Download Chagas Disease

Quality Control of Chagas
diagnostics immunoassays:
Assay characteristics and
manufacturer’s reference
panels.
Gustavo A. Capriotti,
Biochemist, R&D Manager
WHO Consultation Meeting 27-28 January 2009
Antigens used in Conventional Tests for
T. cruzi infection:
 Whole Extracts or Semipurified Fractions of
parasite (epimastigote)
 Purified Proteins
 Synthetic Peptides
 Recombinant Antigens
WHO Consultation Meeting 27-28 January 2009
Conventional serological tests
• Indirect hemagglutination (IHA)
 Parasite lysate
• ELISA
 Parasite lysate
 Recombinant antigens
WHO Consultation Meeting 27-28 January 2009
Conventional serological tests
Performance
Method
IHA
ELISA
Sensitivity
> 97%
> 98%
WHO Consultation Meeting 27-28 January 2009
Specificity
> 98%
> 99%
Kits
 Chagatest HAI
 Chagatest HAI screening A-V
 Chagatest ELISA (lysate)
 Chagatest ELISA recombinante v 3.0
(FDA 510k and CE)
WHO Consultation Meeting 27-28 January 2009
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)
WHO Consultation Meeting 27-28 January 2009
Chagatest ELISA recombinante v.4.0
Recombinant Antigens
System with six recombinant Ags
SAPA, Ag1, Ag2, Ag13, Ag30, Ag36
• Highly sensitive and specific mixture
• Proteins present in the trypomastigote
stage
• Proteins preserved in different parasite
strains/linages
WHO Consultation Meeting 27-28 January 2009
Chagatest ELISA recombinante v.4.0
Recombinant Antigens
Ag1 Ag2 Ag30
Ag13 Ag36
Ag2 Ag13
SAPA
Ag13 Ag 36
SAPA
Chronic
Congenital
Acute
WHO Consultation Meeting 27-28 January 2009
Chagatest ELISA recombinante v.4.0
SENSITIVITY
International performance panels
PANELS
ORIGIN
DETECTED
REACTIVE
SAMPLES
PMT 201
BBI, USA
14/14
100%
PP 0404
Q Panel, Brazil
16/16
100%
PP 0405
Q Panel, Brazil
16/16
100%
PP 0406
Q Panel, Brazil
16/16
100%
Pediatric samples
Endemic area
100/100
100%
Pediatric samples
Rosario
115/116
99.14%
SENSITIVITY %
Reactive sample panels
WHO Consultation Meeting 27-28 January 2009
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 %
WHO Consultation Meeting 27-28 January 2009
Chagatest ELISA recombinante v.4.0
Interference with other pathologies
Parasites
Leishmaniasis
Amebiasis
Toxocariasis
Toxoplasmosis
Hydatidosis
Teniasis
1/10
0/7
0/6
0/5
0/5
0/2
Infectious disease
Hepatitis B
Hepatitis C
Syphilis
HIV
0/22
0/28
0/19
0/25
Other pathologies
Lymphoma
0/1
Myeloma
0/1
Lung tuberculosis 0/4
Autoimmune
disorders
1/4
WHO Consultation Meeting 27-28 January 2009
Recombinant antigens
Advantages in serological diagnosis
 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.
WHO Consultation Meeting 27-28 January 2009
How do we ensure Standardization?
- At
production level
Recombinant 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
WHO Consultation Meeting 27-28 January 2009
How do we ensure Standardization?
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.
WHO Consultation Meeting 27-28 January 2009
How do we ensure Standardization?
ELISAs Calibration
10 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 used
20 negative samples
WHO Consultation Meeting 27-28 January 2009
How do we ensure Standardization?
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.
WHO Consultation Meeting 27-28 January 2009
How do we ensure Standardization?
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
WHO Consultation Meeting 27-28 January 2009
How do we ensure Standardization?
ELISAs Titer verification
- Specificity: 200 sera / fresh plasmas
Acceptance criteria: > 99%.
If < 99%, the conjugate is diluted and
retested.
WHO Consultation Meeting 27-28 January 2009
How do we ensure Standardization?
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
WHO Consultation Meeting 27-28 January 2009
How do we ensure Standardization?
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.
WHO Consultation Meeting 27-28 January 2009
How do we ensure Standardization?
Hemagglutination
Lot preparation
- Internal panel of 20 positive sera
- 100 negative sera
WHO Consultation Meeting 27-28 January 2009
How do we ensure Standardization?
Hemagglutination
Final verification
- Chagas performance panel (QPanel)
- BBI panel
WHO Consultation Meeting 27-28 January 2009
Reference Panel
Considerations (proposal 2007):
• Sample: SERUM (not plasma)
• Number: 10-12 (representing a range of reactivities from nonreactive to strongly reactive)
• Not inactivated by heat (preferably aseptically filtered,
photoinactivation, UV or g 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
WHO Consultation Meeting 27-28 January 2009
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