Environmental and bioclimatic factors influencing yeasts and molds distribution along European shores.

The present study employed data collected during the Mycosands survey to investigate the environmental factors influencing yeasts and molds distribution along European shores applying a species distribution modelling approach. Occurrence data were compared to climatic datasets (temperature, precipitation, and solar radiation), soil datasets (chemical and physical properties), and water datasets (temperature, salinity, and chlorophyll-a concentration) downloaded from web databases. Analyses were performed by MaxEnt software. Results suggested a different probability of distribution of yeasts and molds along European shores. Yeasts seem to tolerate low temperatures better during winter than molds and this reflects a higher suitability for the Northern European coasts. This difference is more evident considering suitability in waters. Both distributions of molds and yeasts are influenced by basic soil pH, probably because acidic soils are more favorable to bacterial growth. Soils with high nitrogen concentrations are not suitable for fungal growth, which, in contrast, are optimal for plant growth, favored by this environment. Finally, molds show affinity with soil rich in nickel and yeasts with soils rich in cadmium resulting in a distribution mainly at the mouths of European rivers or lagoons, where these metals accumulate in river sediments.

Brainstem phaeohyphomycosis due to Curvularia lunata (Cochliobolus lunatus) in a cat.

A 13-year-old female neutered domestic short-hair cat was presented with chronic progressive vestibular ataxia, lethargy and anorexia. Clinical examination revealed bilateral mucopurulent nasal discharge. Neurological examination revealed obtundation, a right head tilt, ambulatory tetraparesis, generalised vestibular ataxia, decreased postural reactions in all limbs, right Horner's syndrome, spontaneous conjugate jerk rotatory nystagmus and right positional ventral strabismus. Neuroanatomical localisation was observed in the right central vestibular system. Computed tomography revealed a solitary ill-defined contrast-enhancing mass lesion at the level of the right cerebellopontine angle. Cerebrospinal fluid (CSF) analysis revealed mild mononuclear pleocytosis and fungal elements. CSF culture was positive for Curvularia spp. Further tests for underlying diseases were all negative. The cat was treated with antibiotic and antifungal treatment, but it deteriorated rapidly and was euthanased. Necropsy of the brainstem mass lesion revealed pyogranulomatous inflammation. Panfungal polymerase chain reaction (PCR) targeting the internal transcribed spacer (ITS) region and subsequent sequencing identified Curvularia lunata in the formalin fixed brain tissue. This is the first report of brainstem phaeohyphomycosis by Curvularia lunata (Pleosporales) in a cat. In addition, this is the first report among animal and humans where fungal elements of Curvularia lunata were found in the CSF cytology. Opportunistic fungal pathogens should be always considered within the differential diagnoses list in cats with neurological signs and advanced imaging findings compatible with solitary mass lesions in the brain. In feline patients with pyogranulomatous meningoencephalitis and a suspicion of a fungal aetiology, panfungal PCR for the ITS region and sequencing should be performed regardless of the absence of fungal elements in histopathology.

Mycosands: Fungal diversity and abundance in beach sand and recreational waters - Relevance to human health.

The goal of most studies published on sand contaminants is to gather and discuss knowledge to avoid faecal contamination of water by run-offs and tide-retractions. Other life forms in the sand, however, are seldom studied but always pointed out as relevant. The Mycosands initiative was created to generate data on fungi in beach sands and waters, of both coastal and freshwater inland bathing sites. A team of medical mycologists and water quality specialists explored the sand culturable mycobiota of 91 bathing sites, and water of 67 of these, spanning from the Atlantic to the Eastern Mediterranean coasts, including the Italian lakes and the Adriatic, Baltic, and Black Seas. Sydney (Australia) was also included in the study. Thirteen countries took part in the initiative. The present study considered several fungal parameters (all fungi, several species of the genus Aspergillus and Candida and the genera themselves, plus other yeasts, allergenic fungi, dematiaceous fungi and dermatophytes). The study considered four variables that the team expected would influence the results of the analytical parameters, such as coast or inland location, urban and non-urban sites, period of the year, geographical proximity and type of sediment. The genera most frequently found were Aspergillus spp., Candida spp., Fusarium spp. and Cryptococcus spp. both in sand and in water. A site-blind median was found to be 89 Colony-Forming Units (CFU) of fungi per gram of sand in coastal and inland freshwaters, with variability between 0 and 6400 CFU/g. For freshwater sites, that number was 201.7 CFU/g (0, 6400 CFU/g (p = 0.01)) and for coastal sites was 76.7 CFU/g (0, 3497.5 CFU/g). For coastal waters and all waters, the median was 0 CFU/ml (0, 1592 CFU/ml) and for freshwaters 6.7 (0, 310.0) CFU/ml (p < 0.001). The results advocate that beaches should be monitored for fungi for safer use and better management.

