INTERNATIONAL WORKING GROUP ON THE STANDARDISATION OF
GENOME AMPLIFICATION TECHNIQUES FOR THE SAFETY TESTING OF
BLOOD, TISSUES AND ORGANS FOR BLOOD BOURNE PATHOGENS
SoGAT XIX 14-15 May 2006
Co-sponsored by Swissmedic, Bern, Switzerland and NIBSC, UK
Report of the meeting prepared by S Baylis (NIBSC), C Morris (NIBSC) and H Holmes
(NIBSC)
The nineteenth meeting of SoGAT was hosted by Swissmedic, the Swiss agency for
Therapeutic Products and was held at the Allegro Kursaal, Bern, Switzerland. The welcome
address was given by Dr Petra Doerr, the head of International Affairs at the Swissmedic, on
behalf of Dr Franz Reigel, a member of the management board of the Swissmedic, and Dr
Philip Minor, the Deputy Director at NIBSC. Dr Minor described the important role that
SoGAT had played in the standardisation of NAT for blood safety and highlighted recent
discussions at NIBSC addressing the future format and timeframe of these meetings and the
possibility of extending this successful formula to encompass areas such as clinical virology.
Presentations from the meeting are available at the following link:
http://www.nibsc.ac.uk/partners/SoGAT/2006_Presentations.html
Session 1: Progress with Standardisation, chaired by Dr Philip Minor, NIBSC, UK
SoGAT, mission completed or work in progress?
Dr Gerold Zerlauth (Baxter AG, Austria) opened this meeting by emphasising the
achievements of SoGAT to date. Whilst the initial ‘goals’ of the group had been achieved, it
was important to maintain support for established standards, as well as having an experienced
forum to address new issues such as emerging blood pathogens. Dr Minor highlighted that
SoGAT is a valuable and unique forum where the use of reference materials can be discussed
prior to proposals made to the WHO Expert Committee on Biological Standardization (ECBS).
He added that the group also has a role in addressing issues such as higher order reference
materials, either biological or synthetic.
Progress on study to validate synthetic standards
Dr Roberta Madej (Roche, USA) described a study to validate the use of synthetic materials
as calibrators and reference reagents by the Industrial Liaison Committee (ILC). Dr Madej
reviewed results from phase 1 of the study and went on to describe the proposal for phase 2.
The new study will consist of a number of reference materials, both synthetic and biological,
from a variety of sources. Results expected later in the year. There was general discussion
about the batch to batch variation of synthetic materials. Dr Minor felt this had not been
addressed previously. Dr Saldanha commented that the synthetic materials, whilst not intended
to replace the biological standards, would be of use for calibration and that phase 2 of the study
was designed to examine issues of commutability. Professor Simmonds questioned whether the
transcripts to be used in the study are full length, since RNA structure of the genome affects
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the ability to quantitate the virus. Dr Madej confirmed that none of the synthetic materials are
full length, as such transcripts would be difficult to manufacture.
Competitive vs. non-competitive internal standards
Dr Kurt Roth, (GFE Blut mbH, Germany) described the critical stages in the NAT process
and contrasted the use of internal controls for each sample and external controls used to
monitor each entire assay run. The advantages and disadvantage of each type of control were
discussed. The type of control used depends on many factors e.g. single virus marker testing or
use of multiplex assays. Dr Saldanha emphasised that external controls should always be
included in assays as in some cases it may be possible to pass an assay where the internal
control has failed but the external controls give valid values.
Update on the P. falciparum standard and the replacement of the HBV and HCV
International Standards
Dr Sally Baylis (NIBSC, UK) summarised the results from the Plasmodium falciparum
collaborative study. Fourteen laboratories took part in the collaborative study. Four
preparations were evaluated and one of which, sample AA was freeze-dried. Sample AA has
been proposed at the 1st International Standard (IS), showing good stability in accelerated
degradation studies and been assigned a potency of 109 IU/ml.
