Hepatitis C Virus Core Antigen And Dried Blood Spots As Simplified .

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 1

Dried blood spot 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 2 1 Cassol S et al. J Clin Microb 1992 2

Strategies to enhance linkage to HCV care Alternative assays: HCV Core antigen Rapid antibody tests HCV RNA point of care 3 Grebely J et al. Int J Drug Policy (2015) 3

Strategies to enhance linkage to HCV care Sample types: Oral fluid Dried blood spot Capillary blood (finger-stick) 4 4

Diagnostic models of care Sexual health NSP services Drug and alcohol clinics Community health centres Clinics Small labs Prisons Primary health care / GPs Central lab Centralised Decentralised 5 5

Diagnostic models of care On-line, self collected DBS for HIV testing 1. Order online 2. DBS kit delivered 9. Referral to care 8. Phone call 3. Self sample 4. Post to lab 7. Reactive? 5. Central lab test HIV testing in comparison with STI clinics (UK) Equal recruitment, return results, and reactivity DBS covered broader geographic area 6 The Terrence Higgins Trust Foundation and Public Health in England 6

DBS and HCV: the Scotland’s action plan (2009) DBS testing introduced into specialised drug services during 2009 Test for HCV Antibody testing DBS Drug services referred 16% of new HCV diagnosed in Scotland during 2009-13 (compared to 1% during 2003-08) 7 McLeod A et al. J Epidemiol Community Health 2014 7

Aim Evaluate the diagnostic performance of HCV core antigen detection in plasma and DBS 8 8

Methodology – sample processing 10mL EDTA blood Collection Plasma collection DBS preparation 50mL whole blood per spot Whatman 903 Protein saver card DBS elution 2 x 10mm spot Eluation in 400mL PBS-0.25% Triton-X100 for 1h, RT Plasma and DBS Storage in -80C freezer HCV RNA levels testing AmpliPrep/COBAS Taqman assay (Roche) HCV core antigen Architect assay ARCHITECT-i2000R Immunoassay Analyser HCVcAg sample volume: Plasma: 108mL DBS eluate: equivalent to 13.5mL plasma 9 9

Result – Setting up Core antigen conditions Plasma dilutions y 0.9213( X 2.269) R 0.997 3 H C V c A g (L o g f m o l/L ) Plasma samples 2 Limit of quantitation 1 Limit of detection 0 2 3 4 5 6 H C V R N A V L ( L O G IU /m L ) Limit of detection 3fmol/L 612IU/mL Limit of quantitation 10fmol/L 2261IU/mL 2 x 10mmDBS 1 x 10mmDBS 1 x 6mm DBS -1.1 log fmol/L -1.4 log fmol/L -2.4 log fmol/L Cutoff value of HCVcAg test in terms of HCV RNA levels (IU/ml) References for studies Ross et al., J Clin Microbiol 2010. Ottiger et al., J Clin Virol 2013 10 500-3,000 IU/ml 3,467 IU/ml 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 10

Result – Specimen characteristics 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 11 11

Result – correlation between plasma and DBS Correlation between HCVcAg in plasma and DBS, with HCV RNA plasma H C V c A g (L o g f m o l/L ) 5 P la s m a 4 DBS 3 2 1 0 0 2 4 6 8 10 H C V R N A V L ( L O G IU /m L ) 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). 12 12

Result – Sensitivity and specificity for paired plasma and DBS HCV RNA 15IU/mL 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%) False positive: HCVcAg: 7.5fmol/L HCV RNA 1000IU/mL 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%) 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 13 13

