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Background: Intracellular Ca2+ modulates several microglial activities, such as proliferation, migration, phagocytosis, and inflammatory mediator secretion. Extracellular ATP, the levels of which significantly change during epileptic seizures, activates specific receptors leading to an increase of intracellular free Ca2+ concentration ([Ca2+]i). Here, we aimed to functionally characterize human microglia obtained from cortices of subjects with temporal lobe epilepsy, focusing on the Ca2+-mediated response triggered by purinergic signaling.Methods: Fura-2 based fluorescence microscopy was used to measure [Ca2+]i in primary cultures of human microglial cells obtained from surgical specimens. The perforated patch-clamp technique, which preserves the cytoplasmic milieu, was used to measure ATP-evoked Ca2+-dependent whole-cell currents.Results: In human microglia extracellular ATP evoked [Ca2+]i increases depend on Ca2+ entry from the extracellular space and on Ca2+ mobilization from intracellular compartments. Extracellular ATP also induced a transient fivefold potentiation of the total transmembrane current, which was completely abolished when [Ca2+]i increases were prevented by removing external Ca2+ and using an intracellular Ca2+ chelator. TRAM-34, a selective KCa3.1 blocker, significantly reduced the ATP-induced current potentiation but did not abolish it. The removal of external Cl- in the presence of TRAM-34 further lowered the ATP-evoked effect. A direct comparison between the ATP-evoked mean current potentiation and mean Ca2+ transient amplitude revealed a linear correlation. Treatment of microglial cells with LPS for 48 h did not prevent the ATP-induced Ca2+ mobilization but completely abolished the ATP-mediated current potentiation. The absence of the Ca2+-evoked K+ current led to a less sustained ATP-evoked Ca2+ entry, as shown by the faster Ca2+ transient kinetics observed in LPS-treated microglia.Conclusions: Our study confirms a functional role for KCa3.1 channels in human microglia, linking ATP-evoked Ca2+ transients to changes in membrane conductance, with an inflammation-dependent mechanism, and suggests that during brain inflammation the KCa3.1-mediated microglial response to purinergic signaling may be reduced. [ABSTRACT FROM AUTHOR]

Background: The human pathogenic mold Aspergillus fumigatus is able to form a complex biofilm embedded in extracellular matrix. Biofilms confer antimicrobial resistance and it is well known that aspergillosis is often refractory to the conventional antifungal therapy. The treatment of biofilm-related infections poses a significant clinical challenge on a daily basis, promoting the search for new therapeutic agents. Our aim was to exploit the modulation of sphingolipid mediators as new therapeutic target to overcome antifungal resistance in biofilm-related infections. Results: Antifungal susceptibility testing was performed on 20 clinical isolates of Aspergillus fumigatus and one reference strain (A. fumigatus Af293) according the EUCAST protocol. Sessile MICs were assessed on 24-h preformed-biofilm by means of XTT-reduction assay. Myriocin (0.25-64 mg/L), a commercial sphingolipid synthesis inhibitor, was used. The MEC50 value (mg/L) of Myriocin was 8 (range 4-16) for both planktonic and sessile cells. Drug-induced morphological alterations were analyzed by optical and electron microscopy (TEM) on 24h preformed A. fumigatus Af293 biofilms. An evident hyphal damage, resulting in short, stubby, and highly branched hyphae was observed by optical microscopy. At 24h, TEM studies showed important morphological alterations, such as invaginations of the cell membrane, modification in the vacuolar system and presence of multilamellar bodies, in some cases within vacuoles. Conclusions: The direct antifungal activity, observed on both planktonic and sessile fungi, suggests that inhibition of sphingolipid synthesis could represent a new target to fight biofilm-related A. fumigatus resistance. [ABSTRACT FROM AUTHOR]

