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PubMed1997

Screening of fungi for the presence of the trichodiene synthase encoding sequence by hybridization to the Tri5 gene cloned from Fusarium poae.

A trichodiene synthase gene (Tri5) was amplified from F. poae by polymerase chain reaction using synthetic primers constructed on the basis of the coding portion of the same gene from F. sporotrichioides. Sequence analysis showed a high degree of similarity with other trichodiene synthase genes. A 378 bp HindIII fragment of the gene that contains the genetic information for the putative active site of the trichodiene synthase enzyme was radiolabelled and used for dot blot analysis. This probe could detect Tri5 hybridization in 1-10 ng DNA of fusaria that have the genetic potentiality to synthesize toxic trichothecene compounds, but gave no reaction with trichothecene nonproducing members of the genus. When other fungi reported to produce trichothecenes (Myrothecium, Stachybotrys, Trichoderma, Trichothecium spp.) were tested, only strains of Myrothecium and Stachybotrys gave strong positive reaction. Faint but consistent hybridization signals were obtained in four species (F. semitectum, F. tricinctum, Trichoderma viride and Trichothecium roseum) indicating the presence of nonhomologous evolutionary variants or inactive remnants of the Tri5 gene in these fungi.

Base Sequence↗
PubMedDate not supplied

A method for detecting fungal contaminants in wall cavities.

This article describes a practical method for detecting the presence of both fungal spores and culturable fungi in wall cavities. Culturable fungi were collected in 25 mm cassettes containing 0.8 microm mixed cellulose ester filters using aggressive sampling conditions. Both culturable fungi and fungal spores were collected in modified slotted-disk cassettes. The sample volume was 4 L. The filters were examined microscopically and dilution plated onto multiple culture media. Collecting airborne samples in filter cassettes was an effective method for assessing wall cavities for fungal contaminants, especially because this method allowed the sample to be analyzed by both microscopy and culture media. Assessment criteria were developed that allowed the sample results to be used to classify wall cavities as either uncontaminated or contaminated. As a criterion, wall cavities with concentrations of culturable fungi below the limit of detection (LOD) were classified as uncontaminated, whereas those cavities with detectable concentrations of culturable fungi were classified as contaminated. A total of 150 wall cavities was sampled as part of a field project. The concentrations of culturable fungi were below the LOD in 34% of the samples, whereas Aspergillus and/or Penicillium were the only fungal genera detected in 69% of the samples in which culturable fungi were detected. Spore counting resulted in the detection of Stachybotrys-like spores in 25% of the samples that were analyzed, whereas Stachybotrys chartarum colonies were only detected on 2% of malt extract agar plates and on 6% of corn meal agar plates.

Aerosols↗
PubMed2005

A regional comparison of mold spore concentrations outdoors and inside "clean" and "mold contaminated" Southern California buildings.

A total of 625 buildings and outdoor locations in the San Diego, California, area were monitored using the Allergenco Sampl-Air MK-3 impaction sampler or the Zefon Air-O-Cell slit bioaerosol cassette. Locations were classified by rigid criteria as clean commercial, commercial with mold growth, clean residential, residential with water staining, and residential with mold growth. In addition, coastal and inland outdoor locations were measured. Seven categories (total spores, Ascospores/Basidiospores, Cladosporium, Smut/Myxomycetes-like, Aspergillus/Penicillium (AS/PE), Alternaria, and Unidentified/Other) were detected frequently enough that maximum likelihood estimate techniques could be used to determine distribution parameters and, thus, treat these as continuous variables. For total counts (no nondetectables) an analysis of variance was used to examine differences in location means. For the other categories Land's confidence limits were generated and visually compared for differences among locations. For 12 other categories (Curvularia, Dreschlera, Epicoccum, Fusarium, Mildew-like, Pithomyces, Rusts, Stachybotrys, Stemphyllium, Torula, Ulocladium, and Zygomycetes-like), detection generally occurred in less than 10% of samples. These genera were treated as dichotomous (detect/nondetect) data, and Chi-square analyses differentiated between locations. For total counts, values were significantly different on the order of clean < outdoor < moldy. There was a large difference between the moldy and other location classes. For AS/PE, moldy location means were clearly higher than those for clean buildings and outdoors, although the clean and outdoor means could not be differentiated. For all other genera the results tend to indicate little or no ability to discriminate location. For example, there were no differences in the probabilities of detecting Stachybotrys among the various locations. In our study only total counts, usually driven by AS/PE concentrations, had value in determining whether a building is mold contaminated employing our set of rigorous location classification criteria.

Air Pollution, Indoor↗
PubMed1999

Microfungal contamination of damp buildings--examples of risk constructions and risk materials.

To elucidate problems with microfungal infestation in indoor environments, a multidisciplinary collaborative pilot study, supported by a grant from the Danish Ministry of Housing and Urban Affairs, was performed on 72 mold-infected building materials from 23 buildings. Water leakage through roofs, rising damp, and defective plumbing installations were the main reasons for water damage with subsequent infestation of molds. From a score system assessing the bioavailability of the building materials, products most vulnerable to mold attacks were water damaged, aged organic materials containing cellulose, such as wooden materials, jute, wallpaper, and cardboard. The microfungal genera most frequently encountered were Penicillium (68%), Aspergillus (56%), Chaetomium (22%), Ulocladium, (21%), Stachybotrys (19%) and Cladosporium (15%). Penicillium chrysogenum, Aspergillus versicolor, and Stachybotrys chartarum were the most frequently occurring species. Under field conditions, several trichothecenes were detected in each of three commonly used building materials, heavily contaminated with S. chartarum. Under experimental conditions, four out of five isolates of S. chartarum produced satratoxin H and G when growing on new and old, very humid gypsum boards. A. versicolor produced the carcinogenic mycotoxin sterigmatocystin and 5-methoxysterigmatocystin under the same conditions.

