Hepatitis C virus core antigen and dried blood spots as ... · 1 Hepatitis C virus core antigen and...
Transcript of Hepatitis C virus core antigen and dried blood spots as ... · 1 Hepatitis C virus core antigen and...
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Hepatitis C virus core antigen and dried
blood spots as simplified hepatitis C virus
diagnostic tools
François MJ Lamoury1, Behzad Hajarizadeh1, Angelica Soker1, Danica Martinez1, Camelia Quek1,
Philip Cunningham2, Beth Catlett2, Gavin Cloherty3, Pip Marks1, Janaki Amin1, Jason Grebely1,
Gregory J. Dore1, Tanya L. Applegate1
1 The Kirby Institute, UNSW Australia, Sydney, Australia. 2 St Vincent’s Applied Medical Research, Darlinghurst,
Sydney, Australia. 3 Abbott Virology, Abbott Park, IL, USA.
Australasian Viral Hepatitis Conference 2016
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1 Cassol S et al. J Clin Microb 1992
Dried blood spot
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• Biosampling where blood samples are blotted and dried on filter paper
• Advantages
Easy and inexpensive
Painless / non-invasive
Less medical training
Easier access to high risk populations
Facilitate testing for remote areas
• Inconvenient
Low sample volume
Analyte degradation if storage condition is not respected1
Assay certification for clinical use – Research tool for specialized lab
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Grebely J et al. Int J Drug Policy (2015)
Strategies to enhance linkage to HCV care
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Alternative assays:
HCV Core antigen
Rapid antibody tests
HCV RNA point of care
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Strategies to enhance linkage to HCV care
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Sample types:
Oral fluid
Dried blood spot
Capillary blood (finger-stick)
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Diagnostic models of care
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Clinics Small labs
CentralisedCentral lab
Drug and alcohol clinics
Primary health care / GPs
Prisons
Community health centres
Decentralised
NSP services
Sexual health
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The Terrence Higgins Trust Foundation and Public Health in England
Diagnostic models of care
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2.
DBS kit
delivered
3.
Self
sample
4.
Post to
lab
5.
Central lab
test
8.
Phone call7.
Reactive?+9.
Referral to
care
1.
Order
online
On-line, self collected DBS for HIV testing
HIV testing in comparison with STI clinics (UK)
• Equal recruitment, return results, and reactivity
• DBS covered broader geographic area
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McLeod A et al. J Epidemiol Community Health 2014
DBS and HCV: the Scotland’s action plan (2009)
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• DBS testing introduced into specialised drug services during 2009
• Test for HCV Antibody testing
• Drug services referred 16% of new HCV diagnosed in Scotland during
2009-13 (compared to <1% during 2003-08)
+DBS
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Aim
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Evaluate the diagnostic performance of HCV core antigen detection
in plasma and DBS
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Methodology – sample processing
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10mL
EDTA blood
Collection
DBS preparation
50mL whole blood
per spotWhatman 903 Protein saver card
Plasma
collection
HCV RNA levels testingAmpliPrep/COBAS Taqman assay (Roche)Plasma and DBS
Storage in -80C freezer
DBS elution
2 x 10mm spot
Eluation in 400mL
PBS-0.25% Triton-X100
for 1h, RT
HCV core antigen Architect assayARCHITECT-i2000R Immunoassay Analyser
HCVcAg sample volume:
Plasma: 108mL
DBS eluate: equivalent to 13.5mL plasma
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2 Ross et al., J Clin Microbiol 48:1161. 2010; 3 Murayama et al., J. Clin. Microbiol. 50: 1943. 2012; 4 Ottiger et al., J Clin Virol 58:535-540. 2013;
Medici et al., J Clin Virol 51: 264. 2011
Result – Setting up Core antigen conditions
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2 3 4 5 6
0
1
2
3
H C V R N A V L (L O G IU /m L )
HC
Vc
Ag
(L
og
fm
ol/
L)
2 3 4 5 6
0
1
2
3
H C V R N A V L (L O G IU /m L )
HC
Vc
Ag
(L
og
fm
ol/
L)
Limit of detection 3fmol/L 612IU/mL
Limit of quantitation 10fmol/L 2261IU/mL
Plasma dilutions
2 x 10mmDBS -1.1 log fmol/L
1 x 10mmDBS -1.4 log fmol/L
1 x 6mm DBS -2.4 log fmol/L
Plasma samples
Cutoff value of HCVcAg test in terms of HCV RNA levels (IU/ml)
References for studies
Ross et al., J Clin Microbiol 2010. 500-3,000 IU/ml
Ottiger et al., J Clin Virol 2013 3,467 IU/ml
Limit of detection
Limit of quantitation
997.0
)269.2(9213.0
R
Xy
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Result – Specimen characteristics
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Characteristics of the paired plasma and venous DBS sample population
Total (n=120) n(%)
PLASMA HCV RNA detected HCV + 95 (79.2)
PLASMA HCV RNA non detected HCV - 25 (20.8)
Median concentration (n=120) (IQR)
LOG HCV RNA IU/mL in PLASMA 5.57 (2.52-6.16)
LOG HCVcAg fmol/L in PLASMA 2.29 (0.07-3.13)
LOG HCVcAg fmol/L in DBS 1.14 (0.00-1.91)∆ 1.15 log
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Result – correlation between plasma and DBS
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Correlation between HCVcAg in plasma and DBS, with HCV RNA plasma
Correlation coefficient
Plasma samples (r=0.89, 95% CI: 0.85 to 0.92, p<0.0001)
DBS samples (r=0.81, 95% CI: 0.73 to 0.86, p<0.0001).
