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DNA extraction from formalin-fixed paraffin-embedded (FFPE) tissues is difficult and requires special protocols in order to extract small amounts of DNA suitable for amplification. Most described methods report an amplification success rate between 60 and 80%; therefore, there is a need to improve molecular detection and identification of fungi in FFPE tissue. Eighty-one archived FFPE tissues with a positive Gomori methenamine silver (GMS) stain were evaluated using five different commercial DNA extraction kits with some modifications. Three different panfungal PCR assays were used to detect fungal DNA, and two housekeeping genes were used to assess the presence of amplifiable DNA and to detect PCR inhibitors. The sensitivities of the five extraction protocols were compared, and the quality of DNA detection (calculated for each kit as the number of housekeeping gene PCR-positive samples divided by the total number of samples) was 60 to 91% among the five protocols. The efficiencies of the three different panfungals used (calculated as the number of panfungal-PCR-positive samples divided by the number of housekeeping gene PCR-positive samples) were 58 to 93%. The panfungal PCR using internal transcribed spacer 3 (ITS3) and ITS4 primers yielded a product in most FFPE tissues. Two of the five DNA extraction kits (from TaKaRa and Qiagen) showed similar and promising results. However, one method (TaKaRa) could extract fungal DNA from 69 of the 74 FFPE tissues from which a housekeeping gene could be amplified and was also cost-effective, with a nonlaborious protocol. Factors such as sensitivity, cost, and labor will help guide the selection of the most appropriate method for the needs of each laboratory.

RATIONALE: The importance of Aspergillus fumigatus sensitisation and colonisation of the airways in patients with asthma is unclear.

OBJECTIVES: To define the relationship between the clinical and laboratory features of A. fumigatus-associated asthma.

METHODS: We studied 79 patients with asthma (89% classed as GINA 4 or 5) classified into 3 groups according to A. fumigatus sensitisation: (1) IgE-sensitised (immediate cutaneous reactivity >3 mm and/or IgE >0.35 kU/L); (2) IgG-only-sensitised (IgG >40 mg/L); and (3) non-sensitised. These were compared to 14 healthy controls. Sputum culture was focused towards detection of A. fumigatus and compared with clinical assessment data.

MEASUREMENTS AND MAIN RESULTS: A. fumigatus was cultured from 63% of IgE-sensitised asthmatics (n=40), 39% of IgG-only-sensitised asthmatics (n=13), 31% of non-sensitised asthmatics (n=26) and 7% of healthy controls (n=14). Patients sensitised to A. fumigatus compared with non-sensitised asthmatics had lower lung function (% predicted, post-bronchodilator FEV1 68% (+/-5) vs 88% (5) p < 0.05), more bronchiectasis (68% versus 35% p < 0.05) and more sputum neutrophils (80.9% (50.1-94.1) vs 49.5% (21.2-71.4) p < 0.01). In a multilinear regression model A. fumigatus-IgE sensitisation and sputum neutrophil differential cell count were important predictors of lung function (p=0.016), supported by culture of A. fumigatus (p=0.046) and eosinophil differential cell count (p=0.024).

CONCLUSIONS: A. fumigatus detection in sputum is associated with A. fumigatus-IgE sensitisation, neutrophilic airway inflammation and reduced lung function. This supports the concept that development of fixed airflow obstruction in asthma is consequent upon the damaging effects of airway colonisation with A. fumigatus.

Allergic bronchopulmonary aspergillosis (ABPA) is caused by a dominant Th2 immune response to antigens derived from the opportunistic mold Aspergillus, most commonly Aspergillus fumigatus. It occurs in 4%-15% of patients with cystic fibrosis (CF); however, not all patients with CF infected with A. fumigatus develop ABPA. Therefore, we compared cohorts of A. fumigatus-colonized CF patients with and without ABPA to identify factors mediating tolerance versus sensitization. We found that the costimulatory molecule OX40 ligand (OX40L) was critical in driving Th2 responses to A. fumigatus in peripheral CD4+ T cells isolated from patients with ABPA. In contrast, CD4+ T cells from the non-ABPA cohort did not mount enhanced Th2 responses in vitro and contained a higher frequency of TGF-beta-expressing regulatory T cells. Heightened Th2 reactivity in the ABPA cohort correlated with lower mean serum vitamin D levels. Further, in vitro addition of 1,25 OH-vitamin D3 substantially reduced DC expression of OX40L and increased DC expression of TGF-beta. This in vitro treatment also resulted in increased Treg TGF-beta expression and reduced Th2 responses by CD4+ T cells from patients with ABPA. These data provide rationale for a therapeutic trial of vitamin D to prevent or treat ABPA in patients with CF.

