Limulus amoebocyte lysate and direct sampling methods for surveillance of operating nebulizers.

The Limulus amoebocyte lysate test for detection of endotoxin (Pyrogent; Mallinckrodt Chemical Co.) and the Easicult method (Orion Diagnostica) for detection of bacteria were compared with direct dilution sampling, a standardized technique for respiratory therapy surveillance previously developed in our laboratory. Tests of 206 reservoirs of nebulizers were done in three hospitals in Georgia. Forty-five percent of all reservoirs sampled were contaminated. Gram-negative, nonfermentative bacilli were the predominant contaminants. The results of the Limulus test and the Easicult system were in agreement with those of the direct dilution sampling tests approximately 84 and 90% of the time, respectively. Direct dilution of water samples onto blood agar plates was the most sensitive, reliable, and informative method for detecting viable bacteria. The Easicult and Limulus systems were sensitive enough to detect greater than or equal to 10(3) colony-forming units per ml. Positive Limulus tests and negative culture tests, reflecting detection of endotoxin but not of viable gram-negative bacteria, occurred in 20 of 206 (9.7%) instances. Positive cultures and negative Limulus tests were noted in 13 of 206 (6.8%) samplings. The Limulus test is a valuable procedure, for it can detect moderate-to-heavy microbial contamination within 1 h of testing and affords the opportunity to remove contaminated equipment from patients within minutes of a positive test result. These results demonstrate the potential value of the Easicult and Limulus tests for selective surveillance of operating nebulizers.     

Comparison of homogenizing, shaking, and blending on the recovery of microorganisms and endotoxins from fresh and frozen ground beef as assessed by plate counts and the Limulus amoebocyte lysate test.

Of three methods studied, brisk shaking of samples in dilution blanks by hand and homogenization by a stomacher were compared relative to their capacity to recover the endotoxins and viable bacteria; blending with a Waring blender was compared with these two methods only on the recovery of viable cells. Aerobic plate counts were essentially the same by the three methods for fresh meats, with the stomacher producing slightly higher aerobic plate counts and significantly higher gram-negative counts determined by violet red bile agar. The stomacher produced significantly higher aerobic plate counts and violet red bile agar results on frozen meats than did shaking. Endotoxins were determined by the Limulus amoebocyte lysate test; results by shaking and stomacher on 15 single samples of frozen meat were identical. Of Limulus amoebocyte lysate-negative beef which was spiked with known endotoxin, a higher percentage of recovery was obtained with the stomacher. Although both aerobic plate counts and violet red bile agar counts were found by shaking and stomacher to decrease significantly in frozen meats, endotoxin content was not significantly affected. The stomacher was found to be the better method overall, especially when meats are to be examined for their content of viable gram-negative bacteria, endotoxins, or both.     

Comparison of homogenizing, shaking, and blending on the recovery of microorganisms and endotoxins from fresh and frozen ground beef as assessed by plate counts and the Limulus amoebocyte lysate test.

Of three methods studied, brisk shaking of samples in dilution blanks by hand and homogenization by a stomacher were compared relative to their capacity to recover the endotoxins and viable bacteria; blending with a Waring blender was compared with these two methods only on the recovery of viable cells. Aerobic plate counts were essentially the same by the three methods for fresh meats, with the stomacher producing slightly higher aerobic plate counts and significantly higher gram-negative counts determined by violet red bile agar. The stomacher produced significantly higher aerobic plate counts and violet red bile agar results on frozen meats than did shaking. Endotoxins were determined by the Limulus amoebocyte lysate test; results by shaking and stomacher on 15 single samples of frozen meat were identical. Of Limulus amoebocyte lysate-negative beef which was spiked with known endotoxin, a higher percentage of recovery was obtained with the stomacher. Although both aerobic plate counts and violet red bile agar counts were found by shaking and stomacher to decrease significantly in frozen meats, endotoxin content was not significantly affected. The stomacher was found to be the better method overall, especially when meats are to be examined for their content of viable gram-negative bacteria, endotoxins, or both.     

