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Aspergillus spp. cause serious invasive lung infections, and Aspergillus fumigatus is the most commonly encountered clinically significant species. Voriconazole is considered to be the drug of choice for treating A. fumigatus infections; however, rising resistance rates have been reported. We evaluated a matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS)-based method for the differentiation between wild-type and non-wild-type isolates of 20 Aspergillus spp. (including 2 isolates of Aspergillus ustus and 1 of Aspergillus calidoustus that were used as controls due their intrinsic low azole susceptibility with respect to the in vitro response to voriconazole). At 30 and 48 h of incubation, there was complete agreement between Cyp51A sequence analysis, broth microdilution, and MALDI-TOF MS classification of isolates as wild type or non-wild type. In this proof-of-concept study, we demonstrated that MALDI-TOF MS can be used to accurately detect A. fumigatus strains with reduced voriconazole susceptibility. However, rather than proving to be a rapid and simple method for antifungal susceptibility testing, this particular MS-based method showed no benefit over conventional testing methods.

In the indoor environment, people are exposed to several fungal species. Evident dampness is associated with increased respiratory symptoms. To examine the immune responses associated with fungal exposure, mice are often exposed to a single species grown on an agar medium. The aim of this study was to develop an inhalation exposure system to be able to examine responses in mice exposed to mixed fungal species aerosolized from fungus-infested building materials. Indoor airborne fungi were sampled and cultivated on gypsum boards. Aerosols were characterized and compared with aerosols in homes. Aerosols containing 10 7 CFU of fungi/m 3 air were generated repeatedly from fungus-infested gypsum boards in a mouse exposure chamber. Aerosols contained Aspergillus nidulans , Aspergillus niger , Aspergillus ustus , Aspergillus versicolor , Chaetomium globosum , Cladosporium herbarum , Penicillium brevicompactum , Penicillium camemberti , Penicillium chrysogenum , Penicillium commune , Penicillium glabrum , Penicillium olsonii , Penicillium rugulosum , Stachybotrys chartarum , and Wallemia sebi . They were all among the most abundant airborne species identified in 28 homes. Nine species from gypsum boards and 11 species in the homes are associated with water damage. Most fungi were present as single spores, but chains and clusters of different species and fragments were also present. The variation in exposure level during the 60 min of aerosol generation was similar to the variation measured in homes. Through aerosolization of fungi from the indoor environment, cultured on gypsum boards, it was possible to generate realistic aerosols in terms of species composition, concentration, and particle sizes. The inhalation-exposure system can be used to study responses to indoor fungi associated with water damage and the importance of fungal species composition.

Invasive fungal infections due to Aspergillus calidoustus with decreased azole susceptibility are emerging in the setting of azole prophylaxis and are associated with poor outcomes. We assessed the in vitro activity of antifungal drugs used alone or in combinations against A. calidoustus and found a synergistic effect between voriconazole and terbinafine at concentrations within the therapeutic range.

Aspergillus ustus is a relatively rare human pathogen causing invasive infections in immunocompromised hosts. In this study isolates originating from clinical and other sources have been examined using molecular, morphological, and physiological approaches to clarify their species assignment. Phylogenetic analysis of partial β-tubulin, calmodulin, actin, and intergenic transcribed spacer sequences indicated that none of the clinical isolates recognized previously as A. ustus belongs to this species. All but two of these isolates formed a well-defined clade related to A. pseudodeflectus based on sequence analysis of protein-coding regions. Morphological and physiological examination of these isolates indicated that they are able to grow above 37°C, in contrast with A. ustus isolates, and give a positive Ehrlich reaction, in contrast with related species including A. granulosus, A. ustus , and A. pseudodeflectus . These isolates are proposed as a new species, A. calidoustus . Antifungal susceptibility testing showed that this species has decreased susceptibilities to several antifungal drugs. The triazoles are inactive in vitro, including the new azole posaconazole.

Molecular approaches are now being developed to provide a more rapid and objective identification of fungi compared to traditional phenotypic methods. Ribosomal targets, especially the large-subunit RNA gene (D1-D2 region) and internal transcribed spacers 1 and 2 (ITS1 and ITS2 regions), have shown particular promise for the molecular identification of some fungi. We therefore conducted an assessment of these regions for the identification of 13 medically important Aspergillus species: Aspergillus candidus , Aspergillus ( Eurotium ) chevalieri , Aspergillus ( Fennellia ) flavipes , Aspergillus flavus , Aspergillus fumigatus , Aspergillus granulosus , Aspergillus ( Emericella ) nidulans , Aspergillus niger , Aspergillus restrictus , Aspergillus sydowii , Aspergillus terreus , Aspergillus ustus , and Aspergillus versicolor . The length of ribosomal regions could not be reliably used to differentiate among all Aspergillus species examined. DNA alignment and pairwise nucleotide comparisons demonstrated 91.9 to 99.6% interspecies sequence identities in the D1-D2 region, 57.4 to 98.1% in the ITS1 region, and 75.6 to 98.3% in the ITS2 region. Comparative analysis using GenBank reference data showed that 10 of the 13 species examined exhibited a ≤1-nucleotide divergence in the D1-D2 region from closely related but different species. In contrast, only 5 of the species examined exhibited a ≤1-nucleotide divergence from sibling species in their ITS1 or ITS2 sequences. Although the GenBank database currently lacks ITS sequence entries for some species, and major improvement in the quality and accuracy of GenBank entries is needed, current identification of medically important Aspergillus species using GenBank reference data seems more reliable using ITS query sequences than D1-D2 sequences, especially for the identification of closely related species.

