Genetic diversity and antifungal susceptibility patterns of Aspergillus nidulans complex obtained from clinical and environmental sources.

The molecular epidemiology and antifungal susceptibility of Aspergillus nidulans species complex has not been well studied. To evaluate the genetic diversity and antifungal susceptibility patterns of clinical and environmental isolates of A. nidulans complex. Sixty clinical and environmental isolates of Aspergillus section Nidulantes were collected from five countries (Iran, The Netherlands, Spain, Portugal and Greece). The species were molecularly identified by sequencing of ß-tubulin gene. The genetic diversity of A nidulans complex isolates (n = 54) was determined with a microsatellite genotyping assay. Antifungal susceptibility profile was determined using EUCAST method. The isolates were classified as A nidulans (46.7%), A spinulosporus (26.6%), A quadrilineatus (10%), A pachycristatus (3.3%), A rugulosus (3.3%), A unguis (5%), A creber, (1.7%), A olivicola (1.7%) and A sydowii (1.7%). Thirty-four sequence types (STs) were identified among the 54 A nidulans complex isolates. A high level of genetic diversity was found among A nidulans sensu stricto strains but low diversity was found among A spinulosporus strains. Amphotericin B showed high MICs to all species. The most active azole was posaconazole (GM = 0.64 mg/L), while itraconazole showed the highest MICs among azoles (GM = 2.95 mg/L). A spinulosporus showed higher MICs than A nidulans sensu stricto for all antifungals except for micafungin and anidulafungin. Interspecies variations may result in differences in antifungal susceptibility patterns and challenge antifungal therapy in infections caused by A nidulans. Differences in the distribution of STs or persistence of multiple STs might be related to the sources of isolation and niche specialisation.

In Vitro Combination of Isavuconazole with Echinocandins against Azole-Susceptible and -Resistant Aspergillus spp.

In vitro combinations of isavuconazole with echinocandins were evaluated against 30 Aspergillus strains with a two-dimensional checkerboard microdilution method and an agar-based diffusion method. With the checkerboard method, the three combinations showed indifferent interactions for all strains. With the agar-based method, indifferent interactions were found for all strains for isavuconazole-micafungin and isavuconazole-anidulafungin. For the isavuconazole-caspofungin combination, indifference was found in 24/30 strains, synergism in 4/30 strains, and antagonism in 2/30 strains.

Fungal osteomyelitis in a patient with chronic granulomatous disease: Case report and review of the literature.

Chronic granulomatous disease (CGD) is the most common of the primary immunodeficiency in children. It is caused by single gene defect resulting in dysfunctional nicotinamide adenine dineucleotide phosphate (NADPH) oxidase complex causing recurrent bacterial and fungal infections. Here we present the case of a 9 year old boy who was a known case of CGD since three years of age. He presented with recent history of fever, left sided pain in the scapular region and difficulty in breathing. Chest imaging revealed developing left upper lobe consolidation and erosion of the 3rd posterior rib. The child underwent video assisted thoracoscopic surgery (VATS) and biopsy of the lesion. Histopathology revealed fungal hyphae which were confirmed to be Aspergillus nidulans on staining. He was successfully treated with voriconazole therapy. We will also review the literature on fungal osteomyelitis in CGD patients.

Chronic granulomatous disease of childhood: an unusual cause of recurrent uncommon infections in a 61-year-old man.

Chronic granulomatous disease (CGD) is a rare congenital immunodeficiency that affects 1 : 250,000 of the population, which is characterized by recurrent bacterial and fungal infections and by granuloma formation. We investigated a 61-year-old man presented with a 20-year history of a relapsing skin rash appearing as mildly pruritic and erythematous plaques affecting various body regions. Cutaneous biopsies were taken and sent for histology and tissue culture. Leucocyte function was assessed by determining the generation of reactive oxygen species. Bactericidal activity was assessed in the presence of autologous and homologous sera. Western blotting was performed for protein analysis of the reduced nicotinamide adenine dinucleotide phosphate oxidase system, and mutation screening was carried out using PCR amplification and sequence analysis. Examination of biopsies obtained from lesional skin indicated a suppurative granulomatous process. Tissue cultures grew Aspergillus nidulans and Aspergillus fumigatus (confirmed by PCR). A. nidulans has often been associated with CGD, and the leucocyte function tests supported this diagnosis. Direct DNA sequencing led to the identification of a hemizygous missense novel mutation in CYBB (c.907C>T), which predicts a p.His303Tyr amino-acid substitution in gp91-phox, thus confirming the diagnosis of CGD. In conclusion, we report a case of a rare inherited immunodeficiency, CGD, in a 61-year-old man, and describe the novel hemizygous missense mutation underlying the condition. Mild forms of usually fatal immunodeficiencies should be considered when assessing the occurrence of unusual infectious diseases in apparently healthy people.

