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 (<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.The human immunodeficiency virus (HIV) epidemic is generally assessed by monitoring seroprevalence i.e., the proportion of persons with HIV antibodies (including recently infected people and people who were infected several years previously). To understand recent changes in the HIV epidemic, it is necessary to estimate the incidence, i.e., the number of newly infected subjects in a defined period. A strategy based on a sensitive/less sensitive testing algorithm was recently used to identify serum samples from recently infected individuals (16). This strategy that uses both a sensitive and a less-sensitive enzyme immunoassay (S/LS EIA), also called a detuned assay, was applied to various situations, providing estimates of HIV incidence (12,14,16,18,24,33,35). One of the major drawbacks of this strategy is that the test is an adaptation of a commercial EIA, which poses problems for long-term availability. It is therefore necessary to develop and to validate simple immunoassays that can continuously be used independent of any commercial source. The knowledge of the anti-HIV type 1 (anti-HIV-1) antibody response (5,8,20,38) and recent studies aimed at identifying antigens to distinguish recent infections (27, 30) allowed us to design a candidate assay to assess persons with recent infection. We report the development and the validation of this assay for the identification of recent HIV-1 infections (EIA-RI) and its application to dried blood spots.

Recently acquired HIV infection in men who have sex with men (MSM) in France, 2003-2008

“…For each case, the laboratories were asked to take a dried serum spot (DSS) from the stored serum sample and send it to the National Reference Center (NRC), to determine the type, group and subtype of HIV by serotyping and to perform a test for RI (EIA-RI) [6,13,14]. The RI assessment was done by an EIA-RI assay able to identify infections acquired less than six months before obtaining the sample.…”

“…In this article, we analyse trends and characteristics of MSM newly diagnosed with HIV from 2003 to the end of 2008 in France and describe factors associated with recent infection (RI) defined as HIV transmission in the last six months preceding diagnosis [6].…”

Estimates of HIV-1 incidence based on serological methods: a brief methodological review

“…16 , including additional data from Bush et al 12 , Chawla et al 34 , Barrin et al 38 and Schüpbach et al 41 . of the HIV infection over time paved the way for all subsequent assessments of HIV incidence based on cross-sectional studies, always profiting from the fact that incidence can be approximately deduced from available data on prevalence and the duration of a given infection (or non-transmissible medical condition).…”

“…In order to overcome issues related to the availability and utilization of commercial kits, an inhouse 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 38 . Among them, the IDE and V3 peptides were selected due to their capacity for discriminating recent (< 6 months) from long-term infections.…”

HIV Diagnostics: Challenges and Opportunities

“…The greater the decrease in assay signal relative to a control sample (to which a neutral buffer is added), the lower the avidity of the anti-HIV antibodies and the more recent the infection. The laboratory-based IDE-V3 EIA employs a series of oligopeptides derived from two conserved immunogenic regions of HIV and uses a mathematical formula that draws on the reactivity of recent and long-standing specimens to the peptides from each region to estimate the recency of infection [52,53]. Detuned assays were the first tests described for detecting incident infections and involve increasing the dilution of a known anti-HIV antibody-positive sample and reducing the incubation time to deliberately lower the sensitivity of an EIA [53].…”