Background: This study assessed HIV–hepatitis B virus (HBV) coinfection in southern Africa in terms of prevalence, viral characteristics, occult HBV, and the effect of lamivudine- versus tenofovir-containing first-line combination antiretroviral treatment (cART) on HBV-related outcomes.
Methods: A multicenter prospective cohort of HIV-infected adults in Zambia and South Africa who initiated cART. Outcomes by month 12 on cART were immunological recovery, hepatitis B surface antigen (HBsAg) loss, viral suppression, and drug resistance. We used descriptive statistics, logistic regression, and linear mixed models.
Results: Of the 1087 participants, 92 were HBsAg seropositive, yielding a sample-weighted prevalence of 7.4% (95% confidence interval: 5.6 to 9.2), with 76% genotype HBV-A1. The estimated CD4 recovery on cART was similar between HIV monoinfection and HIV–HBV coinfection groups and between lamivudine- and tenofovir-treated participants. HBsAg loss was documented in 20% (4/20) of lamivudine-treated and 18% (3/17) of tenofovir-treated participants (P = 0.305). Viral suppression (HBV-DNA < 20 IU/mL) was achieved in 61.5% (16/26) of lamivudine-treated and 71.4% (15/21) of tenofovir-treated participants (P = 0.477). HBV pol sequencing demonstrated M204I (n = 3) and N236T (n = 1) resistance-associated mutations in 4 of 8 (50%) lamivudine-treated participants and none in tenofovir-treated participants. Occult HBV infection was present in 13.3% before cART, but by month 12, HBV-DNA was below the limit of detection (<15 IU/mL) in 90.5% (19/21) of lamivudine-treated and 100% (18/18) of tenofovir-treated participants (P = 0.179).
Conclusions: Tenofovir-containing first-line cART is preferred for HIV–HBV coinfection in Africa because of a superior resistance profile relative to lamivudine monotherapy. Extended follow-up will be needed to determine long-term complications of occult HBV coinfection. Improved access to HBsAg screening and tenofovir is needed.
*Department of Global Health, Academic Medical Center of the University of Amsterdam, Amsterdam, The Netherlands;
†PharmAccess Foundation, Amsterdam, The Netherlands;
‡Amsterdam Institute for Global Health and Development, Amsterdam, The Netherlands;
§Department of Blood-Borne Infections, Sanquin, Amsterdam, The Netherlands;
‖Department of Medical Microbiology, Academic Medical Center of the University of Amsterdam, Amsterdam, The Netherlands;
¶Department of Molecular Medicine and Hematology, University of the Witwatersrand, Johannesburg, South Africa;
#Lancet Laboratories, Johannesburg, South Africa;
**Lusaka Trust Hospital, Lusaka, Zambia;
††Clinical HIV Research Unit, University of the Witwatersrand, Johannesburg, South Africa;
‡‡Muelmed Hospital, Pretoria, South Africa; and
§§Department of Internal Medicine and Infectious Diseases, University Medical Center Utrecht, Utrecht, The Netherlands.
Correspondence to: Raph L. Hamers, MD, PhD, Department of Global Health, Academic Medical Center of the University of Amsterdam, PharmAccess Foundation, Amsterdam Institute for Global Health and Development, Trinity Building C, Pietersbergweg 17, 1105 BM Amsterdam, The Netherlands (e-mail: firstname.lastname@example.org).
Supported by the Ministry of Foreign Affairs of The Netherlands (Grant 12454) and Stichting Aids Fonds.
The authors have no conflicts of interest to disclose.
Part of the data were presented at 19th Conference on Retroviruses and Opportunistic Infections, March 5–8, 2012, Seattle, WA (Abstract 794).
T.F.R.W. was the principal investigator. R.L.H., H.L.Z., C.L.W., I.M.H., W.S., and T.F.R.W. designed the study and developed the protocol. R.L.H. and K.C.E.S. contributed to implementation. M.S., P.I., and M.B. established the cohort and supervised data collection. H.L.Z., C.L.W., and W.S. supervised the laboratory testing. R.L.H. conceived and conducted the data analyses. R.L.H. drafted the manuscript, with assistance from H.L.Z. H.L.Z., C.L.W., K.C.E.S., I.M.H., and T.F.R.W. provided valuable input to interpretation of the data and critically reviewed the manuscript for important intellectual content. All authors reviewed and approved the final version of the manuscript.