Performance of three small-scale wastewater treatment plants. A challenge for possible re use.

The study focused on the assessment of the performance of three WWTPs in Greece by the estimation of the microbiological and chemical quality of influent and effluent sewage. Physicochemical parameters were recorded (temperature, pH, COD, BOD, suspended solids, conductivity), and meteorological data were collected (air temperature, rain). Microbiological parameters were analyzed (Escherichia coli, total coliforms, bacteriophages, Salmonella, human adenoviruses, Candida, Pseudallescheria boydii, helminths, parasites Cryptosporidium ssp., and Giardia spp.). Statistically significant correlations among the various aforementioned parameters were investigated, in an attempt to propose appropriate processing performance indicators. Furthermore, the study aimed to assess current joint ministerial decision (JMD) on wastewater reuse, for irrigation purposes; to evaluate its practicability and its potential for public health protection. In the vast majority, outlet samples from all three studied WWTPs were not appropriate for irrigation reuse purposes based on BOD50 and suspended solids limit values, set by the current JMD, for both limited and unrestricted irrigation applications. Reductions for E. coli, total coliforms, and bacteriophages were found to range between 2-3, 1.5-2.5, and 2-4 log10 values, respectively. Salmonella spp. was detected in outlet sewage samples from Patra (PAT), Arachova (ARH), and Livadeia (LEV), at 23% (3/13), 33% (4/12), and 38% (5/13), respectively. Molds were detected at 92.3% (12/13), 100% (13/13), and 91.6% (11/12), respectively, while Candida was found at 85% (11/13), 67% (8/12), and 46% (6/13). A high prevalence of Pseudallescheria boydii, in outlet samples from all studied WWTPs is an important public health issue, which underlines the need for further studies on this emerging fungal pathogen in wastewater reuse applications. Pseudallescheria boydii was found at 85% (11/13), 67% (8/12), and 46% (6/13), respectively. Helminths were found in both inlet and outlet samples, of all studied WWTPs, at 100%. Human adenoviruses, were detected at high percentages in outlet samples at 76.9% (10/13), 92.3% (12/13), 84.6% (11/13), respectively, while no influence of UV irradiation was recorded on the entry and exit loads of human adenoviruses. No influence of meteorological parameters was found on the microbiological and chemical parameters, with the exception of a weak positive correlation between environmental temperature and bacteriophages. A moderate positive correlation was found between BOD and suspended solids, bacteriophages, and total coliforms, bacteriophages and E. coli, and bacteriophages and adenoviruses. A significant positive correlation was found between total coliforms and E. coli, COD and BOD, and suspended solids and COD. No correlations were proved between human pathogens and bacterial indicator parameters. Collectively, our findings underlined the unsuitability of the current JMD on wastewater reuse in Greece, or public health protection. The study is expected to support the development of a public health risk assessment model based on quantitative risk assessment on the use of treated wastewater for irrigation purposes in Greece.

Invasive Candida infections in surgical patients in intensive care units: a prospective, multicentre survey initiated by the European Confederation of Medical Mycology (ECMM) (2006-2008).

A prospective, observational, multicentre study of invasive candidosis (IC) in surgical patients in intensive care units (ICUs) was conducted from 2006 to 2008 in 72 ICUs in 14 European countries. A total of 779 patients (62.5% males, median age 63 years) with IC were included. The median rate of candidaemia was 9 per 1000 admissions. In 10.8% the infection was already present at the time of admission to ICU. Candida albicans accounted for 54% of the isolates, followed by Candida parapsilosis 18.5%, Candida glabrata 13.8%, Candida tropicalis 6%, Candida krusei 2.5%, and other species 5.3%. Infections due to C. krusei (57.9%) and C. glabrata (43.6%) had the highest crude mortality rate. The most common preceding surgery was abdominal (51.5%), followed by thoracic (20%) and neurosurgery (8.2%). Candida glabrata was more often isolated after abdominal surgery in patients ≥60 years, and C. parapsilosis was more often isolated in neurosurgery and multiple trauma patients as well as children ≤1 year of age. The most common first-line treatment was fluconazole (60%), followed by caspofungin (18.7%), liposomal amphotericin B (13%), voriconazole (4.8%) and other drugs (3.5%). Mortality in surgical patients with IC in ICU was 38.8%. Multivariate analysis showed that factors independently associated with mortality were: patient age ≥60 years (hazard ratio (HR) 1.9, p 0.001), central venous catheter (HR 1.8, p 0.05), corticosteroids (HR 1.5, p 0.03), not receiving systemic antifungal treatment for IC (HR 2.8, p <0.0001), and not removing intravascular lines (HR 1.6, p 0.02).

