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• Is there a need for autopsies in the management of fungal disease? by Knoke M, Bernhardt H, Schwesinger G. Abstract
  

  The autopsy rates in Germany became low like in other European, American and Asian countries. Main reasons for this development are the lack of acceptance of autopsy in the society as well as in the medical profession, the introduction of a requirement for consent, unclear legal position, the public health system, pressure of costs and a change in the field of activity in pathology with much more diagnostics of surgical and biopsy material. The autopsy is missing with respect to the reliability of causes of death and morbidity statistics and other epidemiological studies. Published data indicate that up to 20-30% of patients who die in hospitals have important diseases/lesions that remain undetected before death but that are found at autopsy. For infectious diseases, the data are similar. Therefore, a higher incidence of invasive fungal infections was found. Some rare fungal disorders are diagnosed by autopsy. Only exact death statistics makes specific health care possible and is cost saving in a public health system in the long term. Autopsy remains an important tool for quality control in medical diagnostic and therapeutic activity. It is also essential for fundamental medical education and further training.

• Therapeutic drug monitoring for triazoles by Hope WW, Billaud EM, Lestner J, Denning DW. Abstract
  

  PURPOSE OF REVIEW: Invasive fungal infections are a leading cause of morbidity and mortality in immunocompromised patients, and mechanisms to optimize therapeutic outcomes are urgently required. Therapeutic drug monitoring represents an important component for the routine use of the triazoles. RECENT FINDINGS: Triazoles have revolutionized the prevention and treatment of invasive fungal infections. Increasing data suggest that this class displays important concentration-effect and concentration-toxicity relationships. There has been an increased understanding of the pharmacokinetics and pharmacodynamics of triazoles, and this has facilitated the identification of concentrations (or drug exposures) that are both effective and nontoxic. This review discusses the application of therapeutic drug monitoring to fluconazole, itraconazole, voriconazole and posaconazole. SUMMARY: Therapeutic drug monitoring represents an important mechanism to optimize the outcome of immunocompromised patients receiving triazoles.

• The CYP2C19 ultra-rapid metabolizer genotype influences the pharmacokinetics of voriconazole in healthy male volunteers. by Wang G, Lei HP, Li Z, Tan ZR, Guo D, Fan L, Chen Y, Hu DL, Wang D, Zhou HH. Abstract
  

  AIM: To study the pharmacokinetic characteristics of voriconazole in healthy Chinese male volunteers in relation to cytochrome P450 (CYP) 2C19 genotype status, including ultra-rapid metabolizers (URMs), homozygous extensive metabolizers (EMs), and poor metabolizers (PMs). METHOD: Twenty healthy Chinese male volunteers were recruited for the study. Of these, four were CYP2C19 heterozygous URMs (*1/*17), eight were CYP2C19 homozygous EMs (*1/*1), and eight were CYP2C19 PMs (*2/*2). After a single oral dose of 200 mg voriconazole, plasma concentrations of voriconazole were determined for a 24-h period by liquid chromatography-mass spectrometry/mass spectrometry. RESULT: In Chinese male subjects, the allele frequencies of the CYP2C19*17 and CYP2C19*2 alleles were 0.64 and 35.6%, respectively, and both alleles were in Hardy-Weinberg equilibrium. The area under the concentration-time curve (AUC) from predose to 24 h (AUC(0-24)) and from predose to infinity (AUC(0-infinity)), and apparent oral clearance (CL/F) of voriconazole were statistically different among all three genotypic groups (P < 0.001, respectively). The maximum plasma concentration (C(max)) value of URMs also showed statistically significant differences from those of EMs and PMs (P = 0.036 and P = 0.035, respectively). The elimination half-life (t((1/2))) in URMs was 87% (P = 0.58) of that in EMs and 51% (P= 0.002) of that in PMs. CONCLUSION: Our data indicate that the presence of the CYP2C19*17 allele results in ultra-rapid metabolism of voriconazole after a single oral dose.

• Micafungin alone or in combination with other systemic antifungal therapies in hematopoietic stem cell transplant recipients with invasive aspergillosis. by Kontoyiannis DP, Ratanatharathorn V, Young JA, Raymond J, Laverdière M, Denning DW, Patterson TF, Facklam D, Kovanda L, Arnold L, Lau W, Buell D, Marr KA. Abstract
  

  We describe herein 98 hematopoietic stem cell transplant (HSCT) recipients with invasive aspergillosis (IA) (refractory in 83) who received micafungin either alone (8 patients) or in combination with other licensed antifungal therapies (OLAT) (90 patients). Of the 8 monotherapy patients, 4 were failing OLAT, received de novo micafungin, or were intolerant to prior OLAT (2 patients each). Of the 90 patients treated with combination, 7 had de novo IA and 83 had refractory infection. Most patients (81) had pulmonary IA, 42 (43%) had graft-versus-host disease (GVHD), and 26 (27%) were neutropenic (absolute neutrophil count <500 cells/mm(3)) at onset of treatment. Successful response was seen in 25/98 (26%); an additional 12 patients achieved stable disease. Response was seen in 2/9 (22%) in de novo treatment, 21/87 (24%) in refractory patients, and 2/2 (100%) in toxicity failure patients. Additionally, response was seen in 22 of the 90 (24%) patients treated with combination therapy, and in 3 of 8 (38%) patients who were treated with micafungin alone. No significant differences in responses were found based on type of HSCT, GVHD status, site of IA, or Aspergillus species, and no significant toxicity was seen. Micafungin was well tolerated, even at high doses, and is a reasonable option for treatment of IA in this high-risk patient population.

• Outcome and medical costs of patients with invasive aspergillosis and acute myelogenous leukemia-myelodysplastic syndrome treated with intensive chemotherapy: an observational study. by Slobbe L, Polinder S, Doorduijn JK, Lugtenburg PJ, El Barzouhi A, Steyerberg EW, Rijnders BJ. Abstract
  

  Background. @nbsp; Invasive aspergillosis (IA) is a leading cause of mortality in patients with acute leukemia. Management of IA is expensive, which makes prevention desirable. Because hospital resources are limited, prevention costs have to be compared with treatment costs and outcome. Methods. @nbsp; In 269 patients treated for acute myelogenous leukemia-myelodysplastic syndrome (AML-MDS) during 2002-2007, evidence of IA was collected using high-resolution computed tomography and galactomannan measurement in bronchoalveolar lavage fluid specimens. IA was classified on the basis of updated European Organization for Research and Treatment of Cancer/Mycoses Study Group definitions. Outcome of infection was registered. Diagnostic and therapeutic IA-related costs, corrected for neutropenia duration, were comprehensively analyzed from a hospital perspective. Voriconazole treatment was given orally from day 1 if possible. Results. @nbsp; A total of 80 patients developed IA; 48 (18%) had probable or proven infection, and 32 (12%) had possible IA. Seventy-three patients were treated with voriconazole; 55 (75%) took oral voriconazole from day 1. In patients with IA, the mortality rate 12 weeks after starting antifungal therapy was 22% (16 of 73 patients). The overall mortality rate, registered 12 weeks after neutrophil recovery from the last dose of antileukemic treatment, was 26% in patients with IA versus 16% in patients without IA ([Formula: see text]), reflecting an IA-attributable mortality rate of 10%. In a Cox regression analysis, IA was associated with an increased mortality risk (hazard ratio, 2.4; 95% confidence interval, 1.3-4.4). Total IA-related costs increased to euro8360 and euro15,280 for patients with possible and probable or proven IA, respectively, compared with patients without IA ([Formula: see text]). Conclusions. @nbsp; Early diagnosis and treatment of IA with oral voriconazole result in acceptable mortality rates. Nevertheless, IA continues to have substantial attributable mortality combined with a major impact on hospital resource use, so effective prevention in high-incidence populations has the potential to save lives and costs.

• Outwitting Multidrug Resistance to Antifungals by Brian C. Monk and Andre Goffeau Abstract
  

  The economic cost of fungal infection and its mortality associated with multidrug resistance remain unacceptably high. Recent understanding of the transcriptional regulation of plasma membrane efflux pumps of modest specificity provides new avenues for the development of broad-spectrum fungicides. Together with improved diagnosis and indirect intervention via inhibition of the energy supply for drug efflux, we envisage multifunctional azole analogs that inhibit not only ergosterol biosynthesis and drug efflux-pump activity but also activation of the transcriptional machinery that induces drug efflux-pump expression.

• The fight against fungi by André Goffeau Abstract
  

  Although several drugs are available to combat often-deadly fungal infections, many of these pathogens have acquired multidrug resistance. Discerning how they have achieved this could help us hit back.

• Engineering and Applications of fungal laccases for organic synthesis by Adinarayana Kunamneni, Susana Camarero, Carlos Garcia-Burgos, Francisco Jose Plou , Antonio Ballesteros, Miguel Alcalde. Abstract
  

  Laccases are multi-copper containing oxidases (EC 1.10.3.2), widely distributed in fungi, higher plants and bacteria. Laccase catalyses the oxidation of phenols, polyphenols and anilines by one-electron abstraction, with the concomitant reduction of oxygen to water in a four-electron transfer process. In the presence of small redox mediators, laccase offers a broader repertory of oxidations including non-phenolic substrates. Hence, fungal laccases are considered as ideal green catalysts of great biotechnological impact due to their few requirements (they only require air, and they produce water as the only by-product) and their broad substrate specificity, including direct bioelectrocatalysis. Thus, laccases and/or laccase-mediator systems find potential applications in bioremediation, paper pulp bleaching, finishing of textiles, bio-fuel cells and more. Significantly, laccases can be used in organic synthesis, as they can perform exquisite transformations ranging from the oxidation of functional groups to the heteromolecular coupling for production of new antibiotics derivatives, or the catalysis of key steps in the synthesis of complex natural products. In this review, the application of fungal laccases and their engineering by rational design and directed evolution for organic synthesis purposes are discussed.

• A sterol-regulatory element binding protein is required for cell polarity, hypoxia adaptation, azole drug resistance, and virulence in Aspergillus fumigatus by Willger SD, Puttikamonkul S, Kim KH, Burritt JB, Grahl N, Metzler LJ, Barbuch R, Bard M, Lawrence CB, Cramer RA Jr. Abstract
  

  At the site of microbial infections, the significant influx of immune effector cells and the necrosis of tissue by the invading pathogen generate hypoxic microenvironments in which both the pathogen and host cells must survive. Currently, whether hypoxia adaptation is an important virulence attribute of opportunistic pathogenic molds is unknown. Here we report the characterization of a sterol-regulatory element binding protein, SrbA, in the opportunistic pathogenic mold, Aspergillus fumigatus. Loss of SrbA results in a mutant strain of the fungus that is incapable of growth in a hypoxic environment and consequently incapable of causing disease in two distinct murine models of invasive pulmonary aspergillosis (IPA). Transcriptional profiling revealed 87 genes that are affected by loss of SrbA function. Annotation of these genes implicated SrbA in maintaining sterol biosynthesis and hyphal morphology. Further examination of the SrbA null mutant consequently revealed that SrbA plays a critical role in ergosterol biosynthesis, resistance to the azole class of antifungal drugs, and in maintenance of cell polarity in A. fumigatus. Significantly, the SrbA null mutant was highly susceptible to fluconazole and voriconazole. Thus, these findings present a new function of SREBP proteins in filamentous fungi, and demonstrate for the first time that hypoxia adaptation is likely an important virulence attribute of pathogenic molds

• Introduction to the updated Australian and New Zealand consensus guidelines for the use of antifungal agents in the haematology/oncology setting, 2008 by M. A. Slavin Abstract
  

  The process for development of these consensus Australasian antifungal guidelines for use in adult patients with haematological malignancy is described. New features included, how the guidelines should be applied, the risk assessment tool used and the grading system for evidence and strength of recommendation are discussed.

