Quality Assurance for Quality Assurance for Pneumococcal Assays in Europe Pneumococcal Assays in Europe

Daniel HarrisonDaniel Harrison

IndexIndex

Background to the studyBackground to the study Pneumococcal pathogen Pneumococcal pathogen Pilot pneumococcal serotype studyPilot pneumococcal serotype study

A European QA Scheme for A European QA Scheme for Pneumococcal Assays Pneumococcal Assays

EU funded Consensus conference (Langen 2006) on EU funded Consensus conference (Langen 2006) on Primary Immunodeficiency Diseases (PIDs) outlined: Primary Immunodeficiency Diseases (PIDs) outlined:

A lack of quality assurance for the assays that are used for A lack of quality assurance for the assays that are used for measuring specific antibody assays to common pathogens measuring specific antibody assays to common pathogens and immunization antigens – within PID community!and immunization antigens – within PID community!

A European QA Scheme for A European QA Scheme for pneumococcal assays pneumococcal assays

These assays are an important aid for the diagnosis of These assays are an important aid for the diagnosis of primary antibody deficiencies. primary antibody deficiencies.

Provision of standardized assay protocols with a reliable Provision of standardized assay protocols with a reliable quality assurance scheme will enable progress in quality assurance scheme will enable progress in determinating diagnostic criteria for these diseases.determinating diagnostic criteria for these diseases.

For this reason: A study to develop a quality assurance For this reason: A study to develop a quality assurance scheme for pneumococcal assays in Europe was initiated.scheme for pneumococcal assays in Europe was initiated.

ESID guidelines ESID guidelines

Currently pneumovax unconjugated vaccine is included in Currently pneumovax unconjugated vaccine is included in the ESID guidelines for PIDs. the ESID guidelines for PIDs. E. De Vries 2005E. De Vries 2005

Is used to determine the severity of humoral Is used to determine the severity of humoral immunodeficiency in a patient by way of looking at the immunodeficiency in a patient by way of looking at the result from booster vaccinationsresult from booster vaccinations

However the authors recognise that the protocols will need However the authors recognise that the protocols will need to be revised to be revised

Pneumococcal serotype assays could be incorporated Pneumococcal serotype assays could be incorporated

The Pathogen - The Pathogen - Streptococcus Streptococcus pneumoniae pneumoniae

What Is it?What Is it? Common microflora Common microflora

bacteria that has >90 bacteria that has >90 known serotypesknown serotypes

Because of this variation it Because of this variation it has strong pathogenicityhas strong pathogenicity

Streptococcus pneumoniaeStreptococcus pneumoniae

What does it cause?What does it cause?

Causes pneumonia, meningitis and systemic sepsis. Causes pneumonia, meningitis and systemic sepsis.

Prevalent in individuals at the extremes of agePrevalent in individuals at the extremes of age

1 million deaths are accountable to pneumococcal 1 million deaths are accountable to pneumococcal infections worldwide infections worldwide

This is mostly in developing countriesThis is mostly in developing countries

PIDsPIDs

Are highly susceptible to infections caused Are highly susceptible to infections caused by encapsulated bacteria by encapsulated bacteria

Especially those with X- linked Especially those with X- linked agammaglobulinaemia and common agammaglobulinaemia and common variable immune deficiencyvariable immune deficiency

Invasive Pneumococcal DiseaseInvasive Pneumococcal Disease

What is it and what are the What is it and what are the symptoms?symptoms?

The most severe form of The most severe form of pneumococcal infectionpneumococcal infection

Three major conditions:Three major conditions:

pneumonia, bacteremia, pneumonia, bacteremia, and meningitis. and meningitis.

They are all caused by They are all caused by infection with the same infection with the same bacteria, but have different bacteria, but have different symptoms.symptoms.

