Rapidly progressive HBsAg-positive hepatitis in Italy

274 Journal of Hepatology, 1987; 5:274-281 Elsevier

HEP 00349

Rapidly progressive HBsAg-positive hepatitis in Italy

The role of hepatitis delta virus infection

G. Saracco I , F. R o s i n a 1 , M . R . B r u n e t t o 1 , P. A m o r o s o 2, F. C a r e d d a 3, P. Farci 4, P. P iant ino 1, F. B o n i n o I and M. R izze t to 1

I Dn'tstone di Gastroenterologla, Ospedale Molinette, Torino, -'V Dn,tstone Ospedale D. Cotugno, Napoh, "~Cattedra di Malattie lnfetttve, Ospedale L. Sacco, MUano and 4Clinica Medica I. Universit{~ di Caghari. Cagliari (ItAly)

(Received 10 February, 1987) (Accepted 9 July, 1987)

Summary

Serum or liver markers of hepatitis delta virus (HDV) infection were found in 20 of 22 (90%) Italian patients presenting with an ostensible type B hepatitis that ran an accelerated course to cirrhosis. The features of the illness conformed to a syndrome of HDV infection in young males carrying the hepatitis B surface antigen (HBsAg); a latent HBsAg state was documented in many patients by a history of prior exposure to the hepatitis B virus (HBV) or by the absence of IgM antibodies to the HB core antigen. Characteristic of the disease were the clinical overture as a severe hepatitis, the Iobular involvement by an extensive necroinflammatory reaction, the exuberant expression of intrahepatic hepatitis delta antigen and an atypical HBV profile of inactive infection or accelerated seroconversion from HBeAg to anti-HBe. Superimposed upon HBV infection, HDV may create a rapidly progressive course which resembles very aggressive hepatitis B but is infrequently observed in hepatitis B alone.

Introduction

Infection with the hepatitis delta virus (HDV), a defective pathogen requiring as obligatory symbiont the hepatitis B virus (HBV) [1], has been recognized as the primary or contributing cause of severe forms

of HBsAg-positive liver disease [2]. Within the spectrum of acute HBsAg hepatitis the prevalence of HDV increases in parallel with the severity of the illness, fulminant hepatitis ranking as its most promi- nent clinical expression [3,4]. Studies of chronic HBsAg hepatitis have likewise demonstrated a

This work was supported in part by C.N.R. grants 84.00885.04 and 84.02051.52. Correspondence: Mario Rizzetto M.D., Divisione di Gastroenterologia, Ospedale Molinette, Corso Bramante 88, 10126 Torino, Italy. Tel: 011 - 697.313.

0168-8278/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)

DELTA VIRUS INFECTION 275

higher rate of HDV infection in chronic active hepatitis (CAH) compared to lesser histoiogic forms [5-8].

Chronic active hepatitis, however, is a histoiogic entity corresponding to clinically heterogeneous dis- orders that vary from mild to very aggressive ill- nesses; at the extreme are forms of hepatitis of acute onset that rapidly progress to cirrhosis in a matter of a few months.

To further determine the role of HDV within the spectrum of CAH, we examined the prevalence of HDV markers in Italian patients with this rare but most ominous variant of liver disease.

Materials and Methods

The patients included in this report were involved in prospective studies of the outcome of acute HBsAg-positive hepatitis, carried out at four Medi- cal Centers in Italy.

In over 1000 patients examined, these studies have identified 22 patients whose illness did not resolve but ran a rapidly downhill course toward liver failure (hypoalbuminemia) and/or in whom histology demonstrated cirrhosis in liver biopsies taken within 12 months from the original episode. These 22 patients were retrospectively examined for HDV markers.

Ten patients were enrolled into the study in Naples in the years 1976-1984, and 5, 4 and 3 patients, respectively, in Milan, Turin and Cagliari in the years 1980-1984. All were hospitalized during the initial hepatitic episode and submitted to physical examination and liver function testing bi- or three-monthly after hospital discharge.

The clinical records and liver biopsies of each patient were reviewed. The longitudinal analysis of HBV and HDV serology was performed in the 15 patients enrolled after 1980, including the 12 patients in Milan, Turin and Cagliari and 3 of the 10 patients in Naples. Stored (-20 °C) serum samples taken at admission to hospital and at 2-3-month intervals were available from each of these patients. Sera were tested for HBsAg, antibody to HBsAg (anti-HBs), IgM antibody to HB core antigen (anti-HBc IgM),

HBe antigen (HBeAg), antibody to HBe (anti- HBe), HD antigen (HD-Ag), total (predominantly IgG) antibody to HDV (anti-HD) and IgM-type antibody to HDV (anti-HD IgM).

