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PubMed2000

Gel clot LAL assay in the initial management of peritoneal dialysis patients with peritonitis: a retrospective study.

BACKGROUND: Indiscriminate use of broad-spectrum antibiotic treatment of peritonitis in peritoneal dialysis patients may have either unwanted side-effects or contribute to the development of antibiotic resistance. This may be avoided by improved diagnosis at presentation. The Limulus amoebocyte lysate assay is a convenient test detecting bacterial endotoxins or fungal beta glucans. This study evaluates a qualitative Limulus amoebocyte lysate test as a diagnostic tool used at presentation of a peritoneal dialysis patient with peritonitis. METHODS: One-hundred and eleven episodes of peritonitis in peritoneal dialysis patients have been analysed retrospectively. Limulus amoebocyte lysate results at presentation were compared with culture results. A Limulus amoebocyte lysate assay was performed using a commercial kit by incubating a mixture of dialysate effluent and Limulus amoebocyte lysate reagent at 37 degrees C. The development of a stable solid clot was considered positive. The specificity and sensitivity of the test were calculated. RESULTS: The specificity of the Limulus amoebocyte lysate assay was found to be 98% and the sensitivity 74%. Limulus amoebocyte lysate assay was false-negative in 13 cases of Gram-negative peritonitis (22%). Limulus amoebocyte lysate was positive in three of seven cases of fungal peritonitis. The study included one case each with false-positive Limulus amoebocyte lysate and with culture-negative peritonitis. CONCLUSIONS: The Limulus amoebocyte lysate assay is a convenient and valuable diagnostic tool for excluding Gram-positive peritonitis in peritoneal dialysis patients. This allows more specific antibiotic treatment at presentation and may avoid the development of bacterial resistance. A negative Limulus amoebocyte lysate test is not reliable for the exclusion of Gram-negative peritonitis. In the absence of a positive culture result 48 h after presentation, accompanied by a delayed response to treatment, a positive Limulus amoebocyte lysate assay may indicate the presence of fungus. This justifies early empiric antifungal treatment before definitive culture results are made available. Routine Limulus amoebocyte lysate assay of dialysate effluent from continuous ambulatory peritoneal dialysis patients presenting with peritonitis is recommended.

False Negative Reactions↗
PubMedDate not supplied

Application of the limulus lysate assay in evaluation of disseminated gonorrhea in women.

The limulus lysate assay was utilized as a diagnostic adjunct in the evaluation of three cases of disseminated gonorrhea in women. Although not a specific test for Neisseria gonorrhoeae, the limulus lysate assay, when used with properly diluted endocervical samples, gave results that correlated with conventional diagnostic techniques. If the advantages and limitations of the limulus lysate assay become fully appreciated, it may serve as a useful clinical tool for diagnosis of this syndrome.

Adult↗
PubMed1982

Differentiation of coagulase-positive and coagulase-negative staphylococci by lectins and plant agglutinins.

The screening of staphylococci with a panel of 14 lectins and extracts demonstrating lectin-like activity led to the development of a rapid agglutination slide test for the differentiation of certain coagulase-negative staphylococci and human strains of Staphylococcus aureus. The coagulase-negative staphylococci were agglutinated by agglutinins from Mangifera indica, Triticum vulgaris, and crude Limulus polyphemus. The test is rapid, requiring only 5 to 15 min to identify an unknown strain of staphylococci, as opposed to the 4 to 16 h required to perform the conventional tube coagulase test.

Agglutination Tests↗
PubMed1994

Evaluation of a cow-side test for detection of gram-negative bacteria in milk from cows with mastitis.

A modified Limulus amebocyte lysate (LAL) cow-side test was evaluated under field conditions. The principle of the test is to visualize reactions between test components and endotoxin from the cell wall of Gram-negative bacteria. The practical purpose is to detect such bacteria in mastitic milk. Secretions from 789 udder quarters with clinical mastitis were examined by the LAL-test. Parallel quarter milk samples were sent to a mastitis laboratory of microbiological examination. Eleven veterinary surgeons in three veterinary districts in Norway performed the field investigations. Results of the LAL-test and culture agreed in 93% of all milk samples tested, agreement measured by kappa being 0.63. The sensitivity of the test in detecting Gram-negative bacteria was 63%, while the specificity was 97%. The predictive value of a positive test result was 70%, the figure being somewhat higher (75%) when the material was limited to milk samples without antibiotic residues. The predictive value of a negative test result was 95%. The LAL-test is considered to constitute a valuable cow-side test for the veterinary practitioner, aiding the selection of antibacterial drug of choice for the initial treatment of clinical mastitis.

