Hepatitis B (HBV) and C (HCV) virus coinfections are an important cause of morbidity and mortality in HIV-infected patients. A recent nation-wide survey performed in Spain showed that 61% of all HIV-infected patients were coinfected with HCV and 4.8% were coinfected with HBV . While the epidemiology, natural history and treatment strategies for HBV and HCV coinfections in HIV-infected patients have been extensively studied, much less is known about multiple hepatitis viruses infections.
In triple hepatitis occurring in HIV uninfected individuals, hepatitis D virus (HDV) appears to be the dominant virus because markers of both HCV and HBV replication are inhibited [2,3]. The inhibitory effect of HDV upon HBV replication has been related to the fact that the large delta antigen inhibits the host DNA-dependent RNA polymerase II which is involved in HBV replication [2,4].
In HIV-infected patients the inhibitory effect of HDV upon HBV and HCV has not been studied in detail. In recent recommendations about the care of patients with chronic hepatitis B and HIV infection  the authors state, based on two studies performed before the highly active antiretroviral therapy (HAART) era, that in patients with triple hepatitis high serum HDV RNA titers and delta antigen are often seen in the bloodstream along with markers of HBV or HCV replication. However, we are not aware of any study which have confirmed these results after the advent of HAART.
In order to estimate the prevalence of multiple hepatitis and to study the phenomenon of viral interference in HIV-infected patients with hepatitis BCD we have performed a cross-sectional study.
Patients and methods
During a 5-month-period in the year 2003 we selected all HIV-infected patients seen in the La Paz Hospital HIV clinic who were serum anti-HCV and/or HBsAg positive. Our clinic is located in a university-based public hospital and provides comprehensive medical care to HIV-infected adults. For each patient we reviewed demographics, risk factors for HIV infection, HAART received, current or prior use of tenofovir or lamivudine, current CD4 cell count, current serum HIV-1 RNA and liver function tests. The local Ethics Committee approved the study and all patients gave written informed consent.
Hepatitis was classified as follows: HB, positive serum HBsAg and negative serum HCV and HDV antibodies; HC, positive HCV antibodies and negative HBsAg; HBC, positive HBsAg, positive HCV antibodies and negative HDV total antibodies; HBD, positive HBsAg, positive total HDV antibodies and negative HCV antibodies; HBCD, positive HBsAg, positive HCV antibodies and positive total HDV antibodies. Delta hepatitis was further classified as chronic/replicative if HDV IgM was persistently positive. If HDV total antibodies were positive but HDV IgM was negative HD was subclassified as past/non-replicative. Serum HCV PCR was performed before interferon therapy and was confirmed in two separate visits.
Serologic tests were performed using commercially available products for HBsAg, HBeAg, anti-HBeAg (Enzygnost HBsAg 5.0, Dade Behring, Marburg, Germany), HDV Ag, total HDV antibodies, HDV IgM (DIA.PRO, Milan Italy), and HCV antibodies (Hepatitis C Virus Encoded Antigen Ortho HCV 3.0 ELISA test system with enhanced save. Ortho-Clinical Diagnosis, Rochester, New York, USA). HBV DNA was detected in serum using HBV DNA Assay (Hybrid capture, Abbott, Chicago, Illinois, USA) and by PCR with the HBV DNA COBAS amplicor HBV monitor test (Roche Molecular Systems, Pleasanton, California, USA) with a detection limit of 200 copies/ml. HCV RNA was detected in serum using COBAS Amplicor HCV Monitor Test, Version 2.0 with a detection limit of 600 copies/ml (Roche Diagnostic Systems, Branchburg, New Jersey, USA).
Cirrhosis was defined by one or more of the following: a liver biopsy with grade 4 fibrosis, clinical signs or symptoms of liver failure (ascitis, encephalopathy, bleeding), biochemical data of liver failure (albumin < 3.5 g/dl, prothrombin > 14 s) or signs of portal hypertension and cirrhosis on ultrasound.
Fisher's two-tailed exact test and the chi-square test were used to compare qualitative data. Non-parametric tests (Wilcoxon, Mann–Whitney) were used to compare quantitative variables. Significance was set at P < 0.05.
Of the 423 HIV-infected patients studied, 403 [95.3%; 95% confidence interval (CI), 93.2–97.3%] had single hepatitis (16 B, 387 C), and 20 (4.7%; 95% CI, 2.7–6.7%) had multiple hepatitis (Table 1). Of the 20 patients with multiple hepatitis, five (25%) had dual hepatitis (four BC and one BD) and 15 (75%) had triple hepatitis (BCD). Of the 15 patients with hepatitis BCD, 10 had past/non-replicative hepatitis D (positive total anti-HD, negative HD IgM) and five had chronic/replicative hepatitis D (repeatedly positive HD IgM). All 15 patients with positive total anti-HDV antibodies were serum hepatitis D antigen negative.
