A prospective international Aspergillus terreus survey: an EFISG, ISHAM and ECMM joint study.

OBJECTIVES: A prospective international multicentre surveillance study was conducted to investigate the prevalence and amphotericin B susceptibility of Aspergillus terreus species complex infections. METHODS: A total of 370 cases from 21 countries were evaluated. RESULTS: The overall prevalence of A. terreus species complex among the investigated patients with mould-positive cultures was 5.2% (370/7116). Amphotericin B MICs ranged from 0.125 to 32 mg/L, (median 8 mg/L). CONCLUSIONS: Aspergillus terreus species complex infections cause a wide spectrum of aspergillosis and the majority of cryptic species display high amphotericin B MICs.

Performance of galactomannan, beta-d-glucan, Aspergillus lateral-flow device, conventional culture, and PCR tests with bronchoalveolar lavage fluid for diagnosis of invasive pulmonary aspergillosis.

Galactomannan detection in bronchoalveolar lavage (BAL) fluid samples (GM test) is currently considered the gold standard test for diagnosing invasive pulmonary aspergillosis (IPA). The limitations, however, are the various turnaround times and availability of testing. We compared the performance of GM testing with that of conventional culture, an Aspergillus lateral-flow-device (LFD) test, a beta-d-glucan (BDG) test, and an Aspergillus PCR assay by using BAL fluid samples from immunocompromised patients. A total of 78 BAL fluid samples from 78 patients at risk for IPA (74 samples from Graz and 4 from Mannheim) collected between December 2012 and May 2013 at two university hospitals in Austria and Germany were included. Three patients had proven IPA, 14 probable IPA, and 17 possible IPA, and 44 patients had no IPA. The diagnostic accuracies of the different methods for probable/proven IPA were evaluated. The diagnostic odds ratios were the highest for the GM, PCR, and LFD tests. The sensitivities for the four methods (except culture) were between 70 and 88%. The combination of the GM (cutoff optical density index [ODI], >1.0) and LFD tests increased the sensitivity to 94%, while the combination of the GM test (>1.0) and PCR resulted in 100% sensitivity (specificity for probable/proven IPA, 95 to 98%). The performance of conventional culture was limited by low sensitivity, while that of the BDG test was limited by low specificity. We evaluated established and novel diagnostic methods for IPA and found that the Aspergillus PCR, LFD, and GM tests were the most useful methods for diagnosing the disease by using BAL fluid samples. In particular, the combination of the GM test and PCR or, if PCR is not available, the LFD test, allows for sensitive and specific diagnosis of IPA.

Concentrations of viable airborne fungal spores and trichloroanisole in wine cellars.

In wineries, unwanted microorganisms present not only hygienic problems but also have a negative influence on wine quality. An evaluation of Austrian/Styrian wine cellars with regard to the volume and the composition of the mycoflora is very important both for the process of wine production and for occupational safety. Thirty-six wine cellars of 20 vintners were investigated with regard to microorganisms in the air and on material surfaces. Moreover, the presence of trichloroanisole in the air was determined by means of solid-phase micro-extraction. Microorganisms were sampled using the six-stage Andersen-Cascade impactor. The results showed that the concentrations of xerophilic fungi in the air of cellars with large visible mold areas (> 80%) reached values up to 1.4 × 104 colony forming units per m³. In the wine cellars fourteen predominant fungal genera were found in the indoor air, the most frequent was Penicillium. Trichloroanisole was detected in the air of wine cellars with large visible moldy patches. The spore concentrations in the cellar air were two times higher in cellars with Zasmidium cellare growth than in cellars without Z. cellare. These results will serve as a database for further studies.

Occurrence and hygienic relevance of fungi in drinking water.

Fungi, above all filamentous fungi, can occur almost everywhere, even in water. They can grow in such a quantity in water that they can affect the health of the population or have negative effects on food production. There are several reports of fungal growth in water from different countries, but to our knowledge none from Austria so far. The aim of this study was to gain an overview of the spectrum of filamentous fungi and yeasts in drinking water systems. Thirty-eight water samples from drinking water and groundwater were analysed. Fungi were isolated by using membrane filtration and plating method with subsequent cultivation on agar plates. The different taxa of fungi were identified using routine techniques as well as molecular methods. Fungi were isolated in all water samples examined. The mean value for drinking water was 9.1 CFU per 100 ml and for groundwater 5400 CFU per 100 ml. Altogether 32 different taxa of fungi were found. The taxa which occurred most frequently were Cladosporium spp., Basidiomycetes and Penicillium spp. (74.6%, 56.4% and 48.7%, respectively). This study shows that drinking water can be a reservoir for fungi, among them opportunists, which can cause infections in immunosuppressed patients.

