Factors Associated with Serum HCV RNA Positivity in Anti-HCV Antibody Positive Intravenous Drug Users

J Clin Epidemiol Vol. 51, No. 5, pp. 423–427, 1998 Copyright  1998 Elsevier Science Inc. All rights reserved.

0895-4356/98/$19.00 PII S0895-4356(97)00305-3

Factors Associated with Serum HCV RNA Positivity in Anti-HCV Antibody Positive Intravenous Drug Users Mario Pirisi,1,* Pierluigi Toniutto,1 Carlo Fabris,1 Tiziana Lombardelli,2 Edmondo Falleti,1 Sergio G. Tisminetzky,3 Francisco Baralle,3 and Ettore Bartoli 1 1 Dipartimento di Patologia e Medicina Sperimentale e Clinica, Universita` degli Studi di Udine, Udine, Italy; 2Servizio per le tossicodipendenze, Udine, and 3Molecular Pathology, International Centre for Genetic Engineering and Biotechnology, Trieste, Italy

ABSTRACT. Serum hepatitis C virus (HCV) RNA, HCV genotypes and liver function tests were evaluated in a series of 189 unselected, consecutive anti-HCV positive intravenous drug users (IVDUs). Serum HCV RNA was detected in 106/189 patients. Abnormal liver function tests were associated with alcohol abuse, but not with the presence of serum HCV RNA. Among 109 patients retested after a mean follow-up of 21 months, 41 were intermittently serum HCV RNA positive. Patients persistently negative had more commonly a past history of acute hepatitis. A history of prostitution and/or a pattern of abuse involving .30 injections per week were related to infection by genotype 3a. In conclusion, serum HCV RNA is either transiently or persistently detectable in most anti-HCV positive IVDUs, but bears no association with abnormal liver biochemistry. Infection by HCV-3a is more common in IVDUs with more deviant life styles. In those cases where serum HCV RNA is found repeatedly negative, HCV infection may have been cleared, possibly through an episode of acute hepatitis. j clin epidemiol 51;5:423–427, 1998.  1998 Elsevier Science Inc. KEY WORDS. Hepatitis C virus, intravenous drug users, alcohol, life styles, liver disease, infection

INTRODUCTION Hepatitis C virus (HCV) infection is the cause of most cases of non-A, non-B hepatitis [1] and is estimated to lead to chronic liver damage in approximately 50% of patients [2]. Although the source of infection is often unknown, direct percutaneous exposure (such as transfusion of blood products, transplantation of organs or tissues from infected donors, and sharing of contaminated needles among drug users) is associated with the highest probability of transmission [3]. Intravenous drug users (IVDUs) are at risk of parenterally-acquired viral infections not only because of the practice of sharing needles but also for a life style which involves additional risks, particularly concerning their sexual behavior. The natural history of intravenous drug use is very variable, but it usually starts in adolescence with injections characterized by the sharing of needles and syringes, and ends when IVDUs are in their late 30s and 40s. The transmission of hepatitis occurs predominantly within the first few years [4]. Liver damage in HCV infected IVDUs may have a com*

Address for correspondence: Mario Pirisi, MD, Cattedra di Medicina Interna, Universita` degli Studi di Udine, I-33100 Udine, Italy. Accepted for publication on 8 December 1997.

plex pathogenesis. In fact, IVDUs often take other drugs and alcohol as a complement to narcotics. The reason why alcohol-dependent individuals have a high prevalence of anti-HCV antibodies might be related to the association between alcohol and injection-drug abuse [5]. In alcoholics with chronic hepatitis C, serum HCV RNA is reported to be increased, possibly due to impairment of cellular immunity [6]. On the other hand, those who are not viremic show liver histological features which are indistinguishable from those found in anti-HCV negative alcoholics [7]. The present study was aimed at investigating the factors associated with serum HCV RNA positivity in anti-HCV antibodies positive IVDUs. PATIENTS AND METHODS Study Population During a period of 4 months, all patients attending a clinic for the prevention and treatment of drug dependence were tested for serum anti-HCV antibodies by a third generation enzyme immunoassay (Ortho Diagnostics, Raritan, NJ). Positive test results were always confirmed by a confirmatory test (HCV Matrix, Abbott, Abbott Park, IL). A series of 189 IVDUs (142 male, 47 female, age 29.8 6 5.0, mean 6 SD, range 18–43) were enrolled. Personal consent to partic-

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Serum HCV RNA Determination and HCV Genotyping

