1. Multiplex PCR for detection and quantification of intra- and extra-cellular viral genomes
Lara Isobel Compston, Daniel Candotti, Jean-Pierre Allain
Cambridge Blood Centre, UK
University of Cambridge

2. Introduction
Interaction between viruses with the host is dependant on the interplay between factors related to both the virus and the host
Target cells can be damaged either directly by the virus or by the immune response initiated, and an equilibrium needs to be reached between them
Several viral survival strategies are the result of the virus/host interplay :
1) Clinical recovery due to successful development of humoral and cellular immune responses.
2) Latent viral infection (transient escape of immune response in primary infection and reactivation)
3) Establisment of chronic viral infection with partially effective immune response.

3. New model of viral infection
Recent evidence has emerged that, in common with latent viruses, after recovered infections, common viruses are not eliminated from the host but contained efficiently, persisting in
sanctuaries were they escape the immune response.
Acute infection Sanctuaries: biological portfolio Recipients of TX or organ
Clinical symptoms
Clinical recovery
By immune defenses
Antibodies
White cells
No detectable virus in circulation
Lymph nodes
CMV, HHV-8, EBV, HIV
Liver
HBV, HAV, HCV
Bone marrow
HEV B19
Immunocompetent
Maintain viral control
Undetectable in blood
Immunodeficient
Age, chemo; transplant of BM or organs; HIV
- Increased susceptibility to external pathogens
- Reactivation of past infections ( viral load)
- Severe symptoms

4. Aims of the study
It was hypothesised that the generality of reactivation of common latent and persistent viral genomes may constitute an indicator of the overall immune status of the host.
The objective was to systematically detect and quantify a panel of common viruses in persons who are either immunocompetent or present with varying degrees of immunodeficiency ranging from mild to severe.

5. Screening algorithm Multiplex real-time PCR
undetectable
Sample negative
for specific virus
Multiplex real-time PCR
PCR signal
for specific
viruses No
Yes
Single virus qPCR
Viral load quantification
Final viral load
result (>50 copies)
≤ 50 copies
Confirmation of ≤ 50 copies
Nested-PCR
B19 / HBV/ HHV EBV/CMV/ VZV GBV-C / HAV

6. Development of Standards for qPCR
NIBSC standards:
WHO international standard for Hepatitis B virus 97/746
WHO international standard for Hepatitis A virus 00/560
Plasmid standards:
Constructed for the following targets:
EBV and HHV-8 (cell culture)
CMV (clinical sample)
VZV (oligonucleotide construct of target region)
B19 (received as a gift)
PCR amplicons of target regions
Plasmids purified and quantified by UV spectroscopy, plasmid concentration used to derive copy number by standard conversion for qPCR
Clinical standards:
A high titre clinical sample of GBV-C serially diluted and used as a standard
Unknown initial viral load, therefore dilution giving 100% L.O.D designated as 10 AU (arbitrary units)

7. Dynamic range with NIBSC standards
95% C.I. values shown
HBV
Hepatitis A virus

8. NIBSC standards:
WHO international standard for Hepatitis B virus 97/746
WHO international standard for Hepatitis A virus 00/560
Plasmid standards:
Constructed for the following targets:
EBV and HHV-8 (cell culture)
CMV (clinical sample)
VZV (oligonucleotide construct of target region)
B19 (received as a gift)
PCR amplicons of PCR target region
Plasmids purified and quantified by UV spectroscopy, plasmid concentration used to derive copy number by standard conversion for qPCR
Clinical standards:
A high tire clinical sample of GBV-C serially diluted and used as a standard
Unknown initial viral load, therefore dilution giving 100% L.O.D designated as 10 AU (arbitrary units)

