Thomas Tsaganos

Effect of Clarithromycin in Patients with Sepsis and Ventilator-Associated Pneumonia

BACKGROUND Because clarithromycin provided beneficiary nonantibiotic effects in experimental studies, its efficacy was tested in patients with sepsis and ventilator-associated pneumonia (VAP). METHODS Two hundred patients with sepsis and VAP were enrolled in a double-blind, randomized, multicenter trial from June 2004 until November 2005. Clarithromycin (1 g) was administered intravenously once daily for 3 consecutive days in 100 patients; another 100 patients were treated with placebo. Main outcomes were resolution of VAP, duration of mechanical ventilation, and sepsis-related mortality within 28 days. RESULTS The groups were well matched with regard to demographic characteristics, disease severity, pathogens, and adequacy of the administered antimicrobials. Analysis comprising 141 patients who survived revealed that the median time for resolution of VAP was 15.5 days and 10.0 days among placebo- and clarithromycin-treated patients, respectively (P = .011); median times for weaning from mechanical ventilation were 22.5 days and 16.0 days, respectively (p = .049). Analysis comprising all enrolled patients showed a more rapid decrease of the clinical pulmonary infection score and a delay for advent of multiple organ dysfunction in clarithromycin-treated patients, compared with those of placebo-treated patients (p = .047). Among the 45 patients who died of sepsis, time to death was significantly prolonged in clarithromycin-treated compared with placebo-treated patients (p = .004). Serious adverse events were observed in 0% and 3% of placebo- and clarithromycin-treated patients, respectively (P = .25). CONCLUSIONS Clarithromycin accelerated the resolution of VAP and weaning from mechanical ventilation in surviving patients and delayed death in those who died of sepsis. The mortality rate at day 28 was not altered. Results are encouraging and render new perspectives on the management of sepsis and VAP.

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

IntroductionAlthough 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.MethodsThe 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.ResultsExpression 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.ConclusionsMajor 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.

Effect of the Novel Influenza A (H1N1) Virus in the Human Immune System

Background The pandemic by the novel H1N1 virus has created the need to study any probable effects of that infection in the immune system of the host. Methodology/Principal Findings Blood was sampled within the first two days of the presentation of signs of infection from 10 healthy volunteers; from 18 cases of flu-like syndrome; and from 31 cases of infection by H1N1 confirmed by reverse RT-PCR. Absolute counts of subtypes of monocytes and of lymphocytes were determined after staining with monoclonal antibodies and analysis by flow cytometry. Peripheral blood mononuclear cells (PBMCs) were isolated from patients and stimulated with various bacterial stimuli. Concentrations of tumour necrosis factor-alpha, interleukin (IL)-1beta, IL-6, IL-18, interferon (FN)-alpha and of IFN-gamma were estimated in supernatants by an enzyme immunoassay. Infection by H1N1 was accompanied by an increase of monocytes. PBMCs of patients evoked strong cytokine production after stimulation with most of bacterial stimuli. Defective cytokine responses were shown in response to stimulation with phytohemagglutin and with heat-killed Streptococcus pneumoniae. Adaptive immune responses of H1N1-infected patients were characterized by decreases of CD4-lymphocytes and of B-lymphocytes and by increase of T-regulatory lymphocytes (Tregs). Conclusions/Significance Infection by the H1N1 virus is accompanied by a characteristic impairment of the innate immune responses characterized by defective cytokine responses to S.pneumoniae. Alterations of the adaptive immune responses are predominated by increase of Tregs. These findings signify a predisposition for pneumococcal infections after infection by H1N1 influenza.

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

IntroductionOur 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).MethodsBlood 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.ResultsThe 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 ≤20% (P = 0.041), and patients with sTREM-1 concentrations >180 pg/ml survived longer compared with those patients with sTREM-1 concentrations ≤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.ConclusionEarly 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.