Formate-nitrite transporters: optimisation of expression, purification and analysis of prokaryotic and eukaryotic representatives.

The formate-nitrite transporter family is composed of integral membrane proteins that possess six to eight alpha-helical transmembrane domains. Genes encoding these proteins are observed widely in prokaryotic genomes as well as certain groups of lower eukaryotes. Thus far, no structural information is available for this transporter family. Towards this aim, and to provide protein for biophysical studies, overexpression of a prokaryotic (TpNirC, from the hyperthermophilic archaebacterium Thermofilum pendens) and an eukaryotic (AnNitA, from the fungus Aspergillus nidulans) representative was achieved in Escherichia coli and Pichia pastoris hosts, respectively. The proteins were purified to >95% homogeneity yielding quantities sufficient for crystallisation trials and were shown by Circular Dichroism (CD) spectroscopy to have a highly alpha-helical content as expected from in silico predictions. Preliminary investigations by size exclusion chromatography of the oligomeric state of the purified AnNitA protein suggested that it most likely exists as a tetramer.

Simultaneous detection of Aspergillus nidulans, Aspergillus luchuensis and Lichtheimia sp. in a bovine abortion.

Abortion in dairy cattle may be caused by infectious (viruses, fungi and protozoa) and non-infectious causes mostly related to bad management practices and genetic factors. Recently, the significant contribution of mycotic infection to bovine abortion has been recognized. This report describes an abortion case in a Chianina cow due to Aspergillus nidulans, Aspergillus luchuensis and Lichtheimia sp. diagnosed by histology, cytology, culture and molecular assays. A mixed infection due to more than one fungus in abortion is rarely demonstrated. To our knowledge, this is the first case of bovine abortion caused by co-infection with three different moulds.

Molecular identification, phylogenetic analysis and antifungal susceptibility patterns of Aspergillusnidulans complex and Aspergillusterreus complex isolated from clinical specimens.

OBJECTIVE: Aspergillus sections Terrei and Nidulantes are the less common causes of invasive aspergillosis and pulmonary aspergillosis (PA) in immunocompromised patients when compared to A. fumigatus and A. flavus. Identifying these fungi as the infectious agent is crucial because of the resistance to amphotericin B (AMB) and increased lethality. The aim of this study was to identify the molecular status, evaluate the genetic diversity and examine the antifungal susceptibility profile of the uncommon Aspergillus species. Forty-five uncommon Aspergillus species were identified based on the microscopic and macroscopic criteria. Then, the molecular identification was performed using the sequencing beta tubulin (benA) gene. In vitro antifungal susceptibility to amphotericin B (AMB), itraconazole (ITC), ravuconazole (RAV), voriconazole (VRC), caspofungin (CFG) isavuconazole (ISA) and posaconazole (POS) test was performed according to the CLSI M38-A2 guidelines. RESULTS: A. terreus was the most species detected, followed by A. nidulans, A. latus, A.ochraceus, and A. citrinoterreus, respectively. The analysis of the benA gene showed the presence of 12 distinct genotypes among the A. terreus isolates. The other species did not show any intraspecies variation. CFG exhibited the lowest MEC50/MIC50 (0.007µg/mL), followed by POS (0.125µg/mL), VRC, ITC, ISA (0.25µg/mL), RAV (0.5µg/mL), and AMB (8µg/mL). Among all the isolates, only 15.5% (7/45) were susceptible to AMB. CONCLUSION: Antifungal susceptibility pattern of the uncommon Aspergillus species is useful to improve patient management and increase knowledge concerning the local epidemiology. Moreover, this information is necessary when an outbreak dealing with drug-resistant infections occurs.

Aspergillus mycoviruses are targets and suppressors of RNA silencing.

