Biomedical subjects
Publications and source records attributed to R B Prior.
A regression line for correlating zones of inhibition of a 75-mug mezlocillin disk with minimal inhibitory concentrations of mezlocillin for Enterobacteriaceae and nonfermenters was calculated. Correlation was excellent (r = -0.96) for all species tested except Pseudomonas cepacia.
Three Limulus amoebocyte lysate (LAL) preparations obtained from three different suppliers were comparatively evaluated for sensitivity to native endotoxin contained in urethral exudates from 28 men with gonococcal urethritis and 16 men with nongonococcal urethritis. One LAL preparation was not extracted with organic solvents during manufacture, whereas the other two were extracted with chloroform. All three LAL preparations had equivalent sensitivities (0.06 ng/ml) to an established reference endotoxin standard (EC-2), but significant differences in sensitivities were found among the different LAL preparations when testing clinical specimens. Dilution breakpoints of urethral samples for maximum sensitivity and specificity ranged from 1:400 to 1:1,600, depending on the LAL preparation. The nonextracted lysate was significantly more sensitive to the presence of endotoxin in gonococcal exudates than the other two preparations (P less than 0.001) but not significantly different from one LAL preparation (P greater than 0.05) in detecting endotoxin in nongonococcal exudates. An additional 116 men, 61 with culture-proven gonococcal urethritis and 55 with nongonococcal urethritis, were evaluated with three lots of nonextracted lysate with sensitivities ranging from 0.04 to 0.06 ng/ml, reference endotoxin EC-2. At a dilution breakpoint of 1:1,600, the sensitivity of the LAL test was 100%, and the specificity was 96%.
A test device incorporating Limulus amoebocyte lysate (Mallinckrodt, Inc., St. Louis, Mo.) was developed for the rapid, presumptive diagnosis of gonococcal and nongonococcal disease in males. The device, which was evaluated in 550 men with exudative urethritis, consisted of a specimen collection syringe, a dilution reservoir containing 10 ml of pyrogen-free water, and a Limulus amoebocyte lysate single-test vial. After specimen collection, the syringe was affixed to the dilution reservoir for rapid, accurate dilution of the clinical sample. Contamination of the specimen and potential biohazards to the user were prevented. The diluted sample was then transferred (via the collection syringe) to the lysate test vial for assay of endotoxin. Various incubation times at 37 degrees C were also studied in an additional 301 male patients, and time was reduced from the standard 60 to 30 min while still retaining equivalent predictability of culture results (P less than 0.05). Of the 550 males evaluated with the test device, 366 had positive cultures for Neisseria gonorrhoeae, and 184 were negative. A sensitivity of 99.2% and a specificity of 96.7% were obtained with the test device. Overall ability to predict culture results was 98.4%. Gram-stain sensitivity and specificity were 96.4% and 99.5%, respectively, with an overall accuracy of 97.5%. There were no statistical differences between the Limulus amoebocyte lysate test and Gram stain in predicting cultures (P less than 0.05). Thus, use of the Limulus amoebocyte lysate test device would enable the private physician to make an accurate, presumptive diagnosis of gonococcal and nongonococcal disease in males with exudative urethritis within 30 min without the need of a microscope and to initiate proper therapy during the patient's initial evaluation.
In an evaluation of the limulus lysate assay (LLA) as a method for detecting gonococcal endotoxin in cervical exudates diluted 1:800, positive LLA results were obtained from 17 of 18 patients (94%) with culture-proved gonococcal cervicitis, and negative results were obtained from 22 of 22 patients (100%) with culture-negative specimens. In vitro tests comparing the sensitivity of the LLA for Neisseria gonorrhoeae and other gram-negative organisms showed the LLA to be more sensitive in detecting N. gonorrhoeae (minimum sensitivity, 10(4) organisms per milliliter) than other commonly encountered urogenital gram-negative bacteria (minimum sensitivity, greater than 10(5) organisms per milliliter). Thus, in preliminary studies involving otherwise healthy women, the LLA appeared to correlate with bacteriologic methods for diagnosing gonococcal cervicitis and may aid in identifying nongonococcal cervicitis. In addition, the LLA was easy to perform, with test results available within an hour after the patient's initial examination.
Eighty-one men with exudative urethritis were evaluated on initial visit for gonococcal urethritis by using the standard tube and newly developed microdilution Limulus amoebocyte lysate techniques. Serial dilutions of clinical specimens ranging from 1:100 to 1:102,400 were each tested, and results correlated with Gram stain and culture. Overall accuracy for predicting culture results was 98% for a dilution of 1:200 and 99% for a dilution of 1:400 for the tube and microdilution techniques, respectively. The microdilution technique predicted culture results in 98% of cases for dilutions ranging from 1:400 to 1:1,600, whereas the tube technique was as accurate for dilutions of only 1:100 and 1:200. The microdilution Limulus amoebocyte lysate technique was a rapid, reliable, sensitive, and economical diagnostic aid in the initial evaluation of exudative urethritis in men.
