Prediction of complicated appendicitis risk in children.

OBJECTIVE: We aimed to predict the risk of complicated appendicitis in children, constructing a risk-based prediction tool with the optimal combination of sensitivity and specificity outcomes. PATIENTS AND METHODS: This is a prospective study on a random sample of children with acute appendicitis who underwent appendectomy. Clinical examination, history, routine laboratory tests, Alvarado and pediatric appendicitis scores, operative and histopathological findings were taken into consideration. The predictive ability of the outcome variables was assessed by the Receiver Operating Characteristics (ROC) analysis. The overall predictive ability and determination of the best cut-off value (the higher sum of sensitivity plus specificity) were calculated. A Classification and Regression Tree (CRT) was used to create a multi-level classification algorithm. The model was set to predict the outcome of complicated appendicitis, considering as potential predictors the demographic characteristics, the clinical findings, and the outcome parameters. RESULTS: The various combinations of clinical and laboratory parameters did not improve their overall diagnostic ability. However, the CRT analysis resulted in a short classification algorithm based on the Pediatric appendicitis score, neutrophils percentage and the CRP. This model yielded a significantly better predictive ability than all the other combinations of the outcome parameters. The application of the model would predict complicated appendicitis with 90% sensitivity and 78.6% specificity. CONCLUSIONS: The constructed predictive model may be a useful tool for daily practical use by the clinician, especially in areas where modern diagnostic imaging facilities are absent or not always available. Clinical evaluation and close follow-up remain the more accurate preoperative method to decide the performance and timing of appendectomy.

Postpartum bacteraemia outbreak due to Bacillus cereus in the delivery room.

We report an outbreak Bacillus cereus causing postpartum bacteraemia in the maternity ward and delivery room. Spores transferred by the hands and gloves of the staff in the maternity ward contaminated equipment in these two areas.

Pitfalls in the identification of Enterococcus species and the detection of vanA and vanB genes.

UNLABELLED: The aims were to assess the performance of Vitek 2 in identifying enterococcal species and the implementation of GeneXpert(®) vanA/vanB PCR for the detection of vancomycin-resistant enterococci (VRE). Gram-positive cocci from clinical and environmental specimens (n = 431) suspicious of being enterococci by conventional methods were evaluated by Vitek 2. This system identified 296 Enterococcus faecium, 87 Enterococcus faecalis, 10 Enterococcus villorum, 9 Enterococcus gallinarum, 9 Enterococcus durans, 5 Enterococcus casseliflavus, 1 Enterococcus spp. and 14 isolates as Non-Enterococcus. All strains were submitted to pulsed field gel electrophoresis (PFGE) analysis showing 64 banding patterns. Representative strains from each banding pattern were further characterized to species level by 16S rDNA sequencing. The misidentification rate by Vitek 2 to species level among 429 molecularly identified enterococci was 6% (26 isolates). Additionally, 372 rectal swabs were obtained from critically ill patients. They were evaluated for the presence of VRE by ChromID VRE combined with in-house PCR vs GeneXpert(®) . GeneXpert(®) showed high (>92%) sensitivity, specificity, accuracy for vanA-positive Enterococcus detection, as well as, sensitivity and specificity for vanB-positive strains. Positive predictive value for detection of vanB-positive enterococci by GeneXpert(®) vanA/vanB was low (30%). GeneXpert(®) showed the same efficacy as ChromID VRE in detecting vanA-positive enterococci, but lower for vanB-gene detection. SIGNIFICANCE AND IMPACT OF THE STUDY: The study shows that even though the performance of Vitek 2 Advanced Expert System was good in identifying enterococci to species level, it is important to verify results by a molecular method when phenotypic findings are discordant with epidemiologic patterns. Furthermore, GeneXpert(®) vanA/vanB PCR and ChromID VRE combined with in-house PCR were applied in rectal samples for the detection of VRE colonization among critically ill patients. GeneXpert(®) showed an excellent performance in detecting vanA-positive enterococci, but false-positive results for vanB-gene detection render its application problematic in departments with high incidence of vanB-positive enterococci.

Number of positive blood cultures, biofilm formation, and adhesin genes in differentiating true coagulase-negative staphylococci bacteremia from contamination.

