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Dried Blood Spots in HIV and HCV
Epidemiology and Drug Resistance Testing
National HIV & Retrovirology Labs
Public Health Agency of Canada
Ottawa, Ontario
CANADA
HIV Diagnostics: New Developments and Challenges
Orlando, Florida
Feb 28 - Mar 1, 2005
Dried Blood Spots (DBS)
• use in HIV serological and nucleic acid testing
well-documented & validated
• simple, robust, inexpensive
• two approved EIA tests (GSrLAV & OT
Vironostika)
• surveillance, diagnostic, clinical care
management
DBS - Collection
Good !
Baaad !
Dried Blood Spots (DBS)
(antibody elution-GSrLAV EIA)
1. 1/4" disk is punched into an uncoated plate.
2. 200uL of specimen diluent (normal bovine serum / 0.1%
Proclin added and mixed well.
3. eluted o/n at 40 C.
4. next day samples are brought to RT and mixed well.
5. Mix 40uLsample into 60uL sp.diluent into coated plate.
Rest of assay as per norm.
CBC HIV-1 Western Blot
INNO-LIA HIV
Figure 1. Performance of Cameroon DBS on Confirmatory Tests
HIV testing Algorithm
DBS
HIV-1 screen test
(bioMerieux Vironostika HIV-1 EIA)
Non-reactive
Low Reactive
s/co<3
Report Negative
Reactive
HIV-1 Western Blot
(Bio-Rad GS WB)
Repeat test
Non-Reactive
Reactive
Positive
Report Positive
Negative
Hepatitis C testing Algorithm
(DBS)
HCV screen test
(Ortho HCV EIA)
Non-reactive
Report Negative
Low Reactive
s/co<3
Reactive
Supplementary screen Test
(Innogenetics HCV Inno-test
Repeat screen test
Reactive
Report Positive
Non-reactive
Applications of DBS in NHRL
• Diagnostics
• Surveillance (Prevalence/Incidence)
• V.I.D.U.S. (Vancouver Intrav Drug User Study)
• I-Trak
• M-Trak (MSM in Montreal)
• International
- Kosovo
- Pakistan
Molecular Analysis of HIV & HCV
from DBS
Elute
DBS
Bind to
silica
Wash/Elute
Purified RNA
HIV
HCV
RT-PCR
Sequencing
Table 3. HIV-1 Subtyping Data
(C2-V3 env gene) Cameroon DBS
Sample ID
CN008
CN009
CN010
CN016
CN014
NLHRS#
00-0781
00-0782
00-0783
00-0785
00-0794
cp/ml
Subtype
73 000
210 000
110 000
220 000
1 100 000
A (HIV-1)
A (HIV-1)
A (HIV-1)
A (HIV-1)
D (HIV-1)
Figure 2. HIV-1 Drug Resistance Genotyping on Cameroon DBS
(Visible Genetics) and Subtyping (inset)
HCV-RT-PCR/Sequencing
5’NCR
RT
Core
PCR
Phylogenetic comparison
(LANL HCV Sequence Database)
HCV-Genotype Distribution
(I-TRAK)
I-Track HCV Core Genotypes
0%
3%
HCV 5'NCR Genotype
1% 1% 2%
4% 2%
2%
4%
72%
21%
1a
3a
1b
2a
2b
4%
42%
17%
25%
• high HCV 3a
• high-risk vs low risk populations ?
1a
3a
1b
1b/1a
2a/1a
2b
1c/1a
3b
5a
6b/h/k
Performance of HIV DR Methods
(DBS-RNA)
(26,500)
+
+
-
(154,300)
(31,000)
+
+
+
+
+
(16,600)
+
-
(461,200)
+
+
+
Viral Load
In House
(Nested PCR)
Trugene
(Bayer)
ViroSeq
(ABI)
Nature of Nucleic Acid from DBS
Extraction
+ DNase
+ RNase
Effect of Storage Temperature -DBS
DBS – Viral Load 490,000
DBS – Viral Load 20,000
FTA
FTA
WEEK2 WEEK3 WEEK4 WEEK5
WEEK2 WEEK3 WEEK4 WEEK5
20°C
4 °C
-20°C
-80°C
20°C
4 °C
-20°C
-80°C
903
903
WEEK2 WEEK3 WEEK4 WEEK5
20°C
4 °C
-20°C
-80°C
WEEK2 WEEK3 WEEK4 WEEK5
20°C
4 °C
-20°C
-80°C
Effect of HIGH Humidity/Temp – 903 Paper
(45% Relative Humidity and 37°C)
3
PCRAMP
PCR
Amplification
2
1
DBS10
DBS2 Sample
0
.6
2
K
EE
W K1
EE .9
W K0
EE .6
W K0
EE .3
W K0
EE
W
.3
.9
1
Time (weeks)
2
Effect of HIGH Humidity/Temp – FTA Paper
(45% Relative Humidity and 37°C)
3
2.5
2
PCRPCR
AMP 1.5
Amplification 1
0.5
0
.6
.9
K
EE
W K
EE
W K0
EE .
