An improved fluorescent monoclonal antibody staining kit, Cholera DFA, for direct detection and enumeration of Vibrio cholerae O1 has been developed, employing a highly specific anti-A antigen monoclonal antibody, COLTA, labeled with fluorescein isothiocyanate (FITC). An optimized quantity of anti-photobleaching agent is used in a glycerol mounting medium to retard the rapid fading of immunofluorescent stained cells during fluorescent microscopy, thus enabling prolonged inspection of individual fields, as well as improved photographic recording of results without loss of fluorescence intensity. When tested for specificity, all 30 strains of V. cholerae O1 reacted with Cholera DFA, whereas 100 heterologous species examined did not, yielding 100% specificity for all strains examined in this study. A field trial was conducted in Bangladesh, employing Cholera DFA and the results were compared with those obtained by conventional culture methods. Of 44 diarrheal stool specimens tested, Cholera DFA was positive for V. cholerae O1 in all culture-positive stool specimens and negative for all culture-negative stool specimens. The procedure is sensitive and highly specific, as well as simple, i.e., less complex than the indirect fluorescent assay, requiring only one reagent and less than 30 min to complete the staining process, while retarding rapid fading that often occurs with fluorescent microscopy.

We report on the development and testing of a novel, rapid, colorimetric immunodiagnostic kit, Cholera SMART, for direct detection of the presence of Vibrio cholerae 01 in clinical specimens. Unlike conventional culture methods requiring several days to complete, the Cholera SMART kit can be used directly in the field by untrained or minimally skilled personnel to detect V. cholerae 01 in less than 15 min, without cumbersome laboratory equipment. A total of 120 clinical and environmental bacterial strains, induding both 01 and non-Ol serotypes of V. cholerae isolated from samples collected from a variety of geographical regions, were tested, and positive reactions were observed only with V. cholerae 01. Also, results of a field trial in Bangladesh, employing Cholera SMART, showed 100%o specificity and 96% sensitivity compared with conventional culture methods. Another field trial, in Mexico, showed that Cholera SMART was 100%o in agreement with a recently described coagglutination test when 108 stool specimens were tested.

A monoclonal antibody-based test, Bengal SMART, was developed for rapid detection of Vibrio cholerae O139 synonym Bengal directly from stool specimens. The test, which takes about 15 min to complete, was used to screen 189 diarrheal stool specimens. The results were compared with those of a monoclonal antibody-based coagglutination test (COAT) and the conventional culture methods used as the ''gold standard'' for detection of V. cholerae O139. The Bengal SMART test showed a sensitivity of 100% and a specificity of 97% in comparison with the gold standard. It also fared better than COAT, which had a sensitivity of 96% for rapid detection of V. cholerae O139 synonym Bengal. These results show that Bengal SMART is suitable for use in field settings for rapid diagnosis of cholera caused by V. cholerae O139.

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