• Diagnostic and therapeutic approach to persistent or recurrent fevers of unknown origin in adult stem cell transplantation and haematological malignancy by C. O. Morrissey, P. G. Bardy, M. A. Slavin, M. R. Ananda-Rajah, S. C. Chen, S. W. Kirsa, D. S. Ritchie8, and A. Upton Abstract
  

  Persistent or recurrent fevers of unknown origin (PFUO) in neutropenic patients on broad-spectrum antibiotics have traditionally been treated with empirical antifungal therapy (EAFT). The lack of survival benefit seen with the use of amphotericin B deoxycholate (AmB-D) as EAFT has been attributed to its toxicities. More recently, newer, less toxic and more expensive antifungal agents such as the lipid formulations of AmB, the newer azoles (fluconazole, itraconazole and voriconazole) and caspofungin have been analysed in a number of EAFT trials. Compared with AmB-D the newer agents have superior safety but are of equivalent efficacy. This lack of survival advantage is related to the fact that the trigger for commencement of EAFT is late and non-specific. Thus, alternative approaches are required. New sensitive serological and molecular tests for the detection of Aspergillus antigens and genomic DNA have been developed and evaluated in accuracy studies. These tests have been incorporated into management strategies (i.e. pre-emptive strategies) to direct antifungal therapy. The pre-emptive approach has been shown to be safe and feasible but its impact on clinically important patient outcomes such as survival is less clear. Other advances include the introduction of effective, non-toxic mould-active antifungal prophylaxis and patient risk-group stratification. In this paper we provide new evidence-based algorithms for the diagnosis and treatment of PFUO in adult patients undergoing stem cell transplantation and chemotherapy for haematological malignancy which incorporate these newer diagnostic tests and are directed by the risk category of the patient and type of antifungal prophylaxis the patient is receiving.

• Recommendations for the treatment of established fungal infections by K. A. Thursky, E. G. Playford, J. F. Seymour, T. C. Sorrell, D. H. Ellis, S. D. Guy, N. Gilroy, J. Chu and D. R. Shaw Abstract
  

  Evidence-based guidelines for the treatment of established fungal infections in the adult haematology/oncology setting were developed by a national consensus working group representing clinicians, pharmacists and microbiologists. These updated guidelines replace the previous guidelines published in the Internal Medicine Journal by Slavin et al. in 2004. The guidelines are pathogen-specific and cover the treatment of the most common fungal infections including candidiasis, aspergillosis, cryptococcosis, zygomycosis, fusariosis, scedosporiosis, and dermatophytosis. Recommendations are provided for management of refractory disease or salvage therapies, and special sites of infections such as the cerebral nervous system and the eye. Because of the widespread use newer broad-spectrum triazoles in prophylaxis and empiric therapy, these guidelines should be implemented in concert with the updated prophylaxis and empiric therapy guidelines published by this group.

• Antifungal prophylaxis in adult stem cell transplantation and haematological malignancy by M. A. Slavin, C. H. Heath, K. A. Thursky, C. O. Morrissey, J. Szer, L. M. Ling, S. T. Milliken and A. P. Grigg Abstract
  

  Antifungal prophylaxis can be recommended in patients undergoing induction chemotherapy for acute myeloid leukemia and treatment for grade 2 or greater or chronic extensive graft versus host disease. The evidence for prophylaxis is less clear in other clinical settings although certain groups such as patients with prolonged neutropenia after stem cell transplants using bone marrow or cord blood sources and with impaired cell mediated immunity secondary to treatments such as Alemtuzumab are at high risk. The decision to use prophylaxis and which agent to use will be influenced by effectiveness, number needed to treat and the likelihood of toxicity and drug interactions. The availability of rapid diagnostic tests for fungal infection and institutional epidemiology will also influence the need for and choice of prophylaxis. Whilst prophylaxis can be beneficial, it may impede the ability to make a rapid diagnosis of fungal infection by reducing the yield of diagnostic tests and change the epidemiology of fungal infection. As non-culture based diagnostic tests are refined and become more available there may be a shift from prophylaxis to early diagnosis and treatment.

• Optimizing antifungal drug dosing and monitoring to avoid toxicity and improve outcomes in patients with haematological disorders by L. J. Worth, C. C. Blyth, D. L. Booth, D. C. M. Kong, D. Marriott, M. Cassumbhoy, J. Ray, M. A. Slavin1, and J. R. Wilkes Abstract
  

  Antifungal prophylaxis, empirical therapy and treatment of established fungal infections in the haematology population may be associated with significant toxicity or drug interactions leading to sub-therapeutic antifungal drug concentrations and poorer clinical outcomes. These risks may be minimised by clinical assessment, laboratory monitoring of biochemical or haematological indices, avoidance of particular drug combinations and dose modification in certain circumstances. Specific measures, such as the optimal timing of oral drug administration in relation to meals, use of pre-hydration and electrolyte supplementation may also be required. For certain agents, therapeutic drug monitoring (TDM) is warranted where non-compliance, non-linear pharmacokinetics, a narrow therapeutic window, suspected drug interaction or unexpected toxicity are encountered. Pharmacokinetics and pharmacodynamics of clinical relevance to the haematology population are discussed for the azole, polyene and echinocandin classes of antifungal agents. The evidence supporting an association between TDM and enhanced treatment outcomes is presented for individual antifungal drugs, and recommendations for clinical practice are provided. Further randomised study of newer antifungal agents, such as posaconazole, is required to explore the potential for improved clinical outcomes in association with TDM.

• Preventing invasive fungal infection during hospital building works by C. C. Chang, E. Athan, C. O. Morrissey and M. A. Slavin Abstract
  

  Hospital building works increase the risk of invasive fungal infections. Nosocomial outbreaks have been reported. A pre-emptive strategy for planned building works is paramount. The roles of HEPA filtration, air-sampling and modulation of ‘routine’ antifungal prophylaxis practice are discussed in the context of pre-emptive planning and outbreak management.

• Emergence of Azole Resistance in Aspergillus fumigatus and Spread of a Single Resistance Mechanism by Snelders E., van der Lee H.A.L., Kuijpers J., Rijs A.J.M.M., Varga J., Samson R.A., Mellado E., Donders A.R.T., Melchers W.J.G., Verweij P.E. Abstract
  

  Background Resistance to triazoles was recently reported in Aspergillus fumigatus isolates cultured from patients with invasive aspergillosis. The prevalence of azole resistance in A.fumigatus is unknown. We investigated the prevalence and spread of azole resistance using our culture collection that contained A. fumigatus isolates collected between 1994 and 2007. Methods and Findings We investigated the prevalence of itraconazole (ITZ) resistance in 1,912 clinical A. fumigatus isolates collected from 1,219 patients in our University Medical Centre over a 14-y period. The spread of resistance was investigated by analyzing 147 A. fumigatus isolates from 101 patients, from 28 other medical centres in The Netherlands and 317 isolates from six other countries. The isolates were characterized using phenotypic and molecular methods. The electronic patient files were used to determine the underlying conditions of the patients and the presence of invasive aspergillosis. ITZ-resistant isolates were found in 32 of 1,219 patients. All cases were observed after 1999 with an annual prevalence of 1.7% to 6%. The ITZ-resistant isolates also showed elevated minimum inhibitory concentrations of voriconazole, ravuconazole, and posaconazole. A substitution of leucine 98 for histidine in the cyp51A gene, together with twocopies of a 34-bp sequence in tandem in the gene promoter (TR/L98H), was found to be the dominant resistance mechanism. Microsatellite analysis indicated that the ITZ-resistant isolates were genetically distinct but clustered. The ITZ-sensitive isolates were not more likely to be responsible for invasive aspergillosis than the ITZ-resistant isolates. ITZ resistance was found in isolates from 13 patients (12.8%) from nine other medical centres in The Netherlands, of which69% harboured the TR/L98H substitution, and in six isolates originating from four other countries. Conclusions Azole resistance has emerged in A. fumigatus and might be more prevalent than currently acknowledged. The presence of a dominant resistance mechanism in clinical isolates suggests that isolates with this mechanism are spreading in our environment.

• Postoperative application of amphotericin B nasal spray in chronic rhinosinusitis with nasal polyposis, with a review of the antifungal therapy by Gerlinger I, Fittler A, Fónai F, Patzkó A, Mayer A, Botz L. Abstract
  

  Chronic rhinosinusitis (CRS) affects 1-4% of the adult population. The etiology of this multifactorial, chronic disease, which leads to a significant impairment of the quality of life, often accompanied by nasal polyposis, is not fully understood. In the past decade, it was presumed that the disease, which causes characteristic eosinophilic infiltration of the nasal mucosa, is triggered by an enhanced (but not classical allergic IgE-type) immune response against fungal organisms in the nasal mucus. If this supposition is correct, then it appears obvious that the administration of amphotericin B nasal spray in adequate concentration following endoscopic polypectomy should be advantageous for these patients, and might even reduce the number of recurrent cases. To check on this assumption, we conducted a prospective randomized placebo-controlled trial involving 33 patients, 30 of whom remained in the study throughout. Patients with nasal polyposis were operated on with an endoscopic technique between 1 November 2005 and 1 October 2006; group A (14 randomly selected patients) were treated with a nasal spray containing 5 mg/ml amphotericin B, while the placebo group B (16 randomly selected patients) received a nasal spray lacking amphotericin B. We evaluated our results with the aid of a modified Lund-Mackay CT score, the SNAQ-11 test (which assesses changes in the symptoms), a quality of life test and endoscopy. The SPSS 14.0 for Windows program was utilized to process the data of examinations performed preoperatively and 1 year postoperatively. The CT scores of the group A patients 1 year after the operation exhibited wide scattering, without signs of recovery. The CT scores of the group B patients indicated a slight improvement, though this did not prove significant relative to group A. Both the SNAQ-11 test and the quality of life test revealed a significant improvement in each group, but the degrees of change in these tests did not differ significantly between the two groups of patients. The endoscopic findings indicated a slight improvement to the advantage of the amphotericin B-treated group 12 months after the operation. These results lead to the conclusion that the administration of amphotericin B nasal spray to patients operated on for nasal polyposis does not give rise to a significant alteration in either CT score, clinical symptoms, or quality of life. The more favorable clinical aspects observed in the amphotericin B-treated group during the endoscopic follow-up did not correspond to an improvement in the symptoms. In connection with the conclusions drawn from this study, the authors discuss the controversial data available on the fungal etiology of CRS. They critically analyze the contradictory observations and conclusions of seven recent clinical studies.

