Fungal Keratitis - Improving Diagnostics by Confocal Microscopy

Purpose: Introducing a simple image grading system to support the interpretation of in vivo confocal microscopy (IVCM) images in filamentous fungal keratitis. Setting: Clinical and confocal studies took place at the Department of Ophthalmology, Aarhus University Hospital, Denmark. Histopathological analysis was performed at the Eye Pathology Institute, Department of Neuroscience and Pharmacology, University of Copenhagen, Denmark. Methods: A recent series of consecutive patients with filamentous fungal keratitis is presented to demonstrate the results from in-house IVCM. Based upon our experience with IVCM and previously published images, we composed a grading system for interpreting IVCM images of filamentous fungal keratitis. Results: A recent case series of filamentous fungal keratitis from 2011 to 2012 was examined. There were 3 male and 3 female patients. Mean age was 44.5 years (range 12-69), 6 out of 17 (35%) cultures were positive and a total of 6/7 (86%) IVCM scans were positive. Three different categories of IVCM results for the grading of diagnostic certainty were formed. Conclusion: IVCM is a valuable tool for diagnosing filamentous fungal keratitis. In order to improve the reliability of IVCM, we suggest implementing a simple and clinically applicable grading system for aiding the interpretation of IVCM images of filamentous fungal keratitis

Development of azole resistance in Aspergillus fumigatus during azole therapy associated with change in virulence

Contains fulltext : 88544.pdf (publisher's version ) (Open Access)Four sequential Aspergillus fumigatus isolates from a patient with chronic granulomatous disease (CGD) eventually failing azole-echinocandin combination therapy were investigated. The first two isolates (1 and 2) were susceptible to antifungal azoles, but increased itraconazole, voriconazole and posaconazole MICs were found for the last two isolates (3 and 4). Microsatellite typing showed that the 4 isolates were isogenic, suggesting that resistance had been acquired during azole treatment of the patient. An immunocompromised mouse model confirmed that the in vitro resistance corresponded with treatment failure. Mice challenged with the resistant isolate 4 failed to respond to posaconazole therapy, while those infected by susceptible isolate 2 responded. Posaconazole-anidulafungin combination therapy was effective in mice challenged with isolate 4. No mutations were found in the Cyp51A gene of the four isolates. However, expression experiments of the Cyp51A showed that the expression was increased in the resistant isolates, compared to the azole-susceptible isolates. The microscopic morphology of the four isolates was similar, but a clear alteration in radial growth and a significantly reduced growth rate of the resistant isolates on solid and in broth medium was observed compared to isolates 1 and 2 and to unrelated wild-type controls. In the mouse model the virulence of isolates 3 and 4 was reduced compared to the susceptible ones and to wild-type controls. For the first time, the acquisition of azole resistance despite azole-echinocandin combination therapy is described in a CGD patient and the resistance demonstrated to be directly associated with significant change of virulence

Environmental Study of Azole-Resistant Aspergillus fumigatus and Other Aspergilli in Austria, Denmark, and Spain ▿

A single mechanism of azole resistance was shown to predominate in clinical and environmental Aspergillus fumigatus isolates from the Netherlands, and a link to the use of azoles in the environment was suggested. To explore the prevalence of azole-resistant A. fumigatus and other aspergilli in the environment in other European countries, we collected samples from the surroundings of hospitals in Copenhagen, Innsbruck, and Madrid, flowerbeds in an amusement park in Copenhagen, and compost bags purchased in Austria, Denmark, and Spain and screened for azole resistance using multidish agars with itraconazole, voriconazole, and posaconazole. EUCAST method E.DEF 9.1 was used to confirm azole resistance. The promoter and entire coding sequence of the cyp51A gene were sequenced to identify azole-resistant A. fumigatus isolates. A. fumigatus was recovered in 144 out of 185 samples (77.8%). Four A. fumigatus isolates from four Danish soil samples displayed elevated azole MICs (8%), and all harbored the same TR/L98H mutation of cyp51A. One A. lentulus isolate with voriconazole MIC of 4 mg/liter was detected in Spain. No azole-resistant aspergilli were detected in compost. Finally, A. terreus was present in seven samples from Austria. Multi-azole-resistant A. fumigatus is present in the environment in Denmark. The resistance mechanism is identical to that of environmental isolates in the Netherlands. No link to commercial compost could be detected. In Spain and Austria, only Aspergillus species with intrinsic resistance to either azoles or amphotericin B were found

Aspergillus species and other molds in respiratory samples from patients with cystic fibrosis: A laboratory-based study with focus on Aspergillus fumigatus azole resistance

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

Breakthrough <em>Aspergillus fumigatus </em>and <em>Candida albicans</em> double infection during caspofungin treatment:laboratory characteristics and implication for susceptibility testing

Caspofungin is used for the treatment of acute invasive candidiasis and as salvage treatment for invasive aspergillosis. We report characteristics of isolates of Candida albicans and Aspergillus fumigatus detected in a patient with breakthrough infection complicating severe gastrointestinal surgery and evaluate the capability of susceptibility methods to identify candin resistance. The susceptibility of C. albicans to caspofungin and anidulafungin was investigated by Etest, microdilution (European Committee on Antibiotic Susceptibility Testing [EUCAST] and CLSI), disk diffusion, agar dilution, and FKS1 sequencing and in a mouse model. Tissue was examined by immunohistochemistry, PCR, and sequencing for the presence of A. fumigatus and resistance mutations. The MICs for the C. albicans isolate were as follows: >32 μg/ml caspofungin and 0.5 μg/ml anidulafungin by Etest, 2 μg/ml caspofungin and 0.125 μg/ml anidulafungin by EUCAST methods, and 1 μg/ml caspofungin and 0.5 μg/ml anidulafungin by CLSI methods. Sequencing of the FKS1 gene revealed a mutation leading to an S645P substitution. Caspofungin and anidulafungin failed to reduce kidney CFU counts in animals inoculated with this isolate (P > 0.05 compared to untreated control animals), while both candins completely sterilized the kidneys in animals infected with a control isolate. Disk diffusion and agar dilution methods clearly separated the two isolates. Immunohistochemistry and sequencing confirmed the presence of A. fumigatus without FSK1 resistance mutations in liver and lung tissues. Breakthrough disseminated aspergillosis and candidiasis developed despite an absence of characteristic FKS1 resistance mutations in the Aspergillus isolates. EUCAST and CLSI methodology did not separate the candin-resistant clinical isolate from the sensitive control isolate as well as did the Etest and agar methods

<i>CYP51A</i> mRNA levels in the 4 sequential <i>A. fumigatus</i> isolates and in a control wild type isolate AZN 8196 (WT) and a tri-azole resistant control isolate with over-expression of the CYP51A gene due to the L98H mutation and promoter tandem repeat.

<p><i>CYP51A</i> mRNA levels in the 4 sequential <i>A. fumigatus</i> isolates and in a control wild type isolate AZN 8196 (WT) and a tri-azole resistant control isolate with over-expression of the CYP51A gene due to the L98H mutation and promoter tandem repeat.</p

Growth rate of the 4 sequential isolates expressed as radial growth rate on solid V8 agar (a) and as optical densities in fluent medium (b).

<p>The laboratory strain NCP2109 is included as unrelated comparator. In the radial growth experiment (a) the growth rate is expressed in mean diameter (mm) and Kr (mm/hour) after 24–72 hours of two separate experiments performed in triplicate. In the kinetic evaluation of growth over time in liquid medium (b) growth is expressed as optical densities measured every 10 min.</p