Changes of dermatophytoses in southwestern Greece: an 18-year survey.

The isolation and distribution rate of dermatophytes as causative agents of superficial mycoses of skin, hair, and nails during an 18-year period (1991-2008) at a university hospital are presented. A comparative analysis of epidemiological differences within the first (1991-1999) and the second 9-year period (2000-2008) was performed. Skin scrapings, nail, and hair specimens were examined by a direct microscopic examination and culture. Identification of dermatophyte species was based on macroscopic and microscopic characteristics of colonies. During the complete period (18 years), 5,971 patients with suspected dermatophytosis were examined. Seven hundred and sixty-nine patients (12.8%) were found positive. Among them, 495 cases (64.3%) were of skin dermatophytoses, 91(11.8%) of hair, and 183 (23.7%) of nails. The most frequent etiological agents were Microsporum canis (54%), Trichophyton rubrum (38%), and T. mentagrophytes (6%). Epidermophyton floccosum, T. tonsurans, T. violaceum, and M. gypseum were responsible only for 16 cases (2%) of dermatophytoses. The prevalence of dermatophytoses seems to decrease significantly from 16.2% (1991-1999)-9.6% during the last 9-year period. The most frequent dermatophyte, M. canis, shows decreasing trends during the last period (from 58.5 to 45.7%), whereas T. rubrum shows an increasing isolation rate (from 35 to 43.6%), respectively. The most common form of dermatophytosis among children remains tinea capitis due to M. canis. The most frequent etiological agent of tinea unguium (81%) is T. rubrum.

Eleven-year retrospective survey of candidaemia in a university hospital in southwestern Greece.

The aim of this study was to investigate the isolation and distribution rate of Candida spp. in blood cultures and evaluate antifungal susceptibility during an 11-year period (1998­2008) at a tertiary-care hospital. The causative species were as follows: Candida albicans, 163 strains (64%); Candida parapsilosis, 35 strains (13.7%); Candida glabrata, 25 strains (9.8%); Candida tropicalis, 19 strains (7.4%); and other Candida spp., 13 strains (5.1%). Candidaemia is predominantly caused by C. albicans. C. parapsilosis is the most common non-albicans Candida isolated in neonatal intensive-care units. All Candida isolates remain susceptible to amphotericin B, whereas the highest degree of resistance was observed for azoles.

TNF-alpha induction by Pseudomonas aeruginosa lipopolysaccharide or slime-glycolipoprotein in human monocytes is regulated at the level of Mitogen-activated Protein Kinase activity: a distinct role of Toll-like receptor 2 and 4.

TNF-alpha production has a central role in the development and progression of Pseudomonas aeruginosa septic shock. We have previously shown that P. aeruginosa slime-glycolipoprotein (slime-GLP) is the most potent stimulant compared to P. aeruginosa lipopolysaccharide (LPS), for TNF-alpha production and NF-kB activation in human monocytes. Herein, we show that secretion of TNF-alpha by fresh human monocytes, induced by P. aeruginosa slime-GLP, LPS or viable bacteria, was paralleled by phosphorylation and/or activation of Mitogen-activated Protein Kinases (MAPKs) ERK1/2, p38 as well as c-Jun NH(2)-terminal kinase. TNF-alpha levels were significantly reduced by ERK1/2 inhibitor (PD98059), or p38 inhibitor (SB203580). Combination of both inhibitors almost abolished TNF-alpha induction. Pseudomonas aeruginosa slime-GLP differed from the P. aeruginosa-LPS only regarding the strength of p38 and ERK1/2 activation, with slime-GLP leading to a stronger activation of p38 and ERK1/2. Involvement of TLR2 and TLR4 for phosphorylation of p38 and ERK1/2 was shown using specific blocking anti-TLR2 and anti-TLR4 antibodies. Activation of both p38 and ERK1/2 induced by P. aeruginosa slime-GLP was dramatically reduced in the presence of anti-TLR2 and to a lesser degree in the presence of anti-TLR4, whereas the P. aeruginosa-LPS-induced stimulation was inhibited only in the presence of anti-TLR4. Our data show that P. aeruginosa viable bacteria, through slime-GLP, stimulate specific members of the MAPKs more efficiently than the P. aeruginosa-LPS, involving mainly TLR2.

