Originalia A. Srnnerborg, A. Abebe, O. Stranneg&d
Hepatitis C Virus Infection in Individuals with or without Human Immunodeficiency Virus Type 1 Infection Summary: Serum specimens from 111 human immunodeficiency virus type 1 (HIV-1) infected and 183 HIV-1 seronegative patients were analysed for antibodies to hepatitis C virus (HCV), hepatitis B virus (HBV) and hepatitis A virus (HAV) by enzyme linked immunoassay (ELISA) and radioimmunoassay. Anti-HCV and antiHBV antibodies were found in the vast majority (89 and 83%, respectively) of intravenous drug addicts (IVDA), independent of the type of drug abuse or whether the patients were HIV-t infected or not. Anti-HAV antibodies were found in 60% of the IVDA. Anti-HCV antibodies were found in anti-HIV-1 positive homosexual men (14%) and anti-HIV-1 negative heterosexual persons (8%), but not in HIV-1 seronegative homosexual men. Also anti-HAV antibodies were found to a small extent in these groups. In contrast, anti-HBV antibodies were common in the homosexual men. The absorbance values of the positive reactions in the anti-HCV ELISA were lower for HIV-1 seropositive patients than those for HIV-1 seronegative subjects, particularly in the late stages of HIV-1 infection. These data suggest that HCV infection is transmitted as readily as HBV infection by intravenous drug abuse and that all three types of hepatitis virus infection are common in IVDA. Although transmission of HCV is primarily mediated by blood, sexual transmission may also occur. HIV-1 infection seems to be associated with unusually low levels of anti-HCV antibodies, especially in the late stages of HIV-1 infection. Zusammenfassung: Hepatitis C Virus lnfektionen bei HIV-1 Infizierten und nicht mit HIV-Inifizierten. Serumproben von 111 mit dem menschlichen Immunschwache-
Introduction Hepatitis C virus (HCV) infection is related to transfusionassociated hepatitis and is well known as a major cause of chronic non-A, non-B hepatitis [1-3]. HCV infection has also been reported to be common among other risk groups with frequent parenteral exposure to blood, such as intravenous drug addicts (IVDA) and hemophiliacs [4-7]. Presently there is no method for the detection of infectious HCV by virus culture, but HCV RNA can be demonstrated in blood, serum or liver biopsies by a polymerase chain reaction technique [8]. The genome of HCV has been cloned [1] and based on recombinant proteins derived from these clones a serological assay for anti-HCV antibodies has been developed [2]. In the present study, we have determined the prevalence of
virus Typ 1 (HIV-1) Infizierten und von 183 HIV-1 seronegativen Patienten wurden auf Antikrrper gegen das Hepatitis C Virus (HCV), Hepatitis B Virus (HBV) und Hepatitis A Virus (HAV) mittels enzymgebundenem Immunassay (ELISA) oder Radioimmunassay untersucht. Bei der tiberwiegenden Mehrzahl der i. v. Drogenabh~ingigen fanden sich Antikrrper gegen HCV (89%) und HBV (83%). Dabei fand sich keine Korrelation zur Art der Droge oder HIV-Infektion. 60% der i. v. Drogenabh~ingigen hatten auch HAV-AntikOrper. Unter den antiHIV-1 positiven M~innern wiesen 14% Antikrrper gegen HCV auf. Anti-HIV negative heterosexuelle Personen waren zu 8% anti-HCV positiv. Unter den HIV-1 seronegativen homosexueUen MErmern fanden sich in keinem Fall Antikrrper gegen HCV. Ein kleiner Anteil der Personen dieser Gruppen wies auch Antikrrper gegen HAV auf. Anti-HBV Antikrrper fanden sich h~iufig bei homosexuellen M~innern, wobei keine Abhangigkeit vom HIVSerumstatus bestand. Bei HIV-1 seropositiven Patienten, und zwar besonders ausgepr~igt im Sp~itstadium der HIVInfektionen, waren die Absorptionswerte bei positivem anti-HCV ELISA niedriger als bei HIV-1 seronegativen Personen. Daraus l~iBtsich schliaBen, dab die HCV-Infekdon wie die HBV-Infektion durch intravenrsen DrogenmiBbrauch tibertragen wird und dab alle drei Typen der Virushepatitis bei i.v. Drogenabh'angigen h~iufig sind. Obwohl die HCV-Obertragung haupts~ichlich durch Blut erfolgt, ist eine sexuelle Obertragung ebenfalls mrglich. Bei HIV-1 Infizierten sind die anti-HCV Antikrrperspiegel ungewrhnlich niedrig, vor allem in den Sp~itstadien der Infektion.
antibodies to HCV, hepatitis B virus (HBV) and hepatitis A virus (HAV) in IVDA, homosexual men and heterosexual persons with or without human immunodeficiency virus type 1 (HIV-1) infection. The absorbance values of positive reactions in the enzyme linked immunosorbent assay (ELISA) used to detect anti-HCV antibodies were also analysed in relation to risk group and stage of HIV-1 infection. Received: 15 June 1990/Revisionaccepted: 24 August 1990 Dr. A. Srnnerborg, M. D. Ph. D., A. Abebe, Bsc., Dept. of Virology, the Central Microbiological Laboratory of Stockholm County Council, P. O. Box 70 740, S-107 26 Stockholm, Sweden and the Dept. of Infectious Diseases, Roslagstull Hospital, Karolinska Institute, Stockholm; 0. Strannegdrd, M. D., Ph. D.,Dept. of Virology, the Central Microbiological Laboratory of Stockholm County Council, P. O. Box 70 740, S-107 26 Stockholm, Sweden.
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A. S t n n e r b o r g et al.: HCV Infection and HIV-1 Infected Patients
Table 1: Prevalences of anti-HCV, anti-HBc and anti-HAV antibodies in patients of different risk groups, with or without HIV-1 infection.
Heroin addicts Anti-HIV-1 positive Anti-HIV-1 negative
52 47
94% + 6 79% + 11
45 47
96% + 6 81% + 12
36 47
64% + 20 55% + 20
Central stimulant addicts Anti-HIV-1 negative
38
95% + 7
38
71% + 17
38
63% • 19
All drug addicts
137
89% + 5
130
83% + 7
121
60% + 11
Homosexual men Anti-HIV-i positive Anti-HIV-1 negative
59 48
14% + 9 0
46 48
72% + 13 31% + 13
43 47
28% + 13 19% _+11
Heterosexuals Anti-HIV-1 negative
50
8% + 7
50
8% + 7
50
10% • 8
N = the total number of patients tested in each category. P = proportion of positive patients (per cent + 95% confidence interval).
