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PubMed2001

[Replacement of the pyrogen-test as batch control test for the biological substances aprotinin and urokinase].

In the Pharmeuropa Vol. 10, No. 4, December 1998, the European Pharmacopoeia Commission published an appeal to replace the pyrogen test by the limulus test for a total of 10 biological substances. These substances included the active ingredients aprotinin and urokinase, contained in our sales products Antagosan and Actosolv. After successful validation of the limulus test, we calculated endotoxin limit concentrations for these and submitted these to the European Department for Quality of Medicines (EDQM). We expect - not least on the basis of the data submitted by ourselves - that the pyrogen test will be replaced in future as a quality control test by the limulus test in testing aprotinin and urokinase compounds for the presence of pyrogenic substances. This leads to a reduction in the number of rabbits used as opposed to the current pyrogen test for testing the stated compounds.

Animal Testing Alternatives↗
PubMed1975

Limulus lysate test for gram-negative bacterial meningitis. Bedside application.

The limulus lysate on cerebrospinal fluid was evaluated in 335 infants and children as a method for the rapid diagnosis of Gram-negative bacterial meningitis. Positive limulus tests were obtained within one hour in 33 of 34 cases of Hemophilus influenzae meningitis; four additional patients with Gram-negative meningitis also showed positive limulus lysate tests. Conversely, 13 patients with Gram-positive bacterial meningitis all yielded negative limulus assays. All 48 cases of aseptic meningitis and 236 children with no meningitis showed negative limulus assays. Antibiotic therapy prior to hospitalization did not vitiate the validity of the test. A bedside adaptation of the limulus test, performed by house officers and medical students, showed approximately 98% agreement with the laboratory assay.

Arachnida↗
PubMed1978

Improvement of the micromethod for the limulus lysate test.

Frauch's micro-slide method was improved to facilitate the endpoint-determination of the Limulus test. Two precise observations, by inverted phase contrast microscopy and with a staining procedure, were newly performed as additions to the slide test. The staining procedure was proposed as an improved method for the Limulus test since it is simple and convenient. In the staining method, bromophenol blue (BPB) solution was used as the staining solution. A negative (-), a strong positive (++) and a weak positive reaction (+) were characterized by a "ring" formation, a "cloud-like" spread of gel and a "spot" in the "cloud" respectively. Since the distinction between (-) and (+) reactions was obvious in the proposed method, determination of the endpoint was easier than in the ordinary tube and Frauch's method. The sensitivity of the present method was equal to or higher than that of other methods. Inverted phase contrast microscopy was utilized to confirm the findings obtained by the staining method. The volume of the lysate used in this method was as little as 1/10 of that used in the tube method.

Blood Platelets↗
PubMedDate not supplied

Endotoxin in middle ear effusions tested with Limulus assay.

Endotoxin in middle ear effusion (MEE) from both children and adult patients with otitis media with effusion (OME) was assayed with the Limulus amoebocyte lysate test, in parallel with the detection of microorganisms. Whereas only 17% of MEE from adults, diluted 1:10 showed the presence of endotoxin, endotoxin was found in 69% of MEE from recent and recurrent cases in children, although microorganisms were isolated from only one-quarter of the samples. 83% of mucoid MEE from children contained endotoxin, compared with 41% of serous MEE. In older children, endotoxin was less frequently detected in accordance with the fact that the incidence of OME decreases with age. It is concluded that endotoxin may be one of the important factors in the pathogenesis of onset of, as well as in delayed recovery from OME in children.

Adolescent↗
PubMed1978

Rapid detection of experimental E. coli endophthalmitis by the Limulus lysate test.

Experimental E. coli endophthalmitis was produced in rabbits. The Limulus lysate test was applied to aqueous and vitreous samples at various intervals after the intravitreal injection of E. coli organisms. Results indicated that this test is feasible using vitreous and aqueous samples. The Limulus test was positive for E. coli endotoxin within hours after infection, requiring only 1 hr to determine the presence of endotoxin after sampling. This test may have some value in the rapid diagnosis of gram-negative endophthalmitis.

