Nicola Ciavarella

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

Precipitating antibodies in von Willebrand's disease

WE report the presence of precipitating antibodies in three patients with von Willebrands disease (VWD), an inherited deficiency of factor VIII (anti-haemophilic factor, AHF). To our knowledge this is the first description of patients with complete biological and immunological deficiency of a plasma-clotting protein responding to replacement therapy by the production of antibodies with properties indistinguishable from those elicited in animals. AHF is a high molecular weight (> 106) glycoprotein complex1 associated with two biological activities; one necessary for normal clotting (VIII: C) and the other for normal platelet function (Willebrand factor, VIIIR: WF). Specific antigenic determinants (VIIIR: AG) can be reliably quantified using heterologous antisera2,3. Any concept of the structure or the molecular genetics of AHF needs to incorporate the clinical and immunological findings in the two congenital disorders associated with AHF deficiency: haemophilia A and VWD. The former is a sex-linked deficiency of VIII: C alone whereas the latter represents a deficiency of all three parameters of the AHF complex and is inherited as an autosomal trait. Non-precipitating antibodies to AHF, acquired after replacement therapy, are common in haemophilia A (ref. 4) but only one case has been reported in VWD5,6.

Prevalence of Infection with the Hepatitis C Virus among Italian Hemophiliacs before and after the Introduction of Virally Inactivated Clotting Factor Concentrates: A Retrospective Evaluation

In July 1985, all coagulation factor concentrates were withdrawn from the market in Italy and replaced with virally inactivated concentrates. A retrospective survey comparing the prevalence of the antibody to the hepatitis C virus (anti‐HCV) in hemophiliacs multitransfused with nonvirally inactivated concentrates until 1985 with that in previously untreated hemophiliacs transfused exclusively with virally inactivated concentrates since 1985 has been conducted in 9 Italian hemophilia centers. The centers, which follow about one‐fourth of all the Italian hemophiliacs, provided information about 708 patients infused for the first time before 1985 (group A) and 80 patients infused for the first time between 1985 and 1991 (group B). The prevalence of anti‐HCV was 83% (591/708) in group A and 6% (5/80) in group B. For the 5 anti‐HCV‐seropositive patients from group B, dry heating, hydrophobic interaction chromatography plus dry heating (2 patients), hot vapor and pasteurization were the virucidal methods used for the concentrates implicated in HCV transmission. In the case associated with pasteurization, there is the possibility of intrafamilial transmission of HCV. It appears from this retrospective analysis that there has been a substantial reduction in the risk of HCV transmission since the adoption of virucidal methods. However, these methods do not eliminate completely the risk, which might be further reduced by the recent adoption of anti‐HCV screening for plasma donations used to manufacture concentrates.