Transcript WHO comparative evaluation of serologic assays for Chagas

WHO comparative
evaluation of
serologic assays
for Chagas disease
Journal Club April 2, 2009
– By the triatomine
bug (kissing or
reduviid bug)
– Vertically
– Blood transfusions
– Organ transplants
http://www.cdfound.to.it/img/tryp3.jpg
• caused by the
parasite
Trypanosoma cruzi,
which is
transmitted:
http://www.rso.cornell.edu/bugclub/images/Triatoma%20sanguisuga.jpg
Chagas disease
(American trypanosomiasis)
Epidemiology
• Endemic in Mexico, Central and South
America
– 8-11 million people are infected
• 45,000 fatalities may occur annually
– Those living in rural areas at highest risk
(triatomine bugs love mud walls and thatched
roofs)
• In the US and Canada, infection is
increasing as a result of the immigration
of infected individuals from endemic areas
http://www.aabb.org/Content/Members_Area/Association_Bulletins/ab06-08.htm
Transfusion relevence
• Acute, vector-borne infections are mostly mild
– Chronic, asymptomatic infection results
– acute infection in patients with compromised immune
systems can be very serious
• It has been estimated that there may be as many
as 100,000 legal immigrants in the US and
Canada who are unknowingly infected
• The rate of seropositive blood donors in the US
ranges from 1 in 5400 to 1 in 25,000
– Among high risk populations in Washington and
California, the prevalence was 1 in 500, which 20% able
to transmit infection
– 2 cases of TT-Chagas disease have been identified in
Canada
http://www.aabb.org/Content/Members_Area/Association_Bulletins/ab06-08.htm
http://www.cmaj.ca/cgi/reprint/177/3/242-a
Screening
• In 2006, the US FDA approved a
serologic enzyme-linked
immunosorbent assay (ELISA) for
screening donations
– Ortho-Clinical Diagnostics
Challenges
• Laboratory diagnosis of Chagas disease is
challenging
– Direct detection of the parasites is difficult
• Low parasitemia during chronic phase
• Hence, the laboratory diagnosis is based on serologic
assays
– i.e. looking for T. cruzi antibodies with a lysate of
epimastigotes grown in liquid culture
– There is no accepted/accessible gold standard
• New assays are validated using high-antibody-titre,
consensus positive specimens (and thus their ability
to detect low-antibody-titre cases is untested)
– Sensitivity is overestimated
Purpose
• Evaluation of commercially available
test kits for Chagas disease for use
in blood bank screening is difficult
– Lack of large and well-characterized
panels
• Collaborate effort of Latin American
blood centres and the WHO to
establish a Chagas disease panel
Design
• 437 specimens (Chagas positive and
Chagas negative) were provided in 2000
to the WHO Collaborating Centre in San
Paulo
– 10 Latin American blood centres in 10
countries
– Specimens were assigned a positive or
negative status based on concordant results in
at least three of the four confirmatory assays
• Between 2001 and 2005, this panel was
used to evaluate 19 screening assays
Confirmatory Assays
• Indirect immunofluorescence IF
• Western blot WB
• Radioimmunoprecipitation assay
RIPA
– Only RIPA gave 100% agreement with
the final serologic status of the
specimens
• Recombinant immunoblot IB
– RIBA
Indirect
Immunofluorescence
Anti-human IgGfluorescein conjugate
Donor’s antibodies against parasite
epimastigotes
http://www.biologie.uni-hamburg.de/b-online/ge17/22b.jpg
http://www.molecularstation.com/images/western-blot.jpg
Trypomastigote antigens
Anti-human
IgG
conjugate
The conjugate develops colour,
and the results interpreted by
visual inspection for bands
between 130-200 kDa
RIPA
• Radiolabeled antigen fragments are
combined with the specimen
• Specific antibodies, if present, will
bind these antigen fragments
• The resulting antigen-antibody
complexes are precipitated and
separated by electrophoresis
• The pattern is detected using
autoradiography (the exposure of
sensitive X-ray film by the
radioactive emissions of the bound,
labeled antigens)
Recombinant immunoblot
(RIBA)
• Recombinant
parasite
antigens are
applied to paper
strips
• Exposure to
specimen allows
anti-parasite
antibodies, if
present, to bind
to the antigen
• Anti-human
labelled
conjugate allows
visualization
Results
• 437 specimens
– Positive 39%
– Negative 61%
– Inconclusive 2% (excluded from
analysis)
• Since their true serologic status cannot be
determined, there is no way to conclude
how they might have affected the calculated
sensitivities and specificities
Results
• Screening assays
– Sensitivity and specificity varied
considerably
• Sensitivity 80%-100%
• Specificity 60-100%
• EIAs performed better than other
screening methods
– Four EIAs had specificity and sensitivity
in excess of 99%
Conclusions
• At least four of the commercially available
EIAs are sufficiently sensitive and specific
for use as a single-assay screen of blood
donations
• The majority of commercially available
indirect hemagglutinin assays should not
be used
• Sensitivity and/or specificity was low
• Subject to reader bias
• False negatives reported even for high-titre
specimesn (prozone effect)
• The RIPA is the gold standard
• But it is complex
Prozone effect
Evaluation
• What is the background/rationale for
the investigation?
– There was a clear need for establishing
a Chagas panel
– The report helps to raise awareness
among clinicians/blood bankers about
the challenges of evaluating new assays
Evaluation
• Study design
– The setting was a blend of the small Latin
American endemic countries with the resources
of WHO over several years
• Variables
– Variables and confounders (e.g. concurrent
disease) were left undescribed
– Diagnostic criteria (e.g. diagnostic test) varied
from country to country
Evaluation
• Bias
– Bias is a possible confounder, since
there was no apparent randomization in
the submission of samples
– Centres may have submitted specimens
that were “easy” (clearly positive or
negative), or specimens that were
challenging
• Submitting either a preponderance of easy
or tough specimens could affect the
sensitivity and specificity of the test results
Evaluation
• Study size
– No particular study size was predetermined
Evaluation
• Statistical methods were not
described in detail
– 95% CI were calculated and provided
Evaluation
• Participants
– The number of specimens received was
reported
– The number of specimens eventually excluded
from the study also reported
• Main results
– Sensitivities, specificities and 95% CI were
reported for the 19 screening methods and the
four confirmatory assays
– The “gold standard” was the consensus result
of the four confirmatory assays
Evaluation
• Results
– Four EIA kits were appropriate to be used as
single assay screens
– RIPA is proposed as the gold standard
• Limitations & Generalization
– The specimens provided by the blood services
may differ from those that would be randomly
encountered (especially in countries were
Chagas is not endemic)
– Questions of cost and availability for small
blood services
– Issues of convenience with RIPA technology