• Efficient clearance of Aspergillus fumigatus in murine lungs by an ultrashort antimicrobial lipopeptide, palmitoyl-lys-ala-DAla-lys by Vallon-Eberhard A, Makovitzki A, Beauvais A, Latgé JP, Jung S, Shai Y. Abstract
  

  Aspergillus fumigatus is an opportunistic fungal pathogen responsible for invasive aspergillosis in immunocompromised individuals. The inefficiency of antifungal agents and high mortality rate resulting from invasive aspergillosis remain major clinical concerns. Recently, we reported on a new family of ultrashort cationic lipopeptides active in vitro against fungi. Mode of action studies supported a membranolytic or a detergent-like effect. Here, we screened several lipopeptides in vitro for their anti-A. fumigatus activity. To investigate the therapeutic properties of the selected peptides in vivo, we challenged immunosuppressed C57BL/6 wild-type mice intranasally with DsRed-labeled A. fumigatus conidia and subsequently treated the animals locally with the lipopeptides. Confocal microscopic analysis revealed the degradation of DsRed-labeled hyphal forms and residual conidia in the lungs of the mice. The most efficient peptide was tested further using a survival assay and was found to significantly prolong the life of the treated animals, whereas no mice survived with the current standard antifungal treatment with amphotericin B. Moreover, as opposed to the drug-treated lungs, the peptide-treated lungs did not display any toxicity of the peptide. Our results highlight the potential of this family of lipopeptides for the treatment of pulmonary invasive aspergillosis.

• Generation of large chromosomal deletions in koji molds Aspergillus oryzae and Aspergillus sojae via a loop-out recombination by Takahashi T, Jin FJ, Sunagawa M, Machida M, Koyama Y. Abstract
  

  We established a technique for efficiently generating large chromosomal deletions in the koji molds Aspergillus oryzae and A. sojae by using a ku70-deficient strain and a bidirectional marker. The approach allowed deletion of 200-kb and 100-kb sections of A. oryzae and A. sojae, respectively. The deleted regions contained putative, aflatoxin biosynthetic gene clusters. The large genomic deletions generated by a loop-out deletion method (resolution-type recombination) enabled us to construct multiple deletions in the koji molds by marker recycling. No additional sequence remained in the resultant deletion strains, a feature of considerable value for breeding of food-grade microorganisms. Frequencies of chromosomal deletions tended to decrease in proportion to the length of the deletion range. Deletion efficiency was also affected by the location of the deleted region. Further, comparative genome hybridization analysis showed no unintended deletion or chromosomal rearrangement occurred in the deletion strain. Strains with large deletions that were previously extremely laborious to construct in the wild-type ku70+ strain due to the low frequency of homologous recombination were efficiently obtained from Deltaku70 strains in this study. The technique described here may be broadly applicable for the genomic engineering and molecular breeding of filamentous fungi.

• Characterization of Aspergillus species based on fatty acid profiles by Fraga ME, Santana DM, Gatti MJ, Direito GM, Cavaglieri LR, Rosa CA. Abstract
  

  Cellular fatty acid (FA) composition was utilized as a taxonomic tool to discriminate between different Aspergillus species. Several of the tested species had the same FA composition and different relative FA concentrations. The most important FAs were palmitic acid (C16:0), estearic acid (C18:0), oleic acid (C18:1) and linoleic acid (C18:2), which represented 95% of Aspergillus FAs. Multivariate data analysis demonstrated that FA analysis is a useful tool for differentiating species belonging to genus Aspergillus. All the species analyzed showed significantly FA acid profiles (p < 0.001). Furthermore, it will be possible to distinguish among Aspergillus spp. in the Flavi Section. FA composition can serve as a useful tool for the identification of filamentous fungi.

• Analysis and prediction of gene splice sites in four Aspergillus genomes by Wang K, Ussery DW, Brunak S. Abstract
  

  Several Aspergillus fungal genomic sequences have been published, with many more in progress. Obviously, it is essential to have high-quality, consistently annotated sets of proteins from each of the genomes, in order to make meaningful comparisons. We have developed a dedicated, publicly available, splice site prediction program called NetAspGene, for the genus Aspergillus. Gene sequences from Aspergillus fumigatus, the most common mould pathogen, were used to build and test our model. Compared to many animals and plants, Aspergillus contains smaller introns; thus we have applied a larger window size on single local networks for training, to cover both donor and acceptor site information. We have applied NetAspGene to other Aspergilli, including Aspergillus nidulans, Aspergillus oryzae, and Aspergillus niger. Evaluation with independent data sets reveal that NetAspGene performs substantially better splice site prediction than other available tools. NetAspGene will be very helpful for the study in Aspergillus splice sites and especially in alternative splicing. A webpage for NetAspGene is publicly available at http://www.cbs.dtu.dk/services/NetAspGene.

• Developing Aspergillus as a host for heterologous expression. by Lubertozzi D, Keasling JD. Abstract
  

  Filamentous fungi have long been used for production of a range of valuable products; with the advent of molecular biology, it became apparent that these fungi possess considerable potential as expression hosts for the production of heterologous proteins and small molecules. Aspergillus is an important genus, including well known species of economically significant molds, and widely used for basic genetic research. The development of a genetic engineering "toolkit" for Aspergillus, such as those existing for the simpler yeasts and bacteria, was delayed due to the added complexity of the filamentous fungi, and also to the lesser resources devoted to their study. History of the development of Aspergillus as an expression host, current state of the art and future directions are reviewed, touching on related research in other fungi when discussing the areas of greatest potential for future biotechnological applications, focusing on the large and diverse families of fungal secondary metabolites.

• Overexpression of a novel endogenous NADH kinase in Aspergillus nidulans enhances growth by Panagiotou G, Grotkjær T, Hofmann G, Bapat PM, Olsson L. Abstract
  

  The complete genome sequence of the filamentous fungi Aspergillus nidulans has paved the way for fundamental research on this industrially important species. To the best of our knowledge, this is the first time a gene encoding for ATP-dependent NADH kinase (ATP:NADH 2'-phosphotransferase, EC 2.7.1.86) has been identified. The enzyme has a predicted molecular weight of 49kDa. We characterised the role of this NADH kinase by genomic integration of the putative gene AN8837.2 under a strong constitutive promoter. The physiological effects of overexpressed NADH kinase in combination with different aeration rates were studied in well-controlled glucose batch fermentations. Metabolite profiling and metabolic network analysis with [1-(13)C] glucose were used for characterisation of the strains, and the results demonstrated that NADH kinase activity has paramount influence on growth physiology. Biomass yield on glucose and the maximum specific growth rate increased from 0.47g/g and 0.22h(-1) (wild type) to 0.54g/g and 0.26h(-1) (NADH kinase overexpressed), respectively. The results suggest that overexpression of NADH kinase improves the growth efficiency of the cell by increasing the access to NADPH. Our findings indicate that A. nidulans is not optimised for growth in nutrient-rich conditions typically found in laboratory and industrial fermentors. This conclusion may impact the design of new strains capable of generating reducing power in the form of NADPH, which is crucial for efficient production of many industrially important metabolites and enzymes.

• Galactomannan detection for invasive aspergillosis in immunocompromized patients by Leeflang MM, Debets-Ossenkopp YJ, Visser CE, Scholten RJ, Hooft L, Bijlmer HA, Reitsma JB, Bossuyt PM, Vandenbroucke-Grauls CM. Abstract
  

  BACKGROUND: Invasive aspergillosis (IA) is the most common life-threatening opportunistic invasive mycosis in immunocompromized patients. A test for IA needs to be not too invasive and not too big a burden for the already weakened patient. The serum galactomannan ELISA seems to have potential for both requirements. OBJECTIVES: To obtain summary estimates of the diagnostic accuracy of galactomannan detection in serum for the diagnosis of IA. SEARCH STRATEGY: We searched MEDLINE, EMBASE and Web of Science with both Medical Headings and text words for both aspergillosis and the sandwich ELISA. We checked reference lists of included studies and review articles for additional studies. SELECTION CRITERIA: Cross-sectional studies, case-control designs and consecutive series of patients assessing the diagnostic accuracy of galactomannan detection for the diagnosis of IA in patients with neutropenia or patients whose neutrophils are functionally compromised were included. The reference standard was composed of the criteria given by the European Organization for Research and Treatment of Cancer (EORTC) and the Mycoses Study Group (MSG). DATA COLLECTION AND ANALYSIS: Two review authors independently assessed quality and extracted data MAIN RESULTS: Thirty studies were included in the meta-analyses, with a median prevalence of IA (proven or probable) of 7.7%. Seven of these (901 patients) reported results for an Optical Density Index (ODI) of 0.5 as cut-off value. The overall sensitivity in these studies was 78% (61% to 89%) and overall specificity was 81% (72% to 88%). Twelve studies (1744 patients) reported the results for cut-off value of 1.0 ODI, overall sensitivity was 75% (59% to 86%) and mean specificity 91% (84% to 95%). Seventeen studies (2600 patients) reported the results for cut-off value 1.5 ODI, sensitivity was 64% (50% to 77%) and mean specificity 95% (91% to 97%). AUTHORS' CONCLUSIONS: At a cut-off value 0.5 ODI in a population of 100 patients with a disease prevalence of 8% (overall median prevalence), 2 patients who have IA, will be missed (sensitivity 78%, 22% false negatives), and 17 patients will be treated or further referred unnecessarily (specificity of 81%, 19% false negatives). If we use the test at cut-off value 1.5 in the same population, that will mean that 3 IA patients will be missed (sensitivity 64%, 36% false negatives) and 5 patients will be treated or referred unnecessarily (specificity of 95%, 5% false negatives). These numbers should however be interpreted with caution, because the results were very heterogeneous.

• Detection of circulating Aspergillus fumigatus DNA by real-time PCR assay of large serum volumes improves early diagnosis of invasive aspergillosis in high-risk adult patients under hematologic surveillance. by Suarez F, Lortholary O, Buland S, Rubio MT, Ghez D, Mahé V, Quesne G, Poirée S, Buzyn A, Varet B, Berche P, Bougnoux ME. Abstract
  

  Detection of galactomannan antigen (GMA) in serum is the standard assay for the diagnosis of invasive aspergillosis (IA) in high-risk patients with hematological disorders. Detection of Aspergillus DNA in serum has been proposed, but its sensitivity is lower than that of GMA when small serum volumes (SSV) are used. In this study, we investigated whether extraction of DNA from large serum volumes (LSV) improves diagnostic yield. In a 13-month prospective study, we compared the performances of twice-weekly screening of serum for GMA by an enzyme immunoassay and weekly screening for Aspergillus fumigatus DNA by a real-time PCR (RT-PCR) assay of 1.0 ml (LSV) or 100 mul (SSV) of serum. We included 124 patients (138 treatment episodes), with 17 episodes of EORTC (European Organization for Research and Treatment of Cancer)/MSG (Mycoses Study Group)-documented IA. In all, 1,870 samples were screened for GMA. The sensitivity (Se), specificity (Sp), and positive and negative predictive values (PPV and NPV, respectively) of GMA for IA were 88.2%, 95.8%, 75%, and 98.3%, respectively. We screened 938 samples for Aspergillus DNA by using LSV; 404 of these samples were also tested with SSV. The Se, Sp, PPV, and NPV of RT-PCR were 100%, 96.7%, 81%, and 100%, respectively, with LSV and 76.5%, 96.7%, 81.3%, and 95.6%, respectively, with SSV. DNA detection gave a positive result when performed on LSV in two cases of IA where the GMA assay result remained negative. Furthermore, in four IA cases, DNA was detected earlier than GMA. The use of LSV for extraction improved the performance of the RT-PCR, which appears highly sensitive and specific for the early diagnosis of IA in high-risk patients with hematological disorders.