The Aspergillus nidulans endocytic internalization protein SlaB is essential, in agreement with the key role in apical extension attributed to endocytosis. We constructed, by gene replacement, a nitrate-inducible, ammonium-repressible slaB1 allele for conditional SlaB expression. Video-microscopy showed that repressed slaB1 cells are able to establish but unable to maintain a stable polarity axis, arresting growth with budding yeast-like morphology shortly after initially normal germ-tube emergence. Using GFP-tagged secretory v-SNARE SynA, which continuously recycles to the plasma membrane after being efficiently endocytosed, we establish that SlaB is crucial for endocytosis, although it is dispensable for the anterograde traffic of SynA and of the t-SNARE Pep12 to the plasma and vacuolar membrane, respectively. By confocal microscopy, repressed slaB1 germlings show deep plasma membrane invaginations. Ammonium-to-nitrate medium-shift experiments demonstrated reversibility of the null polarity maintenance phenotype and correlation of normal apical extension with resumption of SynA endocytosis. In contrast, SlaB downregulation in hyphae that had progressed far beyond germ-tube emergence led to marked polarity maintenance defects correlating with deficient SynA endocytosis. Thus, the strict correlation between abolishment of endocytosis and disability of polarity maintenance that we report here supports the view that hyphal growth requires coupling of secretion and endocytosis. However, downregulated slaB1 cells form F-actin clumps containing the actin-binding protein AbpA and thus F-actin misregulation cannot be completely disregarded as a possible contributor to defective apical extension. Latrunculin B treatment of SlaB-downregulated tips reduced the formation of AbpA clumps without promoting growth and revealed the formation of cortical 'comets' of AbpA.

Early recognition and rapid initiation of effective treatment is a prerequisite for successful management of children with invasive fungal infections. The increasing diversity of fungal pathogens in high-risk patients, the differences in the antifungal spectrum of available agents, and the increasing rates of resistance call for identification of the infecting isolate at the species level and for information on drug resistance, in order to provide state of the art patient care. Microscopy and culture of appropriate specimens remain the gold standard of mycological diagnosis, despite difficulties in obtaining appropriate and/or sufficient specimens, long time of culturing and false-negative results. Modern imaging studies, detection of circulating fungal cell wall components and DNA in blood and other body fluids or in affected tissues may improve the laboratory diagnosis of invasive mycoses.

Caspofungin was the first in a new class of antifungal agents (echinocandins) indicated for the treatment of primary and refractory fungal infections. Higher doses of caspofungin may provide another option for patients who have failed caspofungin or other antifungal therapy. This study evaluated the safety, tolerability, and pharmacokinetics of single 150- and 210-mg doses of caspofungin in 16 healthy participants and 100 mg/d for 21 days in 20 healthy participants. Other than infusion site reactions and 1 reversible elevation in alanine aminotransferase (>/=2x and <4x upper limit of normal), caspofungin was generally well tolerated. Geometric mean AUC0-infinity after single 150- and 210-mg doses was 279.7 and 374.9 mug.h/mL, respectively; peak concentrations were 29.4 and 33.5 mug/mL, respectively; and 24-hour postdose concentrations were 2.8 and 4.2 mug/mL, respectively. Steady state was achieved in the third week of dosing. Following multiple 100-mg doses of caspofungin, day 21 geometric mean AUC0-24 was 227.4 mug.h/mL, peak concentration was 20.9 mug/mL, and trough concentration was 4.7 mug/mL. Beta-phase t(1/2) was approximately 8 to approximately 13 hours. Caspofungin pharmacokinetics at these higher doses were dose proportional to and consistent with those observed at lower doses, suggesting a modest nonlinearity of increased accumulation with dose, which was considered not clinically meaningful.

