Multitest

Plasma HIV-1 RNA levels were measured using the RealTime HIV-1 Assay (m2000 RealTime System, Abbott Molecular). EDTA-anticoagulated samples of whole blood were analyzed with the use of the BD Multitest on a FACSCalibur flow cytometer (BD Biosciences).

CD4⁺ T-cell count, CD8⁺ T-cell count, and CD4⁺/CD8⁺ ratio were measured by cytofluorimetry with BD FACS^® Canto II (BD Biosciences, Becton Dickinson and Co, Franklin Lakes, NJ, USA), using BD Multitest™ (BD Biosciences, Becton Dickinson and Co, Franklin Lakes, NJ, USA) and four-color direct immunofluorescence reagent according to the manufacturer’s instructions. The BD Multitest™ includes CD3 fluorescein isothiocyanate (FITC), CD8 phycoerythrin (PE), CD45 peridinin chlorophyll protein (PerCP), CD4 allophycocyanin (APC).
Plasma viral load was measured in 400 µL of plasma with Cobas^® HIV-1 (Roche, Basel, Switzerland) according to the manufacturer’s instructions. The lower limit of detection is 20 cps/mL; the higher limit of detection is 10⁷ cps/mL.
The effectiveness of ART was evaluated by the achievement of an undetectable plasma HIV-RNA viral load (<50 copies/mL) since its measurement became available. Longitudinal observation was stopped on 31 December 2019, that is, before the beginning of the SARS-CoV-2 pandemic. Subjects surviving more than 300 months from HIV diagnosis were defined LTS.

Plasma HIV-1 viral loads (PVL), as well as CD4 and CD8 counts were determined in a laboratory operating under the Clinical Laboratory Improvement Amendment (CLIA). The plasma viral load was measured using the ultrasensitive Quantiplex HIV-1 bDNA version 3.0 (Bayer). CD4⁺ and CD8⁺ T-cell counts were determined by four-color flow cytometry. The BD Multitest (BD Biosciences) that was used includes the following Abs: CD3^FITC (clone SK7); CD4^APC (clone SK3); CD8^PE (clone SK1); and CD45^PerCP (clone 2D1). Samples were acquired on either FACSCalibur or FACSCanto (both BD Biosciences). CD4⁺ cell counts were calculated as percent of CD4⁺ CD3⁺ cells within CD45⁺ lymphocytes divided by 1% of the white blood cell count. The corresponding calculation was performed for CD8⁺ cell counts.

Blood samples were drawn at routine visits at the HIV out-patient clinic and analysed for CD 4 cell counts by flow cytometry using the BD Multitest™ (BD Biosciences, San Jose, CA, USA), and HIV RNA using the AmpliPrep/COBAS, TaqMan HIV-1 test vers. 2.0 (Roche, Branchburg, NJ, USA). Quantitative determination of blood lipids was performed using the Cholesterol gen2 (Roche Diagnostics, GmBh). All blood tests were routinely performed by the hospital laboratory.

The serum immunoglobulin level was measured by nephelometric technique. The lymphocyte subsets from the peripheral blood samples were analyzed by fluorescence‐activated cell sorting using the BD Multitest™ reagents (BD Multitest IMK kit and BD Multitest™ 6‐color TBNK).