Absolute and relative real-time PCR in the quantification of tst gene expression among methicillin-resistant Staphylococcus aureus: evaluation by two mathematical models.

AIM: Absolute and relative quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR) by the use of two mathematical models were applied in order to study the expression of tst gene encoding the toxic shock syndrome toxin-1 (TSST-1), among methicillin-resistant Staphylococcus aureus (MRSA). METHODS AND RESULTS: Thirteen epidemic MRSA belonging to different clones and carrying a variety of toxin genes were selected. tst gene expression was achieved by using absolute and relative quantitative real-time RT-PCR and the SYBR Green I. Absolute RT-PCR showed a statistically significant higher level of tst expression among strains isolated from soft tissue infections. Relative quantification was performed in relation to 23S rRNA expression by the application of two mathematical models, the 2(-DeltaDeltaCt) and the Pfaffl analysis methods. CONCLUSIONS: tst gene expression was best calculated by the relative real-time RT-PCR analysis applying the Pfaffl analysis method, taking into account the reactions' efficiencies. Level of tst expression was related to patients' infection and did not depend on the MRSA genetic profile. SIGNIFICANCE AND IMPACT OF THE STUDY: The results indicate that the application of the Pfaffl analysis method in the evaluation of relative real-time RT-PCR is more adequate.

Methicillin-resistant Staphylococcus aureus producing Panton-Valentine leukocidin as a cause of acute osteomyelitis in children.

Staphylococcus aureus was identified as the cause of acute childhood osteomyelitis in 19 patients. A single clone of community-acquired methicillin-resistant S. aureus (MRSA) carrying the type IV mecA staphylococcal cassette chromosome and the Panton-Valentine leukocidin (PVL) genes was isolated from five patients. Among the remaining 14 patients, two methicillin-sensitive S. aureus (MSSA) isolates were PVL-positive. The maximal erythrocyte sedimentation rate and C-reactive protein values, and the time required for normalisation, were significantly different in patients with PVL-positive strains (MRSA and MSSA), suggesting that the production of PVL is an important factor that contributes to the course of the disease.

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.

Comparison of two commercial methods with PCR restriction fragment length polymorphism of the tuf gene in the identification of coagulase-negative staphylococci.

AIMS: Two commercial methods for the identification of coagulase-negative staphylococci (CNS) were compared with the restriction fragment length polymorphism (RFLP) of the amplified tuf gene, which served as the reference method. METHODS AND RESULTS: One hundred and forty-five CNS were evaluated using the API 32 Staph ID and the Crystal GP/ID BBL systems. The PCR-RFLP of the tuf gene served as the reference method. The APIStaph and the GP/ID BBL had an overall rate of agreement with the molecular method of 58.6% and 46.2% respectively, with the inability of the GP/ID BBL to characterize 11.7% of the isolates. The APIStaph showed higher sensitivity and better agreement than the GP/ID BBL with the PCR-RFLP, except for Staphylococcus hominis and Staphylococcus capitis. CONCLUSIONS: Neither of the commercial systems was as reliable as the PCR-RFLP method for identifying isolates of CNS. Overall the APIStaph had better agreement with the PCR-RFLP than the GP/ID system. SIGNIFICANCE AND IMPACT OF THE STUDY: The results indicate that the PCR-RFLP method is more reliable than the two commercial systems tested, suggesting that it is more reliable for routinely identifying CNS.

Comparison of the manual Mycobacteria Growth Indicator tube and the Etest with the method of proportion for susceptibility testing of Mycobacterium tuberculosis.

BACKGROUND: Clinical microbiology laboratories should provide reliable results on susceptibility testing of Mycobacterium tuberculosis to different agents. METHODS: The manual Mycobacteria Growth Indicator Tube (MGIT) and Etest were compared to the method of proportion (MOP) for susceptibility testing of 88 clinical isolates of M. tuberculosis against isoniazid (INH), rifampin (RIF), streptomycin (STR) and ethambutol (EMB). Isolates were recovered from different patients and were identified at species level by PCR and hybridization. RESULTS: Resistance to INH was detected in 20.5, 29.5 and 12.5% of the isolates, followed by STR resistance (19.3, 26.1 and 1.1%), RIF (9.1, 4.5 and 5.7%) and EMB (2.3, 11.4 and 2.3%) by the MOP, MGIT and Etest, respectively. Sensitivity of the manual MGIT ranged from 37.5% for RIF resistance to 100% for EMB, while Etest sensitivity ranged from 5.9% for STR to 62.5% for RIF. CONCLUSIONS: MOP remains the method of choice, with the manual MGIT showing superior sensitivity at detecting resistance to INH, STR and EMB compared to the Etest.

