HIV-RNA quantification is a sensitive indicator of the effectiveness of highly active antiretroviral therapy (HAART). HIV RNA measurements 2-6 months after treatment initiation correlate with long-term virological outcomes [1van Leth F, Andrews S, Grinsztejn B, et al. The effect of baseline CD4 cell count and HIV-1 viral load on the efficacy and safety of nevirapine or efavirenz-based first-line HAART AIDS 2005; 19: 463-71., 2Guidelines for the use of antiretroviral agents in HIV-infected adults and adolescents U.S Department of Health and Human Services(DHHS), www.aidsinfo.nih.gov [January 29, 2008];]. Successful HAART is generally defined as HIV RNA suppression to below 50 copies/ml although low level replication continues even when HIV RNA is undetectable by conventional assays [3Yerly S, Perneger TV, Vora S, Hirschel B, Perrin L. Decay of cell-associated HIV-1 DNA correlates with residual replication in patients treated during acute HIV-1 infection AIDS 2000; 14: 2805-12.-5Palmisano L, Giuliano M, Nicastri E, et al. Residual viraemia in subjects with chronic HIV infection and viral load < 50 copies/ml: the impact of highly active antiretroviral therapy AIDS 2005; 19: 1843-7.].

Staccato investigated CD4-guided structured treatment interruption (STI) of HAART and found that the rate of virological failure was low (2%) and similar to those who took HAART continuously [6Ananworanich J, Gayet-Ageron A, Le Braz M, et al. CD4-guided scheduled treatment interruptions compared with continuous therapy for patients infected with HIV-1: results of the Staccato randomised trial Lancet 2006; 368: 459-65.]. While most STI patients in our study achieved HIV RNA suppression below 50 copies/ml following HAART re-treatment, it is possible that slow rises in HIV RNA with successive STI cycles occur and result in subsequent virological failure in some patients. In this sub-study, we investigated the value of a modified version of the Roche AMPLICOR Monitor 1.5 protocol with a limit of detection of 5 copies/ml, in predicting virological failure after STI. We hypothesized that compared to patients with HIV RNA < 5 copies/ml, those with HIV RNA between 5-49 copies/ml following HAART re-treatment were more likely to have virological failure after CD4-guided STI.

We included 11 cases and 22 controls. The baseline characteristics of cases and controls are presented in Table 1 and compared to those of the Thai population included into Staccato and randomized in the STI arm. Cases and controls were similar, except that cases had significantly more STI cycles than controls (p=0.02). The case-control population was similar to the entire Thai population included in the STI arm of Staccato, except that Thai patients were significantly older than patients selected in the case-control study (p=0.04).

Table 1.

Characteristics of STI Participants in this Substudy and in the Whole Staccato Thai Cohort

Table 2.

Median HIV RNA in Copies/ml (Interquartile Range) Between Cases and Controls After HAART Re-Treatment

Table 2 shows the median HIV RNA values for the cases and controls, with their interquartile range (IQR), at each STI re-treatment cycle. In cases, the median (IQR) duration of the first and the second STI cycles weres 24 and 36 weeks respectively. In controls, the median duration of the first cycle was 12 weeks, and 24 weeks for the second cycle. Using modified HIV RNA HIV RNA assay with a detection limit of 5 copies/ml, we did not find any difference between cases and controls on log10 HIV RNA at entry (p=0.24). After the first cycle of re-treatment, there was a trend to a higher HIV RNA in cases compared to controls (p=0.06). After the second cycle of re-treatment, there was no significant difference cases and controls regarding the median HIV RNA (p=0.32). When we used the last available HIV RNA below 50 copies/ml for cases and controls, we found that cases had a significantly higher median HIV RNA than the controls (p=0.004). Variations in trend across study time-points showed a significant increase in log10 HIV RNA (p-trend=0.004) only due to a significant increase in cases (p-trend<0.001 vs 0.55 in controls).

By univariate and multivariate conditional logistic regression model, there was a trend for a higher probability of final virological failure when the last available HIV RNA was between 5-49 copies/ml compared to HIV RNA below 5 copies/ml (OR 7.77, 95%CI 0.89-67.5, p=0.06). There was no significant association between the risk to have HIV RNA between 5-49 copies/ml and risk factor, CDC classification and pre-HAART CD4.

With each successive treatment interruption, the differences in median HIV RNA between virological failure cases and controls increased, reaching statistical significance with the last available HIV RNA prior to the protocol-mandated HAART re-treatment at the end of the trial. This finding implies that over the course of multiple interruptions there is a trend towards higher residual viremia amongst those who ultimately experienced treatment failure than in those who maintained low HIV RNA.

