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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• 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
U.S. surveillance of HIV drug resistance using diagnostic sera –CROI Feb 2005
787#624
Bennett et al
HIVDR Surveillance ImplementationChicago Department of Public Health*Colorado Department of Public Health and Environment *District of Columbia Department of HealthFlorida Department of Health Illinois Department of Public Health*Indiana State Department of HealthLouisiana 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 ServicesNYC Department of Health & Mental HygieneNew York State Department of HealthNorth Carolina Department of Health Pennsylvania Department of HealthPuerto Rico Department of HealthSeattle/King County Department*South Carolina Department of Health*Texas Department of HealthVirginia 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
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 200l)• 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)
Rainbow direct in assay or from filter spot
n = 82, r = 0.944
Direct in assay log copies/ml
8765432
Spott
ed o
n fi
lter
log c
opie
s/m
l
8
7
6
5
4
3
2Rsq = 0.9926
thru origin
Utrecht University: Viral Load (plasma vs dried plasma spot)
Genotyping Results with Roche RNA extraction(Cote d’Ivoir)
SpecimenType
Plasma VL log10 RNA copies/mL
PCR Genotyping Mutations
43625 DBS + yes K103N, Y108I
43625 Plasma 4 + yes M84V, K103N, Y108I
44493 DBS + yes M184V, K103N, M36I
44493 Plasma 6.67 + yes M184V, K103N, M36I
44006 DBS + yes M36I
44006 Plasma 4.64 + yes M36I
43900 DBS + yes M36I, L63P
43900 Plasma 5.1 + yes M36I, L63P
SpecimenType
Plasma VL log10 RNA copies/Ml
PCR Genotyping Mutations
43787 DBS 3.07 -
43790 DBS 3.90 +
44316 DBS 3.96 -
44392 DBS 2.72 -
44479 DBS 2.75 -
44497 DBS 4.44 -
44499 DBS 4.46 -
45448 DBS +
Genotyping Results with Roche RNA extraction(Cote d’Ivoir)
VL
16,6201,157231,040
99,9801,06465,332
Plasma
+++
+++
DBS+ RT - RT
+ ++ ++ +
- -- -- -
Panel 1 (-20ºC x 4 yr)
1.11.21.3
Panel 2 (room temp x 4 yr)
2.12.22.3
1-. PCR amplification from Dried Blood Spots
CDC evaluation of 4 year old VQA DBS panels (Garcia-Lerma)
Results
2-. Similarity between plasma and DBS RT-Prot sequences
DBS 1.1DBS 1.2DBS 1.3PLASMA 1.1PLASMA 1.2PLASMA 1.3
DBS 1.1
DBS 1.2
95
DBS 1.3
8889
Plasma 1.1
979386
Plasma 1.2
95998993
Plasma 1.3
88891008689
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
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)
Stability of DBS held at room temperatureFor 2-8 weeks in the Real WorldWHO 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%)
CROI 2005 data on dried fluid testingFrancois 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
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
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”)
Acknowledgements
UMCU Department of Virology Health CanadaRob Schuurman Paul Sandstrom
John KimCDCGerardo Garcia-Lerma Unite de Virologie, RouenWalid Heneine JC PlantierRichard Kline F SimonJoanne MeiLyle McCormick Research Think Tank Inc.Amanda Smith Robert LloydWill WheelerIda OnoratoTim DonderoIrum Zaidi