Hepatitis a virus infection in fulminant hepatitis and chronic active hepatitis

0016-5085/78/7405-0879%02.00/0 GASTROENTEROLOGY 74:879-882, 1978 Copyright 0 1978 by the AmericanGastroenterological Association

Vol. 74,

No. 5, Part 1 Printed In USA.

HEPATITIS A VIRUS INFECTION CHRONIC ACTIVE HEPATITIS JORGE RAKELA, M.D.,

IN FULMINANT

ALLAN G. REDEKER, M.D.,

RICHARD DECKER, PH.D.,

LACY R. OVERBY, PH.D.,

HEPATITIS

AND

VIRGINIA M. EDWARDS, B.S., AND JAMES W. MOSLEY, M.D.

Department of Medicine, University of Southern California School of Medicine; Liver Seruice, Ranch0 Los Amigos County Hospital, Los Angeles, California; and Diagnostics Division, Abbott Laboratories, North Chicago, Illinois

We looked for hepatitis A virus (HAV) infection as an etiological agent among patients with fulminant hepatitis and chronic active hepatitis. Among 42 patients with hepatitis B surface antigen (HBsAg)-negative fulminant disease, we detected seroconversion by immune adherence hemagglutination for antibody to HAV (anti-HAV) in 3 of 10 survivors, as well as an increasing anti-HAV score by immune electron microscopy. In the 32 HBsAg-negative nonsurvivors, we found 3 patients with anti-HAV detectable by immune electron microscopy and radioimmunoassay, but not by immune adherence hemagglutination. We classified the latter cases as presumptively attributable to HAV. In 10 survivors among 30 HBsAg-positive patients with fulminant disease, we did not detect any instances of anti-HAV seroconversion. In the 20 HBsAg-positive nonsurvivors, 1 case had anti-HAV detectable by immune electron microscopy and radioimmunoassay but not by immune adherence hemagglutination. This case was also considered to be presumptively caused by HAV. In addition, we studied anti-HAV in patients with chronic active hepatitis. The incidence of anti-HAV in 13 HBsAg-positive cases was 31%, which did not differ from the 32% found in 22 HBsAg-negative cases. There was one seroconversion for anti-HAV in 1 patient with HBsAg-negative chronic active hepatitis, but this appeared to be an epiphenomenon. We looked for fecal shedding of HAV in 14 patients with HBsAg-negative chronic active hepatitis without success. the appearance of antibody to HAV (anti-HAV) in a patient who was previously anti-HAV negative. On the other hand, persons with chronic active hepatitis could well have detectable levels of fecal excretion’” if HAV is capable of causing that condition. A second method of diagnosing HAV infection is to show conversion from anti-HAV negative to anti-HAV positive. As measured by IEMH, “3 I:’and radioimmunoassay (RIA),” however, anti-HAV becomes detectable before the time when the patient is likely to be seen for clinical symptoms. Furthermore, increases in titer between two or more specimens in the acute stage of infection have not been reliably documented by either IEM or RIA. The immune adherence hemagglutination (IAHA) test, which can be reliably quantitated, does not become positive until convalescence.X, I’%Unfortunately, approximately 75% of patients with fulminant disease succumb during the encephalopathic stage,ld and the reliability of IEM positivity based on a single specimen has been questioned.15 The pattern of IEM positivity, RIA positivity, and IAHA negativity, however, would seem to be a very reasonable basis for a presumptive diagnosis despite the failure of the patient to survive. In chronic active hepatitis, on the other hand, it is difficult to predict what pattern of antibody would be found if HAV were the initiating or perpetuating cause.