Air Pollution, Indoor↗
PubMed1998

Toxigenic fungi in a water-damaged building: an intervention study.

In an investigation of health complaints among employees of a water-damaged office building, the environment showed evidence of fungal contamination with the isolation of Stachybotrys chartarum in one of five bulk samples tested for fungal growth. In response, a public health official recommended that employees be relocated from the building. Employees were subsequently moved to a different environment. A focused environmental investigation of microbial growth within the building followed, revealing moderate to high levels of fungi (Penicillium, Aspergillus versicolor) and bacteria in bulk and surface samples. S. Chartarum was identified in one of 19 (5%) environmental samples using Czapek agar. A health survey of building occupants revealed a high prevalence of multiple symptoms, with the predominance of neurobehavioral and upper respiratory tract complaints. The majority of symptoms were significantly less prevalent after relocation from the water-damaged environment. The initial hypothesis that exposure to toxigenic fungi was responsible for the high prevalence of reported symptoms is difficult to investigate and confirm given the current limits of epidemiological knowledge regarding exposure to these organisms and building-related illness. Future interventions where mycotoxin exposure is suspected should emphasize the importance of risk assessment and risk communication.

Adult↗
PubMed2006

Measurement of xenon diffusing capacity in the rat lung by hyperpolarized 129Xe MRI and dynamic spectroscopy in a single breath-hold.

We used the dual capability of hyperpolarized 129Xe for spectroscopy and imaging to develop new measures of xenon diffusing capacity in the rat lung that (analogously to the diffusing capacity of carbon monoxide or DLCO) are calculated as a product of total lung volume and gas transfer rate constants divided by the pressure gradient. Under conditions of known constant pressure breath-hold, the volume is measured by hyperpolarized 129Xe MRI, and the transfer rate is measured by dynamic spectroscopy. The new quantities (xenon diffusing capacity in lung parenchyma (DLXeLP)), xenon diffusing capacity in RBCs (DLXeRBC), and total lung xenon diffusing capacity (DLXe)) were measured in six normal rats and six rats with lung inflammation induced by instillation of fungal spores of Stachybotrys chartarum. DLXeLP, DLXeRBC, and DLXe were 56 +/- 10 ml/min/mmHg, 64 +/- 35 ml/min/mmHg, and 29 +/- 9 ml/min/mmHg, respectively, for normal rats, and 27 +/- 9 ml/min/mmHg, 42 +/- 27 ml/min/mmHg, and 16 +/- 7 ml/min/mmHg, respectively, for diseased rats. Lung volumes and gas transfer times for LP (TtrLP) were 16 +/- 2 ml and 22 +/- 3 ms, respectively, for normal rats and 12 +/- 2 ml and 35 +/- 8 ms, respectively, for diseased rats. Xenon diffusing capacities may be useful for measuring changes in gas exchange associated with inflammation and other lung diseases.

Animals↗
PubMed1991

Mycotoxin production on different cultivars and lines of broad bean (Vicia faba L.) seeds in Egypt.

One hundred different cultivars and lines of broad bean (Vicia faba L.) seed samples were inoculated with Aspergillus flavus Link (CMI 102135) to determine varietal differences which may support or resist aflatoxin production. Thin-layer chromatographic analysis of the chloroform extracts of the different seed samples revealed that 11 cultivars/lines were highly resistant to seed invasion and aflatoxin production while 9 cultivars/line showed partial resistance. The remaining 80 samples were susceptible to the establishment of A. flavus and aflatoxin accumulation. All the resistant cultivars/lines seed samples were inoculated also with three local isolates of fungi namely; Stachybotrys chartarum (Ehrenb. ex Link) Hughes, Aspergillus ochraceus Wilhelm, and Fusarium oxysporum Schlecht. The resistant seed samples were also resistant for colonization with these fungi and mycotoxin formation.

Aflatoxins↗
PubMed2001

Serum IgE specific to indoor moulds, measured by basophil histamine release, is associated with building-related symptoms in damp buildings.

OBJECTIVE: To study the relationship between basophil histamine release (HRT) to indoor moulds, indicating specific IgE, and building-related symptoms (BRS), asthma, and hay fever in individuals working in damp and mouldy buildings. METHODS: A cross-sectional study was performed among 86 school staff members, who on average had worked 143 months (range: 3-396) in moist buildings with mould growth in the constructions. A questionnaire concerning mucous membrane symptoms, facial skin symptoms, central nervous system symptoms, hay fever, and asthma was fulfilled by the participants, and blood samples were taken. Eight mould species growing on building constructions were identified and cultivated to obtain allergenic materials for testing. The presence in serum of IgE specific to moulds was verified by histamine release test (HRT) based on passive sensitization of basophil leukocytes. The validity of the method was confirmed by parallