Development and validation of an immunoassay for identification of recent human immunodeficiency virus type 1 infections and its use on dried serum spots

Abstract

The objective was to develop and to validate an immunossay to identify recent human immunodeficiency virus type 1 (HIV-1) infections that can be used on dried serum spots (DSS). A single, indirect enzyme-linked immunosorbent assay was developed to quantify antibodies toward four HIV-1 antigens: consensus peptides of the immunodominant epitope of gp41 (IDE), consensus V3 peptides, recombinant integrase, and recombinant p24. The parameters of the logistic regression used to classify the samples were estimated on a training sample (210 serum samples) using resampling techniques to get stable estimates and then applied to a validation sample (761 serum samples). The IDE and V3 peptides were the best able to discriminate between the antibodies present in serum from recently (< or =6 months) infected individuals and those with long-lasting infection. Combined quantification of antibody binding to these two synthetic antigens allowed us to identify recent infections with an area under the receiver operating characteristic curve of 0.949 and a sensitivity of 88.3%, with a specificity of 97.6% in patients with long-term infection (but not AIDS) and 86.0% in patients suffering from AIDS with a threshold of 0.50 in the validation sample. This simple immunoassay can be used to identify recently HIV-1-infected patients. Its performance is compatible with its use in population-based studies including DSS.

FIG. 1.

Distribution of the sample A/A NC ratios for each antigen and each population. The horizontal lines represent the 10th, 25th, 50th, (median), 75th, and 90th percentiles of the ratios. Training sample (TS) ≤ 180, sera collected within 180 days of primary infection (n = 60); TS > 180, sera collected more than 180 days after primary infection (n = 150). Validation sample (VS) ≤ 180, sera from seroconverters collected within 180 days of primary infection (n = 94); VS > 180, sera from seroconverters collected more than 180 days after primary infection (n = 24); VS chronic, sera from patients with established infection not suffering from AIDS (n = 500); VS AIDS, sera from patients suffering from AIDS (n = 143).

FIG. 2.

ROC curves showing the similarity of results obtained with the bootstrap resampling procedure (circles) and the GEE logistic model (squares) for the IDE/V3 combination at the 0.5 threshold. The arrow indicates the results for a threshold of 0.50. Other thresholds are not shown for clarity. The hatched curve is an example of the result that would be obtained with an assay without discriminating power.

FIG. 3.

Distribution of the probability (P) of being classified as a nonrecent seroconverter obtained with the EIA-RI on the validation sample using the serum samples (A) or the dried serum spots (B); and distribution of the probability (P) with the EIA-RI applied to sera collected within (≤180) or beyond (>180) 180 days of primary infection from seroconverters who received early HAART (C). Sc ≤ 180, sera from seroconverters collected within 180 days of primary infection; Sc > 180, sera from seroconverters collected more than 180 days post-primary infection; chronic, sera from patients with established infection not suffering from AIDS; AIDS, sera from patients suffering from AIDS.