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Impact of HAART) on the Natural History of Hepatitis B Virus (HBV) and HIV Coinfection: Relationship between Prolonged Efficacy of HAART and HBV Surface and Early Antigen Seroconversion
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Clinical Infectious Diseases Sept 1, 2007
Patrick Miailhes,1,3,4 Mary-Anne Trabaud,2,3 Pierre Pradat,1,3,4 Bertrand Lebouche,1,3,4 Michele Chevallier,5 Philippe Chevallier,2,3 Fabien Zoulim,1,3,4 and Christian Trepo1,3,4
1Hotel-Dieu, Service d'Hepatologie et de Gastroenterologie, and 2Hopital de la Croix-Rousse, Laboratoire de Virologie, Hospices Civils de Lyon, 3INSERM U871, 4Universite Lyon 1, and 5Laboratoire Marcel Merieux, Lyon, France
(See the article by Gaglio et al. on pages 618-23 and the editorial commentary by Tillman on pages 633-6)
ABSTRACT
Background. Coinfection with hepatitis B virus (HBV) in human immunodeficiency virus (HIV)-infected patients is common. However, little is known about the natural history of chronic hepatitis B in HIV-infected populations, especially the impact of highly active antiretroviral therapy (HAART) on the outcome of HBV early antigen (HBeAg) and HBV surface antigen (HBsAg) status.
Methods. The characteristics of 92 patients coinfected with HIV and HBV were retrospectively assessed before and after HAART and lamivudine treatment to determine the impact of treatment on chronic hepatitis B and factors associated with HBeAg and/or HBsAg seroconversion.
Results. During follow-up, 82 patients received antiretroviral therapy, 79 of whom received HAART. Twenty-eight of the 76 patients who were administered lamivudine therapy developed lamivudine resistance mutations. While receiving antiretroviral therapy, 10 of 59 HBeAg-positive patients developed antibody to HBeAg, 3 of 10 cleared HBsAg, and 2 of 3 developed antibody to HBsAg. Two of 23 HBeAg-negative patients cleared HBsAg and developed antibody to HBsAg. HBeAg and/or HBsAg seroconversion combined with an undetectable HBV DNA level (i.e., an HBV response) correlated with a sustained HIV response (P = .001), shorter duration of antiretroviral therapy (P = .058), and more-severe disease, as evaluated by Centers for Disease Control and Prevention staging (for stage B vs. stage A, P = .029; for stage C vs. stage A, P = .069). For patients with elevated baseline alanine aminotransferase levels, the HBV response correlated significantly with a greater increase in CD4 cell count while receiving HAART.
Conclusions. In HIV-HBV-coinfected patients, HBV response correlated with a sustained HIV response to antiretroviral therapy, usually HAART including lamivudine.
Coinfection with hepatitis B virus (HBV) and HIV is frequent, because both viruses have the same routes of transmission. In western Europe and the United States, chronic HBV infection has been found in 7%-10% of HIV-infected patients, with a higher prevalence in men who have sex with men [1-3]. Before the HAART era, Ockenga et al. [3] revealed that, in HIV-infected patients, coinfection with either HBV or hepatitis C virus (HCV) correlated with a reduced survival rate. As HAART improved the survival of HIV-infected patients, it became obvious that HIV worsened the outcome of chronic hepatitis B and increased the risk of liver-associated morbidity and mortality [4-7]. Therefore, the aim of our study was to describe the natural history of chronic hepatitis B in HIV-infected patients and to assess the impact of HAART on chronic hepatitis B.
PATIENTS AND METHODS
Study population. All patients coinfected with HIV and HBV who were referred to our viral hepatitis and HIV infection reference center (Department of Hepatogastroenterology, Hotel-Dieu Hospital, Lyon, France) from January 1990 through December 2002 and were followed-up for at least 6 months were retrospectively assessed. Inclusion criteria were positive serologic examination results for HIV and seropositivity for hepatitis B surface antigen (HBsAg). Patients entered the study before HAART and lamivudine treatment was initiated. The following baseline (defined as entrance in the study) information was collected: age, sex, ethnicity, HIV risk factors, HIV-related illnesses, classification according to the Centers for Disease Control and Prevention (CDC) 1993 guidelines [8], HIV RNA and HBV DNA levels, CD4 cell count, alanine aminotransferase (ALT) level, hepatitis early antigen (HBeAg) status, HBV genotype, and duration of follow-up for HIV and HBV infections. The impact of HAART, especially of lamivudine therapy, on the evolution of HBeAg and HBsAg status according to the initial HBeAg profile was assessed, as were other factors associated with HBeAg and HBsAg seroconversion. Because the long-term follow-up involved changes in antiretroviral therapy regimens, a sustained HIV response was defined as an undetectable HIV RNA level (<50 copies/mL) during at least 80% of the duration of antiretroviral therapy. An HBV response was defined as HBeAg and/or HBsAg seroconversion combined with an undetectable HBV DNA level (<100 copies/mL). During follow-up, HIV and HBV biological and virological tests were performed every 3-4 months for most patients.