The funders had no role in the study design, data collection, data analysis, data interpretation, decision to publish, or writing of the report. The content of this publication is solely the responsibility of the authors and does not necessarily represent the official views of any of the institutions mentioned above.
Received May 03, 2013
Accepted July 16, 2013
Coinfection with HIV and hepatitis B virus (HBV) is emerging as an important public health problem in sub-Saharan Africa since the scale-up of combination antiretroviral treatment (cART) for HIV has resulted in markedly improved long-term survival.1 Chronic HBV infection has been estimated to be present in up to 15% of HIV-infected persons in the region,2 increasing the risk of cirrhosis, hepatocellular carcinoma, and liver-related death.3–5
Screening for hepatitis B surface antigen (HBsAg) in HIV-infected individuals before cART initiation in resource-limited countries with high HBV prevalence is not routinely performed because of cost. Consequently, HIV-HBV–coinfected persons commonly receive first-line cART regimens that include lamivudine as the sole agent with anti-HBV activity, which is hampered by the rapid selection of drug resistance leading to the potential progression of HBV disease.6,7 By contrast, the use of tenofovir disoproxil fumarate, which has potent anti-HBV activity, has been successful in achieving sustained HBV suppression in both treatment-naive and lamivudine-resistant HIV-HBV–coinfected individuals in studies from Europe and Thailand,4,8–10 thus limiting progressive liver disease.11
To our knowledge, no studies to date have directly compared HBV-related outcomes between lamivudine and tenofovir in HIV-HBV–coinfected populations in sub-Saharan Africa, and only a few studies have assessed the effects of lamivudine.12–14 Furthermore, there is little knowledge on the prevalence and clinical significance of occult HBV infection in African HIV-infected populations, defined as a detectable plasma HBV-DNA in the absence of HBsAg, after the acute phase of infection.15–18
The aims of this study were to determine the prevalence and viral characteristics of HIV–HBV coinfection, to compare HBV-related outcomes and drug-resistance mutations of lamivudine- versus tenofovir-containing cART, and to determine the prevalence and clinical significance of occult HBV coinfections in a large multicenter cohort of antiretroviral-naive patients in Zambia and South Africa.
Study Population and Design
The PharmAccess African Studies to Evaluate Resistance Monitoring study is a multicenter prospective cohort of HIV-1–infected adults receiving cART, as previously profiled.19 HIV-related outcomes have been reported.20,21 The current analysis included all participants at 6 sites in South Africa and Zambia who initiated standard first-line cART containing 2 nucleoside reverse transcriptase inhibitors and a nonnucleoside reverse transcriptase inhibitor because of advanced immunodeficiency (CD4 cell count < 200 cells/μL) or HIV disease [World Health Organization (WHO) clinical stage 3 or 4], in accordance with national guidelines.22,23 Individuals who had previously used any antiretroviral drugs as prophylaxis or treatment were excluded. The collaborating sites did not routinely screen for HBV coinfection before cART initiation. Participants were classified as HIV–HBV coinfected if they were HBsAg seropositive and further classified according to HBV-active therapy: lamivudine monotherapy versus tenofovir (combined with emtricitabine or lamivudine). Participants provided written informed consent at enrollment. The protocols of the PharmAccess African Studies to Evaluate Resistance Monitoring study and HBV substudy were approved by the appropriate national and local research ethics committees.
Data Collection and Outcomes
Participants were followed up as per local standard of care guidelines. Routinely collected clinical and laboratory data were extracted and entered into a central database. Self-reported drug adherence was assessed at each follow-up clinical visit. Plasma was collected in EDTA tubes at the baseline visit and after 12 months of cART (window from 11 to 15 months) and stored at −80°C for retrospective analysis. The end points of interest after 12 months of cART were CD4 lymphocyte recovery, HBsAg loss, HBV-DNA suppression (HBV-DNA < 20 IU/mL), and drug-resistance mutations.
