VL was measured by Nuclisens EasyQ version 2.0 HIV-1 RNA PCR (bioMérieux) with a limit of detection of 20 HIV RNA copies/mL but <100 for low-volume samples. Lysed extracts from thawed peripheral blood mononuclear cells (PBMC) were used to measure total HIV DNA by droplet digital PCR (ddPCR; BioRad) with 5′ long terminal repeat or gag primers and probes, depending on the efficiency of detection in each patient [27 (link)]. The HIV DNA count was normalized using RPP30 housekeeping gene quantification to give a value per million PBMC with a 95% Poisson confidence interval, as estimated across replicates by the QuantaSoft (BioRad) software. A limit of detection for each sample was estimated according to input cell number and where HIV DNA was undetectable, the result was recorded as the limit of detection. Results were excluded if HIV DNA was undetectable and the limit of detection was >25 copies/million PBMC.
Protocol detail
Quantifying Total HIV DNA by ddPCR
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Blood
Cells extracts
Hiv 1
Housekeeping gene
Long terminal repeat
Patient
Peripheral blood mononuclear cells
Primers
Corresponding Organization : Ragon Institute of MGH, MIT and Harvard
Other organizations : Umkhuseli Innovation and Research Management, Africa Health Research Institute, German Center for Infection Research, Ludwig-Maximilians-Universität München, King Edward VIII Hospital, TU Dortmund University, IrsiCaixa, Oxford University Hospitals NHS Trust, John Radcliffe Hospital, Max Planck Institute for Infection Biology, University College London, Universitat de Vic - Universitat Central de Catalunya, Institució Catalana de Recerca i Estudis Avançats, Institut d'Investigació en Ciències de la Salut Germans Trias i Pujol
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Variable analysis
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- Measurement technique: Nuclisens EasyQ version 2.0 HIV-1 RNA PCR (bioMérieux) with a limit of detection of 20 HIV RNA copies/mL but <100 for low-volume samples
- Measurement technique: Droplet digital PCR (ddPCR; BioRad) with 5' long terminal repeat or gag primers and probes
- Viral load (VL) measured by Nuclisens EasyQ version 2.0 HIV-1 RNA PCR
- Total HIV DNA measured by droplet digital PCR (ddPCR)
- Lysed extracts from thawed peripheral blood mononuclear cells (PBMC) used for HIV DNA measurement
- Normalization of HIV DNA count using RPP30 housekeeping gene quantification
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