Search

A rapid (2.5 h) direct enzyme immunoassay (EIA) for Clostridium difficile toxin A was developed for clinical use. Specimen centrifugation and filtration were not required. The EIA detected toxin A levels in patient stool as low as 20 pg (2 ng/ml of stool). The test was 5,000 times more sensitive for toxin A than it was for toxin B and did not react with a panel of other bacterial species with the exception of one highly toxigenic strain of Clostridium sordellii. The EIA was compared with the cytotoxin assay, culture of toxigenic C. difficile (toxigenic culture), and latex agglutination by using 313 fresh stool specimens submitted from patients with suspected C. difficile-associated disease. Results read visually and with a plate reader were similar. Sixty-two specimens were positive by one or more tests, but only 22 (35%) were positive by all four laboratory methods. The EIA was 84.1% sensitive and 98.9% specific when it was compared with the cytotoxin assay. The use of toxigenic culture to referee discrepant results (EIA versus cytotoxin assay) showed the EIA sensitivity and specificity to be 95.1 and 99.3%, respectively, with respect to other laboratory methods. Patient charts were reviewed for antibiotic-associated diarrhea on 108 specimens, including all those that were positive by at least one test method. Of 34 patients determined to have C. difficile-associated disease, 29 (85.3%) were positive by EIA, 32 (94.1%) were positive by the cytotoxin assay, 27 (79.4%) were positive by toxigenic culture, and 20 (58.8%) were positive by latex agglutination. Seven patients with antibiotic-associated diarrhea had a positive latex result, but results were negative by EIA, the cytotoxin assay, and toxigenic culture. The EIA demonstrated high specificity and good sensitivity for C. difficile-associated disease cases. The test can be used alone or in combination with the cytotoxin assay or toxigenic culture to provide rapid and sensitive results.

Lymphomatoid papulosis (LyP) and pityriasis lichenoides et varioliformis acuta (PLEVA) are benign self-healing cutaneous eruptions that may be clinically and histologically similar. However LyP has a 5% to 20% risk of associated lymphoid malignancy, whereas PLEVA does not. To determine whether the immunophenotype of lymphoid cells is useful in the distinction of these two disorders, the pattern of expression of lymphoid cell lineage and activation antigens in nine cases of LyP and seven cases of PLEVA were compared. In all cases of LyP most larger cells expressed the activation antigen Ki-1 (CD30) and lacked expression of the T-cell antigen CD7 and at least one other T-cell antigen (CD2, CD3, CD5). In contrast, CD30-antigen expression was rare or absent in PLEVA, CD3- and CD7-antigen expression was found in all cases, and diminished expression of T-cell antigens (CD2 and CD5) was seen in only one case. Diffuse expression of HLA-DR antigen by epidermal keratinocytes was found in a greater proportion of PLEVA cases (6 of 7) than LyP cases (3 of 6). In addition, CD8+ cells predominated at the dermal/epidermal junction in 3 of 6 cases of PLEVA but in only 1 of 7 cases of LyP. We conclude that LyP and PLEVA can be distinguished immunohistochemically in most, if not all, cases. Furthermore these results suggest that LyP and PLEVA are separate disorders, thus accounting for their variable prognoses.

The Abbott MS-2 system (Abbott Laboratories, Diagnostic Division, Irving, Tex.), equipped with updated bacterial identification software (version 03.02), was used to perform both direct identification and susceptibility tests on gram-negative bacilli from positive BACTEC blood culture bottles. Ninety-eight of 101 Enterobacteriaceae strains, one strain of Acinetobacter calcoaceticus, and two strains of Pseudomonas aeruginosa were correctly identified by following a direct inoculation procedure. One Enterobacter sakazakii isolate was misidentified as Enterobacter cloacae. Ninety-seven percent of 936 direct susceptibility results were identical to the results obtained by using the standard MS-2 susceptibility procedure. Only five tests yielded a sensitive-direct and a resistant-standard MS-2 susceptibility result.

The Rapid Identification Method (RIM) Escherichia coli Kit (Austin Biologicals Laboratories Inc., Austin, TX) was evaluated as a means to rapidly differentiate at reduced cost clinical isolates of Escherichia coli from other members of the Enterobacteriaceae. A group of 408 gram-negative rods that were isolated on primary isolation plates were tested with the use of both the kit and complete bacterial identification. The RIM E. coli Kit identified 92.5% (272 of 294) of the E. coli isolates. Only 1 of 114 non-E. coli isolates was misidentified as E. coli (positive predictive value equal to 99.6). Although the RIM E. coli Kit was sufficiently rapid (less than one hour), any supply cost savings would depend upon the proportion of true E. coli in the group of organisms tested. Savings derived from the lower cost of using the RIM E. coli Kit would be reduced by the extra cost of screening non-E. coli isolates, which would then have to be completely identified.

Attempts were made to use the osmotic shock procedure established for Gram-negative micro-organisms on a Gram-positive micro-organisms, Bacillus megaterium ATCC 19213. The procedure did not appear to shock the cells osmotically. The procedure, in fact, appeared to enhance binding of both glucose and fructose by the organism. The binding of glucose was greater by cells, whether grown on fructose or glucose, than it was for fructose. A procedure was developed for removing and isolating a protein capable of binding glucose. This involved solubilizing the cytoplasmic membrane of the organism with the nonionic detergent Triton X-100, and then isolating it by gel filtration on a Bio-Gel A 1.5 column with 0.05 M pyrophosphate containing 1% (v/v) Triton X-100 as the eluting buffer. This is the first isolation of such a protein in a Gram-positive organism. Isolated protein was demonstrated to have a specificity only for glucose. The protein did not bind to either the monosaccharides fructose and galactose, or the disaccharide sucrose. This was in comparison to whole cells which demonstrated ability to take up fructose. The protein was estimated to have a molecular weight between 24,000 and 30,000 daltons by gel filtration techniques.