Animals↗
PubMed1997

Collaborative study on test systems to assess toxicity of whole cell pertussis vaccine.

A collaborative study has been carried out to establish the precision and accuracy of five test systems for the assessment of the toxicity of whole cell pertussis vaccine. To this end, six vaccines, including both "normal" and "abnormal" products with respect to arbitrary levels of Pertussis toxin and/or potency were tested. The study included in vivo test systems as the Mouse Weight Gain (MWG) test; the current WHO-recommended bioassay to evaluate overall pertussis toxicity and four specific test systems; the Leukocytosis Promotion (LP) test, the Histamine Sensitization (HS) test and in vitro the Chinese Hamster Ovary (CHO) clustering test to estimate pertussis toxin (PT) levels, and the Limulus Amoebocyte Lysate (LAL) test to evaluate endotoxin levels. In addition, participants were also asked to estimate potency by the Mouse Protection test according to Kendrick (MP). Fourteen laboratories in various countries participated in the study. In almost all participating laboratories, the MWG test was not very accurate in evaluating the overall toxicity of whole cell pertussis vaccines. In addition, statistical significant interlaboratory variation was frequently seen. The specific toxicity tests (LP, HS, CHO and LAL test) appeared to be more accurate, but large interlaboratory variation was seen, statistically significant at P < 0.05 for LP test, CHO test and LAL test. Significant variation in test results also occurred in the potency test. Furthermore, the discriminative power of the MP test between different levels of potency was low. It was concluded that, on the condition of optimization and stringent standardisation, HS and CHO test and in particular LP test might be more appropriate to assess PT activity than the MWG test provided that the tests are optimised and stringently standardized. An inhibition ELISA was used to estimate levels of PT. This test could be of value for prescreening purposes. The LAL test should be used to estimate endotoxin activity. The value of the MP test, as a model to assess potency, is disputed.

Animals↗
PubMed2004

[Study of biosensor technology on the detection of endotoxin-neutralizing materials].

OBJECTIVE: To explore the application of biosensor technology in the determination of endotoxin-neutralizing materials. METHODS: After mixing polymyxin B (PMB) with endotoxin in certain concentration, the neutralizing ratio of PMB to endotoxin was assessed by biosensor technique and limulus amebocyte lysate test respectively. The results from the two methods were compared. RESULTS: The neutralizing ratio of PMB to endotoxin as assessed by biosensor technology was 0.35 microg to 1 ng, while that by dynamic turbidimetric and chromogenic limulus amebocyte lysate (LAL) technique was 0.5 mg to 1 ng and 1 mg to 1 ng, respectively. The results obtained by biotechnology were similar to that by biosensor technique. CONCLUSION: Biosensor technology was an accurate, convenient and rapid method for the determination of potency of endotoxin-neutralizing materials.

Bacterial Proteins↗
PubMed1991

Synergistic effect of coumarin (1,2 benzopyrone) and endotoxin in the induction of human interleukin-1.

Coumarin as well as its derivatives 7-OH coumarin and 4-OH coumarin were found to stimulate interleukin-1 beta (IL-1 beta) release from freshly isolated human mononuclear cells (MNC) if the culture medium contained fetal calf serum. Under serum-free conditions, almost no induction of IL-1 beta release was observed and the former effect could be completely eliminated by polymyxin B. Therefore, the combined action of endotoxin and coumarin was tested on MNC IL-1 beta production. The coumarins were able to potentiate human MNC IL-1 beta production by lipopolysaccharide (LPS) in a dose-dependent manner. That the effect was due to the presence of coumarins and not endotoxin contamination was shown by negative Limulus amebocyte lysate tests and pre-incubation of the coumarins with polymyxin B-agarose. The latter procedure was able to block endotoxin induced IL-1 beta production but the synergism between coumarin and endotoxin was not influenced by pre-incubating the coumarins with polymyxin B-agarose. Cycloheximide as well as actinomycin D eliminated the induction of IL-1 release by coumarin and LPS demonstrating that the cytokine was newly synthesized after MNC stimulation. In addition, both the total amount of MNC IL-1 beta (cell-associated + extracellular) and the extracellular portion of the cytokine were synergistically decreased if coumarin or its derivatives were added to endotoxin-stimulated cultures. Synergism of coumarin and endotoxin in the induction of interleukin-6 or tumour necrosis factor-alpha could be observed in a smaller percentage of donors. These findings demonstrate an immunomodulatory effect of coumarin on cytokine production by monocytes in vitro which might help to explain some of the biological activities attributed to the drug upon its application in tumour patients.