TABLE 1. Behavioral characteristics of patients

Characteristic Injections #30 per week .30 per week History of acute hepatitis Yes No Sexual orientation Heterosexual Bisexual Homosexual Prostitution Customary Occasional Never Ethanol consumption #60 g/d .60 g/d History of conviction for criminal charges Yes No

n

%

151 38

80 20

58 131

31 69

182 5 2

96 3 1

16 22 151

8 12 80

138 51

73 27

71 118

38 62

ipate in the study, which was conducted in strict accordance to the principles of the Declaration of Helsinki, was obtained. The average duration of addiction was 9.8 6 5.6 years (range 1–25). All patients were maintained on methadone; however, 162/189 patients declared that they continued to use street drugs, at least occasionally. Although most patients attended the clinic for at least one follow-up examination, many failed to do so on a regular schedule. Detailed information on patients, including patterns of drug and alcohol abuse, sexual orientation, prostitution, and criminal records, were made available in a coded form. These data are summarized in Table 1.

HCV RNA was detected in serum by reverse transcriptasepolymerase chain reaction (RT-PCR) with nucleotide primers derived from the 5′ noncoding region of HCV. The amplified products were visualized by electrophoresis in 1.5% agarose gels and ethidium bromide staining. Both negative (deionized ultrapure water and serum from anti-HCV negative healthy blood donors) and positive controls were used in each run. The sensitivity of this assay was 1 3 10 3 genomes per ml. HCV genotyping was performed by dot blot hybridization of PCR products of HCV RNA with genotype-specific 32 P-labeled oligonucleotide probes. To distinguish subtype 1a from subtype 1b, PCR products were digested with BstU I endonuclease. This restriction enzyme recognizes two restriction sites within HCV-1b products and one in HCV-1a products, thus generating three fragments (of 137 bp, 44 bp, and 30 bp) in the presence of HCV-1b and two fragments (of 167 bp and 44 bp) in the presence of HCV-1a. The digestion products were visualized by electrophoresis on 12% acrylamide gels and ethidium bromide staining [8].

Statistical Analysis Statistical analysis of the data was performed by means of the BMDP/Dynamic statistical software package, release 7.0 (Statistical Software Ltd, Cork, Ireland). Shapiro and Wilk’s W-test was applied to test normality of data (BMDP program 2D). When the data showed a normal or nearly normal distribution, numerical variables were compared between groups by Student’s t-tests (two-tailed) (BMDP program 3D) or one-way analysis of variance (BMDP program 7D); Mann-Whitney rank-sum tests were used (BMDP program 3D) when the data departed significantly from the normal distribution. The associations among ordinal variables were analysed by Pearson’s chi-square tests (BMDP program 4F).

Sample Collection Blood samples were drawn at the beginning of the study for biochemical and virological testing. Serum samples, which had been separated from blood clot, aliquoted and frozen within 2 hours, were conserved at 280°C until use (approximately 12 months later) for HCV RNA and HCV genotype determinations. During a follow-up period which lasted an average of 21 months (range 1–36), 173 patients returned to the clinic at least once (median 4; range 1–14) for a clinical, biochemical, and serological evaluation. Of these latter patients, 109 returned to the clinic approximately 12 months after the first serum HCV RNA determination and underwent a second and final HCV RNA testing. During follow-up, 15 patients underwent percutaneous liver biopsy, which documented minimal hepatic changes in two patients, drug-induced liver disease in one, chronic hepatitis in 10, and cirrhosis in two.

RESULTS At entry into the study, 106/189 patients (56.1%) were found to be serum HCV RNA positive. Of all the demographic, clinical, or laboratory variables evaluated, the only significant difference observed between viremic and nonviremic patients was that past history of acute hepatitis was more frequent in non-viremic patients (Table 2). In contrast, exposure to hepatitis B virus (HBV) (testified by positivity for anti-HBc antibodies) was strongly associated with history of conviction (51/71 vs. 41/118, p , 0.0001), past history of acute hepatitis (46/58 vs. 46/131, p , 0.0001), and anti-HIV seropositivity (17/21 vs. 75/168, p , 0.005). In addition, the anti-HBc antibody positive patients were significantly older than the anti-HBc negative patients (32.3 6 4.2 vs. 27.3 6 4.4, p , 0.0001).

