Changing Epidemiology of Invasive Candidiasis in Children during a 10-Year Period.

Candida species are a common cause of invasive infection in neonates and children. The aim of our study was to evaluate the epidemiology and microbiology of invasive candidiasis (IC) in the largest tertiary Greek pediatric hospital during a 10-year period. A retrospective cohort study was performed from January 2008 to December 2017. Identification of species and antifungal susceptibility testing was performed according to the Clinical and Laboratory Standards Institute (CLSI) methodology. During the study period, 178 cases of IC were recorded. The tissue distribution included blood (87.1%), cerebrospinal (7.9%), peritoneal (3.9%) and pleural fluids (1.1%). Candida albicans and Candida parapsilosis (sensu lato) were the most frequently isolated species (47.8% and 28.7% respectively). From period 2008⁻2012 to period 2013⁻2017, a significant decrease in IC rates was detected (0.21 cases/1000 hospitalization days VS 0.11 cases/1000 hospitalization days, P = 0.040), while median minimum inhibitory concentrations (MICs) of amphotericin B were significantly increased for both C. albicans and C. parapsilosis (sl) (P = 0.037 and P = 0.004 respectively). The decrease in IC rates may reflect the increased awareness as well as the effective infection control initiatives and antifungal interventions. However, the significant increase in the MICs for amphotericin B and echinocandins such as caspofungin, raises concerns about their common use as first-line treatment. Epidemiologic monitoring is, therefore, critically important in order to evaluate and optimize therapeutic protocols for IC in pediatric populations.

Staphylococcus aureus osteoarticular infections in children: an 8-year review of molecular microbiology, antibiotic resistance and clinical characteristics.

PURPOSE: To investigate the clinical, phenotypic and genotypic characteristics of Staphylococcus aureus strains causing osteoarticular infections in a large paediatric series. METHODOLOGY: Medical records of children who were hospitalized with the diagnosis of community-associated S. aureus (CA-SA) osteomyelitis and/or septic arthritis in the two major tertiary paediatric hospitals of Athens during an 8-year period (2007-2015) were reviewed, and S. aureus isolates were analysed regarding antimicrobial resistance, detection of pathogenicity genes and genotyping using SCCmec, agr typing, PFGE and MLST. RESULTS: During the study period, 123 children with CA-SA osteoarticular infections were identified, and methicillin-resistant S. aureus (MRSA) accounted for 44 of these (35.8 %). Children with MRSA infection had a significantly higher admission rate to the ICU (5.7  vs 0 %, P=0.04) and longer duration of hospitalization (21.6 vs 16.7 days, P=0.04). Sixty-eight isolates [42 (methicillin-sensitive S. aureus) MSSA and 26 MRSA] were available for molecular analysis. All MRSA strains were mecA-positive and most carried the SCCmec IV cassette (23/26, 88 %) and belonged to the PFGE type C (24/26, 92.3 %), agr type 3 (24/26, 92.3 %) and the MLST ST80 clone (24/26, 92.3 %). In contrast, MSSA strains showed polyclonality by PFGE and agr typing. Regarding pathogenicity genes, MRSA vs MSSA isolates showed higher detection rates of PVL (96.2 vs 4.8 %, P<0.0001) and fib (80.8 vs 50 %, P=0.02). CONCLUSIONS: In our study a considerable number of S. aureus osteoarticular infections were due to CA-MRSA isolates, most of which belonged to the ST80 clone and had a higher incidence of specific virulence factors, entailing higher ICU admission rates and a longer duration of hospitalization.

Effect of clarithromycin in patients with suspected Gram-negative sepsis: results of a randomized controlled trial.

