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PubMed1979

Clinical forms of endotoxemia in burns.

Two groups of patients in whom endotoxemia was suspected were studied with respect to the laboratory data, clinical picture and autopsy findings. The first group showed the signs of gram-negative septicemia (age range 3 to 54 years with burns of 25 to 80% TBSA) and the second group (age range 20 to 76 years with burns not greater than 20% TBSA) showed an unexpected deterioration of the patient's condition. In 15 patients we established the diagnosis of endotoxemia on positive Limulus test as well as on other laboratory examinations and on the clinical signs. Five different forms of endotoxemia were distinguished varying in the evoking circumstance, clinical manifestation and pathological findings at autopsy: (1) the so called laboratory form--all survived; (2) the pulmonary form displaying the most brief and fulminant course--all died; (3) the form of disseminated intravascular coagulopathy; (4) the form of endotoxic shock due to administration of antibiotics; (5) the form of endotoxic shock as terminal stage of protracted gram-negative septicemia--the fatal outcome was inevitable in all cases. The authors stress the importance of minding and preventing all circumstances that might provoke the endotoxic (septic) shock.

Adolescent↗
PubMed1986

Endotoxin level of sterile injection solutions and substitution fluid for hemofiltration in Japan and Australia.

The endotoxin contamination levels of sterile distilled water and saline solutions used for injection and hemofiltration in Japan and Australia were examined with a colorimetric limulus test using a chromogenic substrate. The endotoxin levels of injection solutions in most products from both countries measured against E. coli 0111:B4 endotoxin or USP reference standard endotoxin were less than 0.2 pg or 0.0006 endotoxin units (EU) X ml-1. Only 2 solutions in 30 from both countries showed higher levels: 7.7 and 10.8 pg for E. coli 0111:B4 endotoxin X ml-1 (0.02 and 0.03 EU X ml-1). Even these higher values were well below the level recommended by the draft guideline published by the United States Food and Drug Administration (FDA). The endotoxin contamination level of a Japanese hemofiltration substitution fluid ranged from 8.2 to 9.2 pg E. coli 0111:B4 endotoxin X ml-1 (0.024-0.027 EU X ml-1).

Australia↗
PubMed1990

[Methodological and clinical evaluation of a microplate assay of endotoxin in whole blood sample].

We have developed a simple, rapid method for determining endotoxin in whole blood using a microtiter plate. The samples were pretreated with 0.66 mol/l nitric acid containing 0.25% Triton X-100; the supernatants were placed in a microtiter plate well, and incubated with endotoxin-specific chromogenic limulus test reagent (Endospecy; Seikagaku Corporation). The calibration curve was linear from 0 to 50 pg/ml of Escherichia coli 0111: B4 endotoxin with a variation coefficient less than 5%. The recovery of endotoxin added to human and experimental animals was about 100% without being affected by dilution. The normal endotoxin level in human whole blood was less than 10 pg/ml in reference to E. coli 0111: B4 endotoxin. The results in 120 patients with various diseases correlated well with those determined by a conventional method using plasma as a sample (r = 0.877; p less than 0.05). Whole blood sample has advantages over plasma in that it allows all endotoxin in a sample to be determined, requires a smaller amount of sample, and has less risk of contamination.

Animals↗
PubMed2004

Potential use of (1,3)-beta-D-glucan as target of diagnosis and treatment of keratomycosis.

