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PubMed1993

[Detection of endotoxin in plasma: specificity and value for development and prognosis of infection].

A number of problems may be involved in the detection of endotoxin in plasma of patients using LAL (Limulus amebocyte lysate). When collecting blood or processing samples, contamination with endotoxin or its adsorption to material must be avoided. In our laboratory a kinetic LAL microtiter assay was developed that takes into account plasma-related interferences with the LAL endotoxin reaction by performing an internal standardization in each sample. Negative results do not absolutely exclude the involvement of endotoxins in the underlying disease. High levels of endotoxins do not necessarily reflect the severeness of the clinical status of the patient. Due to nonendotoxin-specific reactions with some complete lysates, false-positive levels may result, e.g., following immunoglobulin therapy. In spite of these limitations, the LAL test remains a valuable tool in the evaluation of gram-negative infections.

Endotoxins↗
PubMed2005

A rapid highly-sensitive endotoxin detection system.

This paper presents a rapid, highly-sensitive, and low-cost method of endotoxin quantification based on the use of stress-responsive magnetoelastic sensors, that monitor the gel formation (viscosity change) of the Limulus Amoebocyte Lysate (LAL) assay in response to endotoxin. Ribbon-like magnetoelastic sensors, 12.7 mm x 6 mm x 28 microm, were immersed in a LAL assay after mixing with test samples of variable endotoxin concentration, and the decrease in resonance amplitude of the sensor was recorded as a function of time. Experimental results show excellent correlation between endotoxin concentration and the maximum clot rate, determined by taking the minimum point of the first derivative of the amplitude-time curve, as well as the clotting-time, defined as the time that corresponds to the maximum clot rate. Using a LAL gel-clot assay with a sensitivity of 0.06 EU/ml (EU: endotoxin unit), the magnetoelastic sensor based technology can detect the presence of endotoxin at 0.0105 EU/ml in test requiring approximately 20 min. Unlike optical methods used for determining endotoxin concentration, the color of the test solution does not impact the magnetoelastic sensor measurement. Due to the small size of the sensor reader electronics and low cost, the magnetoelastic sensor based endotoxin detection system is ideally suited for wide-spread use in endotoxin screening for sepsis prevention.

Biosensing Techniques↗
PubMed1979

The use of peroxidase-labelled Limulus polyphemus agglutinin for the histochemistry of sialic acid-containing glycoproteins in light microscopy.

The usefulness of a lectin, Limulus polyphemus agglutinin (LPA) has been tested in a series of mammalian tissues with sialic acid-containing glycoproteins. In nearly all the tissues employed, the positive peroxidase-labelled LPA diaminobenzidine (LPA-PO-DAB) reaction of various histological structures was markedly diminished in intensity or abolished, following digestion with neuraminidase. In the same tissues, sialic acid added with LPA-PO abolished the LPA-PO-DAB reaction or notably suppressed its intensity. In the majority of the tissues tested, the LPA-PO-DAB-Alcian Blue (AB) (pH 1.0 or 2.5) procedures appear to be useful dual staining methods which enable one to colour selectively sialic acid-containing and other acidic carbohydrates. In view of the endogenous peroxidase activity in particular histological structures, however, appropriate control staining procedures should be performed when the LPA-PO-DAB procedure is employed, either alone or in combination with AB procedures, to determine the histochemical properties of sialic acid-containing glycoproteins.

Animals↗
PubMed2004

Investigation into the interaction of the bacterial protease OmpT with outer membrane lipids and biological activity of OmpT:lipopolysaccharide complexes.

Outer-membrane proteases T (OmpT) are important defence molecules of Gram-negative bacteria such as Escherichia coli found in particular in clinical isolates. We studied the interaction of OmpT with the membrane-forming lipids phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) from the inner leaflet and lipopolysaccharide (LPS) from the outer leaflet of the outer membrane. These investigations comprise functional aspects of the protein-lipid interaction mimicking the outer-membrane system as well as the bioactivity of LPS:OmpT complexes in the infected host after release from the bacterial surface. The molecular interaction of the lipids PE, PG, and LPS with OmpT was investigated by analysing molecular groups in the lipids originating from the apolar region (methylene groups), the interface region (ester), and the polar region (phosphates), and by analysing the acyl-chain melting-phase behaviour of the lipids. The activity of OmpT and LPS:OmpT complexes was investigated in biological test systems (human mononuclear cells and Limulus amoebocyte lysate assay) and with phospholipid model membranes. The results show a strong influence of OmpT on the mobility of the lipids leading to a considerable fluidization of the acyl chains of the phospholipids as well as LPS, and a rigidification of the phospholipid, but not LPS head groups. From this, a dominant role of the protein on the function of the outer membrane can be deduced. OmpT released from the outer membrane still contains slight contaminations of LPS, but its strong cytokine-inducing ability in mononuclear cells, which does not depend on the Toll-like receptors 2 and 4, indicates an LPS-independent mechanism of cell activation. This might be of general importance for infections induced by Gram-negative bacteria.

