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A Simple and Inexpensive Particle Agglutination Assay for Identifying Recent HIV Infection Niel Constantine 1, Li Hong 2, Kristen Kreisel 1, Anne Sill.

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Presentation on theme: "A Simple and Inexpensive Particle Agglutination Assay for Identifying Recent HIV Infection Niel Constantine 1, Li Hong 2, Kristen Kreisel 1, Anne Sill."— Presentation transcript:

1 A Simple and Inexpensive Particle Agglutination Assay for Identifying Recent HIV Infection Niel Constantine 1, Li Hong 2, Kristen Kreisel 1, Anne Sill 3, Fassil Ketema 1 1 University of Maryland School of Medicine, Baltimore, MD; 2 Visiting Scientist funded by the China Scholarship Council, Yunnan CDC, Kunming, Yunnan, China; 3 Institute of Human Virology, Baltimore, MD. HIV Diagnostics: New Development and Challenges Orlando, Florida March 1, 2005

2 Background Sensitive/less sensitive (S/LS) serologic assays have been developed to differentiate individuals who have recent versus established HIV infection. Because all S/LS assays are based on ELISA technology, they may not be suitable for use in resource-limited countries where stable electricity, technical expertise, financial resources, and infrastructure are lacking. In addition, current S/LS assays are cumbersome to perform. A simpler and more affordable method could address the current limitations.

3 Serodia Particle Agglutination Assay Simple HIV Assay Antigens: recombinant p24, gp41, gp36 Results available in 60 Minutes No electrical equipment required Inexpensive Test (70 cents per Test) Widely used throughout Asia and South America

4 Serodia (Fujirebio) HIV Particle Agglutination Assay

5 Objectives To develop and calibrate a modified Particle Agglutination Assay (PA) as an S/LS test. To determine the concordance of the PA S/LS test with the Vironostika S/LS ELISA.

6 Methods The Serodia HIV-1/HIV-2 particle agglutination assay (PA) (Fujirebio) was modified to act as a S/LS assay to differentiate recent from established HIV infection. Modifications included: (1) diluting the gelatin particles at 1:68, (2) use of 38ul of particles, (3) use of 8 ul of sample, and (4) an overnight incubation before reading. The sera were diluted at intervals from 1:10 to 1:80,000 in specimen diluent to determine antibody titer. The endpoint dilution was assessed for all samples and the dilution interval that yielded the best separation of the two populations by the PA S/LS test was selected and used when determining the concordance of the results with the DV S/LS test.

7 Verification of Sensitivity of the Modified PA S/LS Test Epidemiologic sensitivity verified by testing 200 HIV positive samples Analytical sensitivity verified using 7 seroconversion panels (BBI)

8 Performance of the Modified PA Sensitivity for HIV positive samples = 100% Analytical sensitivity using 7 seroconversion panels: –Equal to majority of ELISAs = 5/7 –Later than the majority of ELISAs = 0/7 –Earlier than the majority of ELISAs= 2/7

9 Calibration D UPS 1:10 1:100 1:1000 1:10000 1:20000 1:40000 R 1:10 1:20 1:40 1:68 1:80

10 Samples for Assessment of the PA S/LS Assay A total of 152 samples were tested by the PA S/LS assay. All were classified as being from persons with recent (n = 75) or established (n = 77) HIV infection using the Vironostika S/LS ELISA. Concordance between the PA S/LS and the Vironostika S/LS tests was determined.

11 RESULTS Concordance with the Vironostika S/LS ELISA Overall concordance = 88% Concordance with Recent Samples = 96% Concordance with Established Samples = 81%

12 Note The concordance between the PA S/LS and the Vironostika S/LS was 88% overall. However, the ability of the Vironostika S/LS to correctly classify recent and established samples may be less than 90% (Constantine et al.; JAIDS:32: 94-103, 2003). We are in the process of testing a large number of seroconversion panels with the PA S/LS test to show its true test indices for classifying recent and established HIV infection

13 Conclusions A commercially available HIV gelatin particle agglutination test was modified as a S/LS test and exhibited a good concordance with an ELISA S/LS test to differentiate persons with recent and established HIV infection. The PA S/LS test offers unparalleled low cost, is simple to perform, and does not require any instrumentation or electrical support. Although we have shown proof of principle, the test must be challenged with a large number of well- characterized sera before being standardized for widespread use.

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