Patients and Methods
Study population and study design: Serum samples were collected in 1988-1989 from a total of 294 adult consecutive Caucasian outpatients (214 males, 80 females; mean 34 years; range 24-56 years), who were examined due to a known HIV-1 infection or due to multiple sexual partners or intravenous drug abuse, at the Department of Infectious Diseases, Roslagstull Hospital, Stockholm, Sweden. One hundred and eleven patients (100 males, 11 females; mean 36 years, range 32-56 years) were infected with HIV-1, as determined by ELISA and Western blotting. Information on which stage of HIV-I infection the patients belonged to was obtained for 101 subjects of whom 55 were asymptomatic HIV-1 seropositive, seven had persistent generalized lymphadenopathy, 18 had AIDS related complex (ARC) and 21 had AIDS. One hundred and thirty-seven patients (87 males. 50 females; mean 34 years, range 23-55) were IVDA. The numbers of individuals with or without HIV-1 infection in the different risk groups and the main drug used by the IVDA are indicated in T a b l e 1. Fifty heterosexual patients (20 males, 30 females; mean 38 years, range 23-54), who denied intravenous drug abuse or homosexual contacts, were also included. Assay of anti-HCV, anti-HBc, anti-HAV antibodies and liver function tests: The serum samples were analysed for anti-HCV antibodies by ELISA (Ortho Diagnostics Raritan, New Jersey, USA), which uses a recombinant HCV antigen polypeptide (c100-3) [2]. The absorbance values were determined by spectrephotometer (Dynatech ELISA Microplate reader) at 490 + 2 nm. The cut-off values were determined according to the manufacturer's instructions. The samples had been stored at -70 °C before being tested. No heat inactivation had been performed. Antibodies to hepatitis B core antigen (anti-HBc) and HAV were determined in 274 patients by radioimmunoassay and ELISA, respectively (CORAB and HAVAB. Abbott Laboratories, Chicago, USA), according to the manufacturers' instructions. Measurements of alanine aminotransferase (ALAT) of 90 HIV-1 seropositive patients were performed by standard chemical tests (normal
32 / 348
value: < 0.70 gkat/1). The liver function test was repeated in 36 patients two to four times during a follow-up period of six months. Statistics: Group comparisons were performed by Mann-Whitney U test. Chi-square test or McNemar's test. Results
Anti-HCV Antibodies among Intravenous Drug Abusers A n t i - H C V antibodies were found in 122 out o f 137 (89%) I V D A ( T a b l e 1 ). N o significant difference was found bet w e e n patients with o r without HIV-1 infection (49/52, 94% versus 73/85, 86%), heroin addicts (86/99, 87%) and central stimulants (CS) addicts (36/38, 95%), m a l e s (80/87, 9 2 % ) and f e m a l e s (42/50, 84%) or in age b e t w e e n individuals with or without a n t i - H C V antibodies. W h e n the a b s o r b a n c e values o f the p o s i t i v e a n t i - H C V reactions were c o m p a r e d , the values w e r e found to be l o w e r in I V D A with HIV-1 infection than in those without (p< 0.02) ( F i g u r e 1 ). This difference was not related to the type o f drug abuse or sex. I V D A in the early stages o f HIV-1 infection had h i g h e r a b s o r b a n c e values than those with A R C or A I D S (p< 0.02), but lower than HIV-1 seronegative I V D A (p< 0.001) (Figure 1 ).
Anti-HBc and Anti-HAV Antibodies in Intravenous Drug, Abusers T h e r e were similar high p r e v a l e n c e s o f a n t i - H B c antibodies as for a n t i - H C V antibodies a m o n g heroin addicts ( T a b l o 1 ). H o w e v e r , a l o w e r p r e v a l e n c e o f a n t i - H B c antib o d i e s (27/38, 71%) than o f a n t i - H C V antibodies (36/38, 9 5 % ) was f o u n d a m o n g I V D A using CS (p< 0.01). N o difference in the prevalences o f a n t i - H B c or a n t i - H A V antib o d i e s was f o u n d b e t w e e n m a l e s and females. I V D A with
Infection 18 (1990) No. 6 © MMV Medizin Verlag GmbH Miinchen. Miinchen 1990
A. S6nuerborg et al.: HCV Infection and HIV-1 Infected Patients
antibodies (0.05< p< 0.1). HBsAg was found in one patient only. Anti-HAV antibodies were found in the majority of the IVDA, although to a lower extent than anti-HCV and anti-HBc antibodies (p< 0.001; p< 0.05) (Table 1 ). All of the 76 IVDA, who were tested for antibodies to all three hepatitis viruses, had antibodies to one or more of the viruses (Table 2).
Absorbance
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Anti-HCV, Anti-HBc and Anti-HA V Antibodies in Homosexual Men
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IVDA
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HlVinfection:
Homosexuals
Heterosexuals
+ orearly stages
late s t a g e s
Figure 1 : Absorbance values in the anti-HCV ELISA in relation to risk behaviour and HIV-1 infection. • = positive reactions, O = negative reactions in HIV-1 seropositive patients, x = negative reactions in HIV-1 seronegative subjects. Horizontal bars indicate median values.
