Walter Buzina

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.

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.

Identification and quantification of fungi and mycotoxins from Pu-erh tea

Pu-erh tea originates from the province of Yunnan in south-western China. As this tea is produced by so called Aspergillus post-fermentation the question arises which molds and mycotoxins may be found in this tea. In total 36 samples of Pu-erh tea were investigated for their content of filamentous fungi and the mycotoxins aflatoxins B1, B2, G1, and G2, fumonisins B1, B2, and B3, and ochratoxin A. Fungi were isolated from all samples in a concentration of 1.0×10(1) to 2.6×10(6) colony forming units (cfu)/g tea, all together 19 fungal genera and 31 species were identified. The most prevalent species were Aspergillus acidus and Aspergillus fumigatus, followed by Zygomycetes and Penicillium species. Aflatoxins and fumonisins were not found in the samples investigated, ochratoxin A was detected in 4 of 36 teas (11.1%).

Gangrenous necrosis of the diabetic foot caused by Fusarium acutatum

Foot infections are common and serious complications of diabetic patients. We report the case of a 68-year-old patient with a diabetic foot infection that developed into a gangrenous necrosis. Fusarium spp. was isolated on two successive occasions with no other associated microorganisms. Histopathology demonstrated invasion of the fungus into the tisssue. These findings suggested an infection rather than colonization. A detailed morphological study showed that the isolate was Fusarium acutatum, which was confirmed by rDNA sequencing. This fungus is found only in Asia and has not been previously reported as a human pathogen.

Der BasidiomyzetSchizophyllum communein den Nasennebenhöhlen

In der medizinischen Literatur der vergangenen 50 Jahre findet man nur 22 Fälle, in denen der weltweit vorkommende Basidiomyzet Schizophyllum commune beschrieben wurde. In einem Zeitraum von drei Jahren haben wir 270 Patienten untersucht, die entweder unter einer chronischen Sinusitis oder unter einer Nasennebenhöhlen‐mykose litten. Dazu wurde entweder Nasenschleim oder Operationsmaterial aus den Nasennebenhöhlen (NNH) kultiviert und die entstandenen Pilzkulturen mikroskopisch identifiziert. Konnten die Pilze nicht eindeutig bestimmt werden, wurde deren DNA zur molekularbiologischen Untersuchung isoliert. Die Internal Transcribed Spacer (ITS) Region des ribosomalen Gen‐Clusters wurde mit pilzspezifischen Primern amplifiziert und das entstandene PCR‐Produkt sequenziert. Zusätzlich wurden alle sterilen, mit weißem Myzel wachsenden Pilze mit dem Primerpaar scom1/scom2r, welches spezifisch für S. commune ist, amplifiziert. Insgesamt konnten wir im Untersuchungszeitraum von drei Jahren in zwölf Fällen S. commune aus unserem Patientengut isolieren. Die präsentierten Methoden zeigen, dass dieser Basidiomyzet in Patienten mit Erkrankungen der NNH viel häufiger vorkommt, als man bisher angenommen hat.

The role of interleukin-16 in eosinophilic chronic rhinosinusitis

Eosinophilic granulocytes (Eos) are found in great numbers both in the tissue and in the mucus of patients suffering from chronic rhinosinusitis with polyposis (ECRS). Interleukin-16 (IL-16) is known as a highly potent chemotactic and chemoattractant molecule (ED 10−11) for Eos. In an open, explorative, controlled study we examined the presence of IL-16 in mucosa tissue, mucus and serum in patients suffering from ECRS and its association to Eos activation. Tissue and nasal mucus specimen from 10 previously untreated, non allergic ECRS-patients undergoing paranasal sinus surgery and from 10 healthy non sinusitis subjects, undergoing nasal surgery because of anatomic nasal obstruction were investigated by real-time (RT-) PCR targeting human IL-16 mRNA. Haematoxylin-eosin (HE) staining and immunohistochemistry of formalin embedded tissue and mucus were applied for detection and determination of the proportion of activated Eos (aEos) and IL-16. Serum IL-16 was analyzed by enzyme-linked-immunosorbent assay (ELISA). IL-16 mRNA and IL-16 protein levels were elevated in nasal mucus, polyp tissue and in the serum of ECRS patients compared to healthy controls. There was a high proportion of aEos in ECRS patients compared to healthy subjects. Serum IL-16, IL-16 mRNA expression and IL-16 protein in mucus and tissue specimens were significantly associated with the presence of aEos in polyps of ECRS patients. Immunohistochemically IL-16 protein was mainly expressed in aEos, mast cells, lymphocytes and epithelial cells. In conclusion our data indicate that IL-16 may stimulate the migration and persistence of activated Eos in ECRS. IL-16 production in ECRS patients is not mediated by Immunglobuline-E (IgE).

