Study On Rapid Test For Detection Of HIV-1Incidence

BackgroundThe development of laboratory assays for the estimation of recent HTV-1infection has more than ten years, most of the assays are based on serological antibody detection. Nowadays, the commonly used methods such as BED-CEIA and avidity assay require standard immunoassay equipment, highly trained personnel and several hours to complete. In order to solve these problems, it is necessary to develop a rapid and accurate method for detection of HIV-1incidence. Dot immunogold filtration assay (DIGFA) is a rapid detection technology which combines the immunogold technology and the solid-phase membrane. With the characteristics of rapid, convenient, economical and visualized, DIGFA has been widely developed and applied. In this study, a rapid HIV test based on immuno-gold-silver staining filtration assay for simultaneous detection of recent HIV-1infection and HIV-1conventional antibody was established, hoping to provide a reliable tool for saving the cost and improve the efficiency of detection of HIV-1incidence.Objectives1. To establish a rapid test for simultaneous detection of HIV-1conventional antibody and recent infection.2. To obtain the mean duration of recent HIV-1infection of the rapid test for incidence calculation.3. To obtain the false recent rate of the rapid test for incidence calculation.4. To evaluate the feasibility and applicability of the rapid test for detection of HIV-1conventional antibody and recent infection, and compare with the existing commercial assays, for providing a technical platform for development of effective method for rapid detection of HIV-1incidence.Methods1. Nitrocellulose membranes were coated by a multisubtype HIV-1gp41recombinant protein (Recombinant immunodominant region of gp41of HIV-1group M, rIDR-M) and common HIV-1gp41recombinant protein for detecting recent HIV-1infection and conventional antibody, respectively. Gold-labeled anti-human IgG conjugate was used as a detection antibody and silver staining was taken for further enhancing the reaction signal, by which the rapid test was established and preliminary assessed.2. Two hundred and fifty seroconversional specimens from two cohort studies (62seroconvertors) were tested and the data was analyzed using SPSS17.0to obtain the mean duration of recency of the rapid test.3. Two hundred and fifty six long-term infection (diagnosed time longer than one year) and antiretroviral therapy naive specimens were tested to calculate the false recent rate of the rapid test.4. Recent infection testing results of582specimens were compared and the agreement, concordance and correlation of the rapid test with BED-CEIA and LAg-Avidity EIA were evaluated. HIV-1antibody testing results of85specimens were compared and the agreement and concordance of the rapid test with western blot results were observed.Results1. The optimal coating concentration of the rIDR-M and common HIV-1gp41recombinant protein was15.3μg/ml and500μg/ml, respectively. When the dosage of the silver staining reagent was50μl and stained for3minutes, the reaction signal got the strongest. The cutoff value of the two probes was3.50and4.08, respectively. The testing results of5positive specimens with seroconversion date and one negative specimen by the rapid test were all correct. The rapid test showed favourable repeatability with coefficient of variation of each probe less than15%.2. When the cutoff value of the rapid test was3.50, the mean duration of recency of this assay was189days. The antiretroviral therapy could affect the results of the rapid test.3. The false recent rate of the rapid test was2.73%(7/256,95%CI0.73%-4.73%), and CD4+T cell counts could not affect the results of the rapid test.4. There was a92.10%(536/582) agreement of final classification (recent or long-term) with the BED assay (kappa=0.65) and a high correlation between the grey value of the rIDR-M probe with the BED ODn (R2=0.9397). While there was a95.36%(555/582) agreement of final classification with the LAg-Avidity EIA (kappa=0.75) and a high correlation between the grey value of the rIDR-M probe with the LAg-Avidity ODn (R2=0.9549). The HIV-1antibody testing results of the rapid test and western blot were in full accord, and the coincidence rates of both positive and negative specimens were100%.Conclusions1. Through the optimization of reaction system, determination of the cutoff value of each probe and development the interpretative criteria of the testing results, a rapid HIV test based on immuno-gold-silver staining filtration assay for simultaneous detection of HIV-1conventional antibody and recent HIV-1infection was established.2. Preliminary obtain the mean duration of recent HIV-1infection of the rapid test was189days.3. Preliminary obtain the false recent rate of the rapid test was2.73%, higher than the false recent rate of the LAg-Avidity EIA and lower than the false recent rate of the BED assay.4. The concordance and correlation of the rapid test with the BED assay and LAg-Avidity EIA for detection of recent infection was satisfactory. There was a high agreement of HIV-1antibody testing results between the rapid test and western blot test. The method described in this study could be a reliable and high-efficiency tool for the field detection and laboratory screening of recent HIV-1infection.