Eight HIV-1 seropositive homosexual men (tour asymptomatic HIV-1 seropositive persons and four men with ARC or AIDS) had detectable antibodies to HCV (Table 1 ). All of the HIV-1 seronegative homosexual men were also seronegative for HCV. Lower absorbance values of the positive anti-HCV antibody reactions were found in the eight homosexual men than in the HIV-1 seropositive IVDA with antibodies to HCV (p< 0.01) (Figure 1 ). A higher prevalence of anti-HBc antibodies than of antiHAV and anti-HCV antibodies was found in HIV-1 serepositive (72%, 28%, 14%, respectively) (p< 0.001, both comparisons) as well as in HIV-1 seronegative homosexual men (31%, 19%, 0%, respectively) (Table 1 ). Seven out of eight anti-HCV antibody positive homosexual men also had anti-HBc antibodies (Table 2), but only one out of six tested had anti-HAV antibodies. HIV-1 seropositive men had antibodies to one or more of the hepatitis viruses more often (76%) than the HIV-1 seronegative men (38%) (p < 0.001) (Table 2). The patients with anti-HBc and antiHAV antibodies were older than those without (p< 0.01, both comparisons). No such difference was found for antiHCV antibodies. HBsAg was found in one patient, who also was HIV-1 and HCV seropositive. Liver Function Tests in HIV-1 Seropositive Patients
antibodies to HAV were older than those without (p< 0.001). A similar tendency was found for anti-HBc
An increased serum ALAT value was found in 36 out of 90 (40%) tested HIV-1 seropositive patients (Table 3), when
Table 2: Concomitant occurrence of anti-HCV (C), anti-HBc (B) and anti-HAV (A) antibodies in intravenous drug addicts (IVDA), homosexual men (HS) and heterosexual persons, with or without HIV-1 infection.
HIV-1 seropositive IVDA HS
22 42
0 10
0 2
0 16
2 1
2 8
0 0
4 4
4 1
54 47
0 29
1 4
1 9
8 0
2 5
4 0
10 0
28 0
50
41
3
1
2
1
0
1
1
HIV-1 seronegative IVDA HS Heterosexuals
- and + = negative and positive reactions. N = total number of individuals in each group. The figures in the other columns indicate the numbers of patients in each category.
Infection 18 (1990) No. 6
© MMV Medizin Verlag GmbH Mfinchen, Miinchen 1990
349 [ 33
A. S6nnerborg et al.: HCV Infection and HIV-1 Infected Patients Table 3: Levels of serum alanine aminotransferase (ALAT) in relation to the presence of anti-HCV antibodies in 90 HIV-1 seropositive individuals.
IVDA
+
IVDA HS
+
HS IVDA + HS IVDA + HS
~"
37
18 (49)
3
2 (67)
7
4 (57)
43
12 (28)
44
22 (50)
46
14 (30)
and + = negative and positivereactions.N = total nnmberof individuals in each group~The figures in the other columns indicate the numbers and the percentageof patients with increasedALAT values.
-
the analysis was performed on blood obtained the same day as the serum for the anti-HCV antibody assay. Anti-HCV antibodies were detected in 22 out of these 36 subjects. A normal liver function test was obtained despite the presence of anti-HCV antibodies in 22 out of 44 (50%) patients. No significant difference in serum ALAT levels was found between 29 HCV seropositive patients in the early stages of HIV-1 infection and 15 HCV seropositive patients with ARC or AIDS. When the analysis of serum ALAT was repeated in 16 HCV seropositive patients with initially normal serum ALAT values, a further four subjects had increased levels. A fluctuating pattern with periods of normal levels was also found in six out of 20 tested HCV seropositive patients with an initially increased serum A L A T value. Thus. altogether ten out of the 36 tested HCV positive patients (28%) showed a pattern with periods of normal and raised values. Repeatedly normal serum ALAT values were found in 12 out of 3 6 H C V seropositive subjects (33%), when tested two to four times during a six month period. None of the patients had serological signs of an acute infection with HCV, HAV, HBV, HIV-1, cytomegalovirus or EpsteinBarr virus.
Anti-HCV, Anti-HBc and Anti-HA V Antibodies in Heterosexuals Anti-HCV antibodies were detected in four heterosexual subjects (two males and two females) of whom two also had anti-HBc antibodies (Tables 1 and 2). All four had an absorbance value of more than 2.0. Anti-HAV antibodies were found in five persons (two males and three females). Discussion
In the present study, high prevalences of anti-HCV antibodies, as well as anti-HBc and anti-HAV antibodies, were
34 /350
found in IVDA, independent of whether HIV-1 infection was presenl or not. In contrast, anti-HCV antibodies were found in a minority of homosexual men as well as in HIV-1 seronegative heterosexuals with multiple sex partners. These data indicate that HCV is readily transmitted by intravenous drug abuse and suggest that sexual transmission may occur to a lesser extent. The frequent occurrence of anti-HCV antibodies among IVDA was not unexpected, although the prevalence was even higher (89%) than earlier reported from other European countries. Thus, in studies performed in Spain, England and Austria, anti-HCV antibodies have been detected in 70%, 81% and 50%, respectively, of IVDA [4, 5, 7]. It has earlier been suggested that HIV-1 infection may facilitate the acquiring of HCV infection since HIV-1 seronegative IVDA had a lower prevalence (36%) of anti-HCV antibodies than those with HIV-1 infection (67%) in one study [5]. In the present study, however, we found that HCV was present to a similar high extent in HIV-1 seronegative as in HIV-1 seropositive IVDA in Stockholm, indicating that an extensive spread of HCV may occur independent of a concomitant HIV-1 infection in this population. There was no significant difference between the prevalences of antibodies to HBc and HCV among the IVDA suggesting that no major difference in contagiousness exists between the two viruses regarding the intravenous route of transmission. Furthermore, there was also a high prevalence of anti-HAV antibodies, as compared to the low prevalence found in other groups of the Scandinavian population. The high prevalence among IVDA in the present study is in concordance with earlier reports of a cyclic pattern o f immunity to hepatitis A among IVDA in Sweden with a high level of immunity the years following outbreaks of hepatitis A [9]. A fluctuating pattern of the serum ALAT levels including normal and pathological values was found in 28% of the HIV-I seropositive patients with anti-HCV antibodies. This finding is concordant with earlier studies on HIV-1 seronegative patients with chronic non-A, non-B hepatitis [10]. Although 33% of the HCV- and HIV-1 seropositive subjects had repeatedly normal values during a six month period in the present study, it should be emphasized that even a protracted period with normal serum A L A T values may occur during the course of a Chronic non-A, non-B hepatitis [10]. It has earlier been suggested that chronic hepatitis B may be less severe when accompanied by HIV-1 infection and that this effect increases with the degree of immune suppression [11]. In our study, no difference was found in serum ALAT values between patients with anfi-HCV antibodies in the early and late stages of HIV-1 infection suggesting that the Clinical course of HCV infection is not markedly influenced by concomitant HIV- 1 infection. There is at present no available confirmatory assay of positive reactions in the anti-HCV ELISA used in our study. Thus, it cannot be excluded that false positive reactions occurred, especially in patients with absorbance values just above cut-off. Patients with ARC or AIDS had lower ab-
Infection 18 (1990) No. 6 © MMV Medizin VertagGmbH Mfinchen,Mfinchen1990
A. Stnnerborg et al.: HCV Infection and HIV-1 Infected Patients sorbance values than subjects in the early stages of HIV-1 infection. Furthermore, the latter patients had lower values than HCV-seropositive subjects without HIV-1 infection. These data suggest that the weakly positive reactions in some HIV-1 seropositive patients are due to a low amount o f anti-HCV antibodies rather than to false positive reactions. It should be noted that H I V - t infected individuals often have unusually low concentrations of antibodies to other pathogens and to the HIV-1 p24 antigen, particularly in the late stages o f the disease [12, 13]. It has earlier been reported that anti-HCV antibodies may disappear with time [4]. It is possible that the immunodeficiency related to HIV-1 infection may enhance this process either by decreasing the ability to produce anti-HCV antibodies or by increasing replication of HCV with a secondary consumption of the specific antibodies, as suggested to occur in HIV-1 infected patients with chronic hepatitis B [11]. The frequent finding of anti-HBc and anti-HAV antibodies in the homosexual men in our study is in line with the well-known fact that H B V and H A V may spread sexually. It has been debated whether HCV also may be sexually
References 1. Choo, Q. L., Kuo, G., Weiner, A. J., Overby, L. R., Bradley, D. W., Houghton, M.: Isolation of a cDNA clone derived from a blood borne non-A, non-B, viral hepatitis genome. Science 244 (1989) 359-362. 2. Kuo, G.,Choo, Q. L, Alter, J.,Gitnick, G.,Redeker, A. G, Purcell, R. H°, Miyamura, T., Dienstag, J. L., Alter, M. J., Stevens, C. E., Tegtmeier, G. E., Bonino, F., Colombo, M., Lee, W-S, Kuo, C., Berger, K., Shuster, J. R.: An assay for circulating antibodies to a major etiological virus of human non-A, non-B hepatitis. Science 244 (t989) 362-364. 3. Alter, H. J, Purcell, R. H., Shill, J. W., Melpolder, J. C., Houghton, M., Choo, Q-L., Kuo G.: Detectionof antibody to hepatitis C virus in prospectively followed transfusion recipients with acute and chronic non-A, non-B hepatitis, N. Engl. J. Med. 321 (1989) 1494-1500. 4. Esteban, J. I., Esteban, R., Viladomio, L., Ltpez-Talavera, J. C., Gonzfilez, A., Hernfindez, J. M., Roger, M., Vargas, V., Genescfi, J., Buff, M., Guardia, J., Houghton, M., Choo, Q-L., Kuo, G.: Hepatitis C virus antibodies among risk groups in Spain. Lancet ii (1989) 294-297. 5. Huemer, H. P., Prodinger, W. M., Larcher, C., M6st, J., Dierich, M. P.: Correlationof hepatitis C virus antibodieswith HIV-1 seropositivity in intravenous drug addicts. Infection 18 (1990) 122. 6. Noel, L., Guerois, C., Maisonneuve, P., Verroust, F., Laurian, Y.: Antibodies to hepatitis C virus in haemophilia. Lancet ii (1989) 560. 7. Mortimer, P. P., Cohen, B.J., Litton, P. A., Vandervelde, E. M.,
transmitted. Anti-HCV antibodies have been detected in 5% of patients with other sexually transmitted diseases [ 14] and to a higher extent in homosexual men with HIV-1 infection (26%) than in those without (4%) [7]. In the present study, anti-HCV antibodies were found in 14% o f the HIV-1 seropositive but not in the HIV-1 seronegative homosexual men. Furthermore, anti-HCV antibodies were also found in 8% of the heterosexual persons with multiple sexual partners. Although it cannot be excluded that these persons had used intravenous drugs without mentioning it, the data are in line with the suggestion that H C V infection can be sexually transmitted [7, 14]. It should be emphasized, however, that the prevalence of anti-HBc antibodies was much higher than that o f anfi-HCV antibodies among the homosexual men suggesting that hepatitis B virus is more readily spread sexually than is HCV. Acknowledgement The assistance of Ms Ulla Magnusson at the Central Microbiological Laboratory of Stockholm County Council is gratefully acknowledged.
Bassendine, M. F., Brind, A. M., Hambling, M. H.: HepatitisC virus antibody. Lancet ii (1989) 798. 8. Weiner, A. J., Kuo, G, Bradly, D. W.: Detection of hepatitis C viral sequences in non-A, non-B hepatitis. Lancet ii (1990) 355 1-3. 9. Wideli, A., Hansson, B. G., Moestrup, T., Nordenfelt, E.: Increased occurrence of hepatitis A with cyclic outbreaks among drug addicts in a Swedish community. Infection 11 (1983) 198-200. 10. Mattson, L., Weiland, O., Glaumann, H.: Chronic non-A, non-B hepatitis developed after transfusions, illicit self injections or sporadically. Liver 9 (1989) 120-t27. 11. Bodsworth, N., Donovan, B., Nightingale, B.: The effect of concurrent human immunodeficiencyvirus infection on chronic hepatitis B: a study of 150 homosexual men. J. Infect. Dis. 160 (1989) 577-582. 12. Converse, P. J., Fehniger, T. E., Ehrnst, A., Stranneg~rd, ()., Britton, S.: Immune responses to fractionated cytomegalovirus (CMV) antigens after HIV infection: Loss of cellular and humoral reactivity to antigens recognized by HIV-, CMV+ individuals. Clin. Expfl. Immunol. (1990) in press. 13. Lange, J., Paul, D., Huisman, H., de Wolf, F., van den Berg, H., Coutinho, R., Danner, S., van der Noordaa, J., Goudsmit, J.: Persistent HIV antigenaemiaand decline of HIV core antibodies associated with transition to AIDS. Br. Meat.J. 293 (1986) 1459-1462. 14. Hess, G., Massing, A., Rossol, S., Schiitt, H., Clemens, R., Meyer zum Biischenfelde.: Hepatitis C virus and sexual transmission. Lancet ii (1989) 987.