Objectives: To characterize the pharmacokinetics and bioavailability of voriconazole in adult lung transplant patients during early post-operative period, identify factors significantly associated with various pharmacokinetic parameters, and make recommendations for adequate dosing regimens. Methods: Thirteen lung transplant patients received two intravenous infusions (6mg/kg, bid) immediately post-transplant followed by oral doses (200mg, bid) for prophylaxis. Blood samples (n=9/interval) were collected during one intravenous and one oral dosing interval from each patient. Voriconazole plasma concentrations were measured by HPLC. NONMEM was used to develop pharmacokinetic models, evaluate covariate relationships and perform Monte Carlo simulations. Results: There was a good correlation (R(2)=0.98) between AUCo-infinity and trough concentrations. A two-compartment model adequately described the data. Population estimates of bioavailability, clearance, Vc and Vp were 45.9%, 3.45L/hr, 54.7L and 143L. Cystic fibrosis (CF) patients exhibited a significantly lower bioavailability (23.7%, n=3) than non-CF patients (63.3%, n=10). Bioavailability increased with post-operative time and reached steady levels in about one week. Vp increased with body weight. Conclusions: Bioavailability of voriconazole is substantially lower in lung transplant patients than non-transplant subjects, but significantly increases with post-operative time. CF patients exhibit significantly lower bioavailability and exposure of voriconazole, and therefore need higher doses. Intravenous administration of voriconazole during the first post-operative day followed by oral doses of 200mg or 400mg appeared to be the optimal dosing regimen. However, voriconazole levels should be monitored and the dose can be individualized based on trough concentrations as a good measure of drug exposure.

Eisosomes are sub-cortical organelles hitherto described only in Saccharomyces cerevisiae as sites implicated in endocytosis. They comprise two homologue proteins, Pil1 and Lsp1, which colocalize with the transmembrane protein Sur7. These proteins are universally conserved in the ascomycetes. We identify in Aspergillus nidulans (and in all the Pezizomycotina) two homologues of Pil1/Lsp1, PilA and PilB, originating from a duplication independent from that extant in the Saccharomycotina. In the Aspergilli there are several Sur7-like proteins in each species, including one strict Sur7orthologue (SurG in A. nidulans). In A. nidulans conidiospores, but not in hyphae, the three proteins colocalize at the cell cortex and form tightly packed punctate structures that appear different from the clearly distinct eisosome patches observed in S. cerevisiae. These structures are assembled late during the maturation of conidia. In mycelia, punctate structures are present, but they are composed only of PilA, while PilB is diffused in the cytoplasm and SurG is located in vacuoles and endosomes. Deletion of each of the genes does not lead to any obvious growth phenotype, except for a moderate resistance to itraconazole. We could not find any obvious association between mycelial (PilA) eisosome-like structures and endocytosis. PilA and SurG are necessary for conidial eisosome organisation in ways that differ from their S. cerevisiae homologues. These data illustrate that conservation of eisosomal proteins within the ascomycetes is accompanied by a striking functional divergence.

Objectives: Resistance to azole antifungal drugs in Aspergillus fumigatus is now a major clinical problem in some locations. Here we update our previous experience with data from 2008–09.

Methods: We tested all A. fumigatus isolates submitted to the Mycology Reference Centre Manchester in 2008 and 2009 for susceptibility to itraconazole, voriconazole and posaconazole. We undertook CYP51A sequencing for most of the azole-resistant isolates.

Results: Of 230 isolates, 64 (28%) were azole resistant. In 2008 and 2009, 14% and 20% of patients had resistant isolates, respectively. During this period 62 of 64 (97%) were itraconazole resistant, 2 of 64 (3%) were only voriconazole resistant and 78% of cases were multi-azole resistant. Forty-three percent of isolates did not carry a cyp51A mutation (previously the most common azole resistance mechanism), indicating that other mechanisms must be responsible and are increasing in frequency.

Conclusions: Azole resistance is evolving and growing in frequency. Established and novel mechanisms may be responsible.

The incidence of invasive fungal infections (IFIs) in nonneutropenic solid organ transplant patients is increasing. We report our clinical experience with the use of interferon-gamma (IFN-gamma) immunotherapy in seven renal transplant patients who developed life threatening, disseminated IFIs refractory to conventional antifungal drug therapy. The infections were all microbiologically and histologically proven. The rapid cure of these disseminated infections with exogenous IFN-gamma injections was not associated with impaired kidney allograft function despite the use of liposomal amphotericin B in all cases. No clinical toxicity from the IFN-gamma immunotherapy was seen and no IFI relapsed during long-term follow-up. Our experience is both uncontrolled and in patients with unpredictable fungal infection-related outcomes. However, compared to standard approaches, the accelerated cure of life threatening, disseminated IFIs with 6 weeks of combination antifungal drug therapy and IFN-gamma immunotherapy saved lives, retained allograft function and led to substantial cost savings in this small patient group.

