M2000 system platform

Macaques: Plasma isolated from EDTA-blood of SIVmac251 infected RM was clarified by centrifugation at 2300 × g for 3 minutes. The clarified plasma (0.1 mL) was then lysed directly in lysis buffer (bioMerieux, Durham, NC, USA). Alternatively, a higher volume of plasma (0.5 to 1 ml) was centrifuged to pellet virus by ultracentrifugation at 49 100 × g for 60 min and the pellet lysed in lysis buffer. Viral RNA load in the lysed samples was quantitated using Real-time NASBA assay as described elsewhere ^{35 (link)}. Plasma SIVmac239/251 viral loads were determined as described, with an assay threshold of 30 copies/mL^{24 (link),25 (link)} Plasma isolated from the blood of RT-SHIV infected challenged macaques was analyzed using previously described methods ^{26 (link)} where the lower limit of detection was 5 copies/ml.
Humans: Plasma HIV viral load was measured using the COBAS Ampliprep/COBAS TaqMan 96 (Roche, Branchburg, NJ, USA) with a linearity range of 20-10,000,000 copies/ml or Abbott m2000 system platform with a linearity range of 40-10,000,000 copies/ml. The Abbott platform was used in the first 12 months of the study and the Roche for all subsequent measures. Both platforms were registered on an external quality assurance program provided by the American Pathologists and Virology Quality Assurance from RUSH University Medical Center.

HIV viral load was measured in plasma using the COBAS Ampliprep/COBAS TaqMan 96 (Roche) with a linearity range of 20–10,000,000 copies/ml or Abbott m2000 system platform with a linearity range of 40–10,000,000 copies/ml (respective lower limits of quantitation 20 and 40 copies/ml). The Abbott platform was used in the first 12 months of the study and the Roche for subsequent measures. Both platforms were registered on an external quality assurance program provided by the American Pathologists and Virology Quality Assurance from RUSH University Medical Center. Blood CD4 and CD8 T cells counts were measured by flow cytometry using FACSCount^™ (Becton Dickinson).