Long-term observation of HCV-positive patients with normal ALT values: persistence of a clinically healthy state

0 INSTITUT PASTEURELSEVIER Paris 1998

Res. Virol. 1998, 149, 277-282

Long-term observation of HCV-positive patients with normal ALT values: persistence of a clinically healthy state F. Morisco (l), G. Del Vecchio Blanc0 (I), C. Tuccillo cl), C. Galli (*), S. Cirino (*) and N. Caporaso (3) (*) (I) Department of Internal Medicine “F. Magrassi”, Second University of Naples, Naples (Italy), 12)Abbott Diagnostics Division, Rome, and 13)Department of Food Science, University of Naples “Federico II “, Naples (Italy)

SUMMARY

The purpose of our investigation was to ascertain the presence of viral replication in subjects positive for antibodies to hepatitis C virus (anti-HCV) with persistently normal values of liver tests, to define their natural history, and to determine whether the immunoglobulin M (IgM) response could be a useful parameter to distinguish viraemic from non-viraemic patients. Twenty-seven subjects were selected based on their anti-HCV positivity and sustained normality (for at least 18 months) of alanine-aminotransferase (ALT) values. They were enrolled into the study and observed for another l-4 years (mean 2.6). Fifteen out of 27 subjects were positive for hepatitis C virus ribonucleic acid (HCV RNA) and 1205 were also positive for IgM. The remaining 12127 patients were negative for both assessments. ALT levels remained in the norm throughout the investigation for all 27 subjects studied; the 15 viraemic patients showed persistent positivity for HCV RNA and the 12 positive for IgM anti-core also maintained their positivity. Patients shown to be negative for HCV RNA and IgM sustained their negativity throughout the study. Our results indicate that some patients remain viraemic while not having and/or developing clinical and biochemical signs of liver damage. IgM anti-HCV seems to be a specific index of viraemia in HCV-positives and could be useful for monitoring these patients. Key-words: ALT, HCV, Chronic hepatitis; HCV RNA, IgM, Viraemia.

INTRODUCTION Hepatitis C virus (HCV) infection is associated with a high chronicity rate leading to the development of chronic hepatitis (CH), cirrhosis, and to its complications which include hepatocellular carcinoma (HCC) (Caporaso et al., 1991). HCV-related chronic diseaseis characterized by fluctuating serum alanine aminotransferase (ALT) levels and by a

slow progression to cirrhosis (Alter et al., 1992). However, in clinical experience, it is quite common to observe anti-HCV-positive subjects without clinical and biochemical signs of liver damage (Brillanti et al., 1992; Coltorti et al., 1995). One of the most recent cross-sectional studies, carried out on a general population in southern Italy in 1996, demonstrated the presence of serum HCV ribonucleic acid (HCV RNA) in a large percentage of anti-HCV

Received September15, 1998. (*) Address for correspondence : Prof. Nicola Caporaso, Via Caravaglios, 36, 80125 Naples, Italy.

278

F. MORISCO

positives even if only a minority of them had raised serum ALT levels (Guadagnino ef al., 1997). Up to now, it still remains questionable as to whether anti-HCV-positive patients with persistently normal ALT levels may be considered chronic “healthy carriers” of HCV (i.e. HCV-RNA-positive with normal ALT values and no histological features of CH despite ongoing viraemia), are affected by HCV-RNA-positive chronic hepatitis despite persistently normal ALT values suggesting low levels of cytolytic activity, or finally, are patients in the process of clearing up their infection. Limited data are available on the natural course of anti-HCV/HCV-RNA-positive subjects without clinical evidence of liver disease. In particular, it is unclear if this clinical picture will remain stable in time, or alternatively, will lead to ongoing or progressive evidence of hepatic injury. This question can only be resolved by a prolonged prospective study that includes strict monitoring of virologic and liver damage parameters. Positivity for specific immunoglobulin M (IgM) directed against HCV core epitopes is a common feature of acute hepatitis C (Giuberti et al., 1994), but this feature is also frequent in chronic hepatitis C (Martinelli et al., 1996; Morisco et aE., 1996) and this marker has been shown to correlate with viraemia (Man&i et al., 1995; Yuki et al., 1995; Kapprell et al., 1996; Martinelli et al., 1996; Ounanian-Parez et al., 1996). The purpose of our investigation was to ascertain the presence of viral replication in HCV-positive subjects with persistently normal values of liver tests, to define their natural history, and to determine whether the IgM response could be a useful parameter to distinguish viraemic from non-viraemic patients.