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TABLE 2. Comparison between HCV RNA positive and HCV RNA negative patients

HCV RNA Patient characteristic

Positive (n 5 106)

Negative (n 5 83)

P-value a

Female sex (n) Age (years) Injections, .30 per week (n) History of acute hepatitis (n) Sexual orientation, homo- or bisexual (n) Prostitution, customary or occasional (n) Ethanol consumption .60 g/d (n) History of convictions (n) Alanine aminotransferase .40 U/L (n) Bilirubin (µmol/L) Seropositivity for anti HIV-1 or -2 Seropositivity for HBcAb

28 29.7 6 5.1 23 24 3 20 30 38 51 10 (9–12) 10 51

19 29.8 6 4.8 15 34 4 18 21 33 40 10 (9–12) 11 41

NS NS NS ,0.01 NS NS NS NS NS NS NS NS

a For ordinal variables, P-values refer to Pearson’s chi-square tests, and, for numerical variables, to Student’s t-tests (data with normal distribution, reported as mean 6 SD) or Mann-Whitney rank-sum tests (data with non-normal distribution, reported as medians and 95% confidence limits of medians).

At entry into the study, the majority of patients had serum transaminases (either alanine aminotransferase or aspartate aminotransferase) below the normal limit (,40 U/L). During the follow-up period, 89/173 (51.4%) patients maintained transaminases within normal limits each time the test was repeated; 75/173 (43.4%) had abnormal transaminases occasionally; and 9 (5.2%) had abnormal transaminases on each retesting. Persistently abnormal values of serum transaminases were associated with a history of high ethanol consumption and a past history of acute hepatitis, but not with detectable serum HCV RNA (Table 3). One-hundred and nine patients returned to the clinic for repeat serum HCV RNA testing approximately 1 year after the first determination. In comparison to the original, larger

population studied, this smaller sample of patients did not differ for serum HCV RNA seropositivity rate (60/109, 55.0%) or for other demographic, clinical, virologic, or laboratory variables. Among these 109 patients, 10/60 who were HCV RNA positive were found on subsequent testing to be HCV RNA negative, whereas 31/49 who originally were HCV RNA negative were later found to be HCV RNA positive. Patients who were found to be HCV RNA negative at both tests had more commonly a past history of acute hepatitis (10/38 vs. 8/71, P , 0.05). HCV genotype, determined at entry in the study, was 1a in 29 patients (27%), 1b in 20 (19%), 2a in 22 (21%), 2b in 5 (5%), 3a in 27 (25%), and 4a in 3 (3%). A history of prostitution and/or a pattern of drug abuse involving .30 injections per week were related to infection by genotype

TABLE 3. Comparisons between patients according to the results of serum transaminases

on repeat testing Serum transaminases

Patient characteristic

Always normal (n 5 89)

Intermittently abnormal (n 5 75)

Always abnormal (n 5 9)

P-value

Female sex (n) Age (years) Injections, .30 per week (n) History of acute hepatitis (n) Sexual orientation, homo- or bisexual (n) Prostitution, customary (n) Ethanol consumption .60 g/d (n) History of conviction (n) Positivity of serum HCV RNA (n) Seropositivity for anti HIV-1 or -2 Seropositivity for HBcAb

24 29.9 6 4.9 17 20 3 17 22 36 52 5 44

18 29.5 6 4.8 16 29 4 17 22 24 41 13 36

3 30.7 6 4.7 2 5 0 2 6 5 5 1 5

NS NS NS ,0.05 NS NS ,0.05 NS NS ,0.10 NS

a For ordinal variables, P-values refer to Pearson’s chi-square tests (reported as frequencies) and, for numerical variables, to Student’s t-tests (reported as mean 6 SD).

