Efficacy, safety and feasibility of intravenous iloprost in the domiciliary treatment of patients with ischemic disease of the lower limbs.

OBJECTIVE: Intravenous iloprost is an important option in the treatment of ischemic disease of the lower limbs; however, the administration of therapy is frequently compromised because of the need for long cycles of infusion in a hospital setting. The aim of the study is to evaluate the efficacy, safety, feasibility, and the economic impact of infusion therapy in the outpatient setting. PATIENTS AND METHODS: Twenty-four consecutive patients were treated with iloprost at their homes where they were administered a slow rate of infusion for 24 hours a day, during 9.9 ± 2.3 days, with a portable syringe pump (Infonde®). RESULTS: The clinical condition of patients evaluated with the modified SVS/ISCVS scale significantly improved after treatment (+1.29 ± 1.04 points vs. baseline, p<0.001). The drug was well tolerated; neither significant adverse events associated with medication nor problems related to venous access were recorded at home. Ninety-six percent of patients successfully completed the entire treatment cycle, and the evaluation questionnaire showed a high acceptance of the therapy. From the perspective of the hospital authority, lower direct medical costs were estimated for the domiciliary infusion process compared with the inpatient infusion setting. CONCLUSIONS: Treatment with iloprost in the outpatient setting is effective, safe, feasible, and more acceptable to patients than infusion at the hospital. In addition, it has a favorable economic and organizational impact on the medical ward.

Survival of Helicobacter pylori in ready-to-eat foods at 4 degrees C.

The survival of Helicobacter pylori (NCTC 11638) in various semiprocessed and fresh, ready-to-eat foods, and one raw chicken was studied at 4 degrees C and under aerobic conditions by experimentally inoculating these with 10(4) CFU. Cells were concentrated by two centrifugation cycles followed by plating onto selective blood agar medium made from Wilkins-Chalgren agar supplemented with 5% whole horse blood, and 30 mg/l colistin methanesulfonate, 100 mg/l cycloheximide, 30 mg/l nalidixic acid, 30 mg/l trimethoprim, and 10 mg/l vancomycin. H. pylori was recovered from spiked pasteurized milk and tofu samples up to 5 days and from spiked leaf lettuce and raw chicken up to 2 days. H. pylori could not be recovered from yogurt after any length of storage time. H. pylori is unlikely to grow in foods; however, it may survive in low acid-high moisture environments under refrigeration and pose a possible risk for transmission of infection via foods.

Incidence of Salmonella in fish and seafood.

Field laboratories of the U.S. Food and Drug Administration collected and tested 11,312 import and 768 domestic seafood samples over a 9-year period (1990 to 1998) for the presence of Salmonella. The overall incidence of Salmonella was 7.2% for import and 1.3% for domestic seafood. Nearly 10% of import and 2.8% of domestic raw seafood were positive for Salmonella. The overall incidence of Salmonella in ready-to-eat seafood and shellfish eaten raw was 0.47% for domestic--one shucked oyster and one shark cartilage powder. The incidence in the 2,734 ready-to-eat import seafood was 2.6%--cooked shrimp, shellfish or fish paste, smoked fish, salted/dried fish, and caviar. The incidence in import shellfish consumed raw was 1% in oyster, 3.4% in clams, and 0% in mussels. The incidence in raw, import fish was 12.2%. Distribution of Salmonella in seafood on a regional basis indicated the incidence to be highest in central Pacific and Africa and lowest in Europe/Russia and North America (12% versus 1.6%). Data on a country basis indicated Vietnam to have the highest (30%) and Republic of Korea the lowest (0.7%). While the most frequent serotypes in import seafood were Salmonella Weltevreden (1st), Salmonella Senftenberg (2nd), Salmonella Lexington, and Salmonella Paratyphi-B (3rd, equal numbers for each serotype), the top 20 list included Salmonella enteritidis (5th), Salmonella Newport (6th), Salmonella Thompson (7th), Salmonella typhimurium (12th), and Salmonella anatum (13th), commonly involved in foodborne illness in the United States. Because the incidence in the present study is based on only a small fraction of the seafood imported into the United States, efforts should be directed toward implementation of hazard analysis and critical control points to reduce the incidence of Salmonella in seafood without relying on testing for Salmonella.

Salmonella enteritidis contamination of ice cream associated with a 1994 multistate outbreak.

