1. Use of dried blood spots (DBS) or dried serum/plasma spots (DSS/DPS) to detect recent HIV-1 seroconversion by the BED-CEIA Bharat Parekh, PhD Centers for Disease Control and Prevention Atlanta, GA

3. Background/Objectives The BED-CEIA was developed and optimized to detect recent HIV-1 infections in serum specimens. Because this test will be performed mainly in central laboratories, blood from outlying areas need to be processed, stored frozen and shipped to the central laboratory Optimal storage and shipping can be very expensive and is not practical in many countries Extending use of the BED-CEIA to DBS, DSS or DPS can have major implications for incidence surveillance worldwide.

4. Initial optimization Done using matched serum and serum spot controls IgG eluted from 6mm punch using PBS/Triton or BSA/PBS/Triton (specimen diluent) Application of the protocol to a small batch of paired specimens Preparation of DBS, DSS/DPS and wider applications Preparation of controls as serum spots NC CAL LPC HPC 1001 1001

5. Use of DBS and serum spots: Optimized procedure Preparation : 50  l of serum or 100  l blood on SS# 903 Dry overnight in the hood, store in sealed bag with desiccant Using 6mm puncher, make one punch into a 96-well blank plate Add 200  l specimen diluent, mix and incubate overnight at 4-8 C for IgG elution Next day, add 50  l of specimen diluent to the BED assay plate. Transfer 50  l of eluant to the BED plate, mix and perform the assay as described starting with specimen incubation Use serum spot controls and CAL for calculating OD-n

6. Schematic of the procedure 1001 1002 + 200 μl of mix, let IgG elute Diluent at 4 C/overnight 1001 1002 6 mm punch DSS or DBS Blank 96-well plate Add 50 μ l of diluent to the BED assay plate and transfer 50 μ l of eluants, Follow the steps as in regular assay starting with specimen incubation

8. Specimens A total of 205 matched DBS and serum specimens were available DSS were prepared as described Serum, DBS and DSS were tested in parallel by the BED assay Serum results compared to DBS and DSS Stability study of DSS

9. Correlation between serum and serum spots

10. Correlation between serum and DBS

11. Correlation between DSS and DBS

12. Overall Concordance RECENTLTTOTAL SERUM35170205 DBS34171205 DSS 32173205 Agreement between diff specimens types Recent LTTotal % Agree SERUM+DBS 2916519494.6 SERUM+DSS 3016819896.6 DBS + DSS 3117020198.0 ALL THREE 2916519494.6

13. Stability of DSS on filter paper Controls and 5 member panel were spotted on SS # 903 as DSS Stability at –20 C, 4 C, 25 C and 37 C Periodic testing and comparison of raw OD and OD-n

14. Stability of DSS at 37 C Raw OD

15. Stability of DSS at 37 C OD-n

16. Serum spots are stable for at least 99 days at 37 C

17. Conclusions We have developed an optimized protocol for use of the DBS or DSS with the BED assay; results are similar to those obtained with matched serum specimens Controls and calibrator are prepared as DSS on # 903 filter paper to match the sample matrix Current data indicate that DSS are stable for >3 months @ 37 C, >5 months @ 25 C, 4 C and -20 C. Additional stability studies are ongoing. These findings may have major implications for extending the use of the BED assay in many settings for HIV-1 incidence estimates.

18. Acknowledgement Susan Phillips Trudy Dobbs Debbie Kuehl