The current goal of antiretroviral therapy is to suppress plasma HIV-1 RNA to below 50 copies/ml, which is the detection limit for commercially available HIV-1 RNA assays. To achieve this goal, life-long therapy with at least three antiretroviral drugs from two different drug classes is recommended . These multi-drug regimens are associated with acute and chronic toxicities, drug–drug interactions, and substantial cost [2–5]. Accordingly, induction therapy followed by simplified maintenance therapy with a single protease inhibitor (PI) boosted with ritonavir is being evaluated. Recent pilot trials of simplified maintenance therapy with a ritonavir-boosted PI have shown promise.
The ‘Only Kaletra’ (OK) study  was a randomized comparison of standard of care (SOC) therapy with two nucleoside reverse transcriptase inhibitors (NRTI) and ritonavir-boosted lopinavir (LPV/r) versus simplified maintenance therapy with LPV/r alone. By intent-to-treat analysis, HIV-1 RNA levels remained suppressed to < 50 copies/ml in 17 of 21 (81%) subjects randomized to LPV/r alone at 48 weeks compared to 20 of 21 (95%) subjects in the SOC arm. In the LPV/r alone arm, there were three virologic failures (confirmed viral load > 500 copies/ml) and one patient was lost to follow-up. In the SOC arm, one patient discontinued the study because of dyslipidemia. Standard genotype analysis of samples from subjects who experienced virologic failure in the LPV/r alone arm did not detect the emergence of new PI resistance mutations.
Ritonavir-boosted atazanavir (ATV/r) has also been evaluated as maintenance therapy in two pilot studies. In the ATARITMO study , 24 subjects simplified therapy to ATV/r alone and 92% remained suppressed below 50 copies/ml at 24 weeks. Similarly, in the ACTG study A5201 , 34 subjects simplified therapy to ATV/r and 31 (91%) remained suppressed < 50 copies/ml through 24 weeks.
Despite these encouraging results, there are concerns that simplifying therapy to a single boosted-PI could reduce inhibition of viral replication, leading to drug resistance and virologic failure. This concern is justified because the majority of patients with HIV-1 RNA suppressed to below 50 copies/ml on standard antiretroviral therapy have persistent viremia that is measurable with more sensitive HIV-1 RNA assays [9,10]. We therefore assessed the impact of simplifying therapy on the level of persistent viremia by testing longitudinal plasma samples from subjects in the OK trial using an HIV-1 RNA assay with a detection limit of 3 copies/ml.
HIV-1 testing was performed on stored plasma samples (2.0 ml) from the OK trial . Plasma samples were obtained at the screening visit, week 0 (study entry and randomization), and weeks 4, 8, 12, 24, and 48. Longitudinal samples from a given subject were batch tested to eliminate inter-assay variation. Results from the screening and week 0 visits were averaged to define baseline viremia.
Modified ultraSensitive HIV-1 RNA assay
All samples were tested using a modified Amplicor HIV-1 RNA assay version 1.5 (Roche Molecular Systems, Branchburg, New Jersey, USA) with a limit of detection of 3 copies/ml [9,11]. The modifications included pelleting of virus from 2 ml of plasma (23 600 × g at 4 °C for 2 h), addition of half the normal volume of quantification standard, and re-suspension of the RNA pellet in 50 μl of diluent. HIV-1 RNA standards (12.5, 6.25 and 3.125 copies/ml) were obtained from the Virology Quality Assurance Laboratory (Rush-Presbyterian-St. Luke's Medical Center, Chicago, Illinois, USA) and were included with each run along with one negative control (0 copies/ml). Samples yielding an OD450 < 0.2 with an internal quantification standard within the expected range were reported as having no detectable HIV-1 RNA (0 copies/ml). For samples yielding an OD450 > 0.2 with a calculated HIV-1 RNA level between 0 and 3 copies, the observed values were used for the analyses.
Student's t test was used to compare mean values for HIV-1 RNA standards and the Wilcoxon–Mann–Whitney test was used to compare median HIV-1 RNA values across treatment arms or patient subgroups (e.g., those with virologic failure versus sustained suppression). A non-parametric test was used because the number of patients in each study arm was small and no assumptions of normality could be made. Quantile regression analysis was performed to assess trends in viremia for each study arm. We used 95% confidence intervals and an alpha value of 0.05 to compare the slope of viremia over time to zero. Analyses were performed using SPSS statistical software v.11.5.0 and Stata statistical software v 8.0. [12,13] Power calculations were performed using the NCSS/PASS software .