Serological methods. HBV serological tests included the detection of HBsAg (Monolisa Ag HBs PLUS kit; Bio-Rad), antibody to HBsAg (anti-HBs) and antibody to HBV core antigen (Architect kits; Abbott), and HBeAg or antibody to HBeAg (anti-HBe; AxSYM HBe 2.0 and anti-HBe 2.0 kits; Abbott). Antibodies to HCV were tested using the Monolisa anti-HCV PLUS 2.0 kit (Bio-Rad). When the results were positive, a second test was performed using the Ortho HCV 3.0 ELISA kit (Ortho-Clinical Diagnostics). Total antibodies to hepatitis D virus were detected using the Eti-AB-Deltak-2 ELISA kit (DiaSorin). For most patients, 2 commercial ELISA kits were used for HIV antibody detection: Genscreen HIV 1/2 (Bio-Rad) and Vironostika HIV Uni-Form II (Organon). However, because HIV infection was diagnosed over a 15-year period, other commercial assays may also have been used. The NewLavBlot kit (Bio-Rad) was used to confirm HIV seropositivity.
Genomic quantification. During follow-up, for routine genomic quantification, branched DNA technology was used to quantify HIV RNA levels (Versant HIV RNA 3.0 kit; Bayer; detection limit, 50 copies/mL) and HBV DNA levels (Versant Quantiplex HBV DNA 1.0; Bayer; detection limit, 700,000 copies/mL) according to the manufacturer's instructions. When HBV DNA levels were below the threshold of the Versant kit, real-time PCR (detection limit, 100 copies/mL) was performed on frozen serum samples as described elsewhere, using primers in the C gene region [9].
HBV genotyping. HBV genotype and resistance to lamivudine were retrospectively determined from frozen serum samples using commercial line probe assays (INNO-LiPA HBV genotyping kit and INNO-LiPA HBV resistance; Innogenetics) according to the manufacturer's instructions. Discrimination between the genotypes or resistance mutations was made possible by hybridization with specific oligonucleotide sequences immobilized on membrane-based strips after amplification in the HBV B-C domains of the pol gene [10]. The presence of polymerase mutations at codons 173, 180, and 204, associated with lamivudine resistance, was explored when HBV load was detectable (>700,000 copies/mL) after a period of undetectability during treatment with lamivudine. This was performed using the first serum sample with a detectable HBV load or, if unavailable, the following sample before a second anti-HBV drug was added to the treatment regimen or lamivudine therapy was discontinued.
Statistical analysis. Statistical analysis was performed using SPSS, version 11.5.1 for Windows (SPSS). Dichotomous variables were compared using Fisher's exact test, and other categorical variables were compared using the Pearson X2 test. For continuous variables, Student's t test was used (with correction if variances were not equal), and the Mann-Whitney U test was used when the number of cases was small. Multivariate Cox regression analysis, with data censored at the time of HBeAg and/or HBsAg seroconversion, was conducted to check for factors that might be related to the HBV response. ORs and 95% CIs were calculated. P < .05 was considered to be statistically significant.
RESULTS
Baseline characteristics of patients. A total of 92 patients coinfected with HIV and HBV were evaluated (table 1). There was no difference between the median baseline HBV DNA level, irrespective of whether the patient's CD4 cell count was <200 cells/uL or >200 cells/uL (8.9 log10 copies/mL vs. 8.4 log10 copies/mL; P = .80). ALT levels were normal in 57 patients (62%) but elevated in 35 (38%). HBV genotyping was performed for 80 patients (87%) (table 1). Most white patients had genotype A or D, and sub-Saharan Africans had equally genotype A or E.