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Hepatitis C Virus Infection in Individuals with or without Human Immunodeficiency Virus Type 1 Infection Summary: Serum specimens from 111 human immunodeficiency virus type 1 (HIV-1) infected and 183 HIV-1 seronegative patients were analysed for antibodies to hepatitis C virus (HCV), hepatitis B virus (HBV) and hepatitis A virus (HAV) by enzyme linked immunoassay (ELISA) and radioimmunoassay. Anti-HCV and antiHBV antibodies were found in the vast majority (89 and 83%, respectively) of intravenous drug addicts (IVDA), independent of the type of drug abuse or whether the patients were HIV-t infected or not. Anti-HAV antibodies were found in 60% of the IVDA. Anti-HCV antibodies were found in anti-HIV-1 positive homosexual men (14%) and anti-HIV-1 negative heterosexual persons (8%), but not in HIV-1 seronegative homosexual men. Also anti-HAV antibodies were found to a small extent in these groups. In contrast, anti-HBV antibodies were common in the homosexual men. The absorbance values of the positive reactions in the anti-HCV ELISA were lower for HIV-1 seropositive patients than those for HIV-1 seronegative subjects, particularly in the late stages of HIV-1 infection. These data suggest that HCV infection is transmitted as readily as HBV infection by intravenous drug abuse and that all three types of hepatitis virus infection are common in IVDA. Although transmission of HCV is primarily mediated by blood, sexual transmission may also occur. HIV-1 infection seems to be associated with unusually low levels of anti-HCV antibodies, especially in the late stages of HIV-1 infection. Zusammenfassung: Hepatitis C Virus lnfektionen bei HIV-1 Infizierten und nicht mit HIV-Inifizierten. Serumproben von 111 mit dem menschlichen Immunschwache-
Introduction Hepatitis C virus (HCV) infection is related to transfusionassociated hepatitis and is well known as a major cause of chronic non-A, non-B hepatitis [1-3]. HCV infection has also been reported to be common among other risk groups with frequent parenteral exposure to blood, such as intravenous drug addicts (IVDA) and hemophiliacs [4-7]. Presently there is no method for the detection of infectious HCV by virus culture, but HCV RNA can be demonstrated in blood, serum or liver biopsies by a polymerase chain reaction technique [8]. The genome of HCV has been cloned [1] and based on recombinant proteins derived from these clones a serological assay for anti-HCV antibodies has been developed [2]. In the present study, we have determined the prevalence of
virus Typ 1 (HIV-1) Infizierten und von 183 HIV-1 seronegativen Patienten wurden auf Antikrrper gegen das Hepatitis C Virus (HCV), Hepatitis B Virus (HBV) und Hepatitis A Virus (HAV) mittels enzymgebundenem Immunassay (ELISA) oder Radioimmunassay untersucht. Bei der tiberwiegenden Mehrzahl der i. v. Drogenabh~ingigen fanden sich Antikrrper gegen HCV (89%) und HBV (83%). Dabei fand sich keine Korrelation zur Art der Droge oder HIV-Infektion. 60% der i. v. Drogenabh~ingigen hatten auch HAV-AntikOrper. Unter den antiHIV-1 positiven M~innern wiesen 14% Antikrrper gegen HCV auf. Anti-HIV negative heterosexuelle Personen waren zu 8% anti-HCV positiv. Unter den HIV-1 seronegativen homosexueUen MErmern fanden sich in keinem Fall Antikrrper gegen HCV. Ein kleiner Anteil der Personen dieser Gruppen wies auch Antikrrper gegen HAV auf. Anti-HBV Antikrrper fanden sich h~iufig bei homosexuellen M~innern, wobei keine Abhangigkeit vom HIVSerumstatus bestand. Bei HIV-1 seropositiven Patienten, und zwar besonders ausgepr~igt im Sp~itstadium der HIVInfektionen, waren die Absorptionswerte bei positivem anti-HCV ELISA niedriger als bei HIV-1 seronegativen Personen. Daraus l~iBtsich schliaBen, dab die HCV-Infekdon wie die HBV-Infektion durch intravenrsen DrogenmiBbrauch tibertragen wird und dab alle drei Typen der Virushepatitis bei i.v. Drogenabh'angigen h~iufig sind. Obwohl die HCV-Obertragung haupts~ichlich durch Blut erfolgt, ist eine sexuelle Obertragung ebenfalls mrglich. Bei HIV-1 Infizierten sind die anti-HCV Antikrrperspiegel ungewrhnlich niedrig, vor allem in den Sp~itstadien der Infektion.
antibodies to HCV, hepatitis B virus (HBV) and hepatitis A virus (HAV) in IVDA, homosexual men and heterosexual persons with or without human immunodeficiency virus type 1 (HIV-1) infection. The absorbance values of positive reactions in the enzyme linked immunosorbent assay (ELISA) used to detect anti-HCV antibodies were also analysed in relation to risk group and stage of HIV-1 infection. Received: 15 June 1990/Revisionaccepted: 24 August 1990 Dr. A. Srnnerborg, M. D. Ph. D., A. Abebe, Bsc., Dept. of Virology, the Central Microbiological Laboratory of Stockholm County Council, P. O. Box 70 740, S-107 26 Stockholm, Sweden and the Dept. of Infectious Diseases, Roslagstull Hospital, Karolinska Institute, Stockholm; 0. Strannegdrd, M. D., Ph. D.,Dept. of Virology, the Central Microbiological Laboratory of Stockholm County Council, P. O. Box 70 740, S-107 26 Stockholm, Sweden.
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A. S t n n e r b o r g et al.: HCV Infection and HIV-1 Infected Patients
Table 1: Prevalences of anti-HCV, anti-HBc and anti-HAV antibodies in patients of different risk groups, with or without HIV-1 infection.
Heroin addicts Anti-HIV-1 positive Anti-HIV-1 negative
52 47
94% + 6 79% + 11
45 47
96% + 6 81% + 12
36 47
64% + 20 55% + 20
Central stimulant addicts Anti-HIV-1 negative
38
95% + 7
38
71% + 17
38
63% • 19
All drug addicts
137
89% + 5
130
83% + 7
121
60% + 11
Homosexual men Anti-HIV-i positive Anti-HIV-1 negative
59 48
14% + 9 0
46 48
72% + 13 31% + 13
43 47
28% + 13 19% _+11
Heterosexuals Anti-HIV-1 negative
50
8% + 7
50
8% + 7
50
10% • 8
N = the total number of patients tested in each category. P = proportion of positive patients (per cent + 95% confidence interval).