9. Dynamic range with plasmid standards
95% C.I. values shown
CMV
VZV
EBV
HHV-8

10. Dynamic range with plasmid standards
95% C.I. values shown
50 % Limit of detection
B19

11. NIBSC standards:
WHO international standard for Hepatitis B virus 97/746
WHO international standard for Hepatitis A virus 00/560
Plasmid standards:
Constructed for the following targets:
EBV and HHV-8 (cell culture)
CMV (clinical sample)
VZV (oligonucleotide construct of target region)
B19 (received as a gift)
PCR amplicons of PCR target regions
Plasmids purified and quantified by UV spectroscopy, Plasmid concentration used to derive copy number by standard conversion for qPCR
Clinical standards:
A high titre clinical sample of GBV-C serially diluted and used as a standard
Unknown initial viral load, therefore dilution giving 100% L.O.D designated as 10 AU (arbitrary units)

12. Dynamic range with clinical standards
95% C.I. values shown
GBV-C

13. Multiplex assays for DNA viruses relevant in Africa
B19 singleplex = limit of detection; 50 IU
Hepatitis B singleplex = limit of detection; 50 IU
Rsq 0.995
Rsq 0.992
Ct of 50 IU = 34.95
Ct of 50 IU = 35.02
Multiplex: limit of detection;
B IU, HBV 50 IU, HHV-8 10 copies
HHV-8 singleplex = limit of detection;10 copies
B19 Rsq 0.939
Ct of 500 IU: 34.59
Rsq 0.977
HBV Rsq 0.996
Ct of 50 IU: 35.43
HHV-8 Rsq 0.953
Ct of 10 copies: 33.67
Ct of 10 copies = 34.04

14. Multiplex assay for DNA viruses relevant worldwide
EBV Singleplex: limit of detection; 214 copies
CMV Singleplex: limit of detection; 350 copies
Rsq 0.995
Rsq 0.941
Ct of 214 copies = 35.28
Ct of 350 copies = 34.46
Multiplex: limit of detection;
VZV 217 copies, EBV 214 copies, CMV 350 copies
VZV Singleplex: limit of detection; 217 copies
EBV Rsq 0.995
Ct of 214 copies = 34.53
Rsq: 0.981
VZV Rsq 0.997
Ct of 217 copies = 39.24
CMV Rsq 0.998
Ct of 350 copies = 37.4
Ct of 217 copies = 36.59

15. RNA virus Duplex Hepatitis A singleplex: limit of detection; 40 IU
Rsq 0.853
Duplex: Limit of detection;
GBV-C 10 AU, HAV 40 IU
HAV Rsq 0.636
Ct of 40 IU = 35.79
Ct of 40 IU = 37.59
GBV-C Rsq 0.880
Ct of 100 AU = 37.0
GBV-C singleplex: limit of detection; 10 AU
Rsq 0.942
Ct of 100 AU = 37.42

16. qPCR results in Ghanaian blood donors
CMV
VZV
EBV
HHV-8
HBV
B19
GBV-C
HAV 1 10
100
1000
10000
100000
1.0x10 07
Limit of detection

17. Background serology and viraemia in Ghanaian blood donors
VZV
CMV
EBV
HHV-8
HBV
B19
GBV-C
HAV
46.3
93.4
97.1
22.8
72.4
13.4
58.4
0.49
3.9
0.99 10 20 30 40 50 60 70 80 90
100
Virus
Viraemia Ab
% Positive

18. Summary
A range of different types of standards where utilised for qPCR, which had similar dynamic ranges, repeatability and reproducibly
NIBSC standards
Plasmid standards
Clinical standards
Triplex assays where developed and optimised to match the sensitivity of the singleplex PCR
This was achieved, except for B19 ( one-log decrease in sensitivity)
The HAV assay could not be utilised as a quantitative assay despite extensive optimisation
The Ghanaian blood donor population, while having high seroprevalence for each virus examined indicating previous exposure, had only very low frequency and level of viraemia
No viraemia was detected with HHV-8, VZV and HAV despite high background seroprevalence
Against this background studies of reactivation in various situations of immunodeficiency can now be conducted

19. Acknowledgements
Division of Transfusion Medicine (University of Cambridge)
Jean-Pierre Allain
Daniel Candotti
Komfo Anokye Teaching Hospital (Kumasi, Ghana)
Ohene Opare-Sem
Francis Sarkodie
Laboratoire de Virologie (Hôpital Armand Trousseau)
A. Garbarg-Chenon