Comparison between indirect immunofluorescence antibody (IFA) and dotblot enzyme
immunoassay (EIA) methods to detect Coxiella burnetii (Q Fever) phase I and II antibodies
C. M. Farris1, B. Kiehl2, J. Samuel3
1Viral and Rickettsial Diseases Department, Naval Medical Research Center, Silver Spring, MD, 2GenBio, San Diego, CA, 3Department of Microbial and Molecular Pathogenesis, College of Medicine,
Texas A&M, Bryan, TX
Abstract
Methods & Materials
Sensitivity
Conclusions
Coxiella burnetii is the causative agent of Q Fever, a worldwide zoonotic disease. Diagnosis of human disease is
typically made using serology. Comparison between the
indirect immunofluorescence antibody (IFA) and dot blot
enzyme immunoassay (EIA) methods is reported.
Materials and Methods: A commercial dotblot
immunoassay, ImmunoDOT™ Coxiella Burnetii, was
developed using a cell-free culture method to prepare
purified phase I and II Coxiella burnetii antigens. IgG and
IgM commercial IFA kits (Focus Diagnostics™) and inhouse prepared IFA tests are compared to the dotblot kit.
Paired sera collected from thirty-eight (38) patients with
symptomatology and laboratory results consistent with C.
burnetii are tested. These serum pairs are part of a C.
burnetii reference serum panel maintained by the
university laboratory. Each of the 76 specimens (38 serum
pairs) is tested using the IFA tests detecting Phase I and II
IgG or IgM. The 76 specimens are also tested using the
ImmunoDOT Coxiella Burnetii test.
Results: Sensitivity, based on paired serum interpretation is
shown in Table 1. There is no significant difference
(p=0.05, Chi-square, confidence limits based on Gart and
Nam's score method with skewness correction) between
assay methods.
Table 1: Sensitivity
Method
Phase 1
Phase 2 Combined
EIA
67-91%
67-91% 67-91%
Commercial IFA 61-87%
67-91% 70-93%
In-house IFA
55-83%
55-83% 67-91%
To evaluate sensitivity, each of the 76
specimens (38 serum pairs) were tested using
three serology tests. All testing was performed
at the university laboratory. ImmunoDOT
interpretation was made following package
insert instructions. IFA interpretation was
made using the following criteria.
There is no significant difference between
assay methods (p=0.05, Chi-square,
confidence limits based on Gart and Nam's
score method with skewness correction).
 No significant difference between IFA and
the ImmunoDOT dotblot method is
detected.
 Unlike the IFA method, ImmunoDOT
uses cell-free Coxiella burnetii Phase I and
II antigens.
 The improved purity of the Phase I
antigen may allow earlier detection.
 Although the Phase II antigen purity is
improved, clinical interpretation using
Phase II results is not affected because Q
fever is endemic in most populations.
That is, approximately one-third of the
population expresses a low level of
Coxiella burnetii Phase II antibody.
 ImmunoDOT offers the advantage that
only one test is required and the test
method can be performed in moderate
complexity laboratories.
Summary: No significant difference between IFA and the
dotblot is detected. In several cases, a second convalescent
specimen is required for IFA sero-diagnosis while EIA
reports a positive result using only a single, acute
specimen. Presumably, phase I antigen purity
improvement contributes to the added diagnostic utility.
Introduction
Q fever, caused by Coxiella burnetii, is a
world-wide zoonotic disease. Infection usually
occurs by aerosol transmission in a rural
(farm, back country, etc.) environment. Like
many respiratory infections, many are
asymptomatic. Disease usually presents as flulike symptoms that may progress to atypical
pneumonia or acute respiratory distress
syndrome (ARDS). Fever may last 1-2 weeks.
The time from infection to disease
expression is several days coinciding with
antibody production. Sero-diagnosis is the
most common laboratory method.
Historically, the indirect
immunofluorescence antibody (IFA) method
has been the most commonly used test and
remains the gold standard for diagnosis.
Comparison to a dot blot enzyme
immunoassay (EIA) method
(ImmunoDOT™) is reported.
Method
Positive Interpretation Criteria
Phase 1 IgG IFA Antibody detected at screening dilution
(titer). Commercial method screening
titer is 1:16 and university method
screening titer is 1:20.
Phase 2 IgG IFA Four-fold dilution (titer) difference
between serum pair specimens or either
serum pair specimen reports antibody
dilution (titer) greater than 1:1000
Phase 1 IgM IFA Antibody detected at screening dilution
(titer). Commercial method screening
titer is 1:16 and university method
screening titer is 1:10.
Phase 2 IgM IFA Antibody detected at screening dilution
(titer). Commercial method screening
titer is 1:16 and university method
screening titer is 1:10.
Paired Serum Specimens: Serum pairs
collected from thirty-eight (38) patients with
clinically defined Q Fever were tested. The
serum pairs are part of a Q fever reference
serum panel maintained by the university
laboratory.
IFA Method: Two indirect
immunofluorescence assay (IFA) methods
were evaluated. One is a commercial test
(Focus Diagnostics, detecting IgG and IgM
Phase 1 and Phase 2 antibody types) and the
other was produced by the university
laboratory using the same antigen. The serial
dilution schemes of the assays differ slightly.
For the university method, antigen is fixed to
the slide using acetone. The procedures are
the same for both methods following the
commercial test’s package insert instructions.
Dot Blot EIA Method: A commercial dotblot
immunoassay (ImmunoDOT™ Coxiella
Burnetii) was developed using Phase I or
Phase II antigens prepared from Coxiella
burnetii propagated using a cell-free culture
method (1). Since the dotblot format
provides a multiplex result, only one test is
required.
Presumptive Negative Specimens: To
evaluate ImmunoDOT specificity, forty-seven
(47) sera collected from prospective U.S.
blood donors were tested.
Method
Dotblot
Commercial IFA
In-house IFA
Phase I
67-91%
61-87%
55-83%
Phase II
67-91%
67-91%
55-83%
Combined
67-91%
70-93%
67-91%
Specificity
Using the ImmunoDOT dotblot method,
forty-seven presumptive negative specimens
collected from healthy, U.S. subjects are
tested. Using package insert instructions,
specificity is 100% (47/47). Sixteen (34%) of
these clinical negative specimens reported
borderline reactivity (see below).
Phase I
Positive
Negative
Positive
Negative
Positive
Negative
Positive
Phase II
3 Dots
3 Dots
2 Dots
2 Dots
1 Dot
1 Dot
No Dots
Percent
0
4% (2)
4% (2)
0
0
26% (12)
0
Reference
1. Omsland, et al, 2009, Host cell-free
growth of Q fever bacterium Coxiella
burnetii, Proc Natl Acad Sci USA, 106
(11):4430-4434.
Disclaimer
The views expressed in this article
are those of the authors and do not
necessarily reflect the official policy
or position of the Department of the
Navy, Department of Defense, nor
the U.S. Government.
Contact Information
Bryan Kiehl (bkiehl@GenBio.com)
Mobile (+1 760-855-8999)