Clinical relevance of leukocyte-associated endotoxins measured by semi-automatic synthetic luminescent substrate method.

A newly developed semi-automatic synthetic luminescence substrate (SALS) method for measuring endotoxin was compared with the existing turbidimetric kinetic assay (TKA) using leukocyte-rich plasma to verify its usefulness. As a result, the endotoxin levels by this method were higher than that by the existing assay in most specimens, and the time required for measurement was much shorter. In addition, the leukocyte-rich plasma endotoxin level minus the plasma endotoxin levels were named leukocyte-associated endotoxin, and these levels per one leukocyte were compared. As a result, those levels were highly correlated with the endotoxin measurement levels of leukocyte-rich plasma. The correlation coefficient of SALS method was superior to the existing TKA method, the endotoxin level by this method may be close to true endotoxin levels.

Basic Verification of ß-D Glucan in Leukocyte-Rich Plasma for the Diagnosis of Deep Mycosis.

BACKGROUND: Currently, supplementary serological testing for ß-D glucan (BDG) is often selected to diagnose deep mycosis in care covered by the health insurance in Japan. The Wako method used by our center has low sensitivity, and different studies have used different cut-off values due to factors that cause false positives and false negatives. One possible cause of false negatives is the use of platelet-rich plasma (PRP) as the sample material. Because phagocytic white blood cells (WBC) are precipitated by centrifugation and only plasma is measured, it seems unlikely that the actual amount of BDG is being measured when using PRP. Further, a frequent cause of false positives is contamination from blood products and gauze containing BDG. To resolve these issues, the blood cell separator, hydroxyethyl starch, is used to precipitate only the red blood cells to obtain leukocyte-rich plasma (LRP). We hypothesized that it might be possible to improve the diagnostic rate of deep mycosis by measuring the BDG content of plasma containing WBC and fungal components and by comparing the BDG content of PRP and LRP measured simultaneously. MATERIALS AND METHODS: Healthy human blood, albumin-added blood, wrung-out gauze fluid-added blood, and fungal solution-added blood were prepared, and PRP and LRP were prepared using hydroxyethyl starch. The BDG content of each sample was measured using the Wako method and compared. In addition, PRP and LRP of fungal-added blood were Gram-stained and examined under a microscope, and the number of WBCs and phagocytosed fungi was counted visually and compared. RESULTS: Measuring the BDG content of LRP confirmed that there were no false positives with LRP, and in vitro experiments comparing albumin-added false-positive blood to fungal-added blood showed significant differences between PRP and LRP only in the fungal-added blood. CONCLUSION: Calculating the BDG-ratio (LRP/PRP) by measuring both LRP and PRP may eliminate false positives and false negatives of true deep mycosis and improve the diagnostic rate.

Auto-brewery syndrome caused by oral fungi and periodontal disease bacteria.

BACKGROUND: Auto-brewery syndrome (ABS) is often caused by fungi in the intestinal tract. We describe a rare case of alcohol production by Candida albicans and periodontal disease bacteria in the oral cavity. CASE PRESENTATION: A man aged in his 60s had a car accident, and alcohol was detected on his breath. At the time, he exhibited alcohol overdose seizures with no alcohol consumption. We carried out a gastrointestinal endoscopy, detected esophageal candidiasis, and diagnosed ABS. His seizures continued despite using miconazole oral gel. Significant tooth decay, periodontal disease, and high C. albicans levels were observed in his oral cavity. Alcohol production was confirmed from periodontal bacteria and C. albicans cultures and alcohol-degrading enzyme functions were poor. Dental treatment and antifungal drugs reduced seizures, and improved his fatty liver. CONCLUSION: Alcohol can be produced by microorganisms in healthy individuals. Therefore, blood alcohol levels and alcohol-degrading enzyme functions should be examined in patients with unexplained liver dysfunction.

Antibody-mediated soluble CD14 stabilization prevents agitation-induced increases in presepsin levels in blood component specimens.

Presepsin is a 13-kDa N-terminal glycoprotein of CD14. Previously, agitation-induced increases in presepsin levels have been reported; however, the mechanism remains poorly understood. In this study, we aimed to reveal the mechanism of presepsin increase. The agitated plasma or serum was separated using gel exclusion chromatography and analyzed by ELISA. The effect of an anti-CD14 antibody (F1024-1-3) was examined. We observed elevated presepsin levels in the agitated plasma and aggregated soluble CD14 (sCD14). However, treatment with F1024-1-3 before agitation prevented the aggregation and the increase in presepsin levels. Depletion of aggregated sCD14 decreased the presepsin levels. Our findings indicate that agitation induces the aggregation of sCD14 and triggers an increase in presepsin. Anti-CD14 antibody prevents an increases in presepsin.

Endotoxin contamination of single-use sterile surgical gloves.

Aim: Strict endotoxin limits are enforced for implants and catheters inserted into the body. However, no standard limit has been set for single-use sterile surgical gloves. Materials & methods: Four types of gloves sold in Japan were dipped in saline and that endotoxin levels were measured. Cytokine producing activity of gloves in blood was also measured. Results: Three of the four types of gloves showed endotoxin contamination. We also confirmed an increase in cytokine production in these gloves except one glove in which anionic surfactants was found. Conclusion: The extent to which detected endotoxins enter the body during surgery is controversial, but strict endotoxin limits need to be established.

Use of Direct Hemoperfusion with Polymyxin B-Immobilized Fiber for the Treatment of Septic Shock Complicated with Lemierre Syndrome Caused by Fusobacterium necrophorum.

We report a case of septic shock treated with PMX-DHP that was complicated with Lemierre syndrome caused by Fusobacterium necrophorum. The patient was a 31-year-old man who was initially diagnosed with influenza. He received treatment; however, because his symptoms gradually worsened, he was transported to our hospital 10 days following his diagnosis. His initial examination revealed symptoms of respiratory distress and an altered level of consciousness. Based on laboratory and imaging results, it became clear that he suffered from septic shock of unknown etiology, disseminated intravascular coagulation, and acute renal and respiratory failure. We initiated treatment with vasopressors, an antibacterial agent, immunoglobulins as well as an appropriate ventilator management; however, his unstable circulatory condition continued. As soon as PMX-DHP was initiated, 2 days following admission to the ICU, his circulatory instability normalized. F. necrophorum was ultimately detected after a culture examination, and contrast-enhanced computed tomography revealed a jugular vein thrombus, which led to the diagnosis of Lemierre syndrome. The patient's condition gradually improved, and he was discharged from the ICU after 19 days.

[Effect of Human Serum Albumin on Endotoxin Scattering Photometry].

PURPOSE: Laser scattering photometry (ESP) is a newly developed plasma endotoxin assay method using horseshoe crab amebocyte lysate (AL) that recognizes small particles produced by polymerization of coagulin under the stirring conditions at 1000rpm. We elucidated the effect of human serum album (HSA) in the ESP method. METHODS: AL was dissolved with 630µL of the specimen and a 200-µL aliquot was used for ESP; this conventional protocol was regarded as the ESP630 method. The ESP210 method was also used, i. e. AL was dissolved with 210µL of the specimen and a 200-µL aliquot was used for ESP. RESULTS: Water induced the agglutination, and HSA prolonged the agglutination time depending on its concentration especially in the ESP630 method. The water-induced agglutination was not inhibited by the addition of anti-factor C monoclonal antibody, and amidinophenyl benzoate hydrochloride, used as a clotting enzyme inhibitor, intensively inhibited the water-induced agglutination. Therefore, the water-induced agglutination was suggested to be a false-positive reaction to non-specific activation of the clotting enzyme. The HSA-induced prolongation of the reaction in the national health insurance-covered turbidimetric kinetic assay was not observed. CONCLUSION: HSA or plasma protein seemed to affect the result, especially in the ESP630 method, and a non-specific reaction was found to occur in the ESP methods.

Evaluation of an endotoxin-specific limulus amebocyte lysate assay using leukocyte-rich plasma for the diagnosis of gram-negative bacterial infection.

We focused our attention on the endotoxin present within and on the surface of white blood cells and attempted to establish a new sample preparation method for endotoxin assays in leukocyte-rich plasma (LRP), taking advantage of the erythrocyte-aggregating property of hydroxyethyl starch. We used an endotoxin-specific turbidimetric kinetic assay, which is the conventional method used to assay endotoxin levels in platelet-rich plasma (PRP). Then, we comparatively assessed the assay results obtained with the endotoxin assay using PRP and LRP. It was found that the sensitivity of endotoxin assay in LRP was 88.5 %, which was superior to 73.1 % of the sensitivity in PRP in the diagnosis of infections caused by gram-negative bacteria. These results suggest that our newly developed LRP endotoxin assay may contribute to an improvement in the rate of sepsis diagnosis.

Sternoclavicular joint septic arthritis following paraspinal muscle abscess and septic lumbar spondylodiscitis with epidural abscess in a patient with diabetes: a case report.

BACKGROUND: Septic arthritis of the sternoclavicular joint (SCJ) is extremely rare, and usually appears to result from hematogenous spread. Predisposing factors include immunocompromising diseases such as diabetes. CASE PRESENTATION: A 61-year-old man with poorly controlled diabetes mellitus presented to our emergency department with low back pain, high fever, and a painful mass over his left SCJ. He had received two epidural blocks over the past 2 weeks for severe back and leg pain secondary to lumbar disc herniation. He did not complain of weakness or sensory changes of his lower limbs, and his bladder and bowel function were normal. He had no history of shoulder injection, subclavian vein catheterization, intravenous drug abuse, or focal infection including tooth decay. CT showed an abscess of the left SCJ, with extension into the mediastinum and sternocleidomastoid muscle, and left paraspinal muscle swelling at the level of L2. MRI showed spondylodiscitis of L3-L4 with a contiguous extradural abscess. Staphylococcus aureus was isolated from cultures of aspirated pus from his SCJ, and from his urine and blood. The SCJ abscess was incised and drained, and appropriate intravenous antibiotic therapy was administered. Two weeks after admission, the purulent discharge from the left SCJ had completely stopped, and the wound showed improvement. He was transferred to another ward for treatment of the ongoing back pain. CONCLUSION: Diabetic patients with S. aureus bacteremia may be at risk of severe musculoskeletal infections via hematogenous spread.

Experimental application of a synthetic luminescent substrate assay using endotoxin-specific limulus amebocyte lysate to human blood.

A synthetic luminescent substrate method, using a mutant-type luciferase whose luminescence intensity is more than ten times as intense as the wild type, was developed recently. We conducted the first basic studies on clinical application of the novel endotoxin measurement method. We assessed and established measurement conditions, including reagent concentrations and reaction time, so that it would be possible to apply the luminescent synthetic substrate method proposed by Noda et al. to measurements in human blood. When we added lipopolysaccharide (LPS) to water, it was possible to measure LPS at a concentration of 0.1 pg/ml, whereas it was possible to measure LPS in tenfold diluted and heated plasma at a concentration of 1 pg/ml. When plasma was further diluted, inhibiting activity decreased considerably. Thus, it will be necessary to completely eliminate the inhibitor present in plasma. However, the shortest time after collecting the specimen in which it was possible to make measurements was 30-40 min, suggesting that if an assay is established, it will be possible to use the method as a novel blood endotoxin assay.

[Study on the leukocyte-rich plasma as the specimen for the measurement of endotoxin].

PURPOSE: In the diagnosis of sepsis, the sensitivity of the endotoxin assay using platelet-rich plasma (PRP) is not as high as expected. In the endotoxin assay using PRP, endotoxin occurring within and on the surface of white blood cells cannot be measured. Thus, we devised a method of preparing leukocyte-rich plasma (LRP) using hydroxyethyl starch (HES) in order to improve the endotoxin assay. METHODS: The endotoxin concentration was measured by an endotoxin-specific Limulus amebocyte lysate assay method: turbidimetric kinetic assay. RESULTS: Only 1% of erythrocytes were recovered in LRP without the hemolysis of erythrocytes. Endotoxin contents in PRP obtained from blood incubated for 0, 30, 60, 120, and 180 min at 37 degrees C in the presence of lipopolysaccharide (50 pg/ml in a final concentration) were 1.18, 1.23, 1.23, 1.39, and 1.25 times higher (mean value from three bloods) than that in PRP, respectively. CONCLUSION: Thus, the superiority of the sensitivity of the LRP-based endotoxin assay over the PRP-based assay was identified. Our newly devised assay method may contribute to improvement of the diagnosis rate of endotoxemia in sepsis.

Retrospective study on the effect of tight glucose control in postoperative sepsis patients using an artificial pancreas.

Tight glucose control (TGC) using a sliding scale based on intermittent blood glucose measurements occasionally can have a fatal outcome as a result of insulin-induced hypoglycemia. The present study was undertaken to examine whether the use of an artificial pancreas to achieve TGC would be possible in postoperative patients with sepsis. The retrospective study was carried out as an exploratory study, focusing on the possibility of precise evaluation of the significance of TGC as a beneficial intervention by serological monitoring of various mediators. TGC was accomplished using an artificial pancreas (STG-22; (Nikkiso, Tokyo, Japan). The patients were divided into two groups: the TGC group (6 patients with sepsis in whom the target blood glucose level set at <150 mg/dl was attempted using the artificial pancreas), and the glucose control (GC) group (6 patients with sepsis in whom glucose control was attempted using a sliding scale; target blood glucose level was set at 200 mg/dl or lower). The mean blood glucose level was 129.7 ± 9.7 mg/dl in the TGC group and 200.9 ± 14.7 mg/dl in the GC group (P < 0.01, ANOVA). No hypoglycemia associated with the artificial pancreas was seen in any of the patients. The serum levels of S100A12 and HMGB-1 tended to decrease, and those of sRAGE tended to increase, in the TGC group. Further data collection from a larger number of cases would be expected to allow a precise assessment of TGC as a potentially beneficial intervention in sepsis patients.

Green Urine Discoloration due to Propofol Infusion: A Case Report.

We present a 19-year-old man who excreted green urine after propofol infusion. The patient was admitted to our hospital for injuries sustained in a traffic accident and underwent surgery. After starting continuous infusion of propofol for postoperative sedation, his urine became dark green. Serum total bilirubin and urine bilirubin were both elevated. We believe that the green discoloration of the urine was caused by propofol infusion and was related to impaired enterohepatic circulation and extrahepatic glucuronidation in the kidneys.