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HIV- Molecular Virology and Bioinformatics Unit, Africa Centre for Health and Population Studies
Centre for HIV/AIDS Networking, Doris Duke Medical Research Institute, and Departments of Dermatology and Medicine, Nelson R. Mandela School of Medicine
Faculty of Health Sciences, University of KwaZulu-Natal, Durban, South Africa
Yale University School of Medicine, New Haven, Connecticut
Invirion Inc., Frankfort, Michigan
Department of Clinical Virology, Stanford University, School of Medicine, Palo Alto, California
Background.
A potential confounding factor in the treatment of human immunodeficiency virus (HIV) infection in Africa is the frequent occurrence of opportunistic infections (OIs). OI-induced immune activation can interfere with HIV-1 clearance by increasing viral replication and target cell availability.
Study design.
Treatment outcomes for patients dually infected with HIV-1 and Mycobacterium tuberculosis or HIV-1 and human herpesvirus (HHV)8 were assessed by measuring changes in viral load and CD4+ cell counts and by determining the time taken to reach undetectable HIV-1 RNA levels, assessed by means of Kaplan-Meier survival analysis. Patients with HIV-1 and Kaposi sarcoma (KS) received generic nevirapine, stavudine, and lamivudine (3TC); patients with HIV-1 and tuberculosis (TB) received standard commercial didanosine, 3TC, and efavirenz.
Results.
Both cohorts exhibited a rapid, near-exponential phase I decline in viral load. Patients with TB and late-stage KS had the steepest decay kinetics. These same patients had the greatest initial increase in CD4+ cell counts. Phase II clearance was slower and more variable. The proportions of patients reaching undetectable plasma HIV-1 levels at days 7, 14, 28, 60, and 90 were, respectively, 15.8%, 30.0%, 52.6%, 78.9%, and 93.8% (Pearson's 2 = 50.5; P < .001) for patients with TB and 0.0%, 5.0%, 22.2%, 64.7%, and 80.0% (Pearson's 2 = 63.6; P < .001) for patients with KS.
Conclusions.
Nucleoside reverse-transcriptase inhibitor/nonnucleoside reverse-transcriptase inhibitorbased treatment regimens are highly effective in clearing rapidly replicating (phase I) virus in African patients dually infected with HIV-1 and either TB or KS.
One of the most dramatic changes in the global AIDS epidemic has been the rapid emergence and devastating spread of HIV-1 subtype C [14]. At the end of 2001, South Africa had an estimated 4.7 million people living with HIV-1/AIDS [5]. By the year 2005, an estimated 250,000 South Africans will have died of AIDS [6]. Young women 2024 years of age have the highest HIV-1 incidence and prevalence rates [7, 8].
Since we are on the brink of introducing a treatment strategy for South Africa, it is urgent that we collect information on the therapeutic response of subtype C viruses. Although it is generally accepted that drugs developed against HIV-1 subtype B will be safe and effective, the efficacy of antiretroviral therapy (ART) for the treatment of HIV-1 subtype C in Africa is largely unproven. In vitro evidence suggests that subtypes B and C are equally susceptible to antiretroviral drugs [9, 10] but that subtype C viruses have natural polymorphisms that facilitate the emergence of drug resistance [1113]. At the patient level, studies of subtype C and of other non-B viruses have been difficult to interpret. In the Ivory Coast, treatment of patients infected with subtype A/G recombinants resulted in a 57.4% prevalence of resistance [14]. However, it is unclear whether this high level of resistance was due to the intrinsic properties of subtype A/G viruses or to poor compliance and suboptimal therapy. In contrast, studies from South Africa [15] and Senegal [16] have reported resistance rates as low as 10%11.8%. At least 4 studies have assessed treatment outcomes in African emigrants living in Europe [1720]. In 1 study, African patients had a lower rate of CD4+ cell recovery [17]; in a second study, African patients were found to have an earlier viral rebound, compared with that in European patients [18]. In the other 2 studies, no difference was detected in the therapeutic response of African versus non-African patients [19, 20]. All of these studies involved a small number of patients infected with a variety of different non-B HIV-1 subtypes.
