Treatment of Experimental Candida Sepsis with a Janus Kinase Inhibitor Controls Inflammation and Prolongs Survival.

Janus kinases (JAK) are intracellular tyrosine kinases that transduce cytokine-mediated signals to the nucleus, promoting gene expression. Cytokines play a major role in microbial sepsis, which is often associated with uncontrolled inflammation leading to death. JAK inhibitors have been used for the treatment of several autoimmune diseases by modulating immune response, but they have never been tested against microbial sepsis. Ruxolitinib is a small-molecule inhibitor of JAK1/2 proteins, which are involved in the downstream signaling pathway of the vast majority of proinflammatory and anti-inflammatory cytokines. We therefore studied the effect of ruxolitinib in a mouse model of sepsis due to Candida albicans. When ruxolitinib therapy (50 mg/kg [of body weight]/day) was started 1 day before infection, the median survival time was reduced by 3 days, the fungal loads in all organs were higher, the inflammation was significantly less, and serum tumor necrosis factor alpha (TNF-α) and interleukin 10 (IL-10) levels and IL-10/TNF-α ratios were higher than in controls. When ruxolitinib therapy (50 to 1.5 mg/kg/day) was started 1 day after infection, an inverted-U relationship was found, with 6.25 mg/kg/day prolonging median survival time by 6 days, resulting in similar fungal loads, less inflammation, and similar cytokine levels but higher IL-10/TNF-α ratios than the controls. The optimal dose of ruxolitinib controlled infection and prolonged survival with less inflammation than in control animals. Administration of JAK inhibitors may be a promising therapeutic adjunct that needs further investigation.

Spectrum of enteropathogens detected by the FilmArray GI Panel in a multicentre study of community-acquired gastroenteritis.

The European, multicentre, quarterly point-prevalence study of community-acquired diarrhoea (EUCODI) analysed stool samples received at ten participating clinical microbiology laboratories (Austria, Finland, France, Germany, Greece, Ireland, Italy, Portugal, Romania, and the UK) in 2014. On four specified days, each local laboratory submitted samples from ≤20 consecutive patients to the Austrian Study Centre for further testing with the FilmArray GI Panel (BioFire Diagnostics, Salt Lake City, UT, USA). Of the 709 samples from as many patients received, 325 (45.8%) tested negative, 268 (37.8%) yielded only one organism, and 116 (16.4%) yielded multiple organisms. Positivity rates ranged from 41% (30 of 73 samples) in France to 74% (59 of 80 samples) in Romania. With the exception of Entamoeba histolytica and Vibrio cholerae, all of the 22 targeted pathogens were detected at least once. Enteropathogenic Escherichia coli, Campylobacter species, toxigenic Clostridium difficile, enteroaggregative E. coli, norovirus and enterotoxigenic E. coli were the six most commonly detected pathogens. When tested according to local protocols, seven of 128 positive samples (5.5%) yielded multiple organisms. Overall, the FilmArray GI Panel detected at least one organism in 54.2% (384/709) of the samples, as compared with 18.1% (128/709) when testing was performed with conventional techniques locally. This underlines the considerable potential of multiplex PCR to improve routine stool diagnostics in community-acquired diarrhoea. Classic culture methods directed at the isolation of specific pathogens are increasingly becoming second-line tools, being deployed when rapid molecular tests give positive results. This optimizes the yield from stool examinations and dramatically improves the timeliness of diagnosis.

Susceptibility breakpoints for amphotericin B and Aspergillus species in an in vitro pharmacokinetic-pharmacodynamic model simulating free-drug concentrations in human serum.