Samples and testing
In the OK study, 42 subjects were randomized to continue standard of care therapy (SOC arm) with LPV/r and two NRTI or discontinue NRTI (LPV/r alone arm). Follow-up was for 48 weeks after randomization. Three virologic failures (confirmed HIV RNA > 500 copies/ml) and one lost to follow-up occurred in the LPV/r alone arm and one treatment discontinuation occurred in the SOC arm. Stored plasma samples from baseline and weeks 4, 8, 12, 24, 48 were available for HIV-1 RNA testing from all patients participating in the trial, including 21 patients from the LPV/r alone arm and 21 from the SOC arm. Samples from one subject in the SOC arm could not be analyzed at any timepoint because the plasma extract inhibited RT–PCR including the quantification standard. Samples were tested in 34 assay runs that included a panel of HIV-1 RNA standards (0, 3.125, 6.25, and 12.5 copies/ml) with each run. Longitudinal samples from a given subject were tested in batch. The mean ± SEM HIV-1 RNA values for the 0, 3.125, 6.25, and 12.5 copies/ml standards across the 34 runs were 0, 2.1 ± 0.34, 5.2 ± 0.5, and 10.9 ± 0.9, respectively (Fig. 1). The differences in mean HIV-1 RNA values for each of the four standards were highly significant (P < 0.001).
Baseline and longitudinal HIV-1 RNA levels across treatment arms
Fifty-seven of 82 (69.5%) samples tested from study entry (41 subjects) had detectable HIV-1 RNA (OD450 > 0.200) with a median value of 3.1 copies/ml [interquartile range (IQR), 0.0–8.3]. Among the 37 patients who continued on study treatment and remained virologically suppressed through 48 weeks, the median level of viremia did not change significantly between baseline and weeks 4, 8, 12, 24 and 48. There were also no significant differences in median HIV-1 RNA levels between the LPV/r alone arm and the SOC arm at any time point (Fig. 2). Specifically, median HIV-1 RNA values for subjects who remained suppressed in the LPV/r arm versus the SOC arm at baseline and weeks 4, 8, 12, 24, and 48 were 5.1 versus 3.0 (P = 0.29), 4.5 versus 2.9 (P = 0.44), 3.3 versus 2.9 (P = 0.99), 1.9 versus 1.0 (P = 0.68), 3.7 versus 3.6 (P = 0.49), and 2.8 versus 1.6 (P = 0.78), respectively. Median HIV-1 RNA values between treatment arms were not significantly different when subjects experiencing virologic failure were included in the analysis. The median (IQR) values for the LPV/r alone arm with failure subjects included at baseline and weeks 4, 8, 12, 24 and 48 were 4.7 (0–10.7), 4.0 (1.3–8.1), 4.2 (0.4–9.39), 3.2 (1.2–6.9), 3.8 (0–33.2) and 2.8 (0.9–4.2), respectively. Quantile regression analysis of HIV-1 RNA values for each treatment arm (not including virologic failures) through 48 weeks showed a slope of −0.035 with a 95% confidence interval (CI) (−0.109 to 0.04) for the LPV/r arm and a slope of −0.039 with a 95% CI (−0.124 to 0.046) for the SOC arm. The slopes were not significantly different between arms or from zero. In addition, there were no differences in the proportion of subjects with undetectable HIV-1 RNA (OD450 < 0.2) between study arms at any time point (Table 1). Based on the variance of the HIV-1 RNA values and the number of patient samples tested, there was 80% power to detect a difference in the overall median values between study arms of greater than 4 copies/ml (alpha, 0.05).
HIV-1 RNA levels in virologic failures versus non-failures
In the LPV/r alone arm, HIV-1 RNA levels were compared in subjects experiencing virologic failure versus those remaining suppressed. At baseline, the median HIV-1 RNA levels were not statistically significantly different between the two subgroups (P = NS). The median (IQR) for the HIV-1 RNA values in the subjects with virologic failure in the LPV/r alone arm during the study at baseline and weeks 4, 8, 12 and 24 were 2.0 (0.0–4.2), 3.7 (3.2–4.7), 11.2 (5.3–16.4), 13.7(4.0–1825.7) and 564 (212–1270), respectively. Those experiencing failure showed a trend toward higher HIV-1 RNA values at week 8 (P = 0.09) that reached statistical significance at week 24 (P = 0.007) compared to non-failures (Fig. 3). In each of the three subjects experiencing virologic failure (confirmed HIV-1 RNA > 500 copies/ml) after discontinuing NRTI, an increase in HIV-1 RNA to between 15 and 50 copies/ml was observed before rising above 50 copies/ml. The interval between the increase above 15 copies/ml and the increase above 50 copies/ml was 4 weeks for two subjects and 8 weeks for the third subject.