ET AL. given informed consent. No subject was an injected-drug user, alcoholic, human immunodeficiency virus (HIV)-positive, or had undergone blood transfusions in the past. All were negative for hepatitis B surface antigen (HBsAg) and autoantibodies and showed no clinical symptoms and/or ultrasonographic signs of liver disease. The anti-HCV positivity resulted either from volunteer blood donation screening or during controls performed on close relatives of antiHCV-positive index cases which are routinely carried out in our centre. Throughout the observation, ALT values were assessed at 3-month intervals To determine HCV RNA, a serum sample was taken from each patient at baseline and every 6 months. Each sample was divided into three 0.5 ml aliquots and then frozen at -70°C under conditions known to best preserve RNA. AI1 patients underwent periodic clinical examinations ; routine liver tests (y-glutamyl-transpeptidase, serum protein, red and white blood cell counts, platelet count, etc.) were assessed every 6 months and an abdominal ultrasonography was performed every 12 months.

Laboratory

tests

In the last five years, we have chosen from patients referred to our Department, 27 anti-HCV-positive subjects (Ortho HCV EIA 3.0) based on persistently normal ALT levels for at least 18 months. The study period began after the patients had been selected, and lasted for a mean of 2.6 years (range l-4). The group included 12 males and 15 females aged 15 to 67 years (mean: 49 years) who had

1) HCV RNA: The first serum aliquot was employed for the assessment of HCV-RNA levels by the branched DNA (bDNA) signal amplification assay (Quantiplex, Chiron Corporation, Emeryville, CA, USA). This assay is based on a specific hybridization between synthetic oligonucleotides and complementary sequences of the 5’ untranslated (5’ UTR) and core regions of the HCV genome which allows the HCV RNA to be captured onto the surface of a well. The limit of detection of this assay is 200,000 copies of HCV genome Eq/ml. The second serum aliquot was employed for the qualitative determination of HCV RNA on samples shown to be negative by bDNA. We employed the commercial RT-PCR method (Amplicor HCV, Roche Molecular Systems, NJ, USA) based on a combined reverse transcription and single amplification reaction carried out by the reverse Thermus thermophiIus (rTth) DNA polymerase. The analytical sensitivity of this assay is 1,000 copies of HCV genome per milliliter. 2) Genotypes and serotypes: HCV genotypes were assessed on the third aliquot by a commercial line-probe assay (Inno-LiPA, Innogenetics S.A., Gent, Belgium) that allows differentiation of the 5 most common genotypes. The LiPA test is a reverse hybridization assay based on

ALT bDNA CH Eq/ml HBsAg HBV HCC HCV

HCV RNA HIV W MNV RT-PCR rTth UTR

MATERIALS

AND METHODS

alanine aminotransferase. branched DNA. chronic hepatitis. equivalent per milliliter. hepatitis B surface antigen. hepatitis B virus. hepatocellular carcinoma. hepatitis C virus.

= = = = = = =

hepatitis C virus ribonucleic acid. human immunodeficiency virus. immunoglobulin M. maximum normal value. reverse transcriptase polymerase chain reaction. reverse Thermus thermophilus. untranslated region.

CLINICALLY

HEALTHY

HCV CARRIER

highly conserved variations in the 5’ UTR. We characterized the HCV serotypes of the samples that were negative for HCV RNA by employing a qualitative immunoenzymatic assay (Chiron RIBA Serotyping SIA, Ortho Diagnostic Systems, Raritan, NJ, USA). This assay utilizes synthetic peptides derived from serotype-specific sequences of the non-structural NS4 and the structural core regions of HCV genotypes 1,2, and 3. 3) HCV ZgM: IgM-class antibodies directed against the immunodominant epitopes expressed by the putative core region of the HCV genome have been determined by a recently developed enzyme immunoassay (HCV-IgM EIA 2.0, Abbott Diagnostics Division, Wiesbaden- Delkenheim, Germany). The IgM anti-core levels are expressed semiquantitatively by an index value that correlates the absorbance of each sample with an internal positive control. The cut-off index ranges from 18 to 22; however, due to an overall assay imprecision of IO- 15 %, samples with a cut-off ratio equal to or greater than 0.85 were considered positive for HCV IgM.