Although conventional amphotericin B was for many years the drug of choice and remains an important agent against invasive aspergillosis, reliable susceptibility breakpoints are lacking. Three clinical Aspergillus isolates (Aspergillus fumigatus, Aspergillus flavus, and Aspergillus terreus) were tested in an in vitro pharmacokinetic-pharmacodynamic model simulating the biphasic 24-h time-concentration profile of free amphotericin B concentrations in human serum with free peak concentrations (fCmax) of 0.1, 0.3, 0.6, 1.2, and 2.4 mg/liter administered once daily. Drug concentrations were measured with a bioassay, and fungal growth was monitored for 72 h with galactomannan production. The fCmax/MIC corresponding to half-maximal activity (P50) was determined for each species, and the percentage of target attainment was calculated for different MICs for the standard (1 mg/kg of body weight) and a lower (0.6-mg/kg) dose of amphotericin B with Monte Carlo simulation analysis. The fCmax/MICs (95% confidence intervals) corresponding to P50 were 0.145 (0.133 to 0.158), 0.371 (0.283 to 0.486), and 0.41 (0.292 to 0.522) for A. fumigatus, A. flavus, and A. terreus, respectively. The median percentages of P50 attainment were ≥88%, 47%, and 0% for A. fumigatus isolates with MICs of ≤0.5, 1, and ≥2 mg/liter, respectively, and ≥81%, 24%, and 0% and ≥75%, 15%, and 0% for A. flavus and A. terreus isolates with MICs of ≤0.25, 0.5, and ≥1 mg/liter, respectively. The lower dose of 0.6 mg/kg would retain efficacy for A. fumigatus, A. flavus, and A. terreus isolates with MICs of ≤0.25, ≤0.125, and ≤0.125 mg/liter, respectively. The susceptibility, intermediate susceptibility, and resistance breakpoints of ≤0.5, 1, and ≥2 mg/liter for A. fumigatus and ≤0.25, 0.5, and ≥1 mg/liter for A. flavus and A. terreus were determined for conventional amphotericin B with a pharmacokinetic-pharmacodynamic model simulating free-drug serum concentrations.

In vitro pharmacokinetic/pharmacodynamic modeling of voriconazole activity against Aspergillus species in a new in vitro dynamic model.

The pharmacodynamics (PD) of voriconazole activity against Aspergillus spp. were studied using a new in vitro dynamic model simulating voriconazole human pharmacokinetics (PK), and the PK-PD data were bridged with human drug exposure to assess the percent target (near-maximum activity) attainment of different voriconazole dosages. Three Aspergillus clinical isolates (1 A. fumigatus, 1 A. flavus, and 1 A. terreus isolate) with CLSI MICs of 0.5 mg/liter were tested in an in vitro model simulating voriconazole PK in human plasma with C(max) values of 7, 3.5, and 1.75 mg/liter and a t(1/2) of 6 h. The area under the galactomannan index-time curve (AUC(GI)) was used as the PD parameter. In vitro PK-PD data were bridged with population human PK of voriconazole exposure, and the percent target attainment was calculated. The in vitro PK-PD relationship of fAUC(0-24)-AUC(GI) followed a sigmoid pattern (global R(2) = 0.97), with near-maximum activities (10% fungal growth) observed at an fAUC(0-24) (95% confidence interval [CI]) of 18.9 (14.4 to 23.1) mg · h/liter against A. fumigatus, 26.6 (21.1 to 32.9) mg · h/liter against A. flavus, and 36.2 (27.8 to 45.7) mg · h/liter against A. terreus (F test; P < 0.0001). Target attainment for 3, 4, and 5 mg/kg-of-body-weight voriconazole dosages was 24% (11 to 45%), 80% (32 to 97%), and 93% (86 to 97%) for A. fumigatus, 12% (5 to 26%), 63% (17 to 93%), and 86% (73 to 94%) for A. flavus, and 4% (2 to 11%), 36% (6 to 83%), and 68% (47 to 83%) for A. terreus. Based on the in vitro exposure-effect relationships, a standard dosage of voriconazole may be adequate for most patients with A. fumigatus but not A. flavus and A. terreus infections, for which a higher drug exposure may be required. This could be achieved using a higher voriconazole dosage, thus highlighting the usefulness of therapeutic drug monitoring in patients receiving a standard dosage.

Staphylococcus lugdunensis strain with a modified PBP1A/1B expressing resistance to ß-lactams.

We describe for the first time the emergence of an mecA-negative Staphylococcus lugdunensis strain with a modified PBP1A/1B that expresses resistance to all ß-lactams. A duplication of the tetrapeptide S(569)AYG, which is part of the transpeptidase domain of PBP1A/1B and closely located to the K(583)TG catalytic motif, was associated with this unusual phenotype.

Mycobacterium arupense pulmonary infection: antibiotic resistance and restriction fragment length polymorphism analysis.

Mycobacterium arupense is a novel mycobacterium species. It was first identified from clinical specimens in 2006 and since then there have been only two reports of its recovery from clinical samples. In the present case M. arupense was isolated from the sputum of a 62-year-old man with a malignant mass in his left kidney, who presented with a one-month history of recurrent fever, dyspnea and haemoptysis. M. arupense was identified with sequencing of hsp65 and 16S rRNA genes. In the present study, its biochemical profile along with its resistance status and hsp65 RFLP analysis is presented.

A newly described vancomycin-resistant ST412 Enterococcus faecium predominant in Greek hospitals.