The planned treatment of large numbers of patients in sub-Saharan Africa has underscored the urgent need to develop simple, standardized drug regimens that have a high barrier to drug resistance and that are affordable and appropriate for the treatment of non-B HIV-1. In addition to being efficacious and compatible with medications used to treat tuberculosis (TB) and other coinfections, each drug in the treatment regimen should have a prolonged half-life, so that it can be combined into a single tablet taken once daily, to reduce the risk of noncompliance. One regimen that has been successfully used to treat HIV-1 B infections in patients with active TB is a combination of didanosine (ddI), lamivudine (3TC), and efavirenz (EFV) [21].
Here, we compare the effects of nonnucleoside reverse-transcriptase inhibitor (NNRTI)based triple combination ART on the dynamics of viral RNA decline and CD4+ cell restoration in South African patients dually infected with HIV-1 and either Mycobacterium tuberculosis or human herpesvirus8 (HHV-8), the causative agent of Kaposi sarcoma (KS). The study is in keeping with international guidelines suggesting that patients with low CD4+ cell counts (<250350 cells/L) and patients with opportunistic infections (OIs) (regardless of their CD4+ cell counts) are likely to derive the most benefit from ART [2224].
MATERIALS AND METHODS
Study design.
Forty-nine HIV-1infected patients attending the Dermatology Clinic at King Edward VIII Hospital (n = 28) and the Cyril Zulu Communicable Diseases Clinic (n = 21) in Durban between January 2002 and August 2003 were selected for study. Eligible patients were treatment-naive adults with sputum-positive pulmonary TB or biopsy-confirmed KS. After counseling had been given and informed consent was received from each patient, participants with KS were randomized into 2 groups. Group A received generic stavudine (D4T) (40 mg), 3TC (200 mg), and nevirapine (NVP) (200 mg) (all manufactured by CIPLA). Group B received the same treatment until day 28, followed by a delayed course of chemotherapy, consisting of bleomycin (10 U/m2, administered intramuscularly [im]), doxorubicin (20 mg/m3, administered intravenously [iv]), and vincristine (1.4 mg/m2, administered iv). Patients in the TB cohort received standard TB treatment (rifampin, isoniazid, pyrazinamide, and ethambutol, once daily for 2 months; then isoniazid and rifampin for 4 months); ART was phased in 2 weeks later. The regimen consisted of weight-adjusted ddI (400 mg), 3TC (300 mg), and EFV (600 mg). These drugs were purchased from standard commercial sources (Bristol-Myers Squibb, Glaxo-Wellcome, and DuPont Pharmaceuticals). Patients with KS were monitored for changes in viral load and CD4+/CD8+ cell count at baseline; at days 4, 7, 14, and 28; and at months 2, 3, and 6. Patients with TB were tested at baseline; at weeks 1, 2, 3, and 4; and monthly thereafter.
Plasma viral load and CD4+/CD8+ subset determination.
RNA was extracted from EDTA blood tubes by use of a guanidinium-silica method (Nuclisens isolation kit; Organon Teknika) and an automated extractor (Organon Teknika). HIV-1 RNA levels were measured using the Nuclisens HIV-1 QT kit. Blood CD4+ and CD8+ lymphocyte percentages were analyzed on a Coulter XL Flow Cytometer, by use of a single-platform Beckman Coulter TetraOne protocol.
Statistical analyses.
The short-term response to ART was assessed by measuring the median change, from baseline levels, in viral load (log10 scale) and in CD4+ and CD8+ cell counts. Correlations among baseline parameters were assessed using linear regression and Spearman's correlations. Repeated-measures analysis of variance (ANOVA) was used to assess the effects of treatment and OIs on changes in these parameters over time. Statistical significance was evaluated using independent 2-tailed t tests for equality of means. Before the analyses were performed, the data were tested for normality. Estimates of the time to CD4+ cell recovery and viral load clearance were determined by Kaplan-Meier analysis. Log-rank tests were used to test equality of survival distributions between groups. All calculations were performed using SPSS software (version 11.5).
RESULTS
Demographics and baseline characteristics of the 2 patient groups.