BACKGROUND: A previous randomized study showed that clarithromycin decreases the risk of death due to ventilator-associated pneumonia and shortens the time until infection resolution. The efficacy of clarithromycin was tested in a larger population with sepsis. METHODS: Six hundred patients with systemic inflammatory response syndrome due to acute pyelonephritis, acute intra-abdominal infections or primary Gram-negative bacteraemia were enrolled in a double-blind, randomized, multicentre trial. Clarithromycin (1 g) was administered intravenously once daily for 4 days consecutively in 302 patients; another 298 patients were treated with placebo. Mortality was the primary outcome; resolution of infection and hospitalization costs were the secondary outcomes. RESULTS: The groups were well matched for demographics, disease severity, microbiology and appropriateness of the administered antimicrobials. Overall 28 day mortality was 17.1% (51 deaths) in the placebo arm and 18.5% (56 deaths) in the clarithromycin arm (P = 0.671). Nineteen out of 26 placebo-treated patients with septic shock and multiple organ dysfunctions died (73.1%) compared with 15 out of 28 clarithromycin-treated patients (53.6%, P = 0.020). The median time until resolution of infection was 5 days in both arms. In the subgroup with severe sepsis/shock, this was 10 days in the placebo arm and 6 days in the clarithromycin arm (P = 0.037). The cost of hospitalization was lower after treatment with clarithromycin (P = 0.044). Serious adverse events were observed in 1.3% and 0.7% of placebo- and clarithromycin-treated patients, respectively (P = 0.502). CONCLUSIONS: Intravenous clarithromycin did not affect overall mortality; however, administration shortened the time to resolution of infection and decreased the hospitalization costs.

Angiopoietin-2 enhances survival in experimental sepsis induced by multidrug-resistant Pseudomonas aeruginosa.

Levels of circulating angiopoietin-2 (Ang-2) increase in sepsis, raising the possibility that Ang-2 acts as a modulator in the sepsis cascade. To investigate this, experimental sepsis was induced in male C57BL6 mice by a multidrug-resistant isolate of Pseudomonas aeruginosa; survival was determined along with neutrophil tissue infiltration and release of proinflammatory cytokines. Survival was significantly increased either by pretreatment with recombinant Ang-2 2 h before or treatment with recombinant Ang-2 30 min after bacterial challenge. Likewise, Ang-2 pretreatment protected against sepsis-related death elicited by Escherichia coli; however, Ang-2 failed to provide protection in lipopolysaccharide (LPS)-challenged mice. The survival advantage of Ang-2 in response to P. aeruginosa challenge was lost in tumor necrosis factor (TNF)-deficient mice or neutropenic mice. Infiltration of the liver by neutrophils was elevated in the Ang-2 group compared with saline-treated animals. Serum TNF-α levels were reduced by Ang-2, whereas those of interleukin (IL)-6 and IL-10 remained unchanged. This was accompanied by lower release of TNF-α by stimulated splenocytes. When applied to U937 cells in vitro, heat-killed P. aeruginosa induced the secretion of IL-6 and TNF-α; low levels of exogenous TNF-α synergized with P. aeruginosa. This synergistic effect was abolished after the addition of Ang-2. These results put in evidence a striking protective role of Ang-2 in experimental sepsis evoked by a multidrug-resistant isolate of P. aeruginosa attributed to modulation of TNF-α production and changes in neutrophil migration. The protective role of Ang-2 is shown when whole microorganisms are used and not LPS, suggesting complex interactions with the host immune response.

Impact of multidrug resistance on experimental empyema by Pseudomonas aeruginosa.

BACKGROUND: Pseudomonas aeruginosa is a cause of infections of the lower respiratory tract among patients with chronic lung disorders. It is questionable whether virulence of this species may be influenced by multidrug resistance (MDR). OBJECTIVES: To define the impact of MDR in experimental lung infection. METHODS: Experimental empyema was induced in rabbits by MDR (group A, n = 16) and by susceptible isolates (group B, n = 10). Pleural fluid was sampled for quantitative culture and estimation of cell apoptosis and of tumor necrosis factor-alpha (TNFα) and malondialdehyde (MDA). Survival was recorded. Cytokine production was stimulated in U937 monocytes by samples of pleural fluid. Whole blood of rabbits was incubated with the isolates; induction of apoptosis was assessed. RESULTS: Survival of group A was prolonged compared to group B. This was accompanied by lower bacterial counts of the inoculated pathogens in pleural fluid and in the lungs of group A compared with group B. Early apoptosis of neutrophils of pleural fluid of group A was lower compared with group B. Pleural fluid concentrations of TNFα and MDA did not differ between the groups. Cytokine production by U937 monocytes after stimulation with pleural fluid was greater in group B than in group A. The susceptible isolate induced apoptosis of neutrophils in vitro at a greater rate than the MDR isolate. CONCLUSIONS: Experimental empyema by susceptible P. aeruginosa is accompanied by greater mortality compared with MDR P. aeruginosa. This phenomenon may be attributed to the different growth pattern of the pathogens or to their interaction with the innate immune system.

