HIV-1 viremia persists at low-levels despite clinically effective antiretroviral therapy (ART). Here we review new methods to quantify and characterize persistent viremia at the single genome level, and discuss the mechanisms of persistence including clonal expansion of infected cells and tissue origins of viremia. A deeper understanding of how viremia persists on ART is critically important to the design of therapies to eliminate viremia and achieve a functional cure for HIV-1.

A real-time quantitative reverse transcriptase PCR assay with single-copy sensitivity targeting the integrase region of HIV-1 (integrase single-copy assay [iSCA] v1.0) has been widely used to quantify plasma viremia in individuals on antiretroviral therapy (ART). iSCA v1.0 requires the use of an ultracentrifuge, and only about half of the nucleic acid extracted from plasma is assayed for HIV-1 RNA. We sought to simplify sample processing using microcentrifugation and improve assay sensitivity by testing more than 75% of the total extracted nucleic acid for HIV-1 RNA (iSCA v2.0). We evaluated the limit of detection (LoD) of iSCA v2.0 by testing replicates of low-copy plasma HIV-1 RNA standards. By probit analysis, the 95% LoD was 1 copy of HIV-1 RNA per milliliter for a 5-ml plasma sample. To compare the sensitivity of iSCA v1.0 and v2.0, we tested plasma samples with both assays from 60 participants on ART with HIV-1 RNA below 20 cps/ml. Of the 31 samples that had no detectable HIV-1 RNA by iSCA v1.0, 17 (55%) were detectable by v2.0 with an HIV-1 RNA mean value of 3.5 cps/ml. Twenty-nine samples were detectable with both assay versions, but average values of HIV-1 RNA cps/ml were 2.7-fold higher for v2.0 than v1.0. These results support the adoption of a new, more sensitive and simpler single-copy HIV-1 RNA assay (iSCA v2.0) to quantify residual viremia on ART and to assess the impact of experimental interventions designed to decrease HIV-1 reservoirs.

Clearance of low-level viremia that persists in most HIV-1 positive individuals on antiretroviral therapy (ART) is an important milestone for efforts to cure HIV-1 infection. The level of persistent viremia on ART is generally below the lower limit of quantification (LOQ) of current FDA-cleared plasma HIV-1 RNA assays (20-40 copies/mL) but can be quantified by RT-PCR assays with single copy sensitivity. Such assays require multistep manual methods and their low throughput limits capacity to monitor the effects of interventions on persistent viremia. Recently, Bakkour, et al. reported the use of multiple replicates and Poisson statistics to infer HIV-1 RNA concentrations below the commercial LOQ of automated platform (Hologic Panther Aptima). Here we evaluate the detection and quantitation of low-level viremia using two adaptions of the automated platform: a multi-replicate strategy [9x] and a single-replicate strategy on plasma concentrated by centrifugation [1x, concentrated]. We compare these new methods to a recently reported manual single copy assay (SCA) targeting integrase (iSCA v2). Using laboratory-generated HIV-1 RNA plasma samples at known concentrations, all three methods had similar sensitivity for HIV-1 RNA detection, although iSCA v2 was most sensitive (95% LOD 2.3 copies/mL), multi-replicate 9x was marginally less sensitive (95% LOD 3.0 copies/mL) and 1x, concentrated was least sensitive (95% LOD 3.9 copies/mL). By contrast, for clinical plasma samples, multi-replicate 9x had greater sensitivity than iSCA v2 (82% of samples were quantifiable compared to 62% of samples by iSCA v2). These results support the multi-replicate 9x method as an acceptable high-throughput alternative to iSCA v2 for quantifying low-level viremia in individuals on ART.

Current recommendations for nanomaterial-specific exposure assessment require adaptation in order to be applied to complicated manufacturing settings, where a variety of particle types may contribute to the potential exposure. The purpose of this work was to evaluate a method that would allow for exposure assessment of nanostructured materials by chemical composition and size in a mixed dust setting, using carbon black (CB) and amorphous silica (AS) from tire manufacturing as an example. This method combined air sampling with a low pressure cascade impactor with analysis of elemental composition by size to quantitatively assess potential exposures in the workplace. This method was first pilot-tested in one tire manufacturing facility; air samples were collected with a Dekati Low Pressure Impactor (DLPI) during mixing where either CB or AS were used as the primary filler. Air samples were analyzed via scanning transmission electron microscopy (STEM) coupled with energy dispersive spectroscopy (EDS) to identify what fraction of particles were CB, AS, or 'other'. From this pilot study, it was determined that ~95% of all nanoscale particles were identified as CB or AS. Subsequent samples were collected with the Dekati Electrical Low Pressure Impactor (ELPI) at two tire manufacturing facilities and analyzed using the same methodology to quantify exposure to these materials. This analysis confirmed that CB and AS were the predominant nanoscale particle types in the mixing area at both facilities. Air concentrations of CB and AS ranged from ~8900 to 77600 and 400 to 22200 particles cm(-3), respectively. This method offers the potential to provide quantitative estimates of worker exposure to nanoparticles of specific materials in a mixed dust environment. With pending development of occupational exposure limits for nanomaterials, this methodology will allow occupational health and safety practitioners to estimate worker exposures to specific materials, even in scenarios where many particle types are present.

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