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Fungi in the order Chaetothyriales are renowned for their ability to cause human infections. Nevertheless, they are not regarded as primary pathogens, but rather as opportunists with a natural habitat in the environment. Extremotolerance is a major trend in the order, but quite different from black yeasts in Capnodiales which focus on endurance, an important additional parameter is advancing toxin management. In the ancestral ecology of rock colonization, the association with metabolite-producing lichens is significant. Ant-association, dealing with pheromones and repellents, is another mainstay in the order. The phylogenetically derived family, Herpotrichiellaceae, shows dual ecology in monoaromatic hydrocarbon assimilation and the ability to cause disease in humans and cold-blooded vertebrates. In this study, data on ecology, phylogeny, and genomics were collected and analyzed in order to support this hypothesis on the evolutionary route of the species of Chaetothyriales. Comparing the ribosomal tree with that of enzymes involved in toluene degradation, a significant expansion of cytochromes is observed and the toluene catabolism is found to be complete in some of the Herpotrichiellaceae. This might enhance human systemic infection. However, since most species have to be traumatically inoculated in order to cause disease, their invasive potential is categorized as opportunism. Only in chromoblastomycosis, true pathogenicity might be surmised. The criterion would be the possible escape of agents of vertebrate disease from the host, enabling dispersal of adapted genotypes to subsequent generations.

In Germany, there are numerous legal regulations that address radioactivity in food, animal feed and environmental sources. With regard to the accident-relevant radionuclides 137Cs and 90Sr, the Radiation Protection Act is of particular importance. When calculating the consumer's exposure to radiation, all relevant entry paths, e.g. air, feed, crop or cow milk must be taken into account. Compliance with the limits of the effective dose and of individual partial body doses is controlled and ensured by a variety of regulations, including, but not limited to strict regulations and requirements for the operation of nuclear facilities. A comprehensive monitoring of radioactivity from various environmental and dietary sources is mandatory in order to ensure consumer’s safety.

Streptococcus anginosus ß‐hemolytic strains harbor CRISPR/Cas systems less frequently than non ß‐hemolytic strains. While in 65% of non‐ß‐hemolytic strains a type II CRSIPR/Cas system was present, it could only be detected in 24% of ß‐hemolytic strains. Streptococcus anginosus is a commensal of the oral mucosa that can cause severe invasive infections. A considerable proportion of Streptococcus anginosus strains are ß‐hemolytic due to the presence of an SLS‐like gene cluster. However, the majority of strains do not display ß‐hemolysis. To investigate ß‐hemolysin heterogeneity in S. anginosus, we determined the presence of sag genes and correlated it with the presence of CRISPR/Cas genes in a collection of ß‐hemolytic and non‐ß‐hemolytic strains. All of the ß‐hemolytic strains carried the sag gene cluster. In contrast to other streptococci, clinical S. anginosus strains that do not display ß‐hemolysis do not harbor sag genes. Phylogenetic analysis of the ß‐hemolytic strains revealed that they belong to two previously defined clusters within S. anginosus. Correlation with CRISPR/Cas genes showed a significant difference for the presence of CRISPR/Cas in ß‐hemolytic versus non‐ß‐hemolytic isolates. The presence of the CRISPR/Cas type IIA or type IIC locus is associated with the absence of sag genes; in 65% of the non‐ß‐hemolytic strains a CRISPR/Cas locus was found, while only 24% of ß‐hemolytic strains carry CRISPR/Cas genes. Further analysis of the spacer content of the CRISPR systems revealed the presence of multiple self‐targeting sequences directed against S. anginosus genes. These results support the hypothesis that horizontal gene transfer is involved in the acquisition of ß‐hemolysin genes and that CRISPR/Cas may limit DNA uptake in S. anginosus., publishedVersion

Current studies focus on cellular and humoral immunity induced by novel SARS-CoV-2 vaccines. Non-responders to vaccinations are not uncommonly encountered in clinical medicine (e.g. in the field of hepatitis B). Whereas vaccine-induced humoral immunity against SARS-CoV-2 is compromised by emerging Variants of Concern (VOCs), cellular immunity against SARS-CoV-2 is emerging as resilient against VOCs. Thus commercially available test kits for diagnostic laboratories designed to evaluate cellular immune responses to SARS-CoV-2 are urgently needed. Here we evaluated the novel QuantiFERON SARS-CoV-2 assay (Qiagen) measuring INF-ɣ release induced by two spike-derived peptide pools (Ag1 and Ag2) in a cohort of health care workers vaccinated with the mRNA-1273 vaccine and confirmed humoral response. Our study indicates the usefulness of this novel assay for routine laboratories to evaluate cellular immunity against SARS-CoV-2 in response to mRNA-1273 vaccination.

Studies on the immune responses to severe acute respiratory syndrome coronavirus 2 vaccines are necessary to evaluate the ongoing vaccination programs by correlating serological response data and clinical effectiveness data. We performed a longitudinal immunological profiling of health care workers vaccinated with mRNA-1273 (Moderna, Cambridge, MA, USA). Half of these vaccinees had experienced a mild coronavirus disease 2019 (COVID-19) infection in the spring of 2020 ("COVID-recovered" cohort), whereas the other half of the vaccinees had no previous COVID-19 infection ("COVID-naive" cohort).Serum was drawn at multiple time points and subjected to assays measuring anti-Spike immunoglobulin G (IgG), avidity of anti-Spike IgG, avidity of anti-receptor binding domain (RBD) IgG, virus neutralizing activity, and interferon-γ release from stimulated lymphocytes.Between both cohorts and within each cohort, we found remarkable inter-individual differences regarding cellular and humoral immune responses to the Moderna mRNA-1273 vaccine.First, our study indicates that the success of mRNA-1273 vaccinations should be verified by serological assays in order to identify "low-responders" to vaccination. Second, the kinetics of anti-S IgG and neutralizing activity correlate well with clinical effectiveness data, thus explaining incipient protection against infection 2 weeks after the first dose of mRNA-1273 in COVID-naive vaccinees. Third, our IgG-avidity data indicate that this incipient protection is mediated by low-avidity anti-RBD IgG and low-avidity anti-S IgG.