PURPOSE: Major problems in the management of keratomycosis stem from the difficulty of its diagnosis and limited choice of antifungal agents. In the present paper we propose a new method of detecting (1,3)-beta-D-glucan, one of the major components of fungal cell wall, in tears from an animal model of keratomycosis. In addition, we investigated the efficacy of topical application of micafungin, a new antifungal agent that inhibits the activity of (1,3)-beta-D-glucan synthase in this animal model. METHOD: Candida albicans (5 x 10(5) organisms) was inoculated into the corneal stroma of 20 New Zealand White rabbits. The animals were randomly assigned to two groups and treated with subconjunctival injection of 0.5 mL of saline or 0.1% micafungin every day for 3 weeks. The clinical course of keratomycosis in both groups was compared. Before and 3 weeks after the injection of saline or micafungin, 5 microL of tears in each eye were collected by capillary tube. The concentration of (1,3)-beta-D-glucan was quantitatively measured by modified Limulus test. RESULTS: The concentration of (1,3)-beta-D-glucan was significantly higher in keratomycosis model animals than in controls (mean +/- SD, 17.4 +/- 9.4 pg/mL and 2.8 +/- 1.8 pg/mL, respectively) at 21 days after treatment. Subconjunctival injection of micafungin had no significant effect on ocular lesions of keratomycosis until 9 days, after which ocular lesions significantly improved. Subconjunctival application of micafungin decreased the concentration of (1,3)-beta-D-glucan in tears to 4.9 +/- 3.0 pg/mL at 21 days after treatment. CONCLUSIONS: Increased levels of (1,3)-beta-D-glucan in tears were detected in this model of keratomycosis. Measuring the concentration of (1,3)-beta-D-glucan in tears may be a reliable noninvasive method for the diagnosis of keratomycosis. Topical application of micafungin was effective in the treatment of keratomycosis.

Animals↗
PubMed1992

Chemical and biological properties of endotoxin from Leptospira interrogans serovars canicola and icterohaemorrhagiae.

1. Endotoxin-like activity was extracted with phenol-chloroform-petroleum either (PCP) from Leptospira interrogans serovars icterohaemorrhagiae and canicola. Chemical analysis of leptospiral cells obtained from the PCP extract indicated the following distribution of lipopolysaccharide (LPS), protein and polysaccharide in mg/ml: 3.0, 4.5 and 1.0 for icterohaemorrhagiae and 3.3, 5.6 and 1.5 for canicola. 2. The preparations presented several biological activities: positive Limulus test (1.0 pg/ml) for icterohaemorrhagiae and canicola PCP extract and 0.5 pg/ml for E. coli O111:B4 LPS, lethality for chicken embryos (LD50 45, 25 and 1.0) for icterohaemorrhagiae, canicola and E. coli O111:B4 LPS, pyrogenicity in rabbits with an average increase in rectal temperature of 0.6 degrees C, 0.9 degrees C and 2.2 degrees C for canicola, icterohaemorrhagiae and E. coli O111:B4 LPS, reacted with complement inhibiting the lysis of sheep red blood cells, 62%, 75% and 90% for 2.0 micrograms/ml of icterohaemorrhagiae, canicola PCP extract and E. coli O111:B4 LPS. The PCP extract showed no cytotoxicity on chicken embryo fibroblasts and epithelial cells. 3. These results demonstrate that Leptospira endotoxin activity is similar to E. coli O111:B4 lipopolysaccharide.

Animals↗
PubMed1979

Platelet and fibrinogen production: relative sensitivities to endotoxin.

The relative sensitivities of platelet and fibrinogen production to endotoxin were determined in male New Zealand rabbits. E. coli endotoxin was administered in single intravenous doses of 0.1 to 50.0 mu/g/kg body mass. 75SeM was injected 5, 12, or 18 hr after endotoxin, and the percent incorporation into platelets and fibrinogen was used to measure thrombopoiesis and fibrinogen synthesis. Leukopenia occurred after the infusion of endotoxin at all dose levels; the lowest dose that caused thrombocytopenia was 0.5 microgram/kg. Endotoxin was detected with the Limulus test in plasma of animals that received 5 to 50 microgram/kg. The stimulation of fibrinogen and platelet production, as well as the postinfusion decrease in platelet count, correlated directly with the log of the dose of endotoxin infused. The lowest dose of endotoxin that stimulated platelet and fibrinogen production was 0.5 microgram/kg. In animals that received this dose of endotoxin, increased fibrinogen production was detected only when 75SeM was injected 5 hr later. In animals injected 5, 12, or 18 hr later. Increased platelet production was detected at these two doses only when 75SeM was injected 18 hr after endotoxin. The data indicate that fibrinogen and platelet production have similar sensitivities to endotoxin, although the time course of stimulation is different for these two blood components.

