Isolated rat hepatocytes as a tool for the determination of antibodies against human liver cell membrane.

Antibodies against liver cell membrane are measured by counting the percentages of fluorescent rat hepatocytes ( FRH ), obtained by an indirect immunofluorescence method after incubation of isolated rat hepatocytes with sera of patients with chronic liver diseases. A close relationship exists between the percentages of FRH and the serological and histological parameters of diseases activity, there was no difference between HBsAg-positive or -negative sera.

Alpha interferon plus thymopentine in treatment of HBV and/or HDV positive patients undergoing liver transplant.

Infection by hepatitis B (HBV) and/or delta virus (HDV), is the most frequent acquired pathology in patients affected by end-stage hepatic disease, candidates for liver transplant. To reduce the risk of virus reactivation after surgery, we used alpha Interferon (IFN) therapy in patients who were HBV-DNA and/or HDV-RNA positives before transplant. Our protocol included alpha IFN at low dosage associated to a thymic hormone that seems to have a synergistic activity with IFN. We have evaluated in four patients, affected by post hepatitic end-stage liver disease, the outcome of HBV and HDV markers in relation to immunological response during treatment. Our interest has been focused on monocyte and natural killer cytotoxic activity. The data show that all patients, before starting therapy, had evidence of active phase viral replication. They also displayed low values of the immunological parameters tested. The study of viral markers showed decrease of HBV and HDV in all patients. The relation between viral markers and natural killer and monocyte cytotoxicity was very interesting; during the treatment we observed a marked increase of both activities. At the same time no relevant modifications in the other immunological parameters tested were found.

Comparison of broth dilution and semisolid agar dilution for in vitro susceptibility testing of Cryptococcus neoformans.

We compared the in vitro activity of amphotericin B, flucytosine, itraconazole, fluconazole, ketoconazole and miconazole against 18 strains of Cryptococcus neoformans by using two methods: microbroth dilution and semisolid agar dilution. By both of the methods minimum inhibitory concentrations (MICs) showed a wide range for all antifungal agents but not for amphotericin B. Statistically significant differences between the two methods were observed only with amphotericin B and flucytosine, p = 0.048 and p = 0.045 respectively. Our study suggests that azole susceptibility testing for C. neoformans may be performed by the broth microdilution as well as the semisolid agar test. The choice of the method when testing amphotericin B and flucytosine is more problematic.

Turbidimetric and visual criteria for in vitro susceptibility testing of Cryptococcus neoformans clinical isolates.

The drug concentration which inhibited 50% of growth (IC50), the lowest drug concentration at which growth was less than 30% of that in a positive control well (IC30), the visual minimal inhibitory concentration (MIC visual), were applied to study the effects of fluconazole, itraconazole, amphotericin B and flucytosine against 27 isolates of Cryptococcus neoformans by a broth microdilution technique. When the recommendations established by NCCLS Subcommittee on Antifungal Susceptibility Test were applied for the visual reading of the microplates, the results were comparable with those obtained by the turbidimetric method. No statistically significant differences between MIC visual and IC30 readings were observed with the azoles. There were, however, differences with amphotericin B and flucytosine. In absolute terms MICs of amphotericin B and flucytosine showed higher values than IC30s and IC50s.

Radioimmunoassay detection of IgM antibodies to the hepatitis B core antigen in HBsAg liver disease.

A radioimmunoassay is described for detection of IgM antibody to the hepatitis B core antigen. The assay is based on the selective absorption of IgM immunoglobulins from test serum by anti-IgM fixed on a solid phase, followed by incubation with HBcAg and radiolabeled anti-HBc of IgG type. IgM anti-HBc was found in high titers in all the patients with acute hepatitis B; in two of four patients whose acute hepatitis progressed to chronicity, IgM anti- HBc disappeared in 4-6 months despite continuing HB viremia. IgM anti-HBc was also found in low titers in 19% of the patients with chronic HBV infection. No relation was noted between the presence of IgM anti-HBc and clinical or serological categories of chronic carriers of the HBsAg. The antibody was not found in carriers with hepatitis caused by superinfection with the hepatitis A virus or the HBV-associated delta agent. IgM anti-HBc is a marker of a recent HBV infection. Its absence in HBsAg-positive individuals with acute hepatitis should rise suspicion that the patients are carriers of the HBsAg experiencing disease caused by factors other than the HBV.

HBV-DNA, HBeAg/anti-HBe serological status in hepatitis B chronic individuals from central Italy.

A population of 488 HBsAg carrier individuals, from central Italy, classified on the basis of biochemical, clinical and histological parameters, was analysed for the presence of HBV-DNA in serum and its relationship with HBeAg/anti-HBe markers. The prevalence of HBV-DNA was 32.8% in chronic patients with biopsy-proven liver disease, and 20 and 4.3% respectively in asymptomatic carriers with and without altered ALT levels. The values in chronic patients were correlated with the histological activity. Concordance of HBV-DNA presence and HBeAg positivity was observed in only 61.4% of cases. However HBV-DNA prevalence in sera of anti-HBe positive individuals was very low in asymptomatic carriers with normal ALT levels (2.5%). Higher values were observed in anti-HBe positive chronic patients (15.8%) and in carriers occasionally found with changes in ALT without any other clinical sign of illness (16.7%). These data would indicate that HBV-DNA is the serological marker which is most closely related to liver disease.