Animals↗
PubMed1980

Detection of endotoxin in ear specimens from patients with chronic otitis media by means of the limulus amebocyte lysate test.

The limulus amebocyte lysate (LAL) test is the most sensitive procedure for the detection of endotoxic lipopolysaccharides. The test was applied to middle ear fluids, cholesteatomas, and granulation tissue specimens from 31 patients in parallel with bacteriologic examination. The LAL test and bacteriologic examination yielded concordant results with 26 out of 28 specimens. A positive LAL test was obtained with 11 specimens containing endotoxin-producing organisms. The test became positive within 60 min of incubation in 10 out of 11 specimens and in 1 specimen between 1 and 24 h, suggesting that, in the majority of specimens, endotoxin was present in the specimen itself and that the result was not due to the in vitro multiplication of the microorganisms. All negative tests remained negative for 24 h. It is conceivable that endotoxin present in ear fluids may contribute to the pathologic changes in chronic otitis media.

Adolescent↗
PubMed1975

Endotoxin, thrombin, and the Limulus amebocyte lysate test.

The Limulus amebocyte lysate, a proteinaceous composite isolated from the hemolymph cells of the horseshoe crab (Limulus polyphemus) is sensitive to picogram quantities of Gram-negative bacterial lipopolysaccharides. However, a controversy currently exists as to whether the Limulus amebocyte lysate is specifically sensitive to Gram-negative bacterial endotoxins as a result of a recent report that the blood coagulation protease, thrombin, can mimic endotoxins in the Limulus amebocyte lysate test. Experiments including those employing two highly purified fractions isolated from the Limulus lystae have provided us with evidence that thrombin per se is unable to mimic endotoxin.

Animals↗
PubMed1984

Detection of free endotoxin in cerebrospinal fluid by the Limulus lysate test.

We used a rabbit model of Escherichia coli meningitis to study the basis for positive Limulus lysate tests in infected cerebrospinal fluid. The results indicated that positive Limulus tests are due to endotoxins in cerebrospinal fluid and not to leukocyte proteases or other possible activators of the Limulus clotting system. The results also suggest that bacteria-free endotoxin may be present in localized gram-negative bacterial infections.

Animals↗
PubMed1984

Hollow-fiber dialyzers and their pyrogenicity testing by Limulus amebocyte lysate.

Aqueous extracts of cellulose hollow fibers (CHF) exhibit positive reactions in some Limulus amebocyte lysate (LAL) tests. However, in spite of LAL activity, the extracts produce no fever reaction in rabbits. A comparison of lysates from different suppliers shows pronounced activity differences when extracts of cuprammonium-derived CHF are tested. One of the lysates, which is fully reactive against standard endotoxin, shows no reaction with such extracts, nor do CHF extracts diminish its sensitivity to standard endotoxin. Investigations of the cuprammonium process have shown that endotoxins introduced by the linters are degraded and washed out. Other endotoxin introduction, particularly by the process water, has been excluded. Oxidative or acidic degradation of cellulose does not result in the formation of LAL-reactive material (LAL-RM). On the other hand, sterile cotton wool shows LAL reactivity, and cellulose acetate regains LAL reactivity when it is saponified. Thus, it appears likely that the LAL-RM found in CHF is of purely cellulosic origin and crossreacts with a number of commercially available lysates.

Animals↗
PubMed1985

Rapid diagnosis of genitourinary infections by use of the Limulus lysate amebocyte test.

