Clostridium perfringens-induced massive hemolysis treatment with blood purification to target toxins: a case report.

Clostridium perfringens can rarely cause severe systemic infections, usually from an abdominal source, associated with massive hemolysis, which is usually fatal. Hemolytic anemia and acute renal injury resulting from toxin action are critical for the development of multiple organ dysfunction syndrome (MODs), making this condition a real emergency, requiring multispecialty skills and aggressive multimodal therapies. We herein describe a case of septic shock from acute cholecystitis with massive hemolysis caused by C. perfringens in a 55 year-old man that was successfully treated with early blood purification and continuous renal replacement therapy (CRRT) along with antibiotic therapy and surgery. The effect of the enormous amount of toxins produced by Clostridium which elicit a strong cytokine response and the damage caused by the hemolysis products are the main pathogenetic mechanisms of this rare but lethal clinical entity. The main goal of treatment is to remove toxins from plasma, block toxin action, and further production by achieving bacterial killing with antimicrobial agents and controlling the infectious focus, remove waste products and prevent or limit multiorgan damage. Blood purification techniques play an important role due to a strong pathophysiological rationale, as they can remove toxins and cytokines as well as cell-free products from plasma and also replace renal function. Although this condition is rare and robust data are lacking, blood purification techniques for C. perfringens-induced massive hemolysis are promising and should be further explored.

Gut microbiota trajectory in pediatric patients undergoing hematopoietic SCT.

Acute GvHD (aGvHD) is the main complication of hematopoietic SCT (HSCT) during the treatment of hematological disorders. We carried out the first longitudinal study to follow the gut microbiota trajectory, from both the phylogenetic and functional points of view, in pediatric patients undergoing HSCT. Gut microbiota trajectories and short-chain fatty acid production profiles were followed starting from before HSCT and through the 3-4 months after transplant in children developing and not developing aGvHD. According to our findings, HSCT procedures temporarily cause a structural and functional disruption of the gut microbial ecosystem, describing a trajectory of recovery during the following 100 days. The onset of aGvHD is associated with specific gut microbiota signatures both along the course of gut microbiota reconstruction immediately after transplant and, most interestingly, prior to HSCT. Indeed, in pre-HSCT samples, non-aGvHD patients showed higher abundances of propionate-producing Bacteroidetes, highly adaptable microbiome mutualists that showed to persist during the HSCT-induced ecosystem disruption. Our data indicate that structure and temporal dynamics of the gut microbial ecosystem can be a relevant factor for the success of HSCT and opens the perspective to the manipulation of the pre-HSCT gut microbiota configuration to favor mutualistic persisters with immunomodulatory properties in the gut.

Multi-residual GC-MS determination of personal care products in waters using solid-phase microextraction.

A multi-residual method is described for the simultaneous determination of 23 personal care products (PCPs), which display a wide range of physicochemical properties, present at trace levels in water samples. A one-step procedure was developed based on solid-phase microextraction (SPME) coupled with GC-MS analysis. A chemometric approach consisting of an experimental design (design of experiments) was applied to systematically investigate how four operating parameters--extraction temperature and time and desorption temperature and time--affect extraction recovery of PCPs in water. The optimum SPME procedure operating conditions, those yielding the highest extraction recovery for all the compounds, were determined; they correspond to an extraction time of 90 min and temperature of 80 °C and a desorption time of 11 min and temperature of 260 °C. Under these optimized conditions, the SPME procedure shows good analytical performance characterized by high reproducibility (RSD% intra-day accuracy varying in the 0.01-1.3% range) as well as good linearity and low detection limits (LODs lower than 2 ppb for most of the investigated PCPs).

Enantiomeric resolution of biomarkers in space analysis: Chemical derivatization and signal processing for gas chromatography-mass spectrometry analysis of chiral amino acids.

