Modification of the Abbott RealTime assay for detection of HIV-1 plasma RNA viral loads less than one copy per milliliter.
ABSTRACT Although commercial tests are approved for detection of HIV-1 plasma viral loads ≥ 20 copies per milliliter (ml), only one specialized research assay has been reported to detect plasma viral loads as low as 1 copy/ml. This manuscript describes a method of concentrating HIV-1 virions from up to 30 ml of plasma, which can be combined with a commercial viral load test to create a widely available, reproducible assay for quantifying plasma HIV RNA levels less than 1 copy/ml. Using this pre-analytically modified assay, samples with a known level of 0.5 copy/ml were detected in 8 of 12 replicates (mean 0.47 copy/ml; 95% confidence interval (CI) 0.14-0.81 copy/ml) and samples with a known level of 1.0 copy/ml were detected in 13 of 13 replicates (mean 1.96 copy/ml; 95% CI 1.42-2.50 copy/ml). By concentrating virus from 30 ml of plasma, HIV RNA could be measured in 16 of 19 samples (84%) from 12 of 12 subjects (mean 2.77 copy/ml; 95% CI 0.86-4.68 copy/ml). The measured viral load correlated inversely (r = -0.78; p = 0.028) with the total duration of viral suppression (viral load<40 copies/ml).
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ABSTRACT: Preventing mucosal transmission of HIV is critical to halting the HIV epidemic. Novel approaches to preventing mucosal transmission are needed. Hyaluronic acid (HA) is a major extracellular component of mucosa and the primary ligand for the cell surface receptor CD44. CD44 enhances HIV infection of CD4(+) T cells, but the role of HA in this process is not clear. To study this, virions were generated with CD44 (HIVCD44) or without CD44 (HIVmock). Exogenous HA reduced HIV infection of unstimulated CD4(+) T cells in a CD44-dependent manner. Conversely, hyaluronidase-mediated reduction of endogenous HA on the cell surface enhanced HIV binding to and infection of unstimulated CD4(+) T cells. Exogenous HA treatment reduced activation of protein kinase C alpha via CD44 on CD4(+) T cells during infection with HIVCD44. These results reveal new roles for HA during the interaction of HIV with CD4(+) T cells that may be relevant to mucosal HIV transmission and could be exploitable as a future strategy to prevent HIV infection.Immunology and Cell Biology advance online publication, 24 June 2014; doi:10.1038/icb.2014.50.Immunology and Cell Biology 06/2014; 92(9). DOI:10.1038/icb.2014.50 · 4.21 Impact Factor
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ABSTRACT: There is intense interest in developing curative interventions for HIV. How such a cure will be quantified and defined is not known. We applied a series of measurements of HIV persistence to the study of an HIV-infected adult who has exhibited evidence of cure after allogeneic hematopoietic stem cell transplant from a homozygous CCR5Δ32 donor. Samples from blood, spinal fluid, lymph node, and gut were analyzed in multiple laboratories using different approaches. No HIV DNA or RNA was detected in peripheral blood mononuclear cells (PBMC), spinal fluid, lymph node, or terminal ileum, and no replication-competent virus could be cultured from PBMCs. However, HIV RNA was detected in plasma (2 laboratories) and HIV DNA was detected in the rectum (1 laboratory) at levels considerably lower than those expected in ART-suppressed patients. It was not possible to obtain sequence data from plasma or gut, while an X4 sequence from PBMC did not match the pre-transplant sequence. HIV antibody levels were readily detectable but declined over time; T cell responses were largely absent. The occasional, low-level PCR signals raise the possibility that some HIV nucleic acid might persist, although they could also be false positives. Since HIV levels in well-treated individuals are near the limits of detection of current assays, more sensitive assays need to be developed and validated. The absence of recrudescent HIV replication and waning HIV-specific immune responses five years after withdrawal of treatment provide proof of a clinical cure.PLoS Pathogens 05/2013; 9(5):e1003347. DOI:10.1371/journal.ppat.1003347 · 8.06 Impact Factor
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ABSTRACT: Low-level viraemia during antiretroviral therapy and its accurate measurement have become increasingly relevant. We present an international collaboration of 4,221 paired plasma viral load (pVL) results from four commercial assays, emphasizing data with low pVL.The assays compared were the Abbott RealTime assay and the Roche Amplicor, TaqMan v1 and v2 assays. Correlation between assays was 0.90-0.97. However, at low pVL, correlation fell to 0.45-0.85. Higher inter-assay concordance was observed when detectability was defined as 200-copies/mL versus 50-copies/mL. A pVL of ∼100-125 copies/mL by TaqMan v1/v2 corresponded best to a 50-copies/mL threshold by Amplicor.Correlation and concordance between viral load assays were lower at low pVL. Clear guidelines are needed on the clinical significance of low-level viraemia.Journal of clinical microbiology 12/2013; 52(2). DOI:10.1128/JCM.02461-13 · 4.23 Impact Factor