Objectives: Aspergillus fumigatus is the most prevalent filamentous fungus in the respiratory tract of patients with cystic fibrosis (CF). The aim of this prospective multicentre study was to investigate the prevalence of azole-resistant A. fumigatus (ARAF) in respiratory secretions from CF patients across Germany and to characterize ARAF isolates by phenotypic and molecular methods. Methods: Twelve tertiary care centres from Germany participated in the study. In total, 2888 A. fumigatus isolates from 961 CF patients were screened for ARAF by using azole-containing agar plates. Antifungal susceptibility testing of isolates was performed by broth microdilution according to EUCAST guidelines. Analysis of mutations mediating resistance was performed using PCR and sequencing of the cyp51A gene. Furthermore, genotyping by microsatellite PCR was performed. Results: Of a total of 2888 A. fumigatus isolates, 101 isolates from 51 CF patients were found to be azole resistant (prevalence per patient 5.3%). The Essen centre had the highest prevalence (9.1%) followed by Munich (7.8%), Munster (6.0%) and Hannover (5.2%). Most ARAF isolates (n = 89) carried the TR34/L98H mutation followed by eight G54E/R, one TR46/Y121F/T289A and one F219S mutation. In two isolates no mutation was found. Genotyping results showed no major clustering. Forty-five percent of CF patients with ARAF had previously received azole therapy. Conclusions: This is the first multicentre study analysing the prevalence of ARAF isolates in German CF patients. Because of a resistance rate of up to 9%, susceptibility testing of A. fumigatus isolates from CF patients receiving antifungal treatment should be part of standard diagnostic work-up.

We describe emergomycosis in a patient in Uganda with HIV infection. We tested a formalin-fixed, paraffin-embedded skin biopsy to identify Emergomyces pasteurianus or a closely related pathogen by sequencing broad-range fungal PCR amplicons. Results suggest that emergomycosis is more widespread and genetically diverse than previously documented. PCR on tissue blocks may help clarify emergomycosis epidemiology.

Recent changes in the Fungal Code of Nomenclature and developments in molecular phylogeny are about to lead to dramatic changes in the naming of medically important molds and yeasts. In this article, we present a widely supported and simple proposal to prevent unnecessary nomenclatural instability.

Objective Detection of hyphomycetes of the Scedosporium apiospermum complex and Lomentospora prolificans ( Sa c- Lp ) is not yet standardized. Prevalence rates in patients with cystic fibrosis (CF) and the resistance pattern of these pathogens in Germany are unknown. Methods In a one-year prospective study 11 laboratories used a selective medium for isolation of Sa c- Lp , examining >11,600 respiratory samples from 2346 patients with CF. Isolates were identified by molecular methods and tested for susceptibility to antifungal drugs. Results The prevalence of Sa c- Lp in patients with CF in Germany varied from 0.0 to 10.5% (mean: 3.1%) among the clinical centres. The benefit of the selective medium SceSel + compared to standard media for fungi was documented for >5000 samples. High antifungal resistance was detected in the S. apiospermum complex, and the multiresistance of L. prolificans was confirmed. Conclusion Microbiology laboratories should be aware of these resistant species in patients with CF and consider using a selective medium.

Microbacterium species (formerly CDC [Centers for Disease Control and Prevention] coryneform group A-4 and A-5 bacteria) are widely distributed in the environment and rarely cause infections in humans. We present a case of endophthalmitis due to Microbacterium species that occurred after accidental trauma and review the literature on microbacterium infections. If the infected tissue or medical device is removed and antimicrobial therapy (preferably with beta-lactams or glycopeptides) is instituted, the prognosis is usually favorable for patients with microbacterium infections.

Various fungi have the ability to colonize surfaces and to form biofilms. Fungal biofilm-associated infections are frequently refractory to targeted treatment because of resistance to antifungal drugs. One fungus that frequently colonises the respiratory tract of cystic fibrosis (CF) patients is the opportunistic black yeast–like fungus Exophiala dermatitidis. We investigated the biofilm-forming ability of E. dermatitidis and its susceptibility to various antiinfective agents and natural compounds. We tested 58 E. dermatitidis isolates with a biofilm assay based on crystal violet staining. In addition, we used three isolates to examine the antibiofilm activity of voriconazole, micafungin, colistin, farnesol, and the plant derivatives 1,2,3,4,6-penta-O-galloyl-b-D-glucopyranose (PGG) and epigallocatechin-3-gallate (EGCG) with an XTT reduction assay. We analysed the effect of the agents on cell to surface adhesion, biofilm formation, and the mature biofilm. The biofilms were also investigated by confocal laser scan microscopy. We found that E. dermatitidis builds biofilm in a strain-specific manner. Invasive E. dermatitidis isolates form most biomass in biofilm. The antiinfective agents and the natural compounds exhibited poor antibiofilm activity. The greatest impact of the compounds was detected when they were added prior cell adhesion. These findings suggest that prevention may be more effective than treatment of biofilm-associated E. dermatitidis infections.

Invasive aspergillosis (IA) is a serious hazard to high-risk haematological patients. There are increasing reports of azole-resistant Aspergillus spp. This study assessed the epidemiology of IA and azole-resistant Aspergillus spp. in patients with acute leukaemia in Germany. A prospective multicentre cohort study was performed in German haematology/oncology centres. The incidence of probable and proven aspergillosis according to the revised EORTC/MSG criteria was assessed for all patients with acute leukaemia [acute myeloid leukaemia (AML) and acute lymphoblastic leukaemia (ALL)]. Cases were documented into a web-based case report form, and centres provided data on standards regarding prophylactic and diagnostic measures. Clinical isolates were screened centrally for azole resistance and, if applicable, underlying resistance mechanisms were analysed. Between September 2011 and December 2013, 179 cases of IA [6 proven (3.4%) and 173 probable (96.6%)] were diagnosed in 3067 patients with acute leukaemia. The incidence of IA was 6.4% among 2440 AML patients and 3.8% among 627 ALL patients. Mortality at Day 84 was 33.8% (49/145) and attributable mortality was 26.9% (39/145). At Day 84, 53 patients (29.6%) showed a complete response, 25 (14.0%) a partial response and 17 (9.5%) a deterioration or failure. A total of 77 clinical Aspergillus fumigatus isolates were collected during the study period. Two episodes of azole-resistant IA (1.1%) were caused by a TR/L98H mutation in the cyp51A gene. With only two cases of IA due to azole-resistant A. fumigatus, a change of antifungal treatment practices in Germany does not appear warranted currently.

