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New commercial techniques for determination of the viral load (VL) in plasma are able to detect as few as 20 copies of HIV-1 RNA/ml. The relevance of this new technical threshold is uncertain. Upon multivariate analysis, factors associated with detection of VLs between 20 and 49 copies/ml by the Cobas TaqMan HIV-1 v2.0 assay in an HIV clinic were the basal VL and time on antiretroviral therapy.
For patients on antiretroviral therapy (ART), the currently accepted threshold for complete suppression of HIV-1 replication is 50 copies (cp) of HIV-1 RNA/ml (3). By ultrasensitive techniques, it is possible to detect viral particles in plasma samples from most patients on suppressive ART. This residual viremia is not considered to represent true replication but particle release from latently infected cells (4). Commercial techniques are now able to detect viral loads (VL) below 50 cp/ml; however, the relevance of detecting viremia at levels below 50 cp/ml in the clinical setting is uncertain. This issue is particularly important since the majority of patients on treatment are now in this category. The Cobas AmpliPrep/Cobas TaqMan HIV-1 v2.0 test is a newly introduced nucleic acid amplification test for the quantitation of HIV-1 RNA in human plasma. The test uses the Cobas AmpliPrep instrument for automated nucleic acid extraction and the Cobas TaqMan analyzer for amplification and quantitation of HIV-1 RNA over the range of 20 to 10,000,000 cp/ml.
In order to study the factors associated with the presence of HIV-1 viremia at levels below 50 RNA cp/ml, we selected a group of patients undergoing suppressive ART and routinely monitored in the HIV clinic of the Hospital Universitario 12 de Octubre, Madrid, Spain, by clinical and laboratory evaluations every 3 to 4 months. Specific inclusion criteria were (i) start of first ART between January 2006 and December 2008, (ii) sustained suppression of viral replication (VL of <50 cp/ml by week 24 of ART and thereafter), and (iii) testing by the Cobas TaqMan HIV-1 v2.0 assay during the first 4 months of its use in the HIV unit (May through August 2009). A single VL determination by the TaqMan HIV v2.0 assay was obtained for each patient and evaluated during this period. Of the 93 patients who fulfilled the inclusion criteria, 10 were excluded due to noncompliance with ART during the follow-up (n = 3) or suspension of therapy at any time since the initiation of therapy (n = 7).
We performed a multivariate regression analysis to assess the factors associated with the presence of HIV loads between 20 and 49 cp/ml. We analyzed the following independent variables: sex, age, previous AIDS diagnosis, HIV load and CD4 cell count at the start of ART, initial ART regimen, ART regiment at the time of VL assessment, and time on ART. For the multivariate model, variables that were associated with HIV loads between 20 and 49 cp/ml in the univariate analysis with a P value of less than 0.1 were included in a forward stepwise fashion. The statistical analyses were carried out using the SPSS 11.0 software package (SPSS Inc., Chicago, IL). For all the patients included in the study, the median age was 39.6 years (interquartile range [IQR], 32.8 to 45.7 years); 68 patients were men, and 45 had a previous AIDS diagnosis. The median CD4 cell count and VL before ART were 253 cells/μl (IQR, 135 to 373 cells/μl) and 5.02 log RNA cp/ml (IQR, 4.68 to 5.32 log RNA cp/ml), respectively. The median time on ART was 83.3 weeks (IQR, 56.9 to 139.6 weeks). The factors associated with VL between 20 and 49 cp/ml in univariate and multivariate analyses are shown in Table Table1.1. In our study, 21 patients (25.3%) undergoing conventional suppressive ART (VL, <50 cp/ml) had viremia detectable by the Cobas TaqMan HIV-1 v2.0 assay (VL, 20 to 49 cp/ml) (Table (Table1).1). This finding is not surprising since a number of studies have found circulating HIV particles in samples from most patients undergoing suppressive ART by using ultrasensitive single-copy assays (SCA) (5-7). The currently accepted concept is that this residual viremia does not represent new cycles of viral replication but particles released from the latently infected cell reservoir. This concept is supported mainly by the lack of temporal structure of residual HIV sequences and the evidence that different intensification strategies do not modify the level of residual viremia (2, 4, 8). The studies providing these data have been performed in the research setting with highly sensitive, locally developed SCA. Recently, the availability of standardized commercial techniques with greater sensitivity allows testing of this hypothesis with a larger population of patients in the clinical setting.
For our group of patients on suppressive therapy, we found that the presence of viremia at levels between 20 and 49 RNA cp/ml was significantly associated with both higher basal VL and less time on ART (Table (Table1).1). In previous studies (5, 6), a significant correlation between pretherapy VL and the level of residual viremia determined by SCA for patients on suppressive ART has been shown clearly. This finding suggests that the size of the cellular compartment infected prior to the initiation of ART is directly related to the level of residual viremia. However, the pattern of decay of VL below 50 cp/ml after treatment is not completely clear. Residual viremia decay in patients with more than 60 weeks of treatment and overall median viremia levels around 3 RNA cp/ml has been studied previously (5, 6). In the study by Maldarelli et al., no significant decline in the level of viremia was found after 60 weeks of suppressive ART (5). However, for samples from weeks 60 to 384, Palmer et al. detected biphasic evolution of residual viremia in an analysis performed by using SCA, suggesting the existence of two cellular reservoirs with different half-lives (6). We did not perform a longitudinal study of VL below 50 cp/ml to measure the evolution of this low-level viremia, and an estimation of the decay rate is thus lacking. The temporal pattern of low-level viremia during suppressive treatment as shown in our study by a commercial technique may be related to particle release from these latent cellular reservoirs; however, the possibility that ART is controlling a low level of viral replication even at VL below 50 cp/ml cannot be excluded, and its consequences may be important. Our findings suggest potential value in quantifying HIV-1 loads below 50 cp/ml. In this regard, the detection of an increase in residual viremia in some patients undergoing simplification regimens by SCA before virologic failure has been reported recently (1, 9). The clinical relevance of the presence of HIV-1 viremia at levels between 20 and 49 RNA cp/ml deserves further investigation, and outcome studies should be performed.
J.F.P.-P. is supported by a grant from the FIS Rio Hortega program (CM07/00216). S.F. is supported by the Fundación de Investigación Biomédica Hospital 12 de Octubre. The study has been supported by grants FIPSE 36749, FIS-PI080806, and Carmusys (EC FP7-PEOPLE-2007-1-1-ITN) to R.D.
Published ahead of print on 24 February 2010.
‡The authors have paid a fee to allow immediate free access to this article.