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