Rapid detection of gram-negative bacterial peritonitis by the Limulus amoebocyte lysate assay.

The chromogenic Limulus amoebocyte lysate test effectively detected 66 (100%) culture-proven gram-negative peritonitis cases among 185 continuous ambulatory peritoneal dialysis patients with clinical evidence of infectious peritonitis.     

Detection of endotoxin in the plasma of patients with gram-negative bacterial sepsis by the Limulus amoebocyte lysate assay.

A total of 120 Limulus amoebocyte lysate (LAL) determinations were made on plasma obtained from normal, healthy human blood donors. Results demonstrated a mean endotoxin level in blood of 0.02 to 1.57 pg/ml. The amount of Escherichia coli endotoxin added to human plasma samples can be quantitated by both nephelometry and turbidimetry. Endotoxin-spiked samples were shown to be significantly different from unspiked samples. When plasma samples were collected from 45 patients hospitalized at three centers, a strong association was demonstrated between a positive Limulus amoebocyte lysate assay and a septic condition. Sensitivity, specificity, and false-positive and false-negative rates for the Limulus amoebocyte lysate assay as a diagnostic test for gram-negative bacteremia were estimated.     

Plasma endotoxin assay by supersensitive reagent for the diagnosis of gram-negative bacteremia

With a novel supersensitive reagent, we evaluated the utility of measuring plasma endotoxin level for the rapid and sensitive diagnosis of gram-negative bacteremia. Subjects were 112 febrile(more than 38 degrees C) patients suspected of having bacterial infection and 170 samples were collected. Venous blood was obtained aseptically before administering antibiotics. Blood culture and endotoxin assays were performed simultaneously with these materials. Plasma endotoxin levels were positive in 64 samples when the cut off index was postulated at 0.35 pg/ml, while only 5 samples when the cut off index was postulated at 5 pg/ml. When the cut off index was at 0.35 pg/ml, sensitivity was 86%, while it was 14% when the cut off index was 5 pg/ml. Gram-negative rods(GNR) were detected by blood culture in 14 cases and the average period to detect GNR was 14.8 hours. The presence of circulating viable bacteria is diagnosed by blood culture, but because of the serious consequence of bacterial sepsis, treatment is initiated even in the absence of an identifiable organism. Since plasma endotoxin level can be assayed in about 2 hours, it will be more practical if we adjust the cut off index according to the clinical situation.     

Single-step, chromogenic Limulus amebocyte lysate assay for endotoxin.

A new reagent for the chromogenic Limulus amebocyte lysate (LAL) assay is described. LAL was formulated for optimal performance in either an endpoint procedure or a kinetic procedure with the chromogenic substrate, buffer, and LAL components colyophilized as a single reagent. The kinetic chromogenic method required an incubating microplate reader coupled to a computer for collection and analysis of data. The kinetic method had a longer incubation time than the endpoint method and spanned a range of over 3 orders of magnitude compared with the 1-order-of-magnitude range of the endpoint assay. The kinetic method was less subject to operator error, since readings were continuous and automatic. The endpoint test was more operator intensive, requiring both addition of acetic acid to stop the reaction and transfer of the sample to the reading device. A single-step chromogenic reagent was also prepared without lyophilization by mixing reconstituted gel clot LAL with a buffer and a chromogenic substrate. The reagent prepared in this manner performed as well as the colyophilized agent.     

Effect of anticoagulants on the chromogenic Limulus lysate assay for endotoxin.

We have examined the effects of anticoagulants on the chromogenic Limulus lysate assay for endotoxin. The results indicate that both heparin and CPD Adenine 1 have a striking dose related inhibitory effect on the assay. At concentrations of heparin as low as about 30 U/ml there was a 90% reduction in detectable endotoxin. The inhibitory effect of up to 100 U/ml of heparin could be neutralised by the addition of protamine sulphate. Anticoagulants appear to be an additional factor which can adversely affect the Limulus chromogenic assay for endotoxin. This further emphasises the need for alternative approaches to the measurement of endotoxin.     

Rapid detection of Gram-negative bacteriuria by Limulus amoebocyte lysate assay.

The Limulus amoebocyte lysate (LAL) test was evaluated for rapid detection of gram-negative bacteriuria in an adult patient population. Time to gelation of a standard LAL preparation was used as a measure of significant (greater than 10(5) bacteria per ml) gram-negative bacteriuria, and the results of 190 LAL assays were compared with quantitative urine cultures. Initially, 33 of 36 urine specimens containing greater than 10(5) gram-negative bacteria per ml were detected by LAL assay. The three false-negative LAL tests were the result of urine pH levels below the pH minimum for LAL gelation; neutralization of these urine specimens resulted in positive LAL assays and 100% correlation with culture results. All 36 bacteriuric urine specimens were LAL positive within 15 min, with the majority of assays (86.1%) being positive after only 10 min of incubation at 37 degrees C. These data compared favorably with gelation times of 15 min when 1 X 10(5) to 2 X 10(5) gram-negative bacteria per ml were added to sterile urine. Two urine samples obtained from male patients with culture-proven gonococcal urethritis yielded positive LAL assays. The LAL assay was shown to correctly differentiate 96.2% of urine specimens as containing less than 10(5) or greater than 10(5) gram-negative bacteria per ml. The results of this study have shown that the LAL test can be used as a rapid, simple, and reliable screening procedure for the diagnosis of clinically significant gram-negative bacteriuria.     

Quantitative Limulus lysate assay for endotoxin and the effect of plasma.

The effects of plasma and chromogenic substrate on the kinetics of the endotoxin-activated Limulus amoebocyte lysate (LAL) assay were determined. A linear correlation was observed between the rate of development of turbidity (optical density 405) with the LAL reagent and the concentration of endotoxin over a four log ten-fold range. Like chromogenic substrate, the addition of dilution and heat treated plasma to the reaction resulted in an increase in optical density proportional to the concentration of plasma present. The presence of the treated plasma also resulted in an accelerated increase in optical density with comparable results when testing plasma at different concentrations and, additionally, serum. This accelerated increase in optical density may not be recognised in assays that monitor the progress of the reaction at a single time point and may confound assays of plasma samples that use chromogenic substrate. Plasma obtained from endotoxin sensitive and resistant strains of mice showed similar effects. The use of kinetic methodology means that a quantitative assay for endotoxin in plasma can be achieved, its variability comparable with that seen with semiquantitative serial dilution but with greater economy of the LAL reagent.     

Chromogenic Limulus amoebocyte lysate assay for rapid detection of gram-negative bacteriuria.

A chromogenic Limulus amoebocyte lysate assay was evaluated as a rapid screening test for the detection of clinically significant gram-negative bacteriuria. The development of a distinctive yellow color after the addition of chromogenic substrate to the Limulus amoebocyte lysate-urine reaction mixture was used to measure greater than or equal to 10(5) gram-negative bacteria per ml. A total of 324 urine specimens were assayed, with 68 gram-negative urinary tract infections identified as defined by quantitative urine colony counts of greater than or equal to 10(5) bacteria per ml. Of these, 68 and 67 of 68 were detected by the chromogenic Limulus amoebocyte lysate assay at urine dilutions of 1:10 and 1:20, respectively. Nine false-positive chromogenic Limulus amoebocyte lysate assay results were observed at both urine dilutions and in the same specimens. At a urine dilution of 1:10, sensitivity and specificity were 100 and 96.6%, respectively, with predictive values of 100% for a negative test and 88.3% for a positive test. At a urine dilution of 1:20, sensitivity and specificity were 98.6 and 96.6%, respectively; predictive values were 99.6% for a negative test and 88.3% for a positive test. These data suggest that chromogenic Limulus amoebocyte lysate assay of urine has potential usefulness as a rapid, reliable, and easily performed and interpreted screening test for the diagnosis of clinically significant gram-negative bacteriuria.     

Laboratory diagnosis of bacterial meningitis.

Bacterial meningitis is relatively common, can progress rapidly, and can result in death or permanent debilitation. This infection justifiably elicits strong emotional reactions and, hopefully, immediate medical intervention. This review is a brief presentation of the pathogenesis of bacterial meningitis and a review of current knowledge, literature, and recommendations on the subject of laboratory diagnosis of bacterial meningitis. Those who work in clinical microbiology laboratories should be familiar with the tests used in detecting bacteria and bacterial antigens in cerebrospinal fluid (CSF) and should always have the utmost appreciation for the fact that results of such tests must always be reported immediately. Academic and practical aspects of the laboratory diagnosis of bacterial meningitis presented in this review include the following: anatomy of the meninges; pathogenesis; changes in the composition of CSF; etiological agents; processing CSF; microscopic examination of CSF; culturing CSF; methods of detecting bacterial antigens and bacterial components in CSF (counter-immunoelectrophoresis, coagglutination, latex agglutination, enzyme-linked immunosorbent assay, Limulus amebocyte lysate assay, and gas-liquid chromatography); use of the polymerase chain reaction; and practical considerations for testing CSF for bacterial antigens.     

Rapid diagnosis of bacterial meningitis by real-time PCR and fluorescence in situ hybridization

Real-time PCR and fluorescence in situ hybridization (FISH) were evaluated as rapid methods for the diagnosis of bacterial meningitis and compared to standard diagnostic procedures. For PCR, a LightCycler approach was chosen, implementing eubacterial and specific PCR assays for the most relevant bacteria. For FISH, a similar probe set containing eubacterial and specific probes was composed of published and newly designed probes. Both methods were evaluated by use of cerebrospinal fluid (CSF) samples from patients with suspected bacterial meningitis. For all microscopy- and culture-positive samples (n = 28), the eubacterial PCR was positive. In addition, all identifiable pathogens were detected with specific PCR assays, according to an algorithm based on the Gram stain. The FISH method detected the pathogen in 13 of 18 positive samples. While the FISH method remained negative for all microscopy- and culture-negative samples (n = 113), the eubacterial PCR was positive for five of these samples. Sequencing of the amplicon revealed the presence of Neisseria meningitidis, Streptococcus agalactiae, and Haemophilus influenzae in three of these five samples. In addition, samples with discordant results by culture and microscopy were successfully investigated by PCR (10 samples) and FISH (5 samples). In conclusion, PCR is a highly sensitive tool for rapid diagnosis of bacterial meningitis. FISH is less sensitive but is useful for the identification of CSF samples showing bacteria in the Gram stain. Based on our results, an approach for laboratory diagnosis of meningitis including PCR and FISH is discussed.     

Counterimmunoelectrophoresis in the diagnosis of bacterial meningitis.

The aim of the present study was to investigate whether counterimmunoelectrophoresis (CIE) would facilitate the rapid, etiological diagnosis of bacterial meningitis when used in parallel with other routine methods in a medical bacteriological laboratory. Of 3,674 consecutive specimens of cerebrospinal fluid (CSF) received at the Department of Diagnostic Bacteriology, Statens Seruminstitu, 283 specimens (each representing one patient) were selected for examination by CIE on the basis of the following criteria: bacteria or pleocytosis or both by microscopy or positive culture or both. CIE was performed with antisera to Neisseria meningitidis (groups A, B and C), Streptococcus pneumoniae (omni-serum and pools A to 1), and Haemophilus influenzae type b. Antigen was detected in 57% (72/126) of specimens in which cultures revealed these three kinds of microorganisms in CSF and in 12% (17/139) of the culture-negative specimens. CSF specimens from 21 patients with bacterial meningitis caused by other species were all negative in CIE, except four, three of which contained Escherichia coli antigen reacting with antiserum to N. meningitidis group B and one E. coli antigen reacting with antiserum to H. influenzae type b. Specific diagnosis was achieved in 60% (170/283) of the specimens studied and could be extablished within 1 h in 85% (145/170) by the combined results of microscopy and CIE. Ten specimens, nine of which showed a reaction with antiserum to N. meningitidis group A, were positive by CIE only.     

Sensitive quantitation of endotoxin by enzyme-linked immunosorbent assay with monoclonal antibody against Limulus peptide C.

Limulus peptide C, a 28-amino-acid fragment of coagulogen formed by the reaction of endotoxin with Limulus amebocyte lysate, was synthesized, and a monoclonal antibody against it was raised. A new microassay for endotoxin was developed, using this antibody in an enzyme-linked immunosorbent assay for generated peptide C-like immunoreactivity. A linear relationship between absorbance and endotoxin concentration was obtained. Control standard endotoxin in water could be detected to a level of 0.001 endotoxin unit per ml. The endotoxin levels in plasma samples from normal humans, rabbit, mice, and guinea pigs were generally found to be below the detection limit of 0.01 endotoxin unit per ml of plasma. The color and turbidity of specimens did not interfere with the assay. The consumption of Limulus amebocyte lysate in the assay was less than 5% of that in the gel-clot and chromogenic assays. With raw lysate, which was much more stable in solution than chloroform-treated lysate, the assay was still highly sensitive to endotoxin but was totally unresponsive to natural glucans. The monoclonal antibody cross-reacted with peptide C-like immunoreactivity generated in Tachypleus amebocyte lysate, which gave equal sensitivity in the endotoxin assay.     

Human endothelial cell-based assay for endotoxin as sensitive as the conventional Limulus Amebocyte Lysate assay

Endotoxin, also known as lipopolysaccharide (LPS) produced by bacteria can be present in any liquid or on any biomaterial even if the material is sterile. Endotoxin in mammals can cause fever, inflammation, cell and tissue damage and irreversible septic shock and death. In the body, endothelial cells making up the blood vasculature and endothelial cells in vitro rapidly react to minute amounts of endotoxin resulting in a rapid induction of the cell adhesion molecule E-selectin. In this study we have used immunofluorescent staining to evaluate the expression of E-selectin on human microvascular endothelial cells from the skin (HDMEC) and human umbilical vein endothelial cells (HUVEC) exposed to various concentrations of LPS. In addition, the sensitivity of detection was compared with the most widely used assay for the presence of endotoxin, the Limulus Amebocyte Lysate assay (LAL). The detection of E-selectin on endothelial cells in the presence of LPS for 4 h was found to be at least as sensitive in detecting the same concentration using the LAL assay. A cell adhesion molecule-enzyme immunosorbent assay was also developed and used to quantify LPS using the endothelial cell model. A comparison of LAL and the immunofluorescent staining method was carried out with solutions, nanoparticles, biomaterial extracts and endothelial cells grown directly on biomaterials. Under all conditions, the endothelial/E-selectin model system was positive for the test samples that were positive by LAL. Thus, we propose the use of this highly sensitive, rapid, reproducible assay for the routine testing of endotoxin in all steps in the manufacturing process of materials destined for use in humans. This can give a rapid feedback and localization of bacterial contamination sources with the LAL being reserved for the testing of the final product.     

Binding and neutralization of endotoxin by Limulus antilipopolysaccharide factor.

In order to examine the ability of Limulus antilipopolysaccharide factor (LALF) to bind lipopolysaccharide (LPS), we purified LALF to homogeneity from Limulus amoebocyte lysate and coupled it covalently to agarose beads. LALF-coupled beads captured more tritiated LPS from rough and smooth strains of gram-negative bacteria than did control human serum albumin-coupled beads. Unlabeled homologous and heterologous LPS competed for the binding of 3H-LPS to LALF-coupled beads. LALF bound LPS in a dose-dependent manner as assessed by the precipitation of LPS-LALF complexes with 50% saturated ammonium sulfate. We also studied the ability of LALF to neutralize LPS. LPS preincubated with LALF was less mitogenic for murine splenocytes, was less pyrogenic in the rabbit fever assay, was less lethal in mice which had been sensitized to LPS with actinomycin D, and induced less fever, neutropenia, and pulmonary hypertension when infused into sheep. Our findings extend prior studies which suggested that LALF binds to and neutralizes LPS from multiple strains of gram-negative bacteria.     

Rapid diagnosis of bacterial meningitis by nanopore 16S amplicon sequencing: A pilot study

Early administration of antibiotics is crucial in the management of bacterial meningitis. Rapid pathogen identification helps to make a definite diagnosis of bacterial meningitis and enables tailored antibiotic treatment. We investigated if the 16S amplicon sequencing performed by MinION, a nanopore sequencer, was capable of rapid pathogen identification in bacterial meningitis. Six retrospective cases of confirmed bacterial meningitis and two prospective cases were included. The initial cerebrospinal fluid (CSF) samples of these patients were used for the experiments. DNA was extracted from the CSF, and PCR was performed on the 16S ribosomal DNA (16S rDNA). Sequencing libraries were prepared using the PCR products, and MinION sequencing was performed for up to 3 h. The reads were aligned to the bacterial database, and the results were compared to the conventional culture studies. Pathogenic bacteria were successfully detected from the CSF by 16S sequencing in all retrospective cases. 16S amplicon sequencing was more sensitive than conventional diagnostic tests and worked properly even in antibiotics-treated samples. MinION sequencing significantly reduced the turnaround time, and even 10 min of sequencing was sufficient for pathogen detection in certain cases. Protocol adjustment could further increase the sensitivity and reduce the turnaround time for MinION sequencing. Finally, the prospective application of MinION 16S sequencing was successful. Nanopore 16S amplicon sequencing is capable of rapid bacterial identification from the CSF of the bacterial meningitis patients. It may have many advantages over conventional diagnostic tests and should therefore be applied in a larger number of patients in the future.