A small collaborative study was described to evaluate a replacement for the 1st International
Standard for hepatitis B virus (HBV) DNA (97/746). The study examined the real-time and
accelerated degradation stability of 97/746 and 97/750 (prepared in parallel to 97/746 and
examined in the original collaborative study to establish the 1st IS). The study concluded that in
terms of potency, there was no significant difference between the two preparations. Both
preparations showed excellent stability with no loss of potency after 4.5 years of storage at
+20ºC. Both studies will be submitted to the 2006 meeting of the WHO ECBS.
The progress in replacing the hepatitis C virus (HCV) IS was reported. Genotype 1a HCV
window period material has been freeze-dried in two batches. This material has potency at least
twice that of the current IS. Prospective collaborative study participants were asked to contact
NIBSC to evaluate these materials.
Session 2: Hepatitis B, chaired by Dr Gerold Zerlauth, Baxter, Austria
Infectious dose and the concentration of HBV DNA in vitro
Professor Yoshizawa (Hiroshima University, Japan) began this session by describing a
study to investigate the minimum concentration of HBV DNA, genotypes A and C, which can
infect chimpanzees. It was reported that as little as 10 copies of HBV DNA can cause hepatitis
in chimpanzees. The doubling times for genotype C viruses were observed to be shorter than
those for genotype A. Professor Jean-Pierre Allain enquired as to the duration of the window
period for the two genotypes studied. Professor Yoshizawa confirmed that the window period
for genotypes A and C were 55-76 days and 35-50 days respectively.
False-negative HBsAg detection in chronic hepatitis B
Dr. Dominique Challine (INSERM, France) presented a study to determine the prevalence
of false negative HBsAg detection in organ tissue and cell donors. Data was presented to show
that HBsAg mutations do not appear to be the principal reason for the lack of detection of
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HBsAg in organ, tissue and cell donors. It was speculated that this was likely to be related to
lack of sensitivity of the assays when HBsAg is present at low levels. Dr. Theo Cuijpers asked
whether samples were derived from non-heart beating donors. Dr Challine confirmed, that with
the exception of cornea donors, all donors were living.
Session 3: NAT assay sensitivity/detection limits and new technology, chaired by Dr
Indira Hewlett, CBER/FDA, USA
Analytical and diagnostic sensitivity of HCV NAT tests
Dr Michael Chudy, (PEI, Germany) reported that since the implementation of HCV NAT
screening in Germany, there has been one reported case of HCV transmission. This occurred
via red cells, with the donor sample being non-reactive using the COBAS Ampliscreen assay in
a pool of 24. This donation contained an HCV genotype 2b virus and was sero-negative. Fresh
frozen plasma was available to further analysis using a range of NAT assays. Qualitative
assays did not perform so well in the detection of this sample compared with some quantitative
real-time PCRs. The implicated donation was likely to have had a titre of less than 10 IU/ml
HCV RNA. Dr Phil Tuke raised the issue of the number of window period donations detected.
Dr Roth stated that during 7 years of HCV NAT testing in Germany, approximately 30 million
samples have been screen, and of these 20 have been confirmed positive for HCV RNA.
Generic automated sample preparation for commercial NAT assays.
Dr Markus Sprenger-Haussels (Qiagen, Germany) described the large variation in starting
matrices and the properties of different viruses. A number of different extraction platforms
were tested in combination with a variety of in-house and commercial NAT assays to
determine compatibility. Data was presented for a number of blood borne viruses. Dr Theo
Cuypers questioned whether this work had incorporated the manufacturer’s internal controls; it
was confirmed this was the case. Dr. Busch enquired whether whole blood and the associated
high background levels of genomic RNA/DNA would be problematic for this method. Dr.
Sprenger-Haussels commented that development is based on cell free systems, more a kin to a
diagnostic situation.