4-Hydroxycoumarins↗
PubMed1985

Lack of utility of a Limulus amoebocyte lysate assay in the diagnosis of urethral discharges in men.

We evaluated a Limulus amoebocyte lysate assay (LALA) test kit for the diagnosis of gonorrhea in 883 unselected men with urethral discharge. Results were compared with those of Gram-stained smears and Martin-Lewis cultures. Of 331 men with gonococcal urethritis and 552 men with nongonoccal urethritis, 125 (37.8%) and 503 (91.1%), respectively, could not be evaluated by LALA owing either to insufficient discharge specimen to perform the test (569 or 64.4%) or to other exclusion criteria (59 or 6.7%). Of 255 LALA-evaluable discharges, LALA correctly diagnosed 252 (98.8%), compared with 244 (95.7%) for the Gram-stained smear. However, the Gram-stained smear also correctly diagnosed 96.5% of 456 men with insufficient discharge for LALA testing. The clinical utility of the LALA test kit is severely limited by performance criteria that exclude the majority of unselected men with urethritis. In addition, it is more technically cumbersome, time consuming, and costly than Gram-stained smears. Further test modifications are unlikely to overcome these inherent disadvantages of LALA.

Adolescent↗
PubMed1982

Release of endotoxic lipopolysaccharide by sensitive strains of Escherichia coli submitted to the bactericidal action of human serum.

Free endotoxin was assayed in filtered samples of E. coli suspensions submitted to the bactericidal and bacteriolytic action of 10% human serum. The Limulus amoebocyte lysate test, a passive hemolysis inhibition assay based on O antigenic specificity and the determination of 3-OH-myristic acid by mass spectrometry were used as assay methods differing from one another with regard to the part of the endotoxin macromolecule involved in the reaction. The biological activity of endotoxin was assessed in a mouse lethality test. The bactericidal and bacteriolytic action of human serum on sensitive strains of E. coli released quantities of endotoxic lipopolysaccharide (LPS) amounting to 3,000-12,000 ng/ml, for an inoculum of 1--3 x 10(8) colony-forming units. The material thus appearing in the medium was shown to react with the Limulus amoebocyte lysate, to be lethal for actinomycin D-sensitized mice and to bear O antigen, as well as 3-OH-myristic acid, a marker of lipid A. Samples of serum depleted of lysozyme by adsorption onto bentonite, and displaying a strictly bactericidal effect, released approximately 80% of the quantity of LPS appearing in the medium in a control experiment performed with untreated serum. The LPS release is therefore mainly linked to the bactericidal effect of antibody and complement. The amount of LPS released depended on the concentration of divalent cations in the medium, being reduced by an increase in the concentration of calcium and magnesium beyond the values optimal for complement activity. This effect was already observed for an increase in the concentration of divalent cations too low to alter the bactericidal or bacteriolytic effects. The significance of the release of endotoxin by complement dependent bactericidal reactions occurring in vivo is discussed.

Antibodies, Bacterial↗
PubMed1980

Japanese minimum requirements for albumin preparations: recent amendments and current problems.

The following amendments have been introduced to the Minimum Requirements for Human Serum Albumin (HSA) and Plasma Protein Fraction (PPF): 1) The upper limit of sodium content was fixed at 3.7 mg/ml; 2) test for chloride content was introduced; 3) sodium and chloride contents were required to be stated on the label; 4) use of zone electrophoresis was introduced in addition to the free-boundary electrophoresis; and 5) albumin content in HSA was lowered from 97% to 96%. Moreover, current problems in quality control which may require further amendments were discussed, namely: 1) limulus amoebocyte lysate test to replace or supplement the pyrogen test in rabbits; 2) attempt of quantitative expression of visual turbidity; 3) establishment of reference A and B blood group substances; 4) gel permeation analysis and scanning at 370-800 nm; and 5) test for prekallikrein activator (PKA).