Earlier articles

We present two cases of aspergillus infection confirmed by blood culture and review 30 other cases of genuine aspergillus fungemia and 34 cases of aspergillus pseudofungemia. Multiple different media and blood culture systems were used to isolated Aspergillus. The median time to positive blood culture was 8.5 days (range, 1-27 days) in the genuine cases. Genuine aspergillus fungemia was observed more often after cardiac surgery (n = 11 [34%]) or during neutropenia (n = 9 [28%]) than in other settings. In a recent series of fungemia during neutropenia, 7.6% of all episodes were due to Aspergillus. Other patients at risk for aspergillus fungemia were similar to those at risk for invasive aspergillosis, including patients with AIDS. Seven (44%) of 19 patients who were treated survived. In the group of patients with aspergillus pseudofungemia, there were no deaths, and cultures of additional specimens from the same patient were not positive. Criteria that may be applied to ascertain whether the isolation of Aspergillus from blood cultures is clinically significant are put forward.

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No abstract. Summary: In einer gedrängten historischen Übersicht wird dargelegt, daß die wesentlichsten Erkenntnisse über die Absiedlung von Schimmelpilzen in der tierischen und menschlichen Lunge bis zum Jahre 1900 bereits bekannt gewesen sind. Neuere Arbeiten haben lediglich das Interesse für die lokalisierten Aspergillusmykosen wieder geweckt und auf diagnostische und operative Möglichkeiten hingewiesen. Die durch Aspergillaceaen verursachte pulmonale Form der Mykose ist mit der Bezeichnung Pneumonomycosis aspergillina durchR. Virchow (1856) sachlich und gemäß der Nomenklaturregeln richtig charakterisiert. Es wird Kritik geübt, insbesondere an Bezeichnungen, die weder pathomorphologisch noch historisch oder sprachlich gerechtfertigt sind und lediglich eine Vorstellung fördern, wonach bestimmte pulmonale Formen der Aspergillusmykose streng lokalisiert seien. Eine Zunahme der Krankheitsfälle wird als unbewiesen verneint, ebenso ein fördernder Einfluß durch Antibiotica und Corticoide. Auch eine primäre Form der pulmonalen Aspergillusmykose wird abgelehnt, ebenfalls die Annahme, die Erkrankung habe in den letzten Jahren einen echten Gestaltwandel erkennen lassen. Zu weitgehende Aufgliederungen in verschiedene Erkrankungstypen sind für die Praxis unbrauchbar, da intra vitam gar nicht diagnostizierbar. Absiedlung und Vermehrung von Aspergillussporen in der Lunge führt stets zu einem echten Parasitismus, bestenfalls zur Prämunition; nicht zur primären Symbiose oder zum Saprophytismus. Bisher sind 13 Aspergillusarten mit pathogenen Eigenschaften aus menschlichen und tierischen Substraten isoliert. Es wird nachgewiesen, daß auch Aspergillus glaucus dazu gehört. Mykologische Probleme werden eingehend besprochen. Auskeimen der Sporen, Hyphen-, Mycel- und Koloniebildung werden durch Vitalbeobachtungen, Dünnschnitte, Züchtung auf Modellstrukturen und cytochemisch untersucht. Beschreibung der monochromen negativen Kontrastmittelfärbung mit Nigrosin. Hinweise zur Pathomorphologie und Klinik der broncho-pneumonischen und generalisierenden Aspergillusinfektionen, auf die lokalisierten Erkrankungen der Bronchien, Bronchusstümpfe, Resektionshöhlen und auf die allergischen und asthmatoiden Formen. Sowohl die Annahme vonA. Brunner, daß eine Sekundärbesiedlung präexistenter Höhlen in der Lunge mit Aspergillaceaen vorkommt als auch die Auffassung vonO. Monod, wonach das Pilzwachstum eine bronchiektasierende Wirkung entfalten kann, müssen als richtig anerkannt werden. Mitteilung von drei einschlägigen Fällen einer Serie von fünf Beobachtungen innerhalb eines Jahres.

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(N.B. The Aspergillus website used to maintain a bibliographic database which was compiled from Medline and Web of Science (GRAsp), but as all users now have access to the former free of charge via the NCBI website and most will have access to Web of Science via their own libraries this resource is currently not being updated. It contains papers dating up to 7th October 2002. Search the GRASp Database here.)