One year of experience with Roche’s COBAS AmpliPrep/COBAS TaqMan96 automated
PCR system in two large blood bank settings
Drs Lutz Pichl and Christine York (DRK-Blutspendedienst West and DRKBlutspendedienst NSTOB) each described the principles of the detection system they used,
the viral markers screened for and the number of donations screened since 2005; both sites
screened for HCV, HIV-1, HBV, HAV and B19. Dr. Pichl explained that due to a larger
population in the Western region, this blood bank utilised 9 detection systems, whereas the
northern region, which is more sparsely populated, only required 6 units. Dr York presented
data showing the percent invalidity rate for each marker and the number of window period
donations detected in each region. 95% detection limits for each marker in each region were
also presented. Pros and cons of each of the systems were discussed.
Evaluation of the Procleix TIGRIS system at the Blood Transfusion Service Berne Ltd.
Dr Martin Stolz (Swissmedic, Switzerland) stated that HCV and HIV NAT testing have been
mandatory since 1999 and 2000 respectively with current discussions on the implementation of
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HBV testing. Dr Stolz described their evaluation of the TIGRIS system, which proved robust
and reliable with a low level of invalidity. The fully automated system was a benefit and the
high throughput opened up the feasibility of single donation testing. However it was
highlighted that a fully automated system requires a large amount of hardware which has
limited flexibility. Dr Stolz concluded his presentation by informing the group that during the
evaluation, a window period HBV donation was detected. This was detected inan individual
donation; however the donation had already been transfused when this was identified. HBV
viremia was detected in the recipient three weeks post transfusion.
Infectivity based risk modelling
Dr Nico Lelie (Chiron, France) discussed analytical standards and the relationship of IU to
genome equivalents/ml with reference to the infectious dose for different blood borne viruses.
Dr Lelie considered the issues of virus detection and infectivity during the window period and
the impact of mini-pool (MP) NAT vs. ID NAT. Data on the relationship between viral nucleic
acid concentration and infectivity titres for HIV, HBV and HCV were reviewed. Documented
cases of transfusion transmitted HIV in which MP-NAT had been implemented were discussed
and the outcome related to viral load transfused.
Session 4: EQAS schemes, chaired by Professor Elizabeth Dax, NRL, Australia
Ongoing quality control programme for testing blood-borne pathogens through an
internet-based application
Professor Elizabeth Dax (NRL, Australia) gave an update on an internet based data reporting
application (EDCNet) which is now used throughout Australia, allowing laboratories to submit
data and view results in real time and look at things such as batch to batch variation of kits.
Graphical data allows aberrant results from laboratories to be clearly identified.
An internet-based application fostering global collaboration of External Quality
Assessment Schemes
Thu-Anh Pham (NRL, Australia) followed on from Professor Dax describing EDCNet in
further detail. In collaboration with HealthMetrx, Canada, an internet based application that
allows on line result submission and analysis, Digital PT has been established. It has been used
in conjunction with the NRL EQAS scheme since 2005 with good uptake in its use as a
reporting tool. Ms Pham outlined the benefits, including quicker turnaround time of final
reports, individualised laboratory printouts and the ability for inter laboratory comparisons.
Update on results of recent EQA panels for blood borne viruses
Dr Vivienne James (HPA, UK) presented data from recent EQA panels for blood borne
viruses, including a pilot study for human cytomegalovirus (CMV) DNA. Participants return
results and these are collated at the HPA. Graphical analysis was presented showing the
distribution of results, with for example ~90% of data for HIV-1 data falling within 1 log10 of
the median concentration, ~83% for HBV DNA and ~60% for CMV for quantitative assays.
HCV RNA assays are analysed qualitatively. Dr Allen enquired about the formulation of the
CMV regents, Dr James confirmed that these were virus spiked plasma. Dr Holmes raised the
problem of detecting ever increasing non B subtypes of HIV-1 and enquired whether the
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NEQAS scheme will include non B subtypes in future. Dr James confirmed this would be the
case. Dr Holmes also questioned whether freeze drying of NEQAS samples resulted in any loss
of titre. Dr James confirmed that whilst this occurs there were no reconstitution issues.
