Evidence of hepatitis E virus (HEV) infection in human and pigs in Sardinia, Italy.

INTRODUCTION: The aim of this study was to determine the seroprevalence of anti-HEV antibodies in humans sera and to study HEV prevalence in swine from different Sardinian farms, testing viral HEV-RNA in bile samples. METHODS: In the first six months of 2008, 532 subjects of whom 402 blood donors and 130 workers at zoonotic risk, were enrolled. Anti-HEV were determined with an enzyme linked immunosorbent assay (ELISA). In positive subjects, RNA was extracted and tested by RT-Nested-PCR. From July 2006 to March 2007, 95 bile samples were collected from randomly selected pigs. RNA was extracted from 250 microl of bile and tested by RT-Nested-PCR. RESULTS: The overall prevalence of anti-HEV antibodies was 4.3%; 5.0% among blood donors and 2.3% among workers at zoonotic risk, with no statistically significant differences between sex, age classes and occupation. The search for HEV-RNA in the subjects positive for antibodies, gave negative results. HEV genome was detected in 6 of the 95 swine bile samples tested. Sequences were clustered within the genotype 3 and are edited on GenBank under accession number: from FJ850960 to FJ850962 and from FJ883000 to FJ883002. DISCUSSION: The overall prevalence of anti-HEV shows that the virus circulates without giving origin to cases of acute hepatitis. The low prevalence value found in workers at zoonotic risk do not apparently support the hypothesis of professional risk. In this study, HEV-RNA was isolated from pigs in Sardinia for the first time confirming the role of swine as HEV reservoir and the possibility of virus transmission to humans.

[Adherence to the screening program for HBV infection in pregnant women and application of the immunoprophylaxis protocol in babies born to HBV carrier women]. / Adesione al programma di screening per Virus dell'epatite B nelle donne gravide ed applicazione del protocollo di immunoprofilassi nel neonato.

The aim of the study was to evaluate the frequency of the screening for HBV infection in pregnant women and the application of immunoprophylaxis in newborns to HBV carrier women. The study, carried out in 2001 for 2 months, involved 1596 pregnant women consecutively recruited in public and private hospitals of the Sardinia. Information has been collected by a questionnaire: 90.5% of the women underwent HBV screening during pregnancy and 2.6% of them were found positive to HBsAg test. Among the newborns to HBsAg carrier mothers, 89.2% received the immunoprophylaxis protocol (specific immunoglobulin and the first dose of vaccine within 24 hours from birth). Two variables resulted statistically associated with the lack of adherence to HBV screening: the original family size of women (more than 4 members in the household) and the delivery in a private hospital. These findings point out a satisfactory adherence to HBV screening during pregnancy and the application of immunisation protocol in most of the newborns to HBsAg carrier mothers. However, the increase of information programs turned to the population is required to reach the total application of the prevention tools available in Italian public health.

TT virus infection in Italy: prevalence and genotypes in healthy subjects, viral liver diseases and asymptomatic infections by parenterally transmitted viruses.

This study was aimed to evaluate TT virus prevalence in subjects with hepatitis B virus (HBV), hepatitis C virus (HCV) and human immunodeficiency virus (HIV) infections in patients affected by hepatitis of unknown origin (non-A-non-E hepatitis) and in healthy subjects who had not been exposed to HBV, HCV and HIV. A total of 317 subjects were tested; 40 were HBsAg asymptomatic carriers, 57 subjects were anti-HCV positive (45 without chronic hepatitis and 12 with HCV-related chronic hepatitis), and 27 had chronic non-A-non-E hepatitis. Fifty-seven subjects were intravenous drug users (IVDUs) (52 with HCV or/and HIV infections), seven patients underwent a liver transplant for fulminant hepatitis and 137 were healthy subjects from the general population. Overall, TTV-DNA was detected in 62 subjects (19.6%): in 17.9% of the HBsAg carriers, in 14% of the anti-HCV-positive patients (in 8.3% and in 15.5% of patients with and without chronic hepatitis, respectively), in 22.2% of non-A-non-E hepatitis patients, in 22.8% of IVDUs, in 57.1% of fulminant hepatitis patients. TTV-DNA was also found in 20.4% healthy subjects. The prevalence in the different subgroups was not statistically different. The genotypes were identified in 40 of the 62 (64.5%) TTV-DNA positive samples: genotype 1a in 17.5%, 1b in 27.5%, genotype 2 in 27.5%, genotype 3 in 15.0%, genotype 4 in 5.0% and genotype 5 in 7.5%; the genotype distribution in the subsets of patients was not significantly different. In conclusion, this study showed that TTV infection is common in Italy; it is widespread throughout the entire population and five genotypes are present in Sardinia. Our results further dismiss the role of TTV as cofactor in influencing the clinical course of infections with other hepatitis viruses as well as the role of HIV in enhancing TTV transmission and replication.