Blood Proteins↗
PubMed1988

[Endotoxin contents of phytopharmaceuticals: correlation with clinically observed side effects].

Four phytopharmaceutics (Carnivora, Pascotox forte-Injectopas, Esberitox N, Iascador M), which sometimes cause side effects after parenteral administration (fever, rigor, nausea), were examined for their endotoxin content by the kinetic turbidometric Limulus-amebocyte-lysate (LAL) microtitre test. Contaminations of over 10(5) EU/ml (endotoxin units; 1 EU = 0.1 ng of the FDA standard EC-5) were found in correlation with the clinical picture. In one preparation (Carnivora) very high endotoxin levels were always found; contaminations were only occasionally found in the others. These endotoxin measurements are supported by tests of endotoxin-dependent parameters in in-vivo experiments (reduction in leukocytes, acute death in hyperreactive mice). These findings underline the urgent need for a widening of the regulations on testing for pyrogens to include those parenteral preparations which now do not have to be tested because of their small volume (less than 15 ml).

Animals↗
PubMed1994

Cuprophane but not synthetic membrane induces increases in serum tumor necrosis factor-alpha levels during hemodialysis.

Cytokine synthesis and secretion by blood mononuclear cells is a well-documented phenomenon in hemodialyzed patients. The present study was conducted in 17 chronically hemodialyzed patients to test the relative effect of uremic toxicity, membrane biocompatibility, dialysate composition, and the risk of endotoxinemia on the serum level of tumor necrosis factor-alpha (TNF-alpha). The only significant parameter that influenced circulating TNF-alpha was the chemical characteristics of the dialyzer membrane. Tumor necrosis factor-alpha levels significantly increased during the session with cuprophane, whereas they decreased with AN69. The TNF-alpha increase was documented whatever the dialysate buffer and the presence or absence (negative Limulus amoebocyte lysate test) of endotoxin in the dialysate. In the subgroup of patients treated with a contaminated dialysate and AN69, none had clinical symptoms and the central body temperature remained constant throughout the session. In these patients, serum TNF-alpha levels did not change after priming the dialyzer with sterile saline. In conclusion, the serum TNF-alpha level during hemodialysis appears to be modulated by biocompatibility, permeability, and binding properties of dialysis membrane rather than dialysate composition. Endotoxin in the dialysate did not result in positive TNF-alpha balance no matter what its possible priming effect on mononucleated blood cells.

Acrylic Resins↗
PubMed1992

Endotoxin of Pseudomonas pseudomallei detected by the body weight-decreasing reaction in mice and comparison of it with those of P. cepacia and P. aeruginosa.

The heat-treated whole cells, culture supernatants, and extracted endotoxin preparations of Pseudomonas pseudomallei were examined for endotoxin by the mouse body weight-decreasing (BWD) test. The experiments were conducted also with those of P. cepacia and P aeruginosa. Endotoxin was detected in all the samples of P. pseudomallei. Endotoxin of P. cepacia was detected in whole cells, but not in culture supernatant. The BWD activity of P. aeruginosa was 30 times as high as that of P. pseudomallei. This result was confirmed by the experiments with endotoxin preparations. In the limulus amebocyte lysate gelation (LAL) test, however, the endotoxin preparations of the two species showed the same level of activity.

Animals↗
PubMed1983

[Rapid diagnosis of purulent meningitis].

The cerebrospinal fluid of 589 subjects, 78 of whom were suffering from a purulent meningitis were examined. Comparatively by classical bacteriological techniques (direct examination and culture) and by electro-immunodiffusion, latex agglutination, and Limulus endotoxin assay. Soluble bacterial Haemophilus influenzae type B, Neisseria meningitidis group A, C, and Streptococcus pneumoniae antigens, were tested by electro-immunodiffusion and latex agglutination, and soluble bacterial N. meningitidis group B, Listeria monocytogenes and Streptococcus agalactiae antigens by electro-immunodiffusion. Specific antigens and endotoxin were found in 75.8 per cent of the specimens with a rapid answer (120 min). The three tests revealed also only the diagnosis in 29.1 per cent of cases of pneumococcal meningitis, in 33.3 per cent of meningococcal meningitis and in 47 per cent of Gram-negative bacteria meningitis. Only five cerebrospinal fluid from the 589 specimens tested were given a non-specific reaction. These two advantages--sensitivity and specificity--of these three tests render them techniques of the future in the diagnosis of purulent meningitis.

