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The biofilm production (BP) of 200 clinical strains of Candida isolated during 2010-2013 were assessed using an in vitro model and a comparison of the results was made between species and between origins of the infections. The BP was assessed using the crystal violet assay, and the strains were classified as low, moderate, or high biofilm producers. Candida tropicalis had the highest values for BP, which varied depending on the origin of the infection. Assessment of BP is a key diagnostic tool that enables us to better understand Candida infections., (Copyright © 2016 Elsevier España, S.L.U. and Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.)

The presence of Clostridium perfringens in water is generally regarded as an indicator of fecal contamination, and exposure to waterborne spores is considered a possible source of infection for animals. We assessed the presence and genetic diversity of C. perfringens in water sources in a zoological park located in Madrid (Spain). A total of 48 water samples from 24 different sources were analyzed, and recovered isolates were toxinotyped, genotyped by fluorophore-enhanced repetitive polymerase chain reaction (rep-PCR) fingerprinting and tested for antimicrobial susceptibility. C. perfringens was recovered from 43.8 % of water samples and 50 % of water sources analyzed. All isolates (n = 70) were type A and 42.9 % were β2-toxigenic (i.e., cpb2+), but none contained the enterotoxin-encoding gene (cpe). Isolates belonged to 15 rep-PCR genotypes and most genetic diversity (88 %) was distributed among isolates obtained from the same sample. Most isolates displayed intermediate susceptibility (57.1 %; MIC = 16 μg ml -1 ) or resistance (5.7 %; MIC ≥ 32 μg ml -1 ) to metronidazole. No resistance to other antimicrobials was detected, although some isolates showed elevated MICs to erythromycin and/or linezolid. Finally, a marginally significant association between absence of cpb2 and decreased susceptibility to metronidazole (MIC ≥ 16 μg ml -1 ) was detected. In conclusion, our results reveal a high prevalence of C. perfringens type A in the studied water reservoirs, which constitutes a health risk for zoo animals. The elevated MICs to metronidazole observed for genetically diverse isolates is a cause of additional concern, but more work is required to clarify the significance of reduced metronidazole susceptibility in environmental strains.

Recent studies suggest that antifungal resistance in yeast isolates of veterinary origin may be an underdiagnosed threat. We tested a collection of 92 ascomycetous yeast isolates that were obtained in Spain from birds, mammals and insects for antifungal susceptibility. MICs to amphotericin B and azoles were low, and no resistant isolates were detected. Despite these results, and given the potential role of animals as reservoirs of resistant strains, continuous monitoring of antifungal susceptibility in the veterinary setting is recommended., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

MALDI-TOF (matrix-assisted laser desorption ionization time-of-flight) mass spectrometry (MS) is becoming an essential tool in most microbiology laboratories. At present, by using a characteristic fungal profile obtained from whole cells or through simple extraction protocols, MALDI-TOF MS allows the identification of pathogenic fungi with a high performance potential. This methodology decreases the laboratory turnaround time, optimizing the detection of mycoses. This article describes the state-of-the-art of the use of MALDI-TOF MS for the detection of human clinical fungal pathogens in the laboratory and discusses the future applications of this technology, which will further improve routine mycological diagnosis., (Copyright © 2016 Elsevier España, S.L.U. All rights reserved.)

We monitored trough voriconazole serum concentrations from 107 patients (n = 258 samples) at 6 hospitals in Madrid. Most of the patients were male (67%) and had the following underlying conditions: hematological cancer (42%), solid organ transplantation (15%), chronic obstructive pulmonary disease (14%), human immunodeficiency virus infection (8.4%), solid cancer (5.6%), and other (29%). The indication for voriconazole administration was aspergillosis treatment (74.6%) and prophylaxis (14%). The main reasons for voriconazole trough drug monitoring were initiation of treatment/prophylaxis (33%), patient monitoring (47%), and suspected toxicity (3.5%). Levels (μg/ml) were subtherapeutic (<1; 18.2%), on-target (1-5.5; 71.3%), and high (>5.5; 10.5%). The samples percentage with on-target levels was significantly lower for the first sample than for subsequent samples (62.6% vs. 77.5%). "Subsequent samples," "admission in nonpediatric wards," "voriconazole used for treatment of invasive aspergillosis," and "use of proton pump inhibitors" were predictors of voriconazole therapeutic levels (≥1 μg/ml)., (© The Author 2016. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Multi-azole resistance acquisition by Candida tropicalis after prolonged antifungal therapy in a dog with urinary candidiasis is reported. Pre- and post-azole treatment isolates were clonally related and had identical silent mutations in the ERG11 gene, but the latter displayed increased azole minimum inhibitory concentrations. A novel frameshift mutation in ERG3 was found in some isolates recovered after resistance development, so it appears unlikely that this mutation is responsible for multi-azole resistance.