Impact of hepatitis C virus infection on healthy subjects on an Italian island.

Field studies in large numbers of subjects unselected for risk factors are needed to assess the true prevalence and health burden of hepatitis C virus (HCV) infection. We assessed the prevalence, virological characteristics, risk factors and evidence of liver disease in a population of healthy subjects from an urban area of Sardinia. Hepatitis B virus (HBV) markers were also studied. The prevalence of antibodies to HCV (anti-HCV) (recombinant immunoblot assay [RIBA]-confirmed positive results) was 3.19% in 3324 workers and 7.11% in 225 elderly subjects, with a cumulative anti-HCV prevalence of 2.70% (95% CI 2.17-3.24). Males were more frequently positive than females (P < 0.01). The age-specific prevalence of HCV infection increased progressively in females. It showed two peaks in males: one in the fourth decade, the other in the seventh decade. HCV RNA was detected in 63.16% of the RIBA-positive sera, in 10% of the RIBA indeterminates and in none of the RIBA-negative specimens. Only 1.75% of anti-HCV-positive subjects had elevated transaminases. The frequency of HCV genotype 1b was 32.79%; of 1a, 21.31%; of 3a, 19.67%; of 4, 13.11%; and of 2a, 13.11%. HBV markers were found in 28.03% of workers. On multivariate analysis, male gender and tattooing were significantly associated with HCV and HBV infections: transfusion and travel with HCV, and age over 40 with HBV. The age prevalence rates of HCV infection in the Cagliari area reflect different risk factors that have been operative at different times. In this urban area, the large majority of HCV infections run a subclinical course.

Sexual behaviour and multiple infections in drug abusers.

We have studied the prevalence and the serological profile of HBV, HCV, HDV and HIV infections in 137 Italian subjects addicted to the intravenous use of heroine and correlated the virological findings with sexual behaviour. HBV and HCV viremia were also measured in 114 patients. Anti-HCV was detected in 81% of the addicts, and one or more markers of HBV infection were detected in 62.8% (4.4% were carriers of HBsAg, 58.4% had evidence of past HBV infection and 13.1% of the latter also had HDV markers). Anti-HIV was positive in 23.4%; 26% of those positive for anti-HCV and 4.6% of those positive for HBV markers had no other viral marker: none had only anti-HIV. HBV-DNA was negative in the carriers of HBsAg, and HCV-RNA was not detected in any of the HBsAg carriers who also had circulating anti-HCV. Overall, 34% of the anti-HCV positive addicts had HCV-RNA in their blood. The prevalence of the virus infection correlated with the duration of drug addiction but not with sexual behaviour, and sexual behaviour did not influence the acquisition of any virus. HCV infection was most frequent and probably the first infection to occur, but exposure to HBV was also common despite a low rate of HBsAg carriage. The prevalence of HDV infection was high (50%) in the HBsAg carriers, while the overall prevalence of HIV was lower (23%) than expected. Lack of HBV-DNA and HCV-RNA in carriers of HBV with anti-HCV in serum may indicate that HBV and HCV mutually inhibit their own replication.

HCV transmission in family members of subjects with HCV related chronic liver disease.

To investigate the risk of sexual and intrafamilial transmission of HCV, 220 family members of 76 patients (index cases) with chronic type C viral liver disease were tested for serological markers of HCV. Of the family members, 129 were offspring, 64 sexual partners, 15 parents and 12 siblings of the index cases. Anti-HCV was tested in all the household contacts; HCV-RNA was tested in antibody positive samples. The serologic markers of HCV were tested in a control group of 168 family members of 81 patients with chronic hepatitis unrelated to HCV. The overall prevalence of anti-HCV was 8.2% compared to 0.6% in the control group (p < 0.001). Sexual partners were anti-HCV positive more frequently than the other contacts (20% vs 2.2%; p < 0.001), without any difference in males or females. No correlation was observed between the occurrence of HCV infection in contacts and age, severity of liver disease or risk factor for the acquisition of HCV in the index cases. Seven of the 18 (39%) anti-HCV positive family contacts had bio-chemical evidence of chronic liver disease, histologically confirmed in the 6 patients who underwent a liver biopsy. Liver chemistry was normal in all the HCV-negative contacts. Ten of the 18 anti-HCV positive contacts (55%) were HCV-RNA positive, Genotypes were the same (1b) in 4 of the 7 viremic couples of subjects: in 3 of the 6 couples of sexual partners and in the only mother/son couple. These data suggest the occurrence of intraspousal transmission of HCV, while intrafamiliar acquisition of HCV in non-sexual contacts seems to be rare.