All patients with multiple hepatitis had acquired HIV through injecting drug use (Table 1). All patients with single hepatitis B had acquired HIV through unprotected sexual contact. When we compared patients with single and multiple hepatitis there were non-statistically significant differences with regard to demographics, CD4 cell count, serum HIV RNA or proportion of patients receiving HAART. Patients with multiple hepatitis had a significantly higher proportion of cirrhosis than patients with hepatitis B or hepatitis C (40% versus 9.7%; P = 0.00002).
Laboratory characteristics are shown in Table 2. Among patients infected with HCV, patients with multiple hepatitis were significantly more likely to have a negative serum HCV PCR. Among patients infected with HBV, patients with multiple hepatitis were significantly more likely to have a negative serum HBeAg and a positive anti-HBeAg.
Among patients with positive serum HBsAg, the percentage of patients receiving a lamivudine or a tenofovir-containing regimen at the time of inclusion in the study were 62% (HB), 75% (HBC), and 46.7% (HBCD). Of the 15 patients with HBCD, seven (four with replicative HD, three with non-replicative HD) had not received lamivudine or tenofovir before serum HBV DNA determination. These seven patients were all serum HBV DNA negative by hybridization. By PCR, four were also negative (three with replicative and one with non replicative HD), one had 4600 copies/ml (patient with replicative HD) one had 27 000 copies/ml (patient with non-replicative HD) and one had 44 000 copies/ml (patient with non-replicative HD).
In our geographical area multiple hepatitis is not rare event in HIV-infected patients. More than half of the HBV chronic infections seen in our series occurred in the context of multiple hepatitis. Chronic HB occurred always as a single hepatitis in patients who acquired HIV through unprotected sexual contact and as part of multiple hepatitis (HBC or, more commonly, HBCD) in patients who acquired HIV through injecting drug use. This finding has important implications in terms of the natural history and treatment of chronic HB in HIV-infected patients. Although significant advances have been made in the treatment of HBV-HIV coinfection, little is known about optimal treatment of patients with HBC or HBCD.
Patients with HBC were four times more likely to have positive serum markers of HD. In a recent study performed in Spain, serologic markers showed that more than 50% of HIV infected patients with a positive serum HBsAg have been exposed to HD . This high prevalence of HD in HIV-infected patients with multiple hepatitis is somewhat unexpected. Although HD is endemic in the Mediterranean area, a number of reports have suggested a significant decrease over the past 20 years in the prevalence of HD in the general population . One study performed in1988 in Spain communicated a prevalence of 67% of positive serum HD markers among patients with HB and a history of injecting drug use . This prevalence is not very different from the prevalence found in our study (79%) suggesting that injecting drug users infected with HIV continue to be a reservoir of HD .
It has been previously shown that in HIV-negative patients coinfected with HBC, the replication of both HBV and HCV is decreased [2,3]. In our study we have only four patients with HBC without serum markers of HDV, consequently it is difficult to speculate about the impact of HBV replication (without HDV) upon HCV replication. Nevertheless, three of the four patients with HBC were HCV serum PCR negative suggesting that HBV plays a dominant role. Similarly, in a recent study Martin-Carbonero et al.  communicated that undetectable serum HCV RNA was significantly more frequent in HBsAg positive than in HBsAg negative HIV-infected patients with HC. However, since HDV tests results were not communicated in this study it is difficult to conclude if HBV, HDV or both caused the inhibitory effect.
As it has been repeatedly reported in HIV uninfected individuals in Europe [2,3], in our study HDV virus seemed to potently inhibit both HBV and HCV replication in HIV-infected patients with HBCD. All patients with chronic/replicative HD were serum HBeAg negative, anti-HBe Ag positive and serum HCV PCR negative. In addition all patients with chronic/replicative HD who were not receiving lamivudine at the time of serum HBV DNA determination were HBV DNA negative by hybridization and negative or with very low viremia by the more sensitive HBV DNA PCR test.
Our study confirms a prior report by Eyster and colleagues in which only one patient out of five with replicative HD (all hemophiliacs also coinfected with HCV) was serum HBV DNA positive by hybridization . Our results apparently contradict those of Pol and colleagues  in which HIV-infected patients with active HD were frequently serum HDAg positive and did not appear to have inhibition of HBV replication. Remarkably, in Pol's study serum HDAg was detectable only if patients had acquired HIV and HDV through unprotected homosexual contact, but not if patients, as it happened in our study, acquired these infections by the intravenous route. In addition, in Pol's study only 27% of patients were coinfected with HCV. These discrepancies suggest that route of transmission, timing and sequence of each hepatitis virus infection, presence of HCV coinfection, or even differences in HDV genotypes  might influence reciprocal interactions between HBV and HDV in HIV infected patients.