A total of 216 colonies of Malassezia pachydermatis from 28 cases of fungal otitis or dermatitis in pets were genotyped by M13 fingerprinting and tested for antifungal susceptibility. A huge genetic diversity was found (157 M13 types in total), with all animals having a polyclonal pattern of infection (5.4 ± 1.5 genotypes/sample). Furthermore, analysis of molecular variance (AMOVA) revealed that most genetic diversity (44%) was found at the within sample level. In contrast, variability in antifungal susceptibility among isolates from the same sample was less important, with different M13 types displaying in most cases identical or very similar MIC results. Most isolates displayed high in vitro susceptibility to amphotericin B, terbinafine and all azoles tested except fluconazole, for which MIC values were always ≥4 μg/ml and a 26.9% of isolates displayed values ≥32 μg/ml. We conclude that although characterization of multiple yeast isolates results in a considerable increase in laboratory workload and expenses, it may help to get a better understanding of the epidemiology of M. pachydermatis in a given patient population., (© The Author 2015. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

In the absence of histopathology studies of lung biopsies, the bronchoalveolar lavage (BAL) sample is preferred for the diagnosis of invasive pulmonary aspergillosis. Isolation of Aspergillus fumigatus from sputum and bronchial secretion samples are commonly interpreted as colonization or laboratory contamination, particularly in nonneutropenic patients. We studied if sputum/bronchial secretions and BAL samples are equally useful for the diagnosis of invasive pulmonary aspergillosis. We retrospectively selected 14 patients with proven (n = 1) or probable (n = 13) invasive pulmonary aspergillosis from whose samples A. fumigatus had been simultaneously isolated in BAL and sputum/bronchial secretions between 2006 and 2012. The isolates were identified by sequencing the β-tubulin gene and genotyped using the STRAf assay. Matches between BAL and sputum/bronchial secretions were observed in patients with identical genotypes in BAL and sputum/bronchial secretions. All patients had clinically suspected pneumonia, before the diagnosis of invasive pulmonary aspergillosis. The sample from which A. fumigatus was initially isolated was collected as a result of the presence of fever (50%), abnormal radiological findings (100%), and/or pneumonia that did not respond to antibiotics (36%). The underlying conditions varied, although the most common predisposing factors were hematological malignancies (21.5%) and COPD (43%). In 13 of the 14 patients (93%), we found matching genotypes in the BAL and the sputum/bronchial secretion samples. Genotyping showed that samples of sputum or bronchial secretions were equally useful as samples of BAL for the diagnosis of invasive pulmonary aspergillosis., (© The Author 2015. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

We studied whether short tandem repeats of Aspergillus fumigatus (STRAf) can differentiate between invasive and colonizing genotypes of A. fumigatus. Of the 395 genotypes detected (n = 1,373 isolates), 50 were clusters and 24 (6% of all genotypes) involved the patients with invasive aspergillosis and those colonized with A. fumigatus, indicating that genotyping cannot discriminate between invasive and colonizing isolates., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

The use of molecular identification techniques has revealed an increasing number of new species within Aspergillus section Terrei. We phenotyped a set of 26 clinical isolates that showed genetic differences from Aspergillus terreus sensu stricto by analyzing sequences from PCR-amplified β-tubulin and calmodulin genes and the internal transcribed spacer region. Since the isolates were phylogenetically and morphologically different from all of the members of Aspergillus section Terrei, they are described here as a new species, Aspergillus citrinoterreus, so named because it produces a diffusible yellowish pigment in agar. A. citrinoterreus isolates were significantly more susceptible to itraconazole, voriconazole, and posaconazole than A. terreus sensu stricto isolates were; in contrast, the amphotericin B MICs for both species were high. A. citrinoterreus was found in clinical samples from patients with proven or probable invasive aspergillosis and colonized patients, none of whom had hematological malignancies as predisposing conditions. However, they did have other underlying conditions such as chronic obstructive pulmonary disease, cirrhosis, and cancer or had received a solid organ transplants and presented not only with invasive pulmonary aspergillosis but also with mediastinitis. A. citrinoterreus isolates were detected for the first time in 2002. In all cases of invasive aspergillosis, A. citrinoterreus was found to be a copathogen, mostly with A. fumigatus., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Many bloodstream infections (BSI) in patients with central venous catheters (CVC) are not catheter-related (CR). Assessment of catheter involvement without catheter withdrawal has not been studied in candidemia. We assessed the value of conservative techniques to evaluate catheters as the origin of candidemia in patients with CVC in a prospective cohort study (superficial Gram stain and culture, Kite technique (Gram stain and culture of the first 1 cm blood drawn from the CVC), proportion of positive blood cultures (PPBCs), differential time to positivity (DTP), and minimal time to positivity (MTP)). All catheters were cultured at withdrawal. From June 2008 to January 2012, 22 cases fulfilled the inclusion criteria. CR-candidemia (CRC) was confirmed in 10. Validity values for predicting CRC were: superficial Gram stain (S, 30%; Sp, 81.83%; PPV, 60%; NPV, 56.3%; Ac, 57.1%), superficial cultures (S, 40%; Sp, 75%; PPV, 57.1%; NPV, 60%; Ac, 59.1%), Kite Gram stain (S, 33.3%; Sp, 66.7%; PPV, 50%; NPV, 50%; Ac, 50%), Kite culture (S, 80%; Sp, 66.7%; PPV, 66.7%; NPV, 80%; Ac, 72.7%), PPBC (S, 50%; Sp, 41.7%; PPV, 41.7%; NPV, 50.0%; Ac, 45.5%), DTP (S, 100%; Sp, 33.3%; PPV, 55.6%; NPV, 100%; Ac, 63.6%), and MTTP (S, 70%; Sp, 58.3%; PPV, 58.3%; NPV, 70%; Ac, 63.6%). While combinations of two tests improved sensitivity and NPV, more than two tests did not improve validity values. Classic tests to assess CR-BSI caused by bacteria cannot be reliably used to diagnose CRC. Combinations of tests could be useful, but more and larger studies are required., (© The Author 2014. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