• Performance characteristics of the Platelia(R) Aspergillus EIA for detection of Aspergillus galactomannan antigen in bronchoalveolar lavage fluid. by Husain S, Clancy CJ, Nguyen MH, Swartzentruber S, Leather H, Lemonte AM, Durkin MM, Knox KS, Hage CA, Bentsen C, Singh N, Wingard JR, Wheat LJ. Abstract
  

  We have evaluated the Platelia(R) Aspergillus enzyme immunoassay for detection of galactomannan in bronchoalveolar lavage (BAL) specimens in solid organ transplant patients with aspergillosis. Precision and reproducibility in serum or BAL to which galactomannan was added were similar. Sensitivity was 81.8% in patients with aspergillosis and specificity was 95.8% in lung transplant patients who underwent BAL for surveillance for infection or rejection. Among transplant controls, positive results were more common in patients who underwent diagnostic BAL performed for evaluation of symptoms or chest CT abnormalities, who had undergone lung transplantation, or were colonized with Aspergillus. Galactomannan testing in BAL is useful for diagnosis of aspergillosis in transplant patients. The significance of positive results in patients without confirmed aspergillosis requires further evaluation.

• H3K9 methylation regulates growth and development in Aspergillus fumigatus by Palmer JM, Perrin RM, Dagenais TR, Keller NP. Abstract
  

  In most species, chromatin remodeling mediates critical biological processes ranging from development to disease states. In fungi within the genus Aspergillus, chromatin remodeling may regulate expression of metabolic gene clusters, but other processes regulated by chromatin structure remain to be elucidated. In many eukaryotic species, methylation of lysine 9 of histone 3 (H3K9) is a hallmark of heterochromatin formation and subsequent gene silencing. The sole H3K9 methyltransferase in Schizosaccharomyces pombe is Clr4. We report that disruption of the Clr4 homolog in the pathogenic mold A. fumigatus (ClrD) - involved in both mono and tri-methylation of H3K9 - results in several growth abnormalities. Developmental defects in DeltaAf-clrD include reduction in radial growth, reduction in conidial production, and delayed conidiation after developmental competence mediated by delayed expression of brlA, the master regulator of conidiophore development. Sensitivity of DeltaAf-clrD to 6-azauracil suggests that ClrD influences transcriptional processing in A. fumigatus. Despite growth abnormalities, macrophage assays suggest ClrD may be dispensable for host interactions.

• Treatment of invasive fungal infections in cancer patients-Recommendations of the Infectious Diseases Working Party (AGIHO) of the German Society of Hematology and Oncology (DGHO). by Böhme A, Ruhnke M, Buchheidt D, Cornely OA, Einsele H, Enzensberger R, Hebart H, Heinz W, Junghanss C, Karthaus M, Krüger W, Krug U, Kubin T, Penack O, Reichert D, Reuter S, Silling G, Südhoff T, Ullmann AJ, Maschmeyer G. Abstract
  

  Invasive fungal infections are a main cause of morbidity and mortality in cancer patients undergoing intensive chemotherapy regimens. Early antifungal treatment is mandatory to improve survival. Today, a number of effective and better-tolerated but more expensive antifungal agents compared to the former gold standard amphotericin B deoxycholate are available. Clinical decision-making must consider results from numerous studies and published guidelines, as well as licensing status and cost pressure. New developments in antifungal prophylaxis improving survival rates result in a continuous need for actualization. The treatment options for invasive Candida infections include fluconazole, voriconazole, and amphotericin B and its lipid formulations, as well as echinocandins. Voriconazole, amphotericin B, amphotericin B lipid formulations, caspofungin, itraconazole, and posaconazole are available for the treatment of invasive aspergillosis. Additional procedures, such as surgical interventions, immunoregulatory therapy, and granulocyte transfusions, have to be considered. The Infectious Diseases Working Party of the German Society of Hematology and Oncology here presents its 2008 recommendations discussing the dos and do-nots, as well as the problems and possible solutions, of evidence criteria selection.

• Toll-like Receptor 4 Polymorphisms and Aspergillosis in Stem-Cell Transplantation by Pierre-Yves Bochud, M.D., Jason W. Chien, M.D., Kieren A. Marr, M.D., Wendy M. Leisenring, Sc.D., Arlo Upton, M.D., Marta Janer, Ph.D., Stephanie D. Rodrigues, Sarah Li, John A. Hansen, M.D., Lue Ping Zhao, Ph.D., Alan Aderem, Ph.D., and Michael Boeckh, M.D. Abstract
  

  Background Toll-like receptors (TLRs) are essential components of the immune response to fungal pathogens. We examined the role of TLR polymorphisms in conferring a risk of invasive aspergillosis among recipients of allogeneic hematopoietic-cell transplants. Methods We analyzed 20 single-nucleotide polymorphisms (SNPs) in the toll-like receptor 2 gene (TLR2), the toll-like receptor 3 gene (TLR3), the toll-like receptor 4 gene (TLR4), and the toll-like receptor 9 gene (TLR9) in a cohort of 336 recipients of hematopoietic- cell transplants and their unrelated donors. The risk of invasive aspergillosis was assessed with the use of multivariate Cox regression analysis. The analysis was replicated in a validation study involving 103 case patients and 263 matched controls who received hematopoietic-cell transplants from related and unrelated donors. Results In the discovery study, two donor TLR4 haplotypes (S3 and S4) increased the risk of invasive aspergillosis (adjusted hazard ratio for S3, 2.20; 95% confidence interval [CI], 1.14 to 4.25; P = 0.02; adjusted hazard ratio for S4, 6.16; 95% CI, 1.97 to 19.26; P = 0.002). The haplotype S4 was present in carriers of two SNPs in strong linkage disequilibrium (1063 A/G [D299G] and 1363 C/T [T399I]) that influence TLR4 function. In the validation study, donor haplotype S4 also increased the risk of invasive aspergillosis (adjusted odds ratio, 2.49; 95% CI, 1.15 to 5.41; P = 0.02); the association was present in unrelated recipients of hematopoietic-cell transplants (odds ratio, 5.00; 95% CI, 1.04 to 24.01; P = 0.04) but not in related recipients (odds ratio, 2.29; 95% CI, 0.93 to 5.68; P = 0.07). In the discovery study, seropositivity for cytomegalovirus (CMV) in donors or recipients, donor positivity for S4, or both, as compared with negative results for CMV and S4, were associated with an increase in the 3-year probability of invasive aspergillosis (12% vs. 1%, P = 0.02) and death that was not related to relapse (35% vs. 22%, P = 0.02). Conclusions This study suggests an association between the donor TLR4 haplotype S4 and the risk of invasive aspergillosis among recipients of hematopoietic-cell transplants from unrelated donors.

• Innate antifungal immunity of human eosinophils mediated by a beta 2 integrin, CD11b by Yoon J, Ponikau JU, Lawrence CB, Kita H. Abstract
  

  Eosinophils produce and release various proinflammatory mediators and also show immunomodulatory and tissue remodeling functions; thus, eosinophils may be involved in the pathophysiology of asthma and other eosinophilic disorders as well as host defense. Several major questions still remain. For example, how do human eosinophils become activated in diseased tissues or at the site of an immune response? What types of host immunity might potentially involve eosinophils? Herein, we found that human eosinophils react vigorously to a common environmental fungus, Alternaria alternata, which is implicated in the development and/or exacerbation of human asthma. Eosinophils release their cytotoxic granule proteins, such as eosinophil-derived neurotoxin and major basic protein, into the extracellular milieu and onto the surface of fungal organisms and kill the fungus in a contact-dependent manner. Eosinophils use their versatile beta(2) integrin molecule, CD11b, to adhere to a major cell wall component, beta-glucan, but eosinophils do not express other common fungal receptors, such as dectin-1 and lactosylceramide. The I-domain of CD11b is distinctively involved in the eosinophils' interaction with beta-glucan. Eosinophils do not react with another fungal cell wall component, chitin. Because human eosinophils respond to and kill certain fungal organisms, our findings identify a previously unrecognized innate immune function for eosinophils. This immune response by eosinophils may benefit the host, but, in turn, it may also play a role in the development and/or exacerbation of eosinophil-related allergic human diseases, such as asthma.

• Molecular characterization of the Aspergillus fumigatus NCS-1 homologue, NcsA by Mota Júnior AO, Malavazi I, Soriani FM, Heinekamp T, Jacobsen I, Brakhage AA, Savoldi M, Goldman MH, da Silva Ferreira ME, Goldman GH. Abstract
  

  Here, we characterize the Aspergillus fumigatus homologue ncsA Neuronal Calcium Sensor. We showed that ncsA is not an essential gene and ncsA growth was decreased in the presence of EGTA and SDS. Furthermore, the ncsA mutant is more resistant to calcium chloride. NcsA:mRFP localizes to the cytoplasm and its cellular localization is not affected by the cellular response to either calcium chloride or EGTA. The ncsA mutant strain is more sensitive to voriconazole, itraconazole, and amphotericin. Polar growth in the DeltancsA mutant was also considerably more affected by lovastatin than in the wild type strain. The Spitzenkörper can be visualized in both strains and although the vacuolar system does not seem to be very different, there is an increase in the staining intensity on the germling surface of the ncsA strain. NcsA promotes pmcA and pmcB expression and therefore there is a reduced expression of these ion pumps in the DeltancsA mutant background, and also of other genes involved in the response to calcium in A. fumigatus. The ncsA inactivation mutation is not causing loss of virulence in a low dose murine infection when compared to the corresponding wild type strain.

• Depletion of the MobB and CotA complex in Aspergillus nidulans causes defects in polarity maintenance that can be suppressed by the environment stress. by Shi J, Chen W, Liu Q, Chen S, Hu H, Turner G, Lu L. Abstract
  

  Polarized growth is a central feature in eukaryotes. Establishment and maintenance of cell polarity are coordinated by signaling pathways. In this study, we have identified MobB is required for the regulation of cell polarity in Aspergillus nidulans. Depletion of MobB by alcA (p) promoter repression or deletion of MobB abolished conidiation completely, and induced severe growth defects. mobB mutants showed abnormal nuclear segregation with increased number of nuclei in spores, but the formation of septa occurred among dividing cells. The phenotype of mobB in A. nidulans is similar to that of cotA. Furthermore, we verified that MobB interacted with CotA to function as a complex. Interestingly, both mobB and cotA deletion mutants clearly exhibited filament elongation by using environmental osmotic stress in the media. However, calcium channel blocker or chelator inhibited phenotype suppression of mobB or cotA mutants. These results suggest that Ca(2+) is potentially involved in the response to the suppression coupled with osmotic stabilizer. This is the first report of the function of MobB in A. nidulans. We propose that the MobB/CotA complex, a component in the conserved RAM-signaling pathway, serves an important role in cell morphogenesis.