Only 1 liver biopsy (demonstrative of cirrhosis) was taken from 8 of these patients; in the other 7, ear- lier biopsies were also obtained during the course of the disease.

No serologic examination other than the HBsAg test had been performed in the 7 patients collected in Naples before 1980.

Further virologic characterization of these patients was performed by immunohistologic analysis of the liver tissue for the HB core antigen (HBcAg) and the HD-Ag. Serial biopsies were available from each, the first obtained within 45 days of admission to hospital for the initial acute HDV illness, and subsequent ones at 4-6-month intervals.

Assays HBsAg, anti-HBs, anti-HBc IgM, HBeAg and

anti-HBe were estimated with commercial radioim- munoassays (RIA) (Ausria II, Ausab, Corab IgM, kit HBe, Abbott Lab., North Chicago, IL, U.S.A.). Total antibody to HDV and anti-HD IgM were measured by RIA [9,10]. The titer of total anti-HD was expressed as the reciprocal 10-fold dilution inhibiting the binding of 50% of the radioactivity detected in the negative control [9].

Sera were tested for anti-HD IgM in serial 10-fold dilutions starting from 1:100 [10]. Titers of anti-HBc IgM and anti-HD IgM were expressed as the reciprocal of the final serum dilution yielding a positive re- sult.

Serum HD-Ag was measured with a solid-phase RIA with the addition of Nonidet P-40 to a final de- tergent concentration of 0.3% [9]. Liver histology was interpreted according to the criteria of De Groote and associates [11].

Intrahepatic HD-Ag was detected by an immunoperoxidase technique in formalin-fixed material after digestion with 0.1% pronase in phosphate-buffered saline (PBS) for 10 min at room temperature; the staining was performed using a horseradish peroxidase-labeled antiserum of human origin [12].

276 G. SARACCO ct al.

Staining for the HBcAg was performed by the per-

oxidase-anti-peroxidase technique. The primary antibody to the HB core antigen (rabbit anti-HBc,

Dako Labometrics, Milan, Italy) was used at 1:500 to 1:1000 dilution; the bridging antibodies, swine anti-

rabbit and rabbit peroxidase-anti-peroxidase (Bio- netic, Kensington, MD, U.S.A.) were used at a 1:40

and a 1:100 dilution, respectively. The peroxidase was visualized on a substrate of diaminobenzidine hydrochloride in PBS.

Tests of liver function were determined with stan- dard laboratory procedures.

Results

Prevalence of HD V infection Markers of HDV infection were detected in serum

or liver of 20 of the 22 patients examined (90%) (Table 1).

Serologic investigation (15 patients) Serum markers of HDV infection were found in 14

patients (Nos. 1-14, Table 2). Hepatitis D antigen

was found in none. Total or IgM anti-HD was found in 11 patients within the first 2 weeks from onset of

the disease. In 3 patients, total or IgM anti-HD was first detected 2-8 weeks after onset of symptoms. In

each case, total or IgM anti-HD increased in a period of 2-5 months to titers of 10 -4-10 -6 and 10 -5-10 -7,

respectively, and persisted unchanged thereafter.

Liver investigation (7patients) Hepatitis D antigen was found in the liver of 6 pa-

tients followed up with multiple biopsies (Nos.

15-20, Table 2). In 3, it was found in the first (acute phase) and subsequent liver biopsies, and in the other 3 it was not found in the acute phase biopsy but was detected in the second biopsy (taken 4-6 months

after onset of the illness) and in subsequent follow-up liver specimens.

Characteristics of the pattents with HD V hepatitis

Demographic and clinical (Table 2) The patients were males, aged 16-33 years (mean

23.6) (Table 2); 10, including the 4 in Turin, the 3 in

Cagliari, 2 in Milan and 1 in Naples were addicted to the intravenous use of illicit drugs. The other 2 pa-

tients in Milan were promiscuous homosexuals. Dis- tinctive demographic features were not identified in

the other 8 patients in Naples.

