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