HCV, HIV, HBV and HDV infections in intravenous drug addicts.

Hepatitis viruses and the acquired immunodeficiency viruses often infect intravenous drug addicts (IVDAs). Our study includes 255 IVDAs (26 females and 229 males, aged 20-35 years) from Cagliari. Of 255 subjects examined, 207 (81.1%) were positive for anti-HCV and 84 (32.9%) for anti-HIV. Nineteen (7.4%) subjects were HBsAg carriers, and 12 of these (63%) had an HDV superinfection. Markers of previous HBV infections were tested in 223 cases and 137 (61.4%) were found positive; of these 14 (10.2%) also had HDV infection. Of the 223 drug addicts examined for all infection markers, 18 (8%) were negative to all markers, 46 (20.6%) were positive to only one, 89 (39.9%) were positive to two, 64 (28.7%) to three and 6 (2.6%) were positive to all. Subjects with a single infection were significantly fewer than those with multiple infections. The correlations studied among the various markers did not point out any statistically significant associations. Even so, a previous HBV infection was more common while active HBV/HDV infections were less common among subjects with anti-HCV; HDV infection was more common among HIV-positive subjects. In HBsAg carriers neither HBV-DNA nor HCV-RNA was detected; HCV-RNA was found more frequently in anti-HIV positive subjects than in subjects with the anti-HCV isolate.

[Epidemiology of hepatitis A in Sardinia: prevalence of anti-HAV in a sample of junior and senior high school students]. / Epidemiologia dell'epatite A in Sardegna: prevalenza di anti HAV in un campione di studenti delle scuole medie inferiori e superiori.

In the developing countries, where socioeconomic and hygienic conditions are inadequate, hepatitis A is mainly a childhood infection. Improvements in environmental conditions cause a decline in the exposure to hepatitis A virus and a change in the age-distribution of the incidence. In the Sardinian population 3-11 years old a study conducted in 1987 showed that 3.8 percent of children were anti-HAV positive. In this research work we studied the anti-HAV prevalence in the population 13-20 years old. The study population consisted of all the children who attended the third year of the junior high school (13-16 years old) and the fifth year of the senior high school (17-20 years old) in 7 health districts of South-Sardinia. All schools were identified in each district. Our list consisted of 455 classes of the last year of secondary schools and of 242 classes of the last year of high schools. Our sample consisted of 20 classes of the last year of junior high schools and of 29 classes of the last year of senior high schools. We used a precoded questionnaire recording age, sex, number of households and father's years of schooling. Sera from 794 students were collected between May and June 1988 and stored at--20 degrees C. A ELISA test (ABBOTT) was used for detection of anti-HAV. The analysis has been carried out in accordance with the manufacturer's instructions. We determined the prevalence of anti-HAV according to age, sex and socioeconomic conditions. We calculated odds' ratios and their 95% confidence limits. We considered as not exposed the group in the most favourable level of exposure (father's highest educational level, lowest number of households). There is a sharp increase from an anti-HAV prevalence of 16.5 percent in students 13-16 years of age to 24.8 percent in students 17-20 years of age. As far as the persons 13-16 years of age are concerned we can recognize a significant difference between females (21.8 percent) and males (10.3 percent). In the other group of age, on the contrary, the difference is not significant.(ABSTRACT TRUNCATED AT 250 WORDS)

[Epidemiology of hepatitis A in Sardinia: mathematical model]. / Epidemiologia dell'epatite A in Sardegna: modello matematico.