Genotype Number of patients Serotype Number of patients Number

levels of HCV-RNA-positive

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 NT = not tested.

lb 2a 2a 2a 2a 2a 2b lb lb NT lb lb 2a NT 2a

Basal 0.75 < 0.20 1.32 2.94 6.18 6.16 < 0.20 0.52 2.87 0.83 < 0.20 4.80 9.75 3.17 < 0.20

lb 5 2 2

2a 7 3 0

NT = not tested.

2b 1 Neg 6

3a NT 0 2

Neg = negative.

All 27 subjects’ serum ALT levels resulted in the normal range at each trimonthly control, with mean values of 0.60 x maximum normal value (MNV) and 0.43 x MNV, respectively in the 15 HCV-RNApositives and in the 12 HCV-RNA-negatives, All 15 viraemic patients remained persistently positive for HCV RNA (table II). The viral titre of patients having a baseline value of < 1 x lo6 Eq/ml (i.e. patients 8 and 11) remained generally stable, while an increase was noticed in patients having higher baseline titre (i.e. pts. 3 and 5). The IgM anti-core response was also consistent during the observation period: The 12 viraemic patients with IgM positivity on their baseline

patients during the follow-up Viraemia

Genotype

= total number.

la 0 1 1

The distribution of genotypes and serotypes respectively for viraemic and non-viraemic patients can be observed in table I.

Fifteen out of 27 patients (55.6 %) were shown to be positive for HCV RNA by RT-PCR on their baseline samples. Values ranged from < 0.2 to 9.75 x lo6 Eq/ml with a mean of 3.57 x 106. In twelve of them (80%), a specific IgM anti-core response was detected with index values ranging from 22 to 140 and a mean of 83.6. All the remaining 12 patients (44.4%) were negative for HCV RNA both by bDNA and RT-PCR and were also negative for IgM.

Patients

279

Table I. Distribution of genotypes of the 15 HCVRNA-positive and of serotypes of the 12 HCVRNA-negative subjects.

RESULTS

Table II. Viraemia

STATE

1 year 6.53 1.17 6.54 14?1 8.73 0.49 0.71 1.21 0.74 0.61 5.31 2.75 -

period.

levels Eq/ml x lo6 2 year 4.11 1.04 8.67 2.60 18.30 6.75 1.95 0.48 0.39 7.10 < 0.20 6.27 9.45

3 year

4 year

19.45 12.61 29.21

30.31

0.57

0.96

6.58 0.84

7.42

7.32 19.06 20.79

280

F. MORISCO

HCV-RNA

positive

HCV-RNA

100

4

60

2 0,

40

negative

30

:;[@31,2,3,4

Jj c s

ET AL.

80

20

6

12

18

24

30

36

42

48

0 6

Months

12

18

24

30

36

42

:o

48

Months

Fig. 1. Courseof IgM anti-HCV levels, respectively, in 15 HCV-RNA-positive and in 12 HCV-

RNA-negative subjects.

sample remained so on all follow-up specimens; only one subject showed a significant decrease in index values (from 140 to 29) while still maintaining positivity. The sameconsistency in the serological picture was also evidenced in the HCVRNA/IgM-negative patients who remained so at all controls (see fig. 1). Liver biopsies were performed at the beginning of the study, after informed consent, in 2 viraemic patients and in 4 non-viraemic patients. Results demonstrated that in the 2 viraemic patients, 1 had mild activity with fibrosis and the other had no activity with moderate fibrosis. In the 4 non-viraemic patients, no activity was found and fibrosis was absent in 2 and mild in 2. Finally, all patients had (both pre-enrollment and throughout the investigation) : normal results in liver tests, showed no clinical signs of liver disease, and had normal ultrasonography results.

