Establishing nationally representative central line-associated bloodstream infection surveillance data for paediatric patients in Greece.

BACKGROUND: Healthcare-associated infections (HCAIs) are associated with increased morbidity and mortality and with excess costs. Central line-associated bloodstream infections (CLABSIs) are the most common HCAIs in neonates and children. AIM: To establish national benchmark data for rates of CLABSI in neonatal and paediatric intensive care units (NICUs and PICUs) and paediatric oncology units (ONCs). METHODS: Active surveillance for CLABSI was conducted from June 2016 to February 2017. A collaborative of 14 NICUs, four PICUs, and six ONCs participated in the programme. Surveillance definitions of central line (CL), central line utilization (CLU) ratio, CLABSI event, and CLABSI rate were based on the Centers for Disease Control and Prevention's 2014 National Healthcare Safety Network criteria. Medical records were assessed daily for calculating CL-days, patient-days, and susceptibility of isolated organisms. FINDINGS: A total of 111 CLABSI episodes were recorded. The overall mean CLABSI rate was 4.41 infections per 1000 CL-days, and the CLU ratio was 0.31. CLABSI rates were 6.02 in NICUs, 6.09 in PICUs, and 2.78 per 1000 CL-days in ONCs. A total of 123 pathogens were isolated. The most common pathogens were Enterobacteriaceae (36%), followed by Gram-positive cocci (29%), non-fermenting Gram-negative bacteria (16%), and fungi (16%). Overall, 37% of Gram-negative pathogens were resistant to third-generation cephalosporins and 37% to carbapenems. CONCLUSION: Nationally representative CLABSI rates were determined for paediatric patients. These data could be used to benchmark and serve as baseline data for the design and evaluation of infection control and antimicrobial stewardship interventions.

Budesonide and formoterol inhibit inflammatory mediator production by bronchial epithelial cells infected with rhinovirus.

BACKGROUND: Rhinoviruses (RVs) are responsible for the majority of acute asthma and chronic obstructive pulmonary disease (COPD) exacerbations. RVs infect the lower airways and induce the production of pro-inflammatory and remodelling-associated mediators. Budesonide (BUD) and formoterol (FORM) synergize in controlling asthma and COPD exacerbations; however, their effects on virus-induced inflammation and remodelling are less known. OBJECTIVE: We investigated whether BUD and FORM synergize in suppressing RV-induced inflammation and remodelling in the airways. METHODS: In vitro models of RV infection of BEAS-2B and primary normal human bronchial epithelial (NHBE) cells were used. We assessed the effects of individual and combined drugs administered post-infection, at a clinically relevant concentration range (10(-6)-10(-10) m), on the production of CCL5, CXCL10, CXCL8, IL-6 and the remodelling-associated VEGF and bFGF, using ELISA and RT-PCR. RESULTS: BUD effectively suppressed RV-mediated induction of all mediators studied, in a concentration-dependent manner. FORM alone suppressed the production of CXCL8 and bFGF. The combination of BUD and FORM had concentration-dependent, additive or synergistic effects in the suppression of RV-induced CCL5, CXCL8 and CXCL10 in both cell types as well as VEGF in NHBE only. Combination treatment also resulted in an enhanced suppression of RV-induced IL-6, and CCL5 at the mRNA level as compared with BUD or FORM alone. CONCLUSION: BUD and FORM suppress RV-induced chemokines and growth factors in bronchial epithelial cells in a concentration-dependent, synergistic or additive manner. These data further support the combined use of BUD and FORM in asthma and COPD and intensification of this therapy during exacerbations.

In vitro susceptibility of Coxiella burnetii to linezolid in comparison with its susceptibilities to quinolones, doxycycline, and clarithromycin.

The in vitro susceptibility to linezolid shown by nine Greek isolates of Coxiella burnetii derived from patients with acute Q fever was investigated. MICs of linezolid were compared with those of pefloxacin, ciprofloxacin, ofloxacin, trovafloxacin, doxycycline, and clarithromycin using the shell vial assay. MICs of linezolid and clarithromycin ranged from 2 to 4 microg/ml; those of doxycycline, trovafloxacin, and ofloxacin ranged from 1 to 2 microg/ml; those of pefloxacin ranged from 1 to 4 microg/ml; and those of ciprofloxacin ranged from 4 to 8 microg/ml. Linezolid was effective in controlling intracellular parasites in cultures of Vero cells infected by C. burnetii. No bactericidal activity by linezolid was obtained against C. burnetii at 8 microg/ml.

Diagnosis of quinolone-resistant Coxiella burnetii strains by PCR-RFLP.

A total of 12 strains of Coxiella burnetii (8 Greek isolates from acute Q-fever patients, two reference strains-Nine Mile and Q212-and two pefloxacin-resistant laboratory strains) were examined for the presence of point mutations in the quinolone resistance determining region (QRDR) of gyrA gene by direct DNA sequencing of the polymerase chain reaction (PCR)-amplified fragments. The gene sequences of all eight Greek isolates and the two reference strains Nine Mile and Q212 [minimal inhibitory concentration (MIC)A) at the corresponding codon 87 of E. coli. This mutation lead to the substitution of Glu (codon GAG) by Lys (codon AAG ). Restriction maps of amplified gyrA gene sequences were determined by GCG Wisconsin PACKAGE, and the MnlI restriction enzyme was found to cut only the sensitive strains sequences and not the resistant ones. The present PCR-RFLP analysis has proved to be a simple, rapid, and useful method for the detection of Coxiella burnetii and, at the same time, for the diagnosis of quinolone-resistant Coxiella burnetii strains.

Colonization of Phlebotomus neglectus (Diptera: Psychodidae), the major vector of visceral leishmaniasis in Greece.