A 5-year (2000-2004) epidemiological survey of Candida and non-Candida yeast species causing vulvovaginal candidiasis in Graz, Austria.

Vulvovaginal candidasis (VVC) is a common disease. The majority of cases is caused by Candida albicans, but in recent years an increase has been observed in the frequency of non-albicans Candida infections, especially due to C. glabrata and C. tropicalis. The aim of the study was to assess the prevalence of non-albicans Candida infections in patients suffering from VVC. Therefore, the statistical data of culture-confirmed VVC ascertained at the Institute of Hygiene (Medical University Graz) have been studied. Altogether, 10,463 samples from patients with vulvovaginal complaints were analysed in the years 2000-2004, a number of 3184 proved to be culture-positive for yeast. Candida albicans was the most prevalent cause in 87.9% of all cases. Non-albicans Candida yeast were detected in 12.1%, mainly C. glabrata and Saccharomyces cerevisiae. During a 1-year period 185 patients showed more than one episode of VVC. Patients aged 21-40 years were significantly more prone to suffer from VVC compared with other age-related groups.

Lethal brain abscess due to the fungus Scedosporium apiospermum (teleomorph Pseudallescheria boydii) after a near-drowning incident: case report and review of the literature.

A 39-year-old healthy man developed a brain abscess weeks after a near-drowning incident. Scedosporium apiospermum, the anamorph of Pseudallescheria boydii, was isolated from the abscess. The patient died 153 days after the accident despite antifungal therapy. We discuss the role of antifungals and review the literature for comparable cases.

Trichophyton rubrum in the external auditory meatus.

We report the case of a 28-year-old immunocompetent male suffering from otitis externa. The right external auditory meatus was filled with cerumen and detritus, the tympanic membrane covered wallpaper-like with layers of fungi. Mycological analysis revealed Trichophyton rubrum. With further examination tinea pedis of plantar and interdigital type and concomitant onychomycosis of the toenails due to T. rubrum could be detected. The auditory meatus was cleaned and treated topically with clotrimazole. Two weeks later the auditory meatus and the tympanic membrane were bare of fungi and the inflammation was resolved. Treatment of tinea pedis and onychomycosis with terbinafine (systemically and topically) is still lasting.

[From when on can fungi be identified in nasal mucus of humans?]. / Ab wann sind Pilze im Nasensekret des Menschen nachweisbar?

BACKGROUND: Fungal spores are frequent in air and their occurrence in the nasal mucus appears to be a common finding within the adult population, as we were able to show in recent studies. 91,3 % of CRS patients but also healthy controls grew positive fungal cultures out of their nasal mucus. The potential role of fungal elements in nasal mucus for the pathogenesis of CRS, with or without polyposis, is currently investigated intensely and discussed very controversially. However, it was still unknown, as of when fungi could be cultured from nasal mucus in humans. We attempted to identify this point of time, in the nasal mucus of neonates. METHODS: In our study we examined nasal mucus from 30 neonates immediately after birth, on the first and fourth day post partum, and after two and four months of life. The samples obtained with sterile cotton swabs were cultured on agar plates. Fungal cultures were identified either conventionally by microscopy or with molecular techniques. To show whether fungi in nasal mucus of newborns were acquired by contamination during birth, mucus of the maternal vagina was examined as well. RESULTS: Just after birth we found in 6 of 30 (20 %) of our neonates positive fungal cultures out of their nasal mucus, in 3 of them Candida albicans, probably due to contamination passing the maternal vagina as cultures of vaginal mucus of their mothers were positive for Candida albicans too. Positive fungal cultures were obtained in 2 of 29 (7 %) neonates on the second and in 4 of 26 (15 %) neonates on the fifth day of life. In all our cases initial presence in nasal mucus contamination just after birth or on the second day of life was limited to one day only. None of the 12 of 30 (40 %) neonates with positive fungal cultures from nasal mucus in the first 5 days of life showed clinical symptoms of nasal fungal colonisation. Besides Candida albicans, Penicillium sp., Cladosporium cladosporioides, Acremonium polychromum, Beauveria bassiana and Epicoccum nigrum could be detected in the first 5 days of life. After the second month of life, examination of nasal mucus yielded positive fungal cultures in 8 of 11 (72 %), after four months even 17 of 18 (94 %) of babies, with a wide array of different species. CONCLUSIONS: Fungi can be cultured from nasal mucus as soon as contact with the environmental air exists. Furthermore, a transfer of fungi from the mother's birth canal into the nose during birth is possible. Presence of fungal spores is common but not persistent in the nose of babies in the first days of life. However, after four months the situation is similar to the one in adults: fungal cultures can be obtained from almost everyone's nose. Therefore fungal spores must be considered a normal content of nasal mucus. Fungal spores are inhaled with every breath, some stick to the mucus, are transported to the nasopharynx and swallowed. This does not cause any clinical symptoms and is therefore not a pathological finding at all.

