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Welcome

Welcome. Total IgG quantity. IgG < 3.5 mg/L. OK. IgG ≥ 3.5 mg/L. Invalid sample. Validation samples. Survey samples. Negative. Positive. Positive/ Indeterminate. Negative. Confirmation with Immuno cromatographic Assay /Western Blotting. Consistency with expected results.

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Welcome

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  1. Welcome

  2. Total IgG quantity IgG < 3.5 mg/L OK IgG ≥ 3.5 mg/L Invalid sample Validation samples Survey samples Negative Positive Positive/ Indeterminate Negative Confirmation with Immuno cromatographic Assay /Western Blotting Consistency with expected results Algorithm for oral fluid testing Vyse et al 2001 Emmons et al, 1997 George et al, 1997 EIA testing

  3. Screening: IgG quantitation : EIA Human IgG quantitation kit (Bethyl Lab. Inc,Texas) NOT FUNCTIONAL Diagnostic tools IgG anti HIV 1/2 quantitation : EIA GENSCREEN HIV 1/2 vs2 (Bio-RAD, Marnes-La Coquette, France) CE marked for serum, FUNCTIONAL Confirmation: IgG anti HIV 1/2 : rapid Immunocromatograpy Determine HIV 1/2 ( Unipath Ltd, Bedford UK) CE marked for serum FUNCTIONAL Western Blotting HIV 1/2 BLOT 2.2 ( MP Biomedical, Singapore, Cina) CE marked for serum, FUNCTIONAL

  4. Cost to benefit considerations Diagnostic tools Screening: IgG quantitation : EIA Human IgG quantitation kit Euro 6.6/ sample IgG anti HIV 1/2 quantitation : EIA GENSCREEN HIV 1/2 vs2 Euro 3/sample Confirmation: IgG anti HIV 1/2 : rapid Immunocromatograpy Determine HIV 1/2 Euro 8.1/sample Western Blotting HIV 1/2 BLOT 2.2 Euro 45/ sample

  5. IgG quantitation : Human IgG quantitation kit Infratest variability of reference curve provided by the kit manufacturer Diagnostic tools

  6. Infratest variability of positive and negative control sera provided by the kit manufacturer Mean 3.06±0.27 Mean 0.16±0.03 IgG anti HIV 1/2 : GENSCREEN HIV 1/2 vs2 (Bio-RAD, Marnes-La Coquette, France) Diagnostic tools External quality control: UK NEQAS HIV serology

  7. Diagnostic tools Confirmation tests : Determine HIV1/2 Blot 2.2 N N P P P P N N P P P P External quality control: UK NEQAS HIV serology

  8. Validation

  9. 263 HIV positive subjects Mean age 39.9 years±10.9 SD, range 20-75 years, 56 F, 205 M 242 HIV negative subjects Mean age 33.6 years±11.2 SD, range 20-75 years, 83 F, 150 M Validation samples Miscollecting 75 ( blood instead of serum ) -included Hemolitic 75 -included Misassignement 7 excluded IgG< 3.5 mg/L 2 OF excluded 0.2% Samples suitable for testing 1008 ( serum plus OF )

  10. IgG concentration in OF of samples of validation HIV Pos : 30.9 mg/L±7.9 HIV Neg: 25.2 mg/L ±12 p<0.01

  11. + - VALIDATION RESULTS Distribution HIV negative subjects HIV positive subjects

  12. False negative OF samples • These samples showed up positive only in Western blotting, by using fivefold amount of regular loading: • Low concentration of functional IgG • IgG degradation? • Bacteria contamination? • Wrong storage? • All of them?

  13. Setting the OF cut-off for OF samples OF from the HIV negative subjects of the validation Mean 0.05±0.03 + 3SD= 0.14 + 4SD= 0.18 ROC analysis 0.18 Serum 0.16 ±0.03 Kit suggestion <0.20

  14. Take home message from validation Oral fluid is a reliable sample for epidemiological studies GENSCREEN HIV 1/2 vs2 is a valuable tool for epidemiological studies

  15. Survey

  16. Samples collected 2841 Mean age 32 years± 9.8 Survey samples IgG< 3.5 mg/L 153 excluded 5.4 % 0.1% in the validation Samples suitable for testing 2688

  17. Survey sample distribution samples

  18. 2688 sample tested 208 EIA above cut-off 151 confirmed positive by Determine HIV 1/2 57 re-assayed by Western Blotting Survey overwiew 47 confirmed HIV positive, 2 indeterminate, 10 negative

  19. Examples of OF Western Blotting

  20. gp160 gp120 p65 p55 gp41 p32 p24 Frequency of HIV reactive bands in Western Blotting of OF samples

  21. Survey results: percent of HIV1/2 positive OF samples

  22. Paola Coato, Giulio fracasso, Claudia Pizzoli Monica Brentegani, Dunia Ramarlii Immunologia Clinica- Microbiologia Azienda Ospedaliera Universitaria Integrata Verona

  23. Follow-up mediante Western blot

  24. Clearance degli anticorpi materni nei nati da madre sieropositiva n.1 Extra UE n.3 UE n.2 Extra UE gp 160 gp 120 p 65 p 55 gp 41 p 32 p 24 0 6 12 8 24 0 6 12 24 0 9 15 mesi Western blotting : test di conferma diagnostico per coinfezioni HIV1/2

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