HBV testing was conducted at the Department of Molecular Medicine and Hematology of the University of the Witwatersrand in Johannesburg, South Africa. All technical failures were retested at Sanquin in Amsterdam, The Netherlands. HBsAg (lower limit of detection 0.05 IU/mL) and Hepatitis B core antibody (anti-HBc) were determined using a commercial enzyme immunoassay (Abbott Architect, Abbott Park, IL). HBV-DNA was assessed quantitatively with Abbott RealTime HBV (lower limit of detection 10 IU/mL) (Abbott, Des plaines, IL) or Cobas AmpliPrep/Cobas TaqMan (lower limit of detection 20 IU/mL) (Roche Molecular Diagnostics, Pleasanton, CA). HBV polymerase gene (including major part of S and part of pre-S2) was amplified and sequenced.24
The study sample size was based on estimated proportions of patients with an HBV drug-resistance mutation after 12 months of cART, at 23% for lamivudine7 and 0% for tenofovir.25 To achieve a statistical power of 80% at a 2-sided significance level of 5%, the sample size was n = 33 for each HBV treatment group, accounting for 10% attrition. HBsAg prevalence was estimated accounting for the sampling weights of the sites, expressed with a 95% confidence interval (CI) based on the normal approximation to the binomial distribution. For comparisons between 2 groups, χ2 test was used for categorical data and t test or Kruskal–Wallis test for continuous data. Logistic regression with robust random errors, accounting for clustering of observations within sites, was used to predict HBV virologic failure (HBV-DNA ≥ 20 IU/mL) by baseline characteristics. All variables were evaluated univariately, and those associated (P < 0.15) with the outcomes were stepwise entered into the multivariate model. Results were expressed as odds ratios (ORs) with 95% CI and P values. Linear mixed models with 4 slopes were used to estimate the gain in CD4 cell counts from cART initiation between the infection (HIV versus HIV–HBV) and treatment groups. The slopes were defined at 3 monthly intervals of follow-up. All CD4 cell counts measured routinely before and after start of ART were used. The model was adjusted for age, sex, baseline CD4 cell count, WHO clinical stage, pretherapy HIV-RNA and HBV-DNA, and types of nucleoside reverse transcriptase inhibitor and nonnucleoside reverse transcriptase inhibitor drugs. Results were expressed as difference in CD4 counts with 95% CI and P values. Reported P values are 2 sided, and a P value <0.05 was considered statistically significant. All analyses were performed using Stata version 11 (StataCorp LP, College Station, TX).
Baseline Characteristics of Study Participants
A total of 1148 study participants were included in the analysis, enrolled between March 2007 and November 2008 (Fig. 1). A pretherapy HBsAg result was available for 1087 (94.7%) participants, of whom 92 were seropositive, yielding an overall sample-weighted prevalence of HIV–HBV coinfection of 7.4% (95% CI: 5.6 to 9.2). Baseline HBsAg results were missing for 61 participants because of unsuitable or missing samples. Site-specific HBV/HIV coinfection prevalence estimates are listed in Table 1. Compared with HIV-monoinfected individuals, HIV-HBV–coinfected individuals were more likely to be men, from Zambia, to have more advanced HIV disease and lower mean body mass index. ALT values were typically low and not higher in HIV-HBV–coinfected individuals (median 22 and 25 IU/L, respectively; P = 0.4131). The median pretherapy CD4 cell count was marginally lower in HIV-HBV–coinfected (96.5 cells/μL) than in HIV-monoinfected (123.5 cells/μL) participants (P = 0.0755) (Table 2).
Of the 92 HIV-HBV–coinfected participants, 54 received lamivudine and 38 tenofovir (Table 3). Compared with the lamivudine group, tenofovir-treated participants were more likely to be men, from Zambia, and to receive efavirenz (instead of nevirapine). Among those for whom an HBV-DNA result was available (n = 70), the median pretherapy HBV-DNA was 5.39 log10 IU/mL [interquartile range (IQR), 2.37–8.83], with similar levels in both treatment groups (P = 0.6098). Low-level baseline viremia, as defined by the internationally accepted threshold of HBV-DNA <2000 IU/mL, was present in 34.3% (24/70) of participants.
Occult HBV Infection
Of the 995 participants who were HBsAg seronegative before cART initiation, 492 (49.5%) participants were anti-HBc positive. Among those for whom an HBV-DNA result was available (414/492), 55 (13.3%) had an HBV-DNA value greater than 15 IU/mL, indicative of occult HBV infection. Thus, the occult HBV prevalence among HIV-infected patients was 13.3% (55/414) overall, 14.3% (36/251) in Zambia, and 11.7% (19/163) in South Africa, with a median HBV-DNA of 1.76 log10 IU/mL (IQR, 1.46–2.16).