Forty-eight patients were infected with HIV-1 subtype C; 1 patient with KS was infected with subtype B. With the exception of this patient, all other patients reported heterosexual contact as the mode of HIV-1 acquisition. As shown in table 1, 24 patients were women, and 25 were men. The mean ages of the TB and KS cohorts were 31 years (range, 1853 years) and 35 years (range, 2749 years), respectively. There was no significant difference in the mean baseline CD4+ (206 cells/L in the TB cohort vs. 223 cells/L in the KS cohort) or CD8+ (1076 cells/L in the TB cohort versus 1235 cells/L in the KS cohort) cell counts between the 2 cohorts. However, the mean baseline viral load was significantly higher in the TB cohort than in the KS cohort (5.41 vs. 4.81 log10 RNA copies/mL, respectively; P = .007). Among patients with KS, a positive correlation was observed between the mean CD4+ and CD8+ cell counts at baseline. This correlation was most pronounced in the subgroup of patients with CD4+ cell counts <150 cells/L. The relationship was statistically significant when tested by parametric and nonparametric correlation analysis (Pearson's correlation coefficient of 0.523, P = .005 at the .01 level [2-tailed]; Spearman's correlation coefficient of 0.637, P = .000 at the .01 level [2-tailed]). In the subgroup of patients with KS who had high CD4+ cell counts (>150 cells/L), a weak positive correlation was observed between the mean baseline CD8+ cell count and the mean log10 viral load at entry into the study (Pearson's correlation, 0.600; P = .030 at the .05 level [2-tailed]).
Viral load decay during the first 3 months of therapy.
Table 2 shows the changes in viral load and CD4+ cell counts from baseline over time. After 3 months, the mean ± SD decrease in HIV-1 RNA level was -3.8 ± 0.8 log10 copies/mL in the TB cohort and -3.1 ± 0.3 log10 copies/mL in the KS cohort. As previously reported for HIV-1 subtype B [2529], this decrease occurred in 2 distinct phases: a rapid, near-exponential decrease, which occurred during the first 7 days of treatment (phase I), followed by a slower, more gradual phase II decline that lasted until the virus became undetectable (figure 1A and 1B). Although the pattern of plasma viral load ("VL" in the following equations) decay was similar in both cohorts, the depth ([VLday 7 - VLbaseline]) and steepness ([VLday 7 - VLbaseline/day 7]) of the phase I decline was greater in patients with TB than in those with KS (decrease, -2.32 vs. -1.55 log10 RNA copies/mL, respectively; slope, -0.33 vs. -0.22 log10 RNA copies/day, respectively). The phase I decline in viral load, measured at day 7, was 99.0% in the TB cohort and 94.6% in the KS cohort. These findings indicate that, even in patients with OIs and severe disease, the vast majority of plasma virus (>94%) is still being produced by actively replicating, short-lived effector CD4+ T cells [2631], with the remaining virus (1.0%5.4%) being derived a small pool of long-lived, chronically infected CD4+ CD45RO+ T cells and monocyte macrophages [3238].
Interesting differences were observed between patients with high (>150 cells/L) baseline CD4+ cell counts and those with low (<150 cells/L) baseline CD4+ cell counts. Within the TB cohort, the magnitude and steepness of the phase I viral load decline was greater in the subgroup of patients with baseline CD4+ cell counts >150 cells/L, consistent with the view that patients with HIV-1/TB who have higher CD4+ counts have a larger, or more active, rapidly replicating CD4+ T cell compartment. Conversely, within the KS cohort, the sharpest and most pronounced phase I viral load decline occurred in patients with baseline CD4+ cell counts <150 cells/L. This finding may reflect the extremely low CD4+ cell counts (<10 cells/L) observed in several of the patients with KS in our study. Patients with OIs and severe disease often have a relatively small phase I compartment, as a result of severe depletion of their CD4+ T cells [36].
CD4+ cell restoration during the first 3 months of therapy.