We determined the in vitro amphotericin B susceptibility of 60 Malassezia pachydermatis isolates by the CLSI broth microdilution method and the Etest using lipid-enriched media. All isolates were susceptible at MICs of ≤ 1 μg/ml, confirming the high activity of amphotericin B against this yeast species. Overall, the essential agreement between the tested methods was high (80% and 96.7% after 48 h and 72 h, respectively), and all discrepancies were regarded as nonsubstantial., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Introduction: Clostridium difficile ribotype 027 (Cd027) has caused outbreaks in the United States, Canada, and Europe since 2001. In Spain, the importance of Cd027 is still unknown. In 2007, we began active surveillance of Cd027 to determine its incidence in our hospital., Methods: From January 2007 to April 2012, isolates of C. difficile by multiplex PCR were studied to detect toxin genes. Binary toxin-positive isolates were characterized using PCR-ribotyping. Cd027 were further characterized by toxino-typing, sequencing of tcdC gene, and MLVA (multilocus-variable-number-tandem-repeat-analysis)., Results: Only 8 strains were Cd027 from 3666 isolates of C. difficile analyzed during the study period. These strains were isolated from 4 patients: a Spanish patient previously hospitalized in the UK, a pregnant laboratory technician, a British tourist, and a Spanish patient without epidemiological antecedents for acquiring Cd027. MLVA typing of Cd027 isolates revealed 4 different patterns. The first patient had 2 episodes of diarrhea caused by different Cd027. The strains from the first episode of patient 1 and the strain from patient 2 were grouped in the same clonal cluster (these cases were previously published as laboratory transmission), while strains from patients 3 and 4 were genetically unrelated to each other, and to the strains from patients 1 and 2., Conclusion: We report the first finding of an autochthonous case of non-severe Cd027 infection. Our results indicate that Cd027 diarrhea is uncommon in our area, and it appears mainly as imported cases. MLVA typing enables us to distinguish different genotypes among our Cd027 isolates., (Copyright © 2013 Elsevier España, S.L. y Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.)

Catheter-related candidemia (CRC) is typically a biofilm related disease, but it is mostly unknown if the production of biofilm is a feature exclusively shown by Candida spp. isolates causing CRC. We performed an in vitro biofilm assay using Candida isolates obtained from the blood of patients with candidemia. We demonstrated that biofilm production was not a good predictor of catheter-related candidemia. Also, we demonstrated that there was no difference in the mortality of candidemia patients infected by biofilm-forming isolates and those in which the infection is caused by nonbiofilm-forming species.

Clostridium difficile is an emerging and potentially zoonotic pathogen, but its prevalence in most animal species, including exhibition animals, is currently unknown. In this study we assessed the prevalence of faecal shedding of C. difficile by zoo animals, and determined the ribotype, toxin profile and antimicrobial susceptibility of recovered isolates. A total of 200 samples from 40 animal species (36.5% of which came from plains zebra, Equus quagga burchellii) were analysed. C. difficile was isolated from 7 samples (3.5% of total), which came from the following animal species: chimpanzee (Pan troglodytes troglodytes), dwarf goat (Capra hircus), and Iberian ibex (Capra pyrenaica hispanica), with one positive sample each; and plains zebra, with 4 positive samples from 3 different individuals. Most recovered isolates (4/7, 57.1%) belonged to the epidemic PCR ribotype 078, produced toxins A and B, and had the genes encoding binary toxin (i.e. A(+)B(+)CDT(+) isolates). The remaining three isolates belonged to PCR ribotypes 039 (A(-)B(-)CDT(-)), 042 (A(+)B(+)CDT(-)) and 110 (A(-)B(+)CDT(-)). Regardless of their ribotype, all isolates displayed high-level resistance to the fluoroquinolones ciprofloxacin, enrofloxacin and levofloxacin. Some isolates were also resistant to meropenem and/or ertapenem. A ribotype 078 isolate recovered from a male zebra foal initially showed in vitro resistance to metronidazole (MIC ≥ 256 μg/ml), but lost that trait after subculturing on non-selective media. We conclude that zoo animals belonging to different species can carry ribotype 078 and other toxigenic strains of C. difficile showing resistance to antimicrobial compounds commonly used in veterinary and/or human medicine., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Previous studies in intensively raised piglets have detected a high prevalence of the epidemic Clostridium difficile PCR ribotype 078. In this article we present a longitudinal survey of C. difficile colonisation in a population of Iberian pigs reared under a free-range system. A total of 160 faecal samples from 20 piglets belonging to different litters were obtained by weekly sampling. C. difficile was recovered from samples collected at different times throughout the survey from a 90% of piglets, resulting in an overall prevalence of 25.6% in the studied samples. Most positive samples (75.6%) came from ≤ 15-day animals, but some piglets shed C. difficile even on day +50. All isolates were ribotype 078, harboured toxin-encoding genes and showed in vitro resistance to several fluoroquinolones. A majority of isolates (80.5%) were also high-level resistant to ertapenem, and four metronidazole heteroresistant isolates (9.8%) were detected. In conclusion, Iberian free-range pigs can be a potential reservoir of epidemic antimicrobial-resistant strains of C. difficile, showing a prevalence rate similar to that found for intensively raised animals., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

We retrospectively studied 22 patients with catheter-related candidemia caused by Candida albicans. Strains isolated simultaneously from blood and catheter tips were genotyped using six microsatellite markers. Matches between genotypes of isolates recovered from both sample sources were found in 20/22 (91%) patients. Consequently, identification of the same species from both the catheter tip and blood could be used to confirm catheter-related candidemia.