Cluster of Fusarium verticillioides bloodstream infections among immunocompetent patients in an internal medicine department after reconstruction works in Larissa, Central Greece.

BACKGROUND: Fusarium spp. can cause disseminated infections, particularly in immunocompromised patients. Fusarium verticillioides is a human pathogen, and sporadic cases of fusariosis have been reported. AIM: To report a nosocomial cluster of F. verticillioides bloodstream infections among seven immunocompetent inpatients following reconstruction works. METHODS: Identification was performed using macroscopic and microscopic morphology, and molecular assays (sequencing the nuclear ribosomal internal transcribed spacer region and translation elongation factor-1α gene). Susceptibility testing was performed in accordance with the guidelines of the Clinical and Laboratory Standards Institute. Environmental surveillance specimens were taken and cultured on Sabouraud dextrose agar plates. FINDINGS: In total, 16 blood cultures obtained from the seven patients were positive for F. verticillioides. All surveillance cultures were negative. CONCLUSIONS: In order to prevent fungaemia, it is important to implement effective infection control measures, before, during and after demolition and construction activities in healthcare settings.

Efficient identification of Malassezia yeasts by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS).

BACKGROUND: Infections caused by Malassezia yeasts are most likely underdiagnosed, because fatty acid supplementation is needed for growth. Rapid identification of Malassezia species is essential for appropriate treatment of Malassezia-related skin infections, fungaemia and nosocomial outbreaks in neonates, children and adults and can be life-saving for those patients. Ma-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been reported to be a rapid and reliable diagnostic tool to identify clinically important yeasts, but so far no data have been reported on identification of Malassezia isolates with this technique. OBJECTIVES: To create an extensive database of main mass spectra (MSPs) that will allow quick identification of Malassezia species by MALDI-TOF MS. METHODS: An in-house library of 113 MSPs was created from 48 reference strains from the CBS-KNAW yeast collection. The in-house library was challenged with two test sets of Malassezia strains, namely 165 reference strains from the CBS collection and 338 isolates collected in Greece, Italy, Sweden and Thailand. RESULTS: MALDI-TOF MS allowed correct identification of all 14 Malassezia spp. MALDI-TOF MS results were concordant with those of sequence analyses of the internal transcribed spacers (ITS1/ITS2) and the D1/D2 domains of the large subunit of the ribosomal DNA. CONCLUSIONS: Implementation of the MALDI-TOF MS system as a routine identification tool will contribute to correct identification of Malassezia yeasts with minimal effort and in a short turnaround time, which is especially important for the rapid identification of Malassezia in skin diseases and nosocomial outbreaks.

Sequence-based identification, genotyping and EUCAST antifungal susceptibilities of Trichosporon clinical isolates from Greece.

Trichosporon yeasts constitute emerging pathogens, implicated in organ-specific and systemic infections. In this first, comprehensive study of Trichosporon clinical isolates in Greece, 42 isolates were identified by sequencing the hypervariable D1/D2 domain of the Large Subunit (LSU) rDNA gene, while Trichosporon asahii were genotyped by sequencing the Intergenic Spacer 1 region, and antifungal susceptibilities were determined by the EDef 7.2 (EUCAST) method, in parallel with the CLSI standard. Trichosporon asahii was the primary species (37 isolates) followed by Trichosporon coremiiforme, Trichosporon dermatis, Trichosporon loubieri and Trichosporon mycotoxinivorans. One strain remained unidentified. Seven T. asahii genotypes were recorded. The major genotypes were: genotypes 4 (29%) and 3 (26%) followed by 1, 5 and 7 (9.5% each). Two novel genotypes were identified designated as 10 and 11. EUCAST MIC ≥2 mg/L was recorded in 58% of the isolates (amphotericin B), 41% (itraconazole), 41% (posaconazole) and 38% (voriconazole). Fluconazole MICs of ≥32 mg/L were recorded in 23.8% of the isolates. Analysis of variance performed on absolute values showed that the amphotericin B, itraconazole, posaconazole and voriconazole MICs of T. asahii were equivalent. Typically higher MIC values were displayed by fluconazole. Antifungal susceptibilities of the seven different genotypes were homogeneous. Agreements between EUCAST and CLSI ranged from 88.1 to 97.62%. Overall, the high MICs recorded among the Trichosporon isolates for all tested drugs justify routine susceptibility testing of clinical isolates.

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.

Outbreak of cutaneous zygomycosis associated with the use of adhesive tape in haematology patients.