W K0
EE .3
W K0
EE
W
.3
DBS10
DBS2 Sample
Time (weeks)
1
2
Stabilization of DBS-RNA by RNAlater
# Successful Amplifications
(n=5)
(85% Relative Humidity and 37°C)
5
4
3
2
1
0
Day 1
Day 3
Day 6
Time
Day 9
Day 14
903 untreated/no desiccant
903 untreated/dessicant
903 treated/no desiccant
903 treated/desiccant
Conclusion/Future Activities - DBS
•
•
•
•
HIV and HCV serology and NA testing
Clinical monitoring – viral load/DR testing
Venipuncture vs DBS sequences
Limits of DBS to ‘extreme’ conditions
“DBS are economical, easy to collect, transport and store. Their ease
of use and versatility make DBS an ideal tool for large scale
surveillance studies, both domestically and abroad”
I-Trak
Enhanced surveillance to track HIV- and HCV- associated risk
behaviours in injecting drug users (IDU).
• Cross-sectional design
• Interviewer administered questionnaire
• Information collected on:
 Demographics
 Injecting and non-injecting substance use
 Injecting, sexual, testing behaviours
• DBS collected for HIV & HCV serological testing
 To describe changing patterns in the prevalence of HIV
and HCV at the national and local level.
Conclusions
1.
I-Track found 21% of infections involving genotype 3, whereas the
database estimates for Canada and the US were 3% and 4%
respectively, and Bernier et al. found 14% in Montreal. Bernier et al.
also found about 30% each 1a and 1b, whereas I-Track was 72% 1a.
This suggests that the observed HCV genotype distribution may be
unique to IDU when compared to the general population.
2.
Although the numbers are still too low to permit complete statistical
interpretation, preliminary univariate analysis suggests that the
emergence of genotypes other than 1a in IDU is a relatively new
phenomenon.
3.
Linking surveillance data to findings of molecular analysis (regional
clusters, genotype distribution) will lead to more targeted approaches
to prevention, helping those who are most susceptible.
4.
DBS are economical, easy to collect, transport and store, and produced
quality HCV sequence data for this study. Their ease of use and
versatility make DBS an ideal tool for large scale surveillance studies,
both here and abroad.
5.
HIV sent in about 10% HCV infected.
Conclusions
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
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)
Improved recovery by pre-treatment of membrane with RNA stabilizer
Future Work
Quantitate HIV RNA on DBS by real time PCR
Establish consensus between DBS and plasma derived sequences
Genotype Distribution
• Genotype 1a is the most prevalent genotype
across all sites, followed by 3a (based on
core region).
• Other genotypes (non-1a/3a) make up less
than 8% of the HCV prevalence among ITrack participants.
• There was a large regional variation in
genotype distribution, although the
numbers are still too low to permit
complete statistical interpretation.
What Next?
• Need to address differences observed between the core
and 5’NCR genetic regions through analysis of other genetic
regions (E1, NS5B).
• The phylogenetic information obtained was often
insufficient to achieve statistical significance of clusters.
Phylogenetic analysis will be repeated with the inclusion of
additional regions as they become available.
• About 10% of HCV I-Track participants were co-infected
with HIV. HIV sequencing (from HCV extracts) is currently
underway to characterize the co-infections using
phylogenetic methods.
Study Design
A laboratory investigation of factors influencing
the durability of DBS particularly under
extreme conditions.
Type of membrane
Viral Load
Storage Temperature
Duration of Storage
Humidity
Sequencing Technology
Nature of viral target found on DBS (RNA/DNA)
Methods
• Blood samples were collected from HIV +ve
patients (with established viral loads) in 10 ml
EDTA vacutainers
• DBS were prepared by spotting 50 ml of blood
(903 cards) or 200 ml of blood (FTA cards)
• Viral RNA was extracted using Nuclisens
extraction system
• DBS (903) or ¼ DBS (FTA) were cut into four equal strips
and lysis buffer added.
Methods
• RT-PCR was performed using 10 ml of isolated viral RNA with
“One Step RT-PCR” (Qiagen)
• Secondary PCR was performed using 4 ml of RT-PCR amplicon
with “Platinum PCR Supermix” (Invitrogen)
• Secondary amplicons were sequenced on a LiCor 4200
sequencer using simultaneous bidirectional sequencing
following manufactures suggested protocol.
• Primers (two sets)
• PR1Forward/RT Protease Inner Reverse (2171-2188 to 2901-2925)
• RT Outer Forward/3’ Half (2813-2836 to 3506-3536)