HBV Genotypes and Pretherapy Drug-Resistance Mutations
HBV genotypes could not be determined for 38 (41%) participants because of low HBV-DNA (n = 22), technical sequence failure (n = 14), or missing samples (n = 2). Observed HBV genotypes were A1 (76%), E (22%), and D (2%). Two individuals had a mutation in HBV pol associated with resistance to entecavir (I169L, T184S).
Clinical, Immunological, and Serological Treatment Outcomes
Patient Retention and Adherence
Eight hundred one (80.5%) of HIV-monoinfected and 66 (71.7%) of HIV-HBV–coinfected participants were retained in care and still on first-line cART after 12 months of follow-up (P = 0.045) (Fig. 1). Among the 92 HIV-HBV–coinfected participants, 9 (9.8%) had a single-drug substitution because of toxicity, intolerance, or other reasons. One patient in Zambia was started on tenofovir-containing cART, which was mistakenly substituted with zidovudine after 3 months; therefore, he was classified in the lamivudine group. Forty (74.1%) lamivudine-treated and 26 (68.4%) tenofovir-treated participants were retained in care and still on first-line ART after 12 months of follow-up (P = 0.553) (Fig. 1). In HIV-HBV–coinfected and HIV-monoinfected participants, 3-day adherence was optimal in 91.3% and 84.5%, respectively (P = 0.081), and 30-day adherence was ≥95% in 84.9% and 82.6%, respectively (P = 0.812). Among HIV-HBV–coinfected participants, 3-day and 30-day adherence did not differ between treatment groups (P = 0.819 and P = 0.379, respectively).
The estimated gain in CD4 cell count from cART initiation was not significantly different across infection and treatment groups (Fig. 2). The estimated increase in CD4 cell count by month 12 was 177 cells per microliter in the HIV-monoinfected and 159 cells per microliter in the HIV-HBV–coinfected participants. Among the HIV-HBV–coinfected participants, the estimated increase in CD4 cell count by month 12 was 155 cells per microliter in the lamivudine-treated and 161 cells per microliter in the tenofovir-treated participants.
Twenty-nine HBsAg outcome results could not be generated because stored samples were unsuitable or not available. HBsAg loss was documented in 20% (4/20) of lamivudine-treated and 18% (3/17) of tenofovir-treated participants (P = 0.305), which compares favorably to the low background rate of spontaneous HBV clearance in untreated patients. Comparison of participants with and without HBsAg results at month 12 demonstrated no differences in age, sex, WHO stage, baseline CD4 cell count, or HIV-RNA. Of note, 1 male patient from Zambia receiving tenofovir, who reported optimal adherence and had HIV-RNA suppression at month 12, seroconverted to HBsAg positivity.
Virological Treatment Outcomes
HBV Viral Suppression
Outcome HBV-DNA was available for 47 of 66 (71%) HIV-HBV–coinfected participants who were retained up to month 12. HBV suppression was achieved in 61.5% (16/26) of lamivudine-treated and 71.4% (15/21) of tenofovir-treated participants (P = 0.477). The median decrease in HBV-DNA from cART initiation was 6.26 log10 IU/mL (IQR, 5.13–7.39) in tenofovir-treated and 4.40 log10 IU/mL (IQR, 3.17–5.63) in lamivudine-treated participants (P = 0.1264). In a multivariate prediction model, HBV virologic failure was positively associated with pretherapy HBV-DNA (OR per log10 IU/mL: 3.69, 95% CI: 2.75 to 4.95; P < 0.0001) and inversely associated with tenofovir, although the latter association did not reach statistical significance (OR, 0.18, 95% CI: 0.015 to 2.05; P = 0.166). HBV virological response was not significantly associated with age, sex, clinical stage of HIV disease, pretherapy CD4 cell count, ALT, or HIV-RNA.
Of the 55 participants who had occult HBV infection, 45 (81.8%) were retained up to 12 months; among the 39 (86.7%) participants for whom an HBV-DNA result was generated, HBV-DNA was below the limit of detection (<15 IU/mL) in 90.5% (19/21) of lamivudine-treated and 100% (18/18) of tenofovir-treated participants (P = 0.179).