We report an outbreak of cutaneous Rhizopus oryzae infection associated with adhesive polyethylene tapes used to stabilize peripheral venous catheters in four patients. All patients were suffering from haematological diseases; the infection severity was proportional to the duration of neutropenia. Intervention with systemic antifungal treatment and surgical debridement was required for resolution of the infection. The entire batch of tapes was withdrawn and the outbreak subsided.

Could Malassezia yeasts be implicated in skin carcinogenesis through the production of aryl-hydrocarbon receptor ligands?

UNLABELLED: Malassezia yeasts are found on the skin of all humans and many warm-blooded animals. In vitro they have the ability to synthesize potent ligands (indolo[3,2-b]carbazole, malassezin and indirubin) of the aryl-hydrocarbon receptor (AhR; synonym: dioxin receptor) when the sweat contained L-tryptophan is used as the single nitrogen source. The production of these AhR-ligands has been associated with pathogenic strains of a certain Malassezia species (Malassezia furfur) but recent evidence shows that this property is widely distributed in almost all currently known Malassezia species. AhR is associated with carcinogenesis and the potential connection of these ubiquitous skin symbionts, and putative pathogens, with skin neoplasia should be evaluated mainly focusing on mechanisms related to the distinctive ability of the yeast to produce potent AhR ligands. HYPOTHESIS: Synthesis of available pertinent data show a possible link between Malassezia produced AhR ligands and skin carcinogenesis, particularly of basal cell carcinoma (BCC). BCCs are almost exclusively observed in animal species colonized by Malassezia. In humans and animals there is overlapping in the skin regions colonized by this yeast and affected by BCC. The potent AhR ligands synthesized by pathogenic Malassezia strains could contribute to tumor promotion by: modification of the UV radiation carcinogenesis, alterations in the salvage/survival of initiated tumor cells, inhibition of cell senescence, interaction with vitamin D metabolism, promotion of immune tolerance and finally pro-carcinogenic modulation of cell cycle progression and apoptosis.

Extended fungal skin infection due to Aureobasidium pullulans.

Black yeasts are a rare cause of infections especially in Europe, yet their pathological significance is increasing, particularly in cases of immunosuppression. We report a 53-year-old immunocompetent woman with an extensive skin infection due to Aureobasidium pullulans, who responded well to treatment with liposomal amphotericin B.

Intracranial haemorrhage possibly related to Tipranavir in an HIV-1 patient with cryptococcal meningitis.

A 55-year-old HIV-infected patient on antiretroviral treatment with Ritonavir-boosted Tipranavir as part of HAART developed intracranial haemorrhage during the acute phase of cryptococcal meningitis. CT scan and MRI confirmed the intracranial haemorrhage. Positive cryptococcal antigen and cultures of both blood and CSF confirmed the diagnosis of meningitis caused by Cryptococcus neoformans. There was no evidence of any bleeding disorder, use of aspirin or antiplatelet agents. The patient was treated with Liposomal Amphotericin B for cryptococcal meningitis. No special treatment was needed for the intracranial haemorrhage, but Tipranavir was discontinued and replaced by Kaletra and Saquinavir. Intracranial haemorrhage could be related to Tipranavir and cryptococcal meningitis was a predisposing factor. Headache stopped 3 days after starting antifungal treatment. To the best of our knowledge, this is the first reported case of intracranial haemorrhage related to Tipranavir treatment after the end of the "RESIST" studies and the only one related to meningitis.

Diagnosis of common dermatophyte infections by a novel multiplex real-time polymerase chain reaction detection/identification scheme.

BACKGROUND: In the absence of a functional dermatophyte-specific polymerase chain reaction (PCR), current diagnosis of dermatophytoses, which constitute the commonest communicable diseases worldwide, relies on microscopy and culture. This combination of techniques is time-consuming and notoriously low in sensitivity. OBJECTIVES: Recent dermatophyte gene sequence records were used to design a real-time PCR assay for detection and identification of dermatophytes in clinical specimens in less than 24 h. PATIENTS AND METHODS: Two assays based on amplification of ribosomal internal transcribed spacer regions and on the use of probes specific to relevant species and species-complexes were designed, optimised and clinically evaluated. One assay was for detecting the Trichophyton mentagrophytes species complex plus T. tonsurans and T. violaceum. The second assayed for the T. rubrum species complex, Microsporum canis and M. audouinii. RESULTS: The analytical sensitivity of both assays was 0.1 pg DNA per reaction, corresponding to 2.5-3.3 genomes per sample. The protocol was clinically evaluated over 6 months by testing 92 skin, nail and hair specimens from 67 patients with suspected dermatophytosis. Real-time PCR detected and correctly identified the causal agent in specimens from which T. rubrum, T. interdigitale, M. audouinii or T. violaceum grew in culture, and also identified a dermatophyte species in an additional seven specimens that were negative in microscopy and culture. CONCLUSIONS: This highly sensitive assay also proved to have high positive and negative predictive values (95.7% and 100%), facilitating the accurate, rapid diagnosis conducive to targeted rather than empirical therapy for dermatophytoses.