An inverse relationship was observed between the pattern and kinetics of viral load decline and CD4+ cell restoration. The overall increase in CD4+ cell count was greater among patients with TB, compared with that among patients with KS (200 vs. 74 cells/L). In the TB cohort, the mean CD4+ cell count increased from 222.6 ± 135.8 cells/L at baseline to 422.6 ± 221.8 cells/L at 3 months posttreatment. In the KS cohort, the increase was from 206 ± 164.9 cells/L at baseline to 280 ± 135.8 cells/L at month 3. The higher level of CD4+ cell restoration in the TB cohort was not unexpected, given that this group had a larger, more dynamic population of highly productive CD4+ cells. For both cohorts, the steepest increase in CD4+ cell count was observed during the first 7 days of treatment, when large amounts HIV-1 were being cleared from plasma and from the short-lived CD4+ compartment. The overall pattern of the CD4+ cell-count increase was similar in both cohorts, consisting of a transient early increase followed by a small decrease and then a second wave of increase after day 30.
Time to undetectable HIV-1 RNA in plasma.
Frequency distribution estimates of the relative proportion of patients reaching undetectable virus levels (<40 copies HIV-1 RNA/mL plasma) at 7, 14, 28, 60, and 90 days were 15.8%, 30.0%, 52.6%, 78.9%, and 93.8% (Pearson's 2 = 50.5; P < .0001), respectively, for the TB cohort, compared with 0%, 5.0%, 22.2%, 64.7%, and 80.0% (Pearson's 2 = 63.6; P < .0001), respectively, for the KS cohort. As determined by Kaplan-Meier analysis, the mean ± SD time required for HIV-1 clearance from the plasma of patients with TB was 62 ± 4 days, compared with 72 ± 4 days for patients with KS (figure 2). This difference was statistically significant when tested for equality of survival distribution (1 df; P = .0333). Within the TB cohort, the time taken to reach undetectable virus levels was significantly longer in patients with CD4+ counts <150 cells/L (79 ± 6 vs. 57 ± 4 days; 1 df; P = .0211). This finding is consistent with patients with late-stage disease having a larger proportion of long-lived phase II cells [38]. Within the KS cohort, the time taken to achieve undetectable viral load levels was significantly shorter in the subgroup of patients with CD4+ cell counts <150 cells/L (66 ± 5 vs. 78 ± 4 days; 1 df; P = .0572).
Time required to achieve a 25% recovery of CD4+ cells.
Although patients with TB had a greater overall increase in CD4+ cell counts, no statistically significant differences were observed with respect to the mean times required to achieve a 25% recovery of CD4+ cells (42 ± 4 days for patients with KS vs. 50 ± 4 days for patients with TB; 1 df; P = .2137). When the results were stratified on the basis of mean CD4+ cell count at entry, no difference was detected in the timing of the 25% CD4+ cell recovery in patients with TB who had low (<150 cells/L) versus high (>150 cells/L) baseline CD4+ cell counts (mean ± SD, 43 ± 7 vs. 53 ± 4 days, respectively; 1 df; P = .1463). Within the KS cohort, the time needed to achieve a 25% restoration of CD4+ cells was significantly shorter (mean ± SD, 29 ± 4 vs. 64 ± 6 days; 1 df; P < .001) in the subgroup of patients with mean baseline CD4+ cell counts <150 cells/L. This finding may be a reflection of the intense homeostatic pressure exerted on patients with severe CD4+ cell depletion.
DISCUSSION
Our findings, together with recent data from Mozambique [39], suggest that treatment of HIV-1 subtype C infections will have significant virological and immunological benefit, even in the context of OIs and low CD4+ cell counts [40, 41]. The ability to successfully treat HIV-1 in Africa is an urgent priority that has far-reaching social and public health implications, in terms of increased life expectancy and productivity, reduced HIV-1 transmission rates, fewer orphaned children, and increased economic stability.