Results from an EDQM/OMCL PTS studies on B19 NAT testing of plasma pools
Dr Karl Heinz Buchheit (EDQM) described the European regulatory requirements for
parvovirus B19 (B19V) testing. The EDQM PTS involves both OMCLs and manufacturers of
plasma products who have received 10 blinded plasma samples in each scheme. All
laboratories correctly identified samples falling below the threshold concentration for B19V
DNA (104 IU/ml). Whilst the majority of laboratories correctly identified genotype 1 B19V
exceeding the threshold concentration, many failed to detect an A6-like high titre genotype 2
B19V. The ICTV have classified the A6 and V9 viruses as strains as parvovirus B19 and in
accordance with the EP, such viruses should be detected. Professor Dax commented that the
programme was revealing and asked if the panels were prepared annually. Dr Buchheit
confirmed that panels were prepared with each new study from a genotype 1 B19V preparation
calibrated against the WHO IS. Whilst this had not been performed for the A6 virus, the high
titre allowed for some uncertainty.
First international proficiency study on West Nile virus (WNV) molecular detection
Dr Matthias Niedrig (Robert Koch Institut, Germany) presented EQA results for a panel of
WNV and related viruses. Viruses were propagated in vitro, diluted into plasma and inactivated
by γ-irradiation and heat treatment at 56ºC, prior to freeze-drying. Virus-positive samples
contained approximately between 1,000 and 1,000,000 RNA copies per ml. Twenty-eight
laboratories enrolled in the study from 20 countries; of these 71% laboratories correctly
detected the virus in all samples with ≥10,000 copies/ml. Only 11 (39.3%) laboratories also
detected WNV of lineage 2 and 10 (37.7%) laboratories found the WNV related Kunjin virus.
Seven laboratories show a very low sensitivity detecting only samples with ≥100,000 copies/ml.
Dr Lelie asked how copy numbers had been determined, and Dr Niedrig confirmed that
samples were tested by three expert laboratories.
Session 5: Emerging – re-emerging pathogens chaired by Dr Micha Nübling, (PEI
Germany)
Detection of dengue viremia in blood donations in Brazil and Honduras
Dr. Mike Busch, Blood Systems Research Institute, USA described a prototype TMA for the
detection of dengue virus RNA in blood donations. More than 7500 samples were collected
from Brazil and Honduras and screened using the automated Procleix TIGRIS system. Positive
samples were sequenced to confirmed genotype. All four genotypes of Dengue virus were
found. Dr Busch concluded that the assay under evaluation could be used to screen for the
presence of Dengue virus. However the prevalence rates identified raised concerns about the
rate of transfusion transmission, which will be addressed in phase II of the study that Dr Busch
outlined. There was general discussion about the identification of cases of transfusion
transmitted dengue and the difficulty in determining genuine transfused cases vs. those
acquired in areas where there are outbreaks and where immunity may be high.
GB virus C (GBV-C), West Nile Virus, and dengue virus in West Africa
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Professor Jean-Pierre Allain, University of Cambridge, UK described a testing algorithm to
screen with a triplex NAT assay to detect GB virus C, West Nile Virus, and dengue virus in
blood samples form Ghana. It was found that genotype 1 GBV-C was prevalent and where
individuals were GBV-C viraemic and HIV-positive, they were found to have lower HIV-1
loads than those with HIV alone. Transmission is normally horizontal for GBV-C in West
Africa. There was no evidence for dengue infection amongst the group studied. Whilst no
WNV RNA could be detected, serological evidence suggested prior infection with WNV.
There was general discussion about serological cross-reactivity and Professor Allain stated that
confirmatory testing was underway.
Session 6: NAT for tissues and organs chaired by Dr John Saldanha (Roche, USA)
Strategies for reducing inhibition of RT-PCR in cadaveric samples
Dr. Simon Carne, National Blood Service, UK gave a detailed account of the strategies used
by the NBS for reducing inhibition of NAT in cadaveric samples whilst implementing
automated systems. Various technical approaches were discussed. Dr Carne emphasised that no
single solution eliminated inhibitory effects observed in the tissues under analysis. It was noted
that tissues collected from certain centres were less inhibitory, suggesting differences in the
procedures implemented. Dr Susan Stramer commented that a small 1 in 5 dilution of
cadaveric samples, whilst resulting in some loss of sensitivity, give valid results. Dr Philip
Minor queried whether the increase in cadaveric testing warrants a change from a system that
had encountered few problems; Dr Carne said that 1000 samples had been received in the last
year. Dr Nico Lelie asked whether all centres collecting tissue used EDTA anticoagulant; Dr
Carne confirmed that was the case. Dr Theo Cuypers suggested that gel barrier EDTA
collection tubes may produce better results.
