Multiplex PCR for detection and quantification of intra- and extra-cellular viral genomes

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
Immunocompetent Maintain viral control Undetectabl
e 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

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
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
10
Dynamic range with plasmid standards
95 C.I. values shown
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 B19 500 IU, HBV 50
IU, HHV-8 10 copies
HHV-8 singleplex limit of detection10 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
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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
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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
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qPCR results in Ghanaian blood donors
Limit of detection
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Background serology and viraemia in Ghanaian
blood donors
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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

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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