Clostridium difficile is an emerging pathogen for humans and animals and there is concern about the possibility that livestock might serve as a reservoir of epidemic strains. In Spain, ribotype 078 is one of the most prevalent in human episodes of C. difficile infection, but the distribution of this and other ribotypes in animals is yet unknown. We present the first report on the ribotype distribution and antimicrobial susceptibility of C. difficile in swine in Spain. A total of 144 isolates were PCR ribotyped, and their MIC values for 13 antimicrobial agents were determined using the Etest. Toxins A and B production was assessed using a commercial immunoassay and, in the case of toxin B, a specific cytotoxicity test. Our results show a high prevalence of the toxigenic 078 ribotype (94.4%) and multidrug resistance (49.3%) among the studied isolates. A minority of isolates (5.6%) belonged to a mostly non-toxinogenic ribotype. All isolates were resistant to the fluoroquinolone ciprofloxacin, but susceptible to daptomycin, linezolid, meropenem, rifampicin, teicoplanin, tigecycline, metronidazole and vancomycin. Resistance to clindamycin, ertapenem, erythromycin and moxifloxacin was common (≥27.8% in all cases). Resistance rates for the different antibiotics tested were in all cases independent from the ribotype of isolates and the host's condition (diarrheic or non-diarrheic), but erythromycin and moxifloxacin resistance was associated with the geographic origin of isolates. Metronidazole heteroresistance was found among animal isolates of C. difficile. Our results highlight the role of livestock as a potential source of epidemic multidrug resistant strains in Spain., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Aspergillus fumigatus complex comprises A. fumigatus and other morphologically indistinguishable cryptic species. We retrospectively studied 362 A. fumigatus complex isolates (353 samples) from 150 patients with proven or probable invasive aspergillosis or aspergilloma (2, 121, and 6 samples, respectively) admitted to the hospital from 1999 to 2011. Isolates were identified using the β-tubulin gene, and only 1 isolate per species found in each sample was selected. Antifungal susceptibility to azoles was determined using the CLSI M38-A2 procedure. Isolates were considered resistant if they showed an MIC above the breakpoints for itraconazole, voriconazole, or posaconazole (>2, >2, or >0.5 μg/ml). Most of the samples yielded only 1 species (A. fumigatus [n = 335], A. novofumigatus [n = 4], A. lentulus [n = 3], A. viridinutans [n = 1], and Neosartorya udagawae [n = 1]). The remaining samples yielded a combination of 2 species. Most of the patients were infected by a single species (A. fumigatus [n = 143] or A. lentulus [n = 2]). The remaining 5 patients were coinfected with multiple A. fumigatus complex species, although A. fumigatus was always involved; 4 of the 5 patients were diagnosed in 2009 or later. Cryptic species were less susceptible than A. fumigatus. The frequency of resistance among A. fumigatus complex and A. fumigatus to itraconazole, voriconazole, and posaconazole was 2.5 and 0.3%, 3.1 and 0.3%, and 4.2 and 1.8%, respectively, in the per-isolate analysis and 1.3 and 0.7%, 2.6 and 0.7%, and 6 and 4% in the per-patient analysis. Only 1 of the 6 A. fumigatus isolates in which the cyp51A gene was sequenced had a mutation at position G448. The proportion of patients infected by azole-resistant A. fumigatus isolates was low.

We report a case of two consecutive episodes of invasive aspergillosis caused by cryptic Aspergillus species in a patient with leukaemia. A first episode of pulmonary infection was caused by Aspergillus calidoustus and Aspergillus novofumigatus, and the second episode by A. novofumigatus and Aspergillus viridinutans. Fungal isolates were identified to species level using traditional and sequencing-based molecular methods.

Diagnosis of invasive aspergillosis (IA) requires increasingly rapid molecular methods that enable sensitive detection and discrimination between species. We designed and evaluated a real-time PCR-based method that combined melting temperature (T(m)) calling analysis of a specific probe with high-resolution melting analysis of the full amplicon. The test correctly identified 78 isolates of Aspergillus section Fumigati and non-Fumigati sections of Aspergillus with a limit of detection of 10(2) conidia/ml (10(2) fg/ml). No cross-reactivity with other fungi was found. The assay was further validated on lower respiratory tract specimens containing Aspergillus or not. It successfully identified Aspergillus to section level in 56 of 59 specimens. With culture as the gold standard, our assay shows 100% sensitivity and specificity and constitutes an efficient alternative for identification of Aspergillus in lower respiratory tract samples.

