Standardisation of Standardisation of P. P. falciparum falciparum HBV, HCV and HBV, HCV and

NATNAT

Sally Baylis, NIBSCSally Baylis, NIBSC

SoGAT XVIIISoGAT XVIII

MalariaMalaria

300 to 500 million cases annually (1.5 to 2.7 million 300 to 500 million cases annually (1.5 to 2.7 million deaths)deaths)

4 plasmodia species cause malaria in man4 plasmodia species cause malaria in man Plasmodium falciparum Plasmodium falciparum causes ~80% of cases (malaria causes ~80% of cases (malaria

tropica)tropica) P. vivax (~15% cases) , P. ovale, P. malariae less severeP. vivax (~15% cases) , P. ovale, P. malariae less severe

Transmitted by night-biting Transmitted by night-biting AnophelesAnopheles mosquitoes mosquitoes

Complex lifecycle with species variationComplex lifecycle with species variation Asexual in manAsexual in man Sexual in mosquitoSexual in mosquito

R. Menard, Nature, 433, 113-4

Malaria cont.Malaria cont.

In non-immune host e.g. visitors to endemic areasIn non-immune host e.g. visitors to endemic areas Incubation period normally 1-3 weeksIncubation period normally 1-3 weeks Seroconversion up to 4 months after infectionSeroconversion up to 4 months after infection Symptoms - fever, chills, malaise, headaches etc.Symptoms - fever, chills, malaise, headaches etc.

In the semi-immune host e.g. immigrants & visitors In the semi-immune host e.g. immigrants & visitors from endemic areas, those taking prophylaxisfrom endemic areas, those taking prophylaxis Delayed onset of illness & mild symptomsDelayed onset of illness & mild symptoms

In the “immune” hostIn the “immune” host Malaria is less severe & asymptomatic in 80% of Malaria is less severe & asymptomatic in 80% of

individualsindividuals

Malaria and TransfusionMalaria and Transfusion

Transfusion-transmitted malaria is rare, but potentially Transfusion-transmitted malaria is rare, but potentially serious consequence of blood transfusionserious consequence of blood transfusion

US, ~ 90 cases since 1963US, ~ 90 cases since 1963 Canada, 3 cases (1994-1999)Canada, 3 cases (1994-1999) UK, 5 cases in last 15 yearsUK, 5 cases in last 15 years

Generally prevention of transfusion transmitted malaria by Generally prevention of transfusion transmitted malaria by donor selection to identify those “at risk” e.g.donor selection to identify those “at risk” e.g.

Born/ lived in endemic areasBorn/ lived in endemic areas Visitors to endemic areasVisitors to endemic areas Had malariaHad malaria

Malaria and Transfusion cont.Malaria and Transfusion cont.

Most recent UK case of transfusion transmitted malaria in Most recent UK case of transfusion transmitted malaria in 2003 (Kitchen 2003 (Kitchen et alet al., ., Vox SangVox Sang. 88, 200-1). 88, 200-1)

Recipient:Recipient: Male (age 50) with sickle cell disease & renal failure became Male (age 50) with sickle cell disease & renal failure became

symptomatic ~ 2 months after transfusionsymptomatic ~ 2 months after transfusion Blood films – Blood films – P. falciparumP. falciparum trophozoites; 5.2% parasitaemia trophozoites; 5.2% parasitaemia No history of travel outside UK since 1957No history of travel outside UK since 1957

Donor:Donor: Female (age 38) from Ghana, migrated to UK & not returned to Female (age 38) from Ghana, migrated to UK & not returned to

Ghana in previous 8 yearsGhana in previous 8 years No history of malariaNo history of malaria Malaria Ab EIA positive serum archive sample (Newmarket)Malaria Ab EIA positive serum archive sample (Newmarket) Follow up blood samples from donor, malarial ab +ve & Follow up blood samples from donor, malarial ab +ve &

P.falciparumP.falciparum DNA was detected by qPCR (~5 parasites/ DNA was detected by qPCR (~5 parasites/μμl - 8 l - 8 months post-donation; months post-donation;

0.0005 parasites/0.0005 parasites/μμl - 12 months post-donation) l - 12 months post-donation)

Screening for MalariaScreening for Malaria

Deferral & antibody screen for donor Deferral & antibody screen for donor reinstatement e.g. the UK – Newmarket EIA; US no reinstatement e.g. the UK – Newmarket EIA; US no approved testsapproved tests However will not detect “window”However will not detect “window”

Stained blood films – routine method for diagnosis Stained blood films – routine method for diagnosis (since 1900)(since 1900) Slow, requiring highly trained microscopistsSlow, requiring highly trained microscopists

Antigen detectionAntigen detection Lacking in sensitivityLacking in sensitivity

Nucleic Acid DetectionNucleic Acid Detection Potentially may not detect very low levels of Potentially may not detect very low levels of

parasitaemiaparasitaemia

NIBSC Proposal for NIBSC Proposal for Production of Standards for Production of Standards for

P. falciparumP. falciparum

For standardisation of NAT assays (qualitative For standardisation of NAT assays (qualitative and quantitative)and quantitative)

Screening of blood for transfusion and tissues: Screening of blood for transfusion and tissues: exclusion of infected donations (run controls); exclusion of infected donations (run controls); determination of levels of parasitaemia where TTIs determination of levels of parasitaemia where TTIs occur; validation of assay sensitivitiesoccur; validation of assay sensitivities Diagnosis and clinical management of malaria: Diagnosis and clinical management of malaria: standardisation of commercial and in-house tests – standardisation of commercial and in-house tests – harmonisation of results to a single reference materialharmonisation of results to a single reference material

Vaccine studies: cross-comparison of studies, parasite Vaccine studies: cross-comparison of studies, parasite loads, efficacy etc.loads, efficacy etc.