Validation and one year experience with HCV-RNA and HIV-RNA detection in small test
pools on cadaveric samples for viral safety of tissue transplants
Dr. Theo Cuijpers, Sanquin Diagnostic Service, The Netherlands presented data on the
detection of HCV and HIV RNA in cadaveric tissues using the NucliSens (Boom) extraction in
conjunction with Roche Ampliscreen. A retrospective study showed there were no technical
problems using frozen samples. Overall the system was robust with good sensitivity and few
issues with inhibition of internal control signals. This testing regime has been in place since
February 2006.
Testing of haemopoietic progenitor cell (HPC) donors
Dr. Susan Stramer, American Red Cross, USA summarised the issues, goals and analysis
methods discussed at a Cell Therapy Liaison meeting that took place earlier in the year. Dr
Stramer then went on to discuss the infectious disease marker incidence rates for donations of
Haematopoietic Progenitor cells (HPC). Studies were described to address the possible
elevated risks associated with MP NAT and ID NAT for HPC than for whole blood donations.
This study focused on the detection of HIV, HCV and HBV in both donation types. Results of
the study suggest that there is no significant difference in incidence rates when HPC are tested
using MP NAT over whole blood donations. Dr Stramer suggested that such data should be
shared with the test kit manufactures.
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Session 7: Bacterial Screening in Blood Banks, chaired by Dr Kurt Roth (GFE Blut mbH,
Germany)
Experience with the detection of bacterial contamination of platelet concentrates with
NAT, need for biological standards to compare the efficacy of NAT test protocols.
Dr. Theo Cuypers, Sanquin Diagnostic Service, The Netherlands raised another area in
need of standardisation, with regard to the detection of bacterial contamination in platelet
concentrates. Dr Cuypers compared real-time PCR and the BacT/Alert culture system. Platelet
pools were spiked with 5 different bacterial strains, and monitored over a 7 day period. Equal
sensitivity and specificity was observed by the real time PCR assay compared with the
BacT/Alert system. However the detection time was dramatically reduced from 24hrs with the
BacT/Alert to 4hrs with the real time PCR. Dr Cuypers concluded by suggesting that while
biological standards are beneficial in the comparison of NAT protocols, some caution needed
to be exercised in the way they are tested. He asked the group to consider that the way forward
would be to assist in the preparation and characterisation of these standards. Dr Busch
commented that it may be difficult to persuade regulators that NAT improved upon the well
established BacT/Alert system.
Controls for blood septicaemia nucleic acid tests
Dr. Mark Manak, SeraCare Life Sciences, USA discussed the use of ACCURUN 500
controls in blood septicaemia NAT assay. Dr Manak presented data that showed the
ACCURUN 500 to be stable and capable of controlling all steps of the assay, from extraction
though to detection. The controls consist of P. aeroginosa, S. aureus, C. albicans, they
demonstrated good reproducibility between control lots, tests runs and test sites. Dr Manak
concluded by suggesting that the regular use of the ACCURUN 500 will allow laboratories to
monitor the long term performance of assays as well as allowing the immediate detection of
testing errors. Dr Kurt Roth questioned the method for determination of colony forming units
(CFU), as concentration may be affected by the inactivation procedure. Dr Manak stated that
all samples were collected at early stages of culture and serial dilutions were performed to
determine the CFU. Dr Vivienne James asked about the homogeneity of the material as
clumping is a common problem with bacterial cultures. Dr Manak confirmed that clumping
was not an issue.