Clostridium difficile infection (CDI) is one of the main causes of diarrhoea associated with antimicrobial therapy. Antibiotics with good 'in vitro' activity against C. difficile could protect patients from developing CDI. In this study, the potential of linezolid to protect patients with ventilator-associated pneumonia (VAP) from developing CDI was assessed. Over a 4-year period, a cohort of patients who developed VAP following major heart surgery (MHS) in Gregorio Marañón General Hospital (Madrid, Spain) was retrospectively analysed. Patients were divided into those who developed CDI in the post-operative period and those who did not. Variables associated with the development of CDI were analysed, including the role of antimicrobial therapy. Overall, 1934 patients underwent MHS; 90 patients were excluded due to intra-operative or early post-operative (first 48h) death, leaving a study population of 1844 patients, of which 105 cases had VAP. Complete clinical data were available in 91 cases. CDI occurred in 22 patients (24.2%). When comparing VAP cases with and without CDI, EuroSCORE and overall antibiotics prescribed were not significantly different. Patients with chronic renal failure (CRF) were more prone to develop CDI than those without CRF (32% vs. 13%; P=0.04), and patients without [corrected] CDI received more doses of linezolid than those with [corrected] CDI [12.4±9.7 defined daily doses (DDDs) vs. 6.7±4.0 DDDs; P=0.007]. Multivariate analysis confirmed that receiving more DDDs of linezolid protects from developing CDI (hazard ratio=0.908, 95% confidence interval 0.83-0.99; P=0.04). This work is retrospective and addresses a very particular population, but it is the first to suggest the potential impact of linezolid against CDI., (Copyright © 2012 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.)

Secondary resistance to azoles in Aspergillus fumigatus isolates from patients taking long-term itraconazole therapy has been described. We studied the acquisition of secondary azole resistance in 20 A. fumigatus isolates with no mutations at codon 54, 98, 138, 220, 432, or 448 in the cyp51A gene. Adjusted conidium inocula (3 × 10(7) CFU/ml) of each isolate were prepared and progressively or directly exposed to increasing itraconazole concentrations, ranging from 0.5 μg/ml to 16 μg/ml. Itraconazole, voriconazole, and posaconazole MICs were determined using the CLSI M38-A2 procedure before (MIC(initial)) and after (MIC(final)) exposure to itraconazole. In both procedures, the MIC(final) was significantly higher than the MIC(initial). However, after progressive exposure to itraconazole, the MICs of the three azoles were higher than after direct exposure. No mutations were found at codon 54, 98, 138, 220, 432, or 448 in the cyp51A gene of isolates growing at the highest concentration of itraconazole. More concentrated conidium inocula (2 × 10(9) CFU/ml) plated in itraconazole at 4 μg/ml revealed the presence of heteroresistant populations in two initially wild-type isolates. These isolates became resistant to itraconazole and posaconazole only after use of the concentrated inoculum. These heteroresistant isolates harbored a mutation at codon G54, and the MICs of itraconazole and posaconazole were >16 μg/ml. In all procedures, A. fumigatus short tandem repeat (STRAf) typing was used to demonstrate that the genotype did not change before or after exposure to itraconazole.

We recently reported an outbreak of invasive aspergillosis in the major heart surgery unit of Hospital Gregorio Marañón, Madrid, Spain (T. Peláez, P. Muñoz, J. Guinea, M. Valerio, M. Giannella, C. H. W. Klaassen, and E. Bouza, Clin. Infect. Dis., in press). Aspergillus fumigatus was isolated from clinical samples from 10 patients admitted to the unit during the outbreak period (surgical wound invasive aspergillosis, n = 2; probable pulmonary invasive aspergillosis, n = 4; colonization, n = 4). In the study described here, we have studied the genotypic diversity of the A. fumigatus isolates found in the air and clinical samples. We used short tandem repeats of A. fumigatus (STRAf) typing to analyze the genotypes found in the 168 available A. fumigatus isolates collected from the clinical samples (n = 109) from the patients and from the environmental samples taken from the air of the unit (n = 59). The genotypic variability of A. fumigatus was higher in environmental than in clinical samples. Intrasample variability was also higher in environmental than in clinical samples: 2 or more different genotypes were found in 26% and 89% of clinical and environmental samples, respectively. We found matches between environmental and clinical isolates in 3 of the 10 patients: 1 patient with postsurgical invasive aspergillosis and 2 patients with probable pulmonary invasive aspergillosis. A total of 7 genotypes from 3 different patients and the air grouped together in 2 clusters. Clonally related genotypes and microvariants were detected in both clinical and environmental samples. STRAf typing proved to be a valuable tool for identifying the source of invasive aspergillosis outbreaks and for studying the genotypic diversity of clinical and environmental A. fumigatus isolates.

We performed molecular identification of 98 Aspergillus fumigatus complex isolates with MICs of 1 to 4 μg/ml for itraconazole and searched for the presence of mutations in cyp51A. Most of the isolates (91%) belonged to A. fumigatus sensu stricto. We found 14 different mutations in nine isolates at codons different from G54, M220, G138, G448, and L98. We report new mutations at positions 165, 262, 479, and 497 (silent). The role of these mutations should be analyzed.

We studied the serotypes, mating-types, AFLP genotypes, and antifungal susceptibility of 58 Cryptococcus neoformans strains causing 56 episodes of cryptococcosis in 55 patients over an 18-year period in a single institution. The underlying conditions of the patients were classified as HIV infection (n = 48) or non-HIV-related immunodeficiency (n = 7). Serotype A (n = 34; 58.9%) predominated, but serotype AD was involved in 23.2% of episodes. Most of the episodes were caused by mating-type α (n = 41; 73.2%) or α/a strains (n = 12; 21.5%). The most common genotype was AFLP1 (n = 26; 44.8%), followed by AFLP3 (n = 21; 36.2%), and AFLP2 (n = 11; 19.0%). In two different patients, we showed the coexistence of different serotypes and/or genotypes in the same episode (AFLP1 and 3). The new triazoles voriconazole, posaconazole and isavuconazole showed high and similar antifungal activity (MICs ≤ 0.125 μg/ml). Fluconazole also had good antifungal activity, but two strains from patients with HIV-infections had an MIC of 16 μg/ml (3.4%). However, these two isolates remained very susceptible to the new triazoles (MICs ≤ 0.062 μg/ml). The remaining strains always showed MICs ≤ 8 μg/ml.

