Transcript PowerPoint-presentatie - 2016 HIV Diagnostics Conference

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
#624
Bennett et al
787
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*
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
* Specimen collection has begun
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 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)
Utrecht University: Viral Load (plasma vs dried plasma spot)
Rainbow direct in assay or from filter spot
n = 82, r = 0.944
8
7
6
5
4
3
Rsq = 0.9926
2
thru origin
2
3
4
5
6
Direct in assay log copies/ml
7
8
Genotyping Results with Roche RNA extraction
(Cote d’Ivoir)
Specimen
Type
Plasma VL
43625 DBS
43625 Plasma
4
44493 DBS
44493 Plasma
6.67
44006 DBS
44006 Plasma
4.64
43900 DBS
43900 Plasma
PCR
Genotyping
Mutations
+
yes
K103N, Y108I
+
yes
M84V, K103N, Y108I
+
yes
M184V, K103N, M36I
+
yes
M184V, K103N, M36I
+
yes
M36I
+
yes
M36I
+
yes
M36I, L63P
+
yes
M36I, L63P
log10 RNA copies/mL
5.1
Genotyping Results with Roche RNA extraction
(Cote d’Ivoir)
Specimen
Type
Plasma VL
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
PCR
log10 RNA copies/Ml
+
Genotyping
Mutations
CDC evaluation of 4 year old VQA DBS panels (Garcia-Lerma)
1-. PCR amplification from Dried Blood Spots
Panel 1 (-20ºC x 4 yr) VL
Plasma
DBS
+ RT
- RT
1.1
1.2
1.3
16,620
1,157
231,040
+
+
+
+
+
+
+
+
+
99,980
1,064
65,332
+
+
+
-
-
Panel 2 (room temp
x 4 yr)
2.1
2.2
2.3
Results
2-. Similarity between plasma and DBS RT-Prot sequences
DBS
1.1
DBS 1.1
DBS 1.2
DBS 1.3
PLASMA 1.1
PLASMA 1.2
PLASMA 1.3
DBS
1.2
DBS
1.3
Plasma Plasma Plasma
1.1
1.2
1.3
95
88
89
97
93
86
95
99
89
93
88
89
100
86
89
3-. Resistance mutations
DBS
ID
Plasma
+ RT
- RT
1.1
D67N, T69N/T, K70R,
M184V, T215T/Y/S/N, K219Q
D67D/N, T69N, K70K/R,
M184V/M, T215T/I/S/F, K219Q/K
D67D/N, K70K/R, M184V/M,
T215T/I/S/F, K219Q/K
1.2
Y181Y/C, M184V
M184V
M184V
1.3
T69N, Y181C
T69N, Y181C
T69N, Y181C
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 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%)
CROI 2005 data on dried fluid testing
Francois Simon:
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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%)
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•
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VL > 100,000
1000 <VL < 100,000
VL < 10,000
protease 17/17 (100%); RT 17/17 (100%)
protease 25/29 (86%);
RT 26/29 (97%)
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
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•
•
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
Rob Schuurman
CDC
Gerardo Garcia-Lerma
Walid Heneine
Richard Kline
Joanne Mei
Lyle McCormick
Amanda Smith
Will Wheeler
Ida Onorato
Tim Dondero
Irum Zaidi
Health Canada
Paul Sandstrom
John Kim
Unite de Virologie, Rouen
JC Plantier
F Simon
Research Think Tank Inc.
Robert Lloyd