Relationship between bacterial counts and endotoxin concentrations in the air of wastewater treatment plants.

The relationship between bacterial counts and endotoxin concentrations in air samples was studied. Selective EMB medium favored the growth of a larger portion of airborne gram-negative bacteria than LES Endo or MacConkey medium and was a good predictor of the endotoxin levels determined with a chromogenic Limulus assay of the air of wastewater treatment plants. The bacterial counts determined with nonselective media correlated poorly with airborne endotoxin levels; however, R2A medium yielded higher viable bacterial counts than TYG medium. Direct counting by epifluorescence microscopy yielded the highest bacterial counts, but no correlation was obtained between total bacterial counts and endotoxin concentrations.     

Relationship between bacterial counts and endotoxin concentrations in the air of wastewater treatment plants.

The relationship between bacterial counts and endotoxin concentrations in air samples was studied. Selective EMB medium favored the growth of a larger portion of airborne gram-negative bacteria than LES Endo or MacConkey medium and was a good predictor of the endotoxin levels determined with a chromogenic Limulus assay of the air of wastewater treatment plants. The bacterial counts determined with nonselective media correlated poorly with airborne endotoxin levels; however, R2A medium yielded higher viable bacterial counts than TYG medium. Direct counting by epifluorescence microscopy yielded the highest bacterial counts, but no correlation was obtained between total bacterial counts and endotoxin concentrations.     

Endotoxin in germfree, gnotobiotic, or conventional-flora Sprague-Dawley rats.

The Limulus assay for bacterial endotoxin was performed on serum and (or) plasma from animals monoassociated with Clostridium species, Staphylococcus aureus, Escherichia coli, Proteus mirabilis, Enterobacter agglomerans, Bacteroides fragilis, Klebsiella pneumoniae, or Candida albicans. Plasma from animals monoassociated with the gram-negative bacteria or C. albicans consistently showed a positive Limulus test while conventional-flora controls, germfree rats, and gnotobiotic animals monoassociated with gram-positive bacteria or E. agglomerans were negative. Germfree and conventional rats were injected (intraperitoneal (i.p.)) with Salmonella typhosa lipopolysaccharide (LPS). Although no endotoxin was detectable in either group prior to the injection, by 1 h post injection endotoxin was in the plasma of all groups. The germfree rats appeared to clear the LPS quicker than their conventional-flora counterparts. Generally, LPS-injected rats (conventional and germfree) showed clumping and decreased number of platelets, a decrease in their lymphocyte counts, and increased polymorphonuclear leukocyte (PMN) counts.     

Studies on the sensitivity and specificity of the Limulus amebocyte lysate test and rabbit pyrogen assays.

The sensitivity and specificity of the Limulus amebocyte lysate test and rabbit pyrogen assay were studied by means of artificially contaminated parenterals. Various gram-negative and gram-positive bacterial strains were used as was one strain of the yeast Candida albicans. The numbers of organisms needed to elicit positive responses in distilled water and normal saline were recorded and compared. The sensitivity and specificity of the Limulus amebocyte lysate assay for the detection of bacterial endotoxin from gram-negative bacteria were demonstrated. Variable results were recorded with gram-positive bacteria and Candida albicans.     

Studies on the sensitivity and specificity of the Limulus amebocyte lysate test and rabbit pyrogen assays

The sensitivity and specificity of the Limulus amebocyte lysate test and rabbit pyrogen assay were studied by means of artificially contaminated parenterals. Various gram-negative and gram-positive bacterial strains were used as was one strain of the yeast Candida albicans. The numbers of organisms needed to elicit positive responses in distilled water and normal saline were recorded and compared. The sensitivity and specificity of the Limulus amebocyte lysate assay for the detection of bacterial endotoxin from gram-negative bacteria were demonstrated. Variable results were recorded with gram-positive bacteria and Candida albicans.     

