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The Use of Dried Blood Spots in HIV Drug Resistance Surveillance

The Use of Dried Blood Spots in HIV Drug Resistance Surveillance. Diane Bennett MD MPH. U.S. HIV drug resistance (HIVDR) surveillance. Remnant HIV diagnostic sera: all individuals newly diagnosed with HIV

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The Use of Dried Blood Spots in HIV Drug Resistance Surveillance

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  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 #624 787 Bennett et al

  4. 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 HIVDR Surveillance Implementation • 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)

  8. Genotyping Results with Roche RNA extraction (Cote d’Ivoir)

  9. CDC evaluation of 4 year old VQA DBS panels (Garcia-Lerma) 1-. PCR amplification from Dried Blood Spots 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 VL 16,620 1,157 231,040 99,980 1,064 65,332 Plasma + + + + + +

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

  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. 14/33 (42%) Pol = 1341 base pairs 25/33 (76%) Gag = 871 base pairs Stability of DBS held at room temperature For 2-8 weeks in the Real World WHO HIV ResNet Mexico Pilot 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 Schuurman Paul Sandstrom • John Kim • CDC • Gerardo Garcia-Lerma Unite de Virologie, Rouen • Walid Heneine JC Plantier • Richard Kline F Simon • Joanne Mei • Lyle McCormick Research Think Tank Inc. • Amanda Smith Robert Lloyd • Will Wheeler • Ida Onorato • Tim Dondero • Irum Zaidi

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