DISCUSSION The existence of “healthy carriers” of HCV has been widely debated over the past years. While normal ALT levels are frequently found in anti-HCVpositive individuals for prolonged periods of time

(Brillanti et aE., 1992; Naito et al., 1994), most of them are viraemic and may or may not show histological signs of chronic liver disease(Albcrti et al., 1992; Brillanti et al., 1992; Fong et al., 1996). These findings have cast a strong doubt on the possible existence of healthy carriers even if the majority of surveys included subjects with slight variations in serum ALT levels (ALT 1.5 x MNV) (Alberti et uE., 1992) and brief monitoring periods of usually one year or less (Fong et al., 1996; Puoti et al., 1997). Very few studies, if any, followed the natural history of these subjects for an extensive period of time while repeatedly testing for ALT, HCV RNA, and anti-HCV IgM as we did. This approach was originally adopted for monitoring chronic hepatitis B virus (HBV) infection and has allowed us to recognize symptomless reactivation characterized by successivepeaks of viral HBV DNA and IgM anti-core (Colloredo-Mels et al., 1994). In most cases, the reactivation may only be detected when sampling takes place at intervals of 2-3 months or less due to the short duration of DNA and ALT increments. This suggests that studies in which patients were tested at intervals of 6 months or more could have seriously underestimated the number and frequency of reactivation episodes. The results of our cohort of HCV-RNA-positive patients demonstrated the absence of episodes of cytolytic reactivation throughout the monitoring per-

CLINICALLY

HEALTHY

iod which was verified both in subjects with stably low viral titre and in those with a progressive increase. This notably differentiates the natural history of these patients from that of chronic HBV infection. Thus we are able to say that an extensive examination period (3-4 years) is long enough to exclude the idea that normal ALT levels signify a prolonged but transitory period of biochemical remission. It is not easy to explain the presence of HCV viraemia in the absence of an ALT increase during a long observation period. The natural history of HCV infection shows remarkably spontaneous variability : in some patients, the progression from acute viral hepatitis to chronic active hepatitis and then to cirrhosis and HCC happens rapidly, while in others only a mild portal inflammation persists after many years from the primary infection and therefore viral persistence is probably compatible with a durable, healthy clinical state. In still other cases, the histological findings only show evidence of mild fibrosis without inflammatory activity; thus it is possible to define the latter subjects as “truly healthy carriers” instead of “clinically healthy carriers.” The histological findings obtained from the clinically healthy carriers of this study show the absence of inflammatory activity in all HCV-RNA-negatives and the absence or mild activity in HCV-RNA-positives. These results were also found in a previous experience of ours (Coltorti et uZ., 1995). It is not known why some viraemic subjects develop chronic liver damage associated with high ALT levels and others do not. We can assume that some HCV genotypes are more aggressive (lb) than others (2a) (Ravaggi et al., 1996). In our group of viraemic patients, even if the prevalence of genotype 2a proved to be much higher when compared to a population of HCV-RNA-positive patients with chronic liver disease from the same geographic area (prevalently genotype lb) (Magrin et al., 1996), the evidence of infection by genotype lb in 5115 (33.3 %) HCV-RNA-positives has left us perplexed. We can hypothesize that the ALT increase is related to viral titre, but our results show that patients with persistently normal ALT display a wide range of viral load. Furthermore, patients with chronic hepatitis C and elevated ALT values usually have a high viral titre; but it is also true that in approximately 20% of these patients viral titre is lower than 0.2 x lo6 Eq/ml (Magrin et al., 1994; Morisco et al., 1995). In HCV-RNA-positives with normal ALT, a particular virus-host interaction most likely occurs, rendering HCV infection less dangerous. Further studies are needed to test the immunological conditions of these subjects. Finally, the IgM results that we have obtained are in agreement with previous observations which

HCV CARRIER

STATE

281

demonstrated the presence and extent of correlation of IgM positivity with both viral replication and degree of chronic liver disease (Caporaso et al., 1994; Mancini et al., 1995; Tanzi et al., 1995; Yuki et a1.,1995). In our study, IgM reactivity was indicative of viral replication with a sensitivity of 80% and this association could be found in all patients throughout the entire observation period. It is possible that the presence of IgM could also be a predictor of underlying liver damage. No IgM positivity was found in HCVRNA-negatives, as previously observed in another Italian study (Man&i et al., 1995). The combination of these findings enables us to suggest that testing for specific anti-core IgM should be considered in all asymptomatic HCV carriers as a indicator of viraemia due to its highly positive predictive value. Based on our results, we are able to conclude that throughout a long-term observation period of antiHCV-positive patients, HCV-RNA-negatives may be considered healed and HCV-RNA-positives may be defined “clinically healthy”. Acknowlegments The authors would like to thank Monique M. Gursky for revising the English of the manuscript. Financial support was provided by a 40 % grant from the MURST (Minister0 dell’Universit21 e della Ricerca Scientifica).

