Application Of A New Limiting-Antigen Avidity Dot Immuno-gold Filtration Assay For HIV-1 Incidence

BackgroundLaboratory assays estimating recent HIV-1 infection in cross-sectional population play an important role in disease surveillance,evaluation of invention strategies,identification of high risk groups and sufficient allocation of limited health resources.Enzyme-linked immunoassay,such as the BED capture enzyme immunoassay(BED-CEIA)and the avidity assay,are commonly used in these fields.However,they are time-consuming,expensive and labor-intensive.The earlier research shows a DIGSSA,which can simultaneously screen HIV-1 antibodies and detect recent HIV-1 infections,was developed.The primary assessment of this rapid test shows a good consistency and correlation with BED-CEIA and the limiting antigen avidity enzyme immunoassay(LAg-Avidity EIA).However,this assay has not reached the step of applications.This study aims at developing an approach that can simultaneously screen HIV-1/2 antibodies and detect recent infections based on the previous study by the optimization of experimental conditions.Then this approach is applied to obtain HIV-1 incidence of the sentinel populations,which facilitate the development of more effective,cost-saving approaches for HIV-1 recent infections.Objectives1.To develop a rapid test that can screen HIV-1/2 antibodies and detect recent infections simultaneously,and improve it to the optimum level.2.To gain two parameters(MDRI and FRR)in calculating HIV incidence and applicable conditions of the new estabalished rapid assay.3.To evaluate the performance of the rapid test in screening HIV-1/2 antibodies and detecting recent infections and compare the results with the commercial assays,that is BED-CEIA and LAg-Avidity EIA,the rapid method will be developed for real-time surveillance of HIV-1 incidence.And to assess the application of the new rapid test by the HIV incidence of sentinel populations from Jiangxi and Yunnan province,for analyzing any possible problems and providing theoretical evidence for future application.Methods1.Development of rapid test.Using a common HIV-1/2 recombinant antigen and a recombinant rIDR-M antigen as probes,a rapid test that detects HIV-1/2 antibodies and recent infections simultaneously based on DIGS SA developed,and further optimize the experimental system and then the rapid test was preliminary assessed.2.Experimental assessment of rapid test.A total of seven panels were established.The seroconversional specimen panels were tested for evaluating the performance of rapid test to detect the recent infection;the regular panels were tested for evaluating the performance of rapid test to detect the diagnostic antibody,the consistency,the correlation,sensitivity,specificity and efficiency were calculated to evaluate the overall performance of the new rapid test.3.Application of rapid test.The samples were collected from HIV sentinels of Jiangxi province in 2014 and Yunnan province in 2011,and included four high risk populations:DUS.MSM.FSW and STD.These samples were tested by rapid test,whose results were compared with LAg-Avidity EIA and BED CEIA.The correlation and consistency among three assays were evaluated.Furthermore,HIV incidences of the 561 samples from Yunnan were compared among three assays.In addition,117 samples collected from patients under antiretroviral therapy(ART)were tested,the misclassification of rapid test was analyzed.Results1.The rIDR-M recombinant protein was identified and purified;when the coating concentration of HIV-1/2 recombinant antigens reached to 0.5 mg/ml,the reaction signal were the best.And the preliminary cutoff values of this probe was 4.08.The results of basic sera panel(including five anti-HIV-1 positive samples with known seroconversion date,one anti-HIV-2 positive samples and one negative samples)tested by rapid test were all classified correctly.The rapid detection card can be stored in 37℃ for 9 days in the seal condition.2.When the cutoff value of the rapid test was 3.50,the rapid test can distinguish recent from long-term infections very well.At this point,MDRI and FRR of rapid assay was 176 days and 2.17%(9/414,95%CI 0.80%-3.58%);the agreement of DIGSSA with the BED and LAg-Avidity assays for distinguishing recent from long-term HIV infections were 96.78%(kappa=0.81,R2=0.9323)and 92.10%(kappa=0.59,R2=0.9678),respectively.The sensitivity,specificity and efficiency were 100%(160/160),98.61%(283/287)and 99.11%(443/447),respectively.3.Through the screening of conventional antibody using rapid test,all samples from Jiangxi HIV sentinels were all classified correctly.Through the detection of recent infection using rapid test,there was a good concordant with LAg-Avidity EIA(Kappa=0.81)and BED CEIA(Kappa=0.73)and a good correlation between the rIDR-M probe grey values of rapid test and LAg-Avidity ODn(R2=0.9475)and BED ODn(R2=0.9234),the same results obtained in MSM population.4.Through the screening of conventional antibody using rapid test,all samples from Yunnan HIV sentinels were all classified correctly.Through the detection of recent infection using rapid test for MSM,DUS,FSW and STD populations,the agreement of final classification with LAg-Avidity EIA ranged from 84.62%to 95.81%,Kappa value ranged from 0.45 to 0.81,with BED CEIA ranged from 83.78%to 93.26%,Kappa value ranged from 0.40 to 0.77.Among them,the highest agreement and coincidence rate were observed in MSM population,while the lowest were observed in DUS population.5.HIV incidences were calculated using two parameters,which were obtained in the second part of this study.By the rapid test,HIV incidences of three risk populations(DUS,MSM and FSW)were 0.61%(95%CI 0.22-1.01).4.04%(95%CI 2.01-6.07)and 0.28%(95%CI 0.10-0.45),respectively.According to Chi-square test,There were no significant differences between by rapid test and LAg-Avidity EIA or BED CEIA(P>0.05).In addition,the misclassification of rapid test was 16.24%(19/117)in ART samples collected from Jiangxi province.Conclusions1.This study has developed a rapid test for simultaneous detection of HIV-1/2 antibody and recent HIV-1 infection based on the immuno-gold-silver staining filtration technology.The rapid test can detect the diagnostic antibody with high sensitivity and high specificity.MDRI and FRR of the rapid test was 176 days and 2.17%,respectively.There was a high agreement and correlation between DIGSSA with LAg-Avidity EIA and the BED CEIA in detecting recent HIV-1 infection.2.The rapid test for simultaneous detection of HIV-1/2 antibody and recent HIV-1 infection can a reliable,convenient and efficient tool for the detection of HIV-1 infection on site,and is is valuable for popularization and application.3.ART could affect the rapid test,and the ART samples should be excluded in cross-sectional population survey.