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3a (15/37 vs. 12/69, Pearson chi-square 6.8, P , 0.01). Intermittently positive test results were more common in patients infected by HCV genotypes other than HCV-1a or HCV-1b (9/10 vs. 25/50, P , 0.05). DISCUSSION Approximately 75% of IVDUs are seropositive for antiHCV antibodies in epidemiologic surveys employing second generation enzyme immunoassays [9]. However, the impact of HCV infection on the morbidity and the mortality of IVDUs remains to be ascertained, since infection by hepatitis viruses is one of several medical complications of drug addiction, and usually not the most severe [4]. It should be noted that post-transfusional hepatitis C, which is associated with more aggressive histological inflammatory activity in comparison with hepatitis C in IVDUs [10], does not seem to result in excess long-term mortality [11,12]. Moreover, the alteration of liver function tests in anti-HCV positive IVDUs cannot be precisely attributed to HCV infection, as it might originate from infection by other viruses as well as from use of drugs or toxins. The present study confirms previous observations that anti-HCV positive IVDUs are often non-viremic [13], and reveals new data that in this population viremia can be intermittent and often not accompanied by biochemical signs of liver disease. In this respect, IVDUs can be considered similar to anti-HCV positive healthy blood donors, in whom PCR amplification of the 5′ untranslated region shows a serum HCV RNA positivity rate close to 70%, often despite normal liver biochemistry [14]. In contrast, antiHCV positive patients with chronic liver disease, who by definition have abnormal liver biochemistry, have detectable serum HCV RNA in more than 90% of cases [15]. It can be hypothesized that these differences might be related to a lower prevalence of infection by more pathogenic viral variants in IVDUs as compared with patients with chronic liver disease. Indeed, HCV types which are not usually found in the general population are over-represented in IVDUs. In particular, there is a high frequency of HCV-3a, which in Europe might be spreading through young subjects from a reservoir constituted by IVDUs [16,17], and which has been considered to be less pathogenic [18]. According to the findings in the present study, infection by HCV-3a is associated in IVDUs with a more deviant behavior, that is, a pattern of drug use involving a larger number of injections per week and greater sexual promiscuity. However, we were not able to demonstrate any association between infection by specific HCV types and abnormal liver biochemistry (data not shown), as was instead the case for ethanol abuse. HCV carriers with morphological evidence of liver disease but normal liver biochemistry have been reported to have higher HCV RNA titers than HCV carriers with normal liver histology [19]. Moreover, HCV RNA titer in-

creases with the progression of histopathological changes of the liver and becomes exponentially greater with prolongation of the interval from infection [20–22]. Thus, the normality of liver biochemistry in HCV RNA positive IVDUs may represent a mere reflection of low viral titer in patients with a short-dated HCV infection. Indeed, anti-HCV positive IVDUs and health care workers have lower HCV RNA titers than patients who acquired HCV infection by blood transfusion [23]. In the present study, the patients for whom liver biopsy was indicated and subsequently performed was too low to allow definite conclusions. In the present study only anti-HCV positive IVDUs were studied. Therefore, it was not possible to compare clinical and demographic factors associated with HBV infection as opposed to HCV infection. However, the strong association found between anti-HBc antibodies and history of conviction is consistent with the fact that HBV can be found in body fluids, such as semen and saliva, at higher titers in comparison to HCV; hence, it is more easily transmitted sexually and by close personal contact than HCV [24]. Interestingly, a past history of acute hepatitis (presumably hepatitis B) was more frequently elicited in patients with persistently negative results of HCV RNA testing. It might have been that concomitant HBV and HCV infection resulted in higher rates of clearance of HCV from blood. In fact, in other studies, patients with concurrent HBV infection have been shown to have lower serum HCV RNA titers [25]. In conclusion, the results of serum HCV RNA determination in anti-HCV positive IVDUs need to be interpreted with caution. Serum HCV RNA can be transiently detected. There are patients who seem to have recovered from HCV infection, possibly through an acute hepatitis. Other patients, formerly HCV RNA positive, are found negative on subsequent testing; more commonly, these intermittently positive results will be found in those infected by a ‘‘favorable’’ genotype. On the other hand, since those who were initially HCV RNA negative are later found positive, it appears that in this population an extended follow-up is required before considering a patient healed by HCV infection. The authors are grateful to Dr. Rodolfo Garcia and Dr. Virginia Canale for useful suggestions and careful reading of the manuscript.

References 1. Choo QL, Kuo G, Weiner AJ, Overby LR, Bradley DW, Houghton M. Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome. Science 1989; 244: 359–362. 2. Rakela J, Redeker AG. Chronic liver disease after acute and chronic non-A, non-B viral hepatitis. Gastroenterology 1979; 77: 1200–1202. 3. Alter MJ. Epidemiology of hepatitis C in the West. Semin Liver Dis 1995; 15: 5–14. 4. Cherubin CE, Sapira JD. The medical complications of drug

Serum HCV RNA in Intravenous Drug Users

5.

6. 7.

8.

9. 10.

11.

12. 13. 14.