International audience; As a result of fundamental changes in the International Code of Nomenclature on the use of separate names for sexual and asexual stages of fungi, generic names of many groups should be reconsidered. Members of the ECMM/ISHAM working group on Pseudallescheria/Scedosporium infections herein advocate a novel nomenclature for genera and species in Pseudallescheria, Scedosporium and allied taxa. The generic names Parascedosporium, Lomentospora, Petriella, Petriellopsis, and Scedosporium are proposed for a lineage within Microascaceae with mostly Scedosporium anamorphs producing slimy, annellidic conidia. Considering that Scedosporium has priority over Pseudallescheria and that Scedosporium prolificans is phylogenetically distinct from the other Scedosporium species, some name changes are proposed. Pseudallescheria minutispora and Petriellidium desertorum are renamed as Scedosporium minutisporum and S. desertorum, respectively. Scedosporium prolificans is renamed as Lomentospora prolificans.

Die Mehrzahl von Pilzinfektionen, die zu mykotischen Tumoren führen und bei Patienten ohne jegliche prädisponierende Vorerkrankung auftreten, wird durch Cryptococcus gattii bzw. C. neoformans oder durch Schwärzepilze, insbesondere durch Cladophialophora bantiana, Ramichloridium mackenziei, Exophiala oder Fonsecaea-Arten verursacht. Beim Nachweis von Hyphen in Granulomen des zentralen Nervensystems (ZNS) sollte in jedem Fall nach einer Eigenpigmentierung der Pilze gesucht werden, welche am besten im HE-, manchmal auch im PAS-gefärbten Schnitt erkennbar ist. Bei Patienten nach einem überlebten Beinaheertrinkungsunfall und mit Abszessen im Gehirn sollte immer an eine Scedosporiose gedacht werden.The majority of mycoses which lead to mycotic tumors in patients without any predisposing underlying disease are either caused by Cryptococcus gattii and C. neoformans or by dematiaceous fungi which include Cladophialophora bantiana, Ramichloridium mackenziei, Exophiala and Fonsecaea species. The detection of hyphae in granuloma in the brain should lead to screening for pigmented fungi, which are recognized best in hematoxylin eosin (HE) or sometimes also in periodic acid-Schiff (PAS) stained sections. In patients who survive a near drowning accident and those who develop brain abscesses, scedosporiosis should always be considered as a possible infection.

Detection and correct identification of fungi causing invasive disease is a major challenge for diagnostic laboratories. However, early and reliable diagnosis is essential to keep mortality to a minimum, whereas delayed or missed diagnosis is typically associated with fatal outcome in the case of invasive fungal infections. Therefore, rapid and reliable results are of importance for successful management. In order to guarantee correct reports, accurate processing of all samples and appropriate methods for detecting the fungal agent as well as quality assurance is mandatory. This article gives an overview of current methods and elucidates quality management measures in medical mycology laboratory.

Mass growth of dark fungal biofilms on water taps and associated habitats was observed in various German drinking water distribution systems recently. Customers of affected drinking water systems are anxious about potential and unknown health risks. These environments are known to harbour a fungal flora also comprising a variety of fungal opportunists that are well known to cause superficial mycoses in humans (Exophiala equina, Exophiala lecanii-corni) but are not known to establish dark biofilms so far. To gain profound insight on composition of respective biofilms, a metagenomic approach using Tag-Encoded FLX Amplicon Pyrosequencing (TEFAP) of the ribosomal internal transcribed spacer 2 region in comparison with a classical cultivation approach using Sabouraud agar with chloramphenicol and erythritol-chloramphenicol-agar was performed. E. lecanii-corni was found to be the major component in 10 of 13 biofilms analysed independently of the method used. Alternaria sp., E. equina, Fusarium spp. and Ochroconis spp. were also relatively abundant. As expected, TEFAP usually revealed a higher diversity than the cultivation approaches. For example, opportunistic species like Candida albicans or Exophiala dermatitidis were detected in very low amounts. In conclusion, TEFAP turned out to be a promising and powerful tool for the semi-quantitative analysis of fungal biofilms. Referring to relevant literature, potential biological hazards caused by fungi of the dark biofilms can be regarded as low.

Wie bei den Hefen verursachen die fllamentos wachsenden Pilze bis auf wenige Ausnahmen nur unter besonderen Umstanden Mykosen beim Menschen. Die Mehrzahl dieser als Opportunisten isolierten Pilze mit Wachstumsvermogen bei 35°C gehort einigen wenigen anamorphen Gattungen (= Hyphomyzeten) an, deren korrespondierende teleomorphen Arten uberwiegend den Askomyzetenzuzuordnen sind (▸ Kap. 77). Nur ein kleiner Anteil

Pilze werden innerhalb der Eukaryonten in ein eigenes Reich (Regnum Fungi) eingruppiert. Sie haben sich vor ca. 1 Mrd. Jahren von den anderen Eukarya abgespalten und sind phylogenetisch mehr mit den Tieren als mit den Pflanzen verwandt (z. B. bilden beide als Speicherstoff Glykogen). Bisher sind ca. 150 000 Arten beschrieben worden, wobei allerdings durch intensive molekulargenetische Analysen standig viele neue Arten hinzukommen. Daher wird vermutet, dass es insgesamt mehr als 1 Mio. Arten gibt. Davon werden ca. 50 Arten haufiger als Mykoseerreger beim Menschen isoliert. Ferner sind bisher etwa weitere 300 Arten als so genannte opportunistische Erreger bei stark immunsupprimierten Patienten als Kranksheitserreger beschrieben worden.