Nine ice cream sample containers, representing three production lots involved in a 1994 outbreak of salmonellosis, were obtained from the manufacturer's distribution warehouse and from consumers. Single 100-g samples from each container were tested initially, with analyses beginning seven weeks after the ice cream was produced. Quantitative salmonella analysis of these samples was performed 14 weeks after the production date using a three-replicate, three-dilution most probable number (MPN) procedure using 100-, 10-, and 1-g samples. The MPN/g estimates ranged from 0.004 to 0.46 Salmonella enteritidis per g with six of nine samples showing an MPN value of 0.093 S. enteritidis per g. The 95% confidence interval for S. enteritidis among these samples was < 0.001 to 2.4 cells/g. The 95% upper limit of S. enteritidis per g was 0.38 for five of six consumer samples. Based on this, the number of S. enteritidis cells per serving (65 g) was 25. Based on the consumption of a single sundae cone (73 g, prepackaged), which caused severe illness in an eight-year-old boy and moderate to mild illness in the adult parents, the infective dose would appear to be no more than 28 cells.

Stability of spores of Bacillus cereus stored on silicagel.

Spores of four different strains of Bacillus cereus were stored on silicagel at 22 degrees C and in physiological saline solution at -20 degrees C for a period of 260 days. At different intervals the spores were tested for survival, heat sensitivity and capacity to germinate. There was no clear change in any of the parameters tested, so storage on silicagel can be a good alternative for storage as a frozen suspension. Spores stored in this way can easily be exchanged for reference material and used for microbiological challenge testing.

Characteristics of Bacillus cereus related to safe food production.

Thirty Bacillus cereus strains, isolated from different sources, were characterized in relation to safe food production. The minimal growth temperatures of the strains varied from < or = 5 degrees C to 11 degrees C. Generation times at 7 degrees C of strains capable of growing at temperatures < or = 5 degrees C were approximately 8.2 h. The D90 degrees C-values of spores of strains with a minimal growth temperature of 11 degrees C determined in phosphate buffer at pH 7.0 ranged from 4.8 to > 200 min. Strains with the capacity to grow at temperatures < or = 9 degrees C, had a D90 degrees C value ranging from 4.6 to 14 min. Addition of either nisin (250 micrograms/ml) or diacetyl (1500 micrograms/ml) to the heating menstruem at the single concentrations investigated seemed not influence the thermal destruction of spores. Germination of spores of almost all strains occurred in all three media tested (Brain Heart Infusion, rice extract and milk) even at temperatures below the minimal growth temperature. All B. cereus strains tested yielded positive results with a commercial test kit for diarrhoeal enterotoxin. The results indicate that strains with the capacity to grow at temperatures < or = 7 degrees C are not essentially different from those with minimal growth temperatures of > 10 degrees C.

A 6 h microslide immunodiffusion assay for confirmed detection of staphylococcal enterotoxins.

Use of appropriately balanced immunological reagents and incubation of microslides at 45 degrees C resulted in confirmed assay results in 6 h for staphylococcal enterotoxins. The sensitivity of the 45 degrees C-6 h assay was comparable to the 37 degrees C-24 h assay: 0.1 micrograms of staphylococcal enterotoxin A/ml.

Rapid Detection of Psychrotrophic Bacteria In Manufacturing Grade Raw Milks.

Methods to rapidly assess the bacteriological quality of raw milk were investigated. Whereas direct microscopic count, modified psychrotrophic plate count, and direct epifluorescent filter technique (DEFT) did not correlate well with initial psychrotrophic bacterial count of raw milk, improvements were obtained after preincubation of the milk samples. The best preincubation conditions were identified as 30°C for 6 h, 21°C for 10 h, 13°C for 15 h, 13°C for 20 h, or 7°C for 37 h. The "square root" equation was applied to the data, and a model was produced for predicting growth of the native microflora of raw milk. Using this equation, a DEFT count after preincubation of the milk at 21°C for 10 h could accurately predict the initial psychrotroph count and the count after storage of the milk at 6°C for 48 h.

Inactivation of staphylococcal enterotoxins by heat and reactivation by high pH treatment.

Inactivation of staphylococcal enterotoxins (SE) added to different media upon heat treatment (80 degrees C or 100 degrees C for 10 min) and reactivation of inactivated SE was studied. In gelatin (pH 4.0), lettuce extract, peas and beans extracts and ovalbumin (pH 5.0) the immunological activity of SE was lost almost completely upon heating. The loss of immunological activity of SEA was accompanied by a concomitant loss of biological activity of this toxin (monkey feeding test). A high pH treatment (pH 11) of the different preparations restored both the immunological and biological activity in most samples tested. Heating at 80 degrees C or 100 degrees C for 10 min of SE containing gelatin (pH 7.0), cauliflower extract (pH 4.0 or pH 7.0), milk (pH 4.0), casein (pH 6.0), rice extract (pH 7.0), noodles extract (pH 4.0) and oat-flakes extract (pH 7.0) had a much lower effect on the immunological activity of the SE (activity greater than or equal to 25%).