The significance of the number of coagulase-negative staphylococci (CNS)-positive blood cultures remains obscure in regards to determining true bacteremia versus contamination. The goal of this study was to determine the predictors of real CNS bloodstream infection among intensive care unit (ICU) patients. ICU patients with at least one CNS-positive blood culture were identified from the microbiology database. Biofilm formation was tested by glass tube and microtiter plate assay. mecA gene, ica operon genes (icaA, icaB, icaD), and adhesin genes (aap, bap, atlE, fbe, fnbA) were detected by polymerase chain reaction (PCR). CNS were recovered from 120 septic episodes, 20 of which were true CNS bacteremias, whereas from the remaining 100 episodes, the isolated CNS were characterized as contaminants. The number of positive blood cultures was significantly associated with true CNS bacteremia. Nineteen true bacteremic Staphylococcus epidermidis strains were compared to 38 contaminants. Biofilm synthesis was documented in 37 isolates associated with the presence of the ica operon (p = 0.048). There were 39, 26, 38, 21, and 10 strains positive for the presence of atlE, bap, fbe, aap, and fnbA genes, respectively. Rifampicin resistance, absence of severe sepsis, number of S. epidermidis-positive blood cultures, and absence of the bap gene were independently associated with true S. epidermidis bacteremia as compared to contaminant strains. The number of positive blood cultures is associated with true CNS bacteremia. The presence of adhesin genes may play a role in differentiating true infection from contamination, whereas absence of the bap gene is associated with true S. epidermidis bacteremia.

Virulence factors among Staphylococcus lugdunensis are associated with infection sites and clonal spread.

Staphylococcus lugdunensis has emerged as a significant human pathogen, with distinct clinical and microbiological characteristics. Our goal was to identify the virulence factors in S. lugdunensis recovered from infected patients of two Greek hospitals during a six-year period (2008-2013). A collection of 38 S. lugdunensis was tested for biofilm formation, antimicrobial susceptibility, clonal distribution, virulence factors (ica operon, fbl, atlL, vwbl, slush) and antibiotic resistance genes (mecA, ermC) carriage. Strains were classified into pulsotypes by pulsed-field gel electrophoresis (PFGE) of SmaI DNA digests. The majority (22) was isolated from skin and soft tissue infections (SSTIs), nine from deep-sited infections (DSIs), including three bacteraemias and seven from prosthetic device-associated infections (PDAIs). All isolates were oxacillin-susceptible, mecA-negative and fbl-positive. The highest resistance rate was detected for ampicillin (50%), followed by erythromycin and clindamycin (18.4%). Fourteen isolates (36.8%) produced biofilm, whereas 26/38 (68.4%) carried the ica operon. Biofilm formation was more frequent in isolates from PDAIs. Thirty-six strains (94.7%) carried atlL and 31 (81.6%) carried vwbl, whereas slush was detected in 15 (39.5%). PFGE revealed a low level of genetic diversity: strains were classified into seven pulsotypes, with two major clones (C: 22 and D: nine strains). Type C strains recovered from all infection sites prevailed in biofilm formation and ermC carriage, whereas type D strains associated with SSTIs and DSIs carried more frequently vwbl, slush or both genes. Despite susceptibility to antimicrobials, the clonal expansion and carriage of virulence factors, combined with biofilm-producing ability, render this species an important pathogen that should not be ignored.

Risk factors for enterococcal infection and colonization by vancomycin-resistant enterococci in critically ill patients.

PURPOSE: Vancomycin-Resistant Enterococci (VRE) are important causes of Intensive Care Unit (ICU) infections. Our goal was to identify the prevalence and risk factors for VRE colonization upon ICU admission and during ICU stay, as well as, their impact in enterococcal infection including vancomycin-susceptible cases (VSE). METHODS: A prospective study regarding patients admitted in ICU (n = 497) was conducted during a 24-month period. Rectal swabs were collected upon admission and during hospitalization and inoculated onto selective medium. Enterococci were phenotypically characterized. van genes were investigated by PCR and clones were identified by Pulsed-Field Gel Electrophoresis and Multilocus Sequence Typing. Epidemiologic data were collected from the ICU database. RESULTS: Risk factors for VRE carriage upon ICU admission (71/497) were: duration of previous hospitalization, glycopeptide administration, chronic heart failure, malignancy, insulin-dependent diabetes mellitus, and previous enterococcal infection (VRE and/or VSE). Risk factors for VRE colonization during ICU stay (36/250) were: quinolone administration, chronic obstructive pulmonary disease, chronic renal failure, and number of VRE-positive patients in nearby beds. Risk factors for enterococcal infection during ICU stay (15/284), including VRE and VSE cases, were: administration of third- or fourth-generation cephalosporins, cortisone use before ICU admission and VRE colonization, whereas, enteral nutrition was a protective factor. CONCLUSIONS: Previous VRE colonization and antibiotic usage are essential parameters for enterococcal infection (by VRE or VSE) during ICU stay. Previous enterococcal infection, co-morbidities and antibiotic usage are associated with VRE colonization upon ICU admission, whereas, patient to patient transmission, co-morbidities and antibiotic usage constitute risk factors for VRE colonization during ICU hospitalization.