Various studies in which hepatitis A virus (HAV) was identified epidemiologically as the agent responsible for acute icteric illness suggested that, in terms of case fatality rate and chronicity, type A hepatitis does not pose the threat associated with type B hepatitis. The fatality rate in type A disease has been estimated to be less than 1 per 1000,’ and attempts to demonstrate a relationship to subsequent chronic liver disease have been generally negative.‘-” The recent development of diagnostic techniques for HAV infection make it possible to assess its etiological role in different liver diseases.‘+9 Our purpose in the present study was to determine if HAV infection were a cause in our patient population of fulminant viral hepatitis and chronic active hepatitis. One method for the diagnosis of HAV infection is the demonstration of virucopria. By the technique of immune elect,ron microscopy (IEM), however, viral particles have generally been found in feces only during the last part of the incubation period and just after onset of symptoms.“‘. ‘I For clinical purposes, therefore, the only practical approach in the acute phase is to demonstrate Received May 31, 1977. Accepted December 19, 1977. Address requests for reprints to: Dr. Jorge Rakela, Ranch0 Los Amigos County Hospital (1100 Building), Downey, California 90242.

7601 E. Imperial Highway,

879

RAKELA ET AL.

880

We studied 72 patients with fulminant disease and 35 patients with chronic active hepatitis to see if HAV could be implicated.

Method of Study Selection of patients. The diagnosis of fulminant hepatitis was based on the presence of acute hepatic encephalopathy in patients with compatible clinical and biochemical findings. Only patients developing coma within 4 weeks of onset of clinical symptoms were accepted. Patients with acute liver failure complicating chronic liver disease and cases suspected of being drug-induced were also excluded. From 125 patients seen before 1968 and 1975 who met these criteria, we were able to retrieve serum samples for 42 cases of fulminant hepatitis that were hepatitis B surface antigen (HBsAgl-negative and, for comparison, 30 HBsAg-positive cases. Ten of 42 patients with HBsAg-negative fulminant hepatitis in our series survived at least 30 days and had follow-up specimens available. Ten of 30 patients with HBsAg-positive disease also survived and could be studied for anti-HAV response. We investigated serologically 35 patients with chronic active hepatitis, of whom 22 were HBsAg-negative and 13 were HBsAg-positive. We also looked for shedding of HAV in fecal samples from 14 HBsAg-negative patients with chronic active hepatitis. All HBsAg-negative patients selected were from those lacking known percutaneous exposures (transfusion and/or illicit self-injection). Laboratory procedures. To detect HAV shedding, we used the IEM procedure of Feinstone and co-workers.” Briefly, a portion of the fecal sample sufficient to give a 2% (w/v) solution was homogenized in a protein-containing medium. A filtrate of this suspension was then incubated with 1:lO dilution of serum known to be strongly reactive against HAV. After 1 hr at room temperature, the mixture was centrifuged, the pellet was resuspended in water, and 1 drop was stained with 2% phosphotungstic acid. At least 10 good quality squares of a 400-mesh grid were systematically searched for “haloed” and aggregated particles before the specimen was called negative. Anti-HAV in patient samples was determined by IEM according to the technique also described by Feinstone and coworkers.” We used a standardized 2% suspension of feces

containing HAV, and evaluated haloing and aggregation of coded preparations on a scale from 0 to 4+. The IAHA procedure for anti-HAV was based on that

described by Mayumi and co-workers”’ for HBsAg. Reagents included: fresh frozen guinea pig complement; human type 0 red blood cells; bovine serum albumin-barbital buffer; and 4 units of HAV antigen (HH-1 strain”). The last had been purified by isopycnic banding with cesium chloride and concentrated by filtration. Hemagglutination was deemed com-

plete after 90 to 120 min at room temperature, and was scored on a scale of o to 4+ (end point for titration, 3+). Each serum was heated at 56°C for 30 min to inactivate complement and then tested at dilutions of l:lO, l:lOO, and 1:lOOO. Specificity of our test was confirmed with preinfection and postinfection chimpanzee sera (National Institute of Allergy and Infectious Diseases reagents V-811-801-573 and V-811-501-573, respectively), and a panel of positive and negative samples also tested for anti-HAV by Dr. Jules L. Dienstag, Bethesda. The RIA procedure for anti-HAV was developed in the Diagnostics Divisions, Abbott Laboratories, North Chicago, Illinois. Plastic beads coated with anti-HAV in the form of convalescent serum were incubated with a standardized 10% suspension of feces collected from a patient 5 days before onset of jaundice. Anti-HAV rich IgG, labeled with lraI according to the method of Hunter and Greenwood,” was derived by ammonium sulfate precipitation from the same convalescent serum used to coat the beads. The serum to be tested and the IgG preparation were then incubated with the beads and fecal suspension. The occurrence of anti-HAV in the serum being evaluated was assessed by inhibition of ““I-anti-HAV binding. The specimen was considered positive if y counts per minute were reduced by 50% or more when compared with a negative control. All samples were tested for HBsAg initially by counterelectrophoresis and, if negative, by RIA (Ausria-II, Abbott Laboratories).