The Limulus amebocyte lysate (LAL) assay has realized numerous clinical applications, including rapid diagnosis of significant Gram negative bacteriuria and gonorrhea. Several published studies have demonstrated that the LAL test detects significant Gram negative bacteriuria with greater than 90% accuracy and that the predictive value of a negative test (97%) is similar to that of other currently available screening methods for bacteriuria. The test has also succeeded when used for presumptive diagnosis of gonorrhea in men, although it has not been shown to be superior to Gram stain examination of urethral exudate. The LAL test appears to offer excellent sensitivity for examination of cervical secretions from women being screened for gonorrhea, although the specificity of the test appears low, especially when a low-risk population is being examined.

Bacteriuria↗
PubMed1990

Correlation between endotoxin-neutralizing capacity of human plasma as tested by the limulus-amebocyte-lysate-test and plasma protein levels.

In the present study the endotoxin-neutralizing capacity of human plasma obtained from healthy volunteers was determined by use of the limulus-amebocyte-lysate test. The extent of the endotoxin-neutralizing capacity which showed a very broad variation was correlated to the plasma levels of some proteins which are believed to contribute to endotoxin transport and detoxification. The plasma levels of alpha 2-macroglobulin and transferrin, the major transport proteins, as well as the levels of IgG, IgA and IgM and apoprotein A were not significantly correlated to the neutralizing capacity. Only the apoprotein B, the major apoprotein of the low density lipoprotein fraction shows a significant correlation. Together with previously published results it should be concluded that low density lipoproteins are involved in the endogenous endotoxin-neutralizing reaction occurring in human plasma, at least when high doses of endotoxin are administered. Nevertheless the endotoxin-neutralizing reaction seems to be a very complex multistep process.

Apolipoproteins A↗
PubMed1994

[Application of a bacterial endotoxin test for parenteral drugs].

The Limulus test, which has been adopted as the Test for Bacterial Endotoxins in the JP XII, can detect or quantitate endotoxins of Gram-negative bacterial origin using blood corpuscle extracts (Limulus amebocyte lysate, LAL) of horseshoe crabs (Limulus polyphemus, Tachypleus tridentatus, etc.). It may be conducted by the gel-clot or spectrophotometric (turbidimetric and colorimetric) techniques, the former being based on gel formation due to the activation of LAL by endotoxins. The turbidimetric technique is based on the LAL turbidity change during the gel formation and the colorimetric technique on activation of peptide hydrolytic enzymes in LAL. The Limulus test has been unofficially utilized as a simple and highly sensitive method for the determination of endotoxins in parenteral drugs in lieu of the in vivo Pyrogen Test using rabbits. For the Bacterial Endotoxins Test of the JP XII, the gel-clot technique alone was adopted, the technique being only allowed for Injection. Although most parenteral drugs show inhibition or enhancement in practice, this test can be most easily conducted by eliminating interfering effects through dilution of specimens by a factor not exceeding the maximum valid dilution (MVD) with water. Since MVD is dependent on the sensitivity of applied methodology, the turbidimetric and colorimetric techniques, which are more sensitive than the gel-clot technique, have a distinct advantage. The JP, as the leading Pharmacopoeia for the international harmonization of Bacterial Endotoxins Testing, has presented a "Draft towards International Harmonization of Bacterial Endotoxins Test", whose main purpose is the introduction of supplementary turbidimetric and colorimetric techniques. Under these circumstances the following subjects are discussed: (1) the proposal that, with a view towards international harmonization of the technical requirements of Pharmacopoeias, both the turbidimetric and colorimetic techniques should be included together with the gel-clot technique, (2) the differences in the testing principles and/or conditions prescribed in the current Bacterial Endotoxins Test of JP, USP and EP, and (3) the worldwide situation for Endotoxin Reference Standards.

Animals↗
PubMed2000

Measurement of blood clearance time by Limulus G test of Candida-water soluble polysaccharide fraction, CAWS, in mice.