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.

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.

Clonality of slime-producing methicillin-resistant coagulase-negative staphylococci disseminated in the neonatal intensive care unit of a university hospital.

Methicillin-resistant coagulase-negative staphylococci (MR-CNS) (n = 132), isolated from pre-term neonates, were analysed to determine their antibiotic resistance patterns, clonal distribution, biofilm production and the presence of the ica operon. All MR-CNS were multiresistant, and 89% produced slime. A major clone was identified (77 isolates) among 115 Staphylococcus epidermidis isolates. Ten of 16 Staphylococcus haemolyticus isolates also belonged to a single clone. Most (80%) slime-positive isolates possessed all the ica genes tested, while the remaining 23 (20%) had a variety of gene combinations. The entire ica cluster was detected in three of 15 slime-negative isolates. One major and two minor slime-positive, multiresistant MR-CNS clones had disseminated among hospitalised pre-term neonates.

Immunization with specific polysaccharide antigen reduces alterations in corneal proteoglycans during experimental slime-producing Staphylococcus epidermidis keratitis.

PURPOSE: Staphylococcus epidermidis is a leading cause of bacterial keratitis associated with corneal damage. Corneal integrity is closely associated with matrix macromolecules, such as proteoglycans (PGs) and collagen. The aim of this study was to examine whether active immunization (AI) using a major immunogenic polysaccharide determinant of slime (20-kDa PS) as antigen, and passive immunization (PI) after administration of specific antibodies toward 20-kDa PS affect the distribution of PGs as well as corneal lesions in an experimental model of slime-producing S. epidermidis keratitis. METHODS: For AI, seven rabbits were immunized with 20-kDa PS, whereas for PI, seven rabbits received specific antibodies against 20-kDa PS. Lesions were graded clinically for a 21-day period. Levels of 20-kDa PS antibodies in serum and aqueous humor in both immunization groups were determined by ELISA. The distribution of certain extracellular matrix PGs during corneal healing was analyzed immunohistochemically. RESULTS: Levels of specific anti-20-kDa PS antibodies in serum and aqueous humor obtained after either AI or PI were significantly higher as compared with those in the respective nonimmunized control groups (p<0.001). Clinical grading showed that both AI and PI rabbits had a significantly less corneal damage as compared with infected nontreated rabbits. Immunohistochemical analyses for PGs exhibited significant differences to the wounded regions as compared with noninfected corneal tissue. Accumulation of keratan sulfate PGs and decorin was observed in the corneal stroma of infected rabbits and of heparan sulfate PGs around the new-formed vessels. This phenomenon was significantly reduced in immunized animals in accordance with macroscopically decreased corneal damage observed in these animals. CONCLUSIONS: Results of this study suggest a key role of 20-kDa PS and its antibodies as prophylactic and therapeutic agents in keratitis caused by slime-producing S. epidermidis.

The emergence of glycopeptide-resistant Enterococcus faecium in a university hospital in southwestern Greece.

BACKGROUND: Enterococci and especially glycopeptide-resistant strains (GRE) are widely distributed in the hospital environment, by acquiring resistance determinants and virulence factors. METHODS: The study included 48 GRE isolated during a 1-year period from different inpatients in a tertiary hospital in southwestern Greece. Antibiotic susceptibility was determined by the Etest, and the presence of resistance and virulence genes was shown by PCR. Clonal types were identified by pulsed-field gel electrophoresis of SmaI DNA digests. RESULTS: All GRE were multi-resistant of the VanA phenotype, verified by the detection of the gene by PCR. Two major clones were distributed in all hospital wards. The majority of the strains (46 of 48) harbored the esp gene, while 27 GRE expressed also the gelE and/or as genes. CONCLUSIONS: The spread of two clones expressing the vanA gene and virulence factors were responsible for the emergence of GRE in the University Hospital of Patras.