The short-term success rate of our study, as shown by the precipitous phase I decline in plasma viremia and as measured by the proportions of patients reaching undetectable HIV-1 RNA levels by month 3 (93.8% of patients with TB and 80.0% of patients with KS), is equivalent to or better than that reported in many studies of HIV-1 subtype B. In a recent study conducted in Germany, only 64% of patients treated with zidovudine, 3TC, and EFV had viral loads <50 copies/mL at month 4 [42]. In a study conducted in West Africa, the proportions of Senegalese patients who achieved viral loads <50 copies/mL after 3 and 6 months of therapy with ddI, 3TC, and EFV were 26% and 78%, respectively [22]. The reason for the good response in our study is not known, but it does not appear to be related to drug dosage, since the patients in our study received less efavirenz (600 compared with 800 mg/day). By month 10, the TB cure rate in patients receiving rifampin and 600 mg/day EFV was 89%. One notable difference was that the patients with TB in our study received their treatment through a directly observed therapy (DOT) program, and, therefore, adherence to medication was assured. Patients with KS were carefully monitored by use of pill counts and self-reporting. Further studies are needed to determine whether the good responses in our study are due to adherence or to differences in the turnover and size of the phase I compartment in patients with OIs. Mathematical modeling has suggested that the rate and slope of the phase I decline is related not only to the efficacy of treatment but also to the number of productively infected CD4+ T cells and their pretreatment replication potential [25, 28]. Rapid, initial clearance of HIV-1 from the phase I compartment is highly desirable, since it minimizes do novo infection of newly activated cell populations. This may be particularly important in the context of KS and other chronic conditions, in which new target cells are continually being activated and seeded by ongoing replication.
Our study also underscores the extensive interpatient variability among HIV-1infected patients with OIs. This heterogeneity, together with the lack of any clear-cut correlations between baseline parameters, suggests a complex interaction between HIV-1 replication, antigen-driven immune activation (either HIV-1 or OI driven), and CD4+ cell depletion. Of particular interest was the lack of a relationship between baseline viral load and CD4+ cell count. Overall, 71.4% of patients with TB and 50.0% of patients with KS had baseline CD4+ cell counts >150 cells/L. Within this subset, 20% of patients with TB and 38.5% of patients with KS had CD4+ cell counts >400 cells/L, including 1 patient with KS who had a CD4+ cell count of 523 cells/L. The detection of severe OIs in the absence of profound CD4+ cell depletion may have important implications for the timing of ART and for the stratification and selection of patients on the basis of CD4+ cell counts at the time of enrollment [23, 24]. At the other end of the spectrum, 4.8% of patients with TB and 10.2% of patients with KS had substantial CD4+ cell depletion in the absence of high-level viremia (i.e., <10,000 HIV-1 RNA copies/mL plasma). The reason for the excessive destruction of CD4+ cells in these patients is not known, but it may be related to enhanced turnover of uninfected CD4+ bystander cells [43, 44]. Several studies have shown that the depletion and turnover of infected (and uninfected) T cells is greatly increased in patients with severe disease and that this process is directly linked to immune activation but only indirectly linked to viral load [45]. Although unproven, the increased steepness and depth of the phase I decline in the TB cohort compared with that in the KS cohort suggests that, at the beginning of ART, patients with TB had a larger, or more dynamic, pool of short-lived HIV-1infected CD4+ cells. Conversely, the more shallow phase I decline followed by a slower, more protracted phase II decline in the KS cohort suggests that these patients had a larger phase II compartment.
Whether the primary agent(s) driving immune activation is HIV-1 alone or a combination of HIV-1 and OI remains to be established [4650]. It is reasonable to expect that different pathogens would produce different activation stimuli and that these stimuli would have diverse effects on the size and kinetics of viral clearance from phase I and II reservoirs. Irrespective of the reason, it is becoming increasingly recognized that an improved understanding of phase II virus and its responsiveness to ART will be critical to the eradication of HIV-1 [5155]. By using high-resolution therapeutic monitoring, it is now possible to begin deciphering the complex interplay between HIV-1 and OIs. When combined with new tools that measure viral replication in specific cell types [5659] and with mathematical modeling of immune activation and target cell availability [2532], this approach will provide a powerful tool for unraveling the impact of OIs on HIV-1 disease. An understanding of these processes is likely to be critical to the optimization of treatment strategies in southern Africa, a region where OIs may play a major role not only in the pathogenesis of HIV-1 disease but also in patient response to therapy. OIs are a major problem for patients failing therapy, and this problem is likely to increase as resistance to antiretroviral drugs becomes more widespread. This study serves to stimulate debate on the role of OI-induced immune activation as a factor contributing to the severity of the AIDS epidemic in Africa and to motivate the development of viral load assays that measure intracellular and tissue reservoirs of HIV-1. Such assays have been developed and are currently undergoing intensive evaluation [38, 5659].
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