Descriptive values were determined for eight antifungal agents within the course of a multi-centre study encompassing 1062 German and Austrian clinical yeast isolates. Candida albicans (54%) was the predominant species isolated followed by Candida glabrata (22%), Candida parapsilosis (6%), Candida tropicalis (5.7%), Candida krusei (4.3%), as well as eleven further candidal and four non-Candida yeast species. While 519 (48.9%) isolates were tested susceptible to all antifungals tested, no isolate was found to exhibit complete cross resistance. For C. albicans, the proportions of susceptible isolates were 93.2% (amphotericin B), 95.6% (flucytosine), 84.3% (fluconazole), 83.8% (posaconazole), 91.8% (voriconazole), 96.5% (anidulafungin), 96.2% (caspofungin) and 97.6% (micafungin). Patterns of complete parallel resistances were observed within azoles (8.8%) and echinocandins (1.7%). While a decreased susceptibility was found infrequently for echinocandins and flucytosine, it was more common for azoles with highest proportions for isolates of C. glabrata (fluconazole, 40.6%; posaconazole, 37.2%), Candida guilliermondii (fluconazole and posaconazole, each 25.0%), C. krusei (posaconazole, 28.3%; voriconazole, 60%), C. parapsilosis (fluconazole, 70.3%) and C. tropicalis (fluconazole, 62.3%). The descriptive values obtained in this study represent a valid basis for the comparison of recent and future epidemiological surveys to analyse the susceptibility of yeast isolates towards major antifungal substances.

Herpotrichiellaceous black yeasts and relatives comprise severe pathogens flanked by nonpathogenic environmental siblings. Reliable identification by conventional methods is notoriously difficult. Molecular identification is hampered by the sequence variability in the internal transcribed spacer (ITS) domain caused by difficult-to-sequence homopolymeric regions and by poor taxonomic attribution of sequences deposited in GenBank. Here, we present a potential solution using short barcode identifiers (27 to 50 bp) based on ITS2 ribosomal DNA (rDNA), which allows unambiguous definition of species-specific fragments. Starting from proven sequences of ex-type and authentic strains, we were able to describe 103 identifiers. Multiple BLAST searches of these proposed barcode identifiers in GenBank revealed uniqueness for 100 taxonomic entities, whereas the three remaining identifiers each matched with two entities, but the species of these identifiers could easily be discriminated by differences in the remaining ITS regions. Using the proposed barcode identifiers, a 4.1-fold increase of 100% matches in GenBank was achieved in comparison to the classical approach using the complete ITS sequences. The proposed barcode identifiers will be made accessible for the diagnostic laboratory in a permanently updated online database, thereby providing a highly practical, reliable, and cost-effective tool for identification of clinically important black yeasts and relatives.

Exophialin, a previously unknown indole alkaloid, was isolated from the human-pathogenic fungus Exophiala dermatitidis upon cultivation on a medium containing tryptophan as sole nitrogen source. Its structure has been elucidated by 1D and 2D NMR experiments and mass spectrometry. When the mutant strain Mel-1 was used in place of the wild-type strain, the new indole alkaloid 8-hydroxyexophialin was generated instead of exophialin. This result indicates that the aromatic moiety of exophialin originates from 2-hydroxyjuglone, a shunt product of the 1,8-dihydroxynaphthalene melanin biosynthesis, which is only produced by the wild-type strain, but not by the mutant. In contrast, the mutant accumulates flavioline, which acts as precursor leading to the generation of 8-hydroxyexophialin instead of exophialin.

Two chromogenic media (Chromagar VRE and chromID VRE [C-ID]) performed equally well in the direct detection of vancomycin-resistant enterococci (VRE) in stool specimens after an overnight enrichment step and a 48-h incubation period, with a sensitivity of 98.2% (56/57) for both and specificities of 96.5% (195/202) and 97.5% (197/202), respectively. However, assigning discriminatory colony color was sometimes difficult, especially on C-ID. In order to facilitate simple species identification, biochemical key reactions were implemented.

The applicability of the PNA FISH (peptide nucleic acid fluorescence in situ hybridization) method for detection of Streptococcus agalactiae [group B streptococci (GBS)] from swab samples was evaluated. Three swab-sample-processing protocols with different time-to-result (TTR) values were compared: (i) direct smearing of fresh swabs onto microscope slides (n=153, TTR 2.5 h), (ii) further extraction and concentration of cells from these same swabs (n=153, TTR 2.7 h), and (iii) short-term LIM broth enrichment culture incubation (7 h, 37 degrees C) of fresh swabs (n=120, TTR 9.5 h). The sensitivity, specificity, positive predictive value and negative predictive value for GBS PNA FISH for sample processing procedures, with TTR values of 2.5, 2.7 and 9.5 h, were 68, 100, 100 and 95 %; 91, 100, 100 and 98 %; and 100, 100, 100 and 100 %; respectively. Improved test results were achieved by subjecting swabs to an extraction procedure or abbreviated LIM broth enrichment culture incubation prior to performing GBS PNA FISH.

Mycological examination of sputum from 121 patients with cystic fibrosis by means of long-term cultures (4 weeks) revealed the occurrence of Candida albicans in low quantities in 70%, Aspergillus fumigatus in 30%, and Exophiala/Wangiella dermatitidis in 9% of the examined patients. A. fumigatus frequently causes the development of allergic bronchopulmonary aspergillosis in patients with cystic fibrosis. The predisposing factors for colonization with the otherwise seldom found fungus E. dermatitidis in these patients and the consequences of these findings are discussed. In conclusion, long-term fungal cultures are advocated for specimens from CF patients.

