Philip William Tuke

HCV window phase infections:Closing the window on HCV

A comparison of two combined Ag / Ab assays with QRT-PCR

Closing the window on HCV

• HCV alone comprises the Hepacivirus genus, a member of the Flavivirus family

• SS +ve RNA virus in a protein core

• Important human pathogen infecting ~3% of the world population1

• >5 million people infected in Europe2

• Prevalence of HCV infection in UK

0.4% England3 1.0% Scotland4

• Blood donor seropositivity .041% new donors, .0016% repeat donors5

Closing the window on HCV

Closing the window on HCV

• HCV potential major cause of post transfusion hepatitis

• Incidence initially dramatically reduced by Ab screening

• Subsequently further reduced by NAT

• Window period = time to detection

• Ab window 51 days

Closing the window on HCV

• NAT and / or Ag testing can be undertaken to close this window

• Testing of donations by NAT may not be feasible or economic

Closing the window on HCV

Samples• Plasma from HCV viraemic Ab neg “window

phase” donors

• Panel 1: 40 HCV NAT positive plasma samples from 17 US donors (11)

• Panel 2: 94 plasma samples from US donors

• Panel 3: 8 /10 NAT positive plasmas from

12,000,000 UK blood donors ’99-’03

Closing the window on HCV

RT-PCR• HCV detected by LightCycler (NTMRL)

or quantified by TaqMan (UCLH)

• Genotyped by sequencing 5’ ncr and in-silico determination of RFLP sites and line probe binding sites

Closing the window on HCV

Serology• Samples confirmed Ab neg by Ortho-HCV v 3.0

• Bio-Rad and Murex combined Ag-Ab assays performed per manufacturer’s instructions

• NOD = OD/cut off (NOD ≥ 1 +ve except BioRad for Panel 3 where modified criterion of 0.5 used)

Closing the window on HCV

Results•142 plasmas 112 (79%) HCV RNA + ve

•Wide range of viral loads (14 - 64,800,000 IU/ml)

•36 detected by Bio-Rad (32%)

•56 detected by Murex (50%)

•45 plasmas with viral load > 1,000,000 IU/ml Bio-Rad 35 +ve (78%) Murex 44 +ve (98%)

Closing the window on HCV

Detection limits interpolated from Panel 1

•Murex 200,000 IU/ml

•BioRad 4,000,000 IU/ml

Bio-Rad failed to detect all genotype 3a in Panels 1 & 2 although 2 donations viraemia >4,000,000 IU/ml

Closing the window on HCV

Murex vs Viral load Panels 1 & 2

0.01

0.1

1

10

100

10 100 1,000 10,000 100,000 1,000,000 10,000,000 100,000,000

IU/ml

NO

D

Genotype 1a

Genotype 1b

Genotype 2

Genotype 3a

Unknown

Closing the window on HCV

Biorad vs Viral load Panels 1 & 2

0.01

0.1

1

10

100

10 100 1,000 10,000 100,000 1,000,000 10,000,000 100,000,000

IU/ml

NO

D

Closing the window on HCV

Distribution of viral loads Panel 2

1

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10,000

100,000

1,000,000

10,000,000

100,000,000

1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51 53 55 57 59 61 63

Viral load

Closing the window on HCV

•Viral loads similar in all 3 panels

•No stat sig diffs.

•2 US Panels 1 and 2, regarded as single pop

•Viral loads of genotypes (1a, 1b, 2 and 3a) in this US pop similar, no sig diff between them (Figure 2).

Closing the window on HCVViral loads

1

10

100

1,000

10,000

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10,000,000

100,000,000

0.5 1 1.5 2 2.5 3 3.5

Panel 1

Panel 2

Panel 3

Closing the window on HCV

Closing the window on HCV

•Greater sensitivity of Murex for each genotype (Figure 2)

•Murex detected ~50% of genotypes 1a, 1b and 2 but 27% of genotype 3a

•Bio-Rad detected ~40% of 1b and 2, 33% of 1a; failed to detect ANY genotype 3a

•Failure to detect 3a indicated Abs used for Ag detection may have a genotypic bias

Closing the window on HCV

•UK 10 NAT window phase in 14,000,000 Ab neg donations 1999-2003, total cost £60 million

•Panel 3 = 8 available for serological testing Bio-Rad detected 5/8 62.5% (NOD 0.5 +ve) Murex detected 6/8 75%

•Very high % of 3a 6/8 (75%)

•UK prevalence data genotype 3a:- 37% 199921

UK HCV National Register N=749 29% 200622 Our departmental data similar 29.4%

Closing the window on HCV•Prevalence 3a in IDUs 54% 23 and 43% 24

•7 of the 10 UK NAT only +ves = genotype 3a ,

•3 reported risk factor, 2 = IDU / partner of IDU

•Possible majority of HCV window phase in blood donors 1999-2003 covertly linked to IDU

•Since 2003 NAT only detection rate 0 in >6 million

•European study of NAT testing within transfusion setting critical of the financial benefits 9

Closing the window on HCV

•Cost benefit analysis not only consideration

•Political implications of change from “best practice” to more economic alternative needs careful consideration

•May be time to resist stampede towards “Gold Standard” of NAT ultimate sensitivity

•Perhaps window closing not just on HCV infections but on NAT testing and a new era of combined Ag Ab assays lies ahead