We compared the in vitro activities of isavuconazole, posaconazole, voriconazole, and fluconazole against Dipodascus capitatus (n = 21), Saccharomyces cerevisiae (n = 20), Rhodotorula mucilaginosa (n = 18), and Trichosporon spp. (n = 15). The MIC(50)s, MIC(90)s, and MIC ranges (in microg/ml) obtained using the CLSI M27-A3 procedure were as follows: isavuconazole, 0.125, 0.5, and < or = 0.015 to 2; posaconazole, 0.5, 2, and < or = 0.015 to > 16; voriconazole, 0.125, 2, and < or = 0.015 to 8; and fluconazole, 4, > 128, and < or = 0.125 to > 128. Isavuconazole showed potent activity against the isolates studied.

We prospectively determined the antifungal susceptibility of yeast isolates causing fungemia using the Etest on direct blood samples (195 prospectively collected and 133 laboratory prepared). We compared the Etest direct (24 h of incubation) with CLSI M27-A3 and the standard Etest methodologies for fluconazole, voriconazole, posaconazole, isavuconazole, caspofungin, and amphotericin B. Strains were classified as susceptible, resistant, or nonsusceptible using CLSI breakpoints (voriconazole breakpoints were used for posaconazole and isavuconazole). Categorical errors between Etest direct and CLSI M27-A3 for azoles were mostly minor. No errors were detected for caspofungin, and high percentages of major errors were detected for amphotericin B. For the azoles, false susceptibility (very major errors) was found in only two (0.6%) isolates (Candida tropicalis and C. glabrata). False resistance (major errors) was detected in 46 (14%) isolates for the three azoles (in 23 [7%] after excluding posaconazole). Etest direct of posaconazole yielded a higher number of major errors than the remaining azoles, especially for C. glabrata, Candida spp., and other yeasts. Excluding C. glabrata, Candida spp., and other yeasts, the remaining species did not yield major errors. Etest direct for fluconazole, voriconazole, isavuconazole, and caspofungin shows potential as an alternative to the CLSI M27-A3 procedure for performing rapid antifungal susceptibility tests on yeast isolates from patients with fungemia. Etest direct is a useful tool to screen for the presence of azole-resistant and caspofungin-nonsusceptible strains.

Objective: Post-surgical invasive aspergillosis (PSIA) is an unusual and underestimated complication of surgery. It may occur after colonization of surgical sites by airborne Aspergillus conidia during surgery, or in the immediate postoperative., Methods: We reviewed 7 cases of PSIA (1997-2006) and checked the air levels of Aspergillus conidia in the operating rooms and/or areas surrounding 5/7 patients., Results: PSIA accounted for 8.4% (n = 83) of all cases of invasive aspergillosis. Patients had no classic predisposing conditions (wound infection (n = 4), mediastinitis (n = 2), and endotipsitis with endocarditis (n = 1)). PSIA occurred sporadically after heart, thoracic, and vascular prosthetic surgery. Aspergillus fumigatus was involved in all cases. Median time from surgery to diagnosis was 25 days. Galactomannan was only positive (> or =1 ng/mL) in 2 patients (endotipsitis with endocarditis and mediastinitis). Mortality was 100% in cases of organ/space post-surgical infections. Although the air of operating rooms taken before surgery was free of Aspergillus, airborne Aspergillus conidia levels were high (>95 CFU/m(3)) in the rooms of 2 patients., Conclusions: PSIA represented almost 10% of all cases of invasive aspergillosis. Our cases were not linked to high levels of Aspergillus conidia in the operating rooms but to postoperative contamination by environmental isolates present in high counts., (Copyright 2009 The British Infection Society. Published by Elsevier Ltd. All rights reserved.)

The fear of candidemia caused by a fluconazole-resistant species of Candida is causing many intensive care units (ICUs) to switch empiric therapy from this drug to broad-spectrum antifungal agents. We studied the epidemiology and antifungal susceptibility of Candida isolates involved in cases of candidemia among adult and pediatric patients in ICUs from 1984 to 2006. We documented 307 episodes of candidemia in 307 patients, of which only eight episodes (2.6%) were caused by a fluconazole-resistant isolate. At least three of the eight patients from whom fluconazole-resistant strains were recovered had recently received fluconazole. Overall, only 1.6% of the episodes of candidemia caused by fluconazole-resistant strains (five patients) occurred in individuals with no evidence of previous fluconazole administration. In conclusion, the use of broad-spectrum antifungal agents is not justified in ICUs with a low proportion of candidemia episodes caused by fluconazole-resistant strains of Candida.