0 2 4 6 8 1 0
0
1
2
3
4
5
H C V R N A V L (L O G IU /m L )
HC
Vc
Ag
(L
og
fm
ol/
L) P la s m a
D B S
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Roche HCV RNA Roche HCV RNA
HCVcAg
plasma + -HCVcAg
DBS + -
+ 87 0 + 81 1
- 7 26 - 13 25
Sensitivity 92.6% (95%CI, 85-97%) Sensitivity 86.2% (95%CI, 77-92%)
Specificity 100% (95%CI, 84-100%) Specificity 96.1% (95%CI, 78-100%)
HCVcAg
plasma + -HCVcAg
DBS + -
+ 87 0 + 81 1
- 3 30 - 9 29
Sensitivity 96.7% (95%CI, 90-99%) Sensitivity 90.0% (95%CI, 81-95%)
Specificity 100% (95%CI, 86-100%) Specificity 96.7% (95%CI, 81-100%)
Result – Sensitivity and specificity for paired plasma and DBS
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False positive: HCVcAg: 7.5fmol/L
False negative: HCV RNA:
1100 / 1200 / 3659IU/mL
False negative: HCV RNA:
1100 / 1200 / 3,659 / 17,600 / 58,600 /
153,800 / 302,200 / 436,800 / 1,686,900IU/mL
HCV RNA > 15IU/mL
HCV RNA > 1000IU/mL
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Conclusion
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• DBS HCVcAg detection showed over one log reduction compared to plasma
• Correlation of HCVcAg compared to plasma HCV RNA satisfactory in
plasma when VL >3 log IU/mL and DBS when VL >4 log IU/mL
• Further work is required to understand potential mechanism of reduced
sensitivity in those undetected by HCVcAg.
• The feasibility of testing Core antigen on DBS should be further assessed as
a diagnostic tool in remote settings, lower and middle-income countries.
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1 A. Hill AASLD 2015, Adapted from Bowden, 2007
Discussion – increasing HCV testing in LMIC
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Expected
HCV POSITIVE: VIRAL LOAD DISTRIBUTION
n = 4020 (July 2007)
Log10 I.U. (Upper load limit for each column)
Rela
tive
Fre
quency
(%
)
0
2
4
6
8
10
12
14
16
18
20
2 3 4 5 6 7 8
Fre
qu
en
cy
HCV RNA (Log10 IU/mL)
3 4 5 6 72Expected
99%95%
100%
Distribution of HCV RNA in chronic HCV infection
HCV RNA Threshold > 25IU/mL >1,000IU/mL >10,000IU/mL
Chronic HCV 100% 99% 95%
population
Assay cost $$$$ $$ $
Access
to testing
Choice of HCV test:
Epidemiology
Infrastructure
Easy to use
Cost
Analytical sensitivity
Nucleic acid testing
Immunoassay
DBS
Rapid diagnostic testing
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1: Hepatitis C Diagnostic Technology Landscape 1st Edition 2015, Unitaid
Other and future application
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• Core antigen as point of care testing1
Daktari™ system
HCVcAg point of care instrument released in 2018
Need a drop of blood to the cartridge
Result in 30 minutes
Cost US$ 15-20 per assay, US$ 8000 for the instrument
• Other use of DBS
HCV RNA testing
Sequencing for genotyping, phylogenetic and resistance studies
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Acknowledgments
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Viral Hepatitis and Clinical Laboratory Program
Behzad Hajarizadeh Angelica Soker Danica Martinez
Camelia Quek Pip Marks Janaki Amin
Jason Grebely Gregory Dore Tanya Applegate
Immunovirology and Pathogenesis Program
Tony Kelleher and team
St Vincent’s Applied Medical Research, Sydney
Philip Cunningham Beth Catlett
Sydpath, St Vincent’s Hospital, Sydney
Hideaki Toji Joymarie Armstrong Spyros Repoussis
Mark Paul Rebecca Collins Lisa Stanton
Abbott:
Gavin Cloherty Wade Foster Andrew StJohn
Funding:
National Health and Medical Research Council (Program grant).