At baseline, 1 patient had isolated antibody to HBV core antigen and then experienced HBV reactivation, with the reappearance of HBsAg and HBeAg. Among the 91 HBsAg-positive patients, 61 (67%) were also HBeAg-positive, 29 (32%) were anti-HBe positive, and 1 was repeatedly negative for both HBeAg and anti-HBe. Anti-HBe status was linked to African origin (19 [90%] of 21 patients), and HBeAg-positive status was linked to white race (61 [87%] of 70 patients). HBeAg status was not correlated with CDC stage: 67% of patients with CDC stage A or B HIV infection were HBeAg positive versus 71% of patients with CDC stage C HIV infection (P = .80). The median serum HBV DNA level was significantly higher in HBeAg-positive patients than in anti-HBe-positive patients (8.98 log10 copies/mL vs. 4.27 log10 copies/mL; P < .001). An elevated ALT level also correlated with an HBeAg-positive status: 30 (49%) of 61 HBeAg-positive patients had elevated ALT levels versus 4 (14%) of 29 anti-HBe-positive patients (P = .002). The median (±SE) ALT levels were 89 ± 14 IU/L and 44 ± 10 IU/L, respectively, in these 2 groups (P < .001). At baseline, only 10 patients (11%) were receiving antiretroviral therapy, and the remaining 82 (89%) had not previously received treatment (figure 1).
HBV disease classification. At baseline, before HAART was initiated, 90 of 91 patients who were HBsAg positive were classified into 3 groups according to chronic hepatitis B disease status. Thirty-four patients (37%) had chronic active hepatitis B (CAHB), which was characterized by an ALT level greater than the upper limit of normal and an HBV DNA level >5 log10 copies/mL. Forty-one patients (45%) were immunotolerant, with repeatedly normal ALT levels and HBV DNA levels >5 log10 copies/mL. Finally, 16 patients (18%) were considered to be inactive HBsAg carriers (i.e., inactive carriers), with both repeatedly normal ALT levels and HBV DNA levels <5 log10 copies/mL. The median baseline CD4 cell counts did not differ among these 3 groups. One patient could not be classified, because the patient had chronic hepatitis B with prolonged HBsAg antigenemia (>1 year) combined with elevated ALT levels and undetectable HBV DNA levels (<100 copies/mL).
Treatment and outcome during follow-up. By December 2002 (the end of the study period), the median duration of follow-up was 5 years (range, 0.5-13.3 years). Four patients in the cohort died during follow-up; 2 died because of uncontrolled HIV infection with an AIDS event (1 had severe pneumocystosis, and the other developed HIV encephalopathy), and the other 2 died of end-stage liver disease. During follow-up, 82 patients (89%) received antiretroviral therapy, of whom 79 received HAART and 76 received lamivudine therapy (figure 1 and table 2). Twenty-eight (37%) of the 76 patients who received lamivudine therapy developed lamivudine resistance mutations (table 3), correlating significantly with longer duration of lamivudine therapy (median, 62 months for patients with the mutation vs. 24 months for patients without the mutation; P < .001), higher baseline HBV DNA level (median, 9.06 log10 copies/mL vs. 7.80 log10 copies/mL; P = .001), and higher baseline CD4 cell count (median, 398 cells/L vs. 235 cells/L; P = .019). Lamivudine resistance was seen only in patients with CAHB and immunotolerance. The median duration of lamivudine treatment was significantly shorter in the 10 treated inactive carriers than in those with CAHB or immunotolerance (18 months vs. 45 months; P = .003). One-half of the patients in the inactive carrier group had an HBV DNA level <3 log copies/mL. Although the others had higher baseline HBV DNA levels (up to 4.50 log copies/mL), no lamivudine resistance was observed. Lamivudine was the only anti-HBV drug used for most of our patients and was the first antiviral administered for 95% of them. Only 3 patients initiated treatment with a combination of lamivudine and tenofovir, for a median period of 4 months (range, 2-7 months). Thirteen others received tenofovir as a component of an HIV treatment regimen that was administered for a median of 6 months (range, 1-20 months). Eight (62%) of these 13 patients also had lamivudine resistance. Finally, only 2 subjects received adefovir therapy, for 12 and 13 months, because of the emergence of lamivudine resistance.
Characteristics of patients who experienced HBeAg and/or HBsAg seroconversion. Of the 82 patients receiving antiretroviral therapy, 10 (17%) of the 59 HBeAg-positive subjects acquired anti-HBe. Three of these 10 patients cleared HBsAg, and 2 of them acquired anti-HBs. Two (9%) of the 23 HBeAg-negative patients cleared HBsAg, and both seroconverted to anti-HBs. At the time of HBeAg and/or HBsAg seroconversion, all patients were receiving antiretroviral therapy. Ten were receiving HAART including lamivudine, 1 was receiving HAART without any anti-HBV drug, and 1 was receiving zidovudine and didanosine combination therapy (table 4). HBsAg or HBeAg seroconversion was neither observed during tenofovir or adefovir therapy nor in patients who did not receive treatment for HIV infection. Overall, HBeAg or HBsAg seroconversion combined with an undetectable HBV DNA level (i.e., an HBV response) was achieved in only 12 patients.
