Hepatitis C virus core antigen and dried blood spots as ... · 1 Hepatitis C virus core antigen and...

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1 Hepatitis C virus core antigen and dried blood spots as simplified hepatitis C virus diagnostic tools François MJ Lamoury 1 , Behzad Hajarizadeh 1 , Angelica Soker 1 , Danica Martinez 1 , Camelia Quek 1 , Philip Cunningham 2 , Beth Catlett 2 , Gavin Cloherty 3 , Pip Marks 1 , Janaki Amin 1 , Jason Grebely 1 , Gregory J. Dore 1 , Tanya L. Applegate 1 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

Transcript of Hepatitis C virus core antigen and dried blood spots as ... · 1 Hepatitis C virus core antigen and...

Page 1: Hepatitis C virus core antigen and dried blood spots as ... · 1 Hepatitis C virus core antigen and dried blood spots as simplified hepatitis C virus diagnostic tools François MJ

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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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Presentation Title // edit 'Header & Footer' to change or remove

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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

8

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

8

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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