The intrahepatic distribution of hepatitis delta virus RNA was studied by in situ hybridization in 33 formalin-fixed, paraffin-embedded biopsies from 17 chronic hepatitis B virus carriers superinfected with hepatitis delta virus. The findings were correlated with the expression of the hepatitis delta antigen, the duration of the hepatitis delta virus infection and the eosinophilic degeneration of the hepatocytes. Intranuclear hepatitis delta virus RNA and antigen were found in 28 specimens, whereas the remaining five were negative for both markers. Hepatitis delta virus RNA and antigen were mostly found within the same cell. In 20 specimens, however, a variable number of hepatocytes showed the presence of hepatitis delta virus RNA alone. The percentage of these over the total number of infected cells was higher in the specimens taken within 1 year from the acute delta hepatitis, whereas their absence was invariably associated with a long-established hepatitis delta virus infection. Interestingly, the vast majority of hepatocytes undergoing eosinophilic degeneration, a change significantly associated with hepatitis delta virus infection, did not show the presence of either hepatitis delta virus RNA or the viral antigen, suggesting a lack of association, at the cellular level, between viral replication and cytopathological change. The specificity of the detection of hepatitis delta virus RNA was confirmed by negative findings in nine specimens from seven chronic hepatitis B virus carriers without evidence of past or current hepatitis delta virus infection. The loss in sensitivity due to the formalin fixation was estimated to be 50% of that obtained in frozen biopsies, as determined by counting autoradiographic grains over infected cells. Consistent results were obtained when sections from the same biopsies were hybridized in separate experiments. Detection of hepatitis delta virus RNA by in situ hybridization in formalin-fixed, paraffin-embedded sections is therefore a rapid, specific, sensitive and reproducible assay for monitoring intrahepatic hepatitis delta virus replication and might have diagnostic relevance. The recent availability of strand-specific RNA probes labeled with 1251 permitted the study of the intracellular localization of HDV nucleic acid sequences of both genomic and antigenomic polarity by in situ hybridization (8, 10). The assay was initially applied to a limited number of frozen human liver samples (11). In this report, the technique was modified in order to detect HDV RNA by in situ hybridization in formalin-fixed, paraffin-embedded liver biopsies. A series of specimens taken at various stages during the course of natural delta infection in humans (12) was studied together with a control group of biopsies from chronic HBV carriers without evidence of HDV infection. The presence and distribution of intrahepatic HDV RNA was then compared to the expression of HDAg, the duration of the HDV infection and t h e eosinophilic degeneration of hepatocytes, a cytopathological change often described in...
