Nicola Schinaia

Transmission of Hepatitis A to Patients with Hemophilia by Factor VIII Concentrates Treated with Organic Solvent and Detergent To Inactivate Viruses

Hepatitis A virus (HAV) is usually transmitted enterically, through contaminated food and water, or directly from person to person. Transmission through transfusion of blood products is rare because although a period of viremia exists during the incubation period of hepatitis A, no chronic carrier state exists. Several Italian centers recently reported a large outbreak of hepatitis A in patients with hemophilia [1]. All of the 52 patients reported so far received, in the 2 months before diagnosis, a large-pool factor VIII concentrate produced by an Italian manufacturer that used a solvent-detergent-based method to inactivate blood-borne viruses [2] and a chromatographic procedure to purify factor VIII [3]. Although the chromatographic procedure should remove all types of viruses, HAV RNA has been detected in factor VIII preparations prepared in this manner [4]. Also, the virucidal process is effective only for lipid-enveloped viruses and hence would not inactivate HAV [2]. Hepatitis A has been reported subsequently in 13 patients with hemophilia from Germany [5], in 17 from Ireland [6], and in 6 from Belgium [7], all of whom were given concentrates produced in Austria or Germany by a German manufacturer that used the same chromatographic and virucidal methods as the Italian manufacturer. That as many as 88 cases have occurred during the past 3 years in four European countries with no concomitant epidemics in the general populations of these countries suggests that this could be a widespread, albeit rare, phenomenon among patients with hemophilia. We describe two approaches to determine whether the factor VIII preparations were the cause of HAV infection in the patients with hemophilia: the results of a casecontrol study carried out in the Italian cohort to determine the source of HAV infection and the results of sequence analyses of HAV cDNA amplified by reverse transcriptase and polymerase chain reactions from specific lots of factor VIII and from serum samples of recipients in whom hepatitis A developed. Description of the Outbreak The first 3 cases of hepatitis A were diagnosed in 1989, 10 cases were identified in 1990, 33 in 1991, and 6 in 1992 at 12 hemophilia centers located in Milan, Trent, Vicenza, Castelfranco Veneto, Bologna, and Pavia in Northern Italy; in Florence, Rome, and Perugia in Central Italy; and in Naples and Bari in Southern Italy. All but 1 of the 52 case patients had severe hemophilia A, and their median age was 24 years (range, 2 to 43 years). Most case patients were identified because jaundice developed (42 of 52 patients; 81%), and hepatitis A was diagnosed when serum samples were positive for IgM anti-HAV antibodies (HAVAB-M EIA; Abbott Laboratories, North Chicago, Illinois). The course of hepatitis was uncomplicated and all patients recovered completely. Within 2 months before the onset of jaundice (the upper limit of the incubation period of hepatitis A), all patients had been infused with a factor VIII concentrate manufactured in Italy from pooled plasma collected in the United States from paid plasmapheresis donors. No blood product other than factor VIII concentrate had been administered to the index case subjects during that period. A virucidal step based on treatment with an organic solvent and a detergent (tri-[n-butyl]-phosphate/polysorbate 80) was incorporated into the manufacturing process of the concentrate [2], followed by an ion-exchange chromatography step to purify factor VIII [3]. At least 20 different concentrate lots were administered to case patients in the 2-month period before jaundice developed, 12 of them to more than 1 patient. Hepatitis A has not been reported among patients with hemophilia receiving factor VIII concentrates treated with other virucidal methods, such as pasteurization [8] or vapor heating, or among patients with hemophilia B (who, in Italy, almost exclusively receive vapor-heated concentrates). No additional cases have been reported since June 1992, but most Italian patients with hemophilia have not been given the solvent-detergent-treated concentrate, and many of them have been vaccinated against hepatitis A since late 1992, when the hepatitis A vaccine became available in Europe. Methods Case-Control Study The first 29 consecutive patients with hepatitis A and jaundice were enrolled. One to three nonjaundiced control patients with hemophilia (n = 71), randomly chosen from the national registry of patients with congenital coagulation disorders (Istituto Superiore di Sanita, Rome, Italy), were matched to each case patient by age, province of residence, hemophilia center, type and severity of hemophilia, and exposure to factor VIII concentrate in the 2 months before the onset of jaundice. For both case patients and control patients we used a questionnaire to gather the following information about the 2-month period before the onset of jaundice in case patients: lots of concentrate and the corresponding virucidal methods used to treat them, number of doses infused, contact with persons who had jaundice or hepatitis A, travel to countries reported to have a high attack rate for hepatitis A, and consumption of raw shellfish. None of the control patients were vaccinated against hepatitis A at the time of the survey. Statistical Analysis Odds ratios and 95% CIs were calculated for matched and unmatched analyses [9]. Polymerase Chain Reaction Analyses The RNA was prepared by a modification of the method of Chomczynski and Sacchi [10]. Briefly, 100 L of serum or 100 L of factor VIII concentrate that had been reconstituted with sterile water to a 10-fold concentration was mixed with 400 L denaturing buffer (5.2 mol/L guanidinium thiocyanate, 0.5% N-lauryl sarcosine, and 0.025 mol/L TRIS, pH 8.0), 50 L (10-fold concentration) phenol extraction buffer (1 mol/L TRIS, pH 8.0; 0.1 mol/L EDTA; and 10% sodium dodecyl sulfate), and 10 g glycogen (Boehringer Mannheim Corp., Indianapolis, Indiana), extracted twice with phenol-chloroform (at 65 C for 30 minutes; at room temperature for 5 minutes) and then with chloroform. After precipitation with isopropanol, RNA pellets were resuspended in 10 L water and the entire sample was used for the synthesis of cDNA. Nested primer sets were used [11]. Set one included 5-AGTGCAGTCAACTTTGAG (positions 1961 to 1978) and 5-ATCTGGAACATTCTGTTCTG (positions 2267 to 2248), and 5-ACAGGTATACAAAGTCAG (positions 2020 to 2037) and 5-CTCCAGAATCATCTCC (positions 2226 to 2211). Set two included 5-TCCCAGAGCTCCATTGAA (positions 2984 to 3001) and 5-CATTATTTCATGCTCCTCAG (positions 3284 to 3265), and 5-CAAATGCCATGTTATCCACTG (positions 3004 to 3024) and 5-GGTGGAAGTGCTTCATTTGAC (positions 3211 to 3191). Before cDNA synthesis, each 10-L RNA sample was annealed with 10 pmol of the reverse external primer at 65 C for 3 minutes. Synthesis of cDNA was performed in a 20-L reaction containing the annealed primer-template, 40 U RNasin (Promega Corp., Madison, Wisconsin), 8 U avian myeloblastosis virus reverse transcriptase (Promega), 1 mmol each of four deoxynucleoside triphosphates (Pharmacia, Piscataway, New Jersey), and polymerase chain reaction buffer (Perkin-Elmer Cetus, Norwalk, Connecticut) at 43 C for 60 minutes. The polymerase chain reaction was performed with each cDNA sample in a 100-L reaction containing 50 pmol of each primer, 2.5 U Taq polymerase (Amplitaq, Perkin-Elmer Cetus), 0.2 mmol each of four deoxynucleoside triphosphates and polymerase chain reaction buffer, and overlaid with mineral oil. The first amplification was for 36 cycles at 94 C for 1 minute, at 40 C for 30 seconds, and at 72 C for 1 minute. Ten L was then amplified with the inner set of primers for 45 cycles at 94 C for 1 minute, at 37 C for 30 seconds, and at 72 C for 1 minute. DNA products were analyzed by electrophoresis through 2% agarose gel and ethidium bromide staining, followed by photography under ultraviolet light. During RNA extraction and polymerase chain reaction amplification, special precautions were taken to avoid false-positive results [12]. In every experiment, one negative control sample was processed and tested in parallel with each test sample. Every sample was tested at least twice, and results were considered valid only if confirmed in repeated experiments. To determine the sensitivity of our polymerase chain reaction assay, we analyzed 10-fold serial dilutions of a laboratory stock of a cell culture-adapted strain of HAV [13]. This HAV stock, harvested from virus-infected cells in culture, contained 107 50% tissue culture infective doses (TCID50) per milliliter of virus, as determined from immunofluorescence assays (data not shown). Hepatitis A viral sequences were detected consistently in 100-L samples of stock diluted to 108, and therefore we estimated that the sensitivity of our assay was 0.01 TCID50. Determination of Nucleotide Sequences Polymerase chain reaction products were purified by fractionation through low-melting-temperature agarose. The DNA bands were excised and DNA was extracted [14]. Sequencing was done with Sequenase (United States Biochemical, Cleveland, Ohio) as previously described [15]. Animal Studies Seronegative juvenile chimpanzees were infused with selected lots of factor VIII, and blood samples were drawn weekly and tested for serum levels of alanine aminotransferase, isocitrate dehydrogenase, and -glutamyltransferase by standard methods (Metpath, Rockville, Maryland), for antibodies to HAV and hepatitis C virus by commercial assays (Abbott), and for antibodies to hepatitis E virus by an enzyme-linked immunosorbent assay using expressed hepatitis E virus nucleocapsid protein [16]. Results Case-Control Study: Implications of Factor VIII from a Specific Manufacturer Case patients ranged in age from 10 to 42 years (median, 22 years), control patients from 11 to 49 years (median, 22 years). Nine of 29 case patients and 19 of 71 control patients were seropositive for antibody to human immunodeficiency virus. Case patients