The prevalence of antibodies to hepatitis A in South-Sardinia was determined in samples of students and workers. In 1987 sera were collected from the children who attended kindergarten and elementary schools, in 1988 from students 13-20 years old (high schools) and from a group of workers who went to the public health office to ask for a certificate of good health. As far as the workers are concerned, the force of infection can be taken as a constant. In childhood, on the contrary, our findings are suggestive for a different immunization model. In this research work the prevalence observed in the population is analyzed using a mathematical model. Our model is based on the assumption that the force of infection increases linearly with age. The estimated fraction of susceptibles to hepatitis A is denoted by Y (X). The parameter h (X) represents the force of infection, the per capita rate at which susceptible population acquires infection. We assume that susceptibility may be lost only by natural infection and that permanent immunity results from first infection. So the fraction of susceptible population is gamma = exp (- x o integral of h (chi) d chi) if the force of infection increases linearly with age h (X) = ax + b then Y = exp (aX2 + bX + c) We can write In Y = y We obtain y = aX2 + bX + c Goodness of fit has been tested using the coefficient of determination R2. The number of cases in the group of age X - X + 1 can be estimated, for 10000 inhabitants, as (Y (X) - Y(X + 1) * 10000) Our findings are suggestive for a linear increasing of the force of infection with the group of age (R2 = 0.974). This could result from an age-dependent force of infection presumably as a consequence of age-related changes in behavioural patterns. Another explication could be a time-dependent force of infection according to improvements in socioeconomic and hygienic conditions. There is clear need for further research to separate the variations attributable to age from those attributable to time. Case notifications records could be very useful, but these data are notoriously unreliable.

[Epidemiology of hepatitis A in Sardinia: prevalence of anti-HAV in a sample of workers required to obtain health certificates]. / Epidemiologia dell'epatite A in Sardegna: prevalenza di anti HAV in un campione di lavoratori soggetto all'obbligo del libretto sanitario.

The prevalence of Hepatitis A in South-Sardinia was determined in two samples of students in 1987 and 1988. In this paper we present the results of a seroepidemiologic survey covering a population of workers who were residents of the city of Cagliari or its suburbs. A sample of 299 workers was randomly drawn from a population of workers who went to the public health office to ask for a certificate of good health. A radioimmunoassay test (ABBOTT) was used for detection of anti-HAV. The analysis has been carried out in accordance with the manufacturer's instructions. We also study a sample of 323 students 14-19 years old. The age-specific prevalence was analyzed using an exponential model. The age is denoted by the variable x. The parameter h is the force of infection, the per capita rate at which susceptibles individuals acquire infection. If we take the force of infection as a constant, the fraction y of susceptibles in a population is y = exp (-h x) The anti-HAV prevalente increase with age: in workers 14-19 years of age 44 percent of the tested individuals were antibody positive. The prevalence was 82.7 percent in the population 25-29 years old. Finally we found a nearly total exposure to HAV in persons over 30 years of age. The workers 14-19 years of age showed a higher prevalence than the students did; the difference was statistically significant. The prevalence that we have observed in our study population is lower the one observed in the city of Sassari (North-Sardinia) in 1981. The siero-epidemiological pattern is similar to that prevailing in Greece and France (1979), in Umbria (1978), in the city of Milan (1973). The prevalence was, on the contrary, higher than that observed in North European Countries (1979) and in Venetia (1987). The data from the study population can be fitted satisfactorily with the exponential model (R2 = 0.994). So we can take the force of infection (h = -0.118, se = 0.005 as a constant.(ABSTRACT TRUNCATED AT 250 WORDS)

Alpha-naphthyl acetate esterase activity in mouse thymus and other lymphoid organs.

Alpha-naphthyl acetate esterase (ANAE) reaction of mouse and rat thymus cells was studied. The majority of cells demonstrated ANAE in the form of multiple scattered granules. A reaction of this type appeared particularly labile, was found only when smears were fixed in formalin vapours and was inhibited by NaF. ANAE staining of thymus cells from mice of different ages was studied: the percentage of cells with single-spotted ANAE reaction was found to increase with age, while cells with granular scattered activity showed a progressive slight decrease. The percentage of ANAE positive cells in spleen appeared to be lower than in thymus, and showed only minor variations in different ages. In mouse and rat thymus and bowel lamina propria clusters of lymphocytes were found showing a sickle-shaped ANAE reaction, surrounding diffusely stained large cells which, on the basis of cytochemistry and phagocytosis ability, could be identified as macrophages. Analogous cell clusters were found in human tonsils and adenoids. These cell clusters were found to be similar to those formed by Ia-positive interdigitating cells which have been previously observed in human thymus, tonsils and bowel. The role of these clusters in the education process of lymphocytes is discussed.