Department of Health and Aging, Australian Government.
Support from Abbott Diagnostics for the supply of reagents.
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HCV Core antigen (HCVcAg)
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• Can detect active infection
• Easy to perform
• Less expensive
• More stable
• Expressed as fmol/L (10-15 mol/L)
• Measured with the HCV Ag ARCHITECT assay on the
ARCHITECT-i2000R Immunoassay Analyser.
Range: 0 – 20000fmol/L
Reactive > 3fmol/L. Quantified > 10fmol/L
Envelope glycoprotein
Envelope Lipid
Core protein
RNA
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1 Chevaliez S et al. Antiviral therapy 2016; 2 Ross et al., J Clin Microbiol 48:1161. 2010; 3 Murayama et al., J. Clin. Microbiol. 50: 1943. 2012; 4
Ottiger et al., J Clin Virol 58:535-540. 2013; Medici et al., J Clin Virol 51: 264. 2011
Result – Comparison between plasma and DBS
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Bland-Altman Bias plot: HCVcAg vs Roche HCV RNA for plasma
HCVcAg levels were converted to log IU/mL based on a conversion factor of 1fmol/L = 500IU/mL1,2,3,4,5
Bland-Altman Bias (95% limits of agreement) mean difference (95%CI)
Plasma 2.46 log IU/mL (-0.50, 5.42) 2.46 log IU/mL (2.19-1.51)
DBS 3.26 log IU/mL (-0.35, 6.86) 3.25 log IU/mL (2.92-3.59)
0 2 4 6
-2
0
2
4
6
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A v e ra g e o f R o c h e H C V R N A a n d A b b o tt H C V c A g
Ro
ch
e H
CV
RN
A -
Ab
bo
tt H
CV
cA
g
9 5 % lim its o f a g re e m e n t
9 5 % lim its o f a g re e m e n t
b ia s
0 2 4 6
-2
0
2
4
6
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A v e ra g e o f R o c h e H C V R N A a n d A b b o tt H C V c A g
Ro
ch
e H
CV
RN
A -
Ab
bo
tt H
CV
cA
g 9 5 % lim its o f a g re e m e n t
9 5 % lim its o f a g re e m e n t
b ia s
Plasma DBS
95% limits of agreement
95% limits of agreement
95% limits of agreement
95% limits of agreement
bias
bias
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13 DBS HCV RNA detected / HCVcAg non-detected
ID: Gt VL (IU/mL):Plasma Core Ag fmol/L
DBS Core Ag fmol/L
8888-61231-339 ND 27 0.7 0
8888-61231-313 1a 57 0 1.4
8888-61231-386 1a 110 0.28 0
8888-61231-388 3a 220 0 0
8888-61231-387 3a 1100 0.09 0
8888-61231-466 1a 1200 1.67 0
8888-61231-317 1a 3659 0.54 0
8888-61231-430 3a 17600 21.38 0.38
8888-61231-444 1b 58600 25.79 0.91
8888-61231-420 3a 153800 5.12 0
8888-61231-304 1b 302200 89.44 0
8888-61231-433 3a 436800 697.04 2.06
8888-61231-407 1b 1686900 26.06 0.1
9 plasma HCV RNA detected / HCVcAg non-detected
ID: Gt VL (IU/mL):Plasma Core Ag fmol/L
DBS Core Ag fmol/L
8888-61231-339 ND 27 0.7 0
8888-61231-313 1a 57 0 1.4
8888-61231-386 1a 110 0.28 0
8888-61231-388 3a 220 0 0
8888-61231-387 3a 1100 0.09 0
8888-61231-466 1a 1200 1.67 0
8888-61231-317 1a 3659 0.54 0
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