Autopsy imaging for aspergillosis in King Penguin, an economically valuable zoo animal.

Autopsy imaging (Ai) was performed for a King Penguin. Ai-computed tomography (CT) revealed air sac membrane thickening, multiple nodules in the cranial air sac, suspected abscess, lung infiltration, and air sac contraction. Based on these findings, respiratory disorder was concerned. Aspergillosis, which is the highly observed in penguins, was considered as the primary differential diagnosis. The cultured sample showed characteristic conidial head of Aspergillus spp., the DNA of which was 100% identical to that of A. fumigatus. The cause of death was determined to respiratory failure due to aspergillosis. Ai-CT findings facilitated the dissection workflow and alerted the pathologist to potential hazards during the autopsy. Ai is useful to determine the cause of death and for readiness and safe pathological dissection.

A simple and low cost tetra-primer ARMS-PCR method for detection triazole-resistant Aspergillus fumigatus.

The mutation at codon L98 accompanied by a tandem repeat of 34 base pairs (TR34/L98H) in the 5´upstream region of cyp51A is the principal mechanism of triazole resistance of Aspergillus fumigatus. We aimed to evaluate a simple and low-cost tetra-primer amplification refractory mutation system (ARMS)-PCR technique for detection of TR34/L98H mutations in the cyp51A gene of azole-resistant A. fumigatus. The tetra-primer ARMS-PCR assay optimized by four primers in one reaction consists of external primers for detection of tandem repeats in the promoter region and internal primers for detection of a point mutation in codon 98 (L98H) in the cyp51A gene of azole-resistant A. fumigatus. The specificity of TR34/L98H mutation detection was assessed by testing 36 clinical and environmental A. fumigatus strains. The tetra-primer ARMS-PCR assay from A. fumigatus, containing wild-type sequence (T allele) and L98H mutation at cyp51A (A allele), yielded two DNA fragments of 908 bp and 740 bp and two of 942 bp and 212 bp, respectively. None of the A. fumigatus isolates without the TR34/L98H mutation yielded false-positive results. The ARMS-PCR assay was 100% concordant with DNA sequencing results. Prevalence and screening of the TR34/L98H mutation in the cyp51A gene in A. fumigatus isolates may now be determined by a fast, low-cost, and simple method in resource-poor settings.

Determination of azole fungal residues in soils and detection of Aspergillus fumigatus-resistant strains in market gardens of Eastern France.

Medical azole antifungals are major compounds used to prevent and to treat invasive aspergillosis (IA). Azole fungicides, called DMI (14-alpha demethylase inhibitors), are also widely used for crop protection and have been reported to be linked to azole-resistant A. fumigatus (aR-Af) development in the environment. The aim of this study was to determine whether or not market gardens that spray DMIs in Eastern France are also affected by the presence of aR-Af. Forty aR-Afs were detected in soils in only two of the four market gardens using DMIs, with 23% (7/30) and 10% (3/30) of soils containing aR-Af. A total of 87.5% of these isolates had the TR34/L98H mutation and 22.5% the TR46/Y121F/T289A mutation on the cyp51A gene. Analyses of residual azole concentrations in soils showed the presence of difenoconazole for up to 2 years after spraying, but only in soils of market gardens where aR-Af was detected. It is very important to identify professional activities that could lead to aR-Af development and to develop preventive measures for at-risk patients living near professional activities using DMIs. We have to better understand why, in some cases, the use of DMI is not linked to aR-Af. Measures should be taken to avoid the use of DMI conferring cross-resistance to preserve the efficiency of human therapeutics.

Simple, low-cost molecular assays for TR34/L98H mutations in the cyp51A gene for rapid detection of triazole-resistant Aspergillus fumigatus isolates.

Simple, low-cost PCR/PCR-restriction fragment length polymorphism (RFLP) assays targeting cyp51A promoter and codon 98 regions were developed for the detection of triazole-resistant Aspergillus fumigatus strains carrying TR34/L98H mutations. The assays were evaluated using 40 itraconazole-susceptible isolates and 35 itraconazole-resistant isolates. The prevalence of TR34/L98H mutations in clinical/environmental A. fumigatus isolates may now be determined easily from resource-poor settings.

Assessment of selection and resistance risk for demethylation inhibitor fungicides in Aspergillus fumigatus in agriculture and medicine: a critical review.

BACKGROUND: An increasing number of publications have claimed that demethylation inhibitor (DMI) fungicides are confronted with resistance development in the fungus Aspergillus fumigatus and that the origin of resistant isolates may also be outside the medical area. For resistance risk assessment and sourcing the origin of DMI resistance, the primary exposure events ofA. fumigatus with DMI treatments have been analysed case by case, resulting in the pathogen exposure risk (PER). RESULTS: The calculated maximum exposure concentrations (MEC) are highest during medical treatments (human and veterinary), certain fruit and seed treatments and wood preservation, and are much lower for crop protection applications. Most agricultural DMIs are intrinsically ∼10-100 times less active than medical DMIs for A. fumigatus control and potential resistance selection. However, imazalil is used in agriculture and veterinary medicine (as enilconazole) expressing strong intrinsic activity against A. fumigatus. The majority of mutations in the target gene, cyp51, of DMI-resistant isolates are different in A. fumigatus(e.g. TR34/L98H) in comparison with plant pathogens (e.g. A379G, I381V). CONCLUSIONS: The assumed selection risk, ASR (MEC × PER) for resistance evolution to DMIs in A. fumigatus is estimated to be highest for human and veterinary applications. However, environmental origin of DMI-resistant spores from certain sites cannot be ruled out.

