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Respiratory tract colonization by molds in patients with cystic fibrosis (CF) were analyzed, with particular focus on the frequency, genotype, and underlying mechanism of azole resistance among Aspergillus fumigatus isolates. Clinical and demographic data were also analyzed. A total of 3,336 respiratory samples from 287 CF patients were collected during two 6-month periods in 2007 and 2009. Azole resistance was detected using an itraconazole screening agar (4 mg/liter) and the EUCAST method. cyp51A gene sequencing and microsatellite genotyping were performed for isolates from patients harboring azole-resistant A. fumigatus. Aspergillus spp. were present in 145 patients (51%), of whom 63 (22%) were persistently colonized. Twelve patients (4%) harbored other molds. Persistently colonized patients were older, provided more samples, and more often had a chronic bacterial infection. Six of 133 patients (4.5%) harbored azole-nonsusceptible or -resistant A. fumigatus isolates, and five of those six patients had isolates with Cyp51A alterations (M220K, tandem repeat [TR]/L98H, TR/L98H-S297T-F495I, M220I-V101F, and Y431C). All six patients were previously exposed to azoles. Genotyping revealed (i) microevolution for A. fumigatus isolates received consecutively over the 2-year period, (ii) susceptible and resistant isolates (not involving TR/L98H isolates) with identical or very closely related genotypes (two patients), and (iii) two related susceptible isolates and a third unrelated resistant isolate with a unique genotype and the TR/L98H resistance combination (one patient). Aspergilli were frequently found in Danish CF patients, with 4.5% of the A. fumigatus isolates being azole nonsusceptible or resistant. Genotyping suggested selection of resistance in the patient as well as resistance being achieved in the environment. more...

A collection of 2,278 isolates belonging to 86 different fungal species was tested with micafungin and eight other drugs using the EUCAST procedures. Micafungin was active against species of Candida and Aspergillus (even azole-resistant species) as well as Penicillium spp., Scedosporium apiospermum, and Acremonium spp. It was inactive for species of Basidiomycota and Mucorales and for multiresistant species such as those of Fusarium. more...

The standardization of antifungal sensitivity tests represents a huge advance in the detection of antifungal drug resistance. Thus, the reference methods of the European Committee on Antibiotic Susceptibility Testing and the Clinical Laboratory Standards Institute have proven capable of detecting strains of yeasts and filamentous fungi with high minimum inhibitory concentrations (MIC) to antifungal agents. This standardization has enabled the genetic alterations responsible for the high MICs to be studied at the molecular level. Furthermore, these strains have been used in experimental models to obtain pharmacokinetic parameters that may allow us to predict clinical response. However, the correlation of the course of the infection in humans with the sensitivity or resistance of the strain is a controversial area with many unanswered questions. We analyze whether the MICs of human pathogenic fungi have clinical relevance, that is, if they affect the course of the infection. more...

We have compared the activities of posaconazole and other currently available antifungal agents against a collection of 3,378 clinical isolates of yeasts and filamentous fungi. A total of 1,997 clinical isolates of Candida spp., 359 of other yeast species, 697 strains of Aspergillus spp., and 325 nondermatophyte non-Aspergillus spp. were included. The average geometric means of the MICs of agents that were tested against Candida spp. were 0.23 microg/ml for amphotericin B, 0.29 microg/ml for flucytosine, 0.97 microg/ml for fluconazole, 0.07 microg/ml for itraconazole, 0.04 microg/ml for voriconazole, 0.15 microg/ml for caspofungin, and 0.03 microg/ml for posaconazole. Voriconazole and posaconazole were active in vitro against the majority of isolates, with resistance to fluconazole and itraconazole, and against Cryptococcus neoformans and other Basidiomycota yeasts. Posaconazole was the most active of antifungal agents tested against Aspergillus spp., with an average geometric mean of 0.10 microg/ml. It was active against Paecilomyces spp., Penicillium spp., Scedosporium apiospermum, and some black fungi, such as Alternaria spp. Multiresistant filamentous fungi, such as Scedosporium prolificans, Scopulariopsis brevicaulis, and Fusarium solani, were also resistant to voriconazole, caspofungin, and posaconazole. Amphotericin B and posaconazole were found to be active against most of the Mucorales strains tested. Posaconazole and currently available antifungal agents exhibit a potent activity in vitro against the majority of pathogenic fungal species. more...

The in vitro activities of ravuconazole against 575 clinical strains of Aspergillus spp. and 348 nondermatophyte non-Aspergillus spp. were analyzed. Ravuconazole was active against Aspergillus spp., other hyaline filamentous fungi, black molds, and some Mucorales. Species such as Scedosporium prolificans, Fusarium spp., and Scopulariopsis spp. were resistant to the triazole. more...

Objectives: The activity in vitro of terbinafine against 442 clinical isolates of several species of filamentous fungi was analysed., Methods: A broth microdilution test was carried out following the National Committee for Clinical Laboratory Standards reference method, with modifications described previously., Results: The geometric mean (GM) of MICs of terbinafine for non-Aspergillus fumigatus species was 0.24 mg/L whereas the GM for A. fumigatus rose as far as 2.92 mg/L. Terbinafine showed a very strong activity in vitro against Penicillium spp., Paecilomyces spp., Trichoderma spp., Acremonium spp. and Arthrographis spp. with GMs <1 mg/L. However, some species such as Scedosporium spp., Fusarium spp., Scopulariopsis brevicaulis, and most of Mucorales exhibited high MICs of the allylamine with GMs >/= 4 mg/L., Conclusions: Overall, the GM of MICs of terbinafine was 1.57 mg/L, but significant differences in susceptibilities were seen between genera and species. more...