Characterization and temperature-dependent quantification of heat shock protein 60 of the immunogenic fungus Alternaria alternata

Heat shock proteins or chaperones are found in mitochondrial and cytosolic compartments of cells. They are responsible for the correct folding of proteins and are up-regulated in reaction to various stressors. In addition, when released or presented on the surface of cells, they may play an important role in inflammatory and immunomodulating processes. To identify and characterize hsp60 in the common environmental mold Alternaria alternata, the fungus was cultivated and incubated at different temperatures to induce a possible heat shock response. Fully automated RNA extraction was followed by quantitative real-time RT-PCR targeting A. alternata specific Hsp60 mRNA and subsequent sequencing of the amplicon. While Hsp60 mRNA was constitutively expressed in all samples tested, a temperature-dependent expression of Hsp60 mRNA was observed. Sequencing revealed an identity of more than 85% to other fungal hsp60, indicating the existence of this protein in A. alternata.

Kurzfassung der AWMF-Leitlinie medizinisch klinische Diagnostik bei Schimmelpilzexposition in Innenräumen

ZusammenfassungDie von der Gesellschaft für Hygiene, Umweltmedizin und Präventivmedizin (GHUP) federführend erstellte Leitlinie „Medizinisch klinische Diagnostik bei Schimmelpilzexposition in Innenräumen“ ist Gegenstand des vorliegenden Abschnitts. Schimmelpilzwachstum im Innenraum ist als ein potenzielles Gesundheitsrisiko zu betrachten, auch ohne dass ein quantitativer und/oder kausaler Zusammenhang zwischen dem Vorkommen einzelner Arten und Gesundheitsbeschwerden gesichert werden kann. Abgesehen von der Allergischen Bronchopulmonalen Aspergillose (ABPA) und den durch Schimmelpilze kausal verursachten Mykosen, liegen lediglich ausreichende Evidenzen für folgende Assoziationen von Feuchte-/Schimmelpilzschäden und unterschiedlichen Gesundheitseffekten vor: allergische Atemwegserkrankungen, Asthma (Manifestation, Progression, Exazerbation), allergische Rhinitis, Exogen Allergische Alveolitis, Begünstigung von Atemwegsinfekten/Bronchitis. Dabei ist das sensibilisierende Potenzial von Schimmelpilzen im Vergleich zu anderen Umweltallergenen deutlich geringer einzuschätzen. Aktuelle Studien zeigen europaweit eine vergleichsweise geringe Sensibilisierungsprävalenz von 3–10 % gemessen an der Gesamtbevölkerung. Eingeschränkte oder vermutete Evidenz für eine Assoziation liegt vor hinsichtlich „mucous membrane irritation“ und Atopischen Ekzems (Manifestation, Progression, Exazerbation). Inadäquate oder unzureichende Evidenz für eine Assoziation liegt vor für COPD, akute idiopathische pulmonale Hämorrhagie bei Kindern, Rheuma/Arthritis, Sarkoidose und Krebserkrankungen. Das Infektionsrisiko von den in Innenräumen regelmäßig vorkommenden Schimmelpilzarten ist für gesunde Personen gering, die meisten Arten sind in die Risikogruppe 1 und wenige in 2 (Aspergillus fumigatus, A. flavus) der Biostoffverordnung eingestuft. Nur Schimmelpilze, die potenziell in der Lage sind, Toxine zu bilden, kommen als Auslöser einer Intoxikation in Betracht. Ob im Einzelfall eine Toxinbildung im Innenraum stattfindet, entscheiden die Umgebungs- und Wachstumsbedingungen und hier vor allem das Substrat. Von Geruchswirkungen und/oder Befindlichkeitsstörungen kann bei Feuchte-/Schimmelpilzschäden im Innenraum grundsätzlich jeder betroffen sein. Hierbei handelt es sich nicht um eine Gesundheitsgefährdung. Prädisponierende Faktoren für Geruchswirkungen können genetische und hormonelle Einflüsse, Prägung, Kontext und Adaptationseffekte sein. Prädisponierende Faktoren für Befindlichkeitsstörungen können Umweltbesorgnisse, -ängste, -konditionierungen und -attributionen sowie eine Vielzahl von Erkrankungen sein. Besonders zu schützende Risikogruppen bezüglich eines Infektionsrisikos sind Personen unter Immunsuppression nach der Einteilung der Kommission für Krankenhaushygiene und Infektionsprävention (KRINKO) beim Robert Koch-Institut (RKI) und Personen mit Mukoviszidose (Zystischer Fibrose), bezüglich eines allergischen Risikos Personen mit Mukoviszidose (Zystischer Fibrose) und Personen mit Asthma bronchiale. Die rationale Diagnostik beinhaltet die Anamnese, eine körperliche Untersuchung, eine konventionelle Allergiediagnostik einschließlich gegebenenfalls Provokationstests, vereinzelt sind auch zelluläre Testsysteme indiziert. Zum Vorgehen bei Schimmelpilzinfektionen wird auf die angemeldete AWMF-Leitlinie Diagnose und Therapie invasiver Aspergillus-Infektionen verwiesen. Hinsichtlich Mykotoxine existieren zurzeit keine brauchbaren und validierten Testverfahren, die in der klinischen Diagnostik eingesetzt werden könnten. Präventivmedizinisch ist wichtig, dass Schimmelpilzbefall in relevantem Ausmaß aus Vorsorgegründen nicht toleriert werden darf. Zur Beurteilung des Schadensausmaßes und zum Vorgehen wird auf den „Schimmelpilzleitfaden“ des Umweltbundesamtes verwiesen.