Treatment of experimental osteomyelitis caused by methicillin-resistant Staphylococcus aureus with a synthetic carrier of calcium sulphate (Stimulan®) releasing moxifloxacin

The objectives of this study were to assess the efficacy of a synthetic semihydrate form of calcium sulphate (Stimulan) in experimental bone infection caused by methicillin-resistant Staphylococcus aureus (MRSA). Osteomyelitis was induced after inoculation of the test pathogen in the left tibia of 72 New Zealand rabbits assigned to the following groups: 18 control rabbits (Group A); 18 rabbits with Stimulan implanted (Group B); and 36 rabbits with moxifloxacin-impregnated Stimulan implanted (Group C). Rabbits were sacrificed at weekly intervals and cancellous bone was harvested for histopathology and for estimation of bacterial growth and concentrations of moxifloxacin. Bacterial growth from cancellous bone of Group C was significantly lower than the respective growth of Groups A and B on all days of sacrifice. The main histological finding of animals in all three groups was a moderate to intense inflammatory reaction accompanied by fibrosis. The degree of fibrosis was higher in Group C compared with both other groups. Infiltration by giant cells was also observed, which was greater in Group C on Day 42. Antibiotic levels in bone were higher for bone samples closer to the site of implantation. In conclusion, Stimulan admixed with 10% moxifloxacin was very effective in achieving complete eradication of the causative pathogen in experimental osteomyelitis caused by MRSA.

Soluble urokinase plasminogen activator receptor (suPAR) for assessment of disease severity in ventilator-associated pneumonia and sepsis

Urokinase plasminogen activator (uPAR) is a receptor mainly expressed on peripheral blood mononuclear cells and neutrophils. The role of its soluble form, namely suPAR, as a predictor of sepsis outcome in a homogenous cohort of 180 septic patients, was investigated. Blood from 180 patients with ventilator-associated pneumonia (VAP) and sepsis was collected for seven consecutive days. suPAR and PCT were measured in serum by an enzyme immunoassay and an immuno-time-resolved amplified cryptate assay respectively. Neutrophils and monocytes were isolated on day 1 and incubated. suPAR levels greater than 10.5 ng/ml had 80% specificity and 77.6% positive predictive value to discriminate between severe sepsis and sepsis. suPAR levels greater than 12.9 ng/ml had 80% specificity and 76.1% positive predictive value for prognosis of unfavorable outcome. suPAR levels were significantly lower among survivors than among non-survivors over follow-up. Step-wise Cox regression analysis found suPAR as an independent factor related with unfavorable outcome (p: 0.026). Concentrations of suPAR in supernatants of neutrophils of patients with sepsis were greater compared to controls. It is concluded that suPAR is a reliable marker of sepsis severity and a strong independent predictor of unfavorable outcome in VAP and sepsis. Neutrophils are involved in release.

Oleuropein: A Novel Immunomodulator Conferring Prolonged Survival In Experimental Sepsis By pseudomonas Aeruginosa

ABSTRACT Oleuropein, a novel immunomodulator derived from olive tree, was assessed in vitro and in experimental sepsis by Pseudomonas aeruginosa. After addition in monocyte and neutrophil cultures, malondialdehyde, TNF-&agr;, IL-6, and bacterial counts were estimated in supernatants. Acute pyelonephritis was induced in 70 rabbits after inoculation of pathogen in the renal pelvis. Intravenous therapy was administered in four groups postchallenge by one multidrug-resistant isolate (A, controls; B, oleuropein; C, amikacin; D, both agents) and in three groups postchallenge by one susceptible isolate (E, controls; F, oleuropein; G, amikacin). Survival was recorded; bacterial growth in blood and organs was counted; endotoxins (LPS), malondialdehyde, total antioxidant status, and TNF-&agr; in serum were estimated. TNF-&agr; and IL-6 of cell supernatants were not increased compared with controls when triggered by LPS and P. aeruginosa. Counts of multidrug-resistant P. aeruginosa were decreased in monocyte supernatants. Median survival of groups A, B, C, D, E, F, and G were 3.00, 6.00, 2.00, 10.00, 1.00, 5.00, and 1.00 days, respectively. Bacteria in blood were lower at 48 h in groups B and D compared with A and in groups F and G compared with E. Total antioxidant status decreased steadily over time in groups A, C, D, and G, but not in groups B and F. TNF-&agr; of groups B, C, and D was lower than A at 48 h. Tissue bacteria decreased in group F compared with E. Oleuropein prolonged survival in experimental sepsis probably by promoting phagocytosis or inhibiting biosynthesis of proinflammatory cytokines.

