Philip William Tuke

1
HCV window phase infectionsClosing the window
on HCV
A comparison of two combined Ag / Ab assays with
QRT-PCR

• Philip William Tuke

2
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
• gt5 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

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Closing the window on HCV
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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

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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

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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

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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

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

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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 gt 1,000,000 IU/ml
• Bio-Rad 35 ve (78)
• Murex 44 ve (98)

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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
  gt4,000,000 IU/ml

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Closing the window on HCV
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Closing the window on HCV
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Closing the window on HCV
Distribution of viral loads Panel 2
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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).

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Closing the window on HCV
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Closing the window on HCV
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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

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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 N749 29 200622
• Our departmental data similar 29.4

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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 gt6
  million
• European study of NAT testing within transfusion
  
• setting critical of the financial benefits 9

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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

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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

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Closing the window on HCV

• Acknowledgements
• This work was funded by the NBS
• Organised 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 was
• supervised by Dr Alan Kitchen
• US samples were provided by Dr Cate Sims BPL

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Closing the window on HCV
1. 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 199980-3. 3. Department
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Department of Health Report No. 40180 . 2004.
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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
20068-15. 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,
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Euro.Surveill 20055-8. 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
1999127-31. 22.Harris HE, Eldridge KP, Harbour
S, Alexander G, Teo CG, Ramsay ME. Does the
clinical outcome of hepatitis C infection vary
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J.Viral Hepat. 2007213-20. 23.Buckton AJ, Ngui
SL, Arnold C, Boast K, Kovacs J, Klapper PE,
Patel B, Lbrahim I, Rangarajan S, Ramsay ME, Teo
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identified by real-time nucleotide sequencing of
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Closing the window on HCV
Genotype viral load regression lines
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Closing the window on HCV
Table 1 Panel 2 results UCLH
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.
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Closing the window on HCV
Table 2 Comparison of results for Panels 1, 2
and 3
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.