The Limulus Lysate Endotoxin Assay as a Test of Microbial Quality of Ground Beef

The Limulus lysate test (LLT) for endotoxin assay has been found to be an excellent, simple and rapid test of microbial quality of refrigerated ground beef. In fresh ground beef held at 5°C for 7–12 d, LLT titres increased from 10²–10⁵ and correlated very highly with extract-release volume (ERV) data and total viable Gram negative counts at both 5° and 30°C. The LLT was negative for fresh beef containing low numbers of bacteria and on aged beef in the absence of increasing numbers of Gram negative bacteria. Of 14 Gram negative meat isolates, all gave a positive LLT while none of eight miscellaneous Gram positive bacteria did. The use of this test provides objective information on the microbial quality of fresh refrigerated ground meats in 1 h. Based upon this study, it is suggested that a 0·1 ml inoculum from a 10³ dilution of good quality ground beef should produce a negative lysate test and thus serve as an additional rapid screening test of meat microbial quality.     

Sodium-Dependent Growth of Azotobacter chroococcum

Selected ion-monitoring gas chromatography-mass spectrometry was used for detection of beta-hydroxy fatty acids as an independent assay for the presence or absence of endotoxin in materials claimed to induce nonspecific activation of Limulus amoebocyte lysate. To this end, suspensions of gram-negative and -positive bacteria, one fungal species, cerebrospinal fluid, and hollow-fiber hemodialyzer rinses were assayed for endotoxin by gas chromatography-mass spectrometry and the Limulus amoebocyte lysate assay. Good qualitative agreement was shown for both methods when suspensions of test organisms were assayed. Two false-negative results were obtained by gas chromatography-mass spectrometry assays of cerebrospinal fluid and were shown to be a result of insufficient endotoxin in the cerebrospinal fluid specimens for detection by gas chromatography-mass spectrometry. Hemodialyzer rinses were Limulus assay positive; however, no beta-hydroxy fatty acids were detected by gas chromatography-mass spectrometry. These data were compared with data obtained from USP rabbit pyrogen tests of the rinse materials (nonpyrogenic) and chemical characterization of the Limulus assay-reactive rinses, which showed the rinses to be cellulosic in nature. It is suggested that beta-hydroxy fatty acids, as assayed by selected ion-monitoring gas chromatography-mass spectrometry, be used as chemical marker molecules for the presence or absence of endotoxin in materials reported to cause nonspecific activation of Limulus amoebocyte lysate.     

Sodium-Dependent Growth of Azotobacter chroococcum

Selected ion-monitoring gas chromatography-mass spectrometry was used for detection of beta-hydroxy fatty acids as an independent assay for the presence or absence of endotoxin in materials claimed to induce nonspecific activation of Limulus amoebocyte lysate. To this end, suspensions of gram-negative and -positive bacteria, one fungal species, cerebrospinal fluid, and hollow-fiber hemodialyzer rinses were assayed for endotoxin by gas chromatography-mass spectrometry and the Limulus amoebocyte lysate assay. Good qualitative agreement was shown for both methods when suspensions of test organisms were assayed. Two false-negative results were obtained by gas chromatography-mass spectrometry assays of cerebrospinal fluid and were shown to be a result of insufficient endotoxin in the cerebrospinal fluid specimens for detection by gas chromatography-mass spectrometry. Hemodialyzer rinses were Limulus assay positive; however, no beta-hydroxy fatty acids were detected by gas chromatography-mass spectrometry. These data were compared with data obtained from USP rabbit pyrogen tests of the rinse materials (nonpyrogenic) and chemical characterization of the Limulus assay-reactive rinses, which showed the rinses to be cellulosic in nature. It is suggested that beta-hydroxy fatty acids, as assayed by selected ion-monitoring gas chromatography-mass spectrometry, be used as chemical marker molecules for the presence or absence of endotoxin in materials reported to cause nonspecific activation of Limulus amoebocyte lysate.     