• Proteomic and transcriptomic analysis of Aspergillus fumigatus on exposure to amphotericin B by Gautam P, Shankar J, Madan T, Sirdeshmukh R, Sundaram CS, Gade WN, Basir SF, Sarma PU. Abstract
  

  Amphotericin B (AMB) is the most widely used polyene antifungal drug for the treatment of systemic fungal infections including invasive aspergillosis. It has been our aim to understand molecular targets of AMB in Aspergillus fumigatus (A. fumigatus) by genomic and proteomic approaches. In transcriptomic analysis, a total of 295 genes were found to be differentially expressed- 165 up-regulated and 130 down-regulated and included many belonging to the ergosterol pathway genes, genes for cell stress proteins, cell wall proteins, transport proteins and hypothetical proteins. Proteomic profiles of A. fumigatus alone or A. fumigatus treated with AMB showed differential expression of 85 proteins- 76 up-regulated and 9 down-regulated. Fourty-eight of them could be identified with high confidence and belonged to the same above categories. Differential expression of Rho-GDP dissociation inhibitor (Rho GDI), secretory pathway GDI, clathrin, Sec 31 (subunit of exocyst complex) and RAB GTPase Ypt51, is being reported for the first time in response to an antifungal drug and may represent specific response of A. fumigatus to AMB. The expression of some of these genes was validated by real-time reverse transcription-PCR (RT-PCR). The AMB responsive genes/proteins observed to be differentially expressed in A. fumigatus, may be further explored for novel drug development.

• Characterization of bZip-type transcription factor AtfA with reference to stress responses of conidia of Aspergillus nidulans. by Hagiwara D, Asano Y, Yamashino T, Mizuno T. Abstract
  

  Microorganisms growing in natural habitats are permanently confronted with a wide variety of external stresses. In fungi, including Aspergillus nidulans, one of the general and ultimate strategies to survive in harsh habitats is the development of stress-tolerant conidia. In this study, we found that one of the basic-region leucine zipper transcription factors, named AtfA, plays a crucial role in the acquisition of tolerance against oxidative and heat stress on conidia, but not on mycelia, although this factor is not involved in asexual development per se.

• Posaconazole as salvage therapy in patients with invasive fungal infections after solid organ transplant by Alexander BD, Perfect JR, Daly JS, Restrepo A, Tobón AM, Patino H, Hardalo CJ, Graybill JR. Abstract
  

  BACKGROUND: The incidence of invasive fungal infections (IFIs) in solid organ transplant (SOT) recipients has increased during the past 20 years and is associated with significant morbidity and mortality. In this post hoc analysis of a large, open-label, multicenter study, we evaluated efficacy and safety of posaconazole, a new extended-spectrum triazole, as salvage therapy for IFIs in SOT recipients. METHODS: Twenty-three SOT recipients with proven or probable IFI and evidence of disease refractory to, or intolerant of, standard antifungal therapies received posaconazole oral suspension (40 mg/mL) 800 mg daily in divided doses. An independent, blinded data-review committee assessed patient diagnosis and outcome. RESULTS: Complete or partial response was documented in 13 of 23 (57%) SOT recipients with proven or probable IFIs, including 1 of 2 (50%) refractory patients, 5 of 8 (63%) intolerant to prior therapy, and 7 of 13 (54%) who were both. Successes by type of IFI included 7 of 12 with invasive aspergillosis, 2 of 2 with invasive fusariosis, 1 of 1 with cryptococcosis, and 1 of 2 with zygomycosis. Treatment-related adverse events (TRAEs) were reported in 12 of 23 patients. Severe TRAEs occurred in 4 of 23 patients including increased levels of cyclosporine or tacrolimus requiring immunosuppressive dose adjustments in three patients and in one, termination of posaconazole. Severe TRAEs associated with renal and liver toxicities were uncommon. CONCLUSION: Posaconazole was well tolerated and effective against IFIs including invasive aspergillosis, zygomycosis, fusariosis, and cryptococcosis in SOT recipients intolerant of or failing other antifungal therapies. Calcineurin inhibitor levels should be closely monitored in patients treated concomitantly with posaconazole to avoid toxicity from drug interaction.

• The effect of temperature on Natural Antisense Transcript (NAT) expression in Aspergillus flavus by Smith CA, Robertson D, Yates B, Nielsen DM, Brown D, Dean RA, Payne GA. Abstract
  

  Naturally occurring Antisense Transcripts (NATs) compose an emerging group of regulatory RNAs. These regulatory elements appear in all organisms examined, but little is known about global expression of NATs in fungi. Analysis of currently available EST sequences suggests that 352 cis NATs are present in Aspergillus flavus. An Affymetrix GeneChip((R)) microarray containing probes for these cis NATs, as well as all predicted genes in A. flavus, allowed a whole genome expression analysis of these elements in response to two ecologically important temperatures for the fungus. RNA expression analysis showed that 32 NATs and 2,709 genes were differentially expressed between 37 degrees C, the optimum temperature for growth, and 28 degrees C, the conducive temperature for the biosynthesis of aflatoxin (AF) and many other secondary metabolites. These NATs correspond to sense genes with diverse functions including transcription initiation, carbohydrate processing and binding, temperature sensitive morphogenesis, and secondary metabolism. This is the first report of a whole genome transcriptional analysis of NAT expression in a fungus.

• Aspergillus fumigatus conidia upregulates NOD2 protein expression both in vitro and in vivo by Zhang HJ, Qu JM, Shao CZ, Zhang J, He LX, Yuan ZH. Abstract
  

  AIM: To determine if NOD2 is involved in host recognition of Aspergillus fumigatus (Af) conidia. METHODS: An Af conidia pulmonary infection murine model was established by intranasal inoculation of Af conidia suspensions. Protein levels of NOD2 in lung tissue were determined by immunohistochemistry. A549 and phorbol-12-myristate 13-acetate (PMA)-activated THP-1 cell lines were treated with heat-killed Af conidia, then the presence of NOD2 protein in these cell lines was detected by Western blotting. The ability of muramyl dipeptide (MDP) to induce the secretion of TNF-alpha after incubation with heatkilled Af conidia was measured by enzyme-linked immunosorbent assay. RESULTS: The expression of NOD2 protein in lung tissue increased after Af conidia infection. Heat-killed Af conidia significantly upregulated NOD2 protein expression in A549 cells and PMA-activated THP-1 cells. Additionally, Af conidia in conjuction with MDP, significantly increased the secretion of TNF-alpha in A549 cells and PMA-activated THP-1 cells. CONCLUSION: Af conidia upregulates NOD2 protein expression in vitro and in vivo. These findings indicate that NOD2 protein may respond to Af conidia.

• Phase-dependent antifungal activity against Aspergillus fumigatus developing multicellular filamentous biofilms. by Mowat E, Lang S, Williams C, McCulloch E, Jones B, Ramage G. Abstract
  

  Objectives Aspergillus fumigatus undergoes morphological transition throughout its growth and development. These changes have direct implications for the effectiveness of antifungal treatment. Here we report the in vitro antifungal activity of voriconazole, amphotericin B and caspofungin against three specific phases of multicellular development of A. fumigatus. Methods A. fumigatus conidia were propagated for 8, 12 and 24 h prior to antifungal challenge. The resultant activity of the three agents tested was determined using an XTT reduction assay to assess both endpoint and time-kill susceptibility profiles. Results Endpoint susceptibility testing demonstrated a time-dependent decrease in efficacy for all three antifungal agents as the complexity of the A. fumigatus hyphal structure developed. Overall, amphotericin B exhibited the best spectrum of activity at each phase of growth, but was comparable to voriconazole against germinated conidial growth (8 h). Later, both voriconazole and caspofungin were ineffective against complex mycelial structures (12 and 24 h). Time-kill studies demonstrated that amphotericin B was significantly more efficacious at reducing A. fumigatus metabolism than both voriconazole and caspofungin for all three growth phases examined, most notably after 1 h of drug exposure (P < 0.001). Conclusions Overall, the data presented demonstrate that treatment of actively growing A. fumigatus cells with antifungal agents is more efficacious than treating mature structures in vitro. Amphotericin B was consistently more effective against each phase and displayed rapid effects, and therefore may be a suitable option for managing patient groups at risk from aspergillosis infections.

• Bioluminescent Aspergillus fumigatus: A new tool for drug efficiency testing and in vivo monitoring of invasive aspergillosis. by Brock M, Jouvion G, Droin-Bergère S, Dussurget O, Nicola MA, Ibrahim-Granet O. Abstract
  

  Aspergillus fumigatus is the main cause of invasive aspergillosis in immunocompromised patients and only a limited number of drugs for treatment are available. A screening method for new antifungal compounds is urgently required, preferably an approach suitable for in vitro and in vivo studies. Bioluminescence imaging is a powerful tool to study the temporal and spatial resolution of the infection and the effectiveness of antifungal drugs. Here, we describe the construction of a bioluminescent A. fumigatus strain by fusing the promoter of the glyceraldehyde-3-phosphate dehydrogenase from A. fumigatus with the luciferase gene from Photinus pyralis to control the expression of the bioluminescent reporter. A. fumigatus transformed with this construct revealed high bioluminescence under all tested growth conditions. Furthermore, light emission correlated with the amount of conidia used for inoculation and with the biomass formed after different incubation times. The bioluminescent strains were suitable to study the effectiveness of antifungals in vitro by several independent methods, including the determination of light emission in a microplate reader and the direct visualisation of light emission in an IVIS 100 system. Moreover, when glucocorticoid treated immunosuppressed mice were infected with a bioluminescent strain, light emission was detected from infected lungs, allowing the visualisation of the progression of invasive aspergillosis. Therefore, this new bioluminescent tool is suitable to study the in vitro effectiveness of drugs, the disease development, localisation and burden of fungi within tissues and may also provide a powerful tool to study the effectiveness of antifungals in vivo.

• Effective lead selection for improved protein production in Aspergillus niger based on integrated genomics. by Jacobs DI, Olsthoorn MM, Maillet I, Akeroyd M, Breestraat S, Donkers S, van der Hoeven RA, van den Hondel CA, Kooistra R, Lapointe T, Menke H, Meulenberg R, Misset M, Müller WH, van Peij NN, Ram A, Rodriguez S, Roelofs MS, Roubos JA, van Tilborg MW, Verkleij AJ, Pel HJ, Stam H, Sagt CM. Abstract
  

  The filamentous fungus Aspergillus niger is widely exploited for industrial production of enzymes and organic acids. An integrated genomics approach was developed to determine cellular responses of A. niger to protein production in well-controlled fermentations. Different protein extraction methods in combination with automated sample processing and protein identification allowed quantitative analysis of 898 proteins. Three different enzyme overproducing strains were compared to their isogenic fungal host strains. Clear differences in response to the amount and nature of the overproduced enzymes were observed. The corresponding genes of the differentially expressed proteins were studied using transcriptomics. Genes that were up-regulated both at the proteome and transcriptome level were selected as leads for generic strain improvement. Up-regulated proteins included proteins involved in carbon and nitrogen metabolism as well as (oxidative) stress response, and proteins involved in protein folding and endoplasmic reticulum-associated degradation (ERAD). Reduction of protein degradation through the removal of the ERAD factor doaA combined with overexpression of the oligosaccharyl transferase sttC in A. niger overproducing beta-glucuronidase (GUS) strains indeed resulted in a small increase in GUS expression.