Upon being questioned about previous disease, 7 patients (Nos. 1,3,6,7,9,19,20, Table 2) reported an

acute hepatitis B infection (presence of HBsAg in serum) in the recent past (6-30 months before the on-

set of the HDV disease). In general, the previous hepatitis B was reported as relatively mild and self-

limited. The HBsAg, however, had remained posi-

tive in patient No. 6, whose serum was tested for this marker during the interval between the two hepatitis

episodes. Three patients (Nos. 4,16,18) were conva-

lescing from a fresh episode of mild hepatitis B that had not required hospitalization. Three patients

were known to be long-term carriers of HBsAg; none of them had experienced clinical hepatitis. No history

of hepatitis emerged in the other 7 patients. The HDV disease began as an icteric HBsAg-posi-

TABLE 1

PREVALENCE OF HDV INFECTION IN 22 PATIENTS WITH RAPIDLY PROGRESSIVE HBsAg+ HEPATITIS

Center of collection Number of patients examined for HDV markers Number of patients positive for

in serum in liver serum antl-HD liver HD-Ag

Milan 5 Turin 4 Cagliari 3 Naples 3 Total 22

4 4 3

7 3 6 20

DELTA VIRLIS INFECTION 277

FABLE 2

DEMOGRAPHIC AND CLINICAL FEATURES OF ATITIS

20 PATIENTS WITH RAPIDLY PROGRESSIVE HBsAg/HDV HEP-

Center of Pattent Sex Age Ep~demiolog~c collection No. (yrs) risk factors

Prior hepatitis B Features of acute Course of Development (months before HDV hepatitis HDV of cirrhosis acute HDV hepat,tis) hepatitis (months after or HBsAg carnage acute hepatitis)

rurin

Mtlan

Caghari

Naples

1 M 20 2 M 21 3 M 19 4 M 25 5 M 23 6 M 25 7 M 3(1 8 M 27 9 M 21

10 M 23 11 M 33 12 M 23 13 M 21 14 M 27 15 M 16 16 M 22 17 M 23 18 M 23 19 M 24 21) M 27

'~ Grade I or II encephalopathy " Fall of prothrombm time lower than 50%. • Death in liver failure a Post-mortem diagnosis.

drug addict drug addict drug addict drug addict drug addict drug addict homosexual homosexual drug addict drug addict drug addict drug addict

6 severe a 8 known HBsAg carrier severe b cholestatlc 6 11 7 3 cholestatlc 9

- severe" h 8 known HBsAg carrier severe h 8

6 cholestatic 6 known HBsAg carrier 12 23 10 - cholestatm 10 _ severe t' cholestatlc c 9 d - severe a 8 known HBsAg carrier 8 - severe a'b 9 _ severe a.b cholestatic c 8 d

2 severe a 6 - severe b 12

3 10 6 severe b 12

18 8

t ive hepa t i t i s a c c o m p a n i e d by a 4 0 - 1 0 0 - f o l d rise of

a l an ine a m i n o t r a n s f e r a s e ( A L T ) act ivi ty . D u r i n g the

acu te i l lness, t r a n s i e n t e n c e p h a l o p a t h y a n d / o r a fall

of p r o t h r o m b i n syn thes i s be low 5 0 % of the con t ro l

o c c u r r e d in 11 of the 20 p a t i e n t s ( 5 5 % ) . In each ,

s y m p t o m s i m p r o v e d in a few weeks , bu t the e l e v a t e d

level of A L T pe r s i s t ed o v e r the fo l lowing m o n t h s , at

va lues 1 5 - 3 0 - t i m e s a b o v e the n o r m a l (Fig. 1). T h e

e n z y m a t i c a l t e r a t i o n was a c c o m p a n i e d by a signifi-

can t p rog re s s ive inc rease of the g a m m a - g l o b u l i n s

and IgG level ( m o r e t h a n 2 5 % a b o v e the ba se l i ne at

the onse t of d , sease ) . All the p a t i e n t s had cons t i t u -

t ional s y m p t o m s , a m o n g which fa t igue and fever

were p r o m i n e n t . T h e i l lness ran a p r o l o n g e d choles -

tatic course ,n 6 pa t i en t s . H y p o a l b u m i n e m i a devel -

oped a f t e r 7-1(I m o n t h s in 4 p a t i e n t s : 2 of t h e m d ied

in hepa t i c c o m a a f t e r 8 and 9 m o n t h s , r espec t ive ly .

Five of the p a t i e n t s were t r e a t e d f rom a few weeks to

a few m o n t h s wi th s t e ro ids a n d / o r a z a t h i o p r i n e with-

ou t s y m p t o m a t i c or b i o c h e m i c a l a m e l i o r a t i o n .

L,ver HD. Ag

2000

looo <¢

hepatitis B

1

acute HBsAg hep CIR

1 1 asc~tes

- - + + + +

/~ lgM. ' tn t i -Hl~ . . . . . . "'" ) , , , "~ . 7-" . . . . .