Patients and Methods
Study population and study design: Serum samples were collected in 1988-1989 from a total of 294 adult consecutive Caucasian outpatients (214 males, 80 females; mean 34 years; range 24-56 years), who were examined due to a known HIV-1 infection or due to multiple sexual partners or intravenous drug abuse, at the Department of Infectious Diseases, Roslagstull Hospital, Stockholm, Sweden. One hundred and eleven patients (100 males, 11 females; mean 36 years, range 32-56 years) were infected with HIV-1, as determined by ELISA and Western blotting. Information on which stage of HIV-I infection the patients belonged to was obtained for 101 subjects of whom 55 were asymptomatic HIV-1 seropositive, seven had persistent generalized lymphadenopathy, 18 had AIDS related complex (ARC) and 21 had AIDS. One hundred and thirty-seven patients (87 males. 50 females; mean 34 years, range 23-55) were IVDA. The numbers of individuals with or without HIV-1 infection in the different risk groups and the main drug used by the IVDA are indicated in T a b l e 1. Fifty heterosexual patients (20 males, 30 females; mean 38 years, range 23-54), who denied intravenous drug abuse or homosexual contacts, were also included. Assay of anti-HCV, anti-HBc, anti-HAV antibodies and liver function tests: The serum samples were analysed for anti-HCV antibodies by ELISA (Ortho Diagnostics Raritan, New Jersey, USA), which uses a recombinant HCV antigen polypeptide (c100-3) [2]. The absorbance values were determined by spectrephotometer (Dynatech ELISA Microplate reader) at 490 + 2 nm. The cut-off values were determined according to the manufacturer's instructions. The samples had been stored at -70 °C before being tested. No heat inactivation had been performed. Antibodies to hepatitis B core antigen (anti-HBc) and HAV were determined in 274 patients by radioimmunoassay and ELISA, respectively (CORAB and HAVAB. Abbott Laboratories, Chicago, USA), according to the manufacturers' instructions. Measurements of alanine aminotransferase (ALAT) of 90 HIV-1 seropositive patients were performed by standard chemical tests (normal
32 / 348
value: < 0.70 gkat/1). The liver function test was repeated in 36 patients two to four times during a follow-up period of six months. Statistics: Group comparisons were performed by Mann-Whitney U test. Chi-square test or McNemar's test. Results
Anti-HCV Antibodies among Intravenous Drug Abusers A n t i - H C V antibodies were found in 122 out o f 137 (89%) I V D A ( T a b l e 1 ). N o significant difference was found bet w e e n patients with o r without HIV-1 infection (49/52, 94% versus 73/85, 86%), heroin addicts (86/99, 87%) and central stimulants (CS) addicts (36/38, 95%), m a l e s (80/87, 9 2 % ) and f e m a l e s (42/50, 84%) or in age b e t w e e n individuals with or without a n t i - H C V antibodies. W h e n the a b s o r b a n c e values o f the p o s i t i v e a n t i - H C V reactions were c o m p a r e d , the values w e r e found to be l o w e r in I V D A with HIV-1 infection than in those without (p< 0.02) ( F i g u r e 1 ). This difference was not related to the type o f drug abuse or sex. I V D A in the early stages o f HIV-1 infection had h i g h e r a b s o r b a n c e values than those with A R C or A I D S (p< 0.02), but lower than HIV-1 seronegative I V D A (p< 0.001) (Figure 1 ).
Anti-HBc and Anti-HAV Antibodies in Intravenous Drug, Abusers T h e r e were similar high p r e v a l e n c e s o f a n t i - H B c antibodies as for a n t i - H C V antibodies a m o n g heroin addicts ( T a b l o 1 ). H o w e v e r , a l o w e r p r e v a l e n c e o f a n t i - H B c antib o d i e s (27/38, 71%) than o f a n t i - H C V antibodies (36/38, 9 5 % ) was f o u n d a m o n g I V D A using CS (p< 0.01). N o difference in the prevalences o f a n t i - H B c or a n t i - H A V antib o d i e s was f o u n d b e t w e e n m a l e s and females. I V D A with
Infection 18 (1990) No. 6 © MMV Medizin Verlag GmbH Miinchen. Miinchen 1990
A. S6nuerborg et al.: HCV Infection and HIV-1 Infected Patients
antibodies (0.05< p< 0.1). HBsAg was found in one patient only. Anti-HAV antibodies were found in the majority of the IVDA, although to a lower extent than anti-HCV and anti-HBc antibodies (p< 0.001; p< 0.05) (Table 1 ). All of the 76 IVDA, who were tested for antibodies to all three hepatitis viruses, had antibodies to one or more of the viruses (Table 2).
Absorbance
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Anti-HCV, Anti-HBc and Anti-HA V Antibodies in Homosexual Men
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Homosexuals
Heterosexuals
+ orearly stages
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Figure 1 : Absorbance values in the anti-HCV ELISA in relation to risk behaviour and HIV-1 infection. • = positive reactions, O = negative reactions in HIV-1 seropositive patients, x = negative reactions in HIV-1 seronegative subjects. Horizontal bars indicate median values.