The combined activity in vitro of amphotericin B/terbinafine and itraconazole/terbinafine was assessed against 17 clinical isolates of Zygomycota using a checkerboard technique. Itraconazole/terbinafine combination exhibited a potent synergistic effect against the most of strains. Amphotericin B/terbinafine combination showed an indifferent interaction for Rhizopus oryzae, and an additive effect for the other species. more...

In the last years, the incidence of the invasive infection by moulds has increased. Also, the characteristics of invasive mycosis are changing due to the description of new pathogenic species and also because strains and species resistant to antifungal drugs are appearing. The development of mould antifungal drug resistance is the inevitable and logical consequence of their clinical use, although the frequency and clinical relevance of this is unknown. In this text current mould antifungal drugs resistance mechanisms are reviewed. The study of molecular mechanisms of antifungal drug resistance is the most valuable strategy to resistance development control and also in helping to develop safer and more active molecules able to avoid them. In the meanwhile is important the correct use of the available tools: epidemiological surveillance of resistance emergence and to use all the efforts towards prompt diagnosis in order to accomplish an adequate and effective treatment. more...

Cryptococcal meningitis is one of the leading causes of death in HIV/ AIDS patients. Our aim was to in order to characterise the epidemiology, antifungal susceptibility pattern and virulence of 28 Cyptococcus sp. strains recovered from 12 AIDS patients during two years in a Spanish single institution. Antifungal susceptibility testing was performed according to the CLSI protocols. Clinical strains were molecularly characterised by serotyping, mating type, PCR fingerprinting (M13 and GACA4 microsatellites) and analysis of two rDNA regions ( IGS1 and ITS). Sequencing of the ERG11 gene was used to explore mechanisms of fluconazole resistance. Differences in virulence between species were studied in a Galleria mellonella infection model. Cryptococcus deneoformans and C. deneoformans x Cryptococcus neoformans hybrids were the most frequent variety (65%) followed by C. neoformans (35%). Strains were categorised according to 13 microsatellite genotypes and mixed infections could be detected in three patients. Twenty-nine per cent of the strains were fluconazole resistant. In one of the patients, the fluconazole resistance phenotype was associated with a point mutation in the ERG11 gene responsible for the amino acid substitution G470R. C. neoformans strains were able to kill G. mellonella larvae more efficiently than C. deneoformans and hybrids between both species. Precisely molecular characterisation of C. neoformans species is important for an accurate patient's management. [ABSTRACT FROM AUTHOR] more...

Aspergillus fumigatus is a saprotrophic mold fungus ubiquitously found in the environment and is the most common species causing invasive aspergillosis in immunocompromised individuals. For A. fumigatus genotyping, the short tandem repeat method (STRAf) is widely accepted as the first choice. However, difficulties associated with PCR product size and required technology have encouraged the development of novel typing techniques. In this study, a new genotyping method based on hypervariable tandem repeats within exons of surface protein coding genes (TRESP) was designed. A. fumigatus isolates were characterized by PCR amplification and sequencing with a panel of three TRESP encoding genes: cell surface protein A; MP-2 antigenic galactomannan protein; and hypothetical protein with a CFEM domain. The allele sequence repeats of each of the three targets were combined to assign a specific genotype. For the evaluation of this method, 126 unrelated A. fumigatus strains were analyzed and 96 different genotypes were identified, showing a high level of discrimination [Simpson’s index of diversity (D) 0.994]. In addition, 49 azole resistant strains were analyzed identifying 26 genotypes and showing a lower D value (0.890) among them. This value could indicate that these resistant strains are closely related and share a common origin, although more studies are needed to confirm this hypothesis. In summary, a novel genotyping method for A. fumigatus has been developed which is reproducible, easy to perform, highly discriminatory and could be especially useful for studying outbreaks. [ABSTRACT FROM AUTHOR] more...

Objective: To describe a nosocomial outbreak of gastric mucormycosis caused by Rhizopus microsporus var. rhizopodiformis in five adult patients admitted to an intensive care unit (ICU).Design: Epidemiological surveillance study.Setting: A 12-bed polyvalent ICU of an acute care teaching hospital in Pamplona, Spain.Patients: Five patients admitted to the ICU requiring artificial ventilation, diagnosis on admission severe pneumonia in four patients and one polytrauma patient, within a 14-week period, were diagnosed with gastric mucormycosis based on microbiological and/or histopathological characteristics. Upper gastrointestinal bleeding was the presenting manifestation in 80% of patients.Interventions: Filamentous fungi isolated at the microbiology laboratory of the hospital were examined at the national Mycology Reference Laboratory in Madrid.Measurements and Results: Rhizopus microsporus var. rhizopodiformis growth was detected in gastric aspiration samples, environmental samples, wooden tongue depressors used to prepare oral medications (and given to patients through a nasogastric catheter), and in some tongue depressors stored in unopened boxes unexposed to the ICU environment. All depressors were purchased from the same supplier. R. microsporus was not isolated from batches purchased at different times from the same supplier and from another supplier. The outbreak terminated when contaminated tongue depressors were withdrawn from use.Conclusions: Wooden tongue depressors contaminated by R. microsporus var. rhizopodiformis used to prepare oral medications caused an outbreak of fungal gastritis with an attributable mortality of 40%. Wooden material should not be used in the hospital setting. [ABSTRACT FROM AUTHOR] more...