Multicentre determination of quality control strains and quality control ranges for antifungal susceptibility testing of yeasts and filamentous fungi using the methods of the Antifungal Susceptibility Testing Subcommittee of the European Committee on Antimicrobial Susceptibility Testing (AFST-EUCAST).

A multicentre study involving seven laboratories was performed using techniques recommended by the Antifungal Susceptibility Testing Subcommittee of the European Committee on Antimicrobial Susceptibility Testing (AFST-EUCAST) to evaluate and propose quality control ranges and strains for susceptibility testing of fermentative yeasts and filamentous fungi. Participating laboratories tested the susceptibilities of a panel of 12 encoded isolates to amphotericin B, flucytosine, fluconazole, itraconazole, voriconazole and posaconazole. In total, 15 lots of assay medium were tested, with one lot being common to all laboratories, and 18 144 MIC values were determined. Intra- and inter-laboratory agreements and intra-class correlation coefficients (ICCs) of the results for each drug/strain/lot combination were calculated. An average value of 85% agreement was selected for validation purposes. The average percentage of intra-laboratory agreement was 90-95%, with ICC values of 0.90-0.95 (p <0.01). Inter-laboratory reproducibility was also high, with 92% agreement and an ICC of 0.97 (p <0.01). The reproducibility was somewhat better with the common lot of assay medium (96% agreement) than with the different lots (91% agreement), but this difference was not significant. Two isolates that showed trailing growth had agreement percentages below the 85% limit selected for validation purposes and were therefore excluded from the panel of quality control strains. The recommended EUCAST methodologies were found to be highly reproducible and reliable for susceptibility testing of yeasts and filamentous fungi. Ten isolates are proposed for use as quality control strains with these EUCAST procedures.

The role of Candida in inflammatory papillary hyperplasia of the palate.

The objective of this study was to investigate the expression by immunohistochemistry of the major basement membrane (BM) components (laminin, collagen type IV, fibronectin) in specimens from the palatal mucosa lesions of patients with complete dentures and diagnosis of inflammatory papillary hyperplasia of the palate (IPHP). Furthermore to evaluate the potential role of candidal infection in patients with IPHP. Biopsies of palatal mucosa were obtained from patients with IPHP, generally healthy/orally healthy patients with dentures, and healthy subjects. Immunohistochemical studies performed with specific antibodies to BM proteins. Scrapings and swaps of oral lesions from all patients and control groups were taken from the palate, and Candida species colonization was assessed with mycology tests. Immunohistochemical expression of BM components revealed thin linear staining in the BM of healthy palatal mucosa. In IPHP discontinuities or disruptions in BM were observed at the interface between epithelium and the underlying connective tissue in the areas of severe inflammatory response. Our findings suggest an interaction between the expression of BM components and Candida involvement in the development of IPHP, a disorder involving inflammatory reaction and modification of soft tissues.

Candiduria in critically ill patients: an early candidemia marker when coupled with multiple blood specimens PCR assays?

BACKGROUND/AIMS: Can repeated blood PCR assays in critically ill patients with culture-confirmed candiduria help in the early detection of candidemia? METHODOLOGY: Urine samples were obtained on the day of admission and if negative were repeated on the 5th ICU day. Upon laboratory confirmation of candiduria, preemptive antifungal therapy was commenced. Whole blood samples were tested simultaneously with conventional cultures and PCR repeated twice weekly for two weeks. RESULTS: In ICU patients with candiduria, preemptive antifungal therapy resulted in candiduria clearance and clinical improvement in 94% of them. Candida DNAemia (detected in 64 out of 136 repeated PCR assays) persisted for 1 or 2 weeks in a significant number of patients. Even after multiple positive PCR assays from each individual candiduric patient, the specificity and positive predictive value of the assay in the early detection of candidemia were low (negative predictive value was 100%). No differences were detected on demographics among patients with or without candiduria. The correlations (PCR with culture) with kappa test presented a poor agreement (K = 0.103, Kmin= -0679, Kmax = 0.103). CONCLUSIONS: PCR in the blood should not be used in early diagnosis of (but it could help in excluding) candidemia, in ICU patients with candiduria.