VaccinesVaccines

2 available pneumococcal vaccines: 2 available pneumococcal vaccines: pneumococcal pneumococcal polysaccharide vaccine (PPV) or Pneumovaxpolysaccharide vaccine (PPV) or Pneumovax®® and the and the pneumococcal conjugate vaccine (PCV) or Prevenar pneumococcal conjugate vaccine (PCV) or Prevenar ®®

PneumovaxPneumovax®® contains chains of polysaccharides that contains chains of polysaccharides that make up the surface capsule of the bacteria. make up the surface capsule of the bacteria.

The 23 types of pneumococci that are included cause 88% The 23 types of pneumococci that are included cause 88% of invasive pneumococcal disease.of invasive pneumococcal disease.

This vaccine is not effective in children younger than 2 This vaccine is not effective in children younger than 2 years in whom 80% of invasive pneumococcal disease in years in whom 80% of invasive pneumococcal disease in childhood occurs – so it is often given to elderly patientschildhood occurs – so it is often given to elderly patients

Vaccines and Serotype Vaccines and Serotype Prevalence in USA and Europe Prevalence in USA and Europe

Prevenar Prevenar ® ® includes purified capsular polysaccharide of seven types of includes purified capsular polysaccharide of seven types of the bacteria conjugated to a harmless variety of diphtheria toxin. the bacteria conjugated to a harmless variety of diphtheria toxin.

The seven types: The seven types: 4, 6B, 9V, 14, 18C, 19F and 23F 4, 6B, 9V, 14, 18C, 19F and 23F account for 86% of account for 86% of bacteremia, 83% of meningitis, and 65% of acute otitis media among bacteremia, 83% of meningitis, and 65% of acute otitis media among children less than six years of age in the United States. children less than six years of age in the United States.

These seven serotypes are responsible for 83% of invasive These seven serotypes are responsible for 83% of invasive pneumococcal disease in children <4 years old in the US and cause the pneumococcal disease in children <4 years old in the US and cause the majority of invasive disease in Europemajority of invasive disease in Europe

All healthy infants and toddler should receive four doses of Prevenar All healthy infants and toddler should receive four doses of Prevenar ® ® vaccinevaccine

Pilot Study Pilot Study

AimsAims To develop a quality assurance scheme for pneumococcal To develop a quality assurance scheme for pneumococcal

assays in Europe that aid the diagnosis of PIDs.assays in Europe that aid the diagnosis of PIDs.

To compare pneumococcal serotype results with those To compare pneumococcal serotype results with those from the existing 23 valent Pneumovax ELISA assay, in from the existing 23 valent Pneumovax ELISA assay, in order to determine the relative usefulness of these assays order to determine the relative usefulness of these assays for test immunisation in the diagnosis of PIDsfor test immunisation in the diagnosis of PIDs

To determine if it is possible to establish levels of To determine if it is possible to establish levels of protection in patients at high risk of invasive pneumococcal protection in patients at high risk of invasive pneumococcal diseasedisease

Pilot StudyPilot Study

MethodsMethods Development of ELISA assays, in accordance with WHO Development of ELISA assays, in accordance with WHO

protocol, for the detectionprotocol, for the detection of IgG reactive to 7 common of IgG reactive to 7 common pneumococcal serotypes (4, 6B, 9V, 14, 18C, 19F and pneumococcal serotypes (4, 6B, 9V, 14, 18C, 19F and 23F) 23F) in human serumin human serum

Acquired and tested 16 control serum samples along with Acquired and tested 16 control serum samples along with 16 known positive pneumovax serum samples16 known positive pneumovax serum samples

Concentrations of specific IgG to certain serotypes, within Concentrations of specific IgG to certain serotypes, within sera, were calculated using the reference serum from sera, were calculated using the reference serum from WHO (89-SF)WHO (89-SF)

Protection LevelProtection Level Needed to have a protection Needed to have a protection

level in order to establish level in order to establish positive and negative resultspositive and negative results