The work compares two GC-MS methods for enantioselective separation of amino acids as suitable candidate for stereochemical analysis of chiral amino acids on board spacecrafts in space exploration missions of solar system body environments. Different derivatization reagents are used: a mixture of alkyl chloroformate-alcohol-pyridine to obtain the alkyl alkoxy carbonyl esters and a mixture of perfluorinated alcohols and anhydrides to form perfluoroacyl perfluoroalkyl esters. 20 proteinogenic amino acids were derivatized with the two procedures and submitted to GC-MS analysis on a Chirasil-l-Val stationary phase. The results were then compared in terms of the enantiomeric separation achieved and intensity of MS response. The combination of methyl chloroformate (MCF) and heptafluoro-1-butanol (HFB) allows separation of 14 enantiomeric pairs, five of which display a resolution (R(s)>or=1.2) supposed to be sufficient to quantify the enantiomeric excess. Three mixtures of trifluoroacetic (TFAA) and heptafluorobutyric (HFBA) anhydrides were combined with the corresponding perfluorinated alcohols - TFE (2,2,2-trifluoro-1-ethanol) and HFB (2,2,3,3,4,4,4-heptafluoro-1-butanol) - to give three different reagents (TFAA-TFE, TFAA-HFB, HFBA-HFB): the derivatives obtained show separation of the same number of proteinogenic amino acids (14 of 20) at a temperature lower than column bleeding limit (200 degrees C) and 8 of them give a separation with R(s)>or=1.2. Linearity study and limit of detection (X(LOD)) computation show that both methods are suitable for quantitative determination of several amino acid diastereomers at trace level (X(LOD) approximately 0.5nmol as derivatized quantity). Both the procedures were coupled with automatic data handling to increase their suitability for space analysis: the simplified data treatment is especially helpful to handle the low quality data recovered from space experiments and labor and time are saved, as imposed by the space experiments requiring a rapid delivery of the results. To achieve this aim, a chemometric approach based on the computation of the Autocovariance Function (ACVF) was applied to extract information on the enantiomeric pairs present in the sample and the enantioseparation achieved on the chiral column.

Gas chromatography-mass spectrometry analysis of amino acid enantiomers as methyl chloroformate derivatives: application to space analysis.

This work describes a GC-MS method for enantioselective separation of amino acids. The method is based on a derivatization reaction which employs a mixture of alkyl chloroformate-alcohol-pyridine, as reagents to obtain the N(O,S)-alkyl alkoxy carbonyl esters of amino acids. Various reaction parameters are investigated and optimized to achieve a reproducible derivatization procedure suitable for separation of amino acid enantiomers on Chirasil-L-Val chiral stationary phase. In particular, the following topics are investigated for 20 proteinogenic amino acids: (i) the proper reagent and reaction conditions to obtain the highest derivative yield; (ii) the amino acid reactivity and the MS properties of the obtained derivatives; (iii) the linearity and sensitivity of the analytical method; (iv) the retention behavior of the derivatives and their enantiomeric separation on the Chirasil-L-Val chiral stationary phase. By combining the resolution power of the Chirasil-L-Val column and the high selectivity of the SIM MS detection mode, the described procedure enables the enantiomeric separation and quantification of 16 enantiomeric pairs of amino acids. The procedure is simple and fast and reproducible. It displays a wide linearity range at ppb detection limits for quantitative determinations: these properties make this derivatization method a suitable candidate for amino acid GC-MS analysis on board of the spacecrafts in space exploration missions of solar system body environments.

Catheter-related bacteremia due to Kocuria kristinae in a patient with ovarian cancer.

We report on the first case of a catheter-related recurrent bacteremia caused by Kocuria kristinae, a gram-positive microorganism belonging to the family Micrococcaceae, in a 51-year-old woman with ovarian cancer. This unusual pathogen may cause opportunistic infections in patients with severe underlying diseases.

Time-dependent efficacy of bacterial filters and infection risk in long-term epidural catheterization.