Pathogen Inactivating Properties and Increased Sensitivity in Molecular Diagnostics by PAXgene, a Novel Non-Crosslinking Tissue Fixative

Background Requirements on tissue fixatives are getting more demanding as molecular analysis becomes increasingly relevant for routine diagnostics. Buffered formaldehyde in pathology laboratories for tissue fixation is known to cause chemical modifications of biomolecules which affect molecular testing. A novel non-crosslinking tissue preservation technology, PAXgene Tissue (PAXgene), was developed to preserve the integrity of nucleic acids in a comparable way to cryopreservation and also to preserve morphological features comparable to those of formalin fixed samples. Methods Because of the excellent preservation of biomolecules by PAXgene we investigated its pathogen inactivation ability and biosafety in comparison to formalin by in-vitro testing of bacteria, human relevant fungi and human cytomegalovirus (CMV). Guidelines for testing disinfectants served as reference for inactivation assays. Furthermore, we tested the properties of PAXgene for detection of pathogens by PCR based assays. Results All microorganisms tested were similarly inactivated by PAXgene and formalin except Clostridium sporogenes, which remained viable in seven out of ten assays after PAXgene treatment and in three out of ten assays after formalin fixation. The findings suggest that similar biosafety measures can be applied for PAXgene and formalin fixed samples. Detection of pathogens in PCR-based diagnostics using two CMV assays resulted in a reduction of four to ten quantification cycles of PAXgene treated samples which is a remarkable increase of sensitivity. Conclusion PAXgene fixation might be superior to formalin fixation when molecular diagnostics and highly sensitive detection of pathogens is required in parallel to morphology assessment.

Health effects of indoor molds

SummaryMolds are found almost everywhere in the environment. Their airborne propagules (conidia, spores, hyphal fragments) can – under certain circumstances – cause a variety of health problems like mycotic infections, allergies, asthma, irritations or toxic syndromes.ZusammenfassungSchimmelpilze findet man nahezu überall in der Umwelt. Unter bestimmten Voraussetzungen können deren luftgetragene Partikel wie Konidien, Sporen und Hyphenbestandteile zu einer Reihe von gesundheitlichen Beeinträchtigungen wie Pilzinfektionen, Allergien, Asthma, Irritationen oder Vergiftungen führen.