,17].
Analysis using archival pathology samples has been reported infrequently. HBV and HCV genomes were successfully amplified in paraffin-embedded liver samples in 1952 through 1960 [18], and HBV and HCV in liver cancer of atomic bomb survivors in Hiroshima and Nagasaki in the period from 1958 to 1987 were demonstrated [19]. Puiu recently identified HBV genome in Italian mummy 450 years earlier [20]. The present study may record the world-oldest detection of hepatitis C virus, we believe.
In Japan, the spread of hepatitis viruses can be linked to two distinct events [21]: The widespread treatment of schistosomiasis with intravenous injection of antimony sodium tartrate since 1923 [22], and the intravenous abuse of methamphetamine during and after the Second World War, often repeatedly sharing needles and

syringes. Another important cause should include vaccination
for tuberculosis and small-pox for school children and regional inhabitants without disposing needles until the 1970's [23]. The Meiji Government of Japan under Emperor Meiji established the public health agenda for small-pox vaccination as early as in 1874, and the inoculation became common in Japan at the end of 19 th century [24].
In these periods of time, tuberculosis and Helicobacter pyloriinduced peptic ulcer prevailed, and after the Second World War, the pulmonary and gastric lesions were surgically removed often with the need of blood transfusion. Many blood donors belonged to paid professionals, and the "Yellow Blood" was often contaminated with hepatitis viruses. Half of the blood recipients received the Yellow Blood, and HCV genotype 1b, probably introduced during the 1880's, started to spread in the 1920's and the 1930's [25].
In conclusion, this should be the first proof for the earliest occurrence of HCV infection in 1909 by analyzing archival autopsied liver specimens. We also demonstrated by nested RT-PCR using type-specific primers that HCV genotypes 1b already existed more than 100 years ago in Japan. HBV was detected in the paraffinembedded liver specimen autopsied in 1907. It is noteworthy that co-infection of HBV and HCV was common since it was seen in three of four HCV-positive cases. In 1956, HCV genotype 1b provoked fulminant transformation in a young patient. Archival pathology specimens are of epidemiological and historical importance and value for detecting hepatitis virus infection.