A total of 359 vancomycin-resistant enterococci (344 Enterococcus faecium and 15 E. faecalis) collected during 2007 from eight tertiary-care hospitals in Greece were analysed for genotypic characteristics. Four common clones, ST412, ST203, ST16 and ST17, were identified among E. faecium and one clone, ST28, among E. faecalis strains.

Mycobacterium thermoresistibile: case report of a rarely isolated mycobacterium from Europe and review of literature.

Mycobacterium thermoresistibile is a non-tuberculous mycobacterium strongly associated with human infections. Since 1966, there have only been six reports of its isolation from clinical samples. We report on the first case from Europe and review all the previous cases. Identification was achieved with sequencing of the 16S rRNA and hsp65 genes. This study presents its phenotypic and biochemical profile, susceptibilities to selected antibiotics and hsp65 polymerase chain reaction-restriction fragment length polymorphism profile with BsteII and Hae III .

In vitro activity of daptomycin against Gram-positive cocci: the first multicentre study in Greece.

A total of 10420 Gram-positive cocci (including staphylococci, enterococci and various groups of streptococci) collected from clinically significant specimens in ten Greek hospitals during 2006--2007 were tested for their susceptibility to daptomycin. The minimum inhibitory concentration (MIC) was determined by the broth microdilution method. Daptomycin demonstrated very high activity against Enterococcus faecalis (MIC at which 50% of the isolates were inhibited (MIC50) = 1mg/L and MIC at which 90% of the isolates were inhibited (MIC90) = 1.36 mg/L), Enterococcus faecium (MIC50 = 1.36 mg/L and MIC90 = 1.90 mg/L), Streptococcus pyogenes (MIC50 = 0.12 mg/L and MIC90 = 0.50mg/L), Streptococcus agalactiae (MIC50 = 0.09 mg/L and MIC90 = 0.12 mg/L), Streptococcus pneumoniae (MIC50 = 0.24 mg/L and MIC90 = 0.5 mg/L) and viridans group streptococci (MIC50 = 0.50 mg/L and MIC90 = 0.89 mg/L). Resistance to linezolid and vancomycin for enterococci and to penicillin for streptococci appears to be independent of reduced susceptibility to daptomycin. On the other hand, daptomycin was also active against meticillin-resistant Staphylococcus aureus (MIC50 = 0.44 mg/L and MIC90 = 0.78 mg/L) and meticillin-resistant coagulase-negative staphylococci (MIC50 = 0.24 mg/L and MIC90 = 0.44 mg/L); however, 0.9% of the staphylococci tested had an MIC > 1mg/L, which is the Clinical and Laboratory Standards Institute breakpoint proposed for susceptibility. For all tested organism groups, resistance to daptomycin was not associated with glycopeptide resistance.

Risk-factors and predictors of mortality in patients colonised with vancomycin-resistant enterococci.

Vancomycin-resistant enterococci (VRE) have emerged as significant nosocomial pathogens. A hospital-wide prevalence study was performed to identify cases with VRE faecal colonisation. A case-control study using two randomly selected VRE-negative controls for each positive case was performed to assess risk-factors for VRE colonisation by univariate and multivariate analysis. VRE faecal colonisation was documented in 53 (14.3%) of 370 patients screened. Previous exposure to anti-anaerobic agents, as well as quinolones, was associated with VRE colonisation (p <0.05). The presence of an invasive device (OR 4.8, p 0.003) and the duration of any antimicrobial treatment before VRE isolation (OR 1.2, p <0.001) predicted VRE colonisation in multivariate models. The crude mortality rate for patients with VRE colonisation was 24.5%, but VRE colonisation was not an independent predictor of mortality in these patients. These results suggest that an active surveillance programme focusing on specific patient groups may help in the identification of VRE-colonised patients. Promptly implemented infection control strategies targeting these groups should help to combat the rising incidence of VRE.

An overview of the epidemiology and epizootology of brucellosis in selected countries of Central and Southeast Europe.

The objective of this paper is to give an overview of the epidemiologic and epizootic status of brucellosis in selected countries of Central and Southeast Europe (Balkan region). Based on dimension of the disease problem, there is a need to establish collaboration in the eradication and prevention of brucellosis between all countries in the region. Although there were no readily accessible data concerning epidemiology and epizootology of brucellosis in these countries, the limited official and published data were analyzed. The incidence of brucellosis caused by Brucella melitensis in sheep, goats and humans is a very significant problem in Macedonia and Greece. In Greece, cattle are also affected either by B. melitensis or B. abortus. The disease is an endemic problem in some regions of Yugoslavia and includes B. suis biovar 2 in pigs and in Croatia, B. melitensis in sheep, goats and human is found occasionally. No problem appears to exist with brucellosis in Bulgaria. Financial well-supported brucellosis control programs of the European Union that will include all countries, regardless of the magnitude of brucellosis incidence, are needed for eradication and control of brucellosis.