An early circulating factor in severe sepsis modulates apoptosis of monocytes and lymphocytes.

We hypothesized that a factor may circulate in serum early during sepsis, modulating apoptosis of monocytes and lymphocytes. Serum was collected from 20 healthy volunteers and from 48 patients with severe sepsis/shock within 12 h from signs of the first failing organ. PBMCs were isolated from 20 healthy volunteers and incubated with collected sera. Apoptosis and expression of CD95 were determined by flow cytometry; experiments were run in the presence of caspase-8 and caspase-9 inhibitors and of CaCl(2). Activity of caspase-3 was determined in cell lysates by a chromogenic kinetic assay. Incubation with serum of patients induced apoptosis of CD4 lymphocytes and inhibited apoptosis of CD14 monocytes. This was attenuated after diluting serum or mixing with healthy serum. Activity of caspase-3 was consistent with these findings. Induced apoptosis of CD4 lymphocytes was greater among nonsurvivors, and it was inhibited in the presence of caspase inhibitors. Inhibitors did not modify the effect of patients' serum on apoptosis of CD14 monocytes. CaCl(2) reversed the inhibitory effect on apoptosis of CD14 moncytes. The above findings support the hypothesis for the existence of an early circulating factor in severe sepsis/shock, modulating apoptosis of CD4 lymphocytes and of CD14 monocytes by interaction with the two apoptotic pathways.

Early alterations of the innate and adaptive immune statuses in sepsis according to the type of underlying infection.

INTRODUCTION: Although major changes of the immune system have been described in sepsis, it has never been studied whether these may differ in relation to the type of underlying infection or not. This was studied for the first time. METHODS: The statuses of the innate and adaptive immune systems were prospectively compared in 505 patients. Whole blood was sampled within less than 24 hours of advent of sepsis; white blood cells were stained with monoclonal antibodies and analyzed though a flow cytometer. RESULTS: Expression of HLA-DR was significantly decreased among patients with severe sepsis/shock due to acute pyelonephritis and intraabdominal infections compared with sepsis. The rate of apoptosis of natural killer (NK) cells differed significantly among patients with severe sepsis/shock due to ventilator-associated pneumonia (VAP) and hospital-acquired pneumonia (HAP) compared with sepsis. The rate of apoptosis of NKT cells differed significantly among patients with severe sepsis/shock due to acute pyelonephritis, primary bacteremia and VAP/HAP compared with sepsis. Regarding adaptive immunity, absolute counts of CD4-lymphocytes were significantly decreased among patients with severe sepsis/shock due to community-acquired pneumonia (CAP) and intraabdominal infections compared with sepsis. Absolute counts of B-lymphocytes were significantly decreased among patients with severe sepsis/shock due to CAP compared with sepsis. CONCLUSIONS: Major differences of the early statuses of the innate and adaptive immune systems exist between sepsis and severe sepsis/shock in relation to the underlying type of infection. These results may have a major impact on therapeutics.

Angiopoietin-2 is increased in septic shock: evidence for the existence of a circulating factor stimulating its release from human monocytes.

We aimed to investigate if angiopoietin-2 (Ang-2) participates in the septic process and what may be the role of monocytes as a site of release of Ang-2 in sepsis. Concentrations of Ang-2 were estimated in sera and in supernatants of monocytes derived form one already described cohort of 90 patients with septic syndrome due to ventilator-associated pneumonia (VAP). Mononuclear cells of 17 healthy volunteers were stimulated by serum of patients in the presence or absence of various intracellular pathway inhibitors. Ang-2 gene expression after stimulation was also tested. Ang-2 was higher in patients with septic shock compared to patients with sepsis, severe sepsis and controls. Ang-2 was significantly increased in non-survivors compared with survivors. Serum levels greater than 9700 pg/ml were accompanied by a 3.254 odds ratio for death (p: 0.033). Ang-2 release from monocytes of septic patients was slightly decreased after stimulation with lipopolysaccharide (LPS) of Escherichia coli O55:B5. Release of Ang-2 from healthy mononuclear cells was stimulated by serum of patients with shock but not by serum of non-shocked patients (p: 0.016). Release was decreased by LPS; increased in the presence of a TLR4 antagonist; and decreased by anti-TNF antibody. RNA transcripts of PBMCs after stimulation with serum of patients with septic shock were higher than those after LPS stimulation. It is concluded that Ang-2 is increased in serum in the event of septic shock and that its increase is related to unfavorable outcome. It seems that a circulating factor may exist in the serum of patients with septic shock that stimulates gene expression and subsequent release of Ang-2 from monocytes. TLR4 and TNFalpha modulate release of Ang-2.

