Over two billion people have been infected with the hepatitis B virus (HBV) worldwide, being 350 million chronic carriers, and therefore at a risk for complications of progressive liver disease [1–3]. Due to shared sexual and blood-borne routes of transmission, the prevalence of chronic hepatitis B is particularly high in HIV-positive individuals. Overall, 4–10% of HIV-infected persons have chronic hepatitis B [4–6]. HIV worsens the natural history of HBV infection, increasing the rate of chronification following exposure, enhancing HBV replication and accelerating liver disease progression [7–9]. Interestingly, effective therapy for HIV may improve HBV outcomes in coinfected individuals, largely throughout immune-mediated mechanisms [10,11].
As several nucleos(t)ide analogues display activity against both HIV and HBV, many HIV/HBV-coinfected individuals have been treated for HBV for long periods [12–15]. Sometimes it has been done inadvertently, ignoring that the HIV patient was positive for HBsAg. Tenofovir displays the most robust and prolonged anti-HBV activity regardless of prior lamivudine exposure [16,17], largely as a result of its unique high barrier to resistance. Conversely, lamivudine therapy selects for HBV resistance in 25–65% of patients treated for 1–5 years [18,19]. This rate is further increased in HIV/HBV-coinfected individuals [20,21]. On the basis of this information, current antiretroviral treatment guidelines recommend the inclusion of tenofovir along with either emtricitabine or lamivudine as part of any triple combination regimen prescribed to HIV/HBV-coinfected patients [22–24]. Herein, we examine longitudinal serum HBV-DNA in a large population of chronic hepatitis B patients on long-term tenofovir therapy, comparing HBV-monoinfected and HIV/HBV-coinfected individuals.
Patients and methods
All patients with chronic hepatitis B (defined as positive serum HBsAg for longer than 6 months) on regular follow-up at four clinics in Spain who had received tenofovir therapy from year 2001 until December 2011 were retrospectively identified. Main demographics (age, sex, risk behaviour), laboratory parameters (HBV genotype, HBeAg, HBsAg and HBV drug resistance), coinfection with other viruses (HCV, delta) and antiviral therapy (for either HIV or HBV) were all recorded. Cross-sectional registers were censored at baseline, at the time of beginning tenofovir and at the last visit for each patient. Data on periodic intermediate registers for HBV-DNA, as well as seroresponse to HBeAg and/or HBsAg, were recorded during the whole study period.
Plasma HIV-RNA and HBV-DNA were measured using commercial methods (VERSANT HIV-1 RNA 1.0; Siemens, Barcelona, Spain and Real Time HBV; Abbott, Madrid, Spain, respectively). The lower limit of viral RNA detection using these tests was 37 copies/ml and 10 IU/ml, respectively. HBV polymerase drug resistance mutations and HBV genotyping were examined using the Abbott HBV Sequencing Assay or a hybridization probe assay (INNO-LiPA; Innogenetics, Barcelona, Spain). Both HBV polymerase drug resistance-associated mutations and HBV genotyping were inferred using the geno2pheno[hbv] interpretation system (http://http://www.geno2pheno.org).
Descriptive data of the whole study population are reported as absolute numbers, percentages and as median and interquartile ranges (IQRs). Baseline and follow-up characteristics were compared using the chi square and Fisher exact tests for categorical variables and the Wilcoxon nonparametric test for continuous variables. Multivariate logistic regression analysis was performed to find which variables predicted the virological response. Significant differences were considered only for P values below 0.05. All statistical analyses were performed using SPSS v15.0 (SPSS Inc., North Chicago, Illinois, USA).
Characteristics of the study population
A total of 176 chronic hepatitis B patients on tenofovir therapy were identified, of whom 38 (22%) were HBV-monoinfected and 138 (78%) coinfected with HIV. Table 1 records the main baseline characteristics of the study population at the time of initiating tenofovir. HIV/HBV-coinfected patients were mostly men (87%) and in a similar proportion former IDUs (38%) or homosexual men (40%). In contrast, HBV-monoinfected individuals were less frequently men (76%), the mechanism of acquisition being unknown in 74%. The rate of HBeAg positivity was similar in HBV-monoinfected and HIV/HBV-coinfected patients (50 vs. 47%; P = 0.7). HBV genotype A predominated in coinfected patients (41%), whereas HBV genotype D was the most common in HBV-monoinfected individuals (61%). Hepatitis delta superinfection was more frequent in coinfected than in HBV-monoinfected patients (20 vs. 3%; P < 0.01), as well as the presence of serum hepatitis C virus (HCV) antibodies (27 vs. 3%; P < 0.01).