Closing the window on HCV

Conclusions

•Combined Ag Ab assays provide a useful improvement on sole reliance on Ab testing

•However, they remain less sensitive than PCR for detecting viraemic donors

•Combined assays may be genotype susceptible

•Now is the time to consider combined assays as a viable alternative to NAT testing

Closing the window on HCVAcknowledgements:

This work was funded by the NBSOrganised jointly by Dr Roger Eglin

Prof Richard Tedder

The work at UCLH was performed by: Dr Philip W Tuke Dr Paul R Grant James Waite

All testing at NTMRL NBS Colindale wassupervised by Dr Alan Kitchen

US samples were provided by Dr Cate Sims BPL

Closing the window on HCV1. World Health Organization. World Health Organization, Weekly Epidemiological Record

1997: 72: 65-72.

2. Trepo C, Pradat P. Hepatitis C virus infection in Western Europe. Journal of Hepatology 1999;80-3.

3. Department of Health. Hepatitis C. Action plan for England. Department of Health Report No.: 40180 . 2004.

4. Hutchinson SJ, Roy KM, Wadd S, Bird SM, Taylor A, Anderson E, Shaw L, Codere G, Goldberg DJ. Hepatitis C virus infection in Scotland: Epidemiological review and public health challenges. Scottish Medical Journal 2006;8-15.

5. Soldan K, Davison K, Dow B. Estimates of the frequency of HBV,HCV and HIV infectious donations entering the blood supply in the United Kingdom, 1996 to 2003. Eurosurveillance 10 (2) (2005) 17-19. 2005.

9. Pillonel J, Laperche S. Trends in risk of transfusion-transmitted viral infections (HIV, HCV, HBV) in France between 1992 and 2003 and impact of nucleic acid testing (NAT). Euro.Surveill 2005;5-8.

21.Harris KA, Gilham C, Mortimer PP, Teo CG. The most prevalent hepatitis C virus genotypes in England and Wales are 3a and 1a. Journal of Medical Virology 1999;127-31.

22.Harris HE, Eldridge KP, Harbour S, Alexander G, Teo CG, Ramsay ME. Does the clinical outcome of hepatitis C infection vary with the infecting hepatitis C virus type? J.Viral Hepat. 2007;213-20.

23.Buckton AJ, Ngui SL, Arnold C, Boast K, Kovacs J, Klapper PE, Patel B, Lbrahim I, Rangarajan S, Ramsay ME, Teo CG. Multitypic hepatitis C virus infection identified by real-time nucleotide sequencing of minority genotypes. Journal of Clinical Microbiology 2006;2779-84.

24.Mohsen AH. The epidemiology of hepatitis C in a UK health regional population of 5.12 million. Gut 2001;707-13.

Closing the window on HCVGenotype viral load regression lines

"X" R2 = 1

Type 3a 117 R2 = 0.9954

Type 1a 111 R2 = 0.9976

Type 1b 122 R2 = 0.9799

Type 2b 157 R2 = 0.9981WHO R2 = 1

Type 3a 135 R2 = 0.9999

1

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1 10 100 1,000 10,000 100,000 1,000,000 10,000,000

X

117 Type3a

111 Type 1a

157 Type 2b

122 Type 1b

WHO

135

Linear (X)

Pow er (117 Type3a)

Pow er (111 Type 1a)

Pow er (122 Type 1b)

157 Type 2b

Pow er (WHO)

Pow er (135)

Closing the window on HCVTable 1 Panel 2 results UCLH

Genotype No Mean Viral Loadx10e6

Proportion detected Murex

Proportion detectedBio-Rad

Mean NODMurex

Mean NODBio-Rad

1a 24 38%

1.88 14/24 58% 6/24 25% 3.10 0.86

1b 14 22%

12.49 8/14 57% 6/14 43% 3.07 1.12

2 12 19%

3.30 5/12 42% 4/12 33% 4.31 1.43

3a 10 16%

0.89 2/10 20% 0/10 0% 1.03 0.09

Unknown 4 6%

0.00003 0/4 0%

0/4 0% 0.33 0.07

All 64 4.19 29/64 45%

16/64 25% 2.82 0.85

Table 1 summarises characeristics of the various genotypes of samples which form Panel 2 in terms of the results obtained at UCLH for viral load and combined antigen antibody tests.

Closing the window on HCV

Table 2 Comparison of results for Panels 1, 2 and 3

Panel Total Nosamples

Mean Viral Loadx10e6

Proportion detected Murex

Proportion detectedBio-Rad

Mean PositiveNODMurex

Mean PositiveNODBio-Rad

1* Samples 40 6.46 53% 38% 7.49 3.46

1† Individuals 17 6.36 71% 41% 6.14 2.93

1‡ 1st bleed 17 4.58 53% 29% 5.89 2.20

2 94 4.2 45% 25% 5.63 2.85

3 8 3.6 75% 63% 1.66 0.79

Table 2 summarises characeristics of the three panels of samples in terms of viral loads and combined antigen antibody test reults. The data from the samples in panel 1 have been presented in three forms; * as a group of the total 40 plasmas, † as the results for individual donors where one positive plasma results in the donor being regarded as positive for that test, and ‡ as the result of the first sample tested on each of the 17 donors.