BACKGROUND: Epidural infection represents a serious albeit infrequent complication of long-term epidural catheterization. The catheter hub is regarded as the main point of entry for microorganisms among the three possible routes (hematogenous, insertion site, hub) of microbial colonization of the inserted catheter. The current study was aimed at evaluating whether frequent changing of antimicrobial filters carries an increased risk of catheter hub contamination and the time-dependent efficacy of commonly used antimicrobial filters after prolonged use. METHODS: In the first part of the study, a microbiologic survey (skin, filter, hub, and catheter tip) was performed weekly in a group of 47 patients with cancer bearing subcutaneously tunneled catheters managed at home. Subsequently, the time-dependent efficacy of 96 micropore filters (32 Portex, 32 Sterifix-Braun, 32 Encapsulon TFX-Medical) differing in surface areas and/or composition of the filtering membrane was evaluated in a laboratory study. Filters were perfused, under the usual conditions of clinical use (flow resistance, injection pressure, temperature), every 8 h up to 60 days, with 5 ml of two different analgesic solutions, either sterile or containing 1.5 x 10(5)/ml of Streptococcus milleri I. Eight filters of each type subsequently were flushed with a S. milleri suspension (0.5 McFarland) after 7, 14, 28, and 60 days of continuous perfusion, and the resulting filtrates were cultured. RESULTS: In 16 of 19 positive hub cultures, the same microorganisms (species, biotype, antibiotype) were cultured from skin and filters. A statistically significant positive trend was found between the number of filter changes and the rate of positive hub cultures (chi 1(2) trend 5.11; P = 0.02). A high correlation coefficient was found between number of positive skin cultures and number of positive filtrates (r = 0.88; P = 0.01) and between number of positive filtrates and number of positive hub cultures (r = 0.93; P = 0.003). Cultures obtained from Portex and Sterifix-Braun filters yielded no bacterial growth (64/64) throughout the study period. Cultures from Encapsulon TFX-Medical filters showed bacterial growth 2/8 at seventh day, 7/8 at the 14th day, and 16/16 from the 28th day onward. CONCLUSIONS: Our data indicate significant correlation between the incidence of catheter hub colonization and the filter-change frequency, when the skin close to the filter-hub connection is contaminated. Our results also show that Portex and Sterifix-Braun bacterial filters, when perfused with reduced volumes at low injection pressures, maintain an unmodified antimicrobial function for at least 60 days. Based on these data, it appears clinically feasible to reduce the frequency of filter changes during long-term epidural catheterization, with a consequent possible decrease of epidural catheter colonization.

Effects of alpha-interferon and steroids on CD23 expression and release in B-cell chronic lymphocytic leukemia.

BACKGROUND: Since high CD23 expression and release have been reported in B-chronic lymphocytic leukemia (B-CLL), we investigated whether alpha-interferon or corticosteroids were able to modulate the expression and/or the release of this factor. METHODS: CD23 expression was determined with FITC-labelled anti-CD23 monoclonal antibody, and sCD23 release with a sandwich enzyme immunoassay. Twenty-one patients affected by B-CLL (stage A or B) were studied before and after three different treatment regimens (alpha-interferon, corticosteroids, alpha-interferon+corticosteroids). RESULTS: CD23 was highly expressed in the B-cells of all patients, and expression was not modified by any of the therapies, sCD23 release from leukemic cells was significantly greater (p < 0.00001) in untreated subjects than controls, and in vitro treatment with phorbol myristate acetate (PMA) led to a 10-fold increase (p < 0.0001) in sCD23 secretion. On the contrary, PMA did not increase sCD23 release in normal B cells. Treatment with corticosteroids (either alone or associated with alpha-interferon) reduced sCD23 secretion from leukemic cells, whereas alpha-interferon alone was not able to modify sCD23 release. CONCLUSIONS: Our data support the hypothesis that CD23 plays a role in the maintenance and progression of B-CLL and that the pharmacological modulation of this receptor/lymphokine could be useful in the therapy of B-CLL.

Phenotypic profile and functional characteristics of human gamma and delta T cells during acute toxoplasmosis.

