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Initial testing of
a first generation, rapid, immunochromatography test for the
detection of H. ducreyi showed it to be highly specific but
only moderately sensitive.
Development of a
rapid immunodiagnostic test for Haemophilus ducreyi.
Patterson K, Olsen B, Thomas C, Norn
D, Tam M, Elkins C.
Journal of Clinical
Microbiology 2002, 40:3694-3702.
Summary:
Question
What is the initial sensitivity and specificity of a first generation,
rapid, immunochromatography (IC) test for H. ducreyi?
Design
This study describes a "proof of principle" test of the
performance of a newly developed IC assay for the detection of H.
ducreyi based on monoclonal antibodies to the hemoglobin receptor
using laboratory and clinical isolates of H. ducreyi and related
bacteria.
Participants
The assay was tested on 26 laboratory strains and clinical isolates of H.
ducreyi, one strain each of S. aureus, E. coli, E. faecalis, K.
pneumoniae, E. aerogenes, and B. fragilis, 14 stains of H.
influenzae (including 3 typeable, 5 nontypeable, and 6 biotype IV
genital isolates), and two laboratory strains of N. gonorrhoeae.
Description of Tests and Diagnostic
Standard
Native H. ducreyi hemoglobin receptor protein was purified under
nondenaturing conditions and used as both the immunogen and the antigen in
a screening ELISA for the development of monoclonal antibodies. A capture
ELISA method was used to determine how broadly cross-reactive each
monoclonal antibody was to a geographically diverse set of 26 H.
ducreyi laboratory isolates. The IC test strips consisted of an NC
membrane (Millipore Corporation, Bedford, MA) containing a monoclonal
antibody conjugated to colloidal gold that bound to the target antigen in
solution, a second antibody immobilized in a line across the strip that
captured the antigen that was bound to the gold-labeled antibody, and goat
anti-mouse IgG immobilized in an upper line that captured the labeled
antibody even if antigen was not present and acted as a positive assay
control. When the immobilized antibodies captured their antigens, a
visible colored line was generated. The appearance of two lines was
interpreted as a positive result, while the appearance of only one line
was interpreted as a negative, yet valid test result. The sensitivity of
the strips was determined by using fivefold dilutions of an H. ducreyi
isolate.
Main Outcome Measures
Test specificity, measured by a positive IC test result using detergent
solubilized H. ducreyi whole cells and a negative result using
other bacteria, and sensitivity, measured by the lowest dilution of
solubilized whole cells of an H. ducreyi stain giving a positive
test result, were calculated.
Main Results
One pair of monoclonal antibodies that recognized all 26 H. ducreyi
strains in the capture ELISA, were noncompetitive for antigenic isotopes,
and gave the strongest signal when used as the capture and detection
antibodies on the IC strips was selected for further testing. All 26 H.
ducreyi strains tested by the IC gave a positive reaction. IC test
results for all 22 non-H. ducreyi bacteria were indistinguishable
from the result for the negative control.
Sensitivity of the IC test against
solubilized whole cells of H. ducreyi was 2 X 106 CFU.
Authors' Conclusions
The IC test has broad reactivity against a
diverse panel of H. ducreyi strains, is highly specific for H. ducreyi,
and has moderate sensitivity.
Source of funding: WHO/UNAIDS grant
and University of North Carolina at Chapel Hill Center for AIDS Research
grant.
For correspondence: Christopher
Elkins, Department of Medicine, Division of Infectious Diseases,
University of North Carolina at Chapel Hill, 547 Burnett-Womack Building,
Chapel Hill, NC 27599-7030. E-mail address: chris_elkins@med.unc.edu.
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