The mycotoxin gliotoxin is produced by fungi of the genus Aspergillus, including the important human pathogen Aspergillus fumigatus. Gliotoxin exerts a broad spectrum of immunosuppressive effects in vitro and is detectable in the sera of patients suffering from invasive aspergillosis. In order to correlate the pathogenic potential of A. fumigatus with the ability to produce gliotoxin and to investigate the taxonomic distribution of gliotoxin-producing Aspergillus strains among clinical isolates, a total of 158 Aspergillus isolates comprising four different species (A. fumigatus, n=100; A. terreus, n=27; A. niger, n=16; A. flavus, n=15) were collected from different medical centers (some originating from probable cases of aspergillosis) and from environmental samples in Germany and Austria. Remarkably, gliotoxin was detected in most culture filtrates of A. fumigatus of both clinical (98%) and environmental (96%) origin. The toxin was also detected, with decreasing frequency, in culture filtrates of A. niger (56%), A. terreus (37%), and A. flavus (13%). The highest gliotoxin concentrations were detected in A. fumigatus strains of clinical (max. 21.35 microg/ml, mean 5.75 microg/ml) and environmental (max. 26.25 microg/ml, mean 5.27 microg/ml) origin. Gliotoxin productivity of other Aspergillus species was significantly lower. Culture supernatants of A. fumigatus strains lacking gliotoxin production showed a significantly lower cytotoxicity on macrophage-like cells and T-cells in vitro. In contrast, lack of gliotoxin production in the other Aspergillus species tested had no significant influence on the cytotoxic effect of culture supernatant on these immune cells.

We evaluated the performance of the Candida albicans / Candida glabrata peptide nucleic acid fluorescent in situ hybridization (PNA FISH) method, a rapid two-color assay for detection of C. albicans and C. glabrata , in a multicenter study. The assay is designed for use directly from positive blood culture bottles in a FISH format. Intact, fixed cells are labeled fluorescent green ( C. albicans ) or fluorescent red ( C. glabrata ) by rRNA hybridization of fluorophore-labeled PNA probes. Results are available C. albicans , and C. glabrata were 98.7% (78/79) and 100% (37/37), respectively, and the specificity for both components of the assay was 100% (82/82). The assay was also evaluated with 70 fungal reference strains and was challenged in the BacT/ALERT microbiological detection system with spiked blood culture bottles. These results support the use of the assay for rapid, simultaneous identification of C. albicans and C. glabrata in positive blood culture bottles. This rapid assay may aid in the selection of initial antifungal drugs, leading to improved patient outcomes.

When provided as the sole nitrogen source tryptophan induces the production of several indole alkaloids, e.g., pityriacitrin, malasseziaindole, pityriaanhydride and pityriarubin with proven biological activity in the lipophilic yeast Malassezia furfur. So far these pigments seem to have been unique and only produced by highly specialized basidiomycetal yeasts of the genus Malassezia. Having surprisingly observed a brown pigmented Candida glabrata isolate as a contaminant on such a pigment inducing culture plate, we systematically analyzed whether this ascomycetal yeast can also synthesize the respective pigments. Therefore, 30 Candida glabrata strains, including the ex-type strain CBS 138, were cultured for 2 weeks on a pigment-inducing medium containing L-tryptophan. This culture medium along with the resultant biomass was then extracted with ethyl acetate. The extracted pigments were separated into six fractions by column chromatography. Each of these fractions was subjected to thin-layer chromatography (TLC) on silica gel and yielded identical pigment bands comparable to those observed with M. furfur. In the case of strain CBS 138, the individual TLC zones were further purified by HPLC and structural analysis of the pure metabolites was performed by mass spectrometry and proton nuclear magnetic resonance ((1)H-NMR), thereby proving the presence of pityriacitrin, malassezia indole, pityriaanhydride and pityriarubin C. Since lineage divergence of Basidiomycota and Ascomycota occurred approximately 600 million years ago, our findings demonstrate that the complex underlying biochemical pathway has not been exclusively evolved in the highly adapted basidiomycetes yeast M. furfur, but instead seems to be rather fundamental and archaic. Therefore, further investigations on the potential biological properties and the genetic regulation of these metabolites are needed to elucidate their hitherto unknown functions.

Invasive Pilzinfektionen sind mit einer hohen Letalitat verbunden, und ihre Inzidenz steigt in den letzten Jahrzehnten an. Die Fungamie mit Candidaspezies und seltener die invasive pulmonale Aspergillose sind dabei die intensivmedizinisch bedeutsamsten Krankheitsbilder. Zu den Risikofaktoren fur systemische Candidainfektionen zahlen z. B. Breitspektrumantibiotika, eine lange intensivmedizinische Behandlung und eine Verletzung/Operation im Bereich des Gastrointestinaltraktes. Immunsupprimierte Patienten sind typischen Risikopatienten fur eine invasive Aspergillusinfektion. Wahrend die Diagnostik von invasiven Pilzinfektionen den Arzt weiterhin vor erhebliche Schwierigkeiten stellt, konnten in den letzten Jahren die Therapieoptionen verbessert werden. Neben Fluconazol, konventionellem und liposomalem Amphotericin B stehen neue Praparate wie Caspofungin und Voriconazol zur Verfugung. Um Antimykotika rational und kosteneffektiv einzusetzen, benotigt der Arzt Kenntnisse uber ihre Indikationen und Grenzen. In diesem Beitrag sollen die Diagnostik und die aktuellen Therapieoptionen von intensivmedizinisch relevanten Aspergillus- und Candidainfektionen dargestellt werden.

Six coryneforms isolated from blood and dialysate fluid were phenotypically similar to Brevibacterium casei , but 16S rRNA gene sequencing and DNA-DNA hybridization indicate that they belong to a new species for which the name Brevibacterium sanguinis is proposed.