Our result is similar to the ISS PART study which showed increased residual HIV viremia between 2.5 to 50 copies/ml in virological failure cases after two years of a step-wise fixed time strategy with five STIs of one, one, 2, 2 and 3 months’ duration, each followed by 3 months of HAART [7Palmisano L, Giugliano M, Bucciardini R, Eds. Residual viremia, but not HIV DNA load, increases in subjects undergoing repeated treatment interruptions: a substudy of ISSPART Clinical Trial [Abstract 515] In: 14th Conference on Retroviruses and Opportunistic Infections; February 15-28; Los Angeles. 2007.]. The ISS PART subjects were different to the Staccato subjects. They were mostly antiretroviral-pre treated and had archived resistance mutations. The Staccato Thai subjects were HAART-naïve and likely had no archived mutations [6Ananworanich J, Gayet-Ageron A, Le Braz M, et al. CD4-guided scheduled treatment interruptions compared with continuous therapy for patients infected with HIV-1: results of the Staccato randomised trial Lancet 2006; 368: 459-65.]. This illustrates that the finding of increased residual viremia after STI may apply to both those with and without pre-existing risk for virological failure.

The treatment failures seen after STI have mostly been attributed to the replication of archived resistant quasispecies that existed in proviral DNA prior to STI. Selection of new mutations have been reported in some studies especially those that use antiretrovirals with low resistance threshold such as lamivudine and non-nucleoside reverse transcriptase inhibitors [8Metzner KJ, Bonhoeffer S, Fischer M, et al. Emergence of minor populations of human immunodeficiency virus type 1 carrying the M184V and L90M mutations in subjects undergoing structured treatment interruptions J Infect Dis 2003; 188: 1433-43., 9Dybul M, Nies-Kraske E, Daucher M, et al. Long-cycle structured intermittent vs continuous highly active antiretroviral therapy for the treatment of chronic infection with human immunodeficiency virus: effects on drug toxicity and on immunologic and virologic parameters J Infect Dis 2003; 188: 388-96.]. The short duration of HAART-retreatment after each STI cycle may be inadequate to control HIV viral replication in viral reservoirs and compartments.

Currently, the goal of HAART is to maintain HIV RNA below 50 copies/ml [2Guidelines for the use of antiretroviral agents in HIV-infected adults and adolescents U.S Department of Health and Human Services(DHHS), www.aidsinfo.nih.gov [January 29, 2008];]. Studies have shown continued viral replication at levels below 50 copies/ml despite HAART [3Yerly S, Perneger TV, Vora S, Hirschel B, Perrin L. Decay of cell-associated HIV-1 DNA correlates with residual replication in patients treated during acute HIV-1 infection AIDS 2000; 14: 2805-12., 4Palmer S, Maldarelli F, Wiegand A, et al. Low-level viremia persists for at least 7 years in patients on suppressive antiretroviral therapy Proc Natl Acad Sci USA 2008; 105: 3879-84.]. Palmer et al. used an HIV-1 RNA assay with single-copy sensitivity to show that 77% of 40 HAART-treated patients with HIV RNA below 50 copies/ml for at least 2 years had detectable low-level viremia, and that all patients had at least one sample with detectable viremia [4Palmer S, Maldarelli F, Wiegand A, et al. Low-level viremia persists for at least 7 years in patients on suppressive antiretroviral therapy Proc Natl Acad Sci USA 2008; 105: 3879-84.]. The significance of this finding is unclear. It may explain the viral blips that occurs frequently but do not appear to lead to virological failure with both protease inhibitor and non-nucleoside reverse transcriptase treatments [10Nettles RE, Kieffer TL, Kwon P, et al. Intermittent HIV-1 viremia (Blips) and drug resistance in patients receiving HAART JAMA 2005; 293: 817-29.-12Martinez V, Marcelin AG, Morini JP, et al. HIV-1 intermittent viraemia in patients treated by non-nucleoside reverse transcriptase inhibitor-based regimen AIDS 2005; 19: 1065-9.]. Continued viremia at varying HIV RNA levels albeit below 50 copies/ml may have different effects on the HIV reservoirs. Persistence of viral reservoir particularly the latently infected CD4+ T cells has been a major obstacle to HIV eradication [13Chun TW, Justement JS, Moir S, et al. Decay of the HIV reservoir in patients receiving antiretroviral therapy for extended periods: implications for eradication of virus J Infect Dis 2007; 195: 1762-4., 14Re MC, Vitone F, Bon I, Schiavone P, Gibellini D. Meaning of DNA detection during the follow-up of HIV-1 infected patients: a brief review New Microbiol 2006; 29: 81-.]. With new HAART becoming more effective, it is possible that in the future suppressing HIV to the lowest possible level using HIV RNA monitoring assay such as that in our study may be desired in an attempt to limit the chance of HIV rebound and the size of HIV reservoir.

Our study is limited by its small sample size and the highly selective nature of the case-control matching which may limit the generalizability of the results to other populations. Nevertheless, by utilizing a modified HIV RNA method, we demonstrated that a higher residual viremia between 5 and 50copies/ml after HAART re-treatment may predict those who would ultimately experienced treatment failure from STI.