1. The Use of Dried Blood Spots in HIV Drug Resistance Surveillance Diane Bennett MD MPH

2. U.S. HIV drug resistance (HIVDR) surveillance Remnant HIV diagnostic sera: all individuals newly diagnosed with HIV Amplification and sequencing of relevant pol gene regions takes place at Stanford University Laboratory, University of Washington Laboratory, or participating state health department laboratories: Florida Maryland Michigan New York State Non-research determination received June 2004; incorporated into routine HIV surveillance July 2004 Hard copy results and sequences returned within a month to health departments (HD) Analyses focus on major mutations associated with HIVDR, HIV-1 subtype Separate analyses for all newly diagnosed persons and the recently infected subset identified by STARHS

3. U.S. surveillance of HIV drug resistance using diagnostic sera –CROI Feb 2005 787 #624 Bennett et al

4. HIVDR Surveillance Implementation Chicago Department of Public Health* Colorado Department of Public Health and Environment * District of Columbia Department of Health Florida Department of Health Illinois Department of Public Health* Indiana State Department of Health Louisiana Office of Public Health Maryland Department of Health& Mental Hygiene* Massachusetts Department of Public Health* Michigan Department of Public Health* * Specimen collection has begun Mississippi State Department of Health* New Jersey Department of Health and Senior Services NYC Department of Health & Mental Hygiene New York State Department of Health North Carolina Department of Health Pennsylvania Department of Health Puerto Rico Department of Health Seattle/King County Department* South Carolina Department of Health* Texas Department of Health Virginia Department of Health* Washington State Department of Health Barriers to implementing HIVDR surveillance include: Lab processing restrictions Centrifuge within 48 hours; freeze within 96 hours of blood draw 1 ml serum minimum Ship on dry ice – labor and expense Oral or rapid testing -> if no confirmatory blood, no specimen for genotyping

5. Dried Blood Spots for HIVDR surveillance DBS seem the ideal specimen type for easy collection, storage and transport: Once dried, no need to rush specimens to lab for quick processing Lower volume required (20  l to 200  l) Easy collection: Fingerstick by non-phlebotomists (training and q.a. important) Can extract blood, plasma, or serum from vacutainer without opening it No laboratory manipulations needed after spotting Simple storage: Short term at ambient temperature Long term storage at –20C Simple transportation at ambient temperature: No dry ice needed (high cost and complicated logistics) DBS can be shipped as non-infectious material (except by US postal service)

6. Utrecht University: Viral Load (plasma vs dried plasma spot)

7. Genotyping Results with Roche RNA extraction (Cote d’Ivoir) Specimen Type Plasma VL log10 RNA copies/mL PCRGenotypingMutations 43625 DBS+yes K103N, Y108I 43625 Plasma4+yesM84V, K103N, Y108I 44493 DBS+yesM184V, K103N, M36I 44493 Plasma6.67+yesM184V, K103N, M36I 44006 DBS+yesM36I 44006 Plasma4.64+yesM36I 43900 DBS+yesM36I, L63P 43900 Plasma5.1+yesM36I, L63P

8. Specimen Type Plasma VL log10 RNA copies/Ml PCRGenotypingMutations 43787 DBS3.07- 43790 DBS3.90+ 44316 DBS3.96- 44392 DBS2.72- 44479 DBS2.75- 44497 DBS4.44- 44499 DBS4.46- 45448 DBS+ Genotyping Results with Roche RNA extraction (Cote d’Ivoir)

9. VL 16,620 1,157 231,040 99,980 1,064 65,332 Plasma + DBS + RT- RT+++--- Panel 1 (-20ºC x 4 yr ) 1.1 1.2 1.3 Panel 2 (room temp x 4 yr) 2.1 2.2 2.3 1-. PCR amplification from Dried Blood Spots CDC evaluation of 4 year old VQA DBS panels (Garcia-Lerma)