Intravenous catheter infections associated with bacteraemia: a 2-year study in a university hospital.

The aim of this retrospective study was to assess the incidence and aetiology of central and peripheral venous catheter (C/PVC) infections during a 2-year period (1999-2000) and to determine the susceptibility of isolated microorganisms to various antimicrobial agents. Catheter tips were processed using the semiquantitative method and blood cultures were performed with the BacT/Alert automated system. Antibiotic susceptibilities were performed by disk agar diffusion and MICs were determined by Etest, according to NCCLS standards. During the study period, samples from 1039 C/PVC infections were evaluated, yielding 384 (37.0%) positive cultures. Blood cultures were also available from 274 patients, of which 155 (56.6%) yielded the same microorganism as from the catheter. No bloodstream infections were detected in 104 C/PVC-positive cases. Methicillin-resistant coagulase-negative staphylococci were the most frequent isolates, followed by Gram-negative bacteria, especially Pseudomonas aeruginosa. Resistance to glycopeptides among staphylococci and enterococci was not detected, whereas 60% of Gram-negative bacilli were resistant to beta-lactams.

Potential use of solid phase immunoassays in the diagnosis of coagulase-negative staphylococcal infections.

Staphylococcus epidermidis is a major nosocomial pathogen, even though it is a member of the normal bacterial flora of skin and the mucous membranes. A major complication is the development of biofilms on implanted medical devices. Diagnosis of coagulase-negative staphylococcal infections relies on the presence of clinical manifestation of infections and on microbiologic evidence, usually obtained after the removal of the biomaterial. Solid-phase immunoassays have not yet been used for routine diagnosis of coagulase-negative staphylococcal infections and distinction between pathogenic and normal cocci. The enzyme immunoassays developed in the last decade are presented in this review article. Serodiagnosis has been attempted by determining antibodies against bacterial cells, mixtures of S. epidermidis slime antigens and discrete slime antigens. Detection or typing of staphylococcal cells has been performed by specific antibodies and lectins. There is still a long way until the application of such assays in the routine clinical laboratory and large clinical studies are necessary.

Does identification of the causal organism of corneal ulcers influence the outcome?

PURPOSE: To investigate whether identification of the causal organism in corneal ulcers influences their outcome. METHODS: We retrospectively studied 114 patients, 72 males and 42 females aged 6-89 years, admitted to this eye clinic during the years 1994-2000 on account of an infectious corneal ulcer. Their examination included a detailed history, visual acuity measurement, and biomicroscopy in everyday follow-up. The ulcers were classified according to their severity and outcome. We assessed the cases where cultures had been done, reviewed the results, and searched for a possible correlation between the outcome and the fact of culturing the ulcer and identifying the causal organism. RESULTS: Of the 114 corneal ulcers studied, 23 were mild, 49 moderate, and 42 severe. Fifty (44%) had not been cultured, but 64 ulcers (56%) had been cultured, with a positive result in 37 cases (58%), Staphylococcus and Pseudomonas species being the most common organisms found. In moderate and severe ulcers, there was a tendency to a higher proportion of successful outcome for cultured ulcers, but with no significant correlation. CONCLUSIONS: Despite a tendency towards favorable results in culture-positive corneal ulcers, the influence of the detection of the organism on their outcome has not been proved. The role of the initial broad-spectrum antibiotic therapy remains important.

Levels of specific antibodies towards the major antigenic determinant of slime-producing Staphylococcus epidermidis determined by an enzyme immunoassay and their protective effect in experimental keratitis.