The aim of this study was to investigate the hypotensive effect of the melatonin analogue, N-butanoyl-2-(2-methoxy-6H-isoindolo[2,1-a]indol-11-yl)ethanamine (IIK7), through MT(2)-melatonin receptors and the involvement of the sympathetic nervous system in this action in New Zealand rabbit eyes. The topical application of melatonin or IIK7 produced a reduction in intraocular pressure of 20.2 +/- 5.3% and 38.5 +/- 3.2% respectively. This effect was concentration-dependent; it was blocked by selective MT(2) receptor antagonists and was severely diminished after chemical sympathectomy. Immunohistochemistry and western blot analysis showed the ciliary processes as the site of this action and no co-localization of MT(2)-melatonin receptor with the sympathetic nervous system was observed. The beta-adrenergic agonists, terbutaline and salbutamol, potentiated the hypotensive effect of IIK7 reducing intraocular pressure (IOP) 41.75 +/- 4.26% and 44.7 +/- 5.6% respectively. Also, IIK7 in presence of the nonspecific protein phosphatase inhibitor okadaic acid, lowered IOP 32.2 +/- 4.5% and in presence of forskolin plus 3-isobutyl-1-methylxanthine decreased IOP in 32.2 +/- 5.47%. These data suggest that the melatonin agonist IIK7 reduces intraocular pressure by acting through MT(2)-melatonin receptors presumably decreasing aqueous humour formation. Also, in the presence of beta-adrenoceptor agonists MT(2)-melatonin receptors activity increase their ability to reduce IOP.

We studied 75 patients with non-hematological conditions from whom Aspergillus spp. were recovered from clinical specimens during the period March 2003 to August 2006. The patients were classified according to EORTC criteria and the presence of galactomannan (Platelia Aspergillus) in their sera was evaluated. Ten of these patients (13.3%) had proven or probable invasive aspergillosis, i.e., chronic obstructive pulmonary disease in five (50%), HIV infection in one (10%), lymphoma in one (10%), liver transplant in one (10%), solid malignancies in one (10%), and corticosteroid treatment in one (10%). The sensitivity, specificity, and positive and negative predictive values for the detection of galactomannan, using cut-offs of > or =0.5 ng/ml and > or =1 ng/ml were 60%/50%, 89.23%/100%, 46.15%/100%, and 93.55%/92.86% (p=0.001 and p<0.001), respectively. The determination of galactomannan in the sera of non-neutropenic patients could prove to be a useful microbiological finding when diagnosing invasive aspergillosis.

We studied the activity of voriconazole against 400 clinical strains of Aspergillus from the pre-voriconazole (1999 to 2002) and post-voriconazole (2003 to 2007) periods. Although the mean MICs of strains from the post-voriconazole period were slightly higher (0.39 versus 0.57 microg/ml; P < 0.001), all strains were susceptible to voriconazole and presented an MIC of

Isavuconazole (BAL4815) is a promising novel broad-spectrum triazole in late-stage clinical development that has proven active in vitro against Aspergillus and Candida species. We compared the in vitro activities of this agent with those of voriconazole and fluconazole by the CLSI (formerly NCCLS) M38-A and M27-A2 procedures against a large collection of 1,007 relevant opportunistic fungi collected from 1986 to 2007: Aspergillus spp. (n = 702), Candida spp. (n = 218), Zygomycetes (n = 45), Scedosporium spp. (n = 22), and Fusarium spp. (n = 20). All Candida isolates were from patients with candidemia. For isavuconazole, these techniques were also compared with the Etest. Isavuconazole and voriconazole had MICs at which 50% and 90% of isolates were inhibited (MIC50 and MIC90), respectively, of 1 and 1 microg/ml and 0.5 and 1 microg/ml against Aspergillus spp. and of < or = 0.015 [corrected] and 0.03 microg/ml and 0.25 and 0.125 microg/ml against Candida spp. (including fluconazole-resistant strains). The MIC50 partial and complete inhibition end points of isavuconazole and voriconazole against the non-Aspergillus molds were as follows: 1 and 2 microg/ml and 16 and >16 mug/ml against Zygomycetes; 1 and 4 microg/ml and 0.25 and 0.5 microg/ml against Scedosporium apiospermum; 4 to 16 and >16 microg/ml and 4 to 8 and 16 to >16 microg/ml (ranges) against Scedosporium prolificans; and 16 and 16 microg/ml and 4 and 4 microg/ml against Fusarium spp. Isavuconazole showed minimal fungicidal concentrations for 50% and 90% of the isolates of 1 and 1 microg/ml against Aspergillus, 16 and >16 microg/ml against Candida, and 4 and >16 microg/ml against Zygomycetes, respectively, and >16 microg/ml against the remaining molds. The Etest proved to be a suitable alternative method for determining the antifungal activities of isavuconazole against Aspergillus and Candida; the Etest results showed 96% and 93% agreement with the results of the CLSI M38-A and M27-A2 methods, respectively.

Background: Fungemia has been historically considered to be a disease caused by a single Candida species; the detection of >1 species of yeast in circulating blood was distinctly uncommon using traditional microbiological procedures. We describe episodes of mixed fungemia (MF), detected between 1985 and 2006, in a large teaching hospital., Methods: The study was divided into 2 periods that were separated by the introduction, in January 2005, of the CHROmagar Candida medium (CHROMagar) for the routine subculturing of blood cultures in which yeast has been identified. Overall, we documented 747 cases of fungemia. During the first period (1985-1994), we identified 217 episodes of fungemia and no single episode of MF; during the second period (1995-2006), 15 episodes of MF were detected among 530 episodes of fungemia (2.8%). Candida albicans was isolated in 13 patients, non-albicans species of Candida in 16 patients, and Saccharomyces cerevisiae in 1 patient. Each episode of MF was compared with 2 control episodes of monomicrobial fungemia., Results: Patients with MF had more frequently experienced organ transplantation (13% vs. 0%) and surgery (60% vs. 27%), had less frequently received parenteral nutrition (40% vs. 70%) or had intravenous lines (80% vs. 100%), and had a lower incidence of shock (6% vs. 37%) and a lower mortality (20% vs. 53%)., Conclusions: Despite the introduction of chromogenic agar, MF is still an uncommon disease and has a less severe outcome than does monomicrobial candidemia.

