New Approaches to Identify Sepsis Biomarkers: The Importance of Model and Sample Source for Mass Spectrometry.

Septic shock is a systemic inflammatory response syndrome associated with circulatory failure leading to organ failure with a 40% mortality rate. Early diagnosis and prognosis of septic shock are necessary for specific and timely treatment. However, no predictive biomarker is available. In recent years, improvements in proteomics-based mass spectrometry have improved the detection of such biomarkers. This approach can be performed on different samples such as tissue or biological fluids. Working directly from human samples is complicated owing to interindividual variability. Indeed, patients are admitted at different stages of disease development and with signs of varying severity from one patient to another. All of these elements interfere with the identification of early, sensitive, and specific septic shock biomarkers. For these reasons, animal models of sepsis, although imperfect, are used to control the kinetics of the development of the pathology and to standardise experimentation, facilitating the identification of potential biomarkers. These elements underline the importance of the choice of animal model used and the sample to be studied during preclinical studies. The aim of this review is to discuss the relevance of different approaches to enable the identification of biomarkers that could indirectly be relevant to the clinical setting.

Urinary neutrophil gelatinase-associated lipocalin for diagnosis of spontaneous bacterial peritonitis.

Cirrhotic patients with ascites are at high risk of developing spontaneous bacterial peritonitis (SBP). After exclusion of patients with acute kidney injury (AKI) or other infections, urinary neutrophil gelatinase-associated lipocalin (NGAL) levels were compared between two matched groups of Egyptian cirrhotic patients with ascites, mostly secondary to hepatitis C infection (98%). Group 1 had SBP (n = 41) and group 2 did not (n = 45). By univariate analysis, urinary-NGAL, high total bilirubin, serum creatinine, international normalised ratio and the Model of End-Stage Liver Disease (MELD) score and low platelet count were all significantly correlated with the presence of SBP, but only urinary-NGAL could independently predict development of SBP (P = 0.001). Urinary-NGAL at a cut-off value of 1225 pg/mL, showed a sensitivity of 95% and a specificity of 76%, and is therefore a most useful tool.

Review article: the utility of reagent strips in the diagnosis of infected ascites in cirrhotic patients.

BACKGROUND: Spontaneous bacterial peritonitis (SBP) can be diagnosed via leucocyte esterase reagent strips, although diagnostic performances vary. AIM: To perform critical review of literature on the use of reagent strips in SBP. METHODS: Nineteen studies were analysed (Medline search), comparing reagent strips in cirrhotic ascites vs. cytobacteriological methods. Diagnostic grades (G) were: GO = 0 leucocytes/mm3; G1 = 15; G2 = 70; G3 = 125; G4 = 500 for Multistix, GO = 0; G1 = 25; G2 = 75; G3 = 500 for Nephur, Combur, UriScan, and GO = 0; G1 = 25; G2 = 75; G3 = 250; G4 = 500 for Aution. RESULTS: Medians per study were: 75 patients (range: 31-1041), 136 ascites (47-2123), 17 SBP (5-117). For Multistix (12 studies), the sensitivities fell within the ranges 64.7-100% (G > or = 1), 45.7-83% (G > or = 2) and 45.3-89% (G > or = 3). For Nephur (n = 2), Combur (n = 6), UriScan (n = 1), sensitivities ranged 80.4-100% (G > or = 1), 63-100% (G > or = 2) and 67.7-97% (G > or = 3). For Aution (n = 3), sensitivities ranged 93-96% (G > or = 2) and 89% (G > or = 3). Nephur, Combur, UriScan displayed higher sensitivities than Multistix. However, in larger studies, sensitivities dramatically fell at 45.3% for Multistix (G > or = 3) if ascites polymorphonuclear count <1000/mm3 and 22.2% for bacterascites or 16.7-25% for asymptomatic patients. CONCLUSION: Use of reagent strips for the diagnosis of SBP cannot be recommended, in view of low sensitivity and a high risk of false negatives, especially in patients with SBP and low polymorphonuclear count.

Collagenase 2/matrix metalloproteinase 8 in critically ill patients with secondary peritonitis.

Secondary peritonitis is an important indication for surgical intensive care admissions, and it is associated with high morbidity and mortality. Collagenase 2/matrix metalloproteinase (MMP) 8 is a tissue matrix-degrading enzyme that is released from leukocytes upon inflammatory stimuli and may thus contribute to peritonitis-associated organ damage. We studied the levels and activity of MMP-8 in the peritoneal fluid of 15 critically ill patients with secondary peritonitis. The MMP-8 levels were measured from the patients' peritoneal fluid, serum, and urine, and from the serum and urine of 10 healthy controls by immunofluorometric assay. Median MMP-8 level in peritoneal fluid supernatant was 1,317 microg/L (interquartile range [IQR]) (1,254-1,359 microg/L) being significantly higher than in the sera of the patients (P=0.008). Molecular forms and isoform distribution of MMP-8, MMP-1, and MMP-13 in peritoneal fluid, assessed by Western immunoblotting, revealed that the neutrophil-type MMP-8 was the major collagenase species in peritoneal fluid, and it was partially in an activated form. Catalytically competent, active MMP-8 produced the characteristic cleavage products from intact human type I collagen. The serum levels of MMP-8 were higher in the patients, 49 microg/L (IQR, 23-214 microg/L), than in the controls, 11 microg/L (IQR, 8-24 microg/L) (P<0.01). The MMP-8 levels in the urine were higher in the patients, 0.27 microg/L (IQR, 0.04-1.89 microg/L), than in the controls, 0.03 microg/L (IQR, 0.0-0.05 microg/L) (P=0.013). Our data demonstrate for the first time that MMP-8 levels are remarkably elevated and in an active and catalytically competent form in the peritoneal fluid samples of patients with secondary peritonitis.