The inflammatory reaction after cell contact with polymer materials is primarily mediated by activated neutrophils and may, in some cases, lead to exhaustion of neutrophil cell function. A direct consequence of this can be impairment of local or even systemic host defense mechanisms, which in turn can result in foreign body infections. Neutrophil activation, as indicated by the up-regulation of the Mac-1 adhesion receptor, is a reliable parameter for estimating the inflammatory risk due to implanted biomaterials. Because at blood contact, biomaterials immediately acquire a material-specific layer of blood proteins on their surface, including fibrinogen, complement, and immunoglobulin G, it is generally believed that after biomaterial contact, neutrophil activation primarily occurs by interaction with this protein layer. In this study, using our recently established polymer bead in vitro assay, we investigated whether complement inhibition alone can reduce biomaterial-mediated neutrophil activation, independent of the type of polymer and, hence, also its surface chemistry. Complement inhibition was achieved by using Compstatin, a recently developed complement inhibitor that binds to the complement component C3 preventing C3 convertase formation. We revealed significantly reduced (p ≤ 0.025) Mac-1 receptor expression levels after 45 min of blood contact with the following polymers (without and with Compstatin): 1. polyurethane, 98.3%, 13.6%; 2. polymethylmetacrylate, 88.5%, 11.0%; and poly-D,L-lactide, 71.8%, 8.4%. Although these three polymer types acquire material-specific protein layers because of their different surface chemistry, complement inhibition by Compstatin alone proved to be sufficient to reduce neutrophil activation after surface contact, thus reducing the risk of biomaterial-mediated inflammatory reaction. © 2003 Wiley Periodicals, Inc. J Biomed Mater Res 66A: 491–499, 2003

A 64-year old farmer developed cough, dyspnoea on exertion, and recurrent febrile episodes. X-ray and CT scan revealed bilateral lower lobe opacities in his lungs. A transbronchial biopsy was performed and histopathological findings were interpreted as consistent with a pulmonary necrotizing clear-cell carcinoma and later as a Pneumocystis carinii pneumonia. Due to persistence of symptoms, six months later another lung biopsy was performed and a mould was cultured which was identified by 18S rDNA sequencing as Emmonsia sp. The patient showed some improvement under itraconazole treatment. This is the first description of a human infection with Emmonsia sp. in Germany.

Analysis of ITS rDNA of the black yeast Exophiala dermatitidis revealed a close phylogenetic relationship to the meristematic fungus Sarcinomyces phaeomuriformis. As most strains of S. phaeomuriformis have a yeast-like phenotype corresponding to the anamorph genus Exophiala, a new combination in Exophiala is proposed. On the basis of ITS sequence, M-13 fingerprint and SSU intron data, two main entities could be distinguished within E. dermatitidis. One of these (B) contained prevalently strains from environmental sources, while the other (A) mainly comprised strains from clinical sources. This may be due to a difference in virulence. All strains from severe brain and disseminated infections in East Asia clustered in group A. However, strains of group A caused a relatively mild fungemia in patients outside East Asia.

Streptococcus urinalis was isolated from a blood culture of a 60-year-old man with a history of urethral stricture. This species has been recently described as a new member of the pyogenic subgroup of streptococci that cause urinary tract infections.

The black yeast Exophiala dermatitidis, an agent of fatal brain infections in East Asia, is common in European steam baths. The related fungi Sarcinomyces phaeomuriformis and Exophiala mesophila were isolated from locations in these complexes with lower ambient temperature and/or moisture. The latter two species had dry, rather than slimy, colonies and lower maximum growth temperatures (38 degrees C, 32 degrees C) than E. dermatitidis (42 degrees C). Exophiala dermatitidis produces abundant extracellular polysaccharide (EPS). The only E. dermatitidis strains lacking EPS were found outside the steam baths. Therefore it is likely that the extracellular polysaccharides commonly produced by E. dermatitidis are significant to survival under hot and moist conditions. Substrates sampled as controls, such as fruit surfaces and human faeces, yielded Exophiala dermatitidis at very low frequency.

Tool support for professional credit-rating is currently dominated by data-oriented approaches. This results in primarily focusing on hard facts in the financial institutions’ process of evaluating credit inquiries of their customers. Content objects and arguments which cannot algorithmically be mapped onto rigid data-tables („soft facts”), however, with the potential of having a decisive impact on credit assessment, tend to be not sufficiently and appropriately placed into consideration. The BONITAS system overcomes this structural lack of information processing in traditional credit management systems in adding to the traditional approach innovative groupware-enabled capabilities. Compound documents in a messaging context are used to be integrative containers for all relevant information. These documents support both information gathering and evaluation of „hard” data and „soft” facts as well as semi-structured information gathering workflows allowing for a continuum of assessment-specific process options. The creation of valid and marketable business knowledge in credit management processes is basically team-driven. Thus, the essential features of groupware-based information management systems, i.e. their outstanding capabilities in communication, collaboration and coordination, represent critical baselines for improving the decision quality in credit-rating. An important goal of the BONITAS system is to aim at creating a constantly improved knowledge base on credit management workflows. This knowledge base is continuously being maintained in the operational progress of customer projects. It enables the financial institution to judge on the financial strength of upcoming clients in a holistic and anticipating evaluation process superior to traditional much more static assessments.

Beta-haemolytic streptococci are important human and animal pathogens: their genetic traits that are associated with the ability to infect human hosts remain, however, unclear. The surface protein, Lmb, mediates the adherence of Streptococcus agalactiae to human laminin. For further analysis of the corresponding gene, the adjacent genomic regions were sequenced. Lmb is localized on a putative composite transposon of 16 kb and is flanked by two copies of a novel insertion sequence element (ISSag2). It harbours the genes scpB and lmb, which are 98% identical with the respective genes of Streptococcus pyogenes. Analysis of the distribution of these genes and ISSag2 among 131 streptococcal strains revealed that all of the human isolates, but only 20% (12 of 61) of the animal isolates, contained scpB and lmb or their homologues. To investigate if the putative transposon can be mobilized, an erythromycin resistance marker was incorporated into the lmb gene of S. agalactiae. Screening for mutant strains with a regained susceptibility for erythromycin identified strains with a deletion of scpB, lmb, and one copy of ISSag2. We hypothesize that a horizontal gene transfer caused the exchange of scpB and lmb and that the ability of S. pyogenes, S. agalactiae and group C and G streptococcal strains to colonize or infect human hosts is dependent on their presence.

Implantation of any medical device normally causes an inflammatory cell interaction with the foreign material. In vitro cell activation of human neutrophils (Mac-1 upregulation) has been taken as one measure to assess the attributable risk of inflammation due to biopolymers before their clinical application. Mac-1 expression has generally been measured by flow cytometric assays, whereas quantification of neutrophil adhesion to the biopolymer surfaces has been performed by separate and time-consuming assays, e.g. microscopically by differential cell counting. However, due to an increasing number of surface-modified novel biopolymers entering clinical usage, effective testing of their inflammatory potential is now mandatory. To facilitate these analyses, we have developed a novel flow cytometric assay permitting simultaneous measurement of biopolymer-mediated neutrophil activation and adhesion. The biopolymers were used as beads (diameter 25+/-10 microm), and were demonstrated to be non-phagocytosable and non-fluorescent before being co-incubated with whole human blood (range of ratio granulocytes/beads from 5:1 to 1:1). Besides flow cytometric measurement of Mac-1 up-regulated neutrophils as fluorescing events, a fluorescence of the bead population indicates the adherence of activated neutrophils to the biopolymer surface.After establishing this assay, we evaluated it by comparing six different biopolymers. We observed high levels of Mac-1 expression (70% of positive control) accompanied by increased adhesiveness (60% of neutrophils) for polyurethane (PUR), polymethylmetacrylate (PMMA), and poly-DL-lactide (PDLLA) beads. Low Mac-1 expression levels (10%) accompanied by a low percentage of adhering neutrophils (10%) were observed for polyethylene (PE), polyisoprene (PI), and silicone (SI) beads.

1,8-Dihydroxynaphthalene (1,8-DHN) is a fungal polyketide that contributes to virulence when polymerized to 1,8-DHN melanin in the cell walls of Wangiella dermatitidis , an agent of phaeohyphomycosis in humans. To begin a genetic analysis of the initial synthetic steps leading to 1,8-DHN melanin biosynthesis, a 772-bp PCR product was amplified from genomic DNA using primers based on conserved regions of fungal polyketide synthases (Pks) known to produce the first cyclized 1,8-DHN-melanin pathway intermediate, 1,3,6,8-tetrahydroxynaphthalene. The cloned PCR product was then used as a targeting sequence to disrupt the putative polyketide synthase gene, WdPKS1 , in W. dermatitidis . The resulting wdpks1 Δ disruptants showed no morphological defects other than an albino phenotype and grew at the same rate as their black wild-type parent. Using a marker rescue approach, the intact WdPKS1 gene was then successfully recovered from two plasmids. The WdPKS1 gene was also isolated independently by complementation of the mel3 mutation in an albino mutant of W. dermatitidis using a cosmid library. Sequence analysis substantiated that WdPKS1 encoded a putative polyketide synthase (WdPks1p) in a single open reading frame consisting of three exons separated by two short introns. This conclusion was supported by the identification of highly conserved Pks domains for a β-ketoacyl synthase, an acetyl-malonyl transferase, two acyl carrier proteins, and a thioesterase in the deduced amino acid sequence. Studies using a neutrophil killing assay and a mouse acute-infection model confirmed that all wdpks1 Δ strains were less resistant to killing and less virulent, respectively, than their wild-type parent. Reconstitution of 1,8-DHN melanin biosynthesis in a wdpks1 Δ strain reestablished its resistance to killing by neutrophils and its ability to cause fatal mouse infections.

To analyze bacteriological treatment failure in streptococcal pharyngitis, 40 consecutive Streptococcus pyogenes isolates from 18 patients were characterized. For 17 patients, isolates were indistinguishable with respect to emm type, random amplified polymorphic DNA pattern, and presence of prtF1 encoding the fibronectin-binding protein F1. prtF1 was detected only in the 11 isolates (4 patients) with emm12 and in the single isolate with emm6. Further analysis by vir(mga) regulon typing, sequencing of sic encoding the streptococcal inhibitor of complement from 19 isolates with emm1 (9 patients), and sequencing of drs (distantly related sic) from 11 isolates with emm12 revealed distinct sic alleles with insertions and/or deletions in sic that corresponded to differences in restriction patterns of the vir(mga) regulon only for paired isolates of 2 patients. Among isolates with emm12, 2 novel drs alleles were found. Analysis of these data suggests that neither the presence of prtF1 nor the diversification of sic / drs is required for the persistence of S. pyogenes in pharyngitis.

Respiratory viruses discovered in the 21st century and human herpes viruses ( N =13) were seldom (4/50) detected in our cystic fibrosis patients although exacerbation frequency (7.75±2.9/a versus 4.45±2.1/a; p =0.03) and colonization with Aspergillus fumigatus ( RR: 2.6; CI95: 1.8–3.7), Pseudomonas aeruginosa (RR: 1.84; CI95: 1.4–2.4), and Staphylococcus aureus (RR: 1.5; CI95: 1.2–1.9) including MRSA (RR: 4.6; CI95: 1.3–16.6) were associated with virus positivity. Further studies should clarify whether this finding reflects non-specific colonization (human Bocavirus) or reactivation (Epstein-Barr virus) or rather an acceleration of lung tissue inflammation.

Summary. A new species, Phialophora europaea, member of the P. verrucosa complex, is introduced. It is distinguished from existing species by reduced, flaring phialidic collarettes and inability to assimilate melibiose as sole source of carbon. Analysis of ITS1 and 2 rDNA of six strains attributed to the species show it to be clearly individualized. All strains originated from cutaneous and nail infections of humans in North-western Europe. A key to morphologically similar taxa is provided.

Summary Despite appropriate antimicrobial and antifungal therapy (amphotericin B), a disseminated infection with Fusarium oxysporum in a 75-year-old immunocompromised patient (acute myeloid leukaemia, minimal leucocyte count of 0.5 giga l−1) led rapidly to death. A similarly fatal course of an F. oxysporum infection has been reported in several cases. Fusarium oxysporum could be isolated shortly before death from blood cultures and from a swab taken from skin efflorescences. An autopsy revealed histopathologically typical fungal infiltrates in the mucosa of the pharynx, epiglottis, trachea, and oesophagus and in the parenchyma of the spleen, the lung and both kidneys. Because of the high risk of a fatal outcome of this infection, the clinician should aim at maximum diagnostic enforcement. We propose both analysis of blood cultures and immediate skin biopsy—with PAS-staining—of suspicious dermal efflorescences for microscopic examination. The treatment of choice is discussed controversially but a beneficial effect has been reported from granulocyte transfusion, subcutaneous administration of GM-CSF and concomitant treatment with amphotericin B.

Candida dubliniensis is often found in mixed culture with C. albicans , but its recognition is hampered as the color of its colonies in primary culture on CHROMagar Candida varies. Furthermore, definite identification of C. dubliniensis is difficult to achieve, time-consuming, and expensive. Therefore, a method to discriminate between these two closely related yeast species by fatty acid methyl ester (FAME) analysis using gas-liquid chromatography (Sherlock Microbial Identification System [MIS]; MIDI, Inc., Newark, Del.) was developed. Although the chromatograms of these two species revealed no obvious differences when applying FAME analysis, a new library (CADLIB) was successfully created using Sherlock Library Generation Software (MIDI). The amount and frequency of FAME was analyzed using library training files ( n = 10 for each species), preferentially those comprising reference strains. For testing the performance of the CADLIB, clinical isolates genetically assigned to the respective species ( C. albicans , n = 32; C. dubliniensis , n = 28) were chromatographically analyzed. For each isolate tested, MIS computed a similarity index (SI) indicating a hierarchy of possible strain fits. When using the newly created library CADLIB, the SIs for C. albicans and C. dubliniensis ranged from 0.11 to 0.96 and 0.53 to 0.93 (for all but one), respectively. Only three isolates of C. albicans (9.4%) were misidentified as C. dubliniensis , whereas all isolates of C. dubliniensis were correctly identified. Resulting differentiation accuracy was 90.6% for C. albicans and 100% for C. dubliniensis . Cluster analysis and principal component analysis of the resulting FAME profiles showed two clearly distinguishable clusters matching up with two assigned species for the strains tested. Thus, the created library proved to be well suited to discriminate between these two species.

Candida dubliniensis is a phylogenetically closely related species to Candida albicans. So far virtually nothing is known about the virulence factors of C. dubliniensis. Cell surface hydrophobicity (CSH) plays a critical role in adhesion of microorganisms to phagocytic cells; hydrophobic cells of C. albicans have been reported to be less sensitive to phagocytic killing than hydrophilic cells. C. dubliniensis displays CSH at 37 degrees C in contrast to C. albicans. To elucidate this issue, we determined levels of phagocytosis, oxidative burst and killing by human neutrophils of C. dubliniensis (n=10) compared to C. albicans (n=10) both cultured at 37 degrees C. Obtained test results revealed no statistically significant differences between these two yeast species for the level of phagocytosis (77.3 vs. 76.2% after 60 min), evoked oxidative burst (64.5 vs. 67.3% after 30 min) and killing (72.7 vs. 73.1% after 240 min). Therefore, human neutrophils can be considered to be equally efficient against these two yeast species.

Candida dubliniensis is often associated with C. albicans in cultures. Easy-to-perform selective isolation procedures for these closely related species do not exist. Therefore, we evaluated previously described discriminatory phenotypic markers for C. dubliniensis . A total of 150 oral rinses from human immunodeficiency virus (HIV)-infected patients were cultured on CHROMagar Candida. Dark green colonies described as being indicative of C. dubliniensis and other green colonies, 170 in total, were isolated. Chlamydospore formation, intracellular β- d -glucosidase activity, ability to grow at 42°C, carbohydrate assimilation pattern obtained by the API ID 32C, and Fourier transform infrared (FT-IR) spectroscopy were used for phenotypic characterization. Sequencing of the 5′ end of the nuclear large-subunit (26S) ribosomal DNA gene was used for definitive species identification for C. dubliniensis. C. dubliniensis was found in 34% of yeast-colonized HIV-infected patients. The color of the colonies on CHROMagar Candida proved to be insufficient for selecting C. dubliniensis , since only 30 of 53 proven C. dubliniensis isolates showed a dark green color in primary cultures. The described typical chlamydospore formation can give only some indication of C. dubliniensis . The assimilation pattern proved to be insufficient to discriminate C. dubliniensis from C. albicans . All C. dubliniensis strains showed no or highly restricted growth at 42°C and a lack of β- d -glucosidase activity. Unfortunately, atypical C. albicans strains can also exhibit these phenotypic traits. FT-IR spectroscopy combined with hierarchical clustering proved to be as reliable as genotyping for discriminating the two species.

Data are presented on the clinically relevant black yeasts and their relatives, i.e., members of the Ascomycete order Chaetothyriales. In order to understand the pathology of these fungi it is essential to know their natural ecological niche. From a relatively low degree of molecular variability of the black yeast Exophiala dermatitidis, potential agent of brain infections in patients from East Asia, it is concluded that this species is an emerging pathogen, currently going through a process of active speciation. It is found to be an oligotrophic fungus in hot, moist environments, such as steambaths. Cladophialophora -, Fonsecaea - and Ramichloridium -like strains, known in humans as agents of chromoblastomycosis, are frequently found on rotten plant material, but the fungal molecular diversity in the environment is much higher than that on the human patient, so that it is difficult to trace the etiological agents of the disease with precision. This approach has been successful with Cladophialophora carri...