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Evaluation of a microplate latex agglutination method (Verotox-F assay) for detecting and characterizing verotoxins (Shiga toxins) in Escherichia coli.

ABSTRACT: The performance of a commercial microplate latex agglutination assay, the Verotox-F assay, was compared with that of the Vero cell assay for the detection and characterization of Escherichia coli verocytotoxins (VTs). Culture filtrates of 68 VT-positive E. coli strains (65 human isolates [33 of serotype O157:H7/H-, 32 of non-O157 serotypes] and 3 reference strains) and 104 VT-negative strains (100 human isolates and 4 reference strains) were investigated. The toxin phenotypes and genotypes of the 68 VT-positive isolates were VT1 only (18 strains), VT2 and/or VT2c (33 strains), and VT1 plus VT2 (17 strains). The Verotox-F assay involved incubation of serial dilutions of culture filtrates with equal volumes of latex particles sensitized with anti-VT1 antibody or anti-VT2 antibody in 96-well microtiter plates with appropriate controls and examination for latex agglutination after 20 to 24 h. Compared to the results of the Vero cell assay, the Verotox-F assay was 100% sensitive and 100% specific for the detection of VTs in culture filtrates and correctly identified the toxin types of all 68 VT producers. By checkerboard titration with purified toxins, the sensitivity of the Verotox-F assay was found to be 14 pg (0.7 ng/ml) for VT1, 12 pg (0.6 ng/ml) for VT2, and 350 pg (17.5 ng/ml) for VT2c; this sensitivity is comparable to that of the bioassay. The anti-VT2 latex reagent detected both VT2 and VT2c and did not cross-react with VT1. The anti-VT1 reagent showed a low-level cross-reaction with VT2c only at levels (>/=4.5 microg/ml) that were about 1,000-fold higher than those found in culture filtrates. We conclude that the Verotox-F assay is highly sensitive and specific for the detection and characterization of VTs in culture filtrates of human E. coli isolates. The test is rapid, reliable, and easy to perform; its results are easy to interpret; and it should allow testing for VT to become more widely performed.

SUBMITTER: Karmali MA

PROVIDER: S-EPMC84318 | biostudies-literature | 1999 Feb

REPOSITORIES: biostudies-literature

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Evaluation of a microplate latex agglutination method (Verotox-F assay) for detecting and characterizing verotoxins (Shiga toxins) in Escherichia coli.

Karmali M A MA Petric M M Bielaszewska M M

Journal of clinical microbiology 19990201 2

The performance of a commercial microplate latex agglutination assay, the Verotox-F assay, was compared with that of the Vero cell assay for the detection and characterization of Escherichia coli verocytotoxins (VTs). Culture filtrates of 68 VT-positive E. coli strains (65 human isolates [33 of serotype O157:H7/H-, 32 of non-O157 serotypes] and 3 reference strains) and 104 VT-negative strains (100 human isolates and 4 reference strains) were investigated. The toxin phenotypes and genotypes of th ...[more]

PMID: 9889226

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Project description:Melioidosis is a life-threatening disease in humans caused by the Gram-negative bacterium Burkholderia pseudomallei. As severe septicemic melioidosis can lead to death within 24 to 48 h, a rapid diagnosis of melioidosis is critical for ensuring that an optimal antibiotic course is prescribed to patients. Here, we report the development and evaluation of a bacteriophage tail fiber-based latex agglutination assay for rapid detection of B. pseudomallei infection. Burkholderia phage E094 was isolated from rice paddy fields in northeast Thailand, and the whole genome was sequenced to identify its tail fiber (94TF). The 94TF complex was structurally characterized, which involved identification of a tail assembly protein that forms an essential component of the mature fiber. Recombinant 94TF was conjugated to latex beads and developed into an agglutination-based assay (94TF-LAA). 94TF-LAA was initially tested against a large library of Burkholderia and other bacterial strains before a field evaluation was performed during routine clinical testing. The sensitivity and specificity of the 94TF-LAA were assessed alongside standard biochemical analyses on 300 patient specimens collected from an area of melioidosis endemicity over 11 months. The 94TF-LAA took less than 5 min to produce positive agglutination, demonstrating 98% (95% confidence interval [CI] of 94.2% to 99.59%) sensitivity and 83% (95% CI of 75.64% to 88.35%) specificity compared to biochemical-based detection. Overall, we show how a Burkholderia-specific phage tail fiber can be exploited for rapid detection of B. pseudomallei. The 94TF-LAA has the potential for further development as a supplementary diagnostic to assist in clinical identification of this life-threatening pathogen. IMPORTANCE Rapid diagnosis of melioidosis is essential for ensuring that optimal antibiotic courses are prescribed to patients and thus warrants the development of cost-effective and easy-to-use tests for implementation in underresourced areas such as northeastern Thailand and other tropical regions. Phage tail fibers are an interesting alternative to antibodies for use in various diagnostic assays for different pathogenic bacteria. As exposed appendages of phages, tail fibers are physically robust and easy to manufacture, with many tail fibers (such as 94TF investigated here) capable of targeting a given bacterial species with remarkable specificity. Here, we demonstrate the effectiveness of a latex agglutination assay using a Burkholderia-specific tail fiber 94TF against biochemical-based detection methods that are the standard diagnostic in many areas where melioidosis is endemic.

Dataset Information

Evaluation of a microplate latex agglutination method (Verotox-F assay) for detecting and characterizing verotoxins (Shiga toxins) in Escherichia coli.

Publications

Evaluation of a microplate latex agglutination method (Verotox-F assay) for detecting and characterizing verotoxins (Shiga toxins) in Escherichia coli.

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