Colonization of Phlebotomus neglectus Tonnoir, the major vector of visceral leishmaniasis, in Greece is reported for the first time. Starting with wild-caught specimens, a small closed colony was established that was maintained for 17 mo or 10 generations. Gonotrophic discordance, stenogamic mating behavior, low fecundity, and dormancy because of low temperature were the most important findings that characterized the colony.

In vitro susceptibility of Coxiella burnetii to trovafloxacin in comparison with susceptibilities to pefloxacin, ciprofloxacin, ofloxacin, doxycycline, and clarithromycin.

The antibiotic susceptibilities of eight Greek isolates of Coxiella burnetii to trovafloxacin were determined by the shell vial assay. MICs of trovafloxacin and ofloxacin ranged from 1 to 2 microg/ml, those of pefloxacin ranged from 1 to 4 microg/ml, those of ciprofloxacin ranged from 4 to 8 microg/ml, those of doxycycline ranged from 1 to 2 microg/ml, and those of clarithromycin ranged from 2 to 4 microg/ml. Trovafloxacin exhibited no activity against C. burnetii at 4 microg/ml.

Q fever in the Greek island of Crete: detection, isolation, and molecular identification of eight strains of Coxiella burnetii from clinical samples.

Over a period of 6 years (1989 to 1995), serum samples from 3,300 patients suspected to be infected by Coxiella burnetii were assayed for the presence of antibodies against antigen phase II of the microorganism by the indirect immunofluorescence antibody technique (IFAT). One hundred fifty-two cases were recorded, and blood samples from 17 patients were cultured for the isolation of the pathogen. By a centrifugation shell vial technique, eight strains were isolated from patients suffering from acute Q fever. The microorganism was detected in the cultures by IFAT, by Gimenez staining, and by the cytopathogenic effect on Vero and human embryonic lung (HEL) cells. PCR followed by restriction fragment length polymorphism analysis was used to confirm the diagnosis and identify the Coxiella burnetii strains within the cell cultures as well as to compare them with reference strains. In order to avoid time-consuming cultures, to achieve direct detection of Coxiella burnetii in clinical samples (blood, buffy coat, etc.), and to increase the specificity and sensitivity of the detection, nested PCR was performed. The first step of DNA extraction was performed with the QIAamp blood kit 250. For the second step of the PCR assays, the conditions of temperature and times of recycling were properly modified, and the microorganism was detected within 4 h. Our study demonstrates that Q fever is an endemic disease in Crete and that the diagnosis of Coxiella burnetii infection can be rapidly achieved by the detection of the microorganism in buffy coat samples by nested PCR. Although the presenting symptoms of the disease in this study differed from those in other studies, the Cretan strains do not differ genotypically from the reference strains (Nine Mile and Q212).

Genotypic identification of murine typhus Rickettsia in rats and their fleas in an endemic area of Greece by the polymerase chain reaction and restriction fragment length polymorphism.

Forty-nine cases of murine typhus were diagnosed in recent years in residents of several communities around the city of Chalkis, the capital of the Prefecture of Evia. (Euboea) Evia is an island connected to central mainland Greece by a bridge. To investigate the endemicity of murine typhus in this area, 226 fleas (Xenopsylla cheopis) and blood samples were collected from 53 rats (Rattus norvegicus) trapped in this area. The polymerase chain reaction followed by restriction fragment length polymorphism analysis (PCR-RFLP) was used to detect and identify Rickettsia typhi, the etiologic agent murine typhus, in the rat blood samples (buffy coat cells) as well as in their fleas. An indirect immunofluorescent antibody (IFA) assay was performed to detect antibodies against R. typhi in rat serum samples. The presence of R. typhi in both fleas and rat blood samples was demonstrated. The frequency of infection for X. cheopis was 4%, while 18% of the rats had buffy coat cells infected, and 92% of the rat sera tested by IFA were positive for anti-R. typhi antibodies. The present work is the first successful application of PCR-RFLP in a field study of naturally infected rats and their fleas in Europe.

Detection of ciprofloxacin resistance mutations in Campylobacter jejuni gyrA by nonradioisotopic single-strand conformation polymorphism and direct DNA sequencing.

A total of 27 strains of Campylobacter jejuni (24 clinical strains and three laboratory strains) were examined for the presence of point mutations in the quinolone resistance determining region (QRDR) of gyrA gene by nonradioisotopic single-strand conformation polymorphism (non-RI SSCP) analysis with silver stain. Direct DNA sequencing of the polymerase chain reaction (PCR)-amplified DNA fragments confirmed the results obtained by non-RI SSCP analysis and revealed that in clinical strains high-level quinolone resistance [minimal inhibitory concentration (MIC) to ciprofloxacin > or = 16 micrograms/ml] was closely associated with one type of single-point mutation at codon 86 (Thr-Ile). Two strains with MICs of 8 and 1 microgram/ml showed point mutations at codons 86 and 70, respectively. Furthermore, transitions at codon 119 of the gyrA QRDR were identified in 17 strains. Six types of bands were separated in a single electrophoretic step with silver stain within 2 hours after PCR amplification of the gyrA QRDR as follows: type I associated to mutation at codon 70 (Ala-Thr), type II to mutation at codon 90 (Asp-Asn), type III to variant with transition at 119, type IV to wild-type, type V to mutation at codon 86 (Thr-Ile), and type VI to mutation at codon 86 (Thr-Ile) and transition at codon 119. Using four DNA extracts from Cambylobacter coli organisms as templates for amplification of the gyrA QRDR, no PCR products were obtained. Non-RI SSCP was proved to be a simple, rapid, and useful screening method for detecting gyrA mutations associated with ciprofloxacin resistance in C. jejuni.