Gamma and delta (gamma delta) T-cell receptor lymphocytes are increased during acute toxoplasmosis. These cells are BB3+ CD45RO+ CD8-. Purified gamma delta T cells failed to proliferate in response to Toxoplasma gondii antigen (stimulation index, 1.4 +/- 0.6) but were responsive to phytohemagglutinin stimulation (stimulation index, 20.8 +/- 1.9). Natural-killer-like cytotoxicity was strongly acquired only after in vitro culture of purified gamma delta T cells with recombinant interleukin 2 (40% +/- 7% specific lysis). Our data show that gamma delta T-cell receptor T cells with a peculiar phenotype are increased during human acute T. gondii infection.

A subset of gamma delta lymphocytes is increased during HIV-1 infection.

The gamma delta T cell receptor (TcR) lymphocytes constitute 3-10% of human peripheral blood lymphocytes. Only a very small fraction of these cells is recognized by the delta TCS1 monoclonal antibody, directed against the V delta 1 chain of the receptor. We describe the immunological, virological and clinical data of a small group of seropositive subjects having high levels of gamma delta TcR T cells in the peripheral blood. Our flow cytometric studies show that most of these cells belong to the delta TCS1+ (V delta 1+), CD8 +/- (dim staining) subset. Patients with high gamma delta TcR T cell numbers were not characterized by the presence of an acute (IgM positive) or reactivated (as defined by high IgG titres against early antigen or IgA titres against viral capsidic antigen) Epstein-Barr virus infection. Cytomegalovirus infection was excluded by serological assays, and other herpes viral infections were not found after clinical examination. HIV p24 antigenaemia was present in two out of 11 subjects. AIDS patients had very high percentages of gamma delta TcR T cells. Altogether these data show that the selective expansion of delta TCS1+ cells in HIV1 seropositive subjects is not related to some exogenous antigen stimulation, but may be related to peculiar pathologic processes involving the immune system.

Phenotypic analysis of a CD2- CD3+ T cell receptor gamma delta lymphocyte subset.

We have identified, in a patient with atopic dermatitis, a consistent population of peripheral blood lymphocytes expressing a CD3+ gamma/delta TCR complex, while being unreactive with CD2. Further immunofluorescence studies showed that these cells almost completely co-express CD29 and CD45RA and have high membrane levels of CD11a compared to the alpha/beta TCR T cells. Neither a genetic influence nor an acute or reactivated herpesvirus infection were found to be related to the expanded gamma/delta T-cell subpopulation. Our data confirm the previous observations regarding the presence in the peripheral blood of an expanded gamma/delta TCR, CD2- subset and show that these cells have a peculiar phenotypic profile. The reasons for this expansion are, however, still unknown.

Critical evaluation of bioluminescence methods for quantifying bacterial adhesion to polystyrene.

A total of 134 strains of Escherichia coli which included 60 fecal and 74 urinary isolates, cultured in liquid and on solid media, were examined for adhesive properties using bioluminescence and haemagglutination methods. The study aimed to verify whether irrespective of the absence or presence of flagella, there is any relation between haemagglutination and bioluminescence test. Examining the results we failed to note any correlation between the two methods: strains bearing MS, MR or MS-MR adhesins adhered to polystyrene at random. Even though it is fast and easy to perform, bioluminescence is not an alternative to traditional methods to reveal MS adhesins.

A method for the determination of the adherence of granulocytes to microtitre plates.

We report a new partially automated method for the measurement of the adherence of PMN in vitro. Adherence to a plastic surface was detected by measuring leukocyte alkaline phosphatase activity of the adherent cells, with a Titertek Multiscan system. Using three different cellular concentrations (1 X 10(6), 5 X 10(5), 2.5 X 10(5) PMN/ml) the response curve was linear to 45 min and adhesion was maximal by 30 min. The specificity of the reaction was acceptable as was the assay reproducibility (intra-assay coefficient of variation less than 8%; inter-assay coefficient of variation less than 11%).