Elevated-temperature, colorimetric, monoclonal, enzyme-linked immunosorbent assay for rapid screening of Salmonella in foods: collaborative study.

A collaborative study was performed by 30 laboratories in 3 sets of trials to validate a modified colorimetric monoclonal enzyme-linked immunosorbent assay (ELISA) method for Salmonella detection. The modifications to the current methodology included incubation of enrichments and post-enrichments at an elevated temperature, addition of novobiocin to the M-broth post-enrichment, and elimination of the centrifugation and agitation steps. Five artificially contaminated foods (nonfat dry milk, milk chocolate, dried egg, ground black pepper, and soy flour) and 1 naturally contaminated food (raw ground turkey) were analyzed. The artificially contaminated foods were inoculated with individual Salmonella serotypes at a high (10-50 cells/25 g) and low (1-5 cells/25 g) contamination level. Results from the modified ELISA method were compared to the Bacteriological Analytical Manual (BAM)/AOAC culture method. In 2 of the food products, milk chocolate and pepper, a number of laboratories isolated Salmonella from un-inoculated control samples, thus invalidating their data. As a result, there were too few laboratories remaining with valid data, and these foods were repeated. In the completed study, there were 11 false negative results obtained by the modified ELISA method, while there were 28 false negatives produced by the BAM/AOAC procedure. There were 11 ELISA positive assays which could not be confirmed by culture methods. Statistically, there were no differences between the modified, colorimetric, monoclonal ELISA and the reference culture method in all foods except raw turkey, where the ELISA method was more productive. The colorimetric monoclonal enzyme immunoassay (Salmonella-Tek) method for detecting Salmonella in all foods has been adopted first action by AOAC INTERNATIONAL.

Comparison of MICRO-ID Listeria method with conventional biochemical methods for identification of Listeria isolated from food and environmental samples: collaborative study.

Fourteen laboratories participated in a collaborative study to evaluate the ability of the MICRO-ID Listeria identification method to correctly identify Listeria isolated from food and environmental sources. Each collaborator received 60 isolates consisting of 51 Listeria and 9 non-Listeria cultures. All isolates were identified by conventional biochemical analyses in the principal laboratory. Cultures were checked for purity by Gram staining and examined for oxidase and catalase activities. Only Gram positive, oxidase negative, catalase positive cultures were tested with the method. Colonies from trypticase soy agar with 0.6% yeast extract were suspended in 4.6 mL physiological saline to a MacFarland No. 1 turbidity standard and used to inoculate the test strip. In addition, the hemolytic reaction of each isolate was determined by using the Christie-Atkins-Munch-Peterson (CAMP) test and by stabbing sheep blood agar. Identification of Listeria is based on the octal code obtained from the strip and the hemolytic reaction of the isolate. The MICRO-ID Listeria method agreed with conventinal biochemical identification for 98.0% of L. monocytogenes, 77.1% of L. seeligeri, 90.0% of L. ivanovii, 96.4% of L. grayi/L. murrayi, 73.9% of L. welshimeri, and 100% of L. innocua isolates. A large percentage of errors in identification of the L. seeligeri and L. ivanovii cultures was caused by inaccurate reading of the CAMP and hemolysis tests rather than errors in the test strip. The method was adopted first action by AOAC International.

Comparison of colorimetric monoclonal enzyme immunoassay screening methods for detection of Salmonella in foods.

A colorimetric enzyme immunoassay (EIA) method for detection of Salmonella in foods has been compared to the AOAC colorimetric monoclonal EIA screening method (986.35, 15th ed.; 46.B21-46.B29, 14th ed.). The assays use the same monoclonal antibodies and have similar reactivity toward Salmonella. However, the new assay uses antibody-coated microtiter wells instead of coated magnetic beads to capture Salmonella antigens. Compared with the bead assay, the coated-well assay format requires significantly less time to complete, and was consistently able to detect lower levels of Salmonella in mixed culture. Compared to the standard AOAC culture method for food samples, the plate assay was as productive. No false negatives were obtained by the immunoassay; the false negative rate was 1.1% by the culture method. The rate of agreement between the 2 methods was 99.1%. The official final action bead assay method for Salmonella in foods, 986.35, and the same assay for use with low-moisture foods, 987.11, have been modified official first action to use antibody-coated microtiter strip-wells.