Nearly half of HIV/HBV-coinfected patients were treatment-naive at baseline, with lamivudine used previously by the rest. Prior exposure to antivirals had occurred in 68% of HBV-monoinfected individuals, being much broader and including lamivudine (63%), adefovir (40%), entecavir (11%) and/or interferon (16%). Along with tenofovir, emtricitabine was given in 91% of HIV/HBV-coinfected patients and 84% of HBV-monoinfected individuals as a coformulation (Truvada). Tenofovir as the only anti-HBV agent was prescribed in two coinfected and three monoinfected patients. A few individuals took tenofovir along with entecavir or lamivudine, with no significant differences between HIV/HBV-coinfected and HBV-monoinfected individuals.
The overall frequency of drug resistance mutations in HBV before beginning tenofovir tended to be greater in HBV-monoinfected than in coinfected patients (46 vs. 30%). However, as noted in Fig. 1, the rate of M204V, the critical change for lamivudine resistance, was present in 24% of coinfected patients compared with 12% of HBV-monoinfected individuals (P = 0.2). Accessory and/or compensatory drug resistance mutations such as L180M and V173L were also more common in HIV/HBV-coinfected individuals. In contrast, as expected, adefovir resistance associated mutations such A181T/V and N236T were more frequent in HBV-monoinfected than in coinfected patients.
Sequencing analysis of the HBV envelope gene suggested that changes associated with vaccine escape mutants were more common in HIV/HBV-coinfected than in HBV-monoinfected patients. This was the case for sE164D (9 vs. 4%, P = 0.5), sI195M (24% vs. 12%, P = 0.2) and sE164D along with sI195M (9 vs. 4%, P = 0.5).
Virological response to tenofovir
The median length on tenofovir therapy was 47 (28–71) months, longer in coinfected [51 (29–77) months] than in HBV-monoinfected [35 (28–47) months] individuals (P = 0.007). The proportion of patients who had initiated tenofovir with undetectable HBV-DNA was higher in coinfected than in HBV-monoinfected patients (23.2 vs. 13.2%, P < 0.04). However, in the subset of viremic patients, median HBV-DNA values tended to be lower in HBV-monoinfected than in coinfected patients [4 (3–7) vs. 6 (4–8) log IU/ml]. In HIV-HBV coinfected patients, median HBV-DNA was higher in HBeAg-positive than in HBeAg-negative individuals [8 (5–8) vs. 5 (3–7) log IU/ml, P < 0.001], whereas it did not differ significantly in HBV-monoinfected patients [4.5 (3–7) vs. 4.0 (3–7) log IU/ml, P = 0.7].
Virological response, defined as an achievement of HBV-DNA of less than 10 IU/ml at either weeks 24, 48 or 96 of tenofovir therapy, is depicted in Fig. 2, split out by both HIV and HBeAg status. Overall, there were no significant differences comparing HBV-monoinfected and HIV/HBV-coinfected patients. However, the rate of undetectable serum HBV-DNA at weeks 24 and 48 was higher in HBeAg-negative than in HBeAg-positive patients with coinfection (100 vs. 45%, P < 0.001; and 86 vs. 71%, P = 0.04; respectively), suggesting that complete viral suppression took longer to be achieved in this subset of patients.
In univariate analysis [odds ratio (OR); 95% confidence interval (CI); P], lower serum HBV-DNA was associated with virological response at weeks 24 and 48 (OR 2.4; 95% CI 1.4–4.0; P = 0.001 and OR 1.4; 95% CI 1.1–2.0; P = 0.04, respectively) in patients who had initiated tenofovir with detectable viremia. On the contrary, negative HBeAg directly correlated with response to tenofovir at week 24 (OR 10; 95% CI 1.2–100; P = 0.03). In contrast, drug resistance mutations, hepatitis C or delta coinfection, or concomitant use of other anti-HBV agents did not influence the virological response to TDF significantly. In multivariate analysis, only lower serum HBV-DNA levels before beginning tenofovir predicted the virological response at weeks 24 (OR 0.4; 95% CI 0.2–0.8; P = 0.005) and 48 (OR 0.7; 95% CI 0.5–0.9; P = 0.02).
We evaluated retrospectively the virological response to long-term tenofovir therapy in a large group of chronic hepatitis B patients, of whom three-quarters were coinfected with HIV. Compared with HBV-monoinfected individuals, those with coinfection were younger, more often seropositive for hepatitis C or delta virus antibodies, and less frequently infected by HBV genotype D. Around half of patients in both groups were HBeAg positive. More than three-quarters of coinfected patients were homosexual men or former IDUs. Before beginning tenofovir, half of HIV/HBV-coinfected patients and more than two-thirds of HBV-monoinfected individuals had been exposed to other anti-HBV agents, lamivudine being by far the most frequent agent. Finally, most patients in our study received tenofovir along with a second anti-HBV agent, emtricitabine being the most common given its convenient coformulation as a single pill (Truvada).