Introduction: An online survey was conducted to characterize current infection management practices in Italian intensive care units (ICUs), including the antibacterial and antifungal drug regimens prescribed for various types of infections. Methods: During February and March 2011, all 450 ICUs in public hospitals in Italy were invited to take part in an online survey. The questionnaire focused on ICU characteristics, methods used to prevent, diagnose, and treat infections, and antimicrobials prescribing policies. The frequency of each reported practice was calculated as a percentage of the total number of units answering the question. The overall response rate to the questionnaire was 38.8% (175 of the 450 ICUs contacted) with homogeneous distribution across the country and in terms of unit type. Results: Eighty-eight percent of the responding facilities performed periodical surveillance cultures on all patients. In 71% of patients, cultures were also collected on admission. Endotracheal/bronchial aspirates were the most frequently cultured specimens at both time points. Two-thirds of the responding units had never performed screening cultures for methicillin-resistant Staphylococcus aureus. Around 67% of the ICUs reported the use of antimicrobial de-escalation strategies during the treatment phase. In general, the use of empirical antimicrobial drug regimens was appropriate. Although the rationale for the choice was not always clearly documented, the use of a combination therapy was preferred over antibiotic monotherapy. The preferred first-line agents for invasive candidiasis were fluconazole and an echinocandin (64% and 25%, respectively). Two-thirds of the ICUs monitored vancomycin serum levels and administered it by continuous infusion in 86% of cases. For certain antibiotics, reported doses were too low to ensure effective treatment of severe infections in critically ill patients; conversely, inappropriately high doses were administered for certain antifungal drugs. Conclusions: Although infection control policies and management practices are generally appropriate in Italian ICUs, certain aspects, such as the extensive use of multidrug empirical regimens and the inappropriate antimicrobial dosing, deserve careful management and closer investigation. [ABSTRACT FROM AUTHOR]

Background: Yeasts are a common cause of invasive fungal infections in critically ill patients. Antifungal susceptibility testing results of clinically significant fungal strains are of interest to physicians, enabling them to adopt appropriate strategies for empiric and prophylactic therapies. We investigated the antifungal susceptibility of yeasts isolated over a 2-year period from hospitalised patients with invasive yeast infections. Methods: 638 yeasts were isolated from the blood, central venous catheters and sterile fluids of 578 patients on general and surgical intensive care units and surgical wards. Etest strips and Sensititre panels were used to test the susceptibility of the isolates to amphotericin B, anidulafungin, caspofungin, fluconazole, itraconazole, posaconazole and voriconazole in 13 laboratories centres (LC) and two co-ordinating centres (CC). The Clinical and Laboratory Standards Institute (CLSI) reference broth microdilution method was used at the CCs for comparison. Results: Etest and Sensititre (LC/CC) MIC90 values were, respectively: amphotericin B 0.5/0.38, 1/1 mg/L; anidulafungin 2/1.5 and 1/1 mg/L; caspofungin 1/0.75 and 0.5/0.5 mg/L; fluconazole 12/8 and 16/16 mg/L; itraconazole 1/1.5, 0.5/0.5 mg/L; posaconazole 0.5 mg/L and voriconazole 0.25 mg/L for all. The overall MIC90 values were influenced by the reduced susceptibility of Candida parapsilosis isolates to echinocandins and a reduced or lack of susceptibility of Candida glabrata and Candida krusei to azoles, in particular fluconazole and itraconazole. Comparison of the LC and CC results showed good Essential Agreement (90.3% for Etest and 92.9% for Sensititre), and even higher Categorical Agreement (93.9% for Etest and 96% for Sensititre); differences were observed according to the species, method, and antifungal drug. No cross-resistance between echinocandins and triazoles was detected. Conclusions: Our data confirm the different antifungal susceptibility patterns among species, and highlight the need to perform antifungal susceptibility testing of clinically relevant yeasts. With the exception of a few species (e. g. C. glabrata for azoles and C. parapsilosis for echinocandins), the findings of our study suggest that two of the most widely used commercial methods (Etest and Sensititre) provide valid and reproducible results. [ABSTRACT FROM AUTHOR]