Animals↗
PubMed2006

Lipopolysaccharide found in aseptic loosening of patients with inflammatory arthritis.

Aseptic loosening of orthopaedic implants occurs in the absence of clinical signs of infection. Nevertheless, bacterial endotoxins derived from subclinical infections, systemic sources, or the implant manufacturing process may contribute to aseptic loosening. Also, the rate of implant infection is greater in patients with inflammatory arthritis than in patients with osteoarthritis. We hypothesized that lipopolysaccharide, the classic endotoxin derived from gram-negative bacteria, is more prevalent in periprosthetic tissue surrounding aseptically loose implants in patients with inflammatory arthritis than in patients with osteoarthritis. To test this, we used a modified Limulus amebocyte assay not affected by beta-glucan-like molecules in mammalian tissues. Lipopolysaccharide rarely was detected in periprosthetic tissue from patients with osteoarthritis and aseptic loosening (one of six patients). In contrast, lipopolysaccharide was detected despite the absence of any clinical signs of infection in peri-prosthetic tissue from all four patients with inflammatory arthritis (rheumatoid arthritis, juvenile rheumatoid arthritis, and systemic lupus erythematosus). Lipopolysaccharide also was detected in two patients with gram-negative infections, who were included as positive control subjects. Endotoxins derived from low-grade or systemic bacteremia may be important contributors to aseptic loosening particularly in patients with autoimmune conditions such as inflammatory arthritis.

Adult↗
PubMed1990

Laboratory methods in genitourinary medicine. Methods of diagnosing gonorrhoea.

Gonorrhoea is normally diagnosed presumptively by the presence of intracellular Gram-negative cocci on a Gram stain and confirmed by culture of the causative organism, Neisseria gonorrhoeae. Alternative methods have been evaluated extensively for the detection of gonococci in clinical specimens including immunological techniques such as ELISA and immunofluorescence, DNA probes, genetic transformation and the limulus lysate assay. Some of these tests have proved as sensitive and specific for the detection of gonorrhoea in symptomatic men as the Gram stain but offer no advantage in time or cost. In women, no test has been found that shows a sensitivity and specificity sufficiently adequate for clinical use. Culture in men and women remains the method of choice for diagnosis. In addition the need to obtain the infecting organism for antibiotic susceptibility testing has not been overcome. In contrast, the rapid identification of N gonorrhoeae can be achieved within four hours using either monoclonal antibodies or by the detection of preformed enzymes. New methods for both the detection and identification of N gonorrhoeae should be carefully evaluated particularly for use in cases of child and sexual abuse where medico-legal problems may arise.

Adult↗
PubMed2000

Induction of tissue factor procoagulant activity in myelomonocytic cells inoculated by the agent of human granulocytic ehrlichiosis.

Human granulocytic ehrlichiosis (HGE) is a recently recognized rickettsial tick-borne febrile illness that may occasionally be complicated by coagulopathy. The agent of HGE (aHGE) is an obligate intracellular pathogen, which replicates in endosomes within neutrophils and their precursors. We hypothesized that aHGE might cause DIC via induction of monocyte tissue factor procoagulant activity (TF PCA). Peripheral blood mononuclear cells (PBMNC) and HL-60 cells were used to model the effect of aHGE infection on monocytes/macrophages. Mononuclear cells inoculated with aHGE in vitro demonstrated approximately a 12-15-fold increase in TF PCA, with peak activity occurring at 8-12 h. HL-60 cells inoculated with aHGE also manifested a 4-6 fold induction of TF PCA, with maximal activity occurring at about 8 h. By comparison, E. Coli lipopolysaccharide (LPS) also induced an increase in TF PCA of an equivalent magnitude, and with a similar time course. Induction of TF did not require inoculation of HL-60 cells with live organism, since heat-inactivated aHGE still stimulated TF PCA expression in the target cells. Furthermore, filtered supernatants from heat-inactivated organisms induced TF PCA suggesting that the effect is due to a soluble mediator produced by the organism. Although aHGE is a gram negative organism, the soluble mediator did not appear to be classic endotoxin in that the supernatants tested negative for endotoxin by the Limulus Amoebocyte assay, and polymixin had no inhibitory effect on aHGE supernatants. We conclude that aHGE induces cells of the myelo-monocytic lineage to synthesize TF, which may contribute to the clinical coagulopathy that can be observed in this condition. An atypical soluble mediator or cellular component of the organism appears to be critically important in TF induction by aHGE.

