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Standardisation of P. falciparum
HBV, HCV and NAT
Sally Baylis, NIBSC
SoGAT XVIII
Malaria
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300 to 500 million cases annually (1.5 to 2.7 million
deaths)
4 plasmodia species cause malaria in man
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Plasmodium falciparum causes ~80% of cases (malaria tropica)
P. vivax (~15% cases) , P. ovale, P. malariae less severe
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Transmitted by night-biting Anopheles mosquitoes
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Complex lifecycle with species variation
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Asexual in man
Sexual in mosquito
R. Menard, Nature, 433, 113-4
Malaria cont.
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In non-immune host e.g. visitors to endemic areas
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In the semi-immune host e.g. immigrants & visitors
from endemic areas, those taking prophylaxis
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Incubation period normally 1-3 weeks
Seroconversion up to 4 months after infection
Symptoms - fever, chills, malaise, headaches etc.
Delayed onset of illness & mild symptoms
In the “immune” host
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Malaria is less severe & asymptomatic in 80% of individuals
Malaria and Transfusion
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Transfusion-transmitted malaria is rare, but potentially
serious consequence of blood transfusion
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US, ~ 90 cases since 1963
Canada, 3 cases (1994-1999)
UK, 5 cases in last 15 years
Generally prevention of transfusion transmitted malaria
by donor selection to identify those “at risk” e.g.
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Born/ lived in endemic areas
Visitors to endemic areas
Had malaria
Malaria and Transfusion cont.
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Most recent UK case of transfusion transmitted malaria in 2003
(Kitchen et al., Vox Sang. 88, 200-1)
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Recipient:
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Male (age 50) with sickle cell disease & renal failure became
symptomatic ~ 2 months after transfusion
Blood films – P. falciparum trophozoites; 5.2% parasitaemia
No history of travel outside UK since 1957
Donor:
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Female (age 38) from Ghana, migrated to UK & not returned to Ghana in
previous 8 years
No history of malaria
Malaria Ab EIA positive serum archive sample (Newmarket)
Follow up blood samples from donor, malarial ab +ve & P.falciparum DNA
was detected by qPCR (~5 parasites/μl - 8 months post-donation;
0.0005 parasites/μl - 12 months post-donation)
Screening for Malaria
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Deferral & antibody screen for donor reinstatement
e.g. the UK – Newmarket EIA; US no approved tests
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Stained blood films – routine method for diagnosis
(since 1900)
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Slow, requiring highly trained microscopists
Antigen detection
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However will not detect “window”
Lacking in sensitivity
Nucleic Acid Detection
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Potentially may not detect very low levels of parasitaemia
NIBSC Proposal for Production
of Standards for P. falciparum
For standardisation of NAT assays (qualitative and
quantitative)
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Screening
of blood for transfusion and tissues: exclusion of
infected donations (run controls); determination of levels of
parasitaemia where TTIs occur; validation of assay
sensitivities
Diagnosis
and clinical management of malaria:
standardisation of commercial and in-house tests –
harmonisation of results to a single reference material
Vaccine
studies: cross-comparison of studies, parasite
loads, efficacy etc.
Candidate P. falciparum Standards
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Freeze-dried blood from patient (~10% parasitaemia)
Liquid preparation of blood from patient (~7%
parasitaemia)
Liquid preparation of P. falciparum cultured in vitro to
~10% ring forms
Liquid preparation of blood from patient (~0.007%
parasitaemia)
R. Menard, Nature, 433, 113-4
Performance of Candidate P. falciparum
Standards
Performance of Candidate Freeze-Dried
P. falciparum Standard
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No significant loss of titre is observed following
freeze-drying of patient blood (~10% parasitaemia)
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Accelerated degradation studies of this material are
in progress
>8 months at -20 ºC, no change in titre
Collaborative Study
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Commenced April 2005
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9 laboratories already received samples
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3 further laboratories are finalising receipt details
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Labs wishing to participate in the collaborative study
should return form in meeting pack to S. Baylis
Replacement of the HBV DNA IS
97/746
The 1st International Standard for HBV DNA was
established by the WHO ECBS in October 1999
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Estimated date of exhaustion of the IS will be
2006 at current rate of usage
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Materials coded AA (97/746) & BB showed no
significant difference in potency in the collaborative
study
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ECBS noted that BB (made from the same stock as
AA) could be reserved for potential future use as a
replacement standard
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Replacement of the HBV DNA IS
97/746 cont.
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Additional stability studies required before BB able
to replace AA
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Propose small collaborative study, similar to that of the HCV
RNA IS replacement
Aim to demonstrate the equivalence of the candidate
replacement (BB) to AA
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Real-time data on AA and BB samples
Accelerated degradation data on AA and BB
Aim to submit report to WHO ECBS by July 2006
Replacement of the HCV RNA IS
96/798
Approximately 1250 vials remain of the 2nd HCV IS
96/798
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Estimated date of exhaustion of the IS will be
2009 at current rate of usage
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Options for replacing the IS
1st & 2nd IS derived from the same genotype 1a
anti-HCV positive donation
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Do we replace the IS with a donation positive for antiHCV or negative for anti-HCV?
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1st & 2nd IS diluted in cryosupernatant
Do we dilute a replacement IS in cryosupernatant or
plasma?
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Acknowledgements
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David Padley, Alan Heath & Nita Shah, NIBSC
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Peter Chiodini, Hospital for Tropical Diseases, London
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Patricia Hewitt, NBS, London
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Claire Swales, LSHTM, London