RNA silencing can function as a virus defense mechanism in a diverse range of eukaryotes, and many viruses are capable of suppressing the silencing machinery targeting them. However, the extent to which this occurs between fungal RNA silencing and mycoviruses is unclear. Here, three Aspergillus dsRNA mycoviruses were partially characterized, and their relationship to RNA silencing was investigated. Aspergillus virus 1816 is related to Agaricus bisporus white button mushroom virus 1 and suppresses RNA silencing through a mechanism that alters the level of small interfering RNA. Aspergillus virus 178 is related to RNA virus L1 of Gremmeniella abietina and does not appear to affect RNA silencing. The third virus investigated, Aspergillus virus 341, is distantly related to Sphaeropsis sapinea RNA virus 2. Detection of mycovirus-derived siRNA from this mycovirus demonstrates that it is targeted for degradation by the Aspergillus RNA silencing machinery. Thus, our results indicate that Aspergillus mycoviruses are both targets and suppressors of RNA silencing. In addition, they suggest that the morphological and physiological changes associated with some mycoviruses could be a result of their antagonistic relationship with RNA silencing.

Sequence analysis of isolates of Aspergillus from patients with chronic and allergic aspergillosis reveals a spectrum of cryptic species.

AIM: To establish the prevalence and antifungal susceptibilities of Aspergillus cryptic species from respiratory samples. Methods: Retrospective susceptibility data on Aspergillus species cultured between 2015 and 2017 by 'high volume culture' (HVC) versus 'conventional' culture techniques. RESULTS: Fifty-six (2.5%) isolates were identified as Aspergillus cryptic species by sequencing of ITS, BenA and CalM gene loci. Recovery was higher in HVCs compared to conventional cultures. Common cryptic species were Aspergillus montevidensis (n = 15), A. creber (n = 11), A. sydowii (n = 5) and A. calidoustus (n = 4). Eighteen (32.1%) isolates had minimum inhibitory concentration (MIC) values ≥4 mg/l to amphotericin B, and 19.1-60.1% had MIC values ≥8 mg/l to the triazoles. CONCLUSION: HVC increases the likelihood of recovery of cryptic species. MIC values to antifungals were high.

Isolation of Aspergillus nidulans from a case of fungal rhinosinusitis: a case report.

A rare species i.e. A. nidulans is reported as a causative agent of allergic fungal rhinosinusitis in this study. It is an increasingly recognized type of chronic recurring hypertrophic sinus disease. There are more than 185 species of aspergillus and over 95% of all infections are caused by A. fumigatus, A. flavus and A. niger. A. fumigatus alone accounts for the large majority of cases of both invasive and non invasive aspergillosis. A young immunocompetent lady presented with bilateral nasal obstruction due to multiple polypoid mass at Sheth Vadilal Sarabhai General Hospital, Ahmedabad. Provisional diagnosis of sinonasal polyposis possibly due to fungal cause with infiltration in to nasal cavity was made. Bilateral functional endoscopic sinus surgery with polypectomy was done. The specimen was examined by standard methods and the fungus was identified as A. nidulans by slide culture.

Isolation of cell wall mutants in Aspergillus nidulans by screening for hypersensitivity to Calcofluor White.

As a first step toward identifying novel genes of wall metabolism in filamentous fungi, we have screened a collection of Aspergillus nidulans mutants for strains exhibiting hypersensitivity toward the chitin binding agent Calcofluor White (CFW). This strategy has been used previously to identify cell wall mutants in Saccharomyces cerevisiae. We have identified 10 mutants representing eight loci, designated calA through calH, for Calcofluor hypersensitivity. All cal mutants are impaired for sporulation at 30 C or 42 C or both, and in eight of the 10 mutations this sporulation defect shows at least partial osmotic remediability. All cal mutants show elevated sensitivity to one or more of the following agents: Caspofungin, Nikkomycin, Tunicamycin, Congo red and SDS, which are recognized wall-compromising agents or have been shown to be inhibitory to wall integrity mutants in yeast. Seven of the 10 cal mutants show swelling at elevated temperature, which in most cases is osmotically remediable. Spore swelling also can be induced at 30 C in all but one of the cal mutants by germination in the presence of one or more of the following: Caspofungin, Nikkomycin or Tunicamycin. Analysis of wall sugars showed no major changes in mutant strains. We also report that the chitin synthase inhibitor Nikkomycin induces excessive spore swelling during germination in all tested strains that have wild type cell wall metabolism (GR5, A4, A28 and AH12) at 42 C but not at 30 C. This effect mimics that of certain temperature-sensitive swollen cell (swo) mutations.

[Diagnosis and treatment of aspergillosis in the patients with chronic granulomatous disease]. / Diagnostyka i leczenie zakazen wywolanych przez Aspergillus spp. u pacjentów z przewlekla choroba ziarniniakowa.

Chronic granulomatous disease is a rare defect of phagocytosis. Increased susceptibility to infections is limited to catalase positive bacteria and fungi. Aspergillus spp was reported as the increased clinical problem and the main cause of the deaths.