Assessment of Aspergillus fumigatus in guinea pig bronchoalveolar lavages and pulmonary tissue by culture and realtime polymerase chain reaction studies.

In this study we pursued a diagnostic target in Aspergillus fumigatus (AF) by using qualitative Realtime PCR combined with proprietary DNA primers and a hydrolysis probe specific for this fungal target. Qualitative Realtime PCR is a diagnostic tool that utilizes Realtime PCR technology and detects the presence or absence target specific DNA within a predetermined detection range. Respiratory tissue and fluids from experimentally infected guinea pigs were tested by extracting DNA from the samples which were amplified and detected using AF specific DNA primers and probe. This study included qualitative evaluations of all specimens for the presence of the DNA of AF. The findings in the tissues after AF infection were compared to the numbers of spores in aerosolized samples used to inoculate the animals. Results demonstrated that the specific probe and primer set could detect the presence or absence of AF DNA in the sample. The qualitative detection limit of the assay ranged from 6 × 10(4) copies to 6 copies. Since blood cultures are rarely positive for Aspergillosis, our data indicate that qualitative Realtime PCR, in combination with the appropriate DNA primers and probe can serve as an effective diagnostic tool in the early detection of fungal infections.

Rapid identification of Aspergillus fumigatus within the section Fumigati.

BACKGROUND: New fungal species that are morphologically similar to Aspergillus fumigatus were recently described and included in section Fumigati. Misidentification of such fungal species, particularly of the human pathogens, Aspergillus lentulus, Neosartorya fischeri, Neosartorya hiratsukae, Neosartorya pseudofischeri and Neosartorya udagawae, has been increasingly reported by numerous clinical labs. Nevertheless, A. fumigatus still accounts for more than 90% of all invasive aspergillosis cases. The purpose of the present study was to develop a rapid method for the molecular identification of A. fumigatus to distinguish it from other species within the section Fumigati. RESULTS: A multiplex PCR was developed using prior information based on ß-tubulin (ßtub) and rodlet A (rodA) partial gene sequences. PCR amplification of ßtub and rodA fragments resulted in a distinctive electrophoretic pattern in A. fumigatus and N. udagawae. The polymorphisms found in the smallest amplified sequence of ßtub (153 bp) and rodA (103 bp) genes were then compared among and within species of this taxonomic section. ßtub was able to differentiate among 13 individual species and two groups of species that included the pathogenic fungus A. lentulus. A more limited number of sequences were available for rodA; nevertheless, we were able to distinguish Aspergillus viridinutans, N. hiratsukae and N. udagawae. CONCLUSIONS: The assay described in the present study proved to be specific and highly reproducible, representing a fast and economic way of targeting molecular identification of the relevant mould, A. fumigatus, in clinical laboratories.

Molecular epidemiology and virulence assessment of Aspergillus fumigatus isolates from white stork chicks and their environment.

Aspergillus fumigatus is a common pathogen in poultry and captive wild birds and an emerging opportunistic fungal pathogen in immunocompromised humans. Although invasive aspergillosis is frequently reported in free-ranging wild birds, the incidence and epidemiology of the disease in a natural setting is unknown. We recently reported endemic outbreaks of invasive aspergillosis at white stork nesting sites close to human habitation in Germany with significant subsequent breeding losses. Therefore, we hypothesized that A. fumigatus strains with higher virulence in birds may have evolved in this environment and performed the first epidemiological analysis of invasive aspergillosis in free-ranging wild birds. Sixty-one clinical and environmental A. fumigatus isolates from six affected nesting sites were genotyped by microsatellite analysis using the STRAf-assay. The isolates showed a remarkable high genomic diversity and, contrary to the initial hypothesis, clinical and environmental isolates did not cluster significantly. Interestingly, storks were infected with two to four different genotypes and in most cases both mating types MAT-1.1 and MAT-1.2 were present within the same specimen. The majority of selected clinical and environmental strains exhibited similar virulence in an in vivo infection model using embryonated chicken eggs. Noteworthy, virulence was not associated with one distinct fungal mating type. These results further support the assumption that the majority of A. fumigatus strains have the potential to cause disease in susceptible hosts. In white storks, immaturity of the immune system during the first three weeks of age may enhance susceptibility to invasive aspergillosis.

Assessment of Aspergillus fumigatus burden in pulmonary tissue of guinea pigs by quantitative PCR, galactomannan enzyme immunoassay, and quantitative culture.