HBV Drug-Resistance Mutations
Of 16 participants with detectable HBV-DNA by month 12, HBV pol sequencing was successful in 12 (8 on lamivudine and 4 on tenofovir); the reason for the repeated technical failures in 4 specimens is possibly because of the suboptimal performance of the primer sets, related to specimen quality or viral diversity. Three lamivudine-treated participants (who reported optimal adherence and had suppressed HIV-RNA by month 12) carried the M204I mutation in HBV pol, associated with resistance to lamivudine, emtricitabine, and telbivudine. One lamivudine-treated participant (who reported suboptimal adherence but had suppressed HIV-RNA by month 12) carried the N236T mutation, associated with resistance to adefovir and possibly tenofovir. Four (50%) lamivudine-treated participants (who reported optimal adherence, 1 with detectable HIV-RNA by month 12) had no resistance mutations. The 4 tenofovir-treated participants (of whom 2 reported suboptimal adherence and 1 had detectable HIV-RNA) did not carry any resistance mutations.
We report the first study in sub-Saharan Africa that prospectively compared HBV-related outcomes between lamivudine- and tenofovir-containing cART for HIV infection. Among HIV-HBV–coinfected antiretroviral-naive individuals in Zambia and South Africa, tenofovir-containing first-line cART provided potent HBV-active therapy with a superior resistance profile to lamivudine monotherapy. Despite the fact that our study did not demonstrate differential HBV suppression or HBsAg loss between tenofovir and lamivudine treatment during the first year of cART, our findings together with previous reports provide support for use of tenofovir over lamivudine monotherapy in HIV-HBV–coinfected persons in resource-limited countries to prevent the development of HBV drug resistance and improve control of viral replication, as advocated in the latest WHO guidelines.26 We found an overall baseline prevalence of HBsAg seropositivity in 7.4% of patients, which is within the range of previous reports (4.8%–22.9%) from South Africa.14,17,18,27
Lamivudine is known to have a low genetic barrier to resistance and is associated with the emergence of mutations in the YMDD (tyrosine, methionine, aspartate, aspartate) motif of HBV pol domain C and with upstream compensatory mutations in polymerase domains A and B that, collectively, reduce treatment effectiveness. Previous studies from industrialized countries in chronic HBV-monoinfected patients have reported lamivudine-resistant mutations in 23% of patients receiving lamivudine monotherapy after 1 year, increasing to 65% after 5 years,7 and YMDD mutations in 49% after a median of 32.4 months of lamivudine monotherapy.6 One report observed significantly more hepatocellular carcinomas shortly after development of lamivudine resistance,28 which is largely explained by the recurrence of viral replication. Moreover, lamivudine resistance induces cross-resistance to emtricitabine, telbivudine, and entecavir, thus reducing the options for subsequent treatment. By contrast, tenofovir has a good resistance profile, and in phase 3 trials in HBV-monoinfected patients, no evidence of tenofovir resistance was shown up to 144 weeks of treatment.25 To date, potential tenofovir resistance, ie, the A194T mutation, has been described in only 2 HIV-HBV–coinfected patients.29 However, the association between this mutation and tenofovir resistance was not confirmed in another study.30
The proportion of tenofovir-treated patients who achieved HBV suppression accords with findings in European cohorts.31–33 A small randomized study in Thailand (n = 16) found that cART with tenofovir–emtricitabine in antiretroviral-naive HIV-HBV–coinfected persons resulted in a greater proportion of patients with HBV suppression at week 48 than emtricitabine monotherapy.10 Another small randomized study in antiretroviral-naive HIV-HBV–coinfected persons in Thailand (n = 36) demonstrated higher virological failure rates and early resistance development for lamivudine monotherapy, compared with tenofovir monotherapy, but did not demonstrate any short-term advantage of HBV dual therapy.8 The Phidisa II trial in South Africa found little additional benefit of lamivudine monotherapy in HIV-HBV–coinfected persons, compared with cART without any HBV activity.14 HBsAg loss rates in this study concur with findings in the Phidisa II trial14 but are higher than reported in studies from Europe33 and Taiwan.34
Chronic HBV coinfection in our cohort was not associated with a reduced CD4 lymphocyte recovery during cART. Several observational studies did not find a sustained negative effect of HBV on CD4 cell count recovery,35–37 although in a report from Nigeria CD4 recovery was delayed in the Hepatitis B e antigen (HBeAg)-positive subgroup.38 The Phidisa II trial in South Africa found no difference in CD4 recovery over 2 years of follow-up between HIV-monoinfected and HIV-HBV–coinfected persons receiving an HBV-active regimen with lamivudine.14 Based on available data, there is no justification to adjust the expected goals for recovery of CD4 cell counts during cART.