10. Results 2-. Similarity between plasma and DBS RT-Prot sequences DBS 1.1 DBS 1.2 DBS 1.3 PLASMA 1.1 PLASMA 1.2 PLASMA 1.3 DBS 1.1 DBS 1.2 95 DBS 1.3 88 89 Plasma 1.1 97 93 86 Plasma 1.2 95 99 89 93 Plasma 1.3 88 89 100 86 89

11. Plasma D67N, T69N/T, K70R, M184V, T215T/Y/S/N, K219Q Y181Y/C, M184V T69N, Y181C + RT D67D/N, T69N, K70K/R, M184V/M, T215T/I/S/F, K219Q/K M184V T69N, Y181C ID 1.1 1.2 1.3 3-. Resistance mutations - RT D67D/N, K70K/R, M184V/M, T215T/I/S/F, K219Q/K M184V T69N, Y181C DBS

12. Conclusions from Health Canada DBS Study: relevance for surveillance (see previous presentation by Health Canada) Using DBS, HIV RNA appears to be preferentially amplified (consistent with plasma) Commercial sequencing kits are compatible although lack of secondary PCR may be problematic for low viral loads No differences in mutations associated with resistance (plasma vs DBS) (data not shown) Similar performance between FTA and 903 under “ideal” conditions Poorer performance for FTA under elevated temperatures and humidity Humidity is detrimental to recovery (desiccant & suitable storage pouches should be required) Improved recovery by pre-treatment of membrane with RNA stabilizer (data not shown)

13. Stability of DBS held at room temperature For 2-8 weeks in the Real World WHO HIV ResNet Mexico Pilot Pol = 1341 base pairs Gag = 871 base pairs 14/33 (42%) 25/33 (76%) Subsequent amplification of smaller fragments of pol: 29/33 (88%)

14. CROI 2005 data on dried fluid testing Francois Simon : Dried Serum Spots from 47 drug naïve and 15 treated patients =62 903 membrane Small DSS volume (20  l ); storage 2 weeks at room temp; no dessicant Overall amplification/sequencing : protease 53/62 (86%); RT 51/62 (82.3%) VL > 100,000 protease 17/17 (100%); RT 17/17 (100%) 1000 <VL < 100,000 protease 25/29 (86%); RT 26/29 (97%) VL < 10,000 protease 11/16 (69%); RT 6/16 (38%) Rob Lloyd : SampleTanker (like a cigarette filter) Up to 1ml serum, plasma, blood – aliquot onto the filter Stable at room temperature for weeks Dessicant and colored warning system included Amplification and sequencing > 90% down to VL 1000 copies/ml

15. Maximizing use of DBS for HIVDR surveillance -DBS, dried serum spots (DSS), dried plasma spots (DPS) all appear promising but data are limited -Minimize humidity Use 903 paper Use of dessicant and proper handling is essential -Freeze or amplify within two weeks -Smaller PCR products may improve amplification Labs using kits may need to partner with labs able to do a nested PCR to amplify smaller fragments

16. CDC/Health Departments collaboration Objectives: Evaluate feasibility of HIVDR surveillance using DBS in selected sites Compare paired sera and DBS in a subset of sites Four health departments funded under PA 4118: Chicago, Los Angeles County, Minneapolis, New York State Three laboratories: Stanford, Minneapolis, New York State DBS will be made on 903 paper: From fingersticks at testing point From confirmatory HIV tests From a red-top tube, must spot immediately or consider DSS Some sites will draw confirmatory specimens in EDTA tubes instead From first clinical specimen (usually for viral load) in selected sites RNA will be targeted ?Pre-treatment of 903 membrane with RNA stabilizer (each spot pre-treated with 50  l “RNA Later”)

17. Acknowledgements UMCU Department of VirologyHealth Canada Rob SchuurmanPaul Sandstrom John Kim CDC Gerardo Garcia-Lerma Unite de Virologie, Rouen Walid HeneineJC Plantier Richard KlineF Simon Joanne Mei Lyle McCormick Research Think Tank Inc. Amanda SmithRobert Lloyd Will Wheeler Ida Onorato Tim Dondero Irum Zaidi