Multiplex PCR for detection and quantification of intra- and extra-
cellular viral genomes
Lara Isobel Compston, Daniel Candotti, Jean-Pierre AllainCambridge Blood Centre, UK
University of Cambridge
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.
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
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.
Screening algorithm
undetectable
Sample negativefor 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
No
Yes
Sample negativefor specific virus
B19 / HBV/ HHV-8 EBV/CMV/ VZV GBV-C / HAV
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)
Dynamic range with NIBSC standards 95% C.I. values shown
Log concentration of external DNA standard as a function of Ct values. HBV assay 5x10e5 - 5x10e1
y = -3.485x + 40.068
R2 = 0.9996
0
5
10
15
20
25
30
35
40
0 1 2 3 4 5 6
Log. Conc. HBV external standard
Mean
Ct
valu
emean CtLinear (mean Ct)
HBV
Hepatitis A virusLog concentration of HAV NIBSC standard 00/560 as a function of
Ct value. HAV assay 5x10e3 - 40 IU
y = -1.1283Ln(x) + 42.176
R2 = 0.9889
0
5
10
15
20
25
30
35
40
45
1 10 100 1000 10000
Log concentration
Me
an
Ct
valu
e
Series1Log. (Series1)
• 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)
Dynamic range with plasmid standards 95% C.I. values shown
Log concentration of external plasmid standard as a function of Ct value.
CMV assay 3.5x10e6-3.5x10e1
y = -1.9945x + 36.448
R2 = 0.9616
0
5
10
15
20
25
30
35
40
0 1 2 3 4 5 6 7Log. conc. (external standard)
Me
an
Ct
valu
e
mean Ct
Linear(meanCt)
CMVLog concentration of external plasmid standard as a function of Ct value. VZV MBP
assay 2.17x10e6 - 2.17x10e2
y = -3.675x + 46.408
R2 = 0.9928
0
5
10
15
20
25
30
35
40
45
0 1 2 3 4 5 6 7
Log Concentration
Mean
Ct
Valu
e
mean Ct
Linear(mean Ct)
VZV
Log concentration of external plasmid standard as a function of Ct value. EBV assay 5.75x10e6 - 5.75x10e2
y = -3.139x + 43.212
R2 = 0.9926
0
5
10
15
20
25
30
35
40
0 1 2 3 4 5 6 7 8
Log concentration
Me
an
Ct
valu
e
Series1
Linear (Series1)
EBV HHV-8log concentration of plasmid standard as a function of Ct value. HHV-8
assay 1x10e5 - 1x10e1
y = -1.5222Ln(x) + 38.779
R2 = 0.9825
0
5
10
15
20
25
30
35
40
1.00E+00 1.00E+01 1.00E+02 1.00E+03 1.00E+04 1.00E+05 1.00E+06
Log Conc. (external plasmid standard)
Mean
Ct
valu
e
Series1
Log. (Series1)
Dynamic range with plasmid standards 95% C.I. values shown
y = -3.192x + 45.414
R2 = 0.9861
0
5
10
15
20
25
30
35
40
45
0 2 4 6 8 10 12 14
Log Concentration (external plasmid standard)
Mean
Ct
Valu
e
mean Ct
Linear(mean Ct)
Log concentration as a function of Ct valueB19 assay (5mM MgCl2)
5x10e11 -5x10e1
5
50 % Limit of detection
B19
•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)
Dynamic range with clinical standards 95% C.I. values shown
Log Concentraion of patent standard as a function of Ct value. GBV-C assay 10,000 AU - 10 AU
y = -2.758x + 38.02
R2 = 0.9991
0
5
10
15
20
25
30
35
40
0 1 2 3 4 5
Log Conc. (Patient standard)
Mean
Ct
Valu
e
mean Ct
Linear (mean Ct)
GBV-C
Multiplex assays for DNA viruses relevant in Africa
B19 singleplex = limit of detection; 50 IU
Rsq 0.992
Ct of 50 IU = 34.95
Hepatitis B singleplex = limit of detection; 50 IU
Rsq 0.995
Ct of 50 IU = 35.02
HHV-8 singleplex = limit of detection;10 copies
Rsq 0.977
Ct of 10 copies = 34.04
Multiplex: limit of detection;B19 500 IU, HBV 50 IU, HHV-8 10 copies
HBV Rsq 0.996Ct of 50 IU: 35.43
HHV-8 Rsq 0.953Ct of 10 copies: 33.67
B19 Rsq 0.939Ct of 500 IU: 34.59
Multiplex assay for DNA viruses relevant worldwide
VZV Singleplex: limit of detection; 217 copies
Rsq: 0.981
EBV Singleplex: limit of detection; 214 copies
Rsq 0.995
CMV Singleplex: limit of detection; 350 copies
Rsq 0.941
Multiplex: limit of detection;VZV 217 copies, EBV 214 copies, CMV 350 copies
VZV Rsq 0.997Ct of 217 copies = 39.24
CMV Rsq 0.998Ct of 350 copies = 37.4
EBV Rsq 0.995Ct of 214 copies = 34.53
Ct of 214 copies = 35.28 Ct of 350 copies = 34.46
Ct of 217 copies = 36.59
RNA virus Duplex
Duplex: Limit of detection;GBV-C 10 AU, HAV 40 IU
Hepatitis A singleplex: limit of detection; 40 IU
Rsq 0.853
Ct of 40 IU = 37.59HAV Rsq 0.636Ct of 40 IU = 35.79
GBV-C Rsq 0.880Ct of 100 AU = 37.0
GBV-C singleplex: limit of detection; 10 AU
Rsq 0.942
Ct of 100 AU = 37.42
qPCR results in Ghanaian blood donors
Limit of detection
CMV VZV EBV HHV-8 HBV B19 GBV-C HAV
1
10
100
1000
10000
100000
1000000
1.0x1007
0
PCR POS/N Risk % viraemia2/246 0.008 0.80/246 0 010/246 0.04 40/246 0 03/246 0.01 1.21/247 0.004 0.41/204 0.005 0.50/39 0 0
0.07 7
HHV-8
HAV
CMV
Culmative viremia
Controls
B19GBV-C
VZVEBV
DNAemia
HBV
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
97.1
13.4
58.4
0 0.493.9
0.49 0.99 00.490.490
10
20
30
40
50
60
70
80
90
100
Virus
Viraemia
Ab% P
osit
ive
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
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