In a cross-sectional study such as ours it is impossible to demonstrate that superinfection with HDV inhibits prior active HBV and HCV replication. Eyster and colleagues  also reported suppression of HCV replication in HIV-infected haemophiliacs with HBCD. Interestingly, two patients showed inhibition of active HCV replication after being infected with HDV . The mechanism behind the inhibitory effect of HDV upon HCV replication has not been elucidated. Animal models of multiple hepatitis could offer an opportunity to investigate in a controlled way the issue of viral interference among hepatotropic viruses.
Patients with multiple hepatitis had a higher prevalence of cirrhosis than patients with single hepatitis. It is known that patients with multiple hepatitis have a worse prognosis than patients with single HB or HC . Optimal treatment for HBC or HBCD has not been established. Liver transplant might be an option for patients with HBCD and end-stage liver disease because, probably due to the viral interference phenomenon, the risk of recurrence of HB and HC in the transplanted liver appears to be decreased .
We thank Dr. Rafael Delgado for his helpful suggestions.
Sponsorship: Supported by Estudio GESIDA 29/02–FIPSE 12185/01 and FIPSE 36465/03.
1. Gonzalez-Garcia JJ, Mahillo B, Hernandez S, Pacheco R, Diz S, Garcia P, et al
. Prevalences of hepatitis virus coinfection and indications for chronic hepatitis C virus treatment and liver transplantation in Spanish HIV-infected patients. The GESIDA 29/02 and FIPSE 12185/01 Multicenter Study. Enferm Infecc Microbiol Clin 2005; 23:340–348.
2. Jardi R, Rodriguez F, Buti M, Costa X, Cotrina M, Galimany R, et al
. Role of hepatitis B, C, and D viruses in dual and triple infection: influence of viral genotypes and hepatitis B precore and basal core promoter mutations on viral replicative interference. Hepatology 2001; 34:404–410.
3. Mathurin P, Thibault V, Kadidja K, Ganne-Carrie N, Moussalli J, El Younsi M, et al
. Replication status and histological features of patients with triple (B, C, D) and dual (B, C) hepatic infections. J Viral Hepat 2000; 1:15–22.
4. Modahl LE, Lai MM. The large delta antigen of hepatitis delta virus potently inhibits genomic but not antigenomic RNA synthesis: a mechanism enabling initiation of viral replication. J Virol 2000; 74:7375–7380.
5. Soriano V, Puoti M, Bonacini M, Brook G, Cargnel A, Rockstroh J, et al
. Care of patients with chronic hepatitis B and HIV co-infection: recommendations from an HIV-HBV International Panel. AIDS 2005; 19:221–240.
6. Gaeta GB, Stroffolini T, Chiaramonte M, Ascione T, Stornaiuolo G, Lobello S, et al
. Chronic hepatitis D: a vanishing Disease? An Italian multicenter study. Hepatology 2000; 32:824–827.
7. Buti M, Esteban R, Jardi R, Allende H, Baselga JM, Guardia J. Epidemiology of delta infection in Spain. J Med Virol 1988; 26:327–332.
8. Navascues CA, Rodriguez M, Sotorrio NG, Sala P, Linares A, Suarez A, et al
. Epidemiology of hepatitis D virus infection: changes in the last 14 years. Am J Gastroenterol 1995; 90:1981–1984.
9. Martin-Carbonero L, Barreiro P, Jimenez R, et al
. Chronic hepatitis B may increase HCV clearance in HIV-HCV co-infected patients. 44th Interscience Conference of Antimicrobial Agents and Chemotherapy
. Washington DC, October-November 2004 [abstract V-1153].
10. Eyster ME, Sanders JC, Battegay M, Di Bisceglie AM. Suppression of hepatitis C virus (HCV) replication by hepatitis D virus (HDV) in HIV-infected hemophiliacs with chronic hepatitis B and C. Dig Dis Sci 1995; 40:1583–1588.
11. Pol S, Wesenfelder L, Dubois F, Roingeard P, Carnot F, Driss F, et al
. Influence of human immunodeficiency virus infection on hepatitis delta virus superinfection in chronic HBsAg carriers. J Viral Hepat 1994; 1:131–137.
12. Fong TL, Di Bisceglie AM, Waggoner JG, Banks SM, Hoofnagle JH. The significance of antibody to hepatitis C virus in patients with chronic hepatitis B. Hepatology 1991; 14:64–67.
13. Taniguchi M, Shakil AO, Vargas HE, Laskus T, Demetris AJ, Gayowski T, et al
. Clinical and virologic outcomes of hepatitis B and C viral coinfection after liver transplantation: effect of viral hepatitis D. Liver Transpl 2000; 6:92–96.