Toxinas Bacterianas

JOURNAL OF CLINICAL MICROBIOLOGY, June 2003, p. 2650–2653
0095-1137/03/$08.00 0 DOI: 10.1128/JCM.41.6.2650–2653.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Vol. 41, No. 6

Evaluation of the Duopath Verotoxin Test for Detection of
Shiga Toxins in Cultures of Human Stools
C. H. Park,1* H. J. Kim,1 D. L. Hixon,1 and A. Bubert2
Inova Fairfax Hospital,Falls Church, Virginia 22042,1 and Merck KGaA, Darmstadt, Germany2
Received 10 December 2002/Returned for modification 1 February 2003/Accepted 25 February 2003

The Duopath Verotoxin test (Merck KgaA, Darmstadt, Germany) is a newly developed immunochromatographic test for the confirmation of Shiga toxin (Stx)-producing Escherichia coli (STEC) strains from food
products. This test detects both Stx 1(Stx1)-positive and Stx2-positive samples individually with the same
device. By modification of the original protocol, the present study evaluated its performance and feasibility for
clinical application with human stool samples, consisting of 41 frozen samples known to contain STEC isolates
(O157:H7 and non-O157 serotypes) and 250 fresh specimens. The test specimens were polymyxin B extractsof
colony sweeps taken from overnight sorbitol-MacConkey agar cultures of stools containing STEC isolates and
other bacteria. All 41 frozen STEC-positive stool samples were positive by the Duopath Verotoxin test, as were
2 fresh stool samples with culture-confirmed STEC O157 infection. Thus, 100% sensitivity and no false-positive
results were obtained when the Premier EHEC assay (MeridianBioscience, Cincinnati, Ohio) was used as the
“gold standard.” The Duopath Verotoxin test is simple to perform and easy to interpret, providing a turnaround time of 24 h. Despite its original intended use, the Duopath Verotoxin test has a great potential for
clinical application.
mier EHEC assay (Meridian Bioscience, Cincinnati, Ohio) and
ProSpecT Shiga Toxin E. coli assay (Alexon-Trend, Ramsey,Minn.), as well as other methods, including the cell culture
cytotoxicity assay and the reverse passive latex agglutination
test (VTEC-RPLA; Denka-Seiken, Tokyo, Japan). The purpose of the investigation described here was to evaluate the
performance of the Duopath Verotoxin (DV) immunochromatographic test (Fig. 1) and its feasibility for clinical applications with human stool specimens. The DVtest is newly developed and was originally intended for use for the
confirmation of STEC strains from food products. The DV test
uses colloidal gold-labeled monoclonal antibodies to detect
Stx1 and Stx2 independently with the same test device. Any
inoculum with Stx1 and/or Stx2 complexes with antibodies in
the reaction zones as the test sample migrates over a membrane, forming a sandwich complexwith immobilized Stx1- and
Stx2-specific antibodies in the detection zones. A positive result appears as a red band within 10 min (Fig. 1). The intensity
of the color is compared to the colors on a chart provided by
the manufacturer showing a scale from 0 to 10 . According to
the scale, 0 (no color) is interpreted as a negative result, while
1 (extremely faint red band) represents a veryweakly positive
result. However, any visible red band in the reaction zone is
considered a positive result, regardless of its intensity. In this
study, clinical stool samples were retrospectively and prospectively tested by the Premier EHEC assay as the “gold standard” for determination of the performance of the DV test.
The methods described here were modified from the original
protocol toprovide a shorter turnaround time with fewer test
steps.
In the retrospective study, 41 known Stx-positive stool specimens (31 STEC O157-positive specimens and 10 non-O157
STEC-positive specimens that had been frozen at 70°C for
up to 10 years) from different patients were thawed and inoculated onto sorbitol-MacConkey agar (SMAC) plates (Becton

Verotoxins or Shiga toxin (Stx) 1 (Stx1) and Stx2...