The study population had markers of highly active hepatitis B disease with a baseline median HBV-DNA above 6 log10 IU/mL, with only 34% having low-level HBV viremia. High values of HBV-DNA have been suggested to be attributable to HIV infection delaying transition to the inactive carrier phase (identified by anti-HBe, absence of HBeAg, and low levels of HBV-DNA) or reactivation with reversion to the immune-active phase during AIDS-related immune suppression.16,39 Serological evidence of a history of HBV exposure (chronic hepatitis B indicated by HBsAg or any exposure indicated by anti-HBc) was present in 53% of participants, which is in agreement with previous studies from Africa.40,41
Compared with HIV-uninfected individuals,16 HIV-infected individuals appear to have increased prevalence of occult HBV infection in studies from South Africa and Cote d’Ivoire, ranging from 10% to 23%.15,17,18,42 In our cohort, HBV-active cART, containing either lamivudine or tenofovir, resulted in HBV viral suppression in a high (≥90%) proportion of patients with occult infection. Although this finding suggests that occult HBV infection may be more a diagnostic than a clinical problem, extended follow-up studies are needed to determine whether it is associated with long-term liver-related complications in regions of high HBV endemicity.43
The role of HBV genotypes in the natural history of the infection and in the response to therapy in patients with HIV coinfection is unclear.44,45 For example, in southern Africa, an increased risk of hepatocellular carcinoma has been linked to genotype A1.46 Therefore, studies that more clearly define the role of genotype on the natural history are warranted.
The study has some limitations. First, participant attrition in our cohort because of mortality and loss to follow-up in the first year of cART was considerable (26%), yet consistent with other reports from the region.47 Also, a number of specimens were unobtainable for retrospective HBV laboratory testing. Second, confounding by treatment indication is likely to be limited because cART regimens were typically prescribed in concordance with national guidelines rather than on an individual basis. Third, participants were assessed neither for hepatitis C virus or hepatitis delta coinfection, which may be associated with lower HBV replication and worse outcomes,48 nor for HBeAg, which precluded a more detailed assessment of HBV immune control during cART. Fourth, population-based sequencing is not able to detect minority resistant viral strains, thus potentially underestimating resistance.
In conclusion, tenofovir-containing cART provides potent anti-HBV therapy with a good resistance profile, relative to lamivudine, for HIV-HBV–coinfected persons in southern Africa. In countries with high HBV endemicity, all HIV-infected individuals should be screened for HBsAg before starting cART. As per WHO recommendations, coinfected patients with an indication for treatment of either HBV or HIV should receive a triple combination of antiretroviral agents, including tenofovir.26 Nonetheless, despite progress, by 2010, only 20% of first-line cART regimens contained tenofovir,47 potentially exposing millions of HIV-HBV–coinfected Africans to suboptimal HBV therapy. Improved access to tenofovir in Africa is an urgent priority. To reduce global HBV-related morbidity and mortality, improved understanding of the epidemiology of chronic HBV in regions with high HBV endemicity and the long-term effectiveness of cART regimens in patients with HIV–HBV coinfection is required.
The authors thank the study participants, the staff at the collaborating clinical sites and reference laboratory, and the support staff at PharmAccess Foundation and Contract Laboratory Services. Special thanks to Moheb Labib, Jack Menke, Francesca Conradie, and Margaret Hardman for supervising data collection and to Kim Steegen and Peter van Swieten for laboratory work. The PharmAccess African Studies to Evaluate Resistance (PASER) is an initiative of PharmAccess Foundation. PASER is part of the LAASER program (Linking African and Asian Societies for an Enhanced Response to HIV/AIDS), a partnership of Stichting Aids Fonds, The Foundation for AIDS Research (amfAR)—TREAT Asia, PharmAccess Foundation, and International Civil Society Support.
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