• The CYPome (Cytochrome P450 complement) of Aspergillus nidulans. by Kelly DE, Kraševec N, Mullins J, Nelson DR. Abstract
  

  The cytochromes P450 (CYPs) are found in all biological kingdoms and genome sequencing projects continue to reveal an ever increasing number. The principle aim of this paper is to identify the complete CYPome of Aspergillus nidulans from the genome sequence version AN.3 deposited at the Broad institute, assign the appropriate CYP nomenclature and define function where possible. The completed analysis revealed a total of 111 CYP genes, 3 of which were previously unknown and 8 pseudogenes, representing 89CYP families, 21 of which are unique. We have identified 28 potential gene clusters associated with one or more CYP genes and discussed those with putative PKS and NRPS associated function. The chromosomal location of the genes, predicted cellular location of the proteins and possible function(s) are discussed.

• Rapid method for testing the susceptibility of Aspergillus fumigatus to amphotericin B, itraconazole, voriconazole and posaconazole by assessment of oxygen consumption by Araujo R, Coutinho I, Espinel-Ingroff A. Abstract
  

  Objectives Antifungal stress conditions affect fungal germination and growth. The assessment of oxygen consumption resulting from the challenge of Aspergillus fumigatus conidia with antifungal agents might be predictive of the susceptibility of this species to the agents evaluated. Methods The antifungal susceptibilities of A. fumigatus to amphotericin B, itraconazole, voriconazole and posaconazole were evaluated for 20 clinical strains by two methods: the rapid assessment of oxygen consumption and the CLSI M38-A2 microdilution method. For the determination of oxygen consumption, conidia were suspended in RPMI 1640 medium with two different concentrations of each antifungal drug (0.25 and 2 mg/L); the oxygen consumption was quantified in a biological oxygen monitor. Results A. fumigatus strains showed a wide spectrum of amphotericin B, itraconazole and voriconazole MICs (0.06 to >16 mg/L), but posaconazole MICs ranged from 0.06 to 1 mg/L. Distinct respiratory kinetics, which corresponded to the MIC results, were found. Strains with the highest itraconazole and voriconazole MICs grew faster, undoubtedly consuming the oxygen available in the liquid medium. The reproducibility of this new method was adequate (87%), as well as the agreement with the CLSI method (85%). Conclusions Although the potential of this new and rapid method (4-8 versus 48 h CLSI method) for evaluating the susceptibility of A. fumigatus to the antifungal agents has been demonstrated by these preliminary results, further collaborative studies with more isolates should better assess the value of this methodology for testing isolates in the clinical laboratory.

• Microbiological investigations of corrosion damages in aircraft by A. Hagenauer, R. Hilpert and T. Hack Abstract
  

  involves the investigation of the involvement of microorganisms in corrosion damagc in aircraft. In a first step, the microflora of corrosion failures was investigated. Swab samples were taken from corroded sites and were cultured on different media under different conditions. Media and incubation conditions were suitable for the growth of acrobic and anaerobic heterotrophic bacteria, sulfate reducing bacteria, Thiobacillus (7:) thiooxidans, T. ferrooxidans. fungi, and yeasts. The isolatcd microorganisms were identified using standard methods like microscopy, physiological tests and the tcst systems API and BIOLOG. From 46 simples, taken from corroded sites of 7 different aeroplanes, a total of 208 microorganisms were isolated (158 bacteria, 36 yeasts, 14 mycelium-forming fungi!. The isolates were cxamined in thc laboratory with regard to their corrosive action towards an aluminium alloy, commonly used in aircraft construction (Al-7075). Material samples were incubated in liquid media. which were inocculated with purc cultures of the respective isolates, over a period of 28 days. The degree of corrosion was determined using quantimetric analysis. Significant damages were examined metallographically, Results show that the corrosive effect of thc isolated microorganisms was quite different. Isolates of the genera Micrococcus, Enterococcus, Staphylococcus, Aerococcus, Bacillus, Aspergillus, and Penicillium provoked strong corrosion towards A1-7075.

• Factors involved in the aerosol transmission of infection and control of ventilation in healthcare premises by J.W. Tang, Y. Li, I. Eames, P.K.S. Chan, G.L. Ridgway Abstract
  

  Summary The epidemics of severe acute respiratory syndrome (SARS) in 2003 highlighted both short- and long-range transmission routes, i.e. between infected patients and healthcare workers, and between distant locations. With other infections such as tuberculosis, measles and chickenpox, the concept of aerosol transmission is so well accepted that isolation of such patients is the norm. With current concerns about a possible approaching influenza pandemic, the control of transmission via infectious air has become more important. Therefore, the aim of this review is to describe the factors involved in: (1) the generation of an infectious aerosol, (2) the transmission of infectious droplets or droplet nuclei from this aerosol, and (3) the potential for inhalation of such droplets or droplet nuclei by a susceptible host. On this basis, recommendations are made to improve the control of aerosol-transmitted infections in hospitals as well as in the design and construction of future isolation facilities.

• The aerobic air microflora in airplanes on various international routes by Năstoiu I, Răduică C, Soitu V, Gavrilă I. Abstract
  

  Aerobic microflora (bacteria, fungi), in the cock pits of the TAROM company (Boeing 707 and Il 62 M) airships flying on various international routes and airports was studied during November 1988-January 1989. 157-8,800 bacteria and 78-1,336 fungi per m3 air were recorded. Except for Staphylococcus aureus (hemolytic and non hemolytic) the greatest part of the isolated microorganisms was nonpathogenic for man: Bacillus, Corynebacterium, Neisseria, Staphylococcus epidermidis, Sarcina, Aspergillus, Penicillium etc. Several airships on the Asian airports contained a higher amount of bacteria and fungi but not higher than in the living rooms. Likewise, in high altitude flights, the microorganism amount was less than on the ground. The taxonomic spectrum of the bacteria and fungi isolated was almost identical on all the 9 international airports, thus suggesting the homogeneous and international character of saprophyte and pathogenic air microflora by means of the passenger and goods air flights.

• Aspergillus fumigatus Majocchi's granuloma in a patient with acquired immunodeficiency syndrome by Saadat P, Kappel S, Young S, Abrishami M, Vadmal MS. Abstract
  

  Majocchi's granuloma, also known as nodular granulomatous perifolliculitis, is an uncommon fungal infection of the skin and subcutaneous tissue. It can occur in healthy and immunocompromised patients. The most common cause of Majocchi's granuloma is Trichophyton rubrum. We report a case of a Majocchi's granuloma caused by Aspergillus fumigatus in a patient with acquired immunodeficiency syndrome

• Hematopoietic stem cell transplantation in patients with active fungal infection: not a contraindication for transplantation by Aki ZS, Sucak GT, Yeğin ZA, Güzel O, Erbaş G, Senol E Abstract
  

  Increasing use of more aggressive treatment approaches in patients with hematologic malignancies leads to an increased frequency of invasive fungal infections, which is a major cause of transplant-related mortality in hematopoietic stem cell recipients. In this respect, the presence of an active fungal infection prior to transplantation may hinder subsequent hematopoietic stem cell transplantation (HSCT); which sometimes is the only curative treatment. We report here the results of 13 consecutive patients transplanted with active fungal infection. Thirteen patients (7 males and 6 females) with a median age of 34 years (range, 16-53 years) underwent 15 HSCT between September 2003 and April 2007. In this group of 15 patients, consisting of hematologic malignancies with high risk of relapse or severe aplastic anemia, 11 (73%) transplants performed in subjects with active invasive fungal infection (IFI) patients survived 30 days after transplantation. Three patients (1 patient with primary disease relapse, 1 patient with graft versus host disease [GVHD] complicated with fungal pneumonia, and 1 patient with severe sinusoidal obstruction syndrome and GVHD complicated with aspiration pneumonia) died on days +66, +74, and +62 posttransplantation, respectively, within the first 100 days of HSCT. After a median follow-up time of 306 days (range, 145-680 days), four of 13 (31%) patients with active IFI were alive and disease free. Among a population of HSCT recipients with a dismal prognosis without transplantation, performing the procedure despite active IFI saved a considerable proportion of the patients. The presence of active IFI did not seem to be an absolute contraindication for HSCT, particularly among high-risk patients in whom a treatment delay could be fatal

• Aspergillus flavus myositis in a patient after liver transplantation by Li DM, Xiu DR, Li RY, Samson RA, de Hoog GS, Wang DL Abstract
  

  We describe the first case of Aspergillus myositis caused by Aspergillus flavus in a liver transplant patient. The patient was a 43-year-old man who underwent liver transplantation because of end-stage hepatic cirrhosis. He experienced pain in his left calf two months after the operation. Nodules with weakness, swelling, and flaring were found in the calf two wk later. Color ultrasonic examination showed uneven resonance in the left gastrocnemius. Needle aspiration and biopsy of the muscle revealed septate hyphae consistent with Aspergillus species and focal necrosis of the muscle cells with inflammatory cell infiltration. A culture subsequently yielded A. flavus, confirming histopathologic diagnosis. Sequencing of the internal transcribed spacer region confirmed the morphologic identification. The patient was first given itraconazole 0.2 g twice daily for one wk and was then switched to terbinafine 0.25 g once a day. A three-month regimen of terbinafine therapy cured the infection, though the cultured fungus showed resistance to a number of antifungal agents. Aspergillus, a genus of ubiquitous molds, may cause invasive and even fatal disease in immunosuppressed patients

• Comparison of the value of measurement of serum galactomannan and Aspergillus-specific antibodies in the diagnosis of canine sino-nasal aspergillosis by Billen F, Peeters D, Peters IR, Helps CR, Huynen P, De Mol P, Massart L, Day MJ, Clercx C. Abstract
  

  Serology is currently used for the diagnosis of canine sino-nasal aspergillosis (SNA). However, the accuracy of serological testing using commercially available, standardized purified antigen preparations of Aspergillus (CAPurAspAg) has only been poorly documented. The aim of the present study was to assess the diagnostic value of an agar-gel double immunodiffusion (AGDD) test and an anti-Aspergillus IgG ELISA, using CAPurAspAg and the commercially available Plateliatrade mark test for the detection of serum galactomannan. Sera from 17 dogs with SNA, 18 dogs with a nasal tumour (NT), 11 dogs with lymphoplasmacytic rhinitis (LPR) and 33 control dogs were tested with the 3 methods. AGDD result was positive in 76.5% of dogs with SNA, whereas all sera from dogs with non-fungal nasal disease and control dogs were negative. A positive IgG ELISA result was obtained in 88% of dogs with SNA and in 18% of dogs with LPR. All patients with NT and control dogs had a negative IgG ELISA result. The Plateliatrade mark test was positive in 24% of dogs with SNA, 11% of dogs with NT, 9% of dogs with LPR and 24% of control dogs. The results of this study suggest that (1) the detection of serum Aspergillus-specific antibodies with AGDD or ELISA, using CAPurAspAg, provides excellent specificity and good sensitivity, (2) the specificity is higher for AGDD (100%) than for ELISA (96.8%) while sensitivity is higher for ELISA (88.2%) than for AGDD (76.5%) and (3) serum galactomannan quantification with the Plateliatrade mark test is unreliable for the diagnosis of canine SNA

• Genomic characterization of a novel partitivirus infecting Aspergillus ochraceus by Liu W, Duns G, Chen J. Abstract
  