Early diagnosis of invasive pulmonary aspergillosis is problematic in some patient groups due to the lack of rapid, sensitive, specific, and reliable diagnostic tests. Fungal burden and therapeutic efficacy were assessed by survival, quantitative culture (CFU counts), galactomannan enzyme immunoassay (GM-EIA), and quantitative PCR (qPCR) in a new guinea pig model of invasive pulmonary aspergillosis using an aerosol challenge. At 1 day postinfection, qPCR determined that the pulmonary fungal burden was 2 log(10) higher than that determined by CFU counting and increased significantly (P < 0.03) over time. In contrast, the tissue burden assessed by CFU counting did not rise over the course of the study. Therapy with the antifungal drug voriconazole produced statistically significant decreases in pulmonary fungal burden, as detected by CFU counting (P < 0.02), qPCR, and GM-EIA (both P < 0.0002). Daily assessment of the progression of fungal infection in serum was performed by qPCR and GM-EIA. GM-EIA demonstrated a statistically significant reduction in the fungal load on days 6 and 7 in voriconazole-treated animals compared to time-matched controls (P < 0.02). Confirmation of fungal tissue burden by two or more methods should provide a more precise account of the burden, allowing improved assessment of diagnostic and therapeutic strategies in invasive pulmonary aspergillosis.

Overproduction, purification and characterization of FgaPT2, a dimethylallyltryptophan synthase from Aspergillus fumigatus.

A putative dimethylallyltryptophan synthase gene, fgaPT2, was identified in the genome sequence of Aspergillus fumigatus. fgaPT2 was cloned and overexpressed in Saccharomyces cerevisiae. The protein FgaPT2 was purified to near homogeneity and characterized biochemically. This enzyme was found to convert L-tryptophan to 4-dimethylallyltryptophan, a reaction known to be the first step in ergot alkaloid biosynthesis. FgaPT2 is a soluble, dimeric protein with a subunit size of 52 kDa, and contains no putative prenyl diphosphate binding site (N/D)DXXD. Km values for L-tryptophan and dimethylallyl diphosphate (DMAPP) were determined as 8 and 4 microM, respectively. Metal ions, such as Mg2+ and Ca2+, enhance the reaction velocity, but are not essential for the enzymic reaction. FgaPT2 showed a relatively strict substrate specificity for both tryptophan and DMAPP. FgaPT2 is the first enzyme in the biosynthesis of ergot alkaloids to be purified and characterized in homogeneous form after heterologous overproduction.

TigrScan and GlimmerHMM: two open source ab initio eukaryotic gene-finders.

UNLABELLED: We describe two new Generalized Hidden Markov Model implementations for ab initio eukaryotic gene prediction. The C/C++ source code for both is available as open source and is highly reusable due to their modular and extensible architectures. Unlike most of the currently available gene-finders, the programs are re-trainable by the end user. They are also re-configurable and include several types of probabilistic submodels which can be independently combined, such as Maximal Dependence Decomposition trees and interpolated Markov models. Both programs have been used at TIGR for the annotation of the Aspergillus fumigatus and Toxoplasma gondii genomes. AVAILABILITY: Source code and documentation are available under the open source Artistic License from http://www.tigr.org/software/pirate

Sequencing the Aspergillus fumigatus genome.

Aspergillus fumigatus is the most common mould pathogen of human beings and unusually causes both invasive disease in immunocompromised patients and allergic disease in patients with atopic immune systems. 4% of patients dying in modern European teaching hospitals have invasive aspergillosis and it is the leading infectious cause of death in leukaemia and bone marrow transplant patients. Until 2001, only two licensed antifungal drugs were available to treat aspergillosis-amphotericin B and itraconazole. Its 28-30Mb genome is being sequenced in an international collaboration, with the Wellcome Trust Sanger Institute (UK) and The Institute for Genomic Research (TIGR, USA) as the two main centres. A whole-genome shotgun approach was adopted and initiated in 2001 with an expected completion date in 2003. The complete sequence will permit identification of pathways specific to pathogenic Aspergillus species, help identify new targets for antifungal drugs, and enable investigations into the basic biology of fungi. Numerous secondary metabolic pathways with biotechnological applications and pharmacological properties are found in the Aspergilli and the genome sequence will facilitate research in this area.

An expression system matures: a highly efficient and cost-effective process for phytase production by recombinant strains of Hansenula polymorpha.

An efficient process was developed for the low-cost production of phytases using Hansenula polymorpha. Glucose or glucose syrups, previously reported as repressive substrates, were used as main carbon sources during fermentation. Glucose was even the most productive substrate for high-level production of phytases. Compared with the process using glycerol, the standard carbon source used for this process until now, the use of glucose led to a reduction of more than 80% in the raw materials costs. In addition, exceptionally high concentrations of active enzyme (up to 13.5 g/L) were obtained in the medium, with phytase representing over 97% of the total accumulated protein. These levels greatly exceed those reported so far for any yeast-based expression system. Very efficient downstream processing procedures were developed with product recovery yields over 90%. Both the fermentation and downstream processing were successfully tested in pilot scale up to 2000 L. As a result, H. polymorpha can be used as a highly competitive system for low-cost phytase production.