Staphylococcus epidermidis is an important cause of bacterial keratitis. Certain S. epidermidis strains produce an extracellular slime layer rich in an acidic polysaccharide with a molecular size of 20 kDa (20-kDa PS). We have demonstrated that the level of 20-kDa PS-specific antibodies significantly rises after establishment of slime-producing S. epidermidis bacteraemia and, furthermore, that rabbit polyclonal antibodies to 20-kDa PS opsonize cells of slime-producing S. epidermidis to a great degree and promote their clearance by polymorphonuclear cells (Arch. Biochem. Biophys. 342 (1997) 389; J. Pharm. Biomed. Anal. 22 (2000) 1029). The purpose of this study was to examine the protective and therapeutic effects both of active immunization, using 20-kDa PS as antigen, and of passive administration of specific antibodies towards the 20-kDa PS in a rabbit keratitis model. For active immunization, 20 rabbits were subcutaneously immunized with 20-kDa PS, whereas for passive immunization specific polyclonal IgG antibodies against 20-kDa PS were administered to 20 rabbits 1 day before induction of infection. Clinical observations were made weekly for 1 month and levels of 20-kDa PS antibodies in serum and aqueous humor in both immunization groups were determined by an enzyme immunoassay. The levels of specific anti-20-kDa PS IgG in serum and aqueous humor following either active or passive immunization were significantly higher as compared with control groups (P<0.001). Although, actively immunized rabbits showed significantly less corneal damage than control animals, passively immunized ones were significantly better protected as compared with both control and those actively immunized. Obtained results suggest that 20-kDa PS plays crucial role in the pathogenesis of S. epidermidis keratitis and that both types of immunization significantly protect against corneal S. epidermidis pathology and damage.

Determination of malondialdehyde by capillary electrophoresis, application to human plasma and relation of its levels with prematurity.

Malondialdehyde (MDA) is considered as the most important marker for monitoring lipid peroxidation, which is strongly associated with the development of serious diseases in adults and premature neonates. In this paper we report a method for determination of free MDA in human plasma using capillary zone electrophoresis. MDA was separated and determined as conjugate with tetrabutylammonium hydrogen sulphate (TBAS). Analysis was performed using 20 mM borare, pH = 9.3, as operating buffer and detection of the MDA-TBAS adduct at 267 nm. The method has a linear range up to 80 microM with a detection limit of 0.2 microM. The method was applied to the analysis of MDA in plasma of healthy adults, normal-gestation infants and of preterm neonates. Plasma proteins were successfully removed following centrifugation through a centricon-3 membrane. Results showed that the method can be easily and accurate applied for the determination of MDA in human plasma and that the level of MDA in pretern neonates is significantly higher (p

Immunoreactivity of 80-kDa peptidoglycan and teichoic acid-like substance of slime producing S. epidermidis and specificity of their antibodies studied by an enzyme immunoassay.

S. epidermidis is considered an important cause of nosocomial bacteraernia in immunocompromized hosts as well as the commonest agent of sepsis in patients with prosthetic devices. Pathogenesis is attributed to adherence and growth on bioniaterials facilitated by production of extracellular slime. The major macromolecules of slime are: a 20-kDa acidic polysaccharide (20-kDa PS) comprising the 60% of carbohydrate-containing slime macromolecules, a peptidoglycan with average molecular size of 80-kDa (30% of slime dry weight) and cell wall teichoic acid-like substance. In this study, antibodies to these macromolecules as well as crude slime were raised in rabbits and their immunological reactivity and specificity were studied by an enzyme immunoassay. All isolated macromolecules induced the production of specific antibodies. 20-kDa PS was less immunogenic than 80-kDa peptidoglycan and teichoic acid-like substance. However, 20-kDa PS was the most potent inhibitor of the reaction of slime with its homologous antibodies revealing that this polysaccharide is the major antigenic determinant of slime. All three antibodies specifically recognize (p < 0.05) and react with slime-producing S. epidermidis in comparison to other staphylococci species. Obtained results indicate that the 20-kDa PS may be distributed in the surface of the slime exposing most of its antigenic determinants to the immune system, whereas those of 80-kDa peptidoglycan and teichoic acid-like substance seem to be less accessible.

Beta-thalassaemia and the prevalence of HCV viraemia.

Twenty-four serum samples from multitransfused patients with beta-thalassaemia major and fourteen positive control samples, (eight haemodialysis patients and six sporadic cases of HCV infection) were selected as anti-HCV-positives by a second-generation ELISA, a confirmatory test (Abbott), and an immunoblot assay (INNO-LIA HCV AbIII, Omicron Medical). Subsequently, by means of the nested polymerase chain reaction technique (PCR), using the set of PT1-4 primers, the RNA of the virus was detected in a total of 29 samples: 22 out of 24 patients with beta-thalassaemia and 7 out of 14 from the control group were positive for the RNA of the virus. The findings show that there is a statistically significant prevalence [chi2 = 6.344, P < or = 0.02] of HCV viraemia in the population of beta-thalassaemia major in Greece, as compared with the positive control group.