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.

HBV serological markers were measured using immune-enzymatic assays (Roche/Cobas Diagnostics, Rotkreuz, Switzerland). Plasma HBV-DNA was identified using real-time polymerase chain reaction (lower limit of quantification: 20 IU/ml) (Roche/Cobas Ampliprep/Cobas Taqman, Rotkreuz, Switzerland). Plasma HIV-RNA levels were measured using a commercial test, with 20 copies/ml of HIV-RNA defined as the lower limit of quantification (COBAS Ampliprep/Cobas Taqman HIV-1 Test, v2.0).

Laboratory investigations were carried out on overnight fasting samples at two certified laboratories with regular system calibration of validated devices and quality measures in place. High sensitivity CRP was measured via Beckman Coulter AU analyser with detection limits of 0.2–160 mg/L. Per hsCRP latex package insert, CVD relative risk is considered as follows: low < 1mg/L, average 1-3mg/L and high > 3mg/L. Urine microalbumin of <3 mg/mmol is considered normal/mildly increased, 3-29mg/mmol is moderately increased, and >30mg/mmol is severely increased. Fasting glucose levels of < 5.6, ≥ 5.6, 6–6.9, ≥ 7 mmol/L are normal, meets metabolic syndrome criteria, impaired fasting glucose and diagnostic of diabetes mellitus, respectively. Levels in mmol/L of serum total cholesterol (TC) > 5, HDL < 1.2, LDL > 3 and triglyceride > 1.7 are abnormal in adult females.
HIV-1 viral load was measured using COBAS AmpliPrep/COBAS TaqMan HIV-1 test. For the purposes of this study, participants with viral load <200 copies/ml (cp/ml) were considered as virally suppressed. CD4 count was determined by the Becton Dickinson Facscalibur flow cytometer.

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)].