WHO Consultation Meeting 27-28 January 2009 Quality Control of Chagas diagnostics immunoassays: Assay characteristics and manufacturers reference panels.

WHO Consultation Meeting 27-28 January 2009

Quality Control of Chagas diagnostics immunoassays: Assay characteristics and manufacturer’s reference

panels.

Gustavo A. Capriotti, Biochemist, R&D Manager


Antigens used in Conventional Tests for T. cruzi infection:

Whole Extracts or Semipurified Fractions of

parasite (epimastigote)

Purified Proteins

Synthetic Peptides

Recombinant Antigens


Conventional serological tests

• Indirect hemagglutination (IHA) Parasite lysate

• ELISA Parasite lysate Recombinant antigens


Conventional serological tests

Performance

Method Sensitivity Specificity

IHA > 97% > 98%

ELISA > 98% > 99%


Kits

Chagatest HAI Chagatest HAI screening A-V Chagatest ELISA (lysate) Chagatest ELISA recombinante v 3.0

(FDA 510k and CE)


Kits

New Chagatest ELISA recombinante v.4.0

(approved in LA/CE market) Chagatest ELISA recombinante for dried

blood spot samples (approved in RA) Rapid test (in development) Colorimetric PCR (in development) Quantitative PCR (to start development this

year)


Chagatest ELISA recombinante v.4.0Recombinant Antigens

• Highly sensitive and specific mixture

• Proteins present in the trypomastigote stage• Proteins preserved in different parasite strains/linages

System with six recombinant Ags

SAPA, Ag1, Ag2, Ag13, Ag30, Ag36


Chagatest ELISA recombinante v.4.0 Recombinant Antigens

Chronic

Ag1 Ag2 Ag30 Ag13 Ag36 Ag2 Ag13

SAPA

Ag13 Ag 36 SAPA

Acute

Congenital


SENSITIVITY

International performance panels

PANELS ORIGINDETECTED REACTIVE SAMPLES

SENSITIVITY %

PMT 201 BBI, USA 14/14 100%

PP 0404 Q Panel, Brazil 16/16 100%



Reactive sample panels

Pediatric samples Endemic area 100/100 100%

Pediatric samples Rosario 115/116 99.14%

Chagatest ELISA recombinante v.4.0


SPECIFICITY

SAMPLES SPECIFICITY %

1192 Blood Bank samples 99.66 %

477 samples from different Health Centers 99.57 %

474 samples from high prevalence population 98.30 %

491 samples with different clinical conditions 98.37 %




Interference with other pathologies

ParasitesLeishmaniasis 1/10Amebiasis 0/7Toxocariasis 0/6Toxoplasmosis 0/5Hydatidosis 0/5Teniasis 0/2

Infectious disease Hepatitis B 0/22Hepatitis C 0/28Syphilis 0/19HIV 0/25

Other pathologies

Lymphoma 0/1Myeloma 0/1Lung tuberculosis 0/4

Autoimmunedisorders 1/4


Standardized system uses a perfectly defined antigen composition.

Antigens expressed in the infected trypomastigote stage of the parasite.

Highly preserved antigens in different strains of the parasite.

SAPA antigen, acute and congenital infection marker.

Recombinant antigens

Advantages in serological diagnosis


How do we ensure Standardization?

- At production levelRecombinant antigens Recombinant antigens well characterized Perfectly defined antigen mix

Parasitic Lysate Characterized lysate by WB Parasite culture under strict growth conditions. Well defined WCB & MCB



The recombinant antigens are tested separately using an ELISA technique with an internal panel of 9 positive samples specific for each antigen and 4 negative samples.



ELISAs Calibration10 weak positive samples (IP between 1.0 and

2.0)10 medium positive samples (IP between 2.0

and 4.0)10 strong positive samples (IP < 4.0) Note: samples diluted in negative or bovine

serum may be used20 negative samples



ELISAs Calibration

A titer verifying the IP coefficient within a range of 0.9 – 1.2 must be selected.

In addition, all negative samples must yield negative results.



ELISAs Titer verification

- Internal panel of reactive samples evaluation: internal panel of 32 samples including weak, medium and strong.

Acceptance criteria: the individual IP coefficient of each sample must be within a range of 0.9 – 1.2



ELISAs Titer verification

- Specificity: 200 sera / fresh plasmas

Acceptance criteria: > 99%.

If < 99%, the conjugate is diluted and retested.



ELISAs Final test

- Internal panel: 2 positive control sera, 3 negative control sera, 6 weak positive, 10 medium and 10 strong samples.

- Commercial panels: Chagas performance panel (QPanel, Brazil); BBI Panel



Hemagglutination- Titration of red blood cells sensitization.The titers are tested with an internal panel of 15

positive sera. Some of them diluted. Panel with 10 negative sera.

Acceptance criteria: a titer where diluted sera match background titer of each sample is selected. Negative sera must yield negative results.



Hemagglutination

Lot preparation

- Internal panel of 20 positive sera

- 100 negative sera



Hemagglutination

Final verification

- Chagas performance panel (QPanel)

- BBI panel


Reference PanelConsiderations (proposal 2007):

• Sample: SERUM (not plasma)

• Number: 10-12 (representing a range of reactivities from non-reactive to strongly reactive)

• Not inactivated by heat (preferably aseptically filtered, photoinactivation, UV or irradiation)

• Preferable without preservatives• Representative from different disease stages and

geographic regions• Selection made based on: IHA, ELISA, Immunoblot• For analytical sensitivity: diluted samples can be used• For clinical sensitivity: undiluted samples


International Biological Reference Preparation for Chagas (2009)

• A known reactivity standard is required to yield consistency lot to lot

• To have a primary Standard of 2 reactive sera, as suggested, seems a good alternative.

• This will allow to determine the analytical sensitivity for each lot, as being used for other international standards.

WHO Consultation Meeting 27-28 January 2009 Quality Control of Chagas diagnostics immunoassays: Assay characteristics and manufacturers reference panels.

Documents

consultation meeting

negative samples

ag36 slide

year slide

ce slide

strong samples

positive samples specific

weak positive samples