Blood Coagulation↗
PubMed1994

Concordance of endotoxemia with gram-negative bacteremia in patients with gram-negative sepsis: a meta-analysis.

The Limulus amebocyte lysate (LAL) assay is a sensitive method for detecting endotoxin. Using gram-negative (GN) bacteremia as the basis for comparison, concordance with endotoxemia in 45 studies could be expressed as an odds ratio. Calculation of summary odds ratios by the Mantel-Haenszel-Peto method indicated that the concordance of the results was no higher by the chromogenic LAL assay than by the gelation version, and the sensitivity was improved by only 11% (62 versus 51%). Endotoxemia was detected in 77 (68%) of 114 patients with bacteremia caused by an organism that was not a member of the family Enterobacteriaceae, whereas endotoxemia was detected in only 120 (45%) of 269 patients with bacteremia caused by a member of the family Enterobacteriaceae or an anaerobe (P < 0.001). This difference was also apparent for patients with GN bacteremia for whom a fatal outcome had been recorded. The prevalence of GN bacteremia in the tested population and the type of etiological agent are critical and previously unrecognized variables which affect the interpretation of the LAL test in patients with suspected sepsis.

Bacteremia↗
PubMed1994

[Evaluation of (1-3)-beta-D-glucan in aspergillosis and cryptococcosis].

(1-3)-beta-D-Glucan (beta-glucan) is a major structural component of fungi. The G test is a direct method to detect beta-glucan using fractionated (1-3)-beta-D-glucan-sensitive component, factor G, eluted from the limulus lysate. Previously, we reported that the G test is a more sensitive method than the mannan detection assay for the serological diagnosis of Candida infection. In this study, we discuss beta-glucanemia in patients with pulmonary aspergillosis and cryptococcosis. The concentration of beta-glucan was less than 10 pg/ml in 9 of 10 cases of pulmonary cryptococcosis, except for one case receiving hemodialysis (16.5 pg/ml). beta-Glucan increased in 3 cases of invasive pulmonary aspergillosis (27-937 pg/ml). Galactomannan antigen was positive in all of those cases. In 8 cases of aspergilloma, which showed fungus ball on roentgenogram, the mean concentration of beta-glucan was 67.1 +/- 92.7 pg/ml. Two of 8 cases were positive for galactomannan antigen. One of three cases of PAIC (productive aspergilloma on the inner wall of a cavity) and one case of chronic necrotizing pulmonary aspergillosis showed slightly increased levels of beta-glucan and positive results of galactomannan antigen test.

Adult↗
PubMed1988

Endotoxaemia: an early predictor of septicaemia in febrile patients.

In 473 consecutive febrile patients a sensitive and rapid chromogenic limulus assay was used to assess the value of endotoxaemia versus bacteraemia for predicting development of the syndrome of septicaemia. In each patient three blood specimens for culture and endotoxin testing were obtained at the onset of fever. Blood pressure, urinary output, and the occurrence of thrombocytopenia and metabolic acidosis were recorded prospectively during three days of follow-up. Septicaemia developed in 19 patients (4%). The sensitivity, specificity, and likelihood ratio for a positive result with the endotoxin assay were 79%, 96%, and 20, respectively. The corresponding indices for bacteraemia were 89%, 78%, and only 4. The results suggest that endotoxaemia is a clinically valid indicator for impending gram-negative septicaemia (positive predictive value 48%) and that the absence of endotoxaemia virtually rules out the risk that septicaemia will ensue (negative predictive value 99%).

Adolescent↗
PubMed1990

Detection of endotoxiuria in polycystic kidney disease patients by the use of the Limulus amebocyte lysate assay.