Results Fulminant hepatitis. Of 42 patients studied who were HBsAg-negative during the period of observation, there were 10 who recovered. Of these 10,3 remained seronegative for anti-HAV by IEM and IAHA at 30 days or more after admission, and 4 were seropositive by both tests at essentially the same levels in the acute and convalescent phases. There were, however, 3 patients who were IEM-positive, RIA-positive, and IAHA-negative in the acute phase, and had IAHA seroconversion after convalescence (table 1). Among 32 HBsAg-negative patients who died in hepatic coma, 7 were anti-HAV negative by IEM and IAHA, and 22 were positive by both techniques. The remaining 3 patients had anti-HAV by IEM, confirmed by RIA-positivity in the 2 for whom there was residual serum for this test (cases 4 to 6, table 2). None of the 3 was anti-HAV positive by IAHA. Of 30 patients with HBsAg-positive hepatitis whom we studied, there were 10 survivors. Of these, 5 were anti-HA negative by IEM and IAHA in the acute and

TABLE 1. Cases of nonfatal fulminant viral hepatitis having seroconversion Onset of encepha- Date of sample lopathy studied

sex

Age

Onset of symp toms

1

M

30

9/l/70

2

M

27

11/2/71

11/14/71

3

M

23

1212174

12/10/74

Case no.

9112170

Vol. 74, No. 5, Part 1

g/12/70 51 6/71 11/15/71 12/16/71 101 5172 12/10/74 11 9175 21 2176

for antibody to hepatitis A virus” HBsAg Neg NT Neg NT NT Neg NT NT

Anti-I-WV IEM

RIA

lABA

1+ 2-3+ 2-3+ 2-3+ 3-4+ 2+ 2+ 3+

Pos NT Pos NT NT Pos NT NT


G Abbreviations are: HBsAg, hepatitis B surface antigen; anti-HAV, antibody to hepatitis A virus (HAV); IEM, immune microscopy; RIA, radioimmunoassay; IAHA, immune adherence hemagglutination; Neg, negative; Pos, positive; NT, not tested.

electron

May 1978

HAV IN FULMINANT TABLE

AND CHRONIC ACTIVE HEPATITIS

881

2. Cases of fatal fulminunt viral hepatitis having antibody to hepatitis A virus by immune electron microscopy andlor radioimmunoassay Onset of encepha- Dat;t;fkkrple lopathy

Case no.

Sex

Age

4

F

27

g/17/69

5 6 7

F F F

17 23 23

4130173 4128175 5128170

” For abbreviations,

g/19/69 51 3173 4130175 61 l/70

(I

Date of death g/20/69 51 4173 51 l/75 61 6170

HBsAg Neg Neg Neg Pos

Anti-HAV IEM l-2+ 2-3+ 2-3+ 2-3+

RIA Pos NT Pos Pos

IAHA
see table 1, footnote a.