Conclusion 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. 14 14

Discussion – increasing HCV testing in LMIC Choice of HCV test: Nucleic acid testing Immunoassay DBS Rapid diagnostic testing 20 99% 18 95% 16 Frequency Epidemiology Infrastructure Easy to use Cost Analytical sensitivity Relative Frequency (%) HCV POSITIVE: VIRAL LOAD DISTRIBUTION Distribution of HCV RNA chronic HCV infection n 4020 (Julyin 2007) 100% 14 12 10 8 6 4 2 0 2 3 2 3 4 4 5 5 6 6 7 7 LogHCV (Upper (Log load limit for each column) 10 I.U. RNA 10 IU/mL) HCV RNA Threshold Chronic HCV population Assay cost 25IU/mL 100% 1,000IU/mL 99% 8 Expected Expected 10,000IU/mL 95% Access to testing 15 1 A. Hill AASLD 2015, Adapted from Bowden, 2007 15

Other and future application 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 16 1: Hepatitis C Diagnostic Technology Landscape 1st Edition 2015, Unitaid 16

Acknowledgments Viral Hepatitis and Clinical Laboratory Program Behzad Hajarizadeh Angelica Soker Camelia Quek Pip Marks Jason Grebely Gregory Dore Danica Martinez Janaki Amin 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 Mark Paul Rebecca Collins Spyros Repoussis Lisa Stanton Abbott: Gavin Cloherty Andrew StJohn Wade Foster 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. 17 17

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HCV Core antigen (HCVcAg) Can detect active infection Envelope glycoprotein Easy to perform Envelope Lipid Core protein Less expensive More stable Expressed as fmol/L (10-15 mol/L) Measured with the HCV Ag ARCHITECT assay on the RNA ARCHITECT-i2000R Immunoassay Analyser. Range: 0 – 20000fmol/L Reactive 3fmol/L. Quantified 10fmol/L 19 19

Result – Comparison between plasma and DBS Bland-Altman Bias plot: HCVcAg vs Roche HCV RNA for plasma 8 95% limits of agreement 6 9 5 % lim it s o f a g re e m e n t bias 4 b ia s 2 0 9 5 % lim it s o f a g re e m e n t 95% limits of agreement -2 R o c h e H C V R N A - A b b o tt H C V c A g R o c h e H C V R N A - A b b o tt H C V c A g DBS Plasma 8 95% limits of agreement 9 5 % lim it s o f a g re e m e n t 6 4 bias b ia s 2 0 9 5 % lim it s o f a g re e m e n t 95% limits of agreement -2 0 2 4 6 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 0 2 4 6 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 HCVcAg levels were converted to log IU/mL based on a conversion factor of 1fmol/L 500IU/mL 1,2,3,4,5 20 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) 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 20

Presentation Title // edit 'Header & Footer' to change or remove 13 DBS HCV RNA detected / HCVcAg non-detected ID: 8888-61231-339 8888-61231-313 8888-61231-386 8888-61231-388 8888-61231-387 8888-61231-466 8888-61231-317 8888-61231-430 8888-61231-444 8888-61231-420 8888-61231-304 8888-61231-433 8888-61231-407 Gt ND 1a 1a 3a 3a 1a 1a 3a 1b 3a 1b 3a 1b VL (IU/mL): 27 57 110 220 1100 1200 3659 17600 58600 153800 302200 436800 1686900 Plasma Core DBS Core Ag Ag fmol/L fmol/L 0.7 0 0 1.4 0.28 0 0 0 0.09 0 1.67 0 0.54 0 21.38 0.38 25.79 0.91 5.12 0 89.44 0 697.04 2.06 26.06 0.1 9 plasma HCV RNA detected / HCVcAg non-detected ID: 8888-61231-339 8888-61231-313 8888-61231-386 8888-61231-388 8888-61231-387 8888-61231-466 8888-61231-317 Gt ND 1a 1a 3a 3a 1a 1a VL (IU/mL): 27 57 110 220 1100 1200 3659 Plasma Core DBS Core Ag Ag fmol/L fmol/L 0.7 0 0 1.4 0.28 0 0 0 0.09 0 1.67 0 0.54 0 21 21

Diagnostic models of care 6 2. DBS kit delivered 3. Self sample 4. Post to lab 5. Central lab test 8. Phone call 7 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