Molecular Diversity of HIV in Albania

Little information is available on circulating human immunodeficiency virus (HIV) subtypes and resistance to antiretroviral drugs in Albania. To fill this gap, we studied 72 plasma samples from HIV-infected individuals from throughout the country. Subtype classification and genotypic resistance analysis were performed on the HIV pol gene region. The analysis was successfully performed on 66 (91.6%) plasma samples and showed that 43 (65.2%) strains were non-B subtypes (mostly subtype A, as determined by analysis of pol gene sequences). No major mutations in the protease gene were found, whereas analysis of the reverse transcriptase gene revealed a few major mutations associated with resistance. In conclusion, non-B subtypes are predominant in Albania, and the prevalence of resistance to antiretroviral drugs is still low.

Epidemiology of hemophilia and of HIV infection in Italy

To evaluate the incidence and prevalence of hemophilia in Italy and the impact of HIV infection on the Italian hemophiliac population, data from a computerized national registry of patients from 95% of the hemophilia care centers in Italy were analyzed. A total of 4643 patients were included in the registry. The prevalence of hemophilia A was 8.2 per 100,000 males, with no significant regional differences; for hemophilia B the corresponding figure was 1.5 per 100,000. Temporal trends in hemophilia incidence suggest that the diagnosis of mild and moderate hemophilia has improved. The overall HIV prevalence was 26% and was significantly (p < 0.001) higher in patients with hemophilia B (47.1%) compared to those with hemophilia A (26.8%) or other diseases (16.5%). The highest rate of HIV seropositivity was among patients 20-29 years of age. The annual amount of clotting factor concentrates received was significantly (p < 0.001) higher in HIV seropositive patients than in those who were seronegative. Antibody testing was never performed on 10.1% of severely affected patients. The number of patients in the Italian registry was similar to the number that would have been expected based on prevalence estimates from other countries. In comparison with other countries, the prevalence of HIV infection recorded in Italy was lower in persons with hemophilia A, but higher in those with hemophilia B. Our study demonstrates the usefulness of a registry in delineating the epidemiology of hemophilia and in studying risk factors for HIV infection. It also underlines the need for continuing surveillance of this population.