Specificity and cross-reactivity of staphylococcal enterotoxin A monoclonal antibodies with enterotoxins B, C1, D, and E.

The cross-reactivity of monoclonal antibodies produced against staphylococcal enterotoxin A with purified and crude enterotoxins B, C1, D, and E and the specificity of such reactions were evaluated by the indirect enzyme-linked immunosorbent assay and immunoblotting of Western blots (from sodium dodecyl sulfate-polyacrylamide gel electrophoresis) followed by autoradiography. Purified and crude enterotoxins B were also tested with polyclonal antibodies. Specificity of reactivity was demonstrated by immunoblotting of crude enterotoxin A, crude enterotoxin A treated with trypsin, crude enterotoxin E, and also with crude A, B, C1, and D that were pretreated with Sepharose-4B-linked normal rabbit immunoglobulin G to remove protein A. A band corresponding to each staphylococcal enterotoxin was seen with monoclonal antibodies under all conditions tested and also with crude and purified enterotoxin B with two different (rabbit and goat) polyclonal antisera.

Nature and reactivity of staphylococcal enterotoxin A monoclonal antibodies.

Monoclonal antibodies from four clones (C5, C3, B2II, and B2I) directed against staphylococcal enterotoxin A were tested by the indirect enzyme-linked immunosorbent assay and double-gel immunodiffusion (micro-Ouchterlony) assay for the nature of heavy and light chain types. The reactivities of monoclonal antibodies were also tested by indirect enzyme-linked immunosorbent assay with various levels of purified staphylococcal enterotoxin A and various levels (dilutions) of monoclonal antibodies and saturation analysis-competitive indirect enzyme-linked immunosorbent assay. The heavy-chain isotype of monoclonal antibodies was found to be an unspecified subclass of immunoglobulin G1, and the light chain was the kappa type. Monoclonal antibodies from all of the clones exhibited high reactivity and nearly the same affinity to staphylococcal enterotoxin A in saturation analysis-competitive enzyme-linked immunosorbent assay. Purified immunoglobulin G from B2I yielded very high absorbance (1.2) at 405 nm with 1 ng of staphylococcal enterotoxin A as the coating antigen in the enzyme-linked immunosorbent assay. Monoclonal antibodies from B2I also neutralized the biological activity of staphylococcal enterotoxin A when tested by the kitten bioassay.

Production of monoclonal antibodies to staphylococcal enterotoxin A.

Spleen cells from mice immunized with staphylococcal enterotoxin A were successfully fused with NS-1 mouse myeloma cells. Two of the four clones studied produced monoclonal antibodies to staphylococcal enterotoxin A in growth medium which showed titers of greater than 10(6) to 10(7) when tested by the indirect enzyme-linked immunosorbent assay. These monoclonal antibodies showed reactivity with enterotoxins A and E in the enzyme-linked immunosorbent assay. However, the reactivity was higher with enterotoxin A than with enterotoxin E. Nanogram quantities of crude staphylococcus enterotoxin A from Staphylococcus aureus growth were detected by the monoclonal antibodies in electroimmunoblots via autoradiography.

Production of enterotoxin(s) by Staphylococcus hyicus.

Five strains of Staphylococcus hyicus (3 of subspecies hyicus and 2 of chromogenes) were tested serologically for their ability to produce known and unknown enterotoxin(s) and were also examined using monkey bioassays. None of the 5 strains produced any detectable level of the known enterotoxins A--E. However, all of them produced emetic responses in 2 or more of 6 cynomologus monkeys when culture growth was fed intragastrically. One of the 5 strains (S. hyicus subspecies hyicus, VII 76) produced emetic responses in 3 of 6 monkeys with 50 ml of culture growth. The other 4 strains required 1 l of culture broth (concentrated 20-fold) to produce an emetic response in at least 2 of 6 monkeys. Enterotoxin production is, therefore, not unique to S. aureus species. Since some of these organisms do not produce coagulase and thermonuclease, they would be ignored in food hazard evaluation.

Characterization of staphylococci.