Multitest system for biochemical identification of Salmonella, Escherichia coli, and other Enterobacteriaceae isolated from foods: collaborative study.

Nine laboratories evaluated the ability of the MICRO-ID test system to correctly identify members of the Enterobacteriaceae. A total of 78 isolates representing 11 genera of enterics that had been previously isolated from foods were used in the collaborative study. The collaborators streaked each isolate on plate count agar and incubated the plates overnight at 35 degrees C to check purity of the isolates. Then they proceeded with the method in which an isolated colony is transferred to a plate count agar slant and is incubated 18-24 h at 35 degrees C. Growth from the slant is emulsified in 3.5 mL physiological saline to a density comparable to a McFarland No. 2 tube and is then used to inoculate the test (MICRO-ID) strip. The strip is incubated 4 h at 35 degrees C and the reactions are read and recorded. Isolates are identified by using an octal code and the test kit manual. The system correctly identified 98.8% of the Salmonella isolates, 97.7% of the E. coli isolates, and 84.6% of the other 9 enteric genera tested. The system has been approved interim official first action as an alternative to conventional biochemical tests (1) for presumptive generic identification of food-borne Salmonella and for screening and elimination of non-Salmonella isolates; (2) for identification of E. coli from foods; and (3) for presumptive generic identification of other Enterobacteriaceae isolated from foods.

Rapid monoclonal antibody-based enzyme-linked immunosorbent assay for detection of Listeria in food products.

A rapid diagnostic test for the detection of Listeria in food products has been created. This test, known as Listeria-Tek, uses 2 monoclonal antibodies specific for Listeria in an enzyme-linked immunosorbent assay (ELISA) format. The test requires only 40 h of broth enrichment with no culturing on solid media. It is extremely simple to perform and easy to interpret, and is at least as sensitive and accurate as the best of the culture methods. The test can be used with dairy products, meat products, and environmental samples. The ELISA test is safely performed on the open bench of the laboratory because no live cultures, no radioactivity, no phage, etc., are necessary. There is no need for special licenses or reserved laboratory space, and no waste disposal problems are encountered. If necessary, one technician could easily perform hundreds of assays per day. A printed data sheet is available for permanent records.

Evaluation of abbreviated enzyme immunoassay method for detection of Salmonella in low-moisture foods.

A modified enzyme immunoassay method (EIA) utilizing an 18 h pre-enrichment, a 6-8 h selective enrichment, and a 14 h M-broth post-enrichment is compared to the standard culture method (AOAC/BAM) on selected low-moisture foods. Tested samples included 238 inoculated, 30 naturally contaminated, and 30 uninoculated foods. By EIA, 235 samples were positive (optical densities greater than 0.2 at 405 nm), 233 of which were confirmed culturally. By the culture methods, 221 samples were positive. The EIA method was more productive in detecting salmonellae in inoculated samples of dry cheese powder, chocolate, and nonfat dry milk, whereas the culture method gave better recovery from naturally contaminated meat and bone meal. The modified EIA could be completed in 40 h and required no centrifugation.

Enzyme immunoassay for detection of Salmonella in low-moisture foods: collaborative study.

A collaborative study was performed in 15 laboratories to evaluate a modification of the enzyme immunoassay (EIA) method for detection of Salmonella in foods (46.B21-46.B29). The modified EIA requires 18-24 h pre-enrichment, 6-8 h selective enrichment, and 14-18 h M-broth post-enrichment prior to performing the assay, which requires 1-2 h. Total assay time is 40-52 h. The modified method was compared with the standard culture method for detection of Salmonella in 5 low-moisture foods: nonfat dry milk, milk chocolate, meat and bone meal, dry whole egg, and ground pepper. The modified method has been adopted official first action for use with low-moisture foods.