Urinary tract infections (UTI) due to gram-negative bacteria are a serious complication in patients with polycystic kidney disease (PKD). Endotoxin, a component of the cell wall of gram-negative bacteria, has been reported to be pro-cystogenic in experimental animals. Because endotoxin levels in urines (endotoxiuria) from PKD patients have not been reported, the Limulus amebocyte lysate (LAL) assay, which detects picogram quantities of endotoxin, was used to probe for this cyst-promoting chemical. Fifteen PKD patients (seven females, eight males), asymptomatic for UTI, were tested and compared with 10 female and 10 male controls. All urines were assessed for (1) evidence of aerobic bacteria by routine quantitative cultures, (2) bacteria and pyuria by microscopic examination of gram-stained urine, and (3) bacterial endotoxin by the LAL assay. LAL tests were positive in 73% (11/15) of PKD patients, but only 25% (5/20) of controls (P = 0.0058). There was no significant difference in test positivity between PKD females (71%) and males (75%). There was no correlation of age, degree of renal dysfunction, or urine osmolality with endotoxiuria. Routine quantitative cultures were negative for gram-negative bacteria in PKD patients and all controls (except one female), as were microscopic findings for intact bacteria and pyuria. Thus endotoxiuria, in the absence of classical signs, symptoms, and microbiological findings of UTI, raises the possibility that endotoxin is available intrarenally to promote cystogenesis even before a potential susceptibility of PKD patients to classical UTI is manifested. Sources of urinary endotoxin observed in PKD patients, such as cryptic intrarenal sites or leakage from the gastrointestinal (GI) tract, remain to be defined.

Adult↗
PubMed1983

New, sensitive rocket immunoelectrophoretic assay for measurement of the reaction between endotoxin and Limulus amoebocyte lysate.

To study the active proteins which participate in the reaction of Limulus amoebocyte lysate (LAL) with lipopolysaccharide, antibody was raised in rabbits against LAL. When LAL was run against rabbit antiserum in crossed immunoelectrophoresis, a complex precipitin pattern appeared. Profound changes took place after reaction of LAL with lipopolysaccharide. The most distinct change was the complete disappearance of the cathodic migrating protein coagulogen, because the antigenicity of coagulogen was lost. Based on this observation, a new rocket immunoelectrophoretic method was developed to detect the disappearance of coagulogen after reaction of LAL with lipopolysaccharide. This assay method was used on clinical specimens (cerebrospinal fluid, plasma, ascites, and urine). It was used as a qualitative test, when a single sample is tested, or as a quantitative assay, when a number of sample dilutions were tested. The new method showed a higher degree of accuracy and sensitivity in comparison with the tube test and it can be used for both research and diagnostic purposes.

Antibodies↗
PubMed2000

Monitoring the microbial contamination of beef carcass tissue with a rapid chromogenic Limulus amoebocyte lysate endpoint assay.

A chromogenic Limulus amoebocyte lysate (LAL) endpoint assay was found to be an accurate and rapid means of gauging levels of beef carcass microbial contamination within 10 min. The assay demonstrated a high correlation with the total mesophilic bacterial and coliform surface populations from inoculated beef carcass surface tissues. This assay was tested on a set of actual beef carcass surface samples (n = 121) demonstrating the utility of the chromogenic LAL test as a means of monitoring carcass microbial contamination in a near real-time fashion. Classifying the chromogenic LAL results into four contamination groups was found to be a sound means of utilizing the resultant chromogenic LAL data for detecting carcasses with high levels of microbial contamination. For beef carcass testing, this assay can be used with no instrumentation other than the required 37 degrees C incubator and, as an option, a microplate reader.

Abattoirs↗
PubMed1996

Usefulness of the endotoxin test for assessing CAPD peritonitis by gram-negative organisms.

The endotoxin (ET) in cloudy peritoneal fluid was measured, and the usefulness of the method was examined. Thirteen continuous ambulatory peritoneal dialysis (CAPD) patients with an episode of peritonitis from December, 1993 to December, 1995 were studied. The peritoneal fluid was collected without contamination from the bags and divided into two batches. The ET was measured in one of them with the limulus amebocyte lysate (LAL) assay, and the other was cultured for bacteria. Among the 13 patients ET was positive in 7 Gram-negative bacteria were identified in all 7 cases. Two of these patients were transferred to hemodialysis. On the other hand, gram-positive bacteria were cultured in only four episodes of the 6 ET-negative patients, all of which were treated successfully with vancomycin. Microbiological culture of the dialysate is still necessary for the treatment of peritonitis. But it takes 24-48 hours to obtain microbiological results. The result of the ET test can be given within one and one half hours (and it has 100% sensitivity and specificity for gram-negative peritonitis) at presentation in the episodes. Therefore, the ET test of the dialysate is very effective for the therapy of peritonitis in CAPD.