References Alberti, A., Morsica, G., Chemello, L., Cavalletto, D., Noventa, F., Pontisso,P. & Ruol, A. (1992), Hepatitis C virus and liver diseasein symptom-freeindividualswith anti-HCV. Lancer,340, 697-698. Alter, M.J., Margolis, H.S., Krawczynski, K., Judson, F.N., Mares, A., Alexander, W.J., Hu, P.Y., Miller, J.K., Gerber, M.A., Sampliner,R.E., Meeks, E.L. & Beach,M.J. for the SentinelCountriesChronic NonA, Non-B HepatitisStudy Team. (1992), The natural history of community acquiredhepatitisC in the US. N. Engl. J. Med., 327,1899-1903. Brillanti, S., Foli, M., Gaiani, S., Masci, C., Miglioli, M. & Barbara, L. (1992), Persistenthepatitis C viraemia without liver disease.Luncet, 341, 464-465. Caporaso, N., Romano, M., Marmo, R., de Sio, I., Morisco, F., Minerva, A. & Coltorti, M. (1991), Hepatitis C virus infection is an additive risk factor for development of hepatocellular carcinoma in patientswith cirrhosis.J. Hepatol., 12, 367-371. Caporaso,N., Morisco, F., Romano,M., Persico,M., De Sio, I. & Tuccillo, C. (1994), IgM anti-HCV core in HCV-RNA positive subjectswith and without chronic liver disease.Ital. J. Gastroenterol., 26, 347-348. Colloredo-Mels, G., Bellati, G., Leandro, G., Brunette, M.R., Vicari, O., Borzio, M., Piantino,P., Fomaciari, G., Scudeller,G., Angeli, G., Bonino, F. & Id& G. (1994), Fluctuationsof viraemia, aminotransferases andIgM antibodyto hepatitisB core antigenin chronic hepatitis B patientswith diseaseexacerbations. Liver, 14, 175-181.

F. MORISCO

Coltorti, M., Romano,M., Persico,M., Morisco, F., Tuccillo, C. & Caporaso,N. (1995) Hepatitis C virus RNA in serumand liver histology in asymptomatic anti-HCV positive subjects.Infection, 23, 33-36. Fong, T.L., Lee, S.R., Briggs, W.K., Valinluk, B., Govindarajan,S., Hoffman, A., Jackzo,B. & Redeker,A.G. (1996),Clinical significanceof hepatitisC viral RNA statusand its correlation to antibodiesto structural HCV antigensin anti-HCV reactive patients with normalliver tests.J. Med. Viral., 49, 253-258. Guadagnino,V., Costantino,A., Rapicetta, M., Kondili, L.A., Menniti-Ippolito, F., Caroleo, B., Costa, C., Grillo, G., Loiacono,L., Pisani,V., Foca, A. & Stroffolini, T. (1997),Prevalence,risk factorsandmolecular epidemiologyof hepatitisC virus infection in the generalpopulation.A community-basedsurvey in a southernItalian town. It. J, Gastroenterol. Hepatol., 29(l), 88. Giuberti, T., Marchelli, S., Rodella,A., Puoti, M., Stellini, R., Almi, P., Rubino, M. & Galli, C. (1994), IgM anti-core antibodiesresponseduring the acute phase of HCV infection. Ital. J. Gastroenterol., 26(suppl2), 79. Kapprell, H.P., Michel, G., Hampl, H., Bonino, F. & Esteban, J-1.(1996), Demonstrationof specific detection of anti-HCV IgM core antibodies.J. Virol. Methods, 59, 121-126. Magrin, S., Craxi, A., Fabiano,C., Simonetti, R.G., Fiorentino, G., Marino, L., Diquattro, O., Di Marco, V., Lo Iacono, O., Volpes, R., Almasio, P., Urdea,M.S., Neuwald,P., Sanchez-Pescador, R., Detmer, J., Wilber, J.C. & Pagliaro,L. (1994), HepatitisC viraemia in chronic liver disease:relationshipto interferon-o or corticosteroidtreatment.Hepatology, 19, 273-279. Magrin, S., Craxl, A,, Fabiano,C., Marino, L., Fiorentino, G., Lo Iacono, O., Volpes, R., Di Marco, V., Almasio, P., Vaccaro, A., Urdea, M.S., Wilber, J.C., Bonura, C., Gianguzza,F., Capursi,V., Filiberti, S., Stuyver, L. & Pagliaro,L. (1996), HCV viraemia is more important than genotype asa predictor of response to interferon in Sicily (Southern Italy). J. Hepatol., 25, 503-590.

