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Journal of Clinical Microbiology, October 2000, p. 3853-3855, Vol. 38, No. 10
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Evaluation of the Alexon-Trend ProSpecT
Campylobacter Microplate Assay
Rita
Tolcin,1
Margaret M.
LaSalvia,1
Barbara A.
Kirkley,1
Emily A.
Vetter,2
Franklin R.
Cockerill III,2 and
Gary W.
Procop1,*
The Cleveland Clinic Foundation, Cleveland,
Ohio,1 and The Mayo Clinic Foundation,
Rochester, Minnesota2
Received 15 February 2000/Returned for modification 31 May
2000/Accepted 17 July 2000
 |
ABSTRACT |
We evaluated stool specimens known to contain or be free of
Campylobacter by traditional culture, using the ProSpecT
Campylobacter microplate assay (Alexon-Trend, Ramsey,
Minn.). This rapid enzyme immunoassay for the detection of
Campylobacter-specific antigens demonstrated 96%
sensitivity and 99% specificity and is an acceptable alternative
method of Campylobacter detection.
| |
TEXT |
Food- and water-borne
bacteria cause gastroenteritis that affects millions of people each
year in the United States, which costs billions of U.S. dollars and
results in thousands of deaths (2, 15, 24).
Campylobacter jejuni is the most common cause of bacterial
gastroenteritis in the United States, surpassing disease caused by
Salmonella and Shigella spp. combined (1, 8-13, 20, 21, 25, 33, 36, 42). The appropriate identification of
the etiologic agent of infectious gastroenteritis is important, since
there are differences in treatment; the possibilities of refractory
disease and postinfectious sequelae also make identification of the
etiologic agent important (3, 20, 32, 35, 38, 40, 43).
Campylobacter species are microaerophilic gram-negative,
curved bacilli that may be detected in stool by direct microscopy, but
more commonly are cultured using selective medium or stool filtration
(5-7, 14, 17, 18, 22, 24, 28, 30, 33, 37, 39, 41). More
recently, nucleic acid amplification methods and enzyme-linked
immunoassays (EIA) have been used to detect these bacteria
(16, 19, 26, 27, 31, 37, 44, 45; A. B. John and
Y. A. Lue, Program Abstr. 98th Gen. Meet. Am. Soc. Microbiol.
1998, abstr. C-263).
We evaluated the ability of the ProSpecT Campylobacter
microplate assay (Alexon-Trend, Ramsey, Minn.) to detect
Campylobacter spp. in clinical stool specimens that were
known to contain or be free of Campylobacter spp. by
traditional culture. Clinical stool specimens were collected and frozen
from three institutions; 50 Campylobacter culture-positive
and 114 Campylobacter culture-negative stools were collected simultaneously.
Campylobacter species were detected and identified by
standard methods (34). Fifteen of the 114 Campylobacter culture-negative stool specimens contained the
following other bacterial enteric pathogens: six Salmonella
spp., three Shigella spp., three Yersinia enterocolitica, and three Escherichia coli O157:H7
strains. All of the stool specimens in this study represented samples
from individual patients; no duplicate specimens were tested.
Seventy-seven of the stool specimens were received in transport medium.
The remaining 87 stool samples were received fresh and frozen
immediately after culture.
The 164 stool specimens were evaluated for the presence of
Campylobacter using the the ProSpecT
Campylobacter microplate assay (Alexon-Trend) according to
the manufacturer's instructions. Stool specimens received
in transport medium were not diluted. For the fresh-frozen stool
specimens, 0.5 ml of stool was mixed with the diluent provided to
obtain the four drops necessary for testing. A positive and negative
control well were also prepared using the positive and negative control
reagents, respectively. The reactions were read both visually and
spectrophotometrically in a single-wavelength spectrophotometer at 450 nm. The validity of each test run was based on appropriate reactions in
the positive and negative control wells. These interpretations were
performed in accordance with the manufacturer's guidelines. Each stool
specimen was tested in duplicate by different medical technologists,
who were blinded to the culture results. Each medical technologist recorded a visual interpretation prior to recording the
spectrophotometric interpretation. The agreement between the two
independent visual interpretations and the two independent
spectrophotometric interpretations was determined. The
agreement was also determined between the manual and
spectrophotometric interpretations. All indeterminate results were
repeated. Repetitively indeterminate specimens were recorded as such
and considered negatives in calculations, since the EIA was unable to
generate a positive result. Upon completion of the study, specimens
with discordant culture and EIA results were retested by EIA, and a
review of the patient's medical record was performed.
The ProSpecT Campylobacter microplate assay correctly
characterized 48 of 50 Campylobacter culture-positive stool
specimens. Two culture-positive stools (one received in transport
medium and one fresh-frozen) were characterized as negative by EIA.
Repeat EIA testing of these two specimens demonstrated one negative
result and one positive result. These specimens were both considered false-negatives, since repeat testing would not have been routinely performed. One hundred and twelve of the 114 Campylobacter
culture-negative stool specimens were characterized by the ProSpecT
Campylobacter microplate assay as negative on initial
testing. Of the remaining two Campylobacter culture-negative
stool specimens, one was repeatedly positive and one was initially
indeterminate but negative upon repeat testing; these were
characterized as false-positive and true-negative, respectively. In
this analysis, the ProSpecT Campylobacter microplate assay
demonstrated 96% sensitivity and 99% specificity. Review of the
medical record, however, revealed that the one "false-positive" EIA
was from a patient diagnosed with infectious enteritis that may have
been campylobacteriosis. It is possible that this could represent a
true-positive EIA and a false-negative stool culture, since viable
organisms are not necessary for detection with the EIA. In this case,
the sensitivity and specificity of the ProSpecT Campylobacter microplate assay would be increased to 96.1 and 100%, respectively. There was excellent interobserver agreement in
both the visual and spectrophotometric test interpretations. Similarly,
there was excellent agreement between the visual and spectrophotometric measurements.
The ProSpecT Campylobacter microplate assay is an EIA that
recognizes a Campylobacter surface antigen which is shared
by C. jejuni and Campylobacter coli (10,
42). This EIA demonstrated at least 96% sensitivity and 99%
specificity using Campylobacter culture-positive and
culture-negative specimens as the reference standard. It was rapid and
easy to use, with excellent agreement between duplicate manual and
duplicate spectrophotometric interpretations. There was no apparent
difference between stool samples received in transport media and those
that were received fresh. Excellent agreement was also found between
the manual and spectrophotometric interpretations. Because one stool
specimen was negative on initial testing but positive on repeat
testing, we recommend that stool specimens be thoroughly mixed prior to
testing. We also suggest that specimens that generate an indeterminate
result be retested. Repetitively indeterminate results were never
encountered in this assessment. The ProSpecT Campylobacter
microplate assay appears to be a reliable method for the detection of
C. jejuni and C. coli.
| |
ACKNOWLEDGMENTS |
This work would not have been possible without the technical
assistance of Amanda Fares, Margaret LaSalvia, Suzanne Schroeder, Parul
Shah, Kathiann Smith, and Rita Tolcin. Their dedication and hard work
on this project are greatly appreciated.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Clinical
Microbiology/L40, 9500 Euclid Avenue, Cleveland, OH 44195. Phone: (216)
444-5879. Fax: (216) 445-6984. E-mail: procopg{at}ccf.org.
| |
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Journal of Clinical Microbiology, October 2000, p. 3853-3855, Vol. 38, No. 10
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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