Serum is the preferred clinical specimen for diagnosis of human brucellosis by PCR.

Human brucellosis poses a significant public health problem in many developing countries and requires fast and accurate diagnosis. A PCR assay amplifying part of the 31-kDa Brucella abortus antigenic protein gene sequence was developed and applied to whole-blood and serum samples from 31 brucellosis patients and 45 healthy individuals. All patients except one had detectable Brucella DNA in either whole blood or serum (combined sensitivity, 97%), but the assay sensitivity was higher with serum samples (94%) than with whole-blood samples (61%). The assay specificity was excellent (100%). A confirmatory PCR assay targeting another Brucella gene region (omp-2) was also developed but lacked sensitivity. Serum is the optimal specimen for the diagnosis of brucellosis by PCR, a choice that leads to assay simplification and shortens turnaround time.

Critical evaluation of the Vitek GNS F6 card results compared to standardized, reference susceptibility test methods.

A large number of Enterobacteriaceae (291 unselected and 30 clinical challenge isolates) were used to evaluate the accuracy of the Vitek GNS-F6 susceptibility testing card for 10 antimicrobial agents (ampicillin, ampicillin/sulbactam, aztreonam, ciprofloxacin, imipenem, mezlocillin, ofloxacin, piperacillin, ticarcillin, and ticarcillin/clavulanate). Results were compared to reference broth microdilution and disk diffusion methods. Highest interpretive error on initial processing were observed with ticarcillin/clavulanate (very major false-susceptible error, 3.4%), imipenem (major false-resistant error, 1.9%), and ampicillin/sulbactam (minor error, 15.9%). Repeat testing resolved many very major errors (21 of 41 results), and the overall accuracy rate was improved from 92.1 to 93.4%. Analysis of all minor interpretive errors demonstrated that the trend for Vitek was to report slightly lower MICs (6 of 10 drugs; 97 of 159 results) in comparison to the reference test results. If a heavy inoculum was used in the Vitek cards, false resistance was observed more frequently with aztreonam and penicillins. These data demonstrate that susceptibility testing accuracy with Vitek GNS-F6 card based on categorical agreement varies from 83.2% (ampicillin/sulbactam) to 99.4% (ciprofloxacin) when testing enteric bacilli. Some antimicrobial agents (beta-lactamase inhibitor/penicillin combinations, antipseudomonal penicillins) may require slight modification of interpretive software. Finally, the overall accuracy of the Vitek System was greater than 91% for 9 of 10 drugs tested with a very low, acceptable rate of false-susceptible error (0.8%).

In vitro susceptibility testing and DNA typing of Saccharomyces cerevisiae clinical isolates.

Saccharomyces spp. are widely distributed in nature and may colonize the normal human gastrointestinal tract. Although Saccharomyces cerevisiae isolates have been previously considered nonpathogenic, they appear to be increasingly associated with infections in immunocompromised or otherwise debilitated patients. The antifungal susceptibility and epidemiology of S. cerevisiae are poorly defined at present. A series of 76 isolates (mostly stool surveillance and throat swab isolates) from 70 bone marrow transplant patients hospitalized at two different medical centers were characterized by antifungal susceptibility testing and restriction endonuclease analysis of chromosomal DNA. For DNA typing, digestion with NotI followed by pulsed-field gel electrophoresis was applied. Typing results revealed 62 distinct DNA types among the 76 clinical isolates. Despite this genomic diversity, clusters of identical isolates were identified among different patients hospitalized concurrently in the same unit, indicating possible nosocomial transmission. The MICs of amphotericin B, 5-fluorocytosine, fluconazole, and itraconazole were determined by a broth microdilution method, as recommended by the National Committee for Clinical Laboratory Standards. The MICs at which 90% of the strains were inhibited were as follows: amphotericin B, 1.0 micrograms/ml; 5-fluorocytosine, 0.25 micrograms/ml; fluconazole, 8.0 micrograms/ml; and itraconazole, 1.0 micrograms/ml. The relative resistance of S. cerevisiae to fluconazole and itraconazole may promote the emergence of this species as a pathogen among immunosuppressed patients.