Screening for Human Immunodeficiency Virus Infection by Use of a Fourth-Generation Antigen/Antibody Assay and Dried Blood Spots: In-Depth Analysis of Sensitivity and Performance Assessment in a Cross-Sectional Study

Abstract

We evaluated the performance of a fourth-generation antigen/antibody (Ag/Ab) assay for detecting HIV-1 infection on dried blood spots (DBS) both in a conventional laboratory environment and in an epidemiological survey corresponding to a real-life situation. Although a 2-log loss of sensitivity compared to that with plasma was observed when using DBS in an analytical analysis, the median delay of positivity between DBS and crude serum during the early phase postacute infection was 7 days. The performance of the fourth-generation assay on DBS was approximately similar to that of a third-generation (antibody only) assay using crude serum samples. Among 2,646 participants of a cross-sectional study in a population of men having sex with men, 428 DBS were found reactive, but negative results were obtained from 5 DBS collected from individuals who self-reported a positive HIV status, confirmed by detection of antiretroviral (ARV) drugs in their DBS. The data generated allowed us to estimate a sensitivity of 98.8% of the fourth-generation assay/DBS strategy in a high-risk population, even including a broad majority of individuals on ARV treatment among those HIV positive. Our study brings additional proofs that DBS testing using a fourth-generation immunoassay is a reliable strategy able to provide alternative approaches for both individual HIV testing and surveillance of various populations.

Keywords: DBS; human immunodeficiency virus.

FIG 1

Reactivity of the Genscreen Ultra HIV Ag-Ab combo assay with DBS compared to that with plasma. An absorbance/cutoff ratio of ≥1.0 designates a reactive sample. A 2-log difference between DBS and plasma was observed for the majority of samples.

FIG 2

Performance of the Genscreen Ultra HIV Ag-Ab combo assay with DBS from HIV-infected individuals on ARV treatment (left) and in the Prevagay study (right). Dots indicate the distribution of signals, where an absorbance/cutoff ratio of ≥1.0 designates a reactive sample. On the left, 91 DBS from ARV-treated HIV-infected individuals provided a positive result, including 85 DBS with strong signals above the saturation signal (≥12.0). On the right, 428 DBS were reactive among 2,646 participants of the Prevagay study. In addition, 5 DBS with a negative test but from individuals reporting an HIV-positive status are represented, including 2 samples just below the threshold.

FIG 3

Performance of the Genscreen Ultra HIV Ag-Ab combo assay with DBS in the context of acute infection. Sequential sera from 12 seroconversion panels (PRB 917 to PRB 952) were tested, with 2 to 5 samples per panel ranging from 2 to 23 days after detection of p24 antigenemia (day 0). We compared the performance of the fourth-generation assay by using DBS or crude serum (4th Gen Ag/Ab DBS and 4th Gen Ag/Ab, respectively) to that of a third-generation assay using crude serum samples (3rd Gen Ab). An absorbance/cutoff ratio of ≥1.0 designates a reactive sample. The time scale is in days.

FIG 4

Western blots for the twelve DBS from individuals who self-reported that they were HIV positive. High Pos, high-positive control; Low Pos, low-positive control; Neg, negative control.