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.

Spread of Staphylococcus aureus clinical isolates carrying Panton-Valentine leukocidin genes during a 3-year period in Greece.

Three collections of Staphylococcus aureus isolates (n = 1,058) were investigated to assess the spread of Panton-Valentine leukocidin (PVL)-producing strains in Greece and their association with skin and soft-tissue infections (SSTIs). The isolates were collected during 2001-2003 from inpatients and outpatients with invasive infections in two distinct geographical areas. Clonal types were identified according to their ClaI-mecA::ClaI-Tn554::pulsed-field gel electrophoresis patterns, and the presence of the lukS-PV and lukF-PV genes was assessed by PCR. In total, 287 (27%) S. aureus isolates carried the PVL genes: 45% of methicillin-resistant S. aureus (MRSA) and 12% of methicillin-sensitive S. aureus (MSSA). All the PVL-positive MRSA isolates belonged to a single clone that was disseminated in the community and hospitals. The PVL-positive MSSA isolates were polyclonal, with 14 of 65 isolates being associated with hospital-acquired infections. The community-acquired isolates were from SSTIs, while the hospital-acquired isolates were associated with surgical wound infections, especially those involving prosthetic devices. Thus, a unique clone of PVL-positive MRSA has spread in both the community and the hospital setting in Greece, and has replaced older clonal types.

Sensitivity of the Cobas Amplicor system for detection of Mycobacterium tuberculosis in respiratory and extrapulmonary specimens.

The Cobas Amplicor PCR system (CA-PCR) was compared with culture and staining for acid-fast bacilli (AFB) for the early detection of Mycobacterium tuberculosis in respiratory clinical specimens and otherwise normal sterile body fluids. The sensitivity, specificity and positive and negative predictive values of CA-PCR were determined with AFB-positive and AFB-negative specimens. The sensitivity of CA-PCR ranged from 73.6% to 100% for AFB-positive samples, while sputa collected after bronchoscopy were the most useful specimens, with 70% sensitivity and 98.6% specificity among the AFB-negative samples.

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.

erm(C) is the predominant genetic determinant for the expression of resistance to macrolides among methicillin-resistant Staphylococcus aureus clinical isolates in Greece.

OBJECTIVES: Macrolide, lincosamide and streptogramin type B (MLS(B)) resistance was determined in Staphylococcus aureus clinical isolates from two University Hospitals. METHODS: Antibiotic resistance was investigated by double disc diffusion and MIC determination. Resistance determinants were detected by PCR and DNA hybridization, while clonal types were identified by pulsed-field gel electrophoresis (PFGE) analysis of SmaI DNA fragments. RESULTS: Among methicillin-susceptible S. aureus (MSSA) isolates, inducible and MS phenotypes were detected, with the predominance of the erm(A) gene, followed by the msr(A) and erm(C) genes. The majority of methicillin-resistant S. aureus (MRSA) isolates expressed the constitutive phenotype and carried the erm(C) gene. PFGE revealed the dissemination of two major clones among the MRSA in both hospitals. CONCLUSIONS: erm(C) is the predominant genetic determinant for the expression of MLS(B) resistance among S. aureus isolates, especially MRSA, in Greece. This is due to the spread of two major clones in the country.

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.

Application of molecular typing methods to characterize nosocomial coagulase-negative staphylococci collected in a Greek hospital during a three-year period (1998-2000).

A total of 143 methicillin-resistant coagulase-negative staphylococci (MR-CNS) collected between 1998 and 2000 at the University Hospital of Patras, Greece, were characterized by antibiogram and genomic typing to define the clonal types endemic in this hospital and their evolution during the 3-year period. These isolates corresponded to 93 methicillin-resistant Staphylococcus epidermidis (MRSE) and 50 other MR-CNS, which were isolated from patients in different wards, exclusively from blood and catheter tips cultures. Pulsed-field gel electrophoresis (PFGE) of SmaI macrofragments and hybridization of ClaI digests with mecA and murE DNA probes were performed. The application of these methodologies demonstrated the existence, persistence and spread of MRSE, MR-Staphylococcus haemolyticus, and MR-Staphylococcus hominis clones in this hospital, whereas the SmaI/murE hybridization pattern was shown to be a valuable tool for the MRSE identification.