This is the first study to quantify HIV-1 RNA below 50 copies/ml in subjects who have simplified therapy from a standard combination regimen to a single ritonavir-boosted PI by discontinuing NRTI. To avoid bias, samples were obtained from a randomized trial of simplified therapy with LPV/r alone versus continued SOC therapy. Samples from 97% of the study participants were tested and analyzed. The results indicate that the majority of individuals suppressed on standard therapy have persistent, quantifiable viremia with a median value of approximately 3 copies/ml, which agrees with recent reports by others [10,15–17]. Importantly, our data also show that the level of persistent viremia did not change after discontinuing NRTI therapy in subjects who remained suppressed < 50 copies/ml. This finding was consistent across multiple analyses that included longitudinal assessments through 48 weeks of mean and median HIV-1 RNA levels, HIV-1 RNA slope, and the proportion of samples with no detectable HIV-1 RNA (OD450 < 0.20).
Three of the subjects randomized to the LPV/r alone arm experienced virologic failure. In each of these subjects, an increase in persistent viremia after discontinuing NRTI above 15 copies/ml was detected before protocol defined virologic failure occurred. The interval between the first detected increase in HIV-1 RNA levels and the rise above 50 copies/ml ranged from 4 to 8 weeks. This confirms that the HIV-1 RNA assay used in this study could detect significant changes in HIV-1 RNA at levels below 50 copies/ml. Virologic failure on the LPV/r alone study arm was associated with incomplete medication adherence . Our analyses did not reveal any difference in baseline HIV-1 RNA levels between the three subjects experiencing virologic failure in the LPV/r alone arm and those who remained suppressed < 50 copies/ml, which is consistent with non-adherence leading to virologic failure, rather than differences in baseline virus suppression. Nevertheless, additional studies are needed to identify baseline factors that predict failure of simplified maintenance therapy.
Our findings also suggest that more sensitive HIV-1 RNA detection methods could influence the management of antiretroviral therapy. Earlier detection of rising HIV-1 RNA levels below 50 copies/ml could provide a warning of impending treatment failure. Interventions at this stage might prevent overt treatment failure and could reduce the development of drug resistance, although additional studies are required to establish this.
Our study findings suggest that long-term suppression of viremia can be maintained with a single boosted-PI/r. Continued use of NRTI may prove to be unnecessary in 80–90% of patients after successful induction therapy. This may spare the costs and long-term side effects associated with NRTI therapy. However, HIV-1 RNA testing of specimens other than plasma, such as genital tract secretions and cerebrospinal fluid, are needed to assess the safety and efficacy of this approach. Additional randomized clinical trials that include sampling of multiple compartments are in development.
The source of persistent viremia in patients on standard antiretroviral therapy is uncertain, but may arise from: (i) ongoing cycles of viral replication in an anatomic or physiologic sanctuary into which antiretroviral penetration is incomplete; (ii) expression of HIV-1 from the latent CD4 memory T-cell reservoir; (iii) continued production of HIV-1 from a long-lived infected cell population, or some combination of these possibilities . The comparable levels of viremia before and after stopping NRTI favor the production of HIV-1 from chronically infected cells, rather than ongoing cycles of HIV-1 replication. If the latter were occurring, the level of viremia would be expected to increase after stopping NRTI, unless viremia originates from a drug ‘sanctuary’ that is not influenced at all by NRTI. Treatment intensification studies with drugs having differing pharmacokinetic properties, especially tissue and cellular penetration, are needed to probe for such a sanctuary.
The higher rate of failure in the LPV/r alone arm warrants cautious evaluation of simplified maintenance therapy and careful identification of appropriate patients for this strategy. Medication adherence is likely to play a critical role in the efficacy of simplified maintenance therapy because missed doses of a single ritonavir-boosted PI alone may well have a greater impact on efficacy than the same number of missed doses of a three drug combination with potentially longer half lives. In addition, baseline virologic and immunologic factors should be sought that are associated with successful simplified therapy to maximize efficacy. Effective simplified maintenance therapy could have important practical and financial benefit, especially in resource-limited settings.
In summary, our findings indicate that a single boosted-PI can maintain suppression of viremia to a similar degree than standard combination antiretroviral therapy in the majority of patients. This provides support for continued studies of simplified maintenance therapy for HIV-1 infection.
Sponsorship: The ‘Only Kaletra’ study was supported by an unrestricted grant from Abbott Laboratories. This study was supported by grants from the Fundación de Investigación Médica Mutua Madrilena (MUTUA 2005-066), the National Cancer Institute (SAIC contract 20XS190A) and from the National Institute of Allergy and Infectious Diseases (T32 AI007333). JEM is the recipient of a K-12 clinical research scholar program award from the National Institute of Health (IK12 HD049109-01).
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Keywords:© 2006 Lippincott Williams & Wilkins, Inc.
antiretroviral therapy; lopinavir/ritonavir maintenance therapy; persistent viremia; HIV-1 RNA; viral load