convalescent phase, and the other 5 were anti-HA positive by both techniques in both sera. None showed seroconversion. Among 20 HBsAg-positive patients dying in hepatic coma, 11 were anti-HAV negative by IEM and IAHA, and 8 were positive by both procedures. There was 1 patient who was anti-HAV positive by IEM and RIA, but negative by IAHA (case 7, table 2). Thus, for HBsAg-negative cases there were 3 presumptive type A infections among the 32 fatal cases, and 3 confirmed instances among the 10 nonfatal. For HBsAg-positive cases there presumably was 1 coincident or superimposed type A infection among the 20 fatal cases, and no confirmed instances among the 10 that were nonfatal. Chronic active hepatitis. We detected anti-HAV in the initial sample taken during the chronic disease in 7 of 22 patients (32%) who were HBsAg-negative and 4 of 13 (31%) who were HBsAg-positive. The interval from these first specimens to the last available ranged from 2 to 6 years. During observation on an outpatient basis, many patients experienced episodes of disease activity as evidenced by distinct increase in serum aminotransferase and bilirubin levels. In instances of anti-HAV negativity, superimposed HAV infection was considered possible. There was, however, only one instance of antiHAV conversion that occurred in the HBsAg-negative group. This was a 35-year-old female who experienced no episodes suggestive of acute viral hepatitis during a 3-year interval. A single exacerbation occurred after the anti-HAV titer by IAHA had begun to rise. We studied single fecal samples from 14 HBsAgnegative patients with chronic active hepatitis. Two were hospitalized at the time of collection for activity of their disease; the remaining 12 were outpatients with abnormal aminotransferase levels. Simultaneous serum samples were available from 7, of whom 3 were antiHAV positive and four were anti-HAV negative. Discussion The pattern of results with the IEM, RIA, and IAHA tests for anti-HAV can provide a presumptive index of type A hepatitis even if the only specimens available are from t,he acute phase of the disease.gr I3This etiological diagnosis must be considered presumptive, however, because of three factors: 1. Antigens other than HAV may be coincidentally present in the standardized fecal suspension used in the IEM and/or RIA procedures. If the serum sample tested for anti-HAV also contains antibody to an extraneous

agent of a size similar to HAV, false positivity occurs. l5 2. The RIA test could give false positivity if the reagent anti-HAV materials and the serum being tested contained antibody to supervenient antigen in the fecal reagent, or in the serum sample being tested. 3. The relative longevity with which anti-HAV remains detectable by IEM, RIA, and IAHA has not been fully evaluated. When only a single specimen can be obtained (as in fatal hepatic necrosis), however, there are ways to minimize the possibility of false-positive results. For IEM, the fecal suspension can be thoroughly evaluated by inoculation of cell cultures in laboratory animals, as well as by accumulated experience with that particular reagent. For RIA, blocking of the reaction with specific antibody of heterologous origin offers confirmation. Even better is testing by both methods, with the reagent HAV and anti-HAV in each derived from different sources. With regard to duration of detectability we have not observed discordance between IEM and IAHA results in testing more than 100 specimens taken months to years after the acute illness (unpublished data). If acute phase IEM and RIA positivity for anti-HAV, accompanied by IAHA negativity, are accepted as a pattern for presumptive diagnosis of acute type A hepatitis, then HAV was responsible for 3 of 32 fatal HBsAg-negative cases. Under any circumstances, it is capable of producing fulminant disease, as evidenced by increasing IEM scores and IAHA seroconversion in 3 of 10 HBsAg-negative survivors. Over-all, it may account for 6 of these 42 cases (14%). If the pattern of anti-HAV tests is presumptuously diagnostic, the one instance of IEM and RIA positivityIAHA negativity among the HBsAg-positiue cases raises an important question. This occurrence could be explained by simultaneous acute infection by HAV and hepatitis B virus, or by HAV infection superimposed upon chronic hepatitis B virus infection. There has been no indication that the latter results in more severe illness than usual-,18 but there may be occasional instances in which that is not true. Unfortunately, without specimens taken before the fulminant episode, there is no way at present to distinguish between superimposed and simultaneous infections. Turning to the question of chronic liver disease produced by HAV, it may presumed earlier that this sequel could occur and be accompanied by intermittent or continuous fecal excretion. Bennett and co-workerP described 2 children who had biochemical abnormality