Black et al. 2000 found that Black et al. 2000 found that >95% of patients receiving the >95% of patients receiving the pneumococcal conjugate pneumococcal conjugate vaccine developed >0.15 vaccine developed >0.15 μμg/ml g/ml after 3after 3rdrd dose dose

Henckaerts et al. 2005 found Henckaerts et al. 2005 found that the putative protection level that the putative protection level was higher (3.5) in the non 22F was higher (3.5) in the non 22F assayassay

Therefore 0.2 Therefore 0.2 μμg/ml was used g/ml was used as a putative protection levelas a putative protection level

ResultsResultsDistribution of Immunity Across All Pneumococcal Serotypes For Control Samples

0

1

2

3

4

5

6

7

8

4 6B 9V 14 18 19F 23F

Pneumococcal Serotype

Sp

ecif

ic Ig

G C

on

cen

trat

ion

in μ

g/m

l

KP

BF

LP

JB

MA

ZW

AS

CD

DH

LA

HC

UT

DS

RS

KT

JW

protection

• Wide range between controls, but also between serotypes

Serotype Specific Immunoglobulin GSerotype Specific Immunoglobulin G Serotype

Control Pneumovax 4 6B 9V 14 18 19F 23F

Range 4.16-104.29 0.04-0.68 0.06-2.07 0.02-7.07 0.53-7.38 0.18-4.47 0.23-6.53 0.07-10.47

JB 104.29 0.42 0.93 0.46 0.69 0.95 3.23 1.11

HC 30.22 0.22 0.07 0.23 0.53 0.52 1.20 1.31

JW 11.22 0.21 0.17 0.02 1.66 0.18 0.31 0.07

ZW 13.24 0.27 1.77 7.07 7.38 1.02 4.08 5.33

Statistical Correlation with 7 serotypes Statistical Correlation with 7 serotypes and Pneumovax Assay:and Pneumovax Assay:

(Kappa Coefficients)(Kappa Coefficients)PNPS 4 6B 9V 14 18C 19F 23F

PNPS --------

4 0.24 ---------

6B 0.35 0.10 ---------

9V 0.26 0.02 0.39 ---------

14 N/A N/A N/A N/A ---------

18C 0.16 -0.12 0.20 0.10 N/A ---------

19F N/A N/A N/A N/A N/A N/A ---------

23F 0.60 0.33 0.43 0.18 N/A 0.33 N/A ---------

Pilot studyPilot study

Next StepsNext Steps Samples with adequate volumes and Samples with adequate volumes and

concentrations will be selected for concentrations will be selected for distribution to 15 centres in Europedistribution to 15 centres in Europe

This is so that our assay can be compared This is so that our assay can be compared and validated with those of our potential and validated with those of our potential European collaborators European collaborators

Pilot studyPilot study

Next StepsNext Steps Will attempt to make a selection of sera Will attempt to make a selection of sera

including 2 with high concentrations of including 2 with high concentrations of specific anti-pneumococcal antibodies, 2 specific anti-pneumococcal antibodies, 2 with low and 2 with intermediate with low and 2 with intermediate concentrationsconcentrations

There are anomalies, but we will inform There are anomalies, but we will inform collaborators of thesecollaborators of these

ConclusionsConclusions Observed high immunity for serotypes 14 and 18C Observed high immunity for serotypes 14 and 18C

Found that immunity against the serotypes varies Found that immunity against the serotypes varies between serotypes and individualsbetween serotypes and individuals

Immunity against most serotypes generally Immunity against most serotypes generally indicates a high pnuemovax result but not alwaysindicates a high pnuemovax result but not always

Compared serotype findings to results obtained Compared serotype findings to results obtained from the existing total Pneumovax ELISA assay from the existing total Pneumovax ELISA assay and found a correlation with 23F serotypeand found a correlation with 23F serotype

AcknowledgementsAcknowledgements

Dr Helen ChapelDr Helen Chapel Dr Berne FerryDr Berne Ferry