Session 8: Parvoviruses, chaired by Dr Kevin Brown (Health Protection Agency, London,
UK)
Overview of parvovirus classification (including detection and seroprevalence of PARV4)
Dr Kevin Brown (HPA, UK) briefly described the taxonomic structure and criteria used in the
classification of the Parvoviridae family. B19V and the adeno-associated viruses were the only
parvoviruses known to infect humans. However two novel, but distinct parvoviruses, termed
PARV4 and human Bocavirus (HBoV) have recently been identified in human sera and
respiratory secretions respectively. Dr Brown went on to describe studies looking at PARV4
DNA pooled plasma and clinical samples, where PARV4 was present infrequently and at low
titre. The use of expressed PARV4 VLPs in an ELISA assay suggests a high seroconversion
rate for this virus.
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Novel and related variant Parvoviruses in human plasma
Dr Jacqueline Fryer (NIBSC, UK) described how PARV4 was originally identified in an
IVDU co-infected with HBV. Dr Fryer described the identification of PARV4 in
approximately 5% plasma pools. Titres of PARV4 DNA ranged from a few hundred to 106
copies/ml. DNA sequence analysis revealed that some pools contained a related virus (PARV5)
that showed 92% nucleotide identity to PARV4. Dr Fryer went on to describe a study carried
out in Dr Eric Delwart’s laboratory at BSRI, San Francisco; here the prevalence of PARV4
was compared in blood donors and in a second group consisting of MSMs, IVDUs and patients
with acute viral infection syndrome. PARV4/5 were found more frequently in the latter group.
Dr Minor (NIBSC, UK) enquired as to the geographical origin of the PARV4 positive pools
that Dr Fryer had identified. Dr Fryer confirmed that the positive pools were from the USA.
Level of viremia and genotype characteristics of parvo B19 in blood donors and patients
Professor Ewa Brojer (Institute of Haematology and Blood Transfusion, Warsaw,
Poland) spoke of the parvovirus B19 testing performed by her laboratory using a combination
of qPCR and serology. Genotype 1 and genotype 2 B19V patients were followed up to
investigate virus persistence. A patient undergoing kidney transplantation was viraemic for
genotype 2 B19V for 1.5 years, viraemia peaked at 1010, and this fell to 104 when chronically
infected. A similar pattern was seen in a BMT patient infected with a genotype 1 B19V.
Distribution of parvovirus B19 genotypes 1-3 in plasma donations; need for a genotype
panel to standardize quantitative parvo B19 DNA assays for screening
Mr. Marco Koppelman, Sanquin Blood Supply Foundation, The Netherlands described
the ability of commercial NAT assay kits to detect different genotypes of B19V. No evidence
has yet been found for genotypes 2 and 3 B19V in Dutch blood and plasma donors (20042006). Screening is performed using a Roche assay, only detecting genotype 1 B19V and an in
house consensus assay that detects all 3 genotypes. Using this approach two genotype 1 B19Vs
were identified that gave aberrant amplification plots in each of the assays. Molecular analysis
identified the likely sequence changes responsible for these unusual plots. Dr Koppelman
proposed that it would be of great value to have reference materials available for the different
genotypes of B19V to validate assays.
Detection of parvovirus B19 variants in Factor VIII concentrates
Dr. Mei-ying Yu, CBER FDA, USA described the identification of a genotype 2 B19V in an
wet-heat treated, intermediate purity Factor VIII concentrate. This was identified using
restriction enzyme digestion of PCR products amplified using consensus primers to genotypes
1-3 of B19V. The vast majority of products were contaminated with genotype 1 B19V. A
similar product was identified that was contaminated with PARV5. Dr Yu concluded that the
prevalence of B19V variants was low, as was the detection of PARV4 and PARV5.
Detection of parvovirus B19 and novel human parvoviruses in high-risk individuals
Professor Peter Simmonds, University of Edinburgh, UK was unable to attend the second
day of the meeting, Dr S. Baylis made the presentation. Professor Simmonds work examined a
cohort of haemophiliacs and a group of clinical respiratory samples. Both groups were
subjected to nested PCR for B19V, PARV4 and HBoV. In the haemophiliac group, 59 samples
were analysed, two of which were positive for PARV4/5. None were positive for B19V or
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HBoV. In the respiratory samples, HBoV was identified most frequently, particularly from
lower respiratory tract infections, with a small number positive for B19V. All the respiratory
samples were negative for PARV4/5.