Presence of mec genes and overproduction of beta-lactamase in the expression of low-level methicillin resistance among staphylococci.

Detection of methicillin-resistant staphylococci is critical for the management of infected patients in the hospital. A total of 55 nonreplicated clinical isolates of staphylococci (31 Staphylococcus aureus and 24 coagulase-negative staphylococci; CNS) collected during a one-year period and expressing low-level resistance to methicillin (oxacillin MIC of 2-4 mg/l for S. aureus and 0.5-4 mg/l for CNS) were studied. mec determinants and overproduction of beta-lactamase were investigated and pulsed-field gel electrophoresis (PFGE) was applied as a typing method. Twenty-four S. aureus isolates and 19 CNS carried the mecA gene. The presence of mecR1/mecI and blaR1/blaI genes correlated with the expression of low-level methicillin resistance in CNS. Four mecA-negative isolates (2 S. aureus and 2 CNS) overproduced beta-lactamase. PFGE revealed the presence of 2 major clonal types in mecA-positive S. aureus isolates, and 3 in CNS. Low-level methicillin resistance of staphylococci is correlated with the presence of the mecA gene and overproduction of beta-lactamase.

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.

Sterile cerebrospinal fluid pleocytosis in young infants with urinary tract infection.

BACKGROUND: During the first 3 months of life febrile infants are subjected to sepsis workup, which includes evaluation for urinary tract infection (UTI) and meningitis. We investigated the existence of concomitant meningeal inflammation in infants younger than 90 days old affected with UTI. METHODS: We reviewed the medical records of all infants younger than 90 days old, who were hospitalized for UTI from January, 1990, to January, 2001. For the diagnosis of sterile cerebrospinal fluid (CSF) pleocytosis, the child's age, the CSF total white blood cell (WBC) count and the CSF absolute neutrophil count were taken into consideration. CSF pleocytosis was defined as the presence of > or = 35, > or = 21 and > or = 15 WBC/mm3 of CSF during the first, second and third month of life, respectively. The CSF Gram-stained smear, latex agglutination test and bacterial culture were negative. RESULTS: Sterile CSF pleocytosis was found in 15 (12.8%) of 117 infants with UTI who had had a lumbar puncture included in their initial laboratory evaluation. The 15 infants had a median age +/- semiinterquartile range of 40 +/- 25 days (range, 4 to 75 days). In these infants the median CSF WBC count +/- semiinterquartile range was 55 +/- 125/mm3 (range, 21 to 1,270/mm3). CONCLUSIONS: Sterile CSF pleocytosis was found in 12.8% of infants younger than 90 days old with UTI. The pathogenesis of this meningeal inflammation is not fully understood. Although bacterial infection of the subarachnoid space, with low bacterial seeding, cannot be excluded, at least in some cases, it is possible that CSF pleocytosis in some of the infants with UTI is mainly caused by the endotoxin of Gram-negative or other inflammation-inducing molecules of Gram-positive urine pathogens.

The possible role of locally produced cytokines in the pathogenesis of peritrochanteric fractures in the elderly.

Sixteen patients with intertrochanteric femoral fractures were studied for possible involvement of the cytokines interleukin-6 (IL-6), interleukin-1 beta (IL-1beta), tumor necrosis factor-alpha, and the disease activity factors C-reactive protein and alpha1-antitrypsin as local bone-resorbing agents. Cytokine and disease activity factor levels were measured in gluteus medius muscle and serum samples and were compared to sera obtained from age- and sex-matched healthy controls. Interleukin-6 and IL-1beta levels were significantly higher (P=.0024 and P=.036, respectively) in the muscle samples from the fractured side than in the samples from the contralateral unaffected side. Levels of IL-6 and IL-1beta also were significantly higher in patients' sera than in the sera of healthy controls. These results support a new hypothesis that may contribute to the pathogenesis of fractures in the elderly: unilaterally locally over-produced IL-6 and IL-1beta may lead to local bone resorption in the intertrochanteric region, which subsequently weakens the femoral bone and increases the risk of unilateral peritrochanteric fractures.