Enzyme immunoassay for detection of Salmonella in foods: collaborative study.

A collaborative study was performed in 25 laboratories to validate an enzyme immunoassay (EIA) procedure utilizing 2 specific monoclonal antibodies for rapid detection of Salmonella in foods. The EIA was compared with the standard culture procedure for detection of Salmonella in 6 food types: ground black pepper, soy isolate, dried whole eggs, milk chocolate, nonfat dry milk, and raw deboned turkey. Uninoculated and inoculated samples were included in each food group analyzed, with the exception of poultry which was naturally contaminated. There was no significant difference in the productivity of the EIA and culture procedures at the 5% level for any of the 6 foods. The enzyme immunoassay screening method has been adopted official first action.

Evaluation of a fluorogenic assay for detection of Escherichia coli in foods.

A fluorogenic assay procedure with 4-methylumbelliferyl-beta-D-glucuronide incorporated into lauryl sulfate broth was evaluated to detect and confirm the presence of Escherichia coli in foods. Fluorescence is indicative of the presence of E. coli; extensive biochemical confirmation is unnecessary with this assay. The 4-methylumbelliferyl-beta-D-glucuronide assay was tested concurrently with our present methodology for detection of E. coli on 270 samples of raw ingredients and powdered food products. Total agreement between the two methods was 94.8%; there was a false-positive rate of 4.8% and no false-negatives. We found the 4-methylumbelliferyl-beta-D-glucuronide assay to be rapid, accurate, simple to perform, and inexpensive.

Enzyme immunoassay in which a myeloma protein is used for detection of salmonellae.

An enzyme immunoassay (EIA) in which an immunoglobulin A monoclonal antibody from a myeloma (MOPC 467) is used was developed to detect the presence of Salmonella organisms. This myeloma protein binds to a flagellar determinant of the organisms but is not directed toward the H antigens. Of 100 strains tested, 94% were detectable with this antibody. The EIA, used with MOPC 467, is quick, sensitive, and specific, showing virtually no cross-reactivity to other enteric organisms. Initial screening of antibody reactivity was performed by Ouchterlony gel diffusion with the supernatants of heat-treated Salmonella cultures. After this, an EIA was performed on the heat extracts with the myeloma protein, which had been directly coupled to alkaline phosphatase. A positive reaction was indicated by the production of a yellow color after the addition of a substrate (p-nitrophenylphosphate), and this was quantitated by determining the absorbance at 405 nm. The EIA proved to be slightly more sensitive than the Ouchterlony analysis. The sensitivity of the EIA is such that as few as 10(6) Salmonella organisms per ml were detected. This concentration was easily obtained after a 24-h preenrichment incubation of the sample. Mixtures of Salmonella strains with a 10 x concentration of Escherichia coli did not prevent detection of the Salmonella strains. This EIA can be successfully used to detect contamination of foods, as it was used to detect the intentional contamination of infant formula in these studies. Indications are that the EIA is sensitive enough to detect Salmonella strains in M broth subcultures taken directly from a preenrichment culture. Testing of samples could thus be completed 36 h after culture initiation, rather than after 96 h, the time currently needed.

Experience of a physician-nurse practitioner team in care of patients in skilled nursing facilities.

The use of a physician-nurse practitioner team is advocated as an approach to delivering better health care to patients in skilled nursing facilities. The application of this approach in a young community with an inadequate supply of primary physicians and 596 extended care beds is discussed. Patients derive benefit from more comprehensive health care delivered with greater attention to individual needs. Staffs of skilled nursing facilities enjoy improved communication with the medical team and better compliance with legal requirements. The team physician is able to use his time more effectively and provide medical supervision for a greater number of patients by sharing responsibilities with a nurse practitioner. Paper compliance, adherence to agency regulations, quality assurance, and payment are some of the problems encountered.