Absence of Toxemia in Clostridioides difficile Infection: Results from Ultrasensitive Toxin Assay of Serum.

Clostridioides difficile infection (CDI) is caused by Toxins A and B, secreted from pathogenic strains of C. difficle. This infection can vary greatly in symptom severity and in clinical presentation. Current assays used to diagnose CDI may lack the required sensitivity to detect the exotoxins circulating in blood. The ultrasensitive single molecule array (Simoa) assay was modified to separately detect toxin A and toxin B in serum with a limit of detection at the low picogram level. When applied to a diverse cohort, Simoa was unable to detect toxins A or B in serum from patients with CDI, including many classified as having severe disease. The detection of toxin may be limited by the inference of antitoxin antibodies circulating in serum. This result does not support the hypothesis that toxemia occurs in C. difficile infection, conflicting with the findings of other published reports.

Zeptomole per milliliter detection and quantification of edema factor in plasma by LC-MS/MS yields insights into toxemia and the progression of inhalation anthrax.

Inhalation of Bacillus anthracis spores can cause a rapidly progressing fatal infection. B. anthracis secretes three protein toxins: lethal factor (LF), edema factor (EF), and protective antigen (PA). EF and LF may circulate as free or PA-bound forms. Both free EF (EF) and PA-bound-EF (ETx) have adenylyl cyclase activity converting ATP to cAMP. We developed an adenylyl cyclase activity-based method for detecting and quantifying total EF (EF+ETx) in plasma. The three-step method includes magnetic immunocapture with monoclonal antibodies, reaction with ATP generating cAMP, and quantification of cAMP by isotope-dilution HPLC-MS/MS. Total EF was quantified from 5PL regression of cAMP vs ETx concentration. The detection limit was 20 fg/mL (225 zeptomoles/mL for the 89 kDa protein). Relative standard deviations for controls with 0.3, 6.0, and 90 pg/mL were 11.7-16.6% with 91.2-99.5% accuracy. The method demonstrated 100% specificity in 238 human serum/plasma samples collected from unexposed healthy individuals, and 100% sensitivity in samples from 3 human and 5 rhesus macaques with inhalation anthrax. Analysis of EF in the rhesus macaques showed that it was detected earlier post-exposure than B. anthracis by culture and PCR. Similar to LF, the kinetics of EF over the course of infection were triphasic, with an initial rise (phase-1), decline (phase-2), and final rapid rise (phase-3). EF levels were ~ 2-4 orders of magnitude lower than LF during phase-1 and phase-2 and only ~ 6-fold lower at death/euthanasia. Analysis of EF improves early diagnosis and adds to our understanding of anthrax toxemia throughout infection. The LF/EF ratio may also indicate the stage of infection and need for advanced treatments.

Effects of acute lipopolysaccharide-induced toxemia model on some neglected blood parameters.

The presence of lipopolysaccharide (LPS) in blood induces an inflammatory response which leads to multiple organ dysfunction and numerous metabolic disorders. Uncontrolled, improper or late intervention may lead to tissue hypoxia, anaerobic glycolysis and a disturbance in the acid -base balance. The effects of LPS-induced toxemia on biological and immunological markers were well studied. However, parameters such as base excess, ions, and acid-base balance were not fully investigated. Therefore, the objective of this study was to examine these blood parameters collectively in LPS-induced inflammatory toxemia in rat's model. After induction of toxemia by injecting LPS at a rate of 5 mg/kg body weight intravenously, blood was collected from the tail vein of twenty rats and immediately analyzed. After 24 hours, the animals were sacrificed and the blood was collected from the caudal vena cava. The results revealed that the levels of pH, bicar- bonate, partial pressure of oxygen, oxygen saturation, Alveolar oxygen, hemoglobin, hematocrit, magnesium (Mg2+), and calcium (Ca2+) were significantly decreased. On the other side, the levels of Base excess blood, Base excess extracellular fluid, partial pressure of carbon dioxide, lactate, Ca2+/Mg2+, potassium, and chloride were significantly increased compared to those found pre toxemia induction. However, sodium level showed no significant change. In conclusion, Acute LPS-toxemia model disturbs acid-base balance, blood gases, and ions. These parameters can be used to monitor human and animal toxemic inflammatory response induced by bacterial LPS conditions to assist in the management of the diagnosed cases.

[The efficiency of reamberinum in infusion therapy of acetonemic syndrome in children].

The current paper presents the results of monitoring of 69 patients with acute respiratory viral infection, acetonemic syndrome. It was shown the effectiveness of inclusion of Reamberinum into complex therapy as a means for reduction of with acetonemic syndrome and for detoxic effect.

[Impact of the combined application of elastic bandaging of the lower extremities and autohemotransfusion on biochemical indices of the blood and severity of endogenous intoxication in patients with concomitant cardiovascular insufficiency].

Venous congestion in abdominal inner organs in surgical diseases in patients with heart insufficiency may additionally impact biochemical indices of the blood and severity of endogenous intoxication (EI). Basing on the data obtained in the investigations, th was established, that the lower extremities bandaging promotes exit of the blood from depot, where it resides in a concentrated state in cellular and biochemical aspects. It promotes more effective accomplishment of hemodilution as well as reduction of the EI severity in taking of the autologous blood.

Host defense against bacterial endotoxemia: mechanism in normal animals.

The present study defines the early response of normal rabbits to the intravenous injection of a single, sublethal dose of endotoxin. Within the first few hours following endotoxin there occurs in the circulating plasma of recipients a decrease in ionized calcium, a threefold increase in the heat-stable, organo-phosphate-resistant esterase level, and a striking increase in the endotoxin-detoxifying capacity. These results are fully consistent with the thesis that circulating plasma represents a principal site of detoxification and that plasma esterases of the nonspecific, carboxylic type are of major concern in defense against circulating endotoxins.

Toxemia in Human Naturally Acquired Botulism.

Human botulism is a severe disease characterized by flaccid paralysis and inhibition of certain gland secretions, notably salivary secretions, caused by inhibition of neurotransmitter release. Naturally acquired botulism occurs in three main forms: food-borne botulism by ingestion of preformed botulinum neurotoxin (BoNT) in food, botulism by intestinal colonization (infant botulism and intestinal toxemia botulism in infants above one year and adults), and wound botulism. A rapid laboratory confirmation of botulism is required for the appropriate management of patients. Detection of BoNT in the patient's sera is the most direct way to address the diagnosis of botulism. Based on previous published reports, botulinum toxemia was identified in about 70% of food-borne and wound botulism cases, and only in about 28% of infant botulism cases, in which the diagnosis is mainly confirmed from stool sample investigation. The presence of BoNT in serum depends on the BoNT amount ingested with contaminated food or produced locally in the intestine or wound, and the timeframe between serum sampling and disease onset. BoNT levels in patient's sera are most frequently low, requiring a highly sensitive method of detection. Mouse bioassay is still the most used method of botulism identification from serum samples. However, in vitro methods based on BoNT endopeptidase activity with detection by mass spectrometry or immunoassay have been developed and depending on BoNT type, are more sensitive than the mouse bioassay. These new assays show high specificity for individual BoNT types and allow more accurate differentiation between positive toxin sera from botulism and autoimmune neuropathy patients.

Hyponatraemia compounding pre-eclamptic toxaemia in a patient with type 1 diabetes.

We report a case of profound, symptomatic hyponatraemia in association with pre-eclamptic toxaemia (PET) in a 38-year-old nulliparous woman with type 1 diabetes mellitus. This patient developed hypertension and proteinuria at 31+6 weeks' gestation and was admitted for management of pre-eclampsia. Severe headache, visual disturbance and nausea were associated with a hyponatraemia of 115 mmol/L followed by ketoacidosis. This was reversed through fluid restriction, supplementation with 1.8%-3.0% hypertonic saline and a volume-reduced variable-rate insulin infusion. Clinical stability was achieved and she was subsequently worked up for an induction of labour for worsening pre-eclampsia. Hyponatraemia in the context of PET has been previously reported as rare. However, it has complications that may significantly compound the sequelae of severe PET. We propose that specific and focused monitoring of serum sodium levels should become common practice in the management of women with this condition to allow for timely, measured correction of abnormalities.

[Endogenous intoxication in patients with stomach cancer observed immediately after surgery].

Severe endogenous intoxication (El) can trigger immediately after surgery many complications right up to multiple organ failure. The evaluation of El according to a degree of toxemia is most objective up to now. The purpose of the case study was to follow up the dynamics of the main El laboratory indices and to evaluate the correlation between them in patients with stomach cancer during the early postoperative period. The El parameters were determined in 2 hours before surgery as well as on days 1, 3, 5, 7 and 14 after surgery. The below indices were determined in blood of patients: toxicity index (TI), count of mean weight molecules (MWM), leukocytic intoxication index (LII) and total count of leukocytes (TCL). The study showed that, when patients with stomach cancer are admitted for surgical treatment, they have pronounced El, which intensifies even more postoperatively. The investigated laboratory indices are independently significant in the evaluation of the El degree. A reliable but faint correlation was found between the parameters of MWM and IT, and MWM and LII. TCL does not correlate reliably with other studied El parameters except for LII. The full-fledged evaluation of the El nature must be used in choosing purposefully detoxication methods with the aim of promoting the quality of postoperative treatment of patients with stomach cancer.

Identification of toxemia in patients with Clostridium difficile infection.

Toxemia can develop in Clostridium difficile-infected animals, and correlates with severe and fulminant disease outcomes. Circumstantial evidence suggests that toxemia may occur in patients with C. difficile infection (CDI), but positive diagnosis is extremely rare. We analyzed the potential for C. difficile toxemia in patients, determined its characteristics, and assessed challenges. C. difficile toxins in serum from patients were tested using an ultrasensitive cell-based assay and further confirmed by Rac1 glucosylation assay. The factors that hinder a diagnosis of toxemia were assessed, including investigation of toxin stability, the level of toxins-specific neutralizing antibodies in sera and its effect on diagnosis limits. CDI patients develop detectable toxemia in some cases (2.3%). Toxins were relatively stable in stored sera. Neutralizing anti-toxin antibodies were present during infection and positively correlated with the diagnosis limits. Thus, the masking effect of toxin-specific neutralizing antibodies is the major obstacle in diagnosing C. difficile toxemia using cell-based bioassays.

Serum corticosterone, interleukin-1 and tumour necrosis factor in rat experimental endotoxaemia: comparison between Lewis and Wistar strains.

1. Circulating corticosterone, interleukin-1 (IL-1) and tumour necrosis factor-alpha (TNF alpha) activities in serum of Lewis and Wistar rats were measured following injection of lipopolysaccharide (LPS). IL-1 was measured as 'lymphocyte activation factor' (LAF) activity following precipitation of inhibitory activity with polyethylene glycol. TNF alpha activity was measured as cytotoxic activity. 2. Compared to the Wistar, the Lewis rat had higher circulating LAF and TNF activities following LPS, and release of both cytokines was prolonged in this strain. 3. Corticosterone increases in response to LPS were less in the Lewis than in the Wistar rat following the initial peak at 1 h; basal corticosterone was lower in the Lewis rat. 4. Adrenalectomized Lewis rats had even greater amounts of circulating LAF and TNF activities following LPS than did intact animals; the effect of adrenalectomy was not however mimicked by acute treatment with the steroid receptor antagonist, RU486, suggesting that endogenous corticosteroids did not acutely control cytokine release. 5. Although in vivo administration of anti-murine IL-1 alpha antiserum significantly lowered LAF activity of serum, circulating corticosterone in response to LPS was not affected. Similarly, treatment with anti-murine TNF alpha monoclonal antibody (mAb) abrogated TNF activity without affecting corticosterone, suggesting that other mediators may be responsible for corticosterone release following LPS. 6. This 'overproduction' of inflammatory cytokines together with lower circulating corticosterone may contribute to the susceptibility of the Lewis rat to diseases such as adjuvant arthritis or experimental allergic encephalomyelitis.

Environmental pollutants in relation to complications of pregnancy.

Certain complications of pregnancy, e.g., threatened spontaneous abortion, toxemia, emesis, and anemia, were studied in pregnant women living in industrial areas contaminated by smelters and the petrochemical industry. Exposure to lead or aromatic hydrocarbons was assessed in parallel by the determination of these agents or their metabolites in blood and urine. Comparison of respective exposure levels was made between women with normal pregnancies and those with complications. Significantly higher levels of lead in blood and increased excretion of the metabolic products of organic solvents were found in women with complicated pregnancies compared to those with normal pregnancies. Threatened spontaneous abortion, toxemia, and anemia were associated with higher lead exposure in the vicinity of smelters. In these patients, evidence of disturbances of blood glutathione equilibrium and increased lipid peroxidation were found indicating a decreased ability to compensate for the effects of exposure. Styrene exposure in a petrochemical industrial area was associated mainly with late toxemia and nephropathy. Patients with these complications also had a tendency to elevated exposure to other aromatic hydrocarbons. It is suggested that complications of pregnancy may be induced by environmental agents at levels lower than those that result in pregnancy loss or preterm birth.

Clinical detection of LPS and animal models of endotoxemia.

The interest in the study of endotoxemia in the clinical area has increased recently as a result of a) improved and simplified endotoxin determination e.g. chromogenic-kinetic microplate methods (also an improved blood sampling tool is available), b) incidence of sepsis has increased due to improvement in early (e.g. posttraumatic) survival, c) interest in and good evidence for gut translocation as a source of endotoxemia, d) agents have developed, which can antagonize endotoxins. There is evidence that patients with positive endotoxin test in the ICU have a higher incidence of organ failure. To study the pathophysiological consequences of endotoxemia and possible ways of intervention animal models are necessary. The choice of the experimental setting depends on the aim of the study e.g. whether prolonged observation is necessary in survival studies or whether hemodynamic variables have to be measured or whether therapeutic agents only crossreact with primates. Since LPS levels are quite low in clinical studies, an important factor for selection of a relevant animal might be LPS sensitivity, or the use of additional sensitization techniques e.g. galactosamine. Another important aspect in this context is whether LPS is given as bolus or infused up to several days. In this review the dose, time, and route of LPS administration is also discussed. For screening purposes rodents are usually used, or sometimes rabbits due to their higher LPS sensitivity. Another very sensitive animal model is the sheep, which can be chronically instrumented and as a specialty allows lung lymph drainage and thus studies of LPS effects on pulmonary permeability. Pigs are used for hemodynamic studies and often in therapeutical studies if species-specificity of the drug tested is not important, in cases where a large animal is necessary. Finally the non-human primates offer a number of advantages due to human-like physiology, due to the cross-reactivity of human assay systems and accordingly also cross-reactivity of human therapeutic agents. While the chimpanzee also shares the LPS sensitivity of humans, baboons are insensitive like rodents. Thus each model serves to provide some useful purpose and the selection must be made to meet the requirements of the specific questions to be asked, with special emphasis of the chosen endotoxin model on relevance for the human sepsis state.

Escherichia coli endotoxemia alters coronary and pulmonary arteriolar responses to platelet products.

In order to examine the effects of Escherichia coli endotoxemia on coronary and pulmonary microvascular responses to serotonin (5-HT) and ADP, arterioles (80-190 micros diameter) were isolated from pigs 3 h after administration of E. coli endotoxin (150 micrograms/kg, intravenously over 1 h, n = 8) or Ringer's lactate (control, n = 8). Arterioles were studied in vitro in a pressurized, partially contracted, no-flow state with video-microscopy. Precontracted (30-50% of baseline diameter) control coronary arterioles dilated in responses to either 5-HT (24 +/- 2%) or ADP (89 +/- 2%). These relaxations were partially inhibited by indomethacin, but were markedly reduced with nitric oxide synthase inhibition. After 3 h of endotoxemia, 5-HT caused contraction of coronary arterioles which was inhibited with indomethacin. In the presence of indomethacin, coronary vessels from endotoxic pigs relaxed slightly, but significantly, more to 5-HT than did control vessels exposed to indomethacin. In contrast, the relaxation response to ADP was unchanged following endotoxemia. Precontracted (15-30% of baseline diameter) pulmonary arterioles dilated in response to 5-HT (13 +/- 1%) or ADP (67 +/- 3%). Following 3 h of endotoxemia, the pulmonary arteriolar relaxation induced by 5-HT was reduced, whereas the response to ADP was not altered. In both coronary and pulmonary arterioles, relaxation induced by the endothelium-independent vasodilator, sodium nitroprusside, was unaffected by endotoxemia. Thus, coronary and pulmonary microvascular relaxation response to ADP are minimally affected by 3 h of endotoxemia, but relaxation responses to 5-HT are significantly reduced or converted to contractile responses.

Effects of acute endotoxemia and glucose administration on circulating leukocyte populations in normal and diabetic subjects.

Effects of intravenous endotoxin and glucose administration on circulating leukocyte populations were compared in seven normal subjects and seven patients with juvenile-onset diabetes by means of automated cytochemical differential counting to quantitate each cell type. Both groups had comparable control cell counts that were unaffected by glucose tolerance testing but altered significantly by endotoxin. Different patterns of response to endotoxin were observed for different circulating cell types. The response of diabetics was parallel to that of normals but showed lower neutrophil and monocyte rebound, longer lasting depression of lymphocytes and eosinophils, and greater rebound of basophils on the day following endotoxin exposure. Characterization of distinctive normal response patterns of circulating leukocyte populations to endotoxin and comparison with responses in diabetes revealed abnormalities under conditions of stress that may impair the diabetic's ability to cope with acute infection.

Leukocyte CD18 monoclonal antibody worsens endotoxemia and cardiovascular injury in canines with septic shock.

We investigated the effects of a murine monoclonal antibody directed against the canine leukocyte CD11/18 adhesion complex (MAb R15.7) in a canine model of septic shock. Awake 2-yr-old purpose-bred beagles were studied 7 days before and 1, 2, 4, and 10 days after intraperitoneal placement of an Escherichia coli-infected fibrin clot. Starting 12 h before clot placement, animals received 0.5-1 mg/kg iv every 12 h (4 doses total) of either MAb R15.7 (MAb group, n = 8) or, as controls, murine serum protein (n = 8). After infected clot placement, all animals received antibiotic (ceftriaxne, 100 mg.kg-1.day-1 for 4 days). Two of eight control animals and four of eight MAb animals died (P = 0.4). During the first 8 h after clot placement, MAb animals, compared with control animals, had greater (P < 0.06) increases in serum endotoxin levels and higher (P < 0.05) neutrophil counts. Day 1 after clot placement, MAb animals, compared with control animals, had decreased (P < 0.05) central venous pressure and arterial pH and increased (P < 0.05) arterial lactate. Day 2 after clot placement, MAb animals, compared with control animals, had decreased (P < 0.05) cardiac index and mean arterial pressure. In summary, MAb R15.7, although associated with increased neutrophil counts, worsened serum endotoxemia, acidosis, and cardiovascular function in this canine model of septic shock. These data suggest that in septic shock, antibody directed against this leukocyte membrane protein complex may be harmful, possibly via impairment of normal leukocyte function.

Role of tissue hypoxia as the mechanism of lactic acidosis during E. coli endotoxemia.

We compared the hemodynamic and metabolic alterations produced in rabbits by similar decreases in cardiac output created by inflating a balloon placed in the right ventricle (n = 6) with those produced by an intravenous bolus of Escherichia coli lipopolysaccharide (LPS; SEP group; n = 6). We measured O2 consumption (VO2), O2 transport (TO2), and O2 extraction ratio (ERO2) for the whole animal and also for the left hindlimb. Both groups experienced similar decreases in cardiac output, systemic TO2, and VO2 and similar increases in ERO2. For the hindlimb, TO2 was similar, but VO2 and ERO2 were lower for the SEP group 30 min after LPS administration (P less than 0.05); however, this difference disappeared during the remainder of the experiment. Arterial lactate concentration was greater (P less than 0.05) for the SEP group. There were no differences in skeletal muscle PO2, measured with a multiwire surface electrode, or in cardiac and skeletal muscle concentrations of high-energy phosphates. We hypothesize that a direct effect of LPS on cellular metabolism may have resulted in greater arterial lactate concentration for the SEP group.

Effects of platelet-activating factor antagonist SRI 63-441 on endotoxemia in sheep.

We investigated whether platelet-activating factor (PAF) mediates endotoxin-induced systemic and pulmonary vascular derangements by studying the effects of a selective PAF receptor antagonist, SRI 63-441, during endotoxemia in sheep. Endotoxin infusion (1.3 micrograms/kg over 0.5 h) caused a rapid, transient rise in pulmonary arterial pressure (Ppa) from 16 +/- 3 to 36 +/- 10 mmHg (P less than 0.001) and pulmonary vascular resistance (PVR) from 187 +/- 84 to 682 +/- 340 dyn.s.cm-5 (P less than 0.05) at 0.5 h, followed by a persistent elevation in Ppa to 22 +/- 3 mmHg and in PVR to 522 +/- 285 dyn.s.cm-5 at 5 h in anesthetized sheep. Arterial PO2 (PaO2) decreased from 341 +/- 79 to 198 +/- 97 (P less than 0.01) and 202 +/- 161 Torr at 0.5 and 5 h, respectively (inspired O2 fraction = 1.0). SRI 63-441, 20 mg.kg-1.h-1 infused for 5 h, blocked the early rise in Ppa and PVR and fall in PaO2, but had no effect on the late phase pulmonary hypertension or hypoxemia. Endotoxin caused a gradual decrease in mean aortic pressure, which was unaffected by SRI 63-441. Infusion of SRI 63-441 alone caused no hemodynamic alterations. In follow-up studies, endotoxin caused an increase in lung lymph flow (QL) from 3.8 +/- 1.1 to 14.1 +/- 8.0 (P less than 0.05) and 12.7 +/- 8.6 ml/h at 1 and 4 h, respectively. SRI 63-441 abolished the early and attenuated the late increase in QL.(ABSTRACT TRUNCATED AT 250 WORDS)

The metabolic effects of platelet-activating factor antagonism in endotoxemic man.

OBJECTIVE: To determine if the inflammatory phospholipid platelet-activating factor (PAF) participated in the symptomatologic, metabolic, and counterregulatory hormonal responses of human endotoxemia. DESIGN: In a double-blind, placebo-controlled study, five subjects received 10 mg of the PAF antagonist Ro 24-4736 orally, while five control subjects received a placebo. Eighteen hours later, all subjects were administered 4 ng/kg of endotoxin (lipopolysaccharide) intravenously. SETTING: The Clinical Research Center of The New York Hospital-Cornell Medical Center. PARTICIPANTS: Healthy male volunteers. MAIN OUTCOME MEASURES: Repeated measurements of vital signs, symptoms, cytokine and hormone levels, resting energy expenditure, platelet aggregation, and bleeding times were performed during a 24-hour period. RESULTS: Subjects who were pretreated with the PAF antagonist experienced fewer symptoms, including rigors at 1 hour (P < .05) and myalgias at 1 through 4 hours (P < .05) after administration of lipopolysaccharide. This was in concert with a diminished peak cortisol level (668 +/- 107 vs 959 +/- 159 nmol/L in controls; P < .05), epinephrine secretion (1057 +/- 165 vs 2029 +/- 431 nmol/L in controls; P < .05), and almost complete inhibition of PAF-induced platelet aggregation ex vivo. CONCLUSIONS: These findings in the face of unaltered circulating cytokines tumor necrosis factor alpha, interleukin 1 beta, and interleukin 6, as well as the tumor necrosis factor receptor-I s, suggest that PAF may influence some endotoxin-induced, counterregulatory hormonal responses and symptoms through cytokine-independent mechanisms. This study further supports the role of PAF antagonists as an adjunct to cytokine blockade in the treatment of gram-negative sepsis.

Prophylactic use of human endotoxin-core hyperimmune gammaglobulin to prevent endotoxaemia in colostrum-deprived, gnotobiotic lambs challenged orally with Escherichia coli.

The efficacy of human IgG polyclonal antibody to endotoxin-core in preventing endotoxaemia and subsequent disease was studied in colostrum-deprived gnotobiotic lambs challenged orally at about 5 h old with 10(9) cfu Escherichia coli. Human endotoxin-core hyperimmune gammaglobulin was given intravenously to 5 lambs at 1.9 g IgG/kg bodyweight prior to challenge. Human albumin was given intravenously to 3 control lambs. Bacteraemia was observed in all lambs, but the incidence was lower (P < 0.01) and the onset later (P < 0.05) in gammaglobulin pre-treated lambs. These lambs showed no signs of disease, whereas clinical endotoxaemia, manifesting as watery mouth disease, was diagnosed in 2 of the 3 control lambs which were killed between 18 and 22 h after challenge. Thus, prophylactic treatment of colostrum-deprived lambs with human IgG enriched in endotoxin-core antibodies was effective in reducing the degree of bacteraemia and preventing endotoxaemia, leukopenia and clinical disease following oral challenge with E. coli.