The first case of fungal endophthalmitis caused by Emericella nidulans after cataract surgery.

Emericella nidulans is a species that has only rarely been implicated in human disease after cataract surgery. Here, we report the first postoperative case in the literature, as far as we know. The patient was a 50-year-old patient presented with mild anterior uveitis one week after cataract surgery, and hypopion developed over the next two days. First microbiological evaluation and the results of direct microscopy and cultures of the anterior chamber and vitreous samples were found to be negative. Despite vigorous topical and intravitreal (vancomycin and amikacin) therapy, the endophthalmitis did not improve. Anterior chamber paracentesis, vitreous tap and finally complete vitrectomy with removal of the capsular bag including the intraocular lens (IOL) were performed. The anterior chamber, vitreous fluid samples and IOL were submitted to the microbiology laboratory: the culture yielded E. nidulans growth. Ocular inflammation resolved and vision improved on intravenous, subconjunctival and long-term oral voriconazole treatment. E. nidulans can be an important cause of ocular fungal infections including endophthalmitis, and voriconazole seems to be effective for the treatment of E. nidulans endophthalmitis.

Cloning of a new bidirectionally selectable marker for Aspergillus strains.

Mutants that lack adenosine triphosphate sulfurylase (ATPsase; EC 2.7.7.4) are unable to use sulfate as sole source of sulfur and are also resistant to selenate. These mutants, denoted sC-, are readily obtained from any strain of Aspergillus niger or Aspergillus nidulans by the strong selection for selenate resistance. We have cloned the gene encoding ATPsase from A. nidulans by complementation of an sC mutant strain of A. nidulans with a gene library and show that plasmids containing this gene transform both A. niger and A. nidulans sC- strains, restoring their ability to grow on sulfate as sole sulfur source. The fact that strong selection for either sC+ or sC- can be applied provides a simple way of delivering genetically engineered constructs to any strain of A. niger including strains of industrial importance. In addition, this system is useful for gene replacements and other genomic DNA manipulations in Aspergillus species.

Quantification of DNA damage and repair in amino acid auxotrophs and UV-sensitive mutants of Aspergillus nidulans using an ELISA.

An ELISA used to investigate DNA repair in mammalian cells has been adapted to investigate mutagen-induced DNA damage and repair in protoplasts of Aspergillus nidulans. The assay shows a reduced rate of repair of DNA damage in methionine and arginine auxotrophs (methG and argB), which were shown previously to be hypersensitive to UV radiation and chemical mutagens. The assay also showed a considerably reduced ability to repair mutagen-induced damage in the uv-sensitive mutants uvsB and uvsH. The increased sensitivity of amino acids auxotrophs to mutagens is, therefore, correlated with a reduced capacity to repair mutagen-induced DNA damage.

Successful elimination of an invasive Aspergillus nidulans lung infection by voriconazole after failure of a combination of caspofungin and liposomal amphotericin B in a boy with chronic granulomatous disease.

Chronic granulomatous disease is an inherited defect in host defense mechanisms mainly affecting neutrophil function. Pulmonary infection with Aspergillus nidulans in a child with chronic granulomatous disease could not be eliminated by a combination of caspofungin and liposomal amphotericin B. Voriconazole was successful in clearing the infection.

Chronic granulomatous disease of childhood. An unusual case of infection with Aspergillus nidulans var. echinulatus.

Aspergillus nidulans var. echinulatus was the sole agent cultured from the left lung, a paraspinal abscess, left ribs, and thoracic vertebral bodies from a patient with chronic granulomatous disease. Hyphal elements were present in histologic sections of lung, vertebral bodies, and infected ribs along with granuloma formation. The patient was treated with two debridement procedures and insertion of a Harrington rod followed by a long course of amphotericin B, flucytosine, and daily white blood cell transfusions.

Intrabronchial Aspergillus nidulans infection in an immunocompetent man.

We describe the first report of intrabronchial Aspergillus nidulans infection in an immunocompetent patient, which fit the description of bronchocentric granulomatosis. The patient had a history of accidental aspiration of light grade oil. Fiberoptic bronchoscopy revealed that the right B4aii alpha was obstructed. Endobronchial biopsy specimens contained fungal hyphae. The fungus was confirmed to be Aspergillus nidulans by culture. We suspected that aspiration of light grade oil had injured the bronchial mucosa, after which airborne Aspergillus nidulans had entered the lesion and multiplied. Intrabronchial fungal infection can occur in a healthy person without immunologic abnormalities, if a bronchial lesion provides an entry portal.