Evidence for the role of gastric mucosa at the secretion of soluble triggering receptor expressed on myeloid cells (strem-1) in peptic ulcer disease.

AIM: To investigate the role of gastric mucosa at the secretion of sTREM-1 in peptic ulcer. METHODS: Seventy two patients were enrolled; 35 with duodenal, 21 with gastric ulcer and 16 with chronic gastritis. Patients were endoscoped and gastric juice was aspirated. Patients with duodenal and gastric ulcer underwent a second endoscopy post-treatment. Biopsies were incubated in the absence/presence of endotoxins or gastric juice. Supernatants were collected and sTREM-1 and TNFalpha were measured by enzyme immunoabsorbent assays. Scoring of gastritis was performed before and after treatment according to updated Sydney score. RESULTS: Patients with duodenal and gastric ulcer and those with chronic gastritis had similar scores of gastritis. sTREM-1 was higher in supernatants of tissue samples of H pylori-positive than of H pylori-negative patients with gastric ulcer. Median (+/- SE) sTREM-1 was found increased in supernatants of patients with gastric ulcer before treatment (203.21 +/- 88.91 pg/1000 cells) compared to supernatants either from the same patients post-treatment (8.23 +/- 5.79 pg/1000 cells) or from patients with chronic gastritis (6.21 +/- 0.71 pg/1000 cells) (P < 0.001 and < 0.001, respectively). Similar differences for sTREM-1 were recorded among LPS-stimulated tissue samples of patients (P = 0.001). Similar differences were not found for TNFalpha. Positive correlations were found between sTREM-1 of supernatants from patients with both duodenal and gastric ulcer before treatment and the degree of infiltration of neutrophils and monocytes. CONCLUSION: sTREM-1 secreted by the gastric mucosa is an independent mechanism connected to the pathogenesis of peptic ulcer. sTREM-1 was released at the presence of H pylori from the inflamed gastric mucosa in the field of gastric ulcer.

Early changes of CD4-positive lymphocytes and NK cells in patients with severe Gram-negative sepsis.

INTRODUCTION: Our aim was to define early changes of lymphocytes and of NK cells in severe sepsis and to correlate them with serum levels of soluble triggering receptor expressed on myeloid cells-1 (sTREM-1). METHODS: Blood was sampled from 49 patients with proven highly suspected infection by Gram-negative pathogens, within 12 hours of the advent of severe sepsis, and was also sampled from six healthy volunteers. White blood cells were targeted with monoclonal antibodies and were analyzed by flow cytometry. The concentrations of sTREM-1 were estimated by ELISA. RESULTS: The presence of CD3/CD4 cells was significantly lower (P < 0.0001) and that of NK cells significantly higher among patients with sepsis compared with controls (P = 0.011). The proportions (median +/- standard error) of ANNEXIN-V/CD4/CD3-positive cells, of ANNEXIN-V/CD8/CD3-positive cells and of ANNEXIN-V/CD14-positive cells of the patient population were 7.41 +/- 2.26%, 7.69 +/- 3.42% and 1.96 +/- 4.22%, respectively. Patients with NK cells >20% survived longer compared with those patients with NK cells < or =20% (P = 0.041), and patients with sTREM-1 concentrations >180 pg/ml survived longer compared with those patients with sTREM-1 concentrations < or =180 pg/ml (P = 0.042). A negative correlation was found between the percentages of ANNEXIN-V/CD4/CD3-positive cells and of CD3/CD4 cells (rs = -0.305, P = 0.049), and a positive correlation was found between the serum sTREM-1 concentration and the percentage of NK cells (rs = +0.395, P = 0.014). NK cells isolated from two healthy volunteers released sTREM-1 upon triggering with endotoxins. CONCLUSION: Early severe sepsis is characterized by CD4-lymphopenia and increased NK cells, providing a survival benefit for the septic patient at percentages >20%. The survival benefit resulting from elevated NK cells might be connected to elevated serum levels of sTREM-1.