Progression to AIDS among Italian HIV-seropositive haemophiliacs

To investigate the interval between HIV-1 infection and the development of clinical AIDS among Italian patients with congenital coagulation disorders, a national cohort study was undertaken in 1988. Information was collected both retrospectively and prospectively on 499 HIV-1-positive patients enrolled in an ongoing national registry of patients with congenital coagulation disorders. Two methods were used to estimate each patients seroconversion date: the mid-point between the last negative (either known or estimated) and the first positive test, and the median under a Weibull distribution, which was assumed to fit seroconversion data. The two methods of estimating the seroconversion time yielded similar results. The actuarial incidence of AIDS was estimated using the Kaplan-Meier survival analysis at 12.8% (95% confidence interval = 9.7–15.9) over 7 years for Italian haemophiliacs. Progression appears to be slow in the first 5 years after the infection, and to rise steadily thereafter. A strong association between faster progression and older age at seroconversion was found. Zidovudine-treated individuals seem to have a slower progression than untreated individuals, after controlling for CD4, but there was no association between progression and type and severity of the congenital disorder.

Prevalence and risk factors for viral hepatitis in the Kosovarian population: implications for health policy

The prevalence of hepatitis infection among the Kosovarian population is largely unknown. The aim of the study was to evaluate the prevalence and risk factors of hepatitis A, B, C, and D (HAV, HBV, HCV, HDV) infection among the general population and in a group of health care workers in the Kosovo region. Overall, 1,287 participants were recruited, 460 males (36%) and 827 females (64%). Health care workers accounted for 253 individuals (20%), 301 were blood donor candidates (23%), 334 were pregnant women (26%), and 399 (31%) were subjects who had been examined in two clinics for routine laboratory testing. The prevalence of total anti‐HAV was 88.6% (95% CI: 86.69–90.25). Prevalence of anti‐HAV among children up to 10 years was 40.5% (95% CI: 29.6–53.15), reaching 70% (95% CI: 62.25–77.10) in the 11–20 age group. Age, living in rural areas and unemployment were factors associated with higher risk of HAV infection. HBsAg was detected in 2.4% (95% CI: 1.57–3.38%) of the study sample, with a significant age trend (P‐value:0.0110). Positivity for total anti‐HBc was detected in 18.4% (95% CI = 16.27–20.59) of the subjects. Ninety‐three subjects (7.2%) were positive for anti‐HBs alone. An association between age, HSV‐2 positivity, working nurses and HBV infection has been observed. One patient was HDV positive. The prevalence for HCV was 0.5% (95% CI: 0.22–1.12%). HAV infection seems to be high‐intermediate, while HBV shows an intermediate endemicity. It is necessary to highlight the importance of an immunization strategy against HAV and HBV in reducing the incidence of the infection. The prevalence for HCV was very low. J. Med. Virol. 80:833–840, 2008.

Helicobacter pylori Seroprevalence in Selected Groups of Albanian Volunteers

Background:Albania is a Mediterranean, South–East European developing country where epidemiological data on infectious diseases are scarce. In this study, the seroprevalence of Helicobacter pylori infection in 1,088 Albanian healthy volunteers (472 females followed-up to a prenatal clinic, 173 recruits, 443 health care workers) was evaluated.Materials and Methods:Sera were tested for immunoglobulin-G (IgG) antibodies against H. pylori using a quantitative enzyme immunosorbent assay.Results:The overall H. pylori seroprevalence was 70.7%. The H. pylori seroprevalence increased by age, from 60.4% in individuals younger than 20 years to 81% among those ‡ 50 years of age with a significant trend of increase by age. The overall seroprevalence was 73.9% for females and 59.5% for males. In addition the seroprevalence was 55.3% for people living in rural areas and 72.3% for people living in urban areas. No significant differences were found according to level of education except for individuals with elementary level of education. Nurses and hospital auxiliaries have significantly higher H. pylori seroprevalence when compared to other health care workers (physicians and office workers). When each variable (age, gender, area of residence, occupation, and education level) was adjusted for the confounding effect of the other variables by stepwise logistic analysis, we observed that age greater than 40 years and female gender remain the only variables independently associated with the presence of H. pylori IgG antibodies.Conclusions:H. pylori is highly prevalent among the Albanian population. Improving living conditions, education in hygiene, and the supply of running water are measures to prevent the transmission of H. pylori infection and other infections spread by the fecal–oral route in Albania.

The use of blood donor data for HIV surveillance purposes.