Validation of the new Sepsis-3 definitions: proposal for improvement in early risk identification

OBJECTIVES Sepsis-3 definitions generated controversies regarding their general applicability. The Sepsis-3 Task Force outlined the need for validation with emphasis on the quick Sequential Organ Failure Assessment (qSOFA) score. This was done in a prospective cohort from a different healthcare setting. METHODS Patients with infections and at least two signs of systemic inflammatory response syndrome (SIRS) were analysed. Sepsis was defined as total SOFA ≥2 outside the intensive care unit (ICU) or as an increase of ICU admission SOFA ≥2. The primary endpoints were the sensitivity of qSOFA outside the ICU and sepsis definition both outside and within the ICU to predict mortality. RESULTS In all, 3346 infections outside the ICU and 1058 infections in the ICU were analysed. Outside the ICU, respective mortality with ≥2 SIRS and qSOFA ≥2 was 25.3% and 41.2% (p <0.0001); the sensitivities of qSOFA and of sepsis definition to predict death were 60.8% and 87.2%, respectively. This was 95.9% for sepsis definition in the ICU. The sensitivity of qSOFA and of ≥3 SIRS criteria for organ dysfunction outside the ICU was 48.7% and 72.5%, respectively (p <0.0001). Misclassification outside the ICU with the 1991 and Sepsis-3 definitions into stages of lower severity was 21.4% and 3.7%, respectively (p <0.0001) and 14.9% and 3.7%, respectively, in the ICU (p <0.0001). Adding arterial pH ≤7.30 to qSOFA increased sensitivity for prediction of death to 67.5% (p 0.004). CONCLUSIONS Our analysis positively validated the use of SOFA score to predict unfavourable outcome and to limit misclassification into lower severity. However, qSOFA score had inadequate sensitivity for early risk assessment.

Kinetics of Angiopoietin-2 in serum of multi-trauma patients: Correlation with patient severity

BACKGROUND Angiopoietin-2 (Ang-2) is considered a proinflammatory mediator promoting vascular leakage. Its participation in the inflammatory process following multiple injuries was investigated. METHODS Blood was sampled on consecutive days from 54 patients with multiple injuries and six healthy volunteers. Ang-2 was estimated in serum by an enzyme immunoassay. RESULTS From the enrolled patients, 10 did not develop any complication; 17 developed systemic inflammatory response syndrome (SIRS); 16 developed sepsis and 11 severe sepsis. Among those who did not develop any complication, all survived. Ang-2 was increased on days 4 and 7 of follow-up in patients with SIRS. Ang-2 was highly increased upon advent of sepsis and of severe sepsis. Patients with serum levels below 15,200 pg/ml survived longer compared to those with levels above 15,200 pg/ml (p=0.015). OR for death with serum Ang-2 above 15,200 pg/ml was 4.00 (p=0.037). CONCLUSIONS Serum levels of Ang-2 in multi-trauma patients are increased upon advent of septic complications and they are connected with bad prognosis. Its exact role in the process of multiple trauma remains to be defined.

Effect of Clarithromycin in Inflammatory Markers of Patients with Ventilator-Associated Pneumonia and Sepsis Caused by Gram-Negative Bacteria: Results from a Randomized Clinical Study

ABSTRACT One recent, double-blind, randomized clinical trial with 200 patients showed that clarithromycin administered intravenously for 3 days in patients with ventilator-associated pneumonia (VAP) accelerated the resolution of pneumonia and decreased the risk of death from septic shock and multiple organ dysfunctions (MODS). The present study focused on the effect of clarithromycin on markers of inflammation in these patients. Blood was drawn immediately before the administration of the allocated treatment and on six consecutive days after the start of treatment. The concentrations of circulating markers were measured. Monocytes and neutrophils were isolated for immunophenotyping analysis and for cytokine stimulation. The ratio of serum interleukin-10 (IL-10) to serum tumor necrosis factor alpha (TNF-α) was decreased in the clarithromycin group compared with the results in the placebo group. Apoptosis of monocytes was significantly increased on day 4 in the clarithromycin group compared with the rate of apoptosis in the placebo group. On the same day, the expression of CD86 was increased and the ratio of soluble CD40 ligand (sCD40L) to CD86 in serum was unchanged. The release of TNF-α, IL-6, and soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) by circulating monocytes after stimulation was greater in the clarithromycin group than in the placebo group. The expression of TREM-1 on monocytes was also increased in the former group. These effects were pronounced in patients with septic shock and MODS. These results suggest that the administration of clarithromycin restored the balance between proinflammatory versus anti-inflammatory mediators in patients with sepsis; this was accompanied by more efficient antigen presentation and increased apoptosis. These effects render new perspectives for the immunotherapy of sepsis.