Endotoxin assay by bioluminescence using mutant firefly luciferase

The Limulus reaction is an application of the defense mechanism of horseshoe crab for endotoxin detection. Endotoxin is a component of the cell wall in the outer membrane of gram-negative bacteria, and causes fever or shock when it enters the human blood stream. For endotoxin detection, gel formation or turbidity of the coagulation factor chromogen or fluorescence-modified peptide is used. However, these conventional methods have problems with regard to their measurement time or sensitivity. We recently obtained a mutant firefly luciferase that has a luminescence intensity over 10-fold higher than that of the wild type. Therefore, we developed a new endotoxin detection method that combines the Limulus reaction and bioluminescence using mutant luciferase. The new method detects 0.0005 EU/ml of endotoxin within 15 min.     

The Triphenyl Tetrazolium Chloride (Uroscreen) Test Alone and in Combination with the Gram Smear as a Screening Procedure for Significant Bacteriuria in Hospital Patients

Of 725 specimens of urine examined by the triphenyl tetrazolium chloride (TTC) [Uroscreen], pour plate and calibrated loop procedures, 30% yielded bacterial colony counts greater than 100,000/ml.; a 100% correlation was obtained among the three methods. Of 539 urine specimens containing more than 100,000 bacteria/ml., 517 (94.06%) gave a positive TTC test.Because of the high correlation between the TTC test and bacterial quantitative counts, the method of TTC in conjunction with smears was adopted as a routine procedure. Specimens which were TTC-negative and smear-negative were discarded. Of 1227 specimens from hospital in-patients and 349 outpatients, 369 urines showed significant bacteriuria (337 from hospital in-patients and 32 from outpatients). There was complete correlation between the TTC test and smear. Of 337 isolations, 27 (8.02%) gave a negative TTC test but a positive smear.     

Investigations on the Specificity of the Limulus Test for the Detection of Endotoxin

Lysates obtained from amoebocytes of Limulus polyphemus, the horseshoe crab, showed gel formation after the addition of bacterial endotoxin. In contrast to living gram-negative bacteria, viable gram-positive microorganisms did not cause gelation of lysate. Nevertheless, peptidoglycan isolated from the cell walls of various gram-positive organisms did induce the reaction. However, the activity of peptidoglycan was 1,000 to 400,000 times less than that of Escherichia coli lipopolysaccharide. After exposure to lysozyme, peptidoglycan no longer gelled amoebocyte lysate, therefore apparently excluding endotoxin contamination. Gelation of amoebocyte lysate by endotoxin or peptidoglycan was inhibited by different concentrations of sodium polystyrolsulfonate. Whereas these studies confirm the specificity of the Limulus test for bacterial endotoxins, they also indicate that other substances of bacterial origin should be investigated for their ability to gel amoebocyte lysate.     

A Comparative Study of the Triphenyltetrazolium Chloride (Uroscreen) Test and Conventional Methods for the Detection of Bacteriuria

Acute urinary tract infection may be preceded by and active pyelonephritis may be associated with asymptomatic bacteriuria. Treatment of asymptomatic bacteriuria may prevent or arrest active, chronic pyelonephritis and its sequelae. Consequently, there is a need for a reliable and simple screening procedure to detect asymptomatic bacteriuria in large segments of the population.The reliability and practicability of tests advocated for the detection of bacteriuria, including the new chemical triphenyltetrazolium chloride (T.T.C.) (Uroscreen) test, were evaluated. Reliability was assessed by correlating results of these tests with bacterial counts of tested urines. Significant bacteriuria is defined as the presence of 100,000 or more organisms per ml. of urine.The T.T.C. (Uroscreen) test was positive in 92.5% of cases of bacteriuria; there were 7.5% false-negative and 2.8% false-positive results. Bacteria on Gram-stained smear were found in 95.5% of the cases of bacteriuria and in 14.6% of those with non-infected urine; pyuria (more than three leukocytes per high-power field), in 60% of those with bacteriuria and in 15.9% of those with presumably non-infected urine. Bacteria were conspicuous in the urinary sediment in 91.1% of cases of bacteriuria and in 3.7% of presumably non-infected urines.The T.T.C. (Uroscreen) test fulfilled the criteria for a reliable and simple screening procedure. It should be used concomitantly with other screening tests when the urine is examined routinely.     