Adult↗
PubMed1985

Chromogenic Limulus amebocyte lysate assay as an aid in the diagnosis of meningitis.

Cerebrospinal fluids (CSFs) from 146 patients with suspected meningitis were screened by a recently introduced, rapid and sensitive chromogenic Limulus amebocyte lysate (CLAL) assay for endotoxin. Fifty-eight of these CSFs were screened as well for bacterial antigens by CIE, latex and/or coagglutination techniques. A total of 68 CSFs were positive by either culture and/or at least one of the antigen screens. The overall sensitivity of the CLAL assay was 93% with a specificity of 100%. However, the assay's sensitivity for community - acquired meningitis due to Haemophilus influenzae type b and Neisseria meningitidis was 100%. In this group the assay's sensitivity was better than those of direct gram stain (62%), culture (80%), and commercially available, rapid antigen detection methods (89%). Used judiciously, the CLAL assay is a cost-effective, helpful adjunct test for the diagnosis of gram-negative meningitis.

Antigens, Bacterial↗
PubMed1991

Detection of experimental Haemophilus influenzae type b bacteremia and endotoxemia by means of an immunolimulus assay.

The rapid quantitation of bacteria in blood was achieved by using a novel assay method for gram-negative bacterial lipopolysaccharide (endotoxin, LPS). The assay involves the capture of specific LPS onto microtiter plates by means of monoclonal antibodies directed against the oligosaccharide region of the LPS, followed by detection of the bound LPS by a chromogenic Limulus amebocyte lysate (LAL) system. This immunolimulus (IML) assay combines the specificity of monoclonal antibodies with the sensitivity of the LAL system to achieve the first specific, sensitive quantitation of bioactive endotoxin in plasma. In the animal model tested, Haemophilus influenzae type b (Hib) bacteremia in infant rats, there was a strong correlation between IML results and the concentration of Hib colony-forming units in blood samples (r = .845, P less than .001). Using antibodies with appropriate specificities, this approach should be useful for rapid detection of a wide range of gram-negative bacteria and endotoxins in blood.

Animals↗
PubMed1981

In vivo studies of dialysis-related endotoxemia and bacteremia.

The transfer of bacteria and endotoxin and the development of fever was monitored during hemodialysis of dogs. Bacterial and endotoxin levels in dialysis fluids exceeded those reportedly associated with pyrogenic reactions, and bacteria-free dialysate filtrates administered intravenously were shown to be pyrogenic in 2 of 2 dogs. During 18 hollow-fiber dialyses, neither bacteria nor endotoxin, as measured in the Limulus lysate assay (LLA), was detected in venous and arterial blood samples. Body temperatures did not increase during or within 1/2 h after dialysis. In experiments with parallel dialyzers, contaminated dialysate was simultaneously ultrafiltered into the second blood compartment. Ultrafiltrates were unreactive in the LLA and the absence of pyrogens was confirmed with the pyrogen test, USP, in five of five experiments. This investigation suggests that the intact cellophane membrane of the hollow-fiber dialyzer is an effective barrier to endotoxin and bacteria.

Animals↗
PubMed1985

Validity of the endotoxin assay in post surgical patients.

In a prospective study 56 patients were studied after major surgery for 10 days to correlate endotoxemia with bacteremia and clinical septicemia. Endotoxemia was detected with a quantitative plasma endotoxin assay using limulus lysate and a chromogenic substrate. Clinical septicemia was defined using a set of predetermined criteria. In 29 patients both bacterial cultures and endotoxin assays were negative. One patient showed clinical signs of septicemia. In 14 patients, low grade endotoxemia (mean 47 +/- 15 pg/ml) was observed in the first three days after surgery. Only three of these patients also showed positive endotoxin tests after this period. Corresponding blood cultures were frequently negative (81%) most likely due to prior and concurrent antibiotic prophylaxis. Two endotoxin positive patients had clinical signs of septicemia. In a total of 16 patients, including two patients from the former group endotoxemia (mean 56 +/- 21 pg/ml) was noted beyond the third post-operative day. Samples for blood cultures collected simultaneously were positive for gram negative bacilli in six patients. Clinical septicemia was present in 12 of the 16 patients. The positive and negative predictive value of the endotoxin test for bacteremia and/or septicemia after the third day of surgery for all patients was found to be 75% and 98% respectively. The results of this small prospective study in surgical patients suggest that this rapid endotoxin test is a useful clinical adjunct for both the detection and exclusion of gram negative bacteremia/septicemia.

Adult↗