The Limulus G test, responsive to beta-1,3-D-glucan, is a well-established method for the detection of invasive fungal infection. We have recently found that Candida albicans released a water-soluble polysaccharide fraction (CAWS) into synthetic medium (Uchiyama et al., FEMS Immunol. Med. Microbiol. 24 (1999) 411-420). CAWS was composed of a mannoprotein-beta-glucan complex and activated Limulus factor G, and thus would be similar to the Limulus active substance in patient's blood. In a preliminary investigation, we have found that CAWS is lethal when administered intravenously in a murine system. In this study, we examined the toxicity and then the fate of CAWS in mice. The lethal toxicity was strain-dependent and strain DBA/2 was the most resistant. The toxicity was, at least in part, reduced by salbutamol sulfate and prednisolone treatment in the sensitive strains. On intravenous administration, the half clearance time (t1/2) was approximately 40 min in mice (DBA/2). On intraperitoneal administration, CAWS appeared in the blood with a peak concentration at 1 h. In order to establish a treatment plan, it is important to demonstrate the onset and the termination of deep-seated mycosis. The Limulus G test is suitable for the above purpose; however, it is necessary to fully understand the fate of beta-1,3-D-glucan in patients' blood.

Animals↗
PubMedDate not supplied

Quantitation of endotoxin in blood from patients with meningococcal disease using a limulus lysate test in combination with chromogenic substrate.

The levels of endotoxin in blood were determined using the Limulus lysate test in combination with a chromogenic substrate. Plasma was analyzed from four patients with fatal meningococcal septicaemia and from one patient who survived meningococcal meningitis. All septicaemia patients showed high levels of endotoxin in their blood during the early stage of their disease. In two of these patients, blood samples collected at intervals of two days revealed a gradual disappearance of measurable endotoxin from the circulation. The patient with meningitis had no clinical signs of circulatory deficiency or coagulopathy and was consistently negative for endotoxin using this test procedure. Pretreating the plasma with heat and alkali and combining the Limulus lysate test with a chromogenic substrate seem to provide a sensible method for the detection and quantitation of endotoxin in blood.

Adolescent↗
PubMed1978

The limulus lysate test. A rapid test for diagnosis of Pseudomonas keratitis or endophthalmitis.

The limulus amebocyte lysate test has been shown to be a highly sensitive indicator of endotoxin. Our studies showed that as little as 5 ng of endotoxin could be detected in aqueous or vitreous humor in vitro, although 10 microgram endotoxin injected into the aqueous could not be detected. Subsequent studies showed that by diluting the aqueous equally with saline solution, this inhibitory effect could be overcome. Detection of endotoxin elaborated from Pseudomonas aeruginosa was made as early as 24 hours after induced Pseudomonas keratitis or endophthalmitis, whereas staphylococcal-induced keratitis or endophthalmitis gave negative results. Positive cultures using trypticase soy broth or agar slants were observed on all infected animals. Thus, this technique should have ready application for rapid detection of Pseudomonas keratitis or endophthalmitis.

Animals↗
PubMed1978

Rapid diagnosis of gram-negative bacterial meningitis by the Limulus endotoxin assay.

The Limulus amoebocyte lysate endotoxin assay was evaluated as a method for rapid diagnosis of acute bacterial meningitis in a series of 305 patients. The results of Limulus assays on cerebrospinal fluid (CSF) samples from these patients were compared with the results for each patient of routine bacterial cultures and Gram stains. Positive Limulus tests were obtained on initial CSF specimens from 84% of patients with culture-proven bacterial meningitis, including all patients with meningitis due to gram-negative organisms. Initial Gram-stained smears revealed the presence of organisms in 68% of the patients. One patient with pneumococcal meningitis had a weakly positive Limulus assay, whereas patients with meningitis due to other gram-positive organisms, those with aseptic meningitis, or patients without meningitis had negative CSF Limulus tests. The Limulus assay also demonstrated the persistence of endotoxin in the CSF of certain patients during antibiotic therapy, especially patients with Haemophilus influenzae meningitis. The Limulus test proved to be a rapid, reliable indicator of the presence of gram-negative organisms in the CSF of patients suspected of acute bacterial meningitis.

Adult↗