[Characteristics of endotoxemia and non-specific adaptive reaction in patients with severe peritonitis in the Far North]. / Pokazateli éndotoksikoza i nespetsificheskoi adaptivnoi reaktsii pri raspostranennom peritonite v usloviiakh Krainego Severa.

We evaluated the endogenous intoxication biochemical characteristics for healthy person residing in Yakuts and assessed the endogenous manifestations and unspecific adaptive reaction in patients with severe surgical abdominal infection. Such assessments of biochemical parameters of the endogenous intoxication syndrome and of the non-specific adaptive reaction were shown to be highly valuable in individual prognostication and treatment for patients with surgical abdominal infection.

Timely initiation of dialysis--single-exchange experience in 39 patients starting peritoneal dialysis.

OBJECTIVE: To establish the effectiveness and patient acceptability of initiating peritoneal dialysis (PD) according to published guidelines. SETTING: A university teaching hospital and a neighboring district general hospital. DESIGN: Nonrandomized prospective pilot study. PATIENTS: 39 patients with a Kt/V > 2.0 attending predialysis clinics at both hospitals agreed to participate in this study. METHODS: Patients were started on a single exchange of dialysate overnight. Dialysis adequacy was monitored at least every 2 months and incremental increases in dialysis were used to maintain combined urinary and dialysis Kt/V above 2.0. Routine laboratory parameters and complications of dialysis were monitored during the follow-up period. RESULTS: The mean weekly Kt/V at initiation of dialysis was 2.09. Median actuarial survival on a single exchange before requiring incremental dialysis was 297 days. At the end of the study period, all patients were still alive: 8 remained on 1 exchange, 18 were on more than 1 exchange, 8 had switched to hemodialysis, and 5 had received renal transplants. During the 12,665 patient-days on single-exchange dialysis, there were 14 hospital admissions of 12 patients. This resulted in a mean of 1.64 hospital days per patient-year for the whole group. During the follow-up period there were 2 episodes of bacterial peritonitis, 3 pleural leaks, 1 patent processus vaginalis, and 1 inguinal hernia that required surgical intervention. The use single daily icodextrin exchanges was associated with a 46% incidence of culture-negative peritonitis. CONCLUSIONS: This pilot study has shown that a timely start of dialysis with a single overnight PD exchange is acceptable to patients. Incremental dialysis as residual renal function falls is easily managed and patients also find this acceptable. Complication and hospitalization rates were low. The presence of residual renal function often allows complications to be managed without the need for hemodialysis. The use of icodextrin as a single-exchange dialysate is associated with sterile peritonitis in a significant proportion of cases.

[Chemoluminescence of blood and urine in diffuse appendicular peritonitis in children]. / Khemoliuminestsentsiia krovi i mochi pri rasprostranennom appendikuliarnom peritonite u detei.

The results of the study of hemiluminescence of native blood, plasma and urine in 56 children with diffuse purulent peritonitis demonstrate that changes of light-sum of the luminescence of the investigated media could reflect aggravation of endogenous intoxication connected with development of organic insufficiency. The usage of blood and urine hemiluminescence for definition of the gravity of endogenous intoxication in diffuse forms of appendicular peritonitis in children is suggested.

The role of I and B in peritonitis associated with the nephrotic syndrome of childhood.

Children with nephrotic syndrome (NS) are susceptible to bacterial infections, including primary bacterial peritonitis. Immunologic abnormalities associated with NS include low serum levels of the complement proteins I and B of the alternative complement pathway. We developed a novel and highly sensitive enzyme immunoassay using murine MAb to I and B to quantitate urinary concentrations of these proteins. We studied 22 patients with minimal change NS, including seven with a history of peritonitis (1.6 +/- 0.3 episodes, mean +/- SEM) and 15 without such a history. The two groups did not differ significantly in age, sex, race, age at onset of disease, or duration of disease. Children with minimal change NS complicated by peritonitis had 1) increased urinary excretion of both I (p < 0.05) and B (p < 0.05) in relapse versus remission, 2) greater excretion of I in both relapse (p < 0.01) and remission (p < 0.05) compared with patients without peritonitis, 3) greater excretion of B in relapse compared with patients without peritonitis (p < 0.05), and 4) decreased plasma levels of I compared with patients without peritonitis and controls (p < 0.01) and decreased plasma levels of B compared with controls. Increased urinary excretion of I correlated with decreased plasma levels of I (r = 0.88, p < 0.001). These data support our initial hypothesis that depressed plasma concentrations of these proteins of the alternative complement pathway may predispose patients with minimal change NS to peritonitis and that urinary loss of these proteins is a tenable mechanism.