Animals↗
PubMed1984

Circulating lipid A antibodies despite absence of systemic endotoxemia in patients with Crohn's disease.

Lipid A is a component common to endotoxins of gram-negative bacteria. It has been suggested that the gut wall of patients with inflammatory bowel disease is more permeable to luminal bacterial macromolecules which may stimulate the gut-associated lymphoid tissue. We therefore investigated 40 patients with Crohn's disease (CD) and 23 patients with ulcerative colitis (UC) with respect to their lipid A antibody titers and presence of endotoxins (Limulus test). Both tests were performed simultaneously using peripheral venous blood. Systemic endotoxin was demonstrated in only two of the patients. The lipid A antibody titers in the CD patients were significantly higher than either in patients with UC or in 42 healthy controls. Lipid A titers of patients with UC did not differ from those of controls. Titers of lipid A were significantly higher in patients with active Crohn's disease than in patients with inactive disease. It is concluded that systemic endotoxemia occurs rarely in patients with inflammatory bowel disease; however, despite this virtual absence of endotoxins in their peripheral blood, patients with CD show an increase in antibody formation against lipid A. This is suggestive of an altered immunologic reactivity against endotoxins in patients with CD and may be relevant to the pathogenesis of this disease.

Adolescent↗
PubMed1995

Plasma endotoxin in typhoid fever.

Plasma endotoxin contents of the patients with sepsis or typhoid fever were measured by two sophisticated chromogenic limulus tests; Endospecy and Toxicolor tests. Endospecy test is the endotoxin-specific test and Toxicolor is responsible for both endotoxin and (1,3)-beta-D-glucan. Plasma was pretreated by our new PCA method which resolved the problem as to the detection of a lesser amount of plasma endotoxin when pretreated by the conventional PCA method. Although Toxicolor values have been reported to exceed more than the Endospecy value, under complicated pathophysiological situations, almost all specimens of these patients had a similar value, except in one expired septic shock case. In 18 typhoid fever cases, Salmonella typhi was isolated only from the bile in 5 cases, however endotoxemia occurred in 11 cases (61.1%). Within the first 4 days, the incidence of endotoxemia was higher (10/14, 71.4%). These results suggest that endotoxin assay seemed to be a useful tool for the diagnosis of typhoid fever.

Bacteremia↗
PubMed1990

Endotoxemia in patients on hemodialysis.

We examined endotoxins and limulus amebocyte lysate-reactive material (LAL-RM) in serum from 87 patients on hemodialysis, using the conventional chromogenic limulus test (CCLT) and a new specific endotoxin assay with factor G-free LAL (endotoxin-specific test: EST). All patients with regenerated cellulose dialyzers had increased CCLT values, whereas the EST values were not higher than in controls. This discrepancy can be explained by the LAL-RM which is cellulose-derived and cross-reacts with LAL via factor G. Using EST for measurements of endotoxin, 6 patients out of 87 had pathological endotoxemia and all of these patients were associated with either cirrhosis, infection, or malignancy. Some patients who had no complications showed fever before or during dialysis, but they did not have high endotoxin levels. EST is useful for the diagnosis of endotoxemia in patients on hemodialysis because of the presence of LAL-RM in serum. Endotoxemia is rare in patients on hemodialysis, if they are not associated with infection, cirrhosis, or other complications.

Adult↗
PubMed1980

Liberation of endotoxin from Escherichia coli by addition of antibiotics.

