Modification of the Abbott RealTime assay for detection of HIV-1 plasma RNA viral loads less than one copy per milliliter

Yukl, Steven A.; Li, Peilin; Fujimoto, Katsuya; Lampiris, Harry; Lu, Chuanyi M.; Hare, C. Bradley; Deeks, Steven G.; Liegler, Teri; Pandori, Mark; Havlir, Diane V.; Wong, Joseph K.

doi:10.1016/j.jviromet.2011.04.015

Cited by 7 publications

(5 citation statements)

References 48 publications

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“…Furthermore, the assays themselves have inherently lower precision, reproducibility, and sensitivity at the lower ends of their dynamic ranges, which likely contributes to interassay disagreement at low viral loads. Assays exist to quantify viral loads even to single-copy levels, and commercial assays can be modified to accommodate low viral loads (43)(44)(45)(46). However, these often require large volumes of blood plasma, up to 30 ml in some cases (43), and this precludes their routine use in the clinic setting.…”

Section: Discussionmentioning

confidence: 99%

Comparative Performances of HIV-1 RNA Load Assays at Low Viral Load Levels: Results of an International Collaboration

et al. 2014

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Low-level viremia during antiretroviral therapy and its accurate measurement are increasingly relevant. Here, we present an international collaboration of 4,221 paired blood plasma viral load (pVL) results from four commercial assays, emphasizing the data with low pVL. The assays compared were the Abbott RealTime assay, the Roche Amplicor assay, and the Roche TaqMan version 1 and version 2 assays. The correlation between the assays was 0.90 to 0.97. However, at a low pVL, the correlation fell to 0.45 to 0.85. The observed interassay concordance was higher when detectability was defined as 200 copies/ml than when it was defined as 50 copies/ml. A pVL of ϳ100 to 125 copies/ml by the TaqMan version 1 and version 2 assays corresponded best to a 50-copies/ml threshold with the Amplicor assay. Correlation and concordance between the viral load assays were lower at a low pVL. Clear guidelines are needed on the clinical significance of low-level viremia.

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Section: Discussionmentioning

confidence: 99%

Comparative Performances of HIV-1 RNA Load Assays at Low Viral Load Levels: Results of an International Collaboration

et al. 2014

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“…Studies investigating patient outcome when residual viraemia is detected were published, as well as intensification trials exploring the effect of an additional antiretroviral drug on full viral suppression. Some authors used methodology enabling to concentrate the HIV-1 RNA by extracting from 4mL up to 30mL of plasma following ultracentrifugation [ 15 - 17 ], or repeat the test 3 times [ 18 ], lowering the detection limits of PCR and reducing the variability in the low copy number range. Those modified assays may be useful to answer scientific questions about low-level viraemia, but cannot be translated into clinical practice because the variability of most recent versions of widespread commercial VL assays is too high.…”

Section: Discussionmentioning

confidence: 99%

HIV-1 low-level viraemia assessed with 3 commercial real-time PCR assays show high variability

Ruelle¹,

Debaisieux

Vancutsem

et al. 2012

BMC Infect Dis

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BackgroundCurrent real-time PCR-based HIV-1 viral load (VL) assays allow the detection of residual viraemia in antiretroviral-treated patients. The clinical outcome of HIV1 patients experiencing low-level replication (<50 cop/mL) in comparison with fully suppressed patients is currently debated. We analysed variability of 3 VL assays <50 cop/mL, and evaluated the reproducibility of viral blips <100 cop/mL.MethodsThree commercial VL assays were tested: Versant HIV-1 RNA 1.0 kPCR (Siemens), Abbott Realtime HIV-1, and Cobas Ampliprep/Cobas Taqman HIV-1 v2.0 (Roche). Ten replicates of a reference sample at 4 low target dilutions were tested to evaluate assay variability. Prospective collection of 181 clinical samples with detectable VL <50 cop/mL was used to evaluate intra-and inter-assay variability by triplicate testing. Samples from 26 patients experiencing a viral blip were retested.ResultsAll assays showed substantial variability at low VL level: the coefficient of variation at 100, 50, 25 and 12 cop/mL ranged respectively from 32 to 44%, 35 to 68%, 41 to 83% and 33 to 77%. In the intra-assay evaluation of repeatability, 52.5 to 57.5% of detectable VL <50 cop/mL tested in triplicate showed at least one fully undetected result. Variability was similar in the inter-assay arm. The VL blips could only be reproduced in 19% of cases.ConclusionsThe most recent versions of widespread commercial VL assays showed substantial variability at low levels and residual viraemia could not be consistently reproduced. Patient outcome studies comparing residual VL to full suppression are therefore biased when using commercial assays.

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“…Plasma HIV RNA was measured in 4 different laboratories using 4 different techniques: 1) Roche Ampliprep assay [19]; 2) single copy assay [7], [20] (SCA); 3) Transcription-Mediated Amplification (TMA) [21], [22]; and 4) a modified Abbott assay that involves pelleting virus from 30 ml of plasma [23] (Table 1). Cell-associated HIV DNA in PBMC was measured in 4 laboratories using qPCR [19], [22], [24], [25] or digital droplet PCR [26] (Table 2).…”

Section: Methodsmentioning

confidence: 99%

Challenges in Detecting HIV Persistence during Potentially Curative Interventions: A Study of the Berlin Patient

et al. 2013

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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.

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Modification of the Abbott RealTime assay for detection of HIV-1 plasma RNA viral loads less than one copy per milliliter

Cited by 7 publications

References 48 publications

Comparative Performances of HIV-1 RNA Load Assays at Low Viral Load Levels: Results of an International Collaboration

Comparative Performances of HIV-1 RNA Load Assays at Low Viral Load Levels: Results of an International Collaboration

HIV-1 low-level viraemia assessed with 3 commercial real-time PCR assays show high variability

Challenges in Detecting HIV Persistence during Potentially Curative Interventions: A Study of the Berlin Patient

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