Effects of positive end-expiratory pressure on splanchnic circulation and function in experimental peritonitis.

Splanchnic and central hemodynamic effects of positive end-expiratory pressure (PEEP) were studied in anesthetized pigs using mechanical ventilatory assistance, with or without sepsis (fecal peritonitis). One hour after sepsis, PEEP (10 cm H2O) was applied (n = 6). Another group (n = 6) had sepsis without PEEP. In one group (n = 6) without sepsis, PEEP was applied after 1 hour, while a fourth group (n = 5), without sepsis or PEEP, served as a control. The group with PEEP and sepsis had reduced cardiac index, portal venous blood flow, and liver surface blood flow. The group with PEEP alone had reduced splanchnic circulation by increasing gastrointestinal vascular resistance, while the group with sepsis alone had increased portal vascular resistance. In a separate series with sepsis, intermittent PEEP, and vigorous fluid resuscitation, it was demonstrated that avoiding hypovolemia did not seem to protect from the PEEP effects on the splanchnic circulation. The combination of sepsis and PEEP was not additive on portal blood flow reduction but reduced bile production.

The urine from patients with peritonitis as a rich source for purifying human acid sphingomyelinase and other lysosomal enzymes.

Milligram amounts of human acid sphingomyelinase (EC 3.1.4.12) were purified to 95% homogeneity using urine from patients with acute peritonitis. The activity of this enzyme is elevated more than 200-times in the urine of these patients. To a lesser extent, levels of some other lysosomal hydrolases are also elevated.

Determination of 3 methyl-L-histidine in human urine by ion exchange high performance liquid chromatography. Applications to patients in post-operative surgical care.

A new chromatographic procedure is proposed for measuring 3 methyl-L-histidine (3 MH) in human urine. The sample was purified on a cation-exchange resin (AGR 50W-X4) and analysed by ion exchange high performance liquid chromatography on a PARTISIL 10 SCX Whatman column in UV light at 210 nm within 16 min. This procedure gave similar outputs of 3 MH to those described in human normal urine (mean +/- SEM = 213 +/- 15 mumol X 24 h-1, n = 19). It was used to measure the urinary outputs of 3 MH of five patients admitted to an intensive surgical care unit, for 48, 28, 25, 15 and 10 days, respectively. The urinary outputs of 3 MH were normal or lower than normal. The 10(3) urinary 3 MH/creatinine molar ratios were also calculated; this new 3 MH analysis could help the reanimator to prescribe an adequate nutritional assessment.

Effect of parenteral nutrition on muscle amino acid output and 3-methylhistidine excretion in septic patients.

The effects of adequate total parenteral nutrition (TPN) on nitrogen excretion, urea N percentage, 3-methylhistidine excretion, and leg amino acid output, were studied during the ten-day period following abdominal surgery for generalized peritonitis in nine patients. The first two postoperative days were without nutritional intake, TPN was started on the third postoperative day (57 cal/KgBW--40% as Intralipid--0.30 g of N/KgBW). Leg amino acid outputs were done before TPN (DO), then two days (D2) and eight days (D8) after TPN. Total nitrogen and urea N percentage did not significantly differ before and after TPN. Between DO and D2 there was a significant reduction of urinary 3-methylhistidine (467 +/- 37 to 280 +/- 29 mumol/24 h-P less than 0.001) and leg amino acid release (604 +/- 103 to 254 +/- 87 nmol/mn/100 g of calf muscle--P less than 0.01) reflecting reduction in muscle hypercatabolism despite the persistence of the septic state. Between D2 and D8, 3-methylhistidine remained stable while leg amino acid release continued to decrease (254 +/- 87 to 68 +/- 40 nmol/mn/100 g--P less than 0.05). This association suggests an increased muscle protein synthesis. A closer examination of the clinical evolution of these patients, especially concerning their septic evolution, shows that only improved patients with recovery from sepsis increased their muscle protein synthesis. Thus, in septic hypercatabolic patients TPN seems to be able to reduce muscle catabolism while the increase in protein synthesis is mainly the consequence of recovery from the septic state. In such patients TPN should be used as a preventive therapeutic measure.

[Postoperative peritonitis. Use of metabolic disorders as criteria for reoperations]. / Les péritonites post-opératoires. Utilisation des perturbations métaboliques comme critères de réintervention

The authors compared three groups of subjects: - the first group (11 patients) : re-operated for post-operative peritonitis; - the second group (6 patients) : re-operated for evisceration without any underlying lesion; - the third group (8 patients) : non-reoperated, complicated abdominal surgery. Study of the different laboratory parameters helped to identify a number of changes which could plead in favor of reintervention, in the group of peritonitis. Among these modifications, the progressive fall in the urinary Na/K ratio and the negativity of the nitrogen balance appear to play a privileged role.