Fungal biodiversity--as found in nasal mucus.

The biodiversity of fungi isolated from the nasal mucus of patients suffering from chronic rhinosinusitis and from healthy persons was monitored over 28 months. Mucus samples were obtained by flushing the noses of patients with saline or by endoscopic sinus surgery. Fungi from mucus were cultivated on agar plates. Identification was performed microscopically and by polymerase chain reaction with subsequent sequencing of the ribosomal internal transcribed spacer region. Altogether, 619 strains of fungi were cultivated from 233 subjects. Eighty-one species were identified, with a maximum of nine different species per person. The most prevalent isolates belonged to the genera Penicillium, Aspergillus, Cladosporium, Alternaria and Aureobasidium. Whereas Aspergillus and Penicillium spp. occurred in more or less the same numbers throughout the year, Cladosporium spp., Alternaria spp. and Aureobasidium pullulans showed a significantly higher occurrence during late summer and early autumn.

[The basidiomycete Schizophyllum commune in paranasal sinuses]. / Der Basidiomyzet Schizophyllum commune in den Nasennebenhöhlen.

In the last fifty years, only 22 medical cases involving the basidiomycetous fungus Schizophyllum commune were reported. In a period of three years we have examined 270 patients suffering from chronic rhinosinusitis as well as from mycoses (fungus balls) within the paranasal sinuses. Either nasal mucus or fungal concrement from the sinuses were cultured and the resulting cultures identified microscopically. In cases, where a reliable identification of the fungi was not possible, DNA was extracted for molecular examination. The internal transcribed spacer (ITS) region of the ribosomal gene-cluster was amplified with fungus specific primers and sequenced thereafter. In addition, DNA of all fungi growing with sterile white mycelium was amplified with the primer pair scom1/scom2r, which is specific for S. commune. Altogether, within a three years period S. commune was isolated in twelve patients. It can be assumed, that with the presented methods S. commune will be found much more frequently in patients suffering from diseases of the nasal sinuses.

[Incidence and detection of fungi and eosinophilic granulocytes in chronic rhinosinusitis]. / Häufigkeit und Nachweis von Pilzen und eosinophilen Granulozyten bei chronischer Rhinosinusitis.

BACKGROUND: Chronic Rhinosinusitis (CRS) is the most common chronic disease in the United States. Though for Europe no data are available, we have to assume that the situation is similar. Although the disease is defined very well by clinical symptoms, up to date the etiology and pathogenesis of chronic rhinosinusitis are unknown. CRS is considered to be multifactorial, with thickening of the mucosa and formation of polyps as an end stage of the disease. Treatment of choice includes corticosteroids and/or endoscopic or microscopic surgery. Antibiotics only help, if there is an acute bacterial exacerbation of the disease. They are not able to cure chronic rhinosinusitis per se. In 1999 Mayo Clinic researchers published data concerning the incidence of so-called "allergic" fungal sinusitis (AFS) in their patients suffering from chronic rhinosinusitis, demonstrating the majority of patients investigated presenting those criteria. Our own initial data from 2000 confirmed their findings. MATERIAL AND METHODS: In an open prospective study fungal cultures were obtained from nasal mucus of 238 consecutive patients suffering from CRS. As control group acted 23 members of our staff, who did not show any evidence for CRS. In addition, in 37 CRS patients surgical specimens (mucus and tissue) were investigated histologically for evidence of eosinophilic granulocytes and fungal elements. RESULTS: Using new techniques for fungal detection in culture and histology as proposed by Mayo Clinic researchers, positive detection of fungal cultures of the mucus of our CRS patients developed from 7 % in the past up to 87 % at present. 91.3 % of our control group yielded a positive result in fungal culture. Histologically, eosinophilic clusters were evident in 94.6 % and fungal elements were detected in 75.5 % within the mucus of 37 surgical CRS patients. Overall, 89.2 % of our surgical patients thus fulfilled the criteria of so-called AFS. Compared to our findings in the past, our latest results show an increase of 80 % in detection of fungal elements in our CRS patients. In all we were able to identify 654 positive fungal cultures in 238 CRS patients and 23 healthy controls respectively. 88 different genera grew, with 2.4 different species per patient and 3.1 different species per healthy control, on average. CONCLUSION: Utilizing new techniques of fungal culturing out of the mucus of CRS patients and healthy controls, the number of positive fungal cultures increased dramatically from 7 % to 87 % in our patients and 91.3 % in healthy controls respectively. To obtain these results it is crucial to perform special techniques of mucus sampling and pretreatment for culturing as well as for histological investigations. Our results show, that with suitable techniques fungi can be identified in almost everybody's nose, CRS patient or healthy. When inhaled, those airborne fungi are only "in transit" through the nose. Positive fungal cultures from nasal secretion therefore have to be considered normal findings. The reason for this delayed recognition has to be attributed to our inadequate methods in the past. In contrast to healthy controls, clusters of eosinophils and fungal elements are present simultaneously within the mucus of CRS patients and appear to be a marker of the disease.

Development of molecular methods for identification of Schizophyllum commune from clinical samples.

In the last 50 years, to our knowledge, only 16 cases of diseases caused by Schizophyllum commune in humans have been reported. Within only 6 months, we found four isolates of this basidiomycetous fungus, obtained from patients suffering from chronic sinusitis. The cultures of the isolated fungi showed neither clamp connections nor fruiting bodies (basidiocarps), which are distinctive features for S. commune, but fast-growing cottony white mycelium only. This was harvested, and DNA was extracted. The internal transcribed spacer region of the ribosomal DNA (rDNA) was amplified with fungus-specific primers, and the PCR products were sequenced. Two strains of S. commune, collected from branches of a European hornbeam (Carpinus betulus) and a tree of heaven (Ailanthus altissima), respectively; four specimens from the herbarium of the Institute of Botany, Karl-Franzens-University Graz; and two strains from internationally known culture collections (CBS 340.81 [ATCC 44201] and CBS 405.96) were investigated in the same way. The sequence data of all strains were compared and showed homology of over 99% in this 660-bp-long fragment of rDNA. With these results, a map of restriction enzyme cutting sites and a primer set specific for S. commune were created for reliable identification of this human pathogenic fungus.

[Molecular biological methods and their consequences in taxonomy and diagnosis of dermatophytes]. / Molekularbiologische Methoden und ihre Konsequenzen für Taxonomie und Diagnostik bei Dermatophyten.

Trichophyton rubrum, T. mentagrophytes, T. verrucosum, Microsporum canis, M. gypseum and Epidermophyton flocoosum represent the cause of human dermatomycoses isolated most often at the University Hospital of Dermatology in Graz, Austria, between 1991 and 1998. So far, identification was mainly based on the cultivation of fungal isolates on special media as well as on the analysis of their microscopic characters. Restriction enzyme length polymorphisms (RFLPs) were now used to identify these human fungal parasites. For this purpose, internal transcribed spacers (ITS) which are located between ribosomal RNA genes have been amplified by using PCR and have afterwards been used to generate species specific RFLP patterns. By this method, a fast and reliable identification of these species was made possible. Nucleotide sequence data of this region not needed for identification have been worked out to show RFLPs in more detail.

[Variability of Trichophyton cerrucosum isolates from vaccinated herds with cattle ringworm]. / Zur Variabilität von Trichophyton verrucosum-Isolaten aus Impfbeständen mit Rindertrichophytie.

Twenty-seven strains of Trichophyton verrucosum from 14 cattle herds in the Federal States of Thuringia and Mecklenburg-Vorpommern were examined by culture morphological and molecular biological (PCR fingerprinting, AFLP analysis sequencing of ITS region) methods. Six reference strains of the same species, among them the so-called album and ochraceum varieties, were also included. Despite great variability in terms of culture morphology, which suggested their possible classification into 4 different colony types, all T. verrucosum isolates were genotypically almost identical. Even the 2 field isolates growing with yellow pigment, which could possibly be regarded as belonging to the ochraceum variety, could not be differentiated using molecular biological methods. The results do not provide indications of a separate taxonomic position of the 3 T. verrucosum varieties. Furthermore, there is no evidence confirming the suspected infection of cattle herds with ochraceum strains as the cause of the failure of immune prophylaxis using various T. verrucosum vaccines. The frequent occurrence of animals not responding to vaccination could not be explained either. It should be assumed that the main factors responsible for this situation include poor handling of the vaccine strains and errors in application, especially the absence of continuous and systematic immune prophylaxis in the herds.