/° so'T

I0"

10" ~ _

I0' ~

1o'

1981 1982 1983 months 1984

Fig. I. Climcal and serological course of pattent No. 2. Table 2. ALT = ahminc ammotransferase: CIR = cirrhosis: hep =

hcpatms

278 G. SARACCO et al.

HB V serum profile (Table 3) The HBsAg persisted in the blood of all 20 pa-

tients; none developed anti-HBs. At presenta t ion, 9

of the 14 patients investigated in serum had H B e A g ,

3 had ant i -HBe and 2 were negative for both mark-

ers. Five of the 9 pat ients (55%) with H B e A g sero-

converted to ant i -HBe after 3 months.

The IgM ant ibody to HB core antigen was found in

7 patients; 1 of these pat ients (No. 6, Table 2) was

known to be an H B s A g carrier and 4 (Nos. 1,3,4,7,

Table 2) to have exper ienced hepati t is B 3-11

months previously. Two of the pat ients had no histo-

ry of previous contact with HBV: high levels of anti-

HBc IgM were found in both (10-5-10-6). Of the 7

anti-HBc IgM-negat ive patients , 3 (Nos. 2,8,13,

Table 2) were long-term carriers of HBsAg, 1 (No. 9,

Table 2) had a history of past hepati t is B, but 3 (Nos.

10,11,12, Table 2) were not aware of pr ior exposure

to HBV.

Liver histology. HD-Ag and HBcAg expression in liver

Focal, confluent and bridging necrosis not distin-

guishable from classical hepati t is B was seen in the 6

biopsies taken from the pat ients in Naples within 45

days of onset of the H D V disease. The intrahepat ic

H D - A g was found in 3 and H B c A g in none. Twelve

biopsies, taken 4 - 8 months after the onset of the

H D V hepati t is (6 as follow-up biopsies from the pa-

tients in Naples , 6 as first biopsies from 4 pat ients in

Turin and 2 in Cagliari) , exhibi ted portal and paren-

chymal damage but did not demons t ra te obvious

nodular t ransformation. This morphological stage

was considered transit ional to the deve lopment of

cirrhosis. The portal tracts were expanded by mono-

nuclear cells and less conspicuously by granuiocytes;

per ipor ta l p iecemeal necrosis was common. The lo-

bular structure was preserved but dis torted by irregu-

lar hepatocel lular regenerat ion and diffuse Kupffer

cell activation, and infil trated by focal aggregates of

macrophages and extensive lymphocytic exudat ion

(Fig. 2).

The inf lammatory changes in the lobule were ac-

companied by a non- inf lammatory eosinophil ic de-

generat ion of single hepatocytes or of groups of hep-

atocytes; free acidophilic bodies were relat ively fre-

quent. In t rahepat ic H D - A g was found in each

biopsy, dis t r ibuted in over 25% of the liver cells.

H B c A G was found in 2 biopsies, from drug addicts

with H B e A g in serum.

Macronodula r cirrhosis was observed at the post-

mor tem of the 2 patients who died, both from Naples.

TABLE 3

SERUM HBV PROFILE OF 14 PATIENTS WITH RAPIDLY PROGRESSIVE HBsAg/HDV HEPATITIS INVESTIGATED IN SERUM

Patients Anti-HBc IgM HBe antigen/antibody system

1 + HBeAg ..... • b anti-HBe (8 months)" 2 - HBeAg '~ 3 + HBeAg ..... • anti-HBe (6 months) 4 + HBeAg" 5 + HBeAg J 6 + . . . . . . • anti-HBe (3 months) 7 + HBeAg ..... • anti-HBe (3 months) 8 - antl-HBe 9 - ant,-HBe

10 - ant~-HBe 11 - HBeAg J 12 - HBeAg ..... • anti-HBe (2 months) 13 . . . . . . . • anu-HBe (3 months) 14 + HBeAg ..... • antl-HBe (2 months)

d Persistent throughout follow-up. b ..... • . seroconversion to anti-HBe.

( ). time interval in months.


Fig. 2. Liver biopsy taken 7 months after onset of rapidly progressive HDV disease in a 19-year-old drug addict. Marked lo- bular inflammation and Kupffer cell activation. Disarray of liver cell structure and eosinophilic degeneration of hepato-

cytes (x 100).

In the other 18 patients, cirrhosis was demonstrated in biopsies taken 6-12 months after the acute HDV hepatitis. These were the second or third liver specimens from 10 previously biopsied patients and the first liver specimen obtained from the remaining 8 patients. HD-Ag was found in all 20 patients, in 5-30% of the hepatocytes.

Due to exhaustion of liver material, intrahepatic HBcAg was determined only in 11 patients and found in a few nuclei of 1.

Discussion

Our study indicts HDV as the protagonist of the most dramatic type of progressive hepatitis, confirm- ing that this infection influences the severity of chronic HBsAg hepatitis, just as it influences the severity of acute hepatitis B [3,4]; markers of HDV were present in 90% of our patients, compared to the 10-50% prevalence in other types of chronic viral hepatitis [13-16].