  Three double-stranded RNA (dsRNA) segments from Aspergillus ochraceus isolate FA 0611, designated as AoR1, AoR2, and AoR3, were cloned and sequenced. AoR1 was identical with AoV dsRNA 1 previously reported from A. ochraceus ATCC 28706, which putatively encoded an RNA-dependent RNA polymerase of an unidentified mycovirus. AoR2 was found to encode a putative viral capsid protein (CP) with 63% similarity to that of Penicillium stoloniferum virus S, which was detected from P. stoloniferum ATCC 14586. The function of AoR3 was unknown. The three segments were found to contain a conserved sequence at their 5' termini, while an identical sequence was only found at the 3' termini of AoR1 and AoR2. It is suggested that AoR1, AoR2, and AoR3 originate from an independent partitivirus infecting A. ochraceus. The novel virus is suggested to be Aspergillus ochraceus virus 1, AoV1

• A simple and reproducible 96-well plate-based method for the formation of fungal biofilms and its application to antifungal susceptibility testing by Pierce CG, Uppuluri P, Tristan AR, Wormley FL Jr, Mowat E, Ramage G, Lopez-Ribot JL. Abstract
  

  The incidence of fungal infections has increased significantly over the past decades. Very often these infections are associated with biofilm formation on implanted biomaterials and/or host surfaces. This has important clinical implications, as fungal biofilms display properties that are dramatically different from planktonic (free-living) populations, including increased resistance to antifungal agents. Here we describe a rapid and highly reproducible 96-well microtiter-based method for the formation of fungal biofilms, which is easily adaptable for antifungal susceptibility testing. This model is based on the ability of metabolically active sessile cells to reduce a tetrazolium salt (2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide) to water-soluble orange formazan compounds, the intensity of which can then be determined using a microtiter-plate reader. The entire procedure takes approximately 2 d to complete. This technique simplifies biofilm formation and quantification, making it more reliable and comparable among different laboratories, a necessary step toward the standardization of antifungal susceptibility testing of biofilms

• Molecular characterisation of cyp51A and cyp51B genes coding for P450 14alpha-lanosterol demethylases A (CYP51Ap) and B (CYP51Bp) from voriconazole-resistant laboratory isolates of Aspergillus flavus by Krishnan-Natesan S, Chandrasekar PH, Alangaden GJ, Manavathu EK. Abstract
  

  Aspergillus flavus is the second most common Aspergillus spp. causing invasive infections in immunocompromised patients. Extensive prophylactic use of voriconazole (VCZ) in immunocompromised patients may enhance the selection of VCZ-resistant clinical isolates of A. flavus, compromising the effectiveness of this antifungal drug against A. flavus infection. To study triazole resistance, we selected A. flavus isolates in the laboratory showing reduced in vitro susceptibility to VCZ. The cyp51A and cyp51B genes coding for P450 14alpha-sterol demethylases A (CYP51Ap) and B (CYP51Bp) were characterised to examine possible drug target modification-dependent resistance to VCZ in this fungus. High-molecular-weight DNA was isolated from 10 A. flavus isolates showing in vitro resistance to VCZ (minimum inhibitory concentration (MIC) range 4-32mug/mL) as well as from the drug-susceptible parent isolate X26728 (MIC=1mug/mL). The cyp51A and cyp51B genes were cloned and the nucleotide sequences were determined. A comparison of the deduced amino acid sequences of CYP51Ap from 10 VCZ-resistant isolates with that of the drug-susceptible parent showed no amino acid variation in six of the ten isolates. CYP51Ap from isolates Afl-VCZ6 and Afl-VCZ46 showed a K197N change, CYP51Ap from isolate Afl-VCZ114 showed Y132N and T469S changes, whereas that from isolate Afl-VCZ45 showed K197N, D282E and M288L changes. These results suggest that VCZ-resistant A. flavus isolates can be readily isolated in the laboratory under selection pressure. Multiple mechanisms, including drug target modification, may be responsible for the in vitro resistance of A. flavus to VCZ

• The level of sulfane sulfur in the fungus Aspergillus nidulans wild type and mutant strains by Wróbel M, Lewandowska I, Bronowicka-Adamska P, Paszewski A. Abstract
  

  The interdependence of the sulfane sulfur metabolism and sulfur amino acid metabolism was studied in the fungus Aspergillus nidulans wild type strain and in mutants impaired in genes encoding enzymes involved in the synthesis of cysteine (a precursor of sulfane sulfur) or in regulatory genes of the sulfur metabolite repression system. It was found that a low concentration of cellular cysteine leads to elevation of two sulfane sulfurtransferases, rhodanase and cystathionine gamma-lyase, while the level of 3-mercaptopyruvate sulfurtransferase remains largely unaffected. In spite of drastic differences in the levels of biosynthetic enzymes and of sulfur amino acids due to mutations or sulfur supplementation of cultures, the level of total sulfane sulfur is fairly stable. This stability confirms the crucial role of sulfane sulfur as a fine-tuning regulator of cellular metabolism

• Sub-telomere directed gene expression during initiation of invasive aspergillosis by McDonagh A, Fedorova ND, Crabtree J, Yu Y, Kim S, Chen D, Loss O, Cairns T, Goldman G, Armstrong-James D, Haynes K, Haas H, Schrettl M, May G, Nierman WC, Bignell E Abstract
  

  Aspergillus fumigatus is a common mould whose spores are a component of the normal airborne flora. Immune dysfunction permits developmental growth of inhaled spores in the human lung causing aspergillosis, a significant threat to human health in the form of allergic, and life-threatening invasive infections. The success of A. fumigatus as a pathogen is unique among close phylogenetic relatives and is poorly characterised at the molecular level. Recent genome sequencing of several Aspergillus species provides an exceptional opportunity to analyse fungal virulence attributes within a genomic and evolutionary context. To identify genes preferentially expressed during adaptation to the mammalian host niche, we generated multiple gene expression profiles from minute samplings of A. fumigatus germlings during initiation of murine infection. They reveal a highly co-ordinated A. fumigatus gene expression programme, governing metabolic and physiological adaptation, which allows the organism to prosper within the mammalian niche. As functions of phylogenetic conservation and genetic locus, 28% and 30%, respectively, of the A. fumigatus subtelomeric and lineage-specific gene repertoires are induced relative to laboratory culture, and physically clustered genes including loci directing pseurotin, gliotoxin and siderophore biosyntheses are a prominent feature. Locationally biased A. fumigatus gene expression is not prompted by in vitro iron limitation, acid, alkaline, anaerobic or oxidative stress. However, subtelomeric gene expression is favoured following ex vivo neutrophil exposure and in comparative analyses of richly and poorly nourished laboratory cultured germlings. We found remarkable concordance between the A. fumigatus host-adaptation transcriptome and those resulting from in vitro iron depletion, alkaline shift, nitrogen starvation and loss of the methyltransferase LaeA. This first transcriptional snapshot of a fungal genome during initiation of mammalian infection provides the global perspective required to direct much-needed diagnostic and therapeutic strategies and reveals genome organisation and subtelomeric diversity as potential driving forces in the evolution of pathogenicity in the genus Aspergillus

• In vitro activity at pH 5.0 and pH 7.0 and in vivo efficacy of flucytosine against Aspergillus fumigatus. by Verweij PE, Te Dorsthorst DT, Janssen WH, Meis JF, Mouton JW. Abstract
  

  The antifungal agent flucytosine was found to be active in vitro against A. fumigatus isolates when the MIC was determined at pH 5.0 instead of pH 7.0. The in vitro MIC at pH 5.0 corresponded with in vivo efficacy of flucytosine monotherapy in a murine model of invasive aspergillosis

• Increased risk for invasive aspergillosis in patients with lymphoproliferative diseases after autologous hematopoietic SCT by Gil L, Kozlowska-Skrzypczak M, Mol A, Poplawski D, Styczynski J, Komarnicki M. Abstract
  

  The risk of invasive aspergillosis (IA) is considered to be low among autologous HSCT recipients, but an increase in the incidence has been observed recently in this setting. The aim of the study was to assess the influence of immunosuppressive drugs (steroids, rituximab, fludarabine, thalidomide), used in treatment of lymphoid malignancies during 6 months of pretransplant period, on IA incidence after autologous HSCT. A total of 109 patients with non-Hodgkin's lymphoma (NHL), Hodgkin's disease (HD) and multiple myeloma (MM), conditioned with carmustine, etoposide, cytarabine, melphalan or melphalan and transplanted with PBSC, were analyzed prospectively. Patients were monitored with twice-weekly galactomannan test. High-resolution computed tomograhy of the chest and bronchoscopy were performed in case of positive galactomanan test, persistent fever or pulmonary infiltrates. Documented IA was diagnosed in nine (8%) patients (three proven, six probable). The incidence of IA was comparable in NHL, HD and MM patients and not influenced by age, advanced disease or conditioning regimen. Factors significant for development of documented IA by univariate analysis were treatment with fludarabine (P=0.008) or rituximab (P=0.039). The only factor predicting documented IA by multivariate analysis was treatment with fludarabine (P=0.008). Patients treated with fludarabine or rituximab in pretransplant period are at risk of IA and require close monitoring and/or anti-mould prophylaxis.Bone Marrow Transplantation advance online publication, 15 September 2008; doi:10.1038/bmt.2008.303

• Aspergillus mycetoma of the Maxillary Sinus Secondary to Overfilling of a Root Canal by Luciano Giardino, MD, DDS, Francesco Pontieri, MD, Enrico Savoldi, MD, DDS, and Federico Tallarigo, MD Abstract
  

  In nonimmunocompromised patients aspergillosis of the paranasal sinuses is a relatively rare disease. Root canal treated teeth with overextension of the root canal sealer or solid materials such as gutta-percha or silver cones into the sinus might be the main etiological factor for aspergillosis of the maxillary sinus in healthy patients. Root-filling materials based zinc oxide-eugenol is considered to be a growth factor for aspergillus. Aspergillus fumigatus needs heavy metals such as zinc oxide for proliferation and metabolism. Prognostic and histological studies showed that instrumentation and obturation should not extend beyond the apical foramen. When the sealer and/or gutta-percha is extruded within the sinus, this produce an inflammatory reaction and then Aspergillus growth. We report one case of healthy 60-yr-old male with overextension of root canal sealer in maxillary sinus. After surgical procedure, microscopic examination revealed aspergillosis. Overextension into the maxillary sinus with root canal cements has to be avoided

• Aspergillosis of the Maxillary Sinus as a Complication of Overfilling Root Canal Material into the Sinus: Report of Two Cases by Pathawee Khongkhunthian, DDS, Dr. med. dent., and Peter A. Reichart, Dr. med. dent. Abstract
  

  Aspergillosis of the maxillary sinus is a relatively rare disease in nonimmunocompromised patients. In recent years a number of cases of aspergillosis of the maxillary sinus have been reported in association with overextension of root canals fillings with certain root canal cements. It has been suggested that zinc oxide-based root canal cements might promote the infection with the Aspergillus species. In particular Aspergillus fumigatus has been found to be associated with the maxillary sinus infection. Radiographically the unique appearance of a dense opacity foreign body reaction in the maxillary sinus was considered a characteristic finding in maxillary sinus aspergillosis. Because this association of overfilling of root canal cements and aspergillosis of the maxillary sinus is not too well known we report two cases of young healthy female patients with the characteristic findings, both radiographically and clinically. In both patients the first maxillary molar was involved. Patients were symptomless and the diagnosis was made accidently. However at surgical inspection both patients revealed aspergillomas, including the overextended root canal cement. The surgical procedure is described as are the microscopic findings in both cases showing the characteristic branching hyphae and conidophores typical of Aspergillus. Overextension into the maxillary sinus with root canal cements has to be avoided; material has to be removed from the sinus because otherwise aspergillosis infection may ensue.