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The effect on antibiotic stability of thawing, with microwave radiation, cefazolin sodium admixtures frozen in polyvinyl chloride minibags was studied. Two brands of cefazolin sodium (Ancef and Kefzol) were reconstituted and placed in 50-, 100- and 250-ml polyvinyl chloride minibags of 5% dextrose in water or 0.9% sodium chloride. The resulting solutions were assayed for antibiotic stability, using an agar disk diffusion technique, and for pH. The solutions were then stored at -20 degrees C for 48 hours, thawed to room temperature in a microwave oven, and kept at room temperature for four hours, after which they were reassayed for potency and pH. The results indicated that after the freeze-thaw process, the cefazolin sodium minibag admixtures retained at least 90% of their initial antimicrobial activity. The minimal pH changes could not be related to changes in antimicrobial activity, and no color changes could be detected visually. Using a microwave oven can greatly reduce thawing time of antibiotic admixtures. To maintain solution stability and prevent accidents, it is important to calibrate the oven, avoid solution overheating, and observe full precautions in oven operation.
The effect of freezing on the stability of several reconstituted antibiotics packaged in disposable hypodermic syringes was studied. The following drugs were reconstituted and, after taking samples for initial assay, were frozen at -20 degrees C in Hy-Pod hypodermic syringes: cephalothin, 1 g/5 ml; cefazolin (with water), 1 g/3 ml; cefazolin (with lidocaine injection), 1 g/3 ml; cefamandole, 1 g/3.5 ml; and nafcillin, 1 g/4 ml. At three, six, and nine months, samples of the frozen solutions were allowed to thaw at room temperature and then were assayed microbiologically. All antibiotics maintained at least 90% potency at the end of nine months. Cefazolin with lidocaine, however, turned cloudy after thawing. The data suggest that cephalothin, cefazolin with water, cefamandole, and nafcillin, when frozen and stored at -20 degrees C in Hy-Pod hypodermic syringes, may be used within nine months of initial freezing.
The Limulus amebocyte lysate (LAL) assay is the most sensitive method currently available for the detection of bacterial endotoxin and is the official test for detecting the presence of pyrogens in pharmaceuticals. Although its value in detecting gram-negative infections is a controversial issue, the test can be used by the practicing physician as an adjunct to accepted diagnostic methods in selected clinical situations.
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Endpoint determinations of endotoxin by the Limulus amoebocyte lysate assay have been facilitated by use of a microdilution procedure.
In an evaluation of the limulus assay as a method for detecting endotoxin in urethral exudates, positive results of urethral samples at a 1/200 dilution were obtained from 73 out of 73 patients with culture-positive gonococcal urethritis while negative results were obtained from 26 out of 27 patients with cuture-negative urethral specimens. A specimen from one patient, which gave negative results on Gram stain and culture, gave positive results to the limulus test. The overall accuracy of the limulus test for predicting culture results was 99% (p less than 0.001). Thus, in preliminary studies of otherwise healthy men, the results of the limulus assay correlated with those of biological methods for diagnosing urethral gonorrhoea; the test may, therefore, be of use in identifying cases of nongonococcal urethritis.
Endocarditis caused by Cardiobacterium hominis was observed in a penicillin-allergic patient with a prosthetic cardiac valve who had received prophylactic therapy with erythromycin for dental extractions. The organism was resistant to erythromycin and vancomycin, with minimal inhibitory concentrations of 12.5 microgram/ml and 25 microgram/ml, respectively, but was sensitive to penicillin G, tetracycline, cephalexin, and cefaclor. This case suggests that currently recommended antibiotic prophylactic therapy for endocarditis, especially in penicillin-allergic patients, may be inadequate for unusual pathogens such as C hominis.
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The in vitro activities of ticarcillin (T) and carbenicillin (C) against 253 strains of Pseudomonas aeruginosa (108 randomly selected clinical isolates and 145 isolates determined to be C resistant by 10 referring hospitals) were evaluated using both broth microdilution and disk diffusion techniques. T activity was usually twofold or greater than C activity for individual strains in both sample populations. The minimal inhibitory concentration (MIC) values of T and C were </=0.4 to >400 (median, 25) and 0.8 to >400 (median, 50) mug/ml, respectively, for the random sample and 1.6 to >400 (median, 50) and 12.5 to >400 (median, 200) mug/ml, respectively, for the select sample. For predicting susceptibility (MIC, </=50 mug of T and </=100 mug of C per ml), 75-mug T and 100-mug C disks were equivalent in accuracy for the random sample (T/T = 92.6%, T/C = 92.0%, C/T = 91.7%, C/C = 89.8%). Regression-line analyses of zones of inhibition versus MIC values for the four combinations showed coefficients of correlation to be -0.81, -0.76, -0.77, and -0.75, respectively. With the sample selected as being resistant to C, T was better than C for predicting T susceptibility (P < 0.05), and T was equivalent to C for predicting C susceptibility (T/T = 84.3%, T/C = 81.0%, C/T = 73.6%, C/C = 80.4%). Coefficients of correlation were -0.70, -0.74, -0.55, and -0.61, respectively. T and C disks were, therefore, equivalent for determining T and C susceptibilities, but the T disk was more reliable for determining T susceptibility in a population skewed with C-resistant strains.
In a multicenter cooperative controlled study, individual test, accuracy, and precision control values were determined for 75-mug ticarcillin disks with Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853.