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Advances in detection and monitoring of plasma viremia in HIV-infected individuals receiving antiretroviral therapy
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Current Opinion in HIV & AIDS: POST AUTHOR CORRECTIONS, 9 January 2013
Palmer, Sarah
Abstract
Purpose of review: This review will describe advances in detection and results of monitoring persistent viremia in patients on long-term suppressive therapy. In addition, the review explores the usefulness of these methods in determining the effectiveness of new HIV-1 eradication strategies in purging persistent HIV-1 reservoirs.
Recent findings: Quantification of plasma HIV-1 RNA levels remains essential for determining the success of combination antiretroviral therapy (cART) in treated patients. Recently, several new platforms with improved sensitivity for quantifying HIV-1 RNA have been developed and the application of these assays has revealed that low-level viremia persists in patients on suppressive therapy. In addition, new technological advances such as digital PCR have been proposed to increase the sensitivity of measuring and characterizing persistent HIV-1 viremia. The application of these assays will be important in determining the effectiveness of future HIV-1 eradication strategies.
Summary: The level of HIV-1 RNA in patient plasma remains an important marker for determining the success of cART. New sensitive assays have found that HIV-1 persists in the plasma of patients on suppressive therapy that may have implications for the clinical management of this disease and strategies for eliminating HIV-1 infection.
Excerpts:
CONCLUSION
The level of HIV-1 RNA in patient plasma remains an important marker for determining the success of cART. New sensitive assays have revealed that HIV-1 persists in the plasma of nearly 80% of patients on suppressive therapy. Understanding the source and dynamics of this persistent viremia is essential for developing effective strategies for HIV-1 eradication. In addition, new technologies may improve the quantification of plasma HIV-1 RNA levels during and after latent reservoir reactivation as part of future HIV-1 eradication efforts.
the new third-generation real-time methodologies, Roche Taqman v2.0 and Abbott RealTime 0.6 ml, have broader reportable ranges and are the most sensitive with lower detection limits of 20 and 40 copies/ml, respectively [20-26]. Additional comparative studies have shown better detection rates of citrated versus EDTA plasma control samples containing 25-50 copies/ml by these new real-time assays [27]. This may be due to EDTA chelating the manganese in the PCR mix after RNA extraction that can affect the efficiency of the PCR reaction. However, these third-generation real-time PCR assays were very specific and no evidence of carryover contamination leading to false positives was detected [27]. Overall, the lower detection limits of the new real-time assays may result in increased measurements of viral blips or transient detectable viral RNA and the impact of these measurements on clinical studies will have to be evaluated [28]. The increased occurrence of measurable low-level HIV-1 RNA by these more sensitive real-time assays should be carefully interpreted. HIV-1 treatment is not curative; rather, the virus persists and is merely suppressed during therapy. Although low-level viral RNA has been found to be a predictor of subsequent suboptimal virological control, it may not lead to virological failure or require a regimen change [29,30].
Application of experimental assays with sensitivities in the range of 10 copies/ml has demonstrated that many patients with viremia suppressed to less than 50 copies/ml have persistently detectable HIV-1 RNA in plasma [31,32]. To investigate the source of residual viremia and to quantify plasma HIV-1 RNA below 50 copies/ml, a more sensitive and precise assay than had previously existed was developed. This assay uses larger plasma sample volumes (7 ml), improved nucleic acid isolation and purification techniques, and real-time RT-PCR to accurately quantify HIV-1 in plasma samples to 1 copy/ml with a limit of detection of one copy in 3.8 ml of plasma, making it 20-50 times more sensitive than currently approved commercial assays [15]. Because the assay has a detection limit of 1 RNA copy it is known as the single copy assay (SCA).......Additional studies using this assay have shown that 77% of samples from patients with plasma HIV-1 RNA consistently below 50 copies/ml had quantifiable viremia......... The clinical significance of low-level viremia, especially between 1 and 10 copies/ml, in patients on longterm suppressive therapy is currently not established. However, these data suggest that there is a constant source of viremia in patients on long-term suppressive therapy....... the source of persistent viremia is long-lived HIV-infected cells and increases in episomal HIV DNA indicating active replication persists in some infected individuals on suppressive therapy [35,36]. These results indicate that low-level viremia could arise from several different sources. These sources may include ongoing replication cycles in cells located in sanctuary sites wherein drug levels are suboptimal, long-lived HIV-infected cells that replicate and produce virus, and/or activation of viral expression from latently infected cell reservoirs [13,33,37-39]. Evidence for the view that long-lived cells are the probable source of persistent viremia includes four studies using a number of different treatment protocols that show no perceptible change in persistent viremia in patients receiving intensified treatment [36,40,41& ,42]. Moreover, genetically homogeneous sub-populations can often be observed in patients under long-term treatment, as well as in the viral population that rebounds during treatment interruption further indicating that persistent viremia arises from long-lived latently infected cells rather than ongoing cycles of replicating virus [43,44]. The role of ongoing HIV-1 replication in tissue compartments and cellular reservoirs, however, remains to be defined as a number of findings suggest that the reservoir is largely established and maintained in tissues, and that the infected cells circulating in blood may not necessarily be representative of the much larger population of infected cells in tissue [45&& ]. For example, sequences of persistent HIV populations in plasma are often not found in peripheral blood resting memory CD4. T cells [43,46]. On the contrary, a recent study of two patients found that HIV-1 sequences isolated from virus outgrowth assays of resting memory CD4. T cells matched the predominant plasma clones [47& ]. These results indicate that persistent viremia is derived in part from reactivation of latent proviruses from resting memory CD4. T cells.