Factors associated with HBV response during antiretroviral therapy. In univariate analysis (table 5), an HBV response correlated significantly with a sustained HIV response (P = .001). In addition, an HBV response correlated significantly with a shorter duration of antiretroviral therapy (18 months vs. 69 months; P = .044), lamivudine therapy (20 months vs. 31 months; P = .018), and HAART (16 months vs. 48 months; P = .003). Other factors, including the baseline and nadir CD4 cell counts, baseline HBV DNA and HIV RNA levels, and HBV genotype, were not related to HBeAg and/or HBsAg seroconversion (table 5).
Remarkably, HBsAg and/or HBeAg seroconversion rates were related to the CDC stage at baseline (3% for stage A, 19% for stage B, and 29% for stage C; P = .014). Moreover, patients with elevated baseline ALT levels had a 4-times greater chance of achieving an HBV response than did patients with normal ALT levels (26.5% vs. 6.3%; P = .023). In addition, among 34 patients with CAHB, we observed a greater increase in the CD4 cell count during HAART in those who experienced an HBV response than in those who did not experience an HBV response (median increase, 232 cells/uL vs. 48 cells/L; P = .03). Conversely, 7 patients with CAHB who experienced failure of HAART developed immunotolerance. In these patients, we observed a median decrease in CD4 cell count of 72 cells/uL (range, -403 cells/L to +58 cells/L).
Using multivariate Cox regression analysis (table 6), with data censored at the time of HBV response, the following factors correlated significantly with HBsAg and/or HBeAg seroconversion: median duration of antiretroviral therapy (OR, 0.41; 95% CI, 0.16-1.03; P = .058) and CDC stage ( for stage B vs. stage A, P = .029; for stage C vs. stage A, P = .069). HIV response could not be included in this model, because none of the patients who did not experience this response had an HBV response, and therefore, the OR could not be calculated.
DISCUSSION
Despite the widespread use of lamivudine since 1996, HBeAg and HBsAg seroconversion has been observed only in a minority of patients initiating HAART regimens [10-13]. In our study, we described 12 instances of HBeAg and/or HBsAg seroconversion during antiretroviral therapy. Most of these seroconversions occurred during HAART including lamivudine and correlated significantly with a sustained HIV response and a shorter duration of antiretroviral therapy. Interestingly, HBeAg or HBsAg seroconversion was not observed in patients who did not receive treatment for HIV infection, in patients who experienced failure of HAART, or in carriers of mutations conferring lamivudine resistance, which suggests that a sustained HIV response to antiretroviral therapy is necessary to control HBV infection. Our study also confirms that powerful and brief, active antiretroviral therapy, combined with anti-HBV drugs, triggers HBeAg and HBsAg seroconversion in patients coinfected with HIV and HBV. Efficient and prolonged HIV therapy after such an HBV response probably hampers the reversibility of HBeAg and/or HBsAg seroconversion.
Unlike the results of 2 small studies evaluating the effects of antiretroviral therapy regimens including lamivudine [10, 12], the results of our study indicate that baseline CD4 cell count and HIV RNA and HBV DNA levels are not associated with either a better outcome during HAART or a higher HBeAg seroconversion rate. Nevertheless, our study suggests that patients in more advanced CDC stages of HIV infection have more chance of seroconverting to anti-HBe or anti-HBs. This finding may suggest that immunorestoration during HAART, even without lamivudine (as noted for 2 of our patients) (table 4), plays a major role in controlling chronic hepatitis B and favors HBeAg and HBsAg seroconversion. Thus, in a subgroup of patients with a CAHB profile, we revealed that immunorestoration during HAART was predictive of HBeAg or HBsAg seroconversion. In the study by Carton et al. [12], 5 patients seroconverted to anti-HBe while receiving HAART including lamivudine, and their CD4 cell counts increased significantly more than did those of patients who did not experience HBeAg seroconversion. Consequently, immunorestoration after the initiation of HAART, together with active anti-HBV therapy, might play a major role in suppressing HBV replication and, thus, enhance the specific immune response to achieve HBeAg or HBsAg seroconversion.
