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  • br Funding The authors disclose

    2022-01-14


    Funding The authors disclose the following support for the research and/or authorship of this article: This work was supported by the Canadian Institutes for Health Research (CIHR). TM is the recipient of a BMS/CTN postdoctoral fellowship and PKQ was funded by a CAHR/CIHR doctoral scholarship. A portion of this project was financially supported by a CAS (grant KSCX2-EW-Q-10) and by the Natural Science Foundation of China (grant 30830115).
    Background Current “kick & kill” HIV cure strategies require more sensitive assays to measure changes in low-level persistent viremia resulting from interventions that reverse latency, i.e. “kick” (increases in viremia expected), or reduce the number of virus expressing cells, i.e. “kill” (decreases in viremia expected) [1,2]. We therefore sought to evaluate a published qRT-PCR method reported to detect eletriptan hbr HIV-1 RNA with single copy sensitivity [3] using samples obtained as part of a phase II clinical trial.
    Objectives The novel integrase single-copy assay (iSCA) [3] was evaluated for its ability to measure low-level persistent viremia in a large number of paired clinical trial samples, and to distinguish between TND and <20 categories of plasma HIV-1 RNA by Cobas-TaqMan.
    Study design In the present analysis, plasma samples were collected from Week 24 visits of a Phase 2 study in previously treatment naïve HIV-1-infected patients treated with an elvitegravir-based complete regimen [4] and cryopreserved at −80 °C. HIV-1 RNA levels were assessed at a central laboratory using HIV-1 Cobas-TaqMan 2.0 Assay (Roche Diagnostics, Indianapolis, IN). The iSCA assay [3,5], was performed in a blinded fashion on matched samples (University of Pittsburgh). For the iSCA assay, ∼3 mL of patient plasma spiked with a known quantity of internal standard RNA (RCAS) was used for viral RNA extraction.
    Discussion In this large sample collection from virologically suppressed HIV-infected adults who were naïve to treatment before being placed on an integrase based HIV treatment regimen, use of iSCA led to detection and quantification of low-level viremia below the limit of detection of the Cobas-TaqMan assay in 77% (117/151) of previously non-quantifiable plasma samples. Within this large dataset (N = 151), the use of 2 standard plasma vials, with combined volume as low as 1.2 mL, was sufficient for successful iSCA quantification. Even though the limit of detection of the assay could be dependent on the sample volume, the low sample volume required for quantification with iSCA observed in our dataset offers a logistical advantage over earlier versions of the assay that required 7 mL of plasma [6] or 6 mL of plasma [3]. As a result, standard sample collection (10 to 15 mL of blood) should be sufficient to accommodate the assay, which could in turn be readily included in standard clinical practice for more sensitive detection of residual viremia, although the clinical significance of viremia <20 copies/mL is not yet defined. In addition to its practicality, the iSCA assay may prove very well suited as a tool to monitor pharmacological effects (PK/PD) of latency reversal agents (LRA) targeting the HIV-1 reservoir, as the magnitude of HIV-1 RNA signal following activation of dormant HIV-1-infected CD4 positive T cells is likely to be very low and out of reach of quantification with the standard viral load assays such as Cobas-TaqMan (Roche) or Realtime HIV-1 viral load (Abbott). Similarly, iSCA has a distinct advantage for measuring reductions in viremia below the limit of quantification of standard viral load assays that result from therapeutic interventions such as broadly-neutralizing monoclonal antibodies designed to promote the clearance of the HIV-1 reservoir. Of note, the detection of low-level HIV-1 RNA with iSCA in all patients in the study after 24 weeks (∼168 days) of treatment with a suppressive regimen correlates with prior studies describing the slow kinetics of decay of viremia (phase III of decay half-life of 273 days) in patients on ART [[7], [8], [9]]. A consequence of the slow kinetics of decay of viremia is that patients in trials of LRA are generally required to have been on suppressive ART for 1–2 years to ensure eletriptan hbr that their viremia is undetectable prior to LRA treatment.