1. Development of an oral fluid assay capable of differentiating recent from established HIV infection Niel Constantine 1, Ligia Peralta 1, Anne Sill 2, Kristen Kreisel 1, Bethany Griffin Deeds 1, and the ATN for HIV/AIDS Intervention. 1 University of Maryland, Baltimore, School of Medicine 2 University of Maryland, Baltimore, Institute of Human Virology Abstract Results Objectives Background Conclusions Methods Background/Objective: Identifying persons who have been recently infected with HIV is important for epidemiologic investigations, including the estimation of incidence and for the enrollment of acutely infected individuals into appropriate intervention programs. Currently, modified serologic methods (sensitive/less-sensitive assays, S/LS) are available to classify recently infected persons (3-6 months), but they require the collection of blood that often limits their application, including among adolescents. Simpler methods of specimen collection are needed to increase compliance and offer an alternative to invasive procedures that increase the risk of occupational exposure to HIV. We sought to develop, calibrate, and validate a S/LS assay that uses easy-to-collect oral fluid. Methods: Serum/oral fluid pairs were collected from 246 HIV-infected adolescents newly enrolled in HIV care programs of the Adolescent Trials Network from 15 sites in the United States and Puerto Rico. The serum was collected by venipuncture, whereas the oral fluid was collected by the OraSure device that targets mucosal transudate containing IgG antibody. Reference testing was performed on all serum specimens by the Vironostika S/LS test using an optical density (OD) cutoff of 1.0 to distinguish recent from established infections. We chose the first 20 recent and 20 established samples, as classified by the serum Vironostika S/LS, and tested the complementary oral fluid specimens at various dilutions to calibrate the modified Vironostika Oral Fluid assay; an optimal dilution of 1:50 and an OD cutoff of 0.238 were selected to yield a concordance of 100% for recent samples and 85% for established samples (OK). Using these parameters, the concordance of the serum S/LS and the oral fluid S/LS assay was then determined using the remaining 206 serum/oral fluid pairs. Of the 206 specimens, 61 were classified as being from recently infected individuals and 145 from those with established HIV infection according to the serum S/LS assay. Results: There was a correlative trend of the OD readings of both assays when testing the 206 serum/oral fluid pairs (r=.658, p<.001). The concordance rate between the serum and oral fluid based S/LS assays using the parameters selected during the calibration phase was lower than anticipated (57% for recents and 92% for established). However, when reassessing the results from the entire specimen pool (n=246 serum/oral fluid pairs), the best agreement between the oral fluid and the serum S/LS results remained at an oral fluid dilution of 1:50, but with a new cutoff of 0.280. This adjustment to the OD cutoff of the oral fluid assay gave the highest kappa value (  =.703), and a concordance rate of 85% for the recent samples and 87% for the established samples as compared with the serum S/LS test. Conclusions: The HIV oral fluid S/LS test has exhibited good concordance with the routinely used serum S/LS test. This S/LS test strategy using oral fluids offers a simple and safe collection method that is suitable for use in a variety of epidemiologic investigations. To develop, calibrate, and validate an oral fluid S/LS EIA method to perform equivalently to the reference S/LS test. Phase I Calibration of the oral fluid S/LS assay with the Vironostika serum S/LS assay Proof of Principle that the oral fluid S/LS acts equivalently to the serum based S/LS assay Phase II Validation of findings of the Phase I calibration Modifications of the oral fluid S/LS assay parameters to enhance concordance Define parameters that yield optimal concordance between the OF S/LS and the serum S/LS tests. Oral fluid assays for HIV are widely available and have been validated as accurate. EIA, Western blot, and rapid tests for HIV using oral fluid for testing are licensed in the U.S. The test medium for oral fluid assays is oral mucosal transudate (rich in IgG). The applications for an oral fluid sensitive/less sensitive assay (OF S/LS) include:  Oral fluid collection is preferred by clients (Peralta, Constantine, et al. Arch Peds & Adol Med, 2001, 155; 838-843)  Collection of samples is easier in populations from which blood is difficult to obtain (IVDU, Obese clients, children) > Oral fluid collection is safer than blood collection. Samples 15 participating Adolescent Trials Network (ATN) sites provided serum/oral fluid pairs Serum was collected by standard venipuncture, whereas the oral fluid was collected using the OraSure device that targets mucosal transudate containing IgG Ab Sample Size (selected on the obtainment of at least 65 samples from recently infected persons) Total sample size = 246 –Phase I = 40 (20 Recents and 20 Establsihed as classified by the serum DV) –Phase II = 206 (62 Recents and 144 Established as classified by the seurm DV) – Phase I and II = 246 (82 Recents and 164 Established as classified by the seurm DV) Tests HIV status Serum – EIA methods and Western blot as per each site Oral Fluid – Oral Fluid Vironostika HIV-1 Microelisa System (sensitive mode) Testing for Recent or Established Infection (S/LS) Serum – Vironostika HIV-1 Microelisa System (less-sensitive mode) Oral Fluid – Oral Fluid Vironostika HIV-1 Microelisa System, modified to a less-sensitive mode Oral Fluid S/LS Calibration Oral fluid dilutions during Phase I calibration: 1:1:10, 1:25, 1:50, 1:60, 1:80, 1:100 Optimal optical density (OD) cutoff determination for separation of recent and established samples  Empirical  ROC The oral fluid (OF) dilution of 1:50 and OF S/LS OD cutoff of 0.280 gave the highest rate of agreement with the reference serum S/LS assay when testing all 246 samples; The OD readings of the oral fluid S/LS assay and the serum S/LS assay when testing 246 oral fluid/serum pairs, showed a correlative trend (r=.658, p<.001). The concordance rate of the OF S/LS assay and the serum S/LS assay was 85% for samples from recently infected persons and 87% from those with established infection. It should be noted that the Vironostika serum S/LS assay itself is believed to be only about 85% accurate when tested on seroconversion panel specimens. (Constantine et al. 2003. JAIDS 32:94-103) It is possible that the true sensitivity for correctly identifying those with recent and established HIV infection using the oral fluid S/LS method could be better, equivalent, or worse than the serum-based S/LS assay. We are in the process of obtaining serum and oral fluid pairs from seroconverting individuals; such samples will yield definitive information on the utility of both tests. The HIV oral fluid S/LS test has exhibited good concordance with the routinely used serum S/LS test. This S/LS test strategy using oral fluids offers a less invasive and safe collection method that is suitable for use in a variety of epidemiologic investigations. The collection of oral fluid will likely increase compliance levels among those being tested. Oral Mucosal Transudate from tooth-gum margin Iterative concordance rates of the oral fluid S/LS and serum S/LS at varying oral fluid cutoffs Considerations RecentEstablished Recent70 (85.4%)22 (13.4%) Established12 (14.6%)142 (86.6%) Serum S/LS Designations Oral fluid S/LS Designations