PLASMA VIREMIA DURING DISRUPTION OF HIV-1 LATENCY IN PATIENTS ON LONG-TERM SUPPRESSIVE THERAPY
One well defined latent HIV-1 reservoir is infected resting memory CD4. T cells carrying an integrated form of the viral genome [48]. The precise molecular mechanisms contributing to the generation and maintenance of this reservoir, however, remain to be elucidated. The scarcity of such cells, even among those carrying integrated HIVDNA, makes these cells extremely difficult to identify, isolate, and study. However, this latent reservoir is now widely recognized as the major obstacle to HIV-1 eradication [49]. Approaches for the reactivation of HIV-1 from resting memory T cells and the subsequent elimination of these cells include T-cell activating cytokines [50-52], T-cell receptor and T-cell receptor signaling pathway agonists [53-57], histone deacetylase inhibitors [58-60], histone methyltransferase inhibitors [61], DNA methylase inhibitors [62,63], and compounds such as 5-hydroxynaphthalene-1,4-dione [64], and disulfiram [65] that reverse latency through mechanisms that are not yet completely understood. Clinical trials of disulfiram and the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) have been initiated [66&&,67&&]. In conducting these two different HIV-1 reactivation trials, HIV-infected patients on longterm suppressive therapy with undetectable plasma viremia (by conventional methods) for 6 months to 1 year have been given: 14 daily doses of disulfiram (500 mg) or two separate doses of SAHA (400 mg) given 4-5 weeks apart. To evaluate the effectiveness of these two interventions in reactivating latent HIV-1, many different analyses were conducted including measuring plasma HIV-1 RNA levels by SCA. An interim analysis of persistent viremia was measured for 14 patients prior, during, and after treatment with disulfiram (15 separate measurements) and an increase in viremia was measured during disulfiram therapy and after therapy was discontinued (53 and 88% increase, respectively) compared with baseline but these increases in plasma viremia were found to be statistically insignificant [67&& ]. For the eight patients enrolled in the SAHA study, no significant change in plasma HIV-1 RNA levels were detected 8-24h after exposure to SAHA compared with baseline, although increased levels of intracellular HIV-1 RNA were measured in resting memory CD4. T cells [66&& ]. To date, reactivation of latent HIV-1 RNA from resting memory cells does not translate into statistically significant increases in plasma HIV-1 RNA. These initial results may be due to the very low levels of persistent plasma viremia in patients on long-term therapy thereby limiting the detection of the brief effects of these new reactivation therapies. Another explanation could be that resting memory CD4. T cells are not the only source of persistent viremia in treated patients.
QUANTIFICATION OF PERSISTENT VIREMIA USING DIGITAL DROPLET PCR METHODOLOGY
In conducting SCA only a limited number of patient samples can be analyzed because each assay run requires two standard curves: one for HIV-1 and one for RCAS [15]. Although the limit of detection of SCA is the most sensitive of all assays to date (0.3 copies/ml in 7 ml plasma), due to the extremely low levels of persistent viremia in the plasma of patients on long-term therapy, new eradication treatment strategies that evoke more than an one log reduction in the size of viral reservoirs contributing to residual viremia may become unmeasurable even by SCA. Digital droplet PCR (ddPCR) is a new technology that quantifies DNA copy number by dividing a 20ml mixture of sample and PCR reagents into 20 000 droplets, each containing a single molecule and fluorescent-labeled probe. PCR amplification of these droplets to end-point amplification is performed in a conventional thermal cycler [68&,69]. For droplets that contain template, specific cleavage of TaqMan probes generates a strong fluorescence signal. Positive droplets containing the DNA target are measured by flowing one droplet at a time through a fluorescence reader. This technology provides an absolute measurement of the DNA target without the use of a standard curve. Recent use of ddPCR for the measurement of HIV-1 RNA, wherein HIV RNA transcripts were serially diluted, reverse transcribed and then quantified using ddPCR, found this method had a broad dynamic range from 0.3 to 1105 copies [70]. Due to saturation of the positive droplets, ddPCR was found to be inaccurate above 1105 copies indicating further dilution would be needed to quantify high copy numbers. In addition, ddPCR detected similar HIV-1 RNA levels when compared with SCA in patients with less than 50 copies/ml (mean 2.0-fold difference). However, viral sequence variation in primer/probe binding sites resulted in varying amplitude of positive droplets causing different signal-to-noise ratios and difficulty in setting a threshold for positive/negative droplets. Although this technology will need further improvement, it holds promise for providing absolute quantification of residual plasma HIV-1 RNA equal to or better than current ultrasensitive assays.
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