Though the overall prevalence of this clinical entity is low, occurring in only 2% of our hepatitis B cases, inferential evidence suggests that it may devel- op in up to 10-15% of cases of acute symptomatic

HDV infections in Italy [17]. The role of HDV outside Italy remains to be estab-

lished, but recent reports of very aggressive hepatitis in Amazonian Indians [18] and drug addicts [19] with HDV infection point to a similar clinical impact else- where where this virus is endemic.

Our patients exhibited features connoting a dis- tinct hepatitis syndrome. They were all young and male. There was evidence in many of prior exposure to HBV that presumably resulted in chronic HBV carriage.

In 65% a past episode of hepatitis B indicates that the HBsAg detected at presentation to our Institu- tions was acquired before the HDV; in 3 of 5 patients without a hepatitis history analyzed for the battery of HBV markers, a latent HBV infection was suggested by a negative test for anti-HBc IgM. Thus prior exposure to HBV appears to be a common predisposing factor linking the patients of this study; among drug addicts, the risk of HBV was parenteral, and among the patients in Naples the risk was probably environ- mental, due to the high local prevalence of HBV infection in the general population [20].

The acute hepatitis heralding the progressive HDV disease was severe. In 30% of the patients a cholestatic picture persisted for many months, and in over half the extent to which the liver was compro- mised during the acute phase was attested by an alarming fall in prothrombin synthesis and/or the development of encephalopathic complications. The HBV pattern was atypical; 35% of the patients whose serum was investigated presented with an inactive HBV infection and 50% of those with the HBeAg seroconverted to anti-HBe within 9 months of follow-up. The accelerated rate of seroconversion, distinctly higher than the 15% yearly rate reported in Italian carriers with HBeAg [21], presumably re- flects the inhibitory action exerted by HDV on the synthesis of HBV; this may also explain the paucity of HBcAg in the liver of our patients. An extensive iobular inflammation accompanied by scattered, eosinophilic necrosis of the hepatocytes was the pre- dominant morphologic pattern at histology; this asso- ciation has been previously reported as being characteristic of HDV disease [22,23].

280 G. SARACCO et al.

Of the mechanisms leading to HDV transmission,

coinfection with HBV or superinfection of HBsAg

carriers [1], the latter is implicated in many of our pa-

tients who had anamnestic evidence of contact with

HBV or were negative upon testing anti-HBc IgM.

As this antibody is invariably raised in acute type B

hepatitis but declines in a few months after primary

exposure to HBV, its absence in a carrier of HBsAg

points to a long-standing HBV infection [24].

In 2 patients without prior exposure to HBV, the

presence of anti-HBc IgM in high titers indicates that

the illness progressed from a simultaneous primary

HBV/HDV infection, thus suggesting that coinfec-

tion, as well as superinfection, can trigger chronic

hepatitis D. This possibility has been contested by

the argument that no case of chronicity occurred in

previous prospective studies of acute hepatitis D;

these studies, however, have included relatively

small numbers of patients [25,26].

It is difficult to establish whether the ominous

course of our patients was determined by an in-

creased virulence of HDV or whether this infection

activated a latent liver disease previously induced by

HBV. Though cirrhotic changes were seen in late but

not in early biopsies taken during the follow-up, sam-

pling variability, the necroinflammatory activity and

observer bias make interpretat ion of histology in ear-

ly biopsies subject to error with regard to pre-existing

hepatitis or cirrhosis. It should be noted, however,

that superimposed on chronic HBV infection, either

non-pathogenic or itself causing latent disease, H D V

created a rapidly progressive course that appeared

clinically as primary severe hepatitis B but which is,

in fact, unfrequently seen in Italy in hepatitis B

alone.

At the florid stage, the true nature of the illness is

recognized from the exuberant expression of the HD

antigen/antibody system. Early diagnosis may be dif-

ficult, as in the post-necrotic phase that follows acute

hepatitis D the expression of the HD antigen in liver

is reduced and the titer of ant i -HD may still be in the

low range compatible with the antibody response of

self-limited HDV disease [27].

Since progression of H D V infection can be sus-

pected from the increase of the total and IgM anti-

body to HDV, patients with unremitt ing, apparently

type B hepatitis should be monitored for these serum

markers; the finding that ant i -HD is increasing is in-

dication for a liver biopsy, in order to confirm the in-

trahepatic presence of the H D - A g diagnostic of pro-

gressive H D V infection [2].

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Rapidly progressive HBsAg-positive hepatitis in Italy