Parvoviruses: a future challenge
Dr. Nicola Boschetti, ZLB Behring AG, Switzerland discussed the particular challenge
facing plasma fractionators in the case of the non-enveloped parvoviruses. Dr Boschetti
described how the highly virulent canine parvovirus emerged through a fairly limited number
of mutations that altered the host range. He also emphasized that using novel molecular
approaches, new bovine and human parvoviruses have been identified and that a proactive
approach by manufacturers is necessary to counter the risk associated with potential
contamination of plasma products.
Maternofetal transmission of human parvovirus B19 genotype 3
Dr. Daniel Candotti, University of Cambridge, UK stated that genotype 3 is the
predominant strain of B19V in Ghana and it exhibits considerable genetic diversity. Three
cases of primary maternal infection with B19V were investigated. Two of these cases resulted
in transmission of the virus to the foetus. There was no evidence for transmission of B19V
where the infection is persistent and specific IgG is present.
Does the presence of B19 DNA in donations correlate with virus infectivity?
Dr. Ruth Laub, CAF-DCF Red Cross, Belgium described the cultivation of B19V in HepG2
cells, expressing globoside, the P-blood group antigen. Virus particles are produced and can reinfect cells. This model has been used to examine neutralization of B19V using antibodies in
B19V DNA positive blood donors. B19V viraemia may be detected in blood donors up to a
year or more following infection. In neutralization experiments using sera from B19V DNA
positive donors, generally neutralization occurs, however in some cases despite the presence of
anti-B19V infectivity may be observed.
Parvovirus B19 infection in seronegative and seropositive individuals – implications for
blood product safety
Dr Sean Doyle, National University of Ireland, Maynooth, Ireland presented data showing
protection in anti-B19V seropositive individuals that were treated with products containing
high titres of B19 DNA. In these individuals pre-existing levels of anti-B19V IgG were found
to rise following re-exposure. In the case of seronegative recipients, it was found that exposure
to pooled plasma containing high titres of B19V DNA (>1010 IU/ml) even in the presence of
high levels of anti-B19V IgG failed to prevent infection in the recipient.
Primer & Probe Design Affects Ability to Detect and Quantify Variant Viruses
Sally Baylis, NIBSC, UK described the analysis of a published real-time PCR assay and an
investigation of its ability to detect different genotypes (1-3; subtypes of genotype3) of B19V
using cloned DNAs. Results were presented for TaqMan and SYBR green formats. The
TaqMan assay was affected by mutations in the probe, particularly for the D91.1 subtype of the
genotype 3 viruses. Here, 4 mismatches were shown to be present between the target sequence
and the probe. Dr Baylis mentioned that plasmid clones for the B19V genotypes would be
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circulated through the EDQM PTS in due course and that efforts were being undertaken to
collect plasma for a genotype reference panel.
Transmissibility of B19 by transfusion: evaluation of donor viremia prior to performing a
linked donor-recipient study
Dr Mike Busch, Blood Systems Research Institute USA described a study that has recently
started to look at the transmissibility of B19V by transfusion, this is being undertaken by the
Retrovirus Epidemiology Donor Study (REDS-II) group. For the B19V study, the REDS
Allogenic Donor and Recipient (RADAR) repository was outlined where plasma and whole
blood samples were collected. Phase I of the study has looked at levels of IgG and IgM
antibodies and B19V viraemia in the donor samples and examined recipient seroprevalence. Dr
Busch outlined the work to be performed in Phase II of the study that will aim to determine the
rate of virus transmission by B19V DNA positive units and correlate this with viral load and
antibody status.