Candidate Candidate P. falciparumP. falciparum Standards Standards

Freeze-dried blood from patient (~10% Freeze-dried blood from patient (~10% parasitaemia)parasitaemia)

Liquid preparation of blood from patient (~7% Liquid preparation of blood from patient (~7% parasitaemia)parasitaemia)

Liquid preparation of Liquid preparation of P. falciparumP. falciparum cultured cultured in in

vitrovitro to ~10% ring forms to ~10% ring forms

Liquid preparation of blood from patient Liquid preparation of blood from patient (~0.007% parasitaemia)(~0.007% parasitaemia)

R. Menard, Nature, 433, 113-4

Performance of Candidate Performance of Candidate P. P. falciparumfalciparum Standards Standards

Performance of Candidate Freeze-Performance of Candidate Freeze-Dried Dried

P. falciparumP. falciparum Standard Standard No significant loss of titre is observed following No significant loss of titre is observed following

freeze-drying of patient blood (~10% freeze-drying of patient blood (~10%

parasitaemia)parasitaemia) Accelerated degradation studies of this material Accelerated degradation studies of this material

are in progressare in progress >8 months at -20 >8 months at -20 ºC, no change in titreºC, no change in titre

Collaborative StudyCollaborative Study

Commenced April 2005Commenced April 2005

9 laboratories already received samples9 laboratories already received samples

3 further laboratories are finalising receipt details3 further laboratories are finalising receipt details

Labs wishing to participate in the collaborative Labs wishing to participate in the collaborative study should return form in meeting pack to S. study should return form in meeting pack to S. Baylis Baylis

Replacement of the HBV Replacement of the HBV DNA IS 97/746 DNA IS 97/746

The 1The 1stst International Standard for HBV DNA was International Standard for HBV DNA was established by the WHO ECBS in October 1999established by the WHO ECBS in October 1999

Estimated date of exhaustion of the IS will be Estimated date of exhaustion of the IS will be 2006 at current rate of usage2006 at current rate of usage

Materials coded AA (97/746) & BB showed no Materials coded AA (97/746) & BB showed no significant difference in potency in the significant difference in potency in the collaborative studycollaborative study

ECBS noted that BB (made from the same stock ECBS noted that BB (made from the same stock as AA) could be reserved for potential future use as AA) could be reserved for potential future use as a replacement standardas a replacement standard

Replacement of the HBV DNA IS Replacement of the HBV DNA IS 97/746 cont.97/746 cont.

Additional stability studies required before BB Additional stability studies required before BB able to replace AAable to replace AA

Propose small collaborative study, similar to that of the Propose small collaborative study, similar to that of the HCV RNA IS replacementHCV RNA IS replacement

Aim to demonstrate the equivalence of the candidate Aim to demonstrate the equivalence of the candidate replacement (BB) to AAreplacement (BB) to AA

Real-time data on AA and BB samplesReal-time data on AA and BB samples Accelerated degradation data on AA and BBAccelerated degradation data on AA and BB

Aim to submit report to WHO ECBS by July 2006Aim to submit report to WHO ECBS by July 2006

Replacement of the HCV Replacement of the HCV RNA IS 96/798 RNA IS 96/798

Approximately 1250 vials remain of the 2Approximately 1250 vials remain of the 2ndnd HCV HCV IS 96/798IS 96/798

Estimated date of exhaustion of the IS will be Estimated date of exhaustion of the IS will be 2009 at current rate of usage2009 at current rate of usage

Options for replacing the ISOptions for replacing the IS

11stst & 2 & 2ndnd IS derived from the same genotype IS derived from the same genotype 1a anti-HCV positive donation1a anti-HCV positive donation

Do we replace the IS with a donation positive for Do we replace the IS with a donation positive for anti-HCV or negative for anti-HCV?anti-HCV or negative for anti-HCV?

11stst & 2 & 2ndnd IS diluted in cryosupernatant IS diluted in cryosupernatant Do we dilute a replacement IS in cryosupernatant Do we dilute a replacement IS in cryosupernatant or plasma?or plasma?

AcknowledgementsAcknowledgements

David Padley, Alan Heath & Nita Shah, NIBSCDavid Padley, Alan Heath & Nita Shah, NIBSC

Peter Chiodini, Hospital for Tropical Diseases, Peter Chiodini, Hospital for Tropical Diseases, LondonLondon

Patricia Hewitt, NBS, LondonPatricia Hewitt, NBS, London

Claire Swales, LSHTM, LondonClaire Swales, LSHTM, London