Eight HIV-1 seropositive homosexual men (tour asymptomatic HIV-1 seropositive persons and four men with ARC or AIDS) had detectable antibodies to HCV (Table 1 ). All of the HIV-1 seronegative homosexual men were also seronegative for HCV. Lower absorbance values of the positive anti-HCV antibody reactions were found in the eight homosexual men than in the HIV-1 seropositive IVDA with antibodies to HCV (p< 0.01) (Figure 1 ). A higher prevalence of anti-HBc antibodies than of antiHAV and anti-HCV antibodies was found in HIV-1 serepositive (72%, 28%, 14%, respectively) (p< 0.001, both comparisons) as well as in HIV-1 seronegative homosexual men (31%, 19%, 0%, respectively) (Table 1 ). Seven out of eight anti-HCV antibody positive homosexual men also had anti-HBc antibodies (Table 2), but only one out of six tested had anti-HAV antibodies. HIV-1 seropositive men had antibodies to one or more of the hepatitis viruses more often (76%) than the HIV-1 seronegative men (38%) (p < 0.001) (Table 2). The patients with anti-HBc and antiHAV antibodies were older than those without (p< 0.01, both comparisons). No such difference was found for antiHCV antibodies. HBsAg was found in one patient, who also was HIV-1 and HCV seropositive. Liver Function Tests in HIV-1 Seropositive Patients
antibodies to HAV were older than those without (p< 0.001). A similar tendency was found for anti-HBc
An increased serum ALAT value was found in 36 out of 90 (40%) tested HIV-1 seropositive patients (Table 3), when
Table 2: Concomitant occurrence of anti-HCV (C), anti-HBc (B) and anti-HAV (A) antibodies in intravenous drug addicts (IVDA), homosexual men (HS) and heterosexual persons, with or without HIV-1 infection.
HIV-1 seropositive IVDA HS
22 42
0 10
0 2
0 16
2 1
2 8
0 0
4 4
4 1
54 47
0 29
1 4
1 9
8 0
2 5
4 0
10 0
28 0
50
41
3
1
2
1
0
1
1
HIV-1 seronegative IVDA HS Heterosexuals
- and + = negative and positive reactions. N = total number of individuals in each group. The figures in the other columns indicate the numbers of patients in each category.
Infection 18 (1990) No. 6
© MMV Medizin Verlag GmbH Mfinchen, Miinchen 1990
349 [ 33
A. S6nnerborg et al.: HCV Infection and HIV-1 Infected Patients Table 3: Levels of serum alanine aminotransferase (ALAT) in relation to the presence of anti-HCV antibodies in 90 HIV-1 seropositive individuals.
IVDA
+
IVDA HS
+
HS IVDA + HS IVDA + HS
~"
37
18 (49)
3
2 (67)
7
4 (57)
43
12 (28)
44
22 (50)
46
14 (30)
and + = negative and positivereactions.N = total nnmberof individuals in each group~The figures in the other columns indicate the numbers and the percentageof patients with increasedALAT values.
-
the analysis was performed on blood obtained the same day as the serum for the anti-HCV antibody assay. Anti-HCV antibodies were detected in 22 out of these 36 subjects. A normal liver function test was obtained despite the presence of anti-HCV antibodies in 22 out of 44 (50%) patients. No significant difference in serum ALAT levels was found between 29 HCV seropositive patients in the early stages of HIV-1 infection and 15 HCV seropositive patients with ARC or AIDS. When the analysis of serum ALAT was repeated in 16 HCV seropositive patients with initially normal serum ALAT values, a further four subjects had increased levels. A fluctuating pattern with periods of normal levels was also found in six out of 20 tested HCV seropositive patients with an initially increased serum A L A T value. Thus. altogether ten out of the 36 tested HCV positive patients (28%) showed a pattern with periods of normal and raised values. Repeatedly normal serum ALAT values were found in 12 out of 3 6 H C V seropositive subjects (33%), when tested two to four times during a six month period. None of the patients had serological signs of an acute infection with HCV, HAV, HBV, HIV-1, cytomegalovirus or EpsteinBarr virus.
Anti-HCV, Anti-HBc and Anti-HA V Antibodies in Heterosexuals Anti-HCV antibodies were detected in four heterosexual subjects (two males and two females) of whom two also had anti-HBc antibodies (Tables 1 and 2). All four had an absorbance value of more than 2.0. Anti-HAV antibodies were found in five persons (two males and three females). Discussion
In the present study, high prevalences of anti-HCV antibodies, as well as anti-HBc and anti-HAV antibodies, were
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found in IVDA, independent of whether HIV-1 infection was presenl or not. In contrast, anti-HCV antibodies were found in a minority of homosexual men as well as in HIV-1 seronegative heterosexuals with multiple sex partners. These data indicate that HCV is readily transmitted by intravenous drug abuse and suggest that sexual transmission may occur to a lesser extent. The frequent occurrence of anti-HCV antibodies among IVDA was not unexpected, although the prevalence was even higher (89%) than earlier reported from other European countries. Thus, in studies performed in Spain, England and Austria, anti-HCV antibodies have been detected in 70%, 81% and 50%, respectively, of IVDA [4, 5, 7]. It has earlier been suggested that HIV-1 infection may facilitate the acquiring of HCV infection since HIV-1 seronegative IVDA had a lower prevalence (36%) of anti-HCV antibodies than those with HIV-1 infection (67%) in one study [5]. In the present study, however, we found that HCV was present to a similar high extent in HIV-1 seronegative as in HIV-1 seropositive IVDA in Stockholm, indicating that an extensive spread of HCV may occur independent of a concomitant HIV-1 infection in this population. There was no significant difference between the prevalences of antibodies to HBc and HCV among the IVDA suggesting that no major difference in contagiousness exists between the two viruses regarding the intravenous route of transmission. Furthermore, there was also a high prevalence of anti-HAV antibodies, as compared to the low prevalence found in other groups of the Scandinavian population. The high prevalence among IVDA in the present study is in concordance with earlier reports of a cyclic pattern o f immunity to hepatitis A among IVDA in Sweden with a high level of immunity the years following outbreaks of hepatitis A [9]. A fluctuating pattern of the serum ALAT levels including normal and pathological values was found in 28% of the HIV-I seropositive patients with anti-HCV antibodies. This finding is concordant with earlier studies on HIV-1 seronegative patients with chronic non-A, non-B hepatitis [10]. Although 33% of the HCV- and HIV-1 seropositive subjects had repeatedly normal values during a six month period in the present study, it should be emphasized that even a protracted period with normal serum A L A T values may occur during the course of a Chronic non-A, non-B hepatitis [10]. It has earlier been suggested that chronic hepatitis B may be less severe when accompanied by HIV-1 infection and that this effect increases with the degree of immune suppression [11]. In our study, no difference was found in serum ALAT values between patients with anfi-HCV antibodies in the early and late stages of HIV-1 infection suggesting that the Clinical course of HCV infection is not markedly influenced by concomitant HIV- 1 infection. There is at present no available confirmatory assay of positive reactions in the anti-HCV ELISA used in our study. Thus, it cannot be excluded that false positive reactions occurred, especially in patients with absorbance values just above cut-off. Patients with ARC or AIDS had lower ab-
Infection 18 (1990) No. 6 © MMV Medizin VertagGmbH Mfinchen,Mfinchen1990
A. Stnnerborg et al.: HCV Infection and HIV-1 Infected Patients sorbance values than subjects in the early stages of HIV-1 infection. Furthermore, the latter patients had lower values than HCV-seropositive subjects without HIV-1 infection. These data suggest that the weakly positive reactions in some HIV-1 seropositive patients are due to a low amount o f anti-HCV antibodies rather than to false positive reactions. It should be noted that H I V - t infected individuals often have unusually low concentrations of antibodies to other pathogens and to the HIV-1 p24 antigen, particularly in the late stages o f the disease [12, 13]. It has earlier been reported that anti-HCV antibodies may disappear with time [4]. It is possible that the immunodeficiency related to HIV-1 infection may enhance this process either by decreasing the ability to produce anti-HCV antibodies or by increasing replication of HCV with a secondary consumption of the specific antibodies, as suggested to occur in HIV-1 infected patients with chronic hepatitis B [11]. The frequent finding of anti-HBc and anti-HAV antibodies in the homosexual men in our study is in line with the well-known fact that H B V and H A V may spread sexually. It has been debated whether HCV also may be sexually
References 1. Choo, Q. L., Kuo, G., Weiner, A. J., Overby, L. R., Bradley, D. W., Houghton, M.: Isolation of a cDNA clone derived from a blood borne non-A, non-B, viral hepatitis genome. Science 244 (1989) 359-362. 2. Kuo, G.,Choo, Q. L, Alter, J.,Gitnick, G.,Redeker, A. G, Purcell, R. H°, Miyamura, T., Dienstag, J. L., Alter, M. J., Stevens, C. E., Tegtmeier, G. E., Bonino, F., Colombo, M., Lee, W-S, Kuo, C., Berger, K., Shuster, J. R.: An assay for circulating antibodies to a major etiological virus of human non-A, non-B hepatitis. Science 244 (t989) 362-364. 3. Alter, H. J, Purcell, R. H., Shill, J. W., Melpolder, J. C., Houghton, M., Choo, Q-L., Kuo G.: Detectionof antibody to hepatitis C virus in prospectively followed transfusion recipients with acute and chronic non-A, non-B hepatitis, N. Engl. J. Med. 321 (1989) 1494-1500. 4. Esteban, J. I., Esteban, R., Viladomio, L., Ltpez-Talavera, J. C., Gonzfilez, A., Hernfindez, J. M., Roger, M., Vargas, V., Genescfi, J., Buff, M., Guardia, J., Houghton, M., Choo, Q-L., Kuo, G.: Hepatitis C virus antibodies among risk groups in Spain. Lancet ii (1989) 294-297. 5. Huemer, H. P., Prodinger, W. M., Larcher, C., M6st, J., Dierich, M. P.: Correlationof hepatitis C virus antibodieswith HIV-1 seropositivity in intravenous drug addicts. Infection 18 (1990) 122. 6. Noel, L., Guerois, C., Maisonneuve, P., Verroust, F., Laurian, Y.: Antibodies to hepatitis C virus in haemophilia. Lancet ii (1989) 560. 7. Mortimer, P. P., Cohen, B.J., Litton, P. A., Vandervelde, E. M.,
transmitted. Anti-HCV antibodies have been detected in 5% of patients with other sexually transmitted diseases [ 14] and to a higher extent in homosexual men with HIV-1 infection (26%) than in those without (4%) [7]. In the present study, anti-HCV antibodies were found in 14% o f the HIV-1 seropositive but not in the HIV-1 seronegative homosexual men. Furthermore, anti-HCV antibodies were also found in 8% of the heterosexual persons with multiple sexual partners. Although it cannot be excluded that these persons had used intravenous drugs without mentioning it, the data are in line with the suggestion that H C V infection can be sexually transmitted [7, 14]. It should be emphasized, however, that the prevalence of anti-HBc antibodies was much higher than that o f anfi-HCV antibodies among the homosexual men suggesting that hepatitis B virus is more readily spread sexually than is HCV. Acknowledgement The assistance of Ms Ulla Magnusson at the Central Microbiological Laboratory of Stockholm County Council is gratefully acknowledged.
Bassendine, M. F., Brind, A. M., Hambling, M. H.: HepatitisC virus antibody. Lancet ii (1989) 798. 8. Weiner, A. J., Kuo, G, Bradly, D. W.: Detection of hepatitis C viral sequences in non-A, non-B hepatitis. Lancet ii (1990) 355 1-3. 9. Wideli, A., Hansson, B. G., Moestrup, T., Nordenfelt, E.: Increased occurrence of hepatitis A with cyclic outbreaks among drug addicts in a Swedish community. Infection 11 (1983) 198-200. 10. Mattson, L., Weiland, O., Glaumann, H.: Chronic non-A, non-B hepatitis developed after transfusions, illicit self injections or sporadically. Liver 9 (1989) 120-t27. 11. Bodsworth, N., Donovan, B., Nightingale, B.: The effect of concurrent human immunodeficiencyvirus infection on chronic hepatitis B: a study of 150 homosexual men. J. Infect. Dis. 160 (1989) 577-582. 12. Converse, P. J., Fehniger, T. E., Ehrnst, A., Stranneg~rd, ()., Britton, S.: Immune responses to fractionated cytomegalovirus (CMV) antigens after HIV infection: Loss of cellular and humoral reactivity to antigens recognized by HIV-, CMV+ individuals. Clin. Expfl. Immunol. (1990) in press. 13. Lange, J., Paul, D., Huisman, H., de Wolf, F., van den Berg, H., Coutinho, R., Danner, S., van der Noordaa, J., Goudsmit, J.: Persistent HIV antigenaemiaand decline of HIV core antibodies associated with transition to AIDS. Br. Meat.J. 293 (1986) 1459-1462. 14. Hess, G., Massing, A., Rossol, S., Schiitt, H., Clemens, R., Meyer zum Biischenfelde.: Hepatitis C virus and sexual transmission. Lancet ii (1989) 987.
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