Ampliprep cobas taqman hiv 1 test

HBsAg screening was completed using the Genescreen HBsAg 3.0 EIA assay (Bio-Rad Laboratories, Redmond, WA). To confirm the presence of HBV and to quantify DNA, we performed in vitro PCR using the Abbott RealTime HBV assay (Abbott Molecular, Des Plaines, IL). Participants who tested positive for HBsAg with detectable HBV DNA were considered HBV-infected.
Whole blood was tested for HIV using a parallel series of rapid tests (Alere Determine, Waltham, MA and Trinity biotech Uni-Gold HIV, Wicklow, Ireland). In cases of discordance, a 3rd rapid test (Chembio Diagnostics HIV-1/2 Stat Pak test, Medford, NY) was used to confirm or exclude HIV infection as outlined by the parallel testing algorithm for at-risk individuals in Nigeria [27 ]. For participants living with HIV, HIV RNA was quantified using the Ampliprep/COBAS TaqMan HIV-1 Test (Roche Molecular Diagnostics, Pleasanton, CA) and CD4 counts using the Partec CyFlow Counter (Sysmex, Kobe, Japan). Voided urine and rectal swabs were tested for Neisseria gonorrhoeae (NG) and Chlamydia trachomatis (CT) using the Aptima Combo 2 CT/ NG Assay (Hologic, San Diego, CA). All participants testing positive for HIV or other STIs were offered appropriate treatment regardless of CD4 count.

Tests for schistosome infections were performed at the National Institute of Medical Research in Mwanza, Tanzania. Microscopic examinations were performed on 10 mL of urine (for S. haematobium) by the filtration technique and on stool (for S. mansoni) following the Kato Katz method. CAA testing was performed as previously described using a luminescent up-converting phosphor technology in combination with a lateral flow-based platform (UCP-LF) [23 (link), 24 (link)]. A 30 pg/mL cutoff threshold was used. Schistosome infection was defined as having either a positive microscopy or CAA test.
Plasma viral load was quantified using the AmpliPrep/COBAS® TaqMan® HIV-1 Test (Roche Molecular Systems Inc., Pleasanton, California, USA) machine at the BMC clinical laboratory, with a lower limit of detection of 20 copies/mL. Virological failure was defined as a viral load above 1000 copies/mL.

Different cell concentrations (fivefold limiting dilutions, i.e., 5× 10⁵, 1 × 10⁵, 2 × 10⁴ and 4 × 10³ cells of sorted viable blood resting memory (CD45RA^-HLA-DR^-CD25^-CD69^-) CD4 T cells from aviremic ART treated HIV-infected individuals were co-cultured with autologous irradiated CD8-depleted blood mononuclear cells (10⁶ cells/ml), as previously described [44 (link)], in presence or in absence of blocking anti-PD-1 Mab, Pembrolizumab, at 5 μg/mL or isotype control (Eureka Therapeutics). As a positive control, sorted cells were stimulated for 3 days with anti-CD3 and anti-CD28 mAb-coated plates (10 μg/ml) in presence of IL-2 (50 units/ml). Supernatants were collected at days 0, 5 and 14. Medium was replaced at day 5. Quantification of replication competent HIV-1 was performed at 14 by assessing HIV-1 RNA levels by COBAS AmpliPrep/TaqMan HIV-1 Test (Roche; Switzerland) as previously described [44 (link)]. Wells with detectable HIV-1 RNA (≥200 HIV-1 RNA copies/ml) were referred to as HIV-1 RNA-positive wells. The frequency of latently HIV-1 infected CD4 T cells reactivated by IC MAbs was estimated by conventional limiting dilution methods using Extreme Limiting Dilution analysis (http://bioinf.wehi.edu.au/software/elda/) [63 (link)] and expressed in RNA-unit per million (RUPM) as previously described [44 (link)].

Viral particles were prepared by transfection of 293 T cells. 10–12 million 293 T cells were seeded per 15 cm tissue culture dishes in DMEM media with 10% FBS and Penicillin-Streptomicin, Gentamicin (50 μg/ml, GIBCO). The next day, cells were transfected with 70 μg of plasmid DNA (Rev, Gag/Pol, VSVG and PD-L1), through Calcium phosphate transfection (Takara) as previously described [89 (link)]. The next day, media were replaced with DMEM containing 5% FCS. After 2 days, media was filtered at 0.45 μM, collected into 38.5mL ultraclear centrifuge tubes (Herolab 253050), centrifuged at 50,000 g for 2 hours at 16°C. Virus pellet was re-suspended in 1 mL PBS, aliquoted and snap frozen in dry ice and then at -80°C for later use. Viral titers were determined by the assessment of HIV p24 antigen by ECL COBAS HIV Ag (Roche; Switzerland), HIV-1 RNA by COBAS AmpliPrep/TaqMan HIV-1 Test (Roche; Switzerland) and the percentage of GFP⁺ CD4 T cells with increasing virus doses at day 3 post-infection by flow cytometry.

Freshly isolated LNMCs were stained with Aqua LIVE/DEAD stain kit (4°C; 15 min) and then with anti-CD1c APC, anti-CD127 PB, anti-CD3-PE, anti-CD4 APC-H7, anti-CD45RA ECD and anti-PD-1 PE-Cy7 (4°C; 25 min). Viable LN memory (CD45RA^-) PD-1⁺ and PD-1^- CD4 T-cell populations and CD1c^highCD127^high DCs were sorted using FACSAria (Beckton & Dickinson). In all sorting experiments the grade of purity of the sorted cell populations was >98%. Sorted CD4 T cell populations (10⁵ cells) were stimulated with anti-CD3/anti-CD28 MAbs (10μg/ml) or co-cultured with autologous DCs (ratio DCs/T 1∶10) in the presence or absence of blocking anti-PD-L1/2 MAbs (10 μg/ml) in 96-well U-bottom plates. All cultures were carried out in the presence of emtricitabine. Supernatants were collected at day 6 and quantification of HIV-1 production was performed by assessing HIV-1 RNA levels by COBAS AmpliPrep/TaqMan HIV-1 Test (Roche; Switzerland) as previously described [44 (link)]. PD-L1, PD-L2 and CD155 expression was assessed on sorted migratory DCs using mass cytometry.

Sorted LN PD-1^high and PD-1^- memory (CD45RA^-) CD4 T-cell populations (2 × 10⁵ cells) were cultured for 6 days at 37°C and 5% CO₂ in 96-well U-bottom plates coated with anti-CD3 (BD) and anti-CD28 (BD) (10 μg/ml) in presence or in absence of recombinant IC ligands at 100 μg/mL in complete RPMI. In some experiments, the combination of two IC-ligands was also tested. Of note, all experiments were performed in presence of 75 nM emtricitabine to prevent re-infection of stimulated CD4 T cells. Supernatants were collected at days 6 and quantification of HIV-1 production was performed by assessing HIV-1 RNA levels by COBAS AmpliPrep/TaqMan HIV-1 Test (Roche; Switzerland) as previously described [44 (link)].