We developed a PCR-based assay to differentiate medically important species of Aspergillus from one another and from other opportunistic molds and yeasts by employing universal, fungus-specific primers and DNA probes in an enzyme immunoassay format (PCR-EIA). Oligonucleotide probes, directed to the internal transcribed spacer 2 region of ribosomal DNA from Aspergillus flavus , Aspergillus fumigatus , Aspergillus nidulans , Aspergillus niger , Aspergillus terreus , Aspergillus ustus , and Aspergillus versicolor , differentiated 41 isolates (3 to 9 each of the respective species; P < 0.001) in a PCR-EIA detection matrix and gave no false-positive reactions with 33 species of Acremonium , Exophiala , Candida , Fusarium , Mucor , Paecilomyces , Penicillium , Rhizopus , Scedosporium , Sporothrix , or other aspergilli tested. A single DNA probe to detect all seven of the most medically important Aspergillus species ( A. flavus , A. fumigatus , A. nidulans , A. niger , A. terreus , A. ustus , and A. versicolor ) was also designed. Identification of Aspergillus species was accomplished within a single day by the PCR-EIA, and as little as 0.5 pg of fungal DNA could be detected by this system. In addition, fungal DNA extracted from tissues of experimentally infected rabbits was successfully amplified and identified using the PCR-EIA system. This method is simple, rapid, and sensitive for the identification of medically important Aspergillus species and for their differentiation from other opportunistic fungi.

A case of invasive pulmonary aspergillosis in an allogeneic bone marrow transplant recipient caused by Aspergillus ustus is presented. A. ustus was also recovered from the hospital environment, which may indicate that the infection was nosocomially acquired. A literature review revealed seven cases of invasive infections caused by A. ustus , and three of these were primarily cutaneous infections. In vitro susceptibility testing of 12 A. ustus isolates showed that amphotericin B and terbinafine had fungicidal activity and that itraconazole and voriconazole had fungistatic activity.

Aspergillus ustus is an uncommon clinical species which is poorly susceptible to antifungals. We report two cases of A. ustus infections that occurred in allogeneic stem cell transplant recipients while they were receiving either voriconazole or caspofungin. Prolonged use of these new antifungal agents may increase the risk of the emergence of resistant organisms.

We report a case of primary cutaneous infection by the emerging fungus Aspergillus ustus in an immunosuppressed patient after a domestic accident. The patient failed to respond to itraconazole and died before receiving a new treatment with amphotericin B. There have been eight other cases reported since 1973, and only two patients survived the infection. In vitro susceptibility testing of seven antifungal drugs showed that terbinafine and the new azole derivative UR-9825 were the most active against this fungus.

The sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns of water-soluble proteins and the randomly amplified polymorphic DNA (RAPD) patterns of whole-cell lysates from 21 Aspergillus ustus isolates, including 11 reference strains and 10 patient and environmental strains from one hospital, were investigated. All isolates showed identical protein patterns. The RAPD assay discriminated between all reference strains. Comparison of hospital isolates showed identical RAPD patterns in some of the patient and environmental isolates. The data indicate that the RAPD technique is useful for fingerprinting A. ustus .

The first case of disseminated aspergillosis caused by Aspergillus ustus in an allogeneic peripheral stem cell transplant patient is described. The patient, a 46-year-old female with a history of myelodysplastic syndrome, underwent high-dose chemotherapy and total body irradiation prior to transplantation. She was released from the hospital 49 days posttransplant (p.t.) in a stable condition with an absolute neutrophil count (ANC) of 2,700 cells per μl. Multiple antimicrobial agents, including itraconazole (ITR), were prescribed during hospitalization and at the time of discharge. Three days after discharge, the patient was readmitted with hemorrhagic cystitis, persistent thrombocytopenia, and bilateral pulmonary consolidation, although no fever was present. The ANC at the time of readmission was 3,500. Upon detection of a pulmonary nodule (day 67 p.t.), a bronchoalveolar lavage was performed; the lavage fluid was positive for both cytomegalovirus and parainfluenza virus and negative for fungus. The patient was placed on ganciclovir. A biopsy specimen from a leg lesion also noted on day 67 p.t. revealed septate hyphae consistent with Aspergillus species, and a culture subsequently yielded Aspergillus ustus . Confirmation detection of A. ustus was made by demonstration of characteristic reproductive structures with the presence of Hülle cells. On day 67 p.t., ITR was discontinued and liposomal amphotericin B (AMB) was initiated. The patient’s condition worsened, and she died 79 days p.t. At the time of autopsy, septate hyphae were present in heart, thyroid, and lung tissues, with lung tissue culture positive for A. ustus . In vitro susceptibility testing indicated probable resistance to AMB but not to ITR. This case supports the need for the development of rapid methods to determine antifungal susceptibility.

A number of species belonging to the genus Aspergillus were evaluated for their toxicity to ducklings and the ability to produce sterigmatocystin. Three new species capable of producing sterigmatocystin were found, namely, Aspergillus aurantio-brunneus, Aspergillus quadrilineatus, and Aspergillus ustus. All three were toxic to ducklings. The production of sterigmatocystin by Aspergillus rugulosus was confirmed, and the toxicity of Aspergillus stellatus and Aspergillus multicolor is described.

Prolyl-2-(1′,1′-dimethylallyl)tryptophyldiketopiperazine and 12,13-dehydroprolyl-2-(1′,1′-dimethylallyl)tryptophyldiketopiperazine, known metabolites of Aspergillus ustus , were produced in low yield by Penicillium italicum in liquid culture and on unsterilized orange peel.