We reviewed the use of HIV prevalence data from blood donors for surveillance purposes and for the estimation of HIV prevalence. Fifty countries with generalized epidemics were considered. Data on HIV prevalence in blood donors were compared with published estimates of adult HIV prevalence. In most of the 19 countries with similar estimates, blood donations came primarily from replacement donors. Our analysis suggests that blood donors are usually not a good proxy for the general population.

Testing practices and spread of HIV among sexual partners of HIV-positive haemophiliacs in Italy

OBJECTIVES To evaluate rates and predictors of testing and HIV positivity among the sexual partners of Italian HIV-positive haemophiliacs. METHODS Our index cases were 602 sexually active HIV-positive haemophiliacs (aged 18 years or more) enrolled in the Italian Registry of Haemophilia. Data on the demographic and clinical status of the haemophiliacs, whether their partners had undergone HIV testing, and the results of these tests were collected. RESULTS To date, 205 (34.1%) partners of HIV-positive haemophiliacs have been tested for HIV, of whom 27 (13.2%) were seropositive. On univariate analysis, haemophiliacs who were unmarried, younger, and asymptomatic were less likely to have partners who had been tested for HIV (P << 0.001). On multivariate analysis, unmarried status [odds ratio (OR), 8.4; 95% confidence interval (CI), 5.4-13.1; P << 0.001] and younger age (OR, 1.9; 95% CI, 1.1-3.2) again predicted a higher rate of non-tested partners. There was no association between the demographic and medical characteristics of HIV-seropositive haemophiliacs and the risk of HIV positivity among their sexual partners. CONCLUSION This study demonstrates that a high proportion of sexual partners of HIV-positive haemophiliacs have not yet been tested for HIV. The single most important predictor of not being tested was the marital status of the index case. These results emphasize the need to strengthen prevention programmes aimed at minimizing the risk of heterosexual HIV transmission, particularly among younger unmarried haemophiliacs.

Psychological status of men with haemophilia and HIV infection: two-year follow-up

This study aimed to assess the psychological status of men with haemophilia and HIV infection and to monitor changes in psychlogical status over time, in order to evaluate the need for psychological support. The study included 24 HIV seropositive men and a control group of 21 HIV seronegative men who attended the Haemophilia Centre in Bari (Italy). Subjects underwent psychological tests (STAl‐Y: State and Trait Anxiety Inventory; SDS: Self‐Rating Depression Scale) and completed a questionnaire on the emotional impact of AIDS. Assessment was repeated at 6‐monthly intervals over a 2‐year period. Contrary to expectation, HIV seronegative men with haemophilia had worse anxiety and depression scores, reported more confusion and fear, and had more reluctance towards the use of blood products (despite their present safety) than HIV seropositives. Possible reasons for these findings are considered, and their implications for clinical practice discussed.

Acute intestinal infections in Europe. A review of reported cases

Acute intestinal infections still constitute one of the leading causes of death in the world and a major cause of morbidity in Europe. Unfortunately, most European countries do not have an information system oriented towards acute intestinal infection surveillance. The present work is an analysis of cases of typhoid fever, Salmonellosis, Shigellosis, foodborne infections, and acute gastroenteritis reported from January 1980 to December 1985. Data from 24 of the 33 countries belonging to WHO Regional Office for Europe (EURO-WHO) were considered. Data for 1980 and 1981 were taken from World Health Organization statistics, while data for 1982–85 were taken from bulletins sent to EURO-WHO and WHOCCHDS by individual countries. Data was entered in an IBM 4341 computer system and a data base was organized using a general purpose inquiry language (IBMs APLDI) Rates per 100,000 were calculated using as a denominator 1983 WHO official population figures. The quality of the data is a great problem, as the only available sources of information are official national reports which underestimate the actual incidence of diseases.For typhoid fever, Mediterranean countries show much higher incidences than the rest of Europe, although a decreasing trend can be seen for all European countries. We estimate that the data for typhoid fever, Salmonellosis and Shigellosis are reliable, while reports of foodborne inflections and acute gastroenteritis represent only a very small percentage of the actual number of cases.