882

RAKELA

during periods of 5 and 15 months, respectively. Their stool specimens at the end of those intervals induced icteric hepatitis in 1 of the 4 subjects to whom each sample was administered experimentally. The incubation periods in the volunteers were 22 and 26 days, respectively. Recent epidemiological observations of relatively isolated populations, however have seemed incompatible with prolonged excretion.lg, y0 Our failure to find HAV by IEM in fecal specimens from 14 patients with chronic active hepatitis does not preclude an etiological relationship of that agent to chronic disease, but it represents initial evidence against that hypothesis. With regard to any evidence of HAV in blood during chronic active hepatitis, the lack of a marker for viremia, such as the excess production of HAV-specified antigens, leaves one without a way of assessing its occurrence (other than experimental infection). A priori, one does not know whether to anticipate presence or absence of anti-HAV if chronic HAV infection does occur. Our finding that the frequency of antiHAV was the same in patients with HBsAg-positive as HBsAg-negative chronic active hepatitis argues against a significant role of HAV in cases at our hospital. REFERENCES Lucke B: The pathology of fatal epidemic hepatitis. Am J Path01 20:471-593, 1944 Zieve L, Hill E, Nesbitt S, et al: The incidence of residuals of viral hepatitis. Gastroenterology 25:495-531, 1953 Neefe JR, Gambescia JM, Durtz CH, et al: Prevalence and nature of hepatic disturbance following acute viral hepatitis with jaundice. Ann Intern Med 43:1-32, 1955 4. Cullinan ER, King RC, Rivers JS: The prognosis of infective hepatitis. A preliminary account of a long-term follow-up. Br Med J 1:1315-1317, 1958 5. Chuttani HK, Sidhu AS, Wig KL, et al: Follow-up study of cases from the Delhi epidemic of infectious hepatitis. Br Med J 2:676679, 1966 6. Feinstone SM, Kapikian AZ, Purcell RH: Hepatitis A: detection by immune electron microscopy of a virus-like antigen associated with acute illness. Science 182:1026-1028, 1973

ET AL.

Vol. 74, No. 5, Part I

7 Miller WJ, Provo,t PJ, McAleer WJ, et al: Specific immune

adherence assay for human hepatitis A antibody. Application to diagnostic and epidemiologic investigations. Proc Sot Exp Biol Med 149:254-261, 1975 8 Moritsugu Y, Dienstag JL, Valdesuso J, et al: Purification of hepatitis A antigen from feces and detection of antigen and antibody by immune adherence hemagglutination. Infect Immun 13:898-908, 1976 9. Bradley DW, Maynard JE, Hindman SH, et al: Serodiagnosis of viral hepatitis A: detection of acute-phase immunoglobulin M anti-hepatitis A virus by radioimmunoassay. J Clin Microbial 5:521-530, 1977 10. Dienstag JL, Feinstone SM, Kapikian AZ, et al: Fecal shedding of hepatitis A antigen. Lancet 1:765-767, 1975 ll. Rakela J, Mosley JW: Fecal excretion of hepatitis A virus in humans. J Infect Dis 135:933-938, 1977 12. Bennett AM, Capps RB, Drake ME, et al: Endemic infectious hepatitis in an infants’ orphanage. II. Epidemiologic studies in infants and small children. Arch Intern Med 90:37-53, 1952 13, Rakela J, Stevenson D, Edwards VM, et al: Antibodies to hepatitis A virus: patterns by two procedures. J Clin Microbial 5:110-111, 1977 14. Redeker AG: Fulminant hepatitis. In The Liver and Its Diseases. Edited by F Schaffner, S Sherlock, CM Leevy. New York, Stratton Intercontinental Medical Book Corporation, 1974, p 149 15. Kapikian AZ, Dienstag JL, Purcell RH: Immune electron microscopy as a method for the detection, identification and characterization of agents not cultivable in a vitro system. Manual of Clinical Immunology. Edited by NR Rose, H Friedman. Washington DC, American Society of Microbiology, 1976, p 467 16 Mayumi M, Okochi K, Nishioka K: Detection of Australia antigen by means of immune adherence hemagglutination test. VOX Sang 26:178-181, 1971 17 Hunter WM, Greenwood FC: Preparation of Iodine-131 labeled human growth hormone of high specific activity. Nature 194:495-496, 1962 18. Dietzman DE, Mathew EB, Madden, et al: The occurrence of epidemic infectious hepatitis in chronic carriers of Australia antigen. J Pediatr 80:577-582, 1972 19. Mosley JW: The epidemiology of viral hepatitis: an overview. Am J Med Sci 270:253-270, 1975 20. Skinhsj P, Mikkelsen F, Hollinger FB: Hepatitis A in Greenland: importance of specific antibody testing in epidemiology surveillance. Am J Epidemiol 105:140-147, 1977