THE JOURNAL OF INFECTIOUS DISEASES. VOL. 135. NO.2. FEBRUARY 1977 @ 1977 by the University of Chicago. All rights reserved.
Hepatitis B in Subjects Treated with a Drug Containing Immunoglobulins From the Institute of Hygiene, University of Genoa, Genoa, Italy
Fernando Luigi Petrilli, Pietro Crovari, and Silvio De Flora
In late 1974 cases of viral hepatitis were observed in Italy among subjects suffering from allergic diseases who had received a drug containing human immunoglobulins. On December 12, 1974, after the suspicion of an iatrogenic cause of the disease emerged, the Italian Ministry of Health ordered nationwide sequestration of the product. During the early months of 1975, however, additional cases of the disease were recorded among individuals who had been treated before December 12, 1974. Official quantitative data are lacking so far about the spread of this outbreak in part because a conclusive relation between the administration of the drug and the appearance of symptoms could not be proven in many cases. However, a rough estimate indicates that there were >100 cases of overt disease.
We have explored a number of cases involved in this outbreak by studying the epidemiologic patterns of the disease and by demonstrating the very likely association between injection of the drug and development of disease among the subjects examined. In fact, the original immunoglobulins, the drug, and the sera of patients were all found to be positive for the surface antigen of hepatitis B virus (HB s Ag) of a subtype that is relatively rare in Italy. Materials and Methods
Drug. Trilergan," the drug that was prescribed for treatment of allergy; is manufactured in Italy. It contains, in one dose (1 ml), 12 mg of human immunoglobulins, 0.15 JLg of histamine hydrochloride, 0.15 JLg of serotonin, 0.1 mg of sodium merthiolate, 9 mg of NaCI, and 20 mg of mannitol. The product is prepared in the lyophilized form and is reconstituted with I ml of bidistilled water before use. It is injected sc in cycles of three to four administrations at intervals of four to five days. Like similar products manufactured and distributed in other European countries, the drug has a vaccine-like mechanism because of formation of antigenic complexes of histamine and serotonin with immunoglobulins. Serotonin and
Received for publication March 22, 1976, and in revised form July 19, 1976. This investigation was supported by grant no. 73.01363.43 from the Italian Centro Nazionale delle Richerche. We acknowledge the assistance of Drs. C. Bennicelli and P. Zanacchi. We thank all of our colleagues who cooperated in providing clinical and epidemiological information, particularly Drs. C. Collotti, F. Croce, M. Furfaro, A. Negro, L. Robert, and G. Veronesi. Please address requests for reprints to Prof. F. L. Petrilli, Institute of Hygiene, University of Genoa, Via Pastore 1, 16132 Genoa, Italy.
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In late 1974 and early 1975, several cases of viral hepatitis were reported in Italy among subjects who had received subcutaneous injections of a drug containing human immunoglobulins that was prescribed for the treatment of allergies. Epidemiologic and laboratory investigations provided evidence that the original immunoglobulins, the series of the drug containing these immunoglobulins, and sera from a number of patients were all positive for hepatitis B surface antigen (HB s Ag) of the adw subtype, which is relatively rare in Italy. Some sera from patients and healthy subjects treated with the HB s Ag-positive drug were also found to be positive for antibody to HB s Ag of the adw subtype. The clinical course of the disease was consistent with typical forms of icteric hepatitis in all patients examined. The average length of the possible incubation period was 111-143 days, and an inverse relation was observed between the number of doses administered and the length of the incubation period. The possibility that immunoglobulins can be responsible for the transmission of viral hepatitis raises a number of theoretical and practical problems concerning control and use of these blood products.
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with purified anti-H'B, (Puritest, ISl\tfSB, Milan, Italy) were used as the solid-phase substrate, and human 125I-labeled anri-Hls, (Abbott Laboratories) was used as labeled antibody. This SPRIA is based upon preincubation of test sera and of negative controls with a fixed dose of a standard HB s Ag (adw and ayw). Radioactivity counts of test sera were determined in relation to those of standard HB s Ag control sera. HB s Ag- and anti-Hb.-positive samples have significantly more and less counts, respectively, than control samples. The end-point dilution of positive sera was determined by plotting of the resulting values on a nomograph, which was drawn according to the findings of a study of SPRIA kinetics [4]. The specificity of positive samples was checked by subtyping of HB s Ag- and anti-H'Bj-positive samples with the aid of previously described SPRIA procedures [4,5]. Reference adw, adr, and ayw antigens and antibodies were supplied by Dr. R. Purcell (National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Md.), and AB, AC, B, and C reagents, corresponding to Le Bouvier's ad, ay, d, and y reagents, respectively, were supplied by Dr. R. Gispen (Laboratory of Virology, National Institute of Public Health, Bilthoven, The Netherlands). Results
Screening for RB s Ag in immunoglobulins and in drug preparations. Examination of the original immunoglobulins contained in the drug, after resuspension in glycine buffer at a concentration of 16% (wt/vol), showed a very weak positivity for HB s Ag. Positivity was rather inconstant and .close to the cut-off point of the method. As shown by the results of the combined SPRIA, small traces of anti-H'B, were also detectable in the product. HB s Ag was neutralized to a greater extent by adw than by ayw antisera. A number of drug lots were screened by tests of the lyophilized product resuspended in 0.2 ml of distilled water (a concentration five times that of the injectable preparation). All lots examined within the series containing the HB s Ag-positive immunoglobulin, including lots no. 49-68 prepared from February to June 1974,