A previous immunologic study involving 5 patients coinfected with HIV and HBV found evidence of the reconstitution of a functionally active HBV-specific CD8 cell response when HAART was combined with an anti-HBV drug [14]. More recently [15], the same group demonstrated that an HBV-specific CD8 cell response had been reconstituted in 50% of the HIV-infected patients receiving HAART.
Information on the natural history of chronic hepatitis B in patients coinfected with HIV and HBV is still limited and mostly comes from studies conducted during the HAART era involving patients previously treated with lamivudine (many of them with lamivudine resistance) [10, 11, 16-18]. In our study, before HAART and lamivudine treatment, most of our patients were HBeAg positive (68%) and had normal ALT levels (62%). Our results are similar to those of previous studies involving both lamivudine-naive [19] and lamivudine-pretreated coinfected patients [17, 20] but differ from the results of a French study involving HBV-monoinfected patients [21], in which a higher prevalence of anti-HBe positivity was reported. In our cohort, the higher prevalence of HBeAg-positive patients was clearly because of the fact that most of the white subjects acquired chronic hepatitis B during adulthood and through sexual activity or injection drug use [22]. Conversely, anti-HBe status occurred more frequently among African subjects, possibly because chronic HBV infection mostly occurred during childhood or even earlier, during the perinatal period. This difference was not reported in previous studies [4, 23], because they concerned only white men who have sex with men or injection drug users.
As noted, with regard to HBV monoinfection [24], the HBV DNA level was higher in HBeAg-positive patients than in anti-HBe-positive patients. As reported by Colin et al. [4], HBV DNA level was not related to CD4 cell count or CDC stage. Immunodepression induced by HIV infection is usually associated with an immunotolerant anti-HBV phase, defined by a normal ALT level and a high HBV DNA level. Nevertheless, in the present study, the baseline median CD4 cell counts were similar in patients with immunotolerance and in those with CAHB. Colin et al. [4] found similar results, with no statistically significant difference in transaminase levels according to CDC stage. However, in some of our patients, evolutions of the immune system and changes in CD4 cell count were clearly associated with different phases of chronic hepatitis B. Thus, the profiles of 7 patients shifted from CAHB to immunotolerance, because their CD4 cell counts decreased because of failure of HAART. Therefore, in certain subjects, there is a dynamic evolution of the chronic hepatitis B pattern that is dependent on changes in the immune system.
In populations coinfected with HIV and HBV, monotherapy with lamivudine for chronic HBV infection is often associated with a rapid appearance of lamivudine resistance [16, 25, 26], with higher risk associated with long-term therapy. We revealed that other factors, such as higher baseline HBV DNA level and both higher baseline and nadir CD4 cell counts, correlated significantly with the occurrence of lamivudine resistance. As with HBV monoinfection, a higher pretreatment HBV DNA level was an important factor explaining this fact [27]. Our routine HBV DNA level quantification method had a detection limit of 700,000 copies/mL, and we could not exclude that some patients had a weaker reduction in HBV DNA level, with persistent low replicative HBV load (10,000-700,000 copies/mL) while receiving lamivudine therapy, which explains the later appearance of lamivudine resistance. However, all of the patients who had prolonged undetectable HBV loads had real-time PCR performed on frozen serum samples (detection limit, 100 copies/mL), which confirmed that HBV DNA levels were <10,000 copies/mL for this subgroup and <100 copies/mL for all 12 HBV seroconverters.
In our study, a low HBV DNA level before lamivudine treatment limited the risk of lamivudine resistance. Consequently, lamivudine resistance was seen only in patients with CAHB and immunotolerance and not in any of the 10 inactive carriers. In the latter group, the median duration of lamivudine treatment was 18 months, which was significantly shorter than that for the other 2 groups. This result should be underlined, because in previous studies involving HIV-HBV-coinfected patients, the rates of lamivudine resistance usually exceeded 20% and 50% at 1 and 2 years, respectively [16, 25, 26]. Here, none of the patients with a baseline HBV DNA level <5.50 log copies/mL developed lamivudine resistance. Moreover, in other studies [10, 16, 18, 25, 26, 28], this low replicative subgroup was usually overlooked.
In summary, our results indicate that HBeAg or HBsAg seroconversion correlates with a sustained HIV response during antiretroviral therapy, which was mostly HAART including lamivudine. Moreover, for patients with an elevated baseline ALT level, the HBV response correlated significantly with immunorestoration following HAART. In practice, our results suggest that a HAART regimen exhibiting dual activity against HIV and HBV is necessary for the majority of patients coinfected with HIV and HBV to optimize the HBV response [1, 29, 30].
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