• Tratamiento de la Aspergilosis: Guı´as para la pra´ctica clı´nica de la Sociedad de Enfermedades Infecciosas de los Estados Unidos de Ame´rica (IDSA) by Thomas J. Walsh, Elias J. Anaissie, David W. Denning, Raoul Herbrecht, Dimitrios P. Kontoyiannis, Kieren A. Marr, Vicki A. Morrison, Brahm H Segal, William J. Steinbach, David A. Stevens, Jo-Anne van Burik, John R. Wingard, Thomas F. Patterson Abstract
  

  RESUMEN EJECUTIVO El ge´nero Aspergillus se ha revelado como una causa importante de infecciones mortales en pacientes inmunocomprometidos. Esta poblacio´n en expansio´n se compone de pacientes que padecen neutropenia prolongada, infeccio´n por VIH avanzada e inmunodeficiencia hereditaria y de pacientes sometidos a trasplante aloge´nico de ce´lulas madre hematopoye´ticas (TCMH) o a trasplante pulmonar. Este documento compila las guı´as de la Sociedad de Enfermedades Infecciosas de Estados Unidos de Ame´rica para el tratamiento de la aspergilosis y reemplaza las guı´as para la pra´ctica clı´nica de Aspergillus publicadas en 2000 [1]. El objetivo de estas guı´as es resumir la evidencia cientı´fica actual para el tratamiento de las distintas formas de aspergilosis. La calidad de la evidencia cientı´fica para el tratamiento se califica segu´n el sistema esta´ndar que utiliza la Sociedad de Enfermedades Infecciosas en otras guı´as. Este documento revisa las guı´as terape´uticas de las 3 principales formas de aspergilosis: aspergilosis invasiva, formas cro´nicas (y saprofı´ticas) de la aspergilosis y formas ale´rgicas de la aspergilosis. Dada la importancia que ha adquirido la aspergilosis invasiva en la salud pu´ blica, se destaca el diagno´ stico, el tratamiento y la prevencio´n de las distintas formas de esta enfermedad, incluyendo la aspergilosis pulmonar invasiva, aspergilosis sinusal, aspergilosis diseminada y diversos tipos de aspergilosis invasiva de o´ rgano u´nico. Son pocos los ensayos aleatorizados sobre el tratamiento de la aspergilosis invasiva. El mayor ensayo controlado aleatorizado demuestra que el voriconazol supera a la anfotericina B deoxycolato (D-AMB) como tratamiento primario para la aspergilosis invasiva. En la mayorı´a de los pacientes se recomienda el voriconazol como tratamiento primario de la aspergilosis invasiva (A-1). Si bien la mayor parte de los casos tratados con voriconazol corresponden a la aspergilosis pulmonar invasiva, este fa´rmaco se ha utilizado en suficientes casos de infecciones extrapulmonares y diseminadas como para que esto permita inferir que el voriconazol es eficaz en estos casos. Un ensayo aleatorizado que T2 • CID 2008:46 (1 February) • Walsh et al. comparo´ 2 dosis de anfotericina B liposomal (L-AMB, por su sigla en ingle´s) demostro´ una eficacia similar en los dos grupos, lo que sugiere que el tratamiento con L-AMB podrı´a considerarse un tratamiento primario alternativo en algunos pacientes (A-I). Para el tratamiento de u´ ltimo recurso, se utilizan formulaciones lipı´dicas de anfotericina (LFAB por su sigla en ingle´s; A-II), posaconazol (B-II), itraconazol (B-II), caspofungina (B-II) o micafungina (B-II). El tratamiento de u´ ltimo recurso para la aspergilosis invasiva plantea desafı´os importantes con vacı´os significativos de conocimiento. En la aspergilosis resistente al voriconazol, existe una escasez de datos que puedan guiar el abordaje terape´utico. Un cambio de clase con el empleo de una formulacio´n con una anfotericina B (AMB) o una equinocandina, como la caspofungina (B-II), se encuentra entre las opciones terape´uticas; una mayor utilizacio´n de azoles debe tener en cuenta los factores relacionados con el anfitrio´n y los aspectos farmacocine´ticos. La infeccio´n resistente puede responder a un cambio de clase de fa´rmaco (B-II) o a una combinacio ´n de agentes (B-II). El papel que desempen˜a la combinacio ´n de opciones terape´uticas en el tratamiento de la aspergilosis invasiva como tratamiento primario o de u´ ltimo recurso es incierto y justifica la realizacio´n de un ensayo clı´nico controlado prospectivo. Es probable que resulte difı´cil evaluar pacientes con aspergilosis resistente al tratamiento. Al hacerlo, es preciso establecer el diagno´ stico de aspergilosis invasiva si previamente era incierto y confirmarlo si se lo conocı´a previamente. Es necesario considerar la dosificacio´n del fa´rmaco. Entre las opciones terape ´uticas se encuentran un cambio a la vı´a intravenosa (IV), determinacio´n de las concentraciones del fa´rmaco, cambio de la clase de fa´rmaco y combinaciones de fa´rmacos. La profilaxis antifu´ ngica con posaconazol es recomendada en el subgrupo de receptores de TCMH con enfermedad del injerto contra el hue´sped (EICH) que tienen alto riesgo de contraer aspergilosis invasiva y en pacientes neutrope´nicos con leucemia mielo´ gena aguda o sı´ndrome mielodispla´sico, todos con un riesgo alto de contraer aspergilosis invasiva (A-I). Los datos del ensayo clı´nico no definen el tratamiento de la aspergilosis invasiva intercurrente en los casos de profilaxis con azoles antifu´ ngicos. Es preciso que se individualice el abordaje de estos pacientes de acuerdo con criterios clı´nicos, tales como inmunosupresio´n del anfitrio´n, enfermedad subyacente y foco infeccioso, ası´ como la consideracio´n de la dosis antimico´ tica, la determinacio´n de las concentraciones de fa´rmacos, un cambio a la vı´a terape´utica IV y un cambio a otra clase de fa´rmaco (B-III). Ciertas variantes de la aspergilosis invasiva justifican que se considere la reseccio´n quiru´ rgica del foco infeccioso. Entre e´stas se encuentran las lesiones pulmonares cercanas al corazo´n o los grandes vasos, invasio´n de la pared tora´cica, osteomielitis, infeccio´n perica´rdica y endocarditis (B-III). Es fundamental lograr la recuperacio´n de las defensas deterioradas del anfitrio´n para mejorar el desenlace de la aspergilosis invasiva (A-III). La recuperacio´n de la neutropenia en los casos de neutropenia persistente o la reduccio´n de los corticoesteroides en un paciente que recibe altas dosis de glucocorticoesteroides es primordial para mejorar el desenlace en la aspergilosis invasiva. Cabe hacer una consideracio´n especial en cuanto a las recomendaciones para tratar la aspergilosis en localizaciones infrecuentes, como la osteomielitis y la endocarditis. Los datos sobre estas infecciones son muy limitados y la mayorı´a considera la D-AMB como tratamiento primario solo por su biodisponibilidad prolongada. El grupo de expertos recomienda el voriconazol para el tratamiento primario de estas manifestaciones muy infrecuentes de la aspergilosis invasiva (B-III), basa ´ndose en la solidez del estudio aleatorizado. El abordaje terape´utico de las formas cro´nicas o saprofı´ticas de la aspergilosis varı´a segu´n la enfermedad. La reseccio´n quiru ´ rgica es el mejor tratamiento para los aspergilomas pulmonares simples (B-III), mientras que el tratamiento farmacolo´ gico a largo plazo es necesario para la aspergilosis pulmonar necrotizante cro´nica y la cavitaria cro´nica (B-III) Las formas ale´rgicas de aspergilosis se tratan con una combinacio ´n de tratamientos me´dicos y antiinflamatorios. Por ejemplo, en el tratamiento de la aspergilosis broncopulmonar ale´rgica (ABPA) se administra itraconazol y corticoesteroides (A-I). INTRODUCCIO´ N Considerado hasta el momento como una causa inusual de infeccio´n, el ge´nero Aspergillus se ha revelado como una causa importante de morbilidad y mortalidad en pacientes inmunocomprometidos [2–4]. En la actualidad, la aspergilosis invasiva constituye la causa ma´s frecuente de mortalidad por neumonı´a infecciosa en pacientes sometidos a TCMH y una causa importante de infeccio´n diseminada y respiratoria oportunista en otros pacientes inmunocomprometidos [5–11]. Adema ´s, el ge´nero Aspergillus tambie´n produce una amplia variedad de enfermedades cro´nicas, saprofı´ticas y ale´rgicas. Si bien otras formas de aspergilosis, como la ABPA, la sinusitis ale´rgica y la infeccio´n saprofı´tica, tambie´n son causas de morbilidad, rara vez son potencialmente mortales. En este documento, las recomendaciones terape´uticas se califican de acuerdo al sistema de calificacio´n esta´ndar de la Sociedad de Enfermedades Infecciosas de los Estados Unidos y del Servicio de Salud Pu´blica de los Estados Unidos para recomendaciones de calificacio´n en guı´as clı´nicas, segu´n el resumen de la tabla 1.

• Factors associated with overall and attributable mortality in invasive aspergillosis by Nivoix Y, Velten M, Letscher-Bru V, Moghaddam A, Natarajan-Amé S, Fohrer C, Lioure B, Bilger K, Lutun P, Marcellin L, Launoy A, Freys G, Bergerat JP, Herbrecht R. Abstract
  

  BACKGROUND: Invasive aspergillosis is associated with high death rates. Factors associated with increased mortality have not yet been identified in a large population of patients with various underlying conditions. METHODS: We retrospectively reviewed 385 cases of suspected or documented aspergillosis that occurred during a 9-year period. We identified 289 episodes that fulfilled the criteria for possible, probable, or proven invasive aspergillosis according to the international definition criteria and that was treated with an anti-Aspergillus active antifungal drug. Clinical and microbiological variables were analyzed for their effects on overall and attributable mortality. Significant variables in univariate analysis were introduced into a multivariate Cox model. RESULTS: Twelve-week overall and disease-specific survival rates were 52.2% (95% confidence interval, 46.5%-57.9%) and 59.8% (95% confidence interval, 54.0%-65.4%), respectively. Receipt of allogeneic hematopoietic stem cell or solid-organ transplant, progression of underlying malignancy, prior respiratory disease, receipt of corticosteroid therapy, renal impairment, low monocyte counts, disseminated aspergillosis, diffuse pulmonary lesions, pleural effusion, and proven or probable (as opposed to possible) aspergillosis are predictors of increased overall mortality. Similar factors are also predictors of increased attributable mortality, with the following exceptions: pleural effusion and low monocyte counts have no impact, whereas neutropenia is associated with a higher attributable mortality. CONCLUSIONS: Identification of predictors of death helps in the identification of patients who could benefit from more-aggressive therapeutic strategies. Initiation of therapy at the stage of possible infection improves outcome, and this finding calls for the development of efficient preemptive strategies to fill the gap between empirical and directed therapy.

(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.)