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histamine are adsorbed to the globulin by mixing of the reactants for 30 min at pH 6.4-7.0. The therapeutic efficacy and the clinical indications for such desensitizing treatment have been studied by a number of investigators [1-3J. The immunoglobulins contained in the implicated lots of the drug were imported from the United States, where they had been released by the Bureau of Biologics of the Food and Drug Administration in May 1973. These immunoglobulins were used exclusively for the preparation of the drug and were incorporated into lots manufactured from February to June 1974. Patients. Subjects receiving the drug had allergic diseases of various types. Many of them were treated at the same time with other antiinflammatory agents. Patients who developed viral hepatitis after injection of the product were hospitalized in various regions of Italy. The course of the disease in 30 subjects hospitalized in northern Italy (16 males and 14 females), aged six to 63 years, was studied by examination of sera collected at various intervals after the onset of symptoms and by exploration of the clinical and epidemiologic characteristics of the cases. Fifty-four additional "blind" serum samples from subjects who were possibly involved in this outbreak were referred to us from various regions of Italy (supplied by Prof. C. Collotti, Istituto Superiore di Sanita, Rome), but enough clinical and epidemiologic information is unavailable so far to interpret the results of the serological screening in these cases. Sera were also available from six healthy subjects who had been treated with the drug and from nine family contacts of patients. Screening for RB s Ag and antibody to RB s Ag. Two solid-phase radioimmunoassay (SPRIA) procedures were used to detect HB s Ag in the original immunoglobulins, in lots of the drug, and in sera from patients and contacts. The first method, which allowed detection of HB s Ag alone, was the commercially available procedure (Ausria II,® Abbott Laboratories, North Chicago, Ill.) and was performed according to the instructions of the manufacturer. The second method was a combined procedure, by which HB s Ag or its antibody (antiHB s ) could be detected quantitatively and reproducibly. Polystyrene tubes (5 X 38 mm) coated
254
HB s ' was also observed in four subjects, and the sera of two additional patients were free of detectable excess HB s Ag and anti-H'B, after the clearance of antigenemia. The last serum sample collected from one patient was found to be weakly positive for both HB s Ag and anti-Hfs., Three subjects who had received the drug of the HB s Ag-positive series, but who did not show any symptoms of viral hepatitis, had circulating anti-Hb, (one anti-HBs/ayw and two anti-HB s/ adyw). The sera of three healthy subjects who had received HB s Ag-negative lots of the drug were negative for both HB s Ag and anti-H'B.; Sera from all family contacts examined so far (nine subjects who included parents and relatives of patients) were negative for HB s Ag. Two of these subjects had circulating anti-HBs/ayw. Of 54 "blind" samples of serum taken from persons possibly involved in the outbreak and sent to us from various locations in Italy, 17 were found to be HB s Ag-positive (all of the adw subtype) and 23 were found to contain anti-H'B, (adw or adyw subtypes) in various titers. Epidemiology of the disease. The epidemiologic features of the disease were explored by screening of the medical records of 30 subjects. From the clinical point of view, the course of the disease was consistent with a typical icteric form of hepatitis B; the disease had no particular distinctive pattern and was accompanied by mild symptoms in all patients. The length of the incubation period was calculated according to dates of drug administration and onset of symptoms. An exact estimate (140 days) could be made for one patient, who received a single vial of the drug, and a rather
Figure 1. Agar gel diffusion with precipitin lines showing that the hepatitis B surface antigen (RB s Ag)positive sera of patients who received the drug was of subtype adw. For both figures, wells no. 1 and 4 contain RB s Ag!adw, well no. 3 contains the serum of patient no. 8, and well no. 6 contains the serum of patient no. 16. Wells no. 2 and 5 contain RB s Ag!ayw (left) or RB s Ag!adr (right). Well no. 7 contains antibody to RB s Ag (anti-RB s)! adw (left) or anti-RBs!adr (right).
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were positive for HB s Ag (lots examined: no. 49, 52, 57, 59, 62, 63, 65, 67, and 68). Conversely, the earlier and later preparations of the drug, which did not contain the implicated globulin, were free of detectable HB s Ag; most of the lots showed a weak positivity for anti-Hfs, (lots examined: no. 20, 26, 27, 32, 35, 39, 40, 43, 44, 46, 47, and 73). Reactivity of the drug was generally more evident than that of the original immunoglobulins, despite the lower protein concentration (6% vs. 16%). All of the antigens could be identified as subtype adw. Screening for HB s Ag and anti-Hli, in subjects receiving the drug. HB s Ag was detected in the serum of 28 of 30 patients for whom epidemiologic information was available. All positive sera were subtype adw, as determined by SPRIA and, for sam ples reacting in the immunoprecipitation tests, also by the agar gel diffusion test (figure 1). Titers of HB s Ag among subjects varied considerably, as measured by the combined SPRIA, and the interval that elapsed between onset of symptoms and collection of serum appeared to affect titers. The end-point dilution ranged from 1:2 to 1: 160,000. In some patients the course of antigenemia was also studied (figure 2). From the two patients who were not HB s Agpositive, sera collected 8.0 and 5.5 months, respectively, after the onset of symptoms were positive for anti-Hfs, (adw specificity). This finding demonstrates that all of the 30 patients had been infected by a hepatitis B virus carrying a, d, and w surface antigenic determinants. Seroconversion, as detected by the appearance of low-level anti-
Petrilli, Crouari, and De Flora
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the incubation period was independent of the age of the patient. An example of the epidemiologic course of the disease is shown in figure 2, in which the number of vials of the drug, the schedule of administration, the length of the incubation period, the persistence of symptoms (which varied considerably, ranging from one to five months), and the titers of HB s Ag in five patients are reported. Discussion
(months)
Figure 2. Course of antigenemia in five patients affected by hepatitis B after administration of a drug prescribed for allergy. Dark lines are titers of hepatitis B surface antigen (HB s Ag). Vertical arrows indicate the injections of the drug, and dotted areas refer to the persistence of symptoms. For patient no. 18 the number of doses and schedule of administration could not be ascertained.
accurate estimate was also made for two additional patients (160-165 and 115-120 days, respectively), who received two vials of the drug at five-day intervals. The possible minimal and maximal incubation periods in the other patients were calculated from the number of days elapsed between onset of symptoms and administration of the last and of the first dose of drug, respectively. The mean incubation period in these cases was 111143 days. An inverse relation was observed between the number of vials administered (one to 16, with a mean value of 5.85 doses per patient) and the length of the incubation period. No statistical correlation, however, could be established because it was not known in some cases whether all of the vials administered to each patient were from the HB s Ag-positive series. The length of
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TIME
The results of the present study demonstrate the very likely association between injection of a drug containing human immunoglobulins and development of hepatitis B in subjects with various allergic diseases. This view is supported by the finding that the original immunoglobulins, the lots of the allergy drug containing these immunoglobulins, and the sera of patients receiving the drug were all found to be positive for HB s Ag of the adw subtype. Moreover, some sera from patients and healthy individuals receiving the drug contained anti-HB s / adw. The adw subtype is relatively rare in Italy, and the ayw subtype is largely predominant, as it is in the whole Mediterranean area [6, 7]. Our previous epidemiologic investigations showed that the adw subtype accounted for only 19.7% of 300 sera positive for HB s Ag or anti-H'B, in the population of various regions of Italy [8, 9]. A tentative estimate of the morbidity from hepatitis among treated subjects can be made on the basis of the approximate numbers of drug vials distributed and presumably used, doses presumably given to each patient, and identified cases of overt disease. On these grounds, the attack rate of hepatitis B attributed to the medication appears to be low, in the range of one of 100 to one of 250. This result may be ascribed to the small infecting dose of virus in the drug and to the preexisting immunity in the treated population. Most patients examined had received multiple injections of the RB s Ag-positive drug. Therefore, it is not possible to know which one of the administered doses was critical in transmission of hepatitis and whether the multiple, parenteral introduction of virus may have favored devel-
256
hepatitis B infection as evaluated by frequency of HB s Ag and anti-Hls, could be revealed in one community of unweaned and preschool children who had received multiple administrations of conventional immunoglobulins prophylactically (authors' unpublished observations). On the other hand, the results of previous investigations [18-21] provided evidence that HB s Ag is also precipitated, although in relatively small amounts, in immunoglobulins prepared from HB s Ag-positive plasma by Cohn fractionation. There are various interpretations for the disagreement between these laboratory findings and the noninfectious character of immunoglobulins as demonstrated by clinical and epidemiologic experience. It is not known whether the infecting particle of hepatitis B virus and the outer coat of the virus (HB s Ag) behave in the same way during cold-ethanol fractionation. Also, the possibility cannot be excluded that this treatment may lead to dissociation of infectivity from antigenicity of the virus. However, neutralization of HB s Ag by an excess of coprecipitated anti-H'B, appears to be the most convincing explanation. In fact, frequency of anti-H'B, in the normal population is considerably higher than the frequency of HB s Ag [8, 9], and this ratio tends to increase further after fractionation of plasma. This view is supported by the finding that no commercial preparation of conventional immunoglobulins that we examined prior to the present study contained HB s Ag, whereas almost all of these preparations had excess anti-H'B., although at low titers [9]. Grady et al. have recently reported that the titers of anti-H'B, in conventional immunoglobulins prepared in Massachusetts have continued to rise since 1971, when screening of blood donors for HB s Ag became mandatory in the United States [22]. Similarly, Hoofnagle et al. [23] have observed a striking overall increase in prevalence and titer of antiHB s in immune serum globulins manufactured in the United States during 1973 and 1974. They examined 1,278 lots that were prepared by 19 manufacturers between 1962 and 1974 and submitted to the Bureau of Biologics of the Food and Drug Administration. Ten lots (0.8%), which had been produced between 1962 and
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opment of the disease. However, transmission of the disease was also observed in a subject who received a single injection of the drug and in two patients who were treated with only two doses at five-day intervals. In these subjects, the incubation period could be established precisely (140, 115-120, and 160-165 days, respectively). The minimal and maximal incubation periods in the remaining patients averaged III and 143 days, respectively, independently of the age of patients. On the whole, the length of the incubation period was considerable as compared, for example, with the mean value of 86 days observed by Krugman and Giles [10] in children experimentally infected via the parenteral route with viruscontaminated material (MS-2 strain). This difference is probably due to the low infecting dose of virus in the implicated drug, as estimated by the content of HB s Ag. In fact, an inverse relation was observed between the number of vials injected and the length of the incubation period, and it is significant that the shortest incubation period (maximum of 80 days) was recorded in a subject who received as many as 16 doses of the product. It is also of interest that HB s Ag could be detected in the serum of a subject during incubation of the disease, i.e., seven weeks before onset of scleral jaundice and six to eight weeks after administration of the drug. The results of these investigations provide clear evidence that immunoglobulins can be responsible for the transmission of hepatitis B. Serious problems arise as a consequence of this finding since this blood product is used widely in medical practice. It has already been recognized by investigators of previous studies that various blood products extracted from plasma by the Cohn method, such as fibrinogen, albumin (unless heated at 60 C for 10 hr), fraction III (thrombin), and fraction IV can create a hazard in the transmission of viral hepatitis [Il17]. Conversely, immunoglobulin preparations have been reported not to transmit hepatitis [16], and the general clinical experience is consistent with the lack of infectivity of this product when prepared from human plasma by the Cohn method. In addition, no increased incidence of
Petrilli, Crovari, and De Flora
Drug-Associated Hepatitis B
physicians that immunoglobulins should be administered only when strictly necessary.
References 1. Gelfand, H. H., Cinder, J. C., Grant, S. F., Soiffer, M. Evaluation of histamine-gamma globulin (histaglobin) in the treatment of various allergic conditions. Ann. Allergy 21: 150-155, 1963. 2. Giacovazzo, M. Physiopathologic premises for a desensitizing treatment against vaso-neuroactive compounds in essential cephalalgies (in Italian). Clin. lrer.67:2l5-247,1973. 3. Numo, R. Clinical and humoral evaluation of an aspecific desensitization by a histamine-serotonin-human imm une serum globulin drug in allergic diseases (in Italian). Folia Allergol. ImmunoI. Clin. 21:182-189, 1974. 4. Crovari, P., De Flora, S. Radioimmunoassay for the simultaneous detection and quantitation of hepatitis B surface antigen and of the homologous antibody. Boll. 1st. Sieroter. Milan. 54:149-160,1975. 5. Petrilli, F. L., Crovari, P., De Flora, S. Quantitative detection and typing of HBs Ag and anti-HB s by radioimmunoassay techniques. Dev. BioI. Stand. 30: 103-110, 1975. 6. Courouce-Pauty, A. M., Soulier, J. P. Further data on HB s subtypes: geographical distribution. Dev. BioI. Stand. 30:137-151,1975. 7. Mazzur, S., Burgert, S., Blumberg, B. S. Geographical distribution of Australia antigen determinants d, y and w. Nature (Lond.) 247:38-40,1974. 8. Petrilli, F. L., Crovari, P., De Flora, S. Seroepidemiology of hepatitis-B infections. Lancet 2:705, 1975. 9. Crovari, P., De Flora, S. Spread of hepatitis-B infection A seroepidemiologic study. Boll. tst. Sieroter. Milan. 54:428-436, 1975. 10. Krugman, S., Giles, J. P. Viral hepatitis: new light on an old disease. J.A.M.A. 212:1019-1029, 1970. 11. Boeve, N. R., Winterscheid, L. C., Merendino, K. A. Fibrinogen transmitted hepatitis in the surgical patients. Ann. Surg. 170:833-838, 1969. 12. Cronberg, S., Belfrage, S., Nilsson, 1. M. Fibrinogentransmitted hepatitis. Lancet 1:967-969, 1963. 13. Gellis, S. S., Neefe, J. R., Stokes, J., Strong, L. E., Janeway, L. A., Scatchard, G. Chemical, clinical, and immunological studies on the products of human plasma fractionation. XXXVI. Inactivation of the virus of homologous serum hepatitis in solution of normal human serum albumin by means of heat. J. Clin. Invest. 27:239-244, 1948. 14. Hsia, D. Y. Y., Kennell, J. H., Gellis, S. S. Homologous serum-hepatitis following use of fraction IV prepared from postpartum plasma. A report of fOUT cases. Am. J. Med. Sci. 226:261-264, 1953. 15. Le Bouvier, G. L., McCollum, R. W. Australia (hepatitis-associated) antigen: physico-chemical and
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1965, were found to be weakly positive for HB s Ag when retrospectively tested by radioimmunoassay. However, no epidemiologic correlation with hepatitis could be demonstrated. These data confirm that there may be excess HB s Ag in immunoglobulin preparations, but this event occurs only rarely under natural conditions when various samples are mixed and enter a fractionated plasma pool. In addition, the results of the present study show the possible relation between positivity for HB s Ag and infectivity of immunoglobulins. Despite the important significance of the event and its serious consequences on public health, it is difficult to pinpoint the responsibility for this iatrogenic accident. In fact, screening of each donation of human blood, plasma, or serum to be used in preparation of a biologic product is mandatory in the United States (according to the Code of Federal Regulations), but so far there are no required techniques for screening for HB s Ag. It is therefore conceivable that the original plasma samples used in preparation of HB s Ag-positive immunoglobulins escaped identification since highly sensitive techniques probably were not available or could not be used. On the other hand, there are no regulations for the screening of blood products, and such screening would be technically difficult. Positivity of immunoglobulins in the drug responsible for the transmission of hepatitis B was weak even at the time of our retrospective study and could be detected only by SPRIA. This method is suitable for HB s Ag screening of only serum or recalcified plasma and not of products of different density and composition, but we could overcome this difficulty by performing confirmatory and subtyping tests. The hepatitis outbreak caused by the drug has prompted the Italian Istituto Superiore di Sanita to appoint a committee of experts (in which we are participating), who are studying the problem of applying radioimmunoassay procedures to HB s Ag screening of immunoglobulins and other plasma derivatives. The serious consequences of this episode and the demonstrated possibility that immunoglobulins can act as a vehicle for transmission of hepatitis B, albeit rarely, should warn attending
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19. Berg, R., Bjorling, H., Berntsen, K., Espmark, A. Recovery of Australia antigen from human plasma products separated by a modified Cohn fractionation. Vox Sang. 22:1-13,1972. 20. Leveque, P., Drouet, J., Schmitthaeusler, R., North, M. L., Amouch, P., Malgras, J. HB s antigen in human plasma fractions. Vox Sang. 28:1-8, 1975. 21. Zuckermann, A. j. Human viral hepatitis. Hepatitis associated antigen and viruses. North-Holland, Amsterdam, 1972.422 p. 22. Grady, G. F., Rodman, M., Larsen, L. H. Hepatitis B antibody in conventional ')'-globulin. J. Infect. Dis. 132:474-477, 1975. 23. Hoofnagle, J. H., Gerety, R. J. Barker, L. F. Antibody to the hepatitis B surface antigen in immune serum globulin. Transfusion 15:408-413, 1975.
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immunological characteristics. Adv. Virus Res. 16: 357-396, 1970. 16. Penell, R. B. The distribution of certain viruses in the fractionation of plasma. In F. W. Hartmann, G. A. Lo Grippo, E. M. Mateer, and S. Baron red]. Hepatitis frontiers. Little, Brown, Boston, 1957, p. 297310. 17. Porter, J. E., Shapiro, M., Maltby, G. L., Drake, M. E., Barondness, J. A., Bashe, W. J., Stokes, J., Jr., Oliphant, J. W., Diefenbach, W. C. L., Murray, R. R., Leone, N. C. Human thrombin as vehicle of infection in homologous 'serum hepatitis. J.A.M.A. 153:1719,1953. 18. Crovari, P., De Flora, S., Castagna, R. Distribution of hepatitis B surface antigen in plasma fractions extracted by Cohn method (in Italian). G. Ig. Med. Prev., 1975 (in press).
Petrilli, Crouari, and De Flora
Hepatitis B in Subjects Treated with a Drug Containing Immunoglobulins From the Institute of Hygiene, University of Genoa, Genoa, Italy
Fernando Luigi Petrilli, Pietro Crovari, and Silvio De Flora
In late 1974 cases of viral hepatitis were observed in Italy among subjects suffering from allergic diseases who had received a drug containing human immunoglobulins. On December 12, 1974, after the suspicion of an iatrogenic cause of the disease emerged, the Italian Ministry of Health ordered nationwide sequestration of the product. During the early months of 1975, however, additional cases of the disease were recorded among individuals who had been treated before December 12, 1974. Official quantitative data are lacking so far about the spread of this outbreak in part because a conclusive relation between the administration of the drug and the appearance of symptoms could not be proven in many cases. However, a rough estimate indicates that there were >100 cases of overt disease.
We have explored a number of cases involved in this outbreak by studying the epidemiologic patterns of the disease and by demonstrating the very likely association between injection of the drug and development of disease among the subjects examined. In fact, the original immunoglobulins, the drug, and the sera of patients were all found to be positive for the surface antigen of hepatitis B virus (HB s Ag) of a subtype that is relatively rare in Italy. Materials and Methods
Drug. Trilergan," the drug that was prescribed for treatment of allergy; is manufactured in Italy. It contains, in one dose (1 ml), 12 mg of human immunoglobulins, 0.15 JLg of histamine hydrochloride, 0.15 JLg of serotonin, 0.1 mg of sodium merthiolate, 9 mg of NaCI, and 20 mg of mannitol. The product is prepared in the lyophilized form and is reconstituted with I ml of bidistilled water before use. It is injected sc in cycles of three to four administrations at intervals of four to five days. Like similar products manufactured and distributed in other European countries, the drug has a vaccine-like mechanism because of formation of antigenic complexes of histamine and serotonin with immunoglobulins. Serotonin and
Received for publication March 22, 1976, and in revised form July 19, 1976. This investigation was supported by grant no. 73.01363.43 from the Italian Centro Nazionale delle Richerche. We acknowledge the assistance of Drs. C. Bennicelli and P. Zanacchi. We thank all of our colleagues who cooperated in providing clinical and epidemiological information, particularly Drs. C. Collotti, F. Croce, M. Furfaro, A. Negro, L. Robert, and G. Veronesi. Please address requests for reprints to Prof. F. L. Petrilli, Institute of Hygiene, University of Genoa, Via Pastore 1, 16132 Genoa, Italy.
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In late 1974 and early 1975, several cases of viral hepatitis were reported in Italy among subjects who had received subcutaneous injections of a drug containing human immunoglobulins that was prescribed for the treatment of allergies. Epidemiologic and laboratory investigations provided evidence that the original immunoglobulins, the series of the drug containing these immunoglobulins, and sera from a number of patients were all positive for hepatitis B surface antigen (HB s Ag) of the adw subtype, which is relatively rare in Italy. Some sera from patients and healthy subjects treated with the HB s Ag-positive drug were also found to be positive for antibody to HB s Ag of the adw subtype. The clinical course of the disease was consistent with typical forms of icteric hepatitis in all patients examined. The average length of the possible incubation period was 111-143 days, and an inverse relation was observed between the number of doses administered and the length of the incubation period. The possibility that immunoglobulins can be responsible for the transmission of viral hepatitis raises a number of theoretical and practical problems concerning control and use of these blood products.
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Drug-Associated Hepatitis B
with purified anti-H'B, (Puritest, ISl\tfSB, Milan, Italy) were used as the solid-phase substrate, and human 125I-labeled anri-Hls, (Abbott Laboratories) was used as labeled antibody. This SPRIA is based upon preincubation of test sera and of negative controls with a fixed dose of a standard HB s Ag (adw and ayw). Radioactivity counts of test sera were determined in relation to those of standard HB s Ag control sera. HB s Ag- and anti-Hb.-positive samples have significantly more and less counts, respectively, than control samples. The end-point dilution of positive sera was determined by plotting of the resulting values on a nomograph, which was drawn according to the findings of a study of SPRIA kinetics [4]. The specificity of positive samples was checked by subtyping of HB s Ag- and anti-H'Bj-positive samples with the aid of previously described SPRIA procedures [4,5]. Reference adw, adr, and ayw antigens and antibodies were supplied by Dr. R. Purcell (National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Md.), and AB, AC, B, and C reagents, corresponding to Le Bouvier's ad, ay, d, and y reagents, respectively, were supplied by Dr. R. Gispen (Laboratory of Virology, National Institute of Public Health, Bilthoven, The Netherlands). Results
Screening for RB s Ag in immunoglobulins and in drug preparations. Examination of the original immunoglobulins contained in the drug, after resuspension in glycine buffer at a concentration of 16% (wt/vol), showed a very weak positivity for HB s Ag. Positivity was rather inconstant and .close to the cut-off point of the method. As shown by the results of the combined SPRIA, small traces of anti-H'B, were also detectable in the product. HB s Ag was neutralized to a greater extent by adw than by ayw antisera. A number of drug lots were screened by tests of the lyophilized product resuspended in 0.2 ml of distilled water (a concentration five times that of the injectable preparation). All lots examined within the series containing the HB s Ag-positive immunoglobulin, including lots no. 49-68 prepared from February to June 1974,
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histamine are adsorbed to the globulin by mixing of the reactants for 30 min at pH 6.4-7.0. The therapeutic efficacy and the clinical indications for such desensitizing treatment have been studied by a number of investigators [1-3J. The immunoglobulins contained in the implicated lots of the drug were imported from the United States, where they had been released by the Bureau of Biologics of the Food and Drug Administration in May 1973. These immunoglobulins were used exclusively for the preparation of the drug and were incorporated into lots manufactured from February to June 1974. Patients. Subjects receiving the drug had allergic diseases of various types. Many of them were treated at the same time with other antiinflammatory agents. Patients who developed viral hepatitis after injection of the product were hospitalized in various regions of Italy. The course of the disease in 30 subjects hospitalized in northern Italy (16 males and 14 females), aged six to 63 years, was studied by examination of sera collected at various intervals after the onset of symptoms and by exploration of the clinical and epidemiologic characteristics of the cases. Fifty-four additional "blind" serum samples from subjects who were possibly involved in this outbreak were referred to us from various regions of Italy (supplied by Prof. C. Collotti, Istituto Superiore di Sanita, Rome), but enough clinical and epidemiologic information is unavailable so far to interpret the results of the serological screening in these cases. Sera were also available from six healthy subjects who had been treated with the drug and from nine family contacts of patients. Screening for RB s Ag and antibody to RB s Ag. Two solid-phase radioimmunoassay (SPRIA) procedures were used to detect HB s Ag in the original immunoglobulins, in lots of the drug, and in sera from patients and contacts. The first method, which allowed detection of HB s Ag alone, was the commercially available procedure (Ausria II,® Abbott Laboratories, North Chicago, Ill.) and was performed according to the instructions of the manufacturer. The second method was a combined procedure, by which HB s Ag or its antibody (antiHB s ) could be detected quantitatively and reproducibly. Polystyrene tubes (5 X 38 mm) coated
254
HB s ' was also observed in four subjects, and the sera of two additional patients were free of detectable excess HB s Ag and anti-H'B, after the clearance of antigenemia. The last serum sample collected from one patient was found to be weakly positive for both HB s Ag and anti-Hfs., Three subjects who had received the drug of the HB s Ag-positive series, but who did not show any symptoms of viral hepatitis, had circulating anti-Hb, (one anti-HBs/ayw and two anti-HB s/ adyw). The sera of three healthy subjects who had received HB s Ag-negative lots of the drug were negative for both HB s Ag and anti-H'B.; Sera from all family contacts examined so far (nine subjects who included parents and relatives of patients) were negative for HB s Ag. Two of these subjects had circulating anti-HBs/ayw. Of 54 "blind" samples of serum taken from persons possibly involved in the outbreak and sent to us from various locations in Italy, 17 were found to be HB s Ag-positive (all of the adw subtype) and 23 were found to contain anti-H'B, (adw or adyw subtypes) in various titers. Epidemiology of the disease. The epidemiologic features of the disease were explored by screening of the medical records of 30 subjects. From the clinical point of view, the course of the disease was consistent with a typical icteric form of hepatitis B; the disease had no particular distinctive pattern and was accompanied by mild symptoms in all patients. The length of the incubation period was calculated according to dates of drug administration and onset of symptoms. An exact estimate (140 days) could be made for one patient, who received a single vial of the drug, and a rather
Figure 1. Agar gel diffusion with precipitin lines showing that the hepatitis B surface antigen (RB s Ag)positive sera of patients who received the drug was of subtype adw. For both figures, wells no. 1 and 4 contain RB s Ag!adw, well no. 3 contains the serum of patient no. 8, and well no. 6 contains the serum of patient no. 16. Wells no. 2 and 5 contain RB s Ag!ayw (left) or RB s Ag!adr (right). Well no. 7 contains antibody to RB s Ag (anti-RB s)! adw (left) or anti-RBs!adr (right).
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were positive for HB s Ag (lots examined: no. 49, 52, 57, 59, 62, 63, 65, 67, and 68). Conversely, the earlier and later preparations of the drug, which did not contain the implicated globulin, were free of detectable HB s Ag; most of the lots showed a weak positivity for anti-Hfs, (lots examined: no. 20, 26, 27, 32, 35, 39, 40, 43, 44, 46, 47, and 73). Reactivity of the drug was generally more evident than that of the original immunoglobulins, despite the lower protein concentration (6% vs. 16%). All of the antigens could be identified as subtype adw. Screening for HB s Ag and anti-Hli, in subjects receiving the drug. HB s Ag was detected in the serum of 28 of 30 patients for whom epidemiologic information was available. All positive sera were subtype adw, as determined by SPRIA and, for sam ples reacting in the immunoprecipitation tests, also by the agar gel diffusion test (figure 1). Titers of HB s Ag among subjects varied considerably, as measured by the combined SPRIA, and the interval that elapsed between onset of symptoms and collection of serum appeared to affect titers. The end-point dilution ranged from 1:2 to 1: 160,000. In some patients the course of antigenemia was also studied (figure 2). From the two patients who were not HB s Agpositive, sera collected 8.0 and 5.5 months, respectively, after the onset of symptoms were positive for anti-Hfs, (adw specificity). This finding demonstrates that all of the 30 patients had been infected by a hepatitis B virus carrying a, d, and w surface antigenic determinants. Seroconversion, as detected by the appearance of low-level anti-
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the incubation period was independent of the age of the patient. An example of the epidemiologic course of the disease is shown in figure 2, in which the number of vials of the drug, the schedule of administration, the length of the incubation period, the persistence of symptoms (which varied considerably, ranging from one to five months), and the titers of HB s Ag in five patients are reported. Discussion
(months)
Figure 2. Course of antigenemia in five patients affected by hepatitis B after administration of a drug prescribed for allergy. Dark lines are titers of hepatitis B surface antigen (HB s Ag). Vertical arrows indicate the injections of the drug, and dotted areas refer to the persistence of symptoms. For patient no. 18 the number of doses and schedule of administration could not be ascertained.
accurate estimate was also made for two additional patients (160-165 and 115-120 days, respectively), who received two vials of the drug at five-day intervals. The possible minimal and maximal incubation periods in the other patients were calculated from the number of days elapsed between onset of symptoms and administration of the last and of the first dose of drug, respectively. The mean incubation period in these cases was 111143 days. An inverse relation was observed between the number of vials administered (one to 16, with a mean value of 5.85 doses per patient) and the length of the incubation period. No statistical correlation, however, could be established because it was not known in some cases whether all of the vials administered to each patient were from the HB s Ag-positive series. The length of
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TIME
The results of the present study demonstrate the very likely association between injection of a drug containing human immunoglobulins and development of hepatitis B in subjects with various allergic diseases. This view is supported by the finding that the original immunoglobulins, the lots of the allergy drug containing these immunoglobulins, and the sera of patients receiving the drug were all found to be positive for HB s Ag of the adw subtype. Moreover, some sera from patients and healthy individuals receiving the drug contained anti-HB s / adw. The adw subtype is relatively rare in Italy, and the ayw subtype is largely predominant, as it is in the whole Mediterranean area [6, 7]. Our previous epidemiologic investigations showed that the adw subtype accounted for only 19.7% of 300 sera positive for HB s Ag or anti-H'B, in the population of various regions of Italy [8, 9]. A tentative estimate of the morbidity from hepatitis among treated subjects can be made on the basis of the approximate numbers of drug vials distributed and presumably used, doses presumably given to each patient, and identified cases of overt disease. On these grounds, the attack rate of hepatitis B attributed to the medication appears to be low, in the range of one of 100 to one of 250. This result may be ascribed to the small infecting dose of virus in the drug and to the preexisting immunity in the treated population. Most patients examined had received multiple injections of the RB s Ag-positive drug. Therefore, it is not possible to know which one of the administered doses was critical in transmission of hepatitis and whether the multiple, parenteral introduction of virus may have favored devel-
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hepatitis B infection as evaluated by frequency of HB s Ag and anti-Hls, could be revealed in one community of unweaned and preschool children who had received multiple administrations of conventional immunoglobulins prophylactically (authors' unpublished observations). On the other hand, the results of previous investigations [18-21] provided evidence that HB s Ag is also precipitated, although in relatively small amounts, in immunoglobulins prepared from HB s Ag-positive plasma by Cohn fractionation. There are various interpretations for the disagreement between these laboratory findings and the noninfectious character of immunoglobulins as demonstrated by clinical and epidemiologic experience. It is not known whether the infecting particle of hepatitis B virus and the outer coat of the virus (HB s Ag) behave in the same way during cold-ethanol fractionation. Also, the possibility cannot be excluded that this treatment may lead to dissociation of infectivity from antigenicity of the virus. However, neutralization of HB s Ag by an excess of coprecipitated anti-H'B, appears to be the most convincing explanation. In fact, frequency of anti-H'B, in the normal population is considerably higher than the frequency of HB s Ag [8, 9], and this ratio tends to increase further after fractionation of plasma. This view is supported by the finding that no commercial preparation of conventional immunoglobulins that we examined prior to the present study contained HB s Ag, whereas almost all of these preparations had excess anti-H'B., although at low titers [9]. Grady et al. have recently reported that the titers of anti-H'B, in conventional immunoglobulins prepared in Massachusetts have continued to rise since 1971, when screening of blood donors for HB s Ag became mandatory in the United States [22]. Similarly, Hoofnagle et al. [23] have observed a striking overall increase in prevalence and titer of antiHB s in immune serum globulins manufactured in the United States during 1973 and 1974. They examined 1,278 lots that were prepared by 19 manufacturers between 1962 and 1974 and submitted to the Bureau of Biologics of the Food and Drug Administration. Ten lots (0.8%), which had been produced between 1962 and
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opment of the disease. However, transmission of the disease was also observed in a subject who received a single injection of the drug and in two patients who were treated with only two doses at five-day intervals. In these subjects, the incubation period could be established precisely (140, 115-120, and 160-165 days, respectively). The minimal and maximal incubation periods in the remaining patients averaged III and 143 days, respectively, independently of the age of patients. On the whole, the length of the incubation period was considerable as compared, for example, with the mean value of 86 days observed by Krugman and Giles [10] in children experimentally infected via the parenteral route with viruscontaminated material (MS-2 strain). This difference is probably due to the low infecting dose of virus in the implicated drug, as estimated by the content of HB s Ag. In fact, an inverse relation was observed between the number of vials injected and the length of the incubation period, and it is significant that the shortest incubation period (maximum of 80 days) was recorded in a subject who received as many as 16 doses of the product. It is also of interest that HB s Ag could be detected in the serum of a subject during incubation of the disease, i.e., seven weeks before onset of scleral jaundice and six to eight weeks after administration of the drug. The results of these investigations provide clear evidence that immunoglobulins can be responsible for the transmission of hepatitis B. Serious problems arise as a consequence of this finding since this blood product is used widely in medical practice. It has already been recognized by investigators of previous studies that various blood products extracted from plasma by the Cohn method, such as fibrinogen, albumin (unless heated at 60 C for 10 hr), fraction III (thrombin), and fraction IV can create a hazard in the transmission of viral hepatitis [Il17]. Conversely, immunoglobulin preparations have been reported not to transmit hepatitis [16], and the general clinical experience is consistent with the lack of infectivity of this product when prepared from human plasma by the Cohn method. In addition, no increased incidence of
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Drug-Associated Hepatitis B
physicians that immunoglobulins should be administered only when strictly necessary.
References 1. Gelfand, H. H., Cinder, J. C., Grant, S. F., Soiffer, M. Evaluation of histamine-gamma globulin (histaglobin) in the treatment of various allergic conditions. Ann. Allergy 21: 150-155, 1963. 2. Giacovazzo, M. Physiopathologic premises for a desensitizing treatment against vaso-neuroactive compounds in essential cephalalgies (in Italian). Clin. lrer.67:2l5-247,1973. 3. Numo, R. Clinical and humoral evaluation of an aspecific desensitization by a histamine-serotonin-human imm une serum globulin drug in allergic diseases (in Italian). Folia Allergol. ImmunoI. Clin. 21:182-189, 1974. 4. Crovari, P., De Flora, S. Radioimmunoassay for the simultaneous detection and quantitation of hepatitis B surface antigen and of the homologous antibody. Boll. 1st. Sieroter. Milan. 54:149-160,1975. 5. Petrilli, F. L., Crovari, P., De Flora, S. Quantitative detection and typing of HBs Ag and anti-HB s by radioimmunoassay techniques. Dev. BioI. Stand. 30: 103-110, 1975. 6. Courouce-Pauty, A. M., Soulier, J. P. Further data on HB s subtypes: geographical distribution. Dev. BioI. Stand. 30:137-151,1975. 7. Mazzur, S., Burgert, S., Blumberg, B. S. Geographical distribution of Australia antigen determinants d, y and w. Nature (Lond.) 247:38-40,1974. 8. Petrilli, F. L., Crovari, P., De Flora, S. Seroepidemiology of hepatitis-B infections. Lancet 2:705, 1975. 9. Crovari, P., De Flora, S. Spread of hepatitis-B infection A seroepidemiologic study. Boll. tst. Sieroter. Milan. 54:428-436, 1975. 10. Krugman, S., Giles, J. P. Viral hepatitis: new light on an old disease. J.A.M.A. 212:1019-1029, 1970. 11. Boeve, N. R., Winterscheid, L. C., Merendino, K. A. Fibrinogen transmitted hepatitis in the surgical patients. Ann. Surg. 170:833-838, 1969. 12. Cronberg, S., Belfrage, S., Nilsson, 1. M. Fibrinogentransmitted hepatitis. Lancet 1:967-969, 1963. 13. Gellis, S. S., Neefe, J. R., Stokes, J., Strong, L. E., Janeway, L. A., Scatchard, G. Chemical, clinical, and immunological studies on the products of human plasma fractionation. XXXVI. Inactivation of the virus of homologous serum hepatitis in solution of normal human serum albumin by means of heat. J. Clin. Invest. 27:239-244, 1948. 14. Hsia, D. Y. Y., Kennell, J. H., Gellis, S. S. Homologous serum-hepatitis following use of fraction IV prepared from postpartum plasma. A report of fOUT cases. Am. J. Med. Sci. 226:261-264, 1953. 15. Le Bouvier, G. L., McCollum, R. W. Australia (hepatitis-associated) antigen: physico-chemical and
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1965, were found to be weakly positive for HB s Ag when retrospectively tested by radioimmunoassay. However, no epidemiologic correlation with hepatitis could be demonstrated. These data confirm that there may be excess HB s Ag in immunoglobulin preparations, but this event occurs only rarely under natural conditions when various samples are mixed and enter a fractionated plasma pool. In addition, the results of the present study show the possible relation between positivity for HB s Ag and infectivity of immunoglobulins. Despite the important significance of the event and its serious consequences on public health, it is difficult to pinpoint the responsibility for this iatrogenic accident. In fact, screening of each donation of human blood, plasma, or serum to be used in preparation of a biologic product is mandatory in the United States (according to the Code of Federal Regulations), but so far there are no required techniques for screening for HB s Ag. It is therefore conceivable that the original plasma samples used in preparation of HB s Ag-positive immunoglobulins escaped identification since highly sensitive techniques probably were not available or could not be used. On the other hand, there are no regulations for the screening of blood products, and such screening would be technically difficult. Positivity of immunoglobulins in the drug responsible for the transmission of hepatitis B was weak even at the time of our retrospective study and could be detected only by SPRIA. This method is suitable for HB s Ag screening of only serum or recalcified plasma and not of products of different density and composition, but we could overcome this difficulty by performing confirmatory and subtyping tests. The hepatitis outbreak caused by the drug has prompted the Italian Istituto Superiore di Sanita to appoint a committee of experts (in which we are participating), who are studying the problem of applying radioimmunoassay procedures to HB s Ag screening of immunoglobulins and other plasma derivatives. The serious consequences of this episode and the demonstrated possibility that immunoglobulins can act as a vehicle for transmission of hepatitis B, albeit rarely, should warn attending
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19. Berg, R., Bjorling, H., Berntsen, K., Espmark, A. Recovery of Australia antigen from human plasma products separated by a modified Cohn fractionation. Vox Sang. 22:1-13,1972. 20. Leveque, P., Drouet, J., Schmitthaeusler, R., North, M. L., Amouch, P., Malgras, J. HB s antigen in human plasma fractions. Vox Sang. 28:1-8, 1975. 21. Zuckermann, A. j. Human viral hepatitis. Hepatitis associated antigen and viruses. North-Holland, Amsterdam, 1972.422 p. 22. Grady, G. F., Rodman, M., Larsen, L. H. Hepatitis B antibody in conventional ')'-globulin. J. Infect. Dis. 132:474-477, 1975. 23. Hoofnagle, J. H., Gerety, R. J. Barker, L. F. Antibody to the hepatitis B surface antigen in immune serum globulin. Transfusion 15:408-413, 1975.
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immunological characteristics. Adv. Virus Res. 16: 357-396, 1970. 16. Penell, R. B. The distribution of certain viruses in the fractionation of plasma. In F. W. Hartmann, G. A. Lo Grippo, E. M. Mateer, and S. Baron red]. Hepatitis frontiers. Little, Brown, Boston, 1957, p. 297310. 17. Porter, J. E., Shapiro, M., Maltby, G. L., Drake, M. E., Barondness, J. A., Bashe, W. J., Stokes, J., Jr., Oliphant, J. W., Diefenbach, W. C. L., Murray, R. R., Leone, N. C. Human thrombin as vehicle of infection in homologous 'serum hepatitis. J.A.M.A. 153:1719,1953. 18. Crovari, P., De Flora, S., Castagna, R. Distribution of hepatitis B surface antigen in plasma fractions extracted by Cohn method (in Italian). G. Ig. Med. Prev., 1975 (in press).
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