Session 9: Genotypes: HIV and other viruses chaired by Dr. Harvey Holmes (NIBSC,
UK) Dr Holmes opened this session by highlighting the different genotypes associated with
different blood-transmitted viruses. In particular he focused on HIV and showed a series of
graphs which demonstrated the inefficiencies of many HIV RNA NAT assays at detecting
diverse members of group M and the outliers forming groups N and O. Dr Holmes briefly
described plans for a second international reference panel for HIV-1 genotypes featuring
representatives of the less common subtypes and circulating recombinant forms (CRFs) and
emphasised the difficulties experienced in obtaining field isolates of these diverse groups. He
asked if SoGAT delegates would be willing to donate such reagents should they have access to
them.
HIV variants and a nanoparticle based assay for detection of HIV-1 p24 and RNA
Dr. Indira Hewlett, CBER FDA, USA emphasised the global distribution of HIV genotypes
and it was highlighted that although the central African region contains a diverse population of
genotypes and circulating recombinant forms, these are no longer restricted to this region as
they can now be found in Asia, South America and beyond. Dr Hewlett presented details of a
collaborative study conducted in Cameroon in collaboration with the Cameroon Ministry of
Health and the US CDC. Blood samples were collected from sites around the Cameroon capital
and initially tested in an HIV rapid assay and then by FDA licensed assays. Discordant samples
were further analysed in house for group O.
Dr Hewlett also introduced the application of nanotechnology with regards to diagnostics; The
assay, based on chemical probes labelled with nanoparticles and magnetic microparticles, has
been demonstrated to be highly sensitive without the use of enzymes for amplification. Data
presented by Dr Hewlett suggests that the BCA assay could be up to 100 fold more sensitive
than current ELISA techniques.
HIV CRFs
Dr. Richard Myers, UCL Medical School, UK described a study to analyse the
recombination events in the HIV-1 pol gene using a three stage process. Dr Myers presented
data whereby 10,545 pol sequences taken from the UK national resistance database and
analyzed for recombination events. Of these sequences 70% were from a Genotype B, however
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the remainder were of non genotype B origin which is considered high for the UK. CRF01 and
CFR02 were present in large numbers and only 10% of recombination sequences conformed to
known CRF sequences. Dr Myers concluded this presentation with the suggestion that the
classification of HIV is out dated; such a system was more suited to virologists than the nature
of the virus.
What about genotypes? (viral genotypes and their relevance in testing)
Dr Matthias Gessner (Baxter AG, Austria) asked whether it was necessary to detect all
known genotypes. Dr Gessner also highlighted the importance of using well characterised
samples so accurate demonstration of detection is possible. It was suggested that genotype
samples should be characterised based on standardised protocols, resulting standards should
then be readily available. Dr Gessner proposed that SoGAT should identify ways of reviewing
the significance of newly identified genotypes and to enable ways to standardise them.
Concluding session/summing up
Dr Gerold Zerlauth opened the meeting by asking whether SoGAT had completed its mission.
Following two days of presentations, Dr Zerlauth concluded that whilst much had been
achieved, there are still issues for SoGAT to address, such as the replacement of ISs, the
development of new standards including synthetic standards, the sensitivity of NAT, the
relationship of IUs to copies/genome equivalents, the potency of replacement standards and
the harmonisation of testing. His recommendation to the group was that SoGAT has new
challenges to address and therefore should continue to meet.
Dr John Saldanha summed up the meeting by reiterating the salient points raised by each
session. Dr Stephen Inglis (NIBSC, UK) gave a final word of thanks to the organising
committee for a successful agenda program; he also thanked the hosts, Swiss Medic, in
particular Dr Michael Sanger and Pamela Lane of NIBSC for her assistance in co-ordinating
the event. Dr Inglis emphasised that NIBSC remains committed to the mission of SoGAT and
gave credit to the group’s achievements in the standardisation of NAT for blood pathogens
over the preceding 19 meetings. Looking ahead, he noted that there was still important work to
be done in the blood safety field, but that there are also other emerging areas where NAT
standardisation is of crucial importance to public health, such as diagnostic virology, and that
this represented a major opportunity for SoGAT to continue its excellent work in the future.
DRAFT SoGAT minutes, June 2006
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