We evaluated the activities of amphotericin B, itraconazole, voriconazole, caspofungin, and posaconazole against zygomycetes by CLSI M38-A, Etest and Sensititre. The most active drug was posaconazole, followed by amphotericin B and itraconazole. The correlation of the Etest and Sensititre with CLSI M38-A was moderate for posaconazole but poor for the others.

The in vitro activities of amphotericin B, itraconazole, and voriconazole against 283 clinical isolates of Aspergillus fumigatus were studied by comparing the E test method with the reference procedure NCCLS (CLSI) M-38A. The methods were considered to agree when the results of the MICs by E test were within +/-2 dilutions of the MICs obtained by CLSI M-38 A. Agreement of readings at 24, 48, and 72 h of incubation was as follows: amphotericin B: 4.2%, 98%, and 40.6%; itraconazole: 92.5%, 100%, and 89.9%; and voriconazole: 98.9%, 100%, and 99.7%. The correlation between methods to classify the strains as "susceptible" or "resistant" was very good for itraconazole and voriconazole after 48 h of incubation, but 23% of the strains were incorrectly classified by E test for amphotericin B. The E test is an efficacious method for antifungal susceptibility testing in A. fumigatus for itraconazole and voriconazole when the plates are read after 48 h of incubation. The use of the E test to study the activity of amphotericin B should be avoided.

The in vitro activities of amphotericin B, itraconazole, and voriconazole against 279 clinical isolates of Aspergillus fumigatus were studied by comparing Sensititre YeastOne with the reference method NCCLS (CLSI) M38-A. The methods were considered to agree when the results of the MICs by Sensititre were within 2 dilutions of the MICs obtained by NCCLS M38-A. Agreement of readings at 24, 48, and 72 h was as follows: amphotericin B (0%, 31.5%, 82.1%), itraconazole (86.7%, 98.3%, 98.2%), and voriconazole (92.8%, 98.3%, 98.9%). Sensititre YeastOne is a suitable alternative for the determination of the MICs of itraconazole and voriconazole against A. fumigatus when results are read at 48 h of incubation.

Unfortunately, little information is available on the 'normal' air and water load of Aspergillus spores and their seasonal changes. We describe the air and water load of Aspergillus spores across the province of Madrid (Spain). We collected samples of air and water (332 samples of air and 148 of water) from selected points and taps (urban and rural environment) in summer, autumn, winter and spring. Temperature, wind speed and humidity at each point were obtained. We collected a total of 369 Aspergillus spp. isolates: 200 A. fumigatus, 94 A. niger, 40 A. flavus, 16 A. nidulans, two A. terreus, and 17 Aspergillus spp. We always found more isolates in the urban environment than in the rural environment (P=0.11). This was also true of A. fumigatus (P=0.014). The autumn collection yielded more isolates than that of the other seasons. The level of conidia in air varied from 0 to 85 c.f.u./m3: 6.4 c.f.u./m3 in summer, 12 in autumn, 2.5 in winter and 1.3 in spring. Changes in atmospheric parameters influenced the levels in air. None of the water samples were positive. Counts of Aspergillus spp. spores were always under 85 c.f.u./m3. Public water does not seem to contain Aspergillus spores.

We present two cases of typical psychosis related to methadone withdrawal. The two cases, appearing in the adult age, are not related to previous personal or familiar psychiatric illness and do not show the classical symptoms of methadone withdrawal. The appearance of this type of case is an experience repeated in different groups of researchers. In the line of previous investigators, we propose an alteration of neuromodulation in central opiate-dopamine system.

We compared the efficiency of Czapeck agar and Sabouraud dextrose agar irradiated to detect Aspergillus conidia in air. Both media were equally suitable. Czapeck agar should be used in institutions interested only in Aspergillus surveys, and Sabouraud agar should be used in those institutions interested in all sorts of filamentous fungi.

Background: Saccharomyces cerevisiae is well known in the baking and brewing industry and is also used as a probiotic in humans. However, it is a very uncommon cause of infection in humans., Methods: During the period of 15-30 April 2003, we found 3 patients with S. cerevisiae fungemia in an intensive care unit (ICU). An epidemiological study was performed, and the medical records for all patients who were in the unit during the second half of April were assessed., Results: The only identified risk factor for S. cerevisiae infection was treatment with a probiotic containing Saccharomyces boulardii (Ultralevura; Bristol-Myers Squibb). This probiotic is used in Europe for the treatment and prevention of Clostridium difficile-associated diarrhea. The 3 patients received the product via nasograstric tube for a mean duration of 8.5 days before the culture result was positive, whereas only 2 of 41 control subjects had received it. Surveillance cultures for the control patients admitted at the same time did not reveal any carriers of the yeast. Strains from the probiotic capsules and the clinical isolates were identified as S. cerevisiae, with identical DNA fingerprinting. Discontinuation of use of the product in the unit stopped the outbreak of infection. A review of the literature identified another 57 cases of S. cerevisiae fungemia. Overall, 60% of these patients were in the ICU, and 71% were receiving enteral or parenteral nutrition. Use of probiotics was detected in 26 patients, and 17 patients died., Conclusions: Use of S. cerevisiae probiotics should be carefully reassessed, particularly in immunosuppressed or critically ill patients.