Agglutination Tests↗
PubMed1996

Immunological properties of meningococcal lipopolysaccharide from serogroups A, B & C.

The aim of the study was to measure and compare the oxidative burst, chemotaxis and cytokine production of human white blood cells, stimulated with meningococcal lipopolysaccharides (LPS) extracted from three different serogroups (A, B and C) of Neisseria meningitidis, and to evaluate whether convalescent sera from patients with meningococcal disease could modify cell stimulation of LPS. All three preparations of LPS from groups A, B and C were tested using the Limulus amoebocyte lysate assay (LAL), and the KDO concentrations of the LPS extracts were measured. Equivalent amounts of biologically active LPS, judged by LAL, and LPS with the same KDO concentration were assayed. IL-1alpha, IL-1beta, IL-6 and TNF-alpha production was stimulated by all three LPS preparations. All three preparations stimulated oxidative burst in monocytes (MNC). Only group A LPS stimulated neutrophil chemotaxis, while none of the three LPS stimulated superoxide production. Pooled convalescent sera from five patients with meningococcal disease suppressed the activity of neutrophils stimulated with LPS from groups B and C (p<0.05, Mann-Whitney U-test).

Cells, Cultured↗
PubMedDate not supplied

Evaluation of the limulus amebocyte lysate assay for presumptive diagnosis of gonorrhea in men at a clinic for sexually transmitted diseases.

Specimens of urethral exudate from 200 men with uncomplicated urethritis were tested for Neisseria gonorrhoeae by the limulus amebocyte lysate assay, culture on modified Thayer-Martin medium, and gram-stained smear. As compared with cultures, the sensitivity and specificity of the limulus assay were 94.8% and 89.3%, respectively, while the sensitivity and specificity of the gram stain were 98.8% and 100.0%, respectively. The accuracy of prediction of the presence of N. gonorrhoeae was significantly better by gram-stained smear than by the limulus assay. Unless the limulus assay is modified, as it was in this study, it does not appear to have a role in the presumptive diagnosis of the gonorrhea in men at a clinic for sexually transmitted diseases.

Adolescent↗
PubMed1997

Is the activity of soluble CD14 enhanced following major trauma?

BACKGROUND: The molecule CD14 acts as a receptor for the protein-bound endotoxin (lipopolysaccharide [LPS]) complex and mediates the cellular effects of LPS. The soluble formation, sCD14, is supposed to neutralize circulating LPS (i.e., LPS antagonist) or transfer LPS effects to endothelial cells (i.e., LPS agonist). OBJECTIVE: To elucidate the release of sCD14 per se in patients with major trauma in the early posttrauma period. Our a priori hypothesis was that sCD14 release depends on the plasma LPS concentration simultaneously measured. PATIENTS: In a prospective study, 65 patients with multiple injuries (Injury Severity Score, 9-75) were enrolled. The patients were rescued by the medical helicopter service and directly admitted to our clinics. The plasma concentrations of sCD14 (enzyme immunoassay) and LPS (chromogenic limulus amebocyte lysate test) were analyzed. The first blood sample was collected immediately at the accident site. The following samples were drawn at intervals from 2 hours to daily for 2 weeks. RESULTS: Sixty-one patients survived the observation time. Immediately after trauma, their mean sCD14 level was not different from that of healthy individuals. Two hours later, a pronounced increase of sCD14 was observed and sustained throughout the observation period. Even nonsurvivors showed an increased sCD14 release, but less pronounced. In all patients, plasma LPS levels were elevated during the first 12 hours. CONCLUSIONS: Major trauma caused an increased release of sCD14. This elevation, however, was not correlated to LPS levels or to the severity of trauma (estimated by trauma scores). We found no evidence that sCD14 levels are of prognostic value regarding survival. Furthermore, the release of sCD14 did not occur in an LPS-neutralizing manner, but rendered possible an LPS-independent mechanism.

Adolescent↗