Serum immunoglobulin M antibody to hepatitis D as a surrogate marker of hepatitis D in interferon-treated patients and in patients who underwent liver transplantation.

The kinetics of the immunoglobulin (Ig) M type antibody to the hepatitis D virus (IgM anti-HD) were investigated in hepatitis B surface antigen (HBsAg) carriers with chronic hepatitis D treated with interferon (IFN) and in patients with terminal hepatitis delta virus (HDV) cirrhosis who underwent liver transplantation. The IgM antibody disappeared in each of 8 patients who responded to IFN therapy with the persistent normalization of aminotransferases and with the clearance of serum HBsAg and HDV-RNA. The IgM reactivity did not decline in the 45 treated patients who did not respond to the cytokine or who experienced a relapse after responding while on therapy. The antibody rapidly disappeared from serum post-transplantation in each of 10 examined patients with HDV who underwent transplantation. In 5 patients who underwent transplantation and who became reinfected with HDV, the antibody remained undetectable during the early reinfection phase, as marked by HDV replication and by the absence of liver damage; however, it rapidly raised to pre-transplantation levels with the recurrence of hepatitis D (HD) in the liver graft. Monomeric 7S IgM anti-HD predominated over pentameric 19S antibody in each of the two patients examined for IgM anti-HD molecular species. The IgM antibody to HDV raises in response to HDV-induced damage and represents a valid surrogate marker of liver damage which is immunopathologically related to HDV infection. Besides providing diagnostic information, it provides the best predictor of impending resolution of chronic HDV disease, whether spontaneous or IFN-induced.

Familiar clustering and spreading of hepatitis delta virus infection.

The prevalence of hepatitis delta virus (HDV) infection was significantly higher among the relatives of 79 carriers of HBsAg with antibody to HDV (index cases) than among relatives of 111 carriers without serological evidence of HDV infection (controls). Antibody to HDV was found in 45 of the 80 (56%) carriers of HBsAg in families of index cases but only in 2 of 59 (3%) carriers in families of controls (P less than 0.0001). During follow-up new HDV infection developed in 31% of 13 susceptible carriers in families of index cases, but only in 1.2% of 162 susceptible carriers in families of controls (P less than 0.001). None of the family members previously unexposed to the hepatitis B virus had HDV markers in serum or developed this infection during the follow-up. Familial clustering shows that HDV is transmitted by personal contacts, presumably through the inapparent permucosal or percutaneous passage of virus during close or intimate contact. The family model indicates that endemic HDV is maintained and spread through the network of carriers in the community, and that HBsAg carriers in contact with HBsAg/HDV carriers are at high risk of contracting HDV.

Recurrence of hepatitis D (delta) in liver transplants: histopathological aspects.

BACKGROUND: The viral/pathological correlates of recurrent hepatitis delta virus (HDV) disease in orthotoptic liver transplants are reported. METHODS: We examined the histological features of recurrent HDV disease in nine patients with transplants for terminal HDV cirrhosis were examined; intrahepatic HDV and hepatitis B virus (HBV) antigens were detected by immunoperoxidase techniques. Sera were tested for the battery of HDV and HBV markers. RESULTS: In four patients, HDV reinfection was accompanied by the recurrence of an HBV infection with features of active viral replication. In the other five, HDV reinfection was accompanied by an atypical recurrence of HBV infection without evidence of active HBV replication (no expression of intrahepatic hepatitis B core antigen). In four of the latter patients, the atypical HBV pattern changed during the follow-up into a pattern of active viral replication accompanied by chronic necroinflammation detected during histology. CONCLUSION: The pattern of recurrent HBV infection can influence the pathological aspects of the relapses of HDV disease in liver grafts.

HBV infection in Italian family groups.

Intrafamilial spread of HBV infection was evaluated in 85 Italian family groups. Secondary infected cases were observed in 43% of these families, of which 73% showed clusters of HBs positive members, with a prevalence among the family members (children-siblings) of the mother/wife index cases, and no strict relation to their "e" system. The presence of the delta agent in the index case was correlated to a higher percentage of secondary HBs-positive cases, but the intrafamilial spread of delta agent was a rare event. A similar higher incidence of HBV markers was observed among the relatives of the index cases positive for auto-antibodies. The evidence presented suggests the importance of genetic factors in the acquisition and clearance of HBV and delta infections.

Liver transplantation in hepatitis delta virus disease.

Seven patients with hepatitis delta virus (HDV) cirrhosis underwent liver transplantation. In every case the HDV infection was florid but accompanied by an inactive hepatitis B virus (HBV) infection. The patients were given anti-HB surface antigen (HBsAg) serum globulins and HBV vaccine. Two patients cleared the HBsAg and the HDV, and are alive and well 14 and 15 months, respectively, after transplantation. HDV infection recurred in the other five patients: hepatitis developed in three, another died, and the fifth was re-transplanted for causes unrelated to viral hepatitis (reinfection was shown by the presence of HD antigen in the graft). Liver transplantation is feasible in patients with HDV disease but involves a high risk of HDV reinfection that cannot be predicted by the virological pattern of the native HBV infection or prevented by conventional HBV prophylaxis.