A total of 158 Staphylococcus strains from various sources were characterized by biochemical, physiological, and morphological tests. Numerical taxonomy was applied by using these features. Taxonomic analysis was done with programs run under the MVS-TSO system of the IBM 370 complex and PDP-10 system of the National Institutes of Health. DNA-DNA hybridization with nitrocellulose filters was done to compare selected atypical cultures with American Type Culture Collection reference strains. We found that the use of the nomenclature of Bergey's Manual (8th edition) to identify these strains by species was not adequate. DNA homology values supported the formation of Staphylococcus hyicus subsp. hyicus separate from Staphylococcus aureus, Staphylococcus epidermidis, and Staphylococcus saprophyticus. The three tests that best separated these strains into four species were (i) tube coagulase (6-h or 24-h porcine plasma or 24-h Difco rabbit plasma), (ii) production of acetoin or acid aerobically from ribose, maltose, or trehalose, and (iii) growth in the presence of novobiocin. Four strains of S. hyicus subsp. hyicus (VII76, VII113, VII131, and VA519) gave typical enterotoxigenic responses in monkey-feeding tests but were negative for enterotoxins A through E, suggesting the presence of one or more new enterotoxins. Two coagulase-negative, heat-stable DNase-positive strains (D143 and ARM) could not be classified by either DNA-DNA hybridization or numerical taxonomy, and D143 was enterotoxigenic as measured by the monkey-feeding bioassay. DNA homology showed that strain FRI-698M was more closely related to S. epidermidis than to S. aureus, yet it produced enterotoxin D. These data suggest the occurrence of coagulase-negative enterotoxigenic strains that are not S. aureus; nonetheless, a positive tube coagulase test and heat-stable DNase test should together be useful for routine screening of most potentially enterotoxigenic staphylococci in foods.

Quantitative estimates of beta-lactam residues in raw milk around a reference standard: collaborative study.

A collaborative study was conducted to determine the reliability of a Bacillus stearothermophilus disc assay method for differentiating various concentrations of penicillin in raw milk. Participating laboratories tested 10 different samples (including one negative) in blind duplicate. Triplicate standards were alternated with triplicate unknowns around the periphery of each of 5 different plates. Zone diameters were measured and the difference in zone size of pairs of adjacent standard and unknown samples were analyzed by a paired t-test. Penicillin concentrations 0.003 IU/mL different from the reference concentrations were consistently distinguishable at a 95% confidence level. Such discriminatory power was determined to be possible with as few as 3 plates (9 replicates) per unknown. The method has been adopted official first action.

Incidence of ketamine-induced emesis in cynomologus monkeys (Macaca fascicularis) used for staphylococcal enterotoxin bioassay.

Ten (24%) of 41 cynomologus monkeys (Macaca fascicularis) showed emetic response to 2.5-20 mg/Kg of ketamine injected i.m. Reduction of the levels of ketamine to one half or less of the emetic level resulted in faster recovery from sedation yet provided adequate time for intubation and subsequent intragastric feeding of staphylococcal enterotoxin (SE) in only 6 of the 10 monkeys without emesis. The onset of the first emetic episode with ketamine was similar to that induced by staphylococcal enterotoxin A (SEA). Cynomologus monkeys showing emetic response to ketamine could still be used for SE bioassay if an experimentally determined non-emetic dose for individual monkeys is employed for sedation.

Biologic activity of cell-associated staphylococcal enterotoxin A.

Cell associated staphylococcal enterotoxin A, released by lysostaphin treatment of Staphylococcus aureus cells, was found to be biologically active in cynomolgus monkeys. The activity was comparable to that of extracellular enterotoxin A; six of six monkeys vomited within 5 h in response to extracellular enterotoxin and four of six also vomited in response to the same serologic level (4.8 micrograms per monkey) of cell-associated enterotoxin. Feeding S. aureus cells containing cell-associated enterotoxin A to cynomolgus monkeys resulted in emesis in three of five monkeys within 3 h. This suggests that consumption of S. aureus cells could lead to staphylococcal intoxication.

Quantitative Assay of Beta-Lactam Residues in Raw Milk Using a Disc Assay Method.

Numerous methods have been developed to determine presence of antibiotics in raw milk. Until recently, major effort had been placed on qualitative considerations, and primarily for detecting presence of penicillin (beta-lactam) residues. Only one method, the Sarcina lutea Cylinder Plate (CP) procedure, has been modified to provide for quantitative estimates. The CP method is a rather long, tedious test, requiring considerable technical skill. Need for a simpler, faster quantitative method was apparent. This paper describes a method for making quantitative estimates of beta-lactam residues around a fixed reference standard. The method uses Bacillus stearothermophilus in a disc assay test. Quantitative estimates above or below the reference level of antibiotic are computed through a paired-t statistical analysis. The test can be completed within 3 h.

Interpretation of the tube coagulase test for identification of Staphylococcus aureus.

The tube coagulase test is a valid means of identifying Staphylococcus auerus, provided that only a firm clot that does not move when the tube is tipped is considered a positive reaction. The widely promulgated interpretation that all degrees of clotting in coagulase plasma are a positive identification of S. auerus was disproved by the use of other tests such as anaerobic glucose fermentation, thermonuclease production, and lysostaphin sensitivity. It was found that the source of supply of the coagulase plasma is a factor in the occurrence of false-positive coagulase test results. The use of a mixture of pig and rabbit plasma in the tube coagulase test is also discussed.