Mancini, C., Rivanera,D., Lilli, D., Di Cuonzo, G., Angeletti, S., Lorino, G., De Sanctis, G.M., Barbacini, LG., Leonetti, G., Bianchi, P., Chircu, L.V. & Galli, C. (1995) IgM anti-hepatitisC virus in patientswith

ET AL.

chronic non-A, non-B hepatitisandtheir relationship to viral replication. Clin. Diagn. Viral., 4, 293-299. Martinelli, A.C., Brown, D., Braun, H.B., Michel, G. & Dusheiko, G.M. (1996), Quantitative assessment of hepatitisC virus RNA and IgM antibodiesto hepatitis C core in chronic hepatitisC. J. Hepatol., 24, 2126. Morisco, F., Del Vecchio Blanco, G., Tuccillo, C., Iasevoli, P., Sessa,G. & Caporaso,N. (1995), Levels of HCV-RNA in subjectswith andwithout HCV-related chronic liver disease.J. Hepatol., 23(suppl1),178. Morisco, F., Tuccillo, C., Del Vecchio Blanco, G., Sessa, G., Iasevoli, P., Crafa, E., Brunasso,G., De Luise, G., Dente, B., Galli, C. & Caporaso,N. (1996), Clinical significanceof immunoglobulinM antibody to hepatitis C virus core. Ital. J. Gastroenterol., 28(suppl5), 17-19. Naito, M., Hayashi, N., Hagiwara, H., Hiramatsu, N., Kasahara,A., Fusamoto,H. & Kamada,T. (1994), SerumhepatitisC RNA quantity andhistologicalfeatures of hepatitis C virus carriers with persistently normalALT levels. Hepatology, 19, 871-875. Ounanian-Parez,A., Morel-Baccard,C., Barlet, V., Gueddah, N., Schweizer, B., Bensa,J.C., Zarski, J.P. & Seigneurin, J.M. (1996), HCV-infection in blood donors:associationbetweenanti-HCV core IgM antibodiesand serumHCV RNA. VOX Sang, 70,139-143. Puoti, C., Magrini, A., Stati, T., Montagnese,F., Rossi, P. & Resta, S. (1997), Virological and histological statesof HCV RNA carriers with persistently normal ALT levels. it. J. Gastroenterol. Hepatol., 29(l), 88-89. Ravaggi, A., Rossini, A., Mazza, C., Puoti, M., Marin, M.G. & Cariani, E. (1996), Hepatitis C virus genotypes in Northern Italy: clinical and virological features.J. Clin. Microbial., 34, 2822-2825. Tanzi, E., Roman& L., Bellati, G., Id&o, G., Cargnel,A., Viganb, P., Galli, C. & Zanetti, A.R. (1995), Predictors of long-term sustainedresponseto IFN therapy in chronic hepatitisC. J. Hepatol., 23(suppl),185. Yuki, N., Hayashi,N., Ohkawa, K., Hagiwara,H., Oshita, M., Katayama, K., Sasaki,Y., Kasahara,A., Fusamoto, H. & Kamada,T. (1995), The significanceof immunoglobulinM antibody responseto hepatitisC virus core proteinin patientswith chronic hepatitisC. Hepatology,

22, 403-406.