Despite differences comparing HIV/HBV-coinfected and HBV-monoinfected patients at the time of beginning tenofovir, the proportion of baseline HBV viremic individuals who achieved undetectable serum HBV-DNA at weeks 24, 48 and 96 did not differ significantly comparing HBV-monoinfected and coinfected patients. However, in the subset of HBeAg-positive individuals, HIV coinfection was associated with a slower virological response. Whereas more than 80% of HBeAg+, HBV-monoinfected individuals had undetectable serum HBV-DNA at weeks 24, 48 and 96 of tenofovir therapy, these numbers were lower in HBeAg+ coinfected patients (45, 71 and 81%, respectively).
Multivariate analysis provided a clue on the negative impact of HIV coinfection and positive HBeAg on virological response to tenofovir in the whole chronic hepatitis B population. The amount of serum HBV-DNA was the only significant determinant of treatment response. Patients with positive HBeAg and/or HIV coinfection had the greatest HBV-DNA levels, which most likely explained their longer time to reach undetectability, as recently shown by others .
Despite being present in 30% of viremic HIV/HBV-coinfected patients and in 46% of viremic HBV-monoinfected individuals, there was no recognizable effect of drug resistance mutations on response to tenofovir in our study population. Although lamivudine as the only anti-HBV agent is now considered as suboptimal to treat chronic hepatitis B , this drug as a part of triple antiretroviral regimens has been widely used for many years in HIV/HBV-coinfected patients. As it occurred in our series, a large proportion of these individuals harbour HBV-resistant strains [14,15]. Apart from lamivudine, other anti-HBV agents, such as adefovir or entecavir, had been used to treat our HBV-monoinfected individuals, which explains the recognition of drug resistance mutations other than those classically associated with lamivudine in our series. Differences in prior pharmacological history and the distinct profile of drug resistance mutations exhibited by HIV/HBV-coinfected and HBV-monoinfected patients did not translate into a different antiviral response to tenofovir in our study. These results are reassuring and support that tenofovir activity against HBV does not seem to be impaired significantly by prior exposure to other anti-HBV agents and/or the presence of drug resistance mutations in HBV. Our findings are in agreement with the results from other studies [27,28], some of which further highlighted that adding another anti-HBV agent to tenofovir did not result in greater antiviral responses than prescription of tenofovir alone [29,30].
Given that coinfection with hepatitis C or delta viruses is relatively common in chronic hepatitis B patients and complex viral interference phenomena exist between these viruses [31,32], we explored whether HBV suppression using tenofovir could be influenced in chronic hepatitis B patients coinfected with other hepatitis viruses. We could not find any evidence for it. However, patients with delta hepatitis had lower median serum HBV-DNA levels than the rest and accordingly should achieve undetectability faster on tenofovir.
All 38 HBV-monoinfected individuals and 85% out of 78 viremic HIV/HBV-coinfected patients achieved undetectable HBV-DNA by week 96 of tenofovir therapy. The trend in HBV-DNA decline in the subset of coinfected individuals still viremic at 4 years of tenofovir therapy suggests that they might eventually also reach complete viral suppression, as shown in a recent study that examined virological responses to tenofovir therapy in a large group of chronic hepatitis B patients with very high baseline viral load .
Prior studies have shown that the yearly rate of spontaneous HBeAg clearance in chronic hepatitis B patients ranges from 8 to 12% . In our series, HBeAg loss occurred in 15% of monoinfected and 7% of coinfected patients under an average of 4 years of tenofovir therapy. Clearance of serum HBsAg is the most ambitious objective of HBV therapy , but rarely occurs either spontaneously or as a result of antiviral therapy with nucleos(t)ide analogues. In our series, HBsAg clearance was seen in nine individuals treated with tenofovir (5.1%). Moreover, it is noteworthy that all were coinfected with HIV, which might partially be explained by the longer exposure to tenofovir in this group than in HBV-monoinfected individuals.
In summary, the antiviral efficacy of tenofovir is high and does not differ significantly comparing HIV/HBV-coinfected and HBV-monoinfected patients. Most patients achieve undetectable serum HBV-DNA at week 96 of tenofovir therapy. Baseline serum HBV-DNA is the major determinant of virological response to tenofovir, with no significant influence of HBeAg, drug resistance mutations nor coinfection with hepatitis C or delta viruses.
This work was supported in part by grants from Fundación Investigación y Educación en SIDA (FIES); NEAT (European AIDS Treatment Network; LSHM-CT-2006–037570); European Community's Seventh Framework Program [FP7/2007–2013 project ‘Collaborative HIV and Anti-HIV Drug Resistance Network (CHAIN)’ – no. 223131]; Red de Investigación en SIDA (ISCIII-RETIC-RD12/0017/0031) and Fondo de Investigación Sanitaria (projects CP08/00214, PI10/02166).
E.P. and V.S. did the study design. P.B., A.A., A.M., S.T., J.dR., J.P., C.R., E.V. and V.S. contributed with blood specimens and clinical data. Z.P., C.R., A.A. and R.S.-E. did the virological work. Z.P., E.P. and V.S. wrote the first draft of the manuscript. All authors revised and provided critical comments.
Conflicts of interest
There are no conflicts of interest.
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