The liberation of endotoxin from Escherichia coli by the addition of antibiotics was investigated using a new method of limulus test, i.e. dry up method. The addition of the bactericidal antibiotic, aminobenzylpenicillin or streptomycin sulfate, to the suspension of Escherichia coli (about 10(6)/ml) increased the concentration of endotoxin in the suspension about eight to nine times during a 3-hour period after the addition of antibiotic accompanying with the concomitant decrease of viable cell counts. The addition of the bacteriostatic antibiotic, tetracycline hydrochloride, also increased the endotoxin level, but this increase was rather mild. On the other hand, in the case of the addition of polymyxin B sulfate, there was no evident increase of the endotoxin level. This ability of polymyxin, i.e. the ability to suppress the elevation of endotoxin levels, was shown when polymyxin in the concentration of more than 2.5 micrograms/ml was added against about 10(6)/ml Escherichia coli and was also shown by the combinational use of polymyxin with aminobenzylpenicillin or streptomycin. Furthermore, the endotoxin-inactivating ability of polymyxin was shown in the study where extracted lipopolysaccharide was used in place of viable cells.

Anti-Bacterial Agents↗
PubMed1997

(1-->3)-beta-D-glucan determination in rat organs with limulus coagulation factor G.

One cause of a false positive limulus test after surgery or hemodialysis has been identified as extrinsic (1-->3)-beta-D-glucan which was derived from surgical gauze or cellulose dialyzer. However, there have been no investigations concerning intrinsic factors and the presence of (1-->3)-beta-D-glucan in mammalian organs. In this study, (1-->3)-beta-D-glucan in homogenate of various rat organs and stool was measured by a Gluspecy test (G test) using factor G, which specifically reacts with (1-->3)-beta-D-glucan. In small intestine and lung, large amounts of factor G-activating substance were observed and identified as (1-->3)-beta-D-glucan by a digestion study using end-(1-->3)-beta-D-glucanase. However, only very small amounts of (1-->3)-beta-D-glucan were found in the kidney, spleen, vena cava, aorta, thymus, heart and liver. In serum and plasma, no (1-->3)-beta-D-glucan was observed. On the other hand, extremely large amounts of (1-->3)-beta-D-glucan were found in stool. Minute amounts of (1-->3)-beta-D-glucan were observed in a variety organs except for the small intestine and lung. High levels of (1-->3)-beta-D-glucan found in the small intestine might be traced to contamination by stool in the small intestine, and such levels in the lung might derive from macrophages which have trapped (1-->3)-beta-D-glucan in the air.

Animals↗
PubMed1989

Perioperative change of plasma endotoxin levels in early infants.

As a preliminary study to elucidate the relationship of endotoxemia to postoperative morbidity, the plasma endotoxin levels in 64 surgical neonates were quantitated by the chromogenic limulus test (Toxicolor test; Seikagaku Kogyo, Tokyo, Japan). The preoperative levels of plasma endotoxin were 64 +/- 59 pg/mL in the group of infants with perforated peritonitis (n = 9), 63 +/- 51 pg/mL in the group of infants with gastroschisis (n = 7), and 15 +/- 16 pg/mL in the group of infants with ileus (n = 28), while the mean level was 6 +/- 5 pg/mL in the remaining 20 surgical neonates who had no signs of ileus or peritonitis. In the serial determination of plasma endotoxin in 28 neonates, the levels on the first postoperative day increased significantly compared with the preoperative levels (16 +/- 18 pg/mL to 46 +/- 25 pg/mL, P less than .01). They decreased gradually to 8 +/- 5 pg/mL within a week in 15 neonates who had no postoperative complications. However, in 13 neonates who had postoperative complications such as wound infection or postoperative ileus, the postoperative levels of plasma endotoxin increased to a much higher level and remained there. In this article the relationship of clinical endotoxemia to postoperative thrombocytopenia and hyperbilirubinemia is analyzed, and the usefulness of evaluating endotoxemia in surgical neonates is discussed.

Bacterial Infections↗
PubMed1988

[Study on mechanism of onset of endogenous endotoxemia during obstructive jaundice--with special reference to involvement of biliary infection].