Simple Disposable Method for Quantitative Cultures of Urine

A disposable kit was tested as a means of detecting significant bacteriuria by quantitative culture of urine. The total error in 3,563 specimens tested by five investigators was less than 1%. The method was very effective in differentiating significant bacteriuria, i.e., more than 100,000 bacteria per ml of urine from uninfected urine. In specimens from patients with urinary tract abnormalities who had mixed bacterial flora, the absolute numbers obtained with the dip-inoculum method had a 10% variation when compared to results obtained by calibrated loop or dilution pour plate methods. Therefore, the main utility of the kit is for screening and following patients after therapy. A significant delay in time between inoculation of the medium in the kit with the freshly voided urine and incubation of the kit to promote growth did not affect the reliability of the kit as a method of doing quantitative urine cultures to detect bacteriuria.     

Comparison of Limulus assay, standard plate count, and total coliform count for microbiological assessment of renovated wastewater.

The Limulus endotoxin assay was compared to the standard plate count and total coliform count for assessment of the bacteriological quality of reclaimed wastewater. A total of 48 water samples from an advanced waste treatment plant in Dallas, Tex. were examined by the three techniques. Limulus assays were technically simpler to perform and provided results much sooner than conventional culture methods. However, the endotoxin values did not correlate extremely well with determinations of viable bacterial numbers. This lack of correlation may have been due to alterations in the normal ratio of viable gram-negative cells to endotoxin caused by water reclamation procedures.     

Comparison of Limulus assay, standard plate count, and total coliform count for microbiological assessment of renovated wastewater.

The Limulus endotoxin assay was compared to the standard plate count and total coliform count for assessment of the bacteriological quality of reclaimed wastewater. A total of 48 water samples from an advanced waste treatment plant in Dallas, Tex. were examined by the three techniques. Limulus assays were technically simpler to perform and provided results much sooner than conventional culture methods. However, the endotoxin values did not correlate extremely well with determinations of viable bacterial numbers. This lack of correlation may have been due to alterations in the normal ratio of viable gram-negative cells to endotoxin caused by water reclamation procedures.     

Limulus amoebocyte lysate assay for detection and quantitation of endotoxin in a small-volume parenteral product.

A Limulus amoebocyte lysate gel-clotting method for the determination of endotoxin in a small-volume parenteral product has been described. Sample dilution with 0.1 M potassium phosphate monobasic buffer (pH 8.0) effectively eliminated assay interference, whereas dilution with water did not. The threshold pyrogenic dose for Escherichia coli EC-2 and O127:B8 endotoxins was determined to be 1.0 ng of endotoxin per kg of body weight. Not more than 1.0 ng of endotoxin (the threshold pyrogenic dose) per the highest recommended human dose or the USP pyrogen test dose per kg of body weight, whichever dose is more stringent, is a logical limit for the quantity of bacterial endotoxin in small-volume parenteral products. Excellent correlation was attained when this criterion was used to compare the Limulus amoebocyte lysate assay with the USP pyrogen test.     

Rapid Detection of Contaminated Intravenous Fluids Using the Limulus In Vitro Endotoxin Assay

Intravenous fluids and administration sets may become contaminated with gram-negative bacteria during use and result in a life-threatening situation to the patient. The Limulus in vitro assay for endotoxin was used in two patients whose parenteral fluids had become contaminated with Pseudomonas aeruginosa. This test allowed rapid detection of the contaminated intravenous fluids and demonstrated a concomitant endotoxemia in both patients. The same strains of pseudomon were subsequently cultured from each patient's blood, intravenous catheter tip, and parenteral fluid and administration set. A different serotype of pseudomonas was unique to each patient, indicating two separate and unrelated cases of accidental contamination of the administration sets. Endotoxin-like activity was also demonstrated from several brands of commercial human serum albumin, which may contribute low-level activity detectable by the Limulus assay.