15.

addiction and the medical assessment of the intravenous drug user: 25 years later. Ann Intern Med 1993; 119: 1017–1028. Verbaan H, Andersson K, Eriksson S. Intravenous drug abuse—the major route of hepatitis C virus transmission among alcohol-dependent individuals? Scand J Gastroenterol 1993; 28: 714–718. Oshita M, Hayashi N, Kasahara A, et al. Increased serum hepatitis C virus RNA levels among alcoholic patients with chronic hepatitis C. Hepatology 1994; 20: 1115–1120. Fong T-L, Kanel GC, Conrad A, Valinluck B, Charbonfau F, Adkins RH. Clinical significance of concomitant hepatitis C infection in patients with alcoholic liver disease. Hepatology 1994; 19: 554–557. Toniutto P, Pirisi M, Tisminetzky SG, et al. Discordant results from hepatitis C virus genotyping by procedures based on amplification of different genomic regions. J Clin Microbiol 1996; 34: 2382–2385. McHutchison JG, Person JL, Govindarajan S, et al. Improved detection of hepatitis C virus antibodies in high-risk populations. Hepatology 1992; 15: 19–25. Gordon SC, Elloway RS, Long JC, Dmuchowski CF. The pathology of hepatitis C as a function of mode of transmission: Blood transfusion vs. intravenous drug use. Hepatology 1993; 18: 1338–1343. Seef LB, Buskell-Bales Z, Wright EC, Durako SJ, Alter HJ, Iber FL. Long-term mortality after transfusion-associated Non-A, Non-B hepatitis. N Engl J Med 1992; 327: 1906– 1911. Koretz RL, Abbey H, Coleman E, Gitnick G. Non-A, Non-B post-transfusion hepatitis. Looking back in the second decade. Ann Intern Med 1993; 119: 110–115. Simmonds P, Zhang LQ, Watson HG, et al. Hepatitis C quantification and sequencing in blood products, haemophiliacs, and drug users. Lancet 1990; 336: 1469–1472. McGuinness PH, Bishop GA, Lien A, Wiley B, Parsons C, McCaughan GW. Detection of serum hepatitis C virus RNA in HCV antibody-seropositive volunteer blood donors. Hepatology 1993; 18: 485–490. Marin MG, Bresciani S, Puoti M, et al. Clinical significance

427

16.

17. 18. 19.

20.

21.

22.

23. 24. 25.

of serum hepatitis C virus (HCV) RNA as a marker of HCV infection. J Clin Microbiol 1994; 32: 3008–3012. Pawlotsky J-M, Tsakiris L, Roudot-Thoraval F, et al. Relationship between hepatitis C virus genotypes and sources of infection in patients with chronic hepatitis C. J Infect Dis 1995; 171: 1607–1610. Silini E, Bono F, Cividini A, et al. Molecular epidemiology of hepatitis C virus infection among intravenous drug users. J Hepatol 1995; 22: 691–695. Silini E, Bono F, Cividini A, et al. Differential distribution of hepatitis C virus genotypes in patients with and without liver function abnormalities. Hepatology 1995; 21: 285–290. Naito M, Hayashi N, Hagiwara H, et al. Serum hepatitis C virus RNA quantity and histological features of hepatitis C virus carriers with persistently normal ALT levels. Hepatology 1994; 19: 871–875. Kato N, Yokosuka O, Hosoda K, Ito Y, Ohto M, Omata M. Quantification of hepatitis C virus by competitive reverse transcription-polymerase chain reaction: Increase of the virus in advanced liver disease. Hepatology 1993; 18: 16–20. Gretch D, Corey L, Wilson J, et al. Assessment of hepatitis C virus RNA levels by quantitative competitive RNA polymerase chain reaction: High-titer viremia correlates with advanced stage of disease. J Infect Dis 1994; 169: 1219–1225. Yousuf M, Nakano Y, Tanaka E, Sodeyama T, Kiyosawa K. Persistence of viremia in patients with type-C chronic hepatitis during long-term follow-up. Scand J Gastroenterol 1992; 27: 812–816. Lau JYN, Davis GL, Kniffen J, et al. Significance of serum hepatitis C virus RNA levels in chronic hepatitis C. Lancet 1993; 341: 1501–1504. Tor J, Llibre JM, Carbonell M, et al. Sexual transmission of hepatitis C virus and its relationship with hepatitis B virus and HIV. BMJ 1990; 301: 1130–1133. Franc¸ois M, Roingeard P, Dubois F, Bacq Y, Goudeau A, Barin F. Lower serum hepatitis C virus RNA titers in patients with concurrent chronic hepatitis B virus infection. J Infect Dis 1994; 169: 1411–1412.