To clarify how biliary infections affect onset of endogenous endotoxemia during obstructive jaundice, I tried to review the clinical result of 104 cases of obstructive jaundice, and conducted a Limulus test of portal and peripheral blood in 20 cases of obstructive jaundice and an animal study using 38 rabbits. In cases of obstructive jaundice complicated by biliary infections, clinical improvement of jaundice became significantly unfavorable, and the outcome of surgical operation was significantly inferior to the cases without biliary infections. The endotoxin positive rate in the portal blood of obstructive jaundice was 65% (13 of 20 cases), among which 10 cases (79.6%) was also positive in the peripheral blood. Of these 10 cases, 7 cases manifested endogenous endotoxemia with no infectious focus, and prognosis of these cases was poor. The endotoxin positive rate in portal blood of obstructive jaundice group was also significantly higher than that of non-jaundice group in animal study, and when the reticuloendothelial system was blocked, the endotoxin positive rate in the peripheral blood showed an increasing tendency. In the animal group with experimental cholangitis, all the endotoxin positive animals in the portal blood were also positive in the peripheral blood. This result suggests that biliary infections accelerate a decrease in the reticuloendothelial function during obstructive jaundice. From these results, endogenous endotoxemia seems to affect the onset of various complications during obstructive jaundice and unfavorable prognosis.

Adult↗
PubMed1986

Clearance of endotoxin from blood of rabbits injected with staphylococcal toxic shock syndrome toxin-1.

This study was undertaken to examine some of the properties of staphylococcal toxic shock syndrome toxin 1 (TSST-1) with regard to the clearance of endotoxin from blood. The concentration of endotoxin in blood was measured by using a chromogenic limulus test and modified perchloric acid method. When TSST-1, which produces fever in rabbits, was injected (100 ng/ml or 100 micrograms/ml per kg) intravenously (i.v.) into the animals, no measurable level of endotoxin was detected in the blood. In control animals, which were given 5 micrograms of endotoxin per ml per kg i.v., endotoxin could be detected in the blood at rapidly declining levels. These results suggested that TSST-1 might not lead bacterial endotoxin from other body sites into the blood. When the animals were given TSST-1 (1 to 100 ng/ml per kg) i.v. and then endotoxin (5 micrograms/ml per kg) i.v. 4 h later, endotoxin was detected in the blood at a high level, depending on the dose of TSST-1 injected. These results showed that TSST-1 inhibited the clearance of endotoxin in the blood; this clearance is thought to be mainly done by the reticuloendothelial system. In the animals given TSST-1 (100 ng/ml per kg) and endotoxin (5 micrograms/ml per kg) simultaneously, the endotoxin level in the blood was found to be higher than that in control animals given endotoxin only but lower than that in the animals given TSST-1 and then endotoxin at the same doses.

Animals↗
PubMed1995

Portal and peripheral endotoxins in patients with esophageal varices undergoing surgery.

Under the hypothesis that portal endotoxemia, which has been considered evidence of endogenous endotoxemia, is actually a false-positive reaction of the Limulus test, the conventional method of determination, Toxicolor (TOX) and a new endotoxin-specific method, Endospecy (ES) were investigated, whereby portal endotoxemia was reexamined. Peripheral and portal blood samples were collected from 12 patients at various intervals during surgery for esophageal varices, and then evaluated by TOX and ES, the normal values of which are under 60.0 pg/ml and under 9.8 pg/ml, respectively. The mean peripheral and portal endotoxin (Et) levels by TOX were 23.1 pg/ml and 38.9 pg/ml, 1.5 h after the start of surgery, which continued to increase thereafter, the corresponding levels being 48.1 pg/ml and 58.7 pg/ml 8 h after the start of surgery, respectively. The portal Et levels were significantly higher by ES, indicating portal endotoxemia, the mean peripheral and portal Et levels being 6.9 pg/ml, and 6.5 pg/ml, 1.5 h after the start of surgery, these levels showing changes within a similar range. The levels determined by the conventional TOX method increased with time, to a portal Et level of 5.0 pg/ml 8 h after the start of surgery, whereas those determined by ES changed within the same range suggesting that the onset of portal endotoxemia primarily involves the G-factors and that endogenous endotoxemia does not occur.

Blood Specimen Collection↗
PubMed2004

Microbial dustiness and particle release of different biofuels.

Exposure to organic dust originating from biofuels can cause adverse health effects. In the present study we have assessed the dustiness in terms of microbial components and particles of various biofuels by using a rotating drum as a dust generator. Microbial components from straw, wood chips, wood pellets and wood briquettes were quantified by several methods. Excellent correlations (r >/= 0.85, P < 0.0001) were found: between lipopolysaccharide (LPS) (as determined by 3-hydroxy fatty acid analysis) and endotoxin (as determined by a Limulus test), cultivable bacteria, total number of bacteria and muramic acid; between endotoxin and cultivable bacteria, total number of bacteria and muramic acid; between total number of bacteria and muramic acid; between cultivable fungi and total number of fungi. Straw was dustier than the other biofuels in terms of actinomycetes, bacteria, muramic acid, endotoxin, LPS, particle mass and number of particles. One of the wood chips studied and the straws had comparatively high dustiness in terms of fungi, while both wood pellets and wood briquettes had comparatively low dustiness in terms of all microbial components. An initially high particle generation rate of straw and wood chips decreased over time whereas the particle generation rate of wood briquettes and wood pellets increased during a 5 min rotation period. Particles of non-microbial origin may be the determining factor for the health risk in handling briquettes and pellets. Straw dust contained significantly more microorganisms per particle than did wood chip dust, probably because bacteria were most abundant in straw dust. The concentrations of endotoxin and fungi were high in wood and straw dust; dust from one of the straws contained 3610 EU/mg and dust from one of the chips contained 7.3 x 10(6) fungal spores/mg. An exposure to 3 mg of straw or wood chips dust/m(3) (the Swedish and Danish OEL of unspecific inhalable dust) could cause exposures to endotoxin and fungi higher than levels were health symptoms are seen to develop. The very different levels of dustiness in terms of particles and microbial components of different biofuels shows that dustiness is an important health-relevant factor to consider when choosing among biofuels and when designing worksites for handling of biofuels.

Air Microbiology↗
PubMed2003

A silkworm larvae plasma test for detecting peptidoglycan in cerebrospinal fluid is useful for the diagnosis of bacterial meningitis.

The silkworm larvae plasma (SLP) test has been established based on a cascade reaction triggered by either peptidoglycan or (1, 3)-beta-D-glucan to produce melanin. We applied this test to the diagnosis of bacterial meningitis. Cerebrospinal fluid (CSF) obtained from patients with bacterial meningitis due to gram-positive bacteria, gram-negative bacteria, or fungi, showed positive reactions to the test. In contrast, CSF from patients with viral meningitis or noninfectious illnesses gave negative reactions. Therefore, this test seems to be useful for diagnosis of bacterial and fungal meningitis. When this test was used together with two types of limulus tests, an endotoxin-specific test, and a conventional test, meningitis was further characterized as gram-positive, gram-negative or fungal meningitis. The SLP test requires a computerized instrument for quantitative colorimetric measurement. A qualitative alternative of this test also can be accomplished by visually observing the darkening color. Thus, this method can be applied for simple and rapid diagnosis of meningitis.

Adolescent↗
PubMed1977

Studies on the toxicity of a variety of human vaccines as measured by laboratory tests.

The results obtained from our laboratory tests for pyrogenic activity on 8 bacterial vaccines are compared with the clinical reactogenicity of the same 8 vaccines in man. It is concluded that, although none of the tests correlate exactly with clinical reactogenicity, the rabbit pyrogenicity test provides the best indication of such activity. The limulus test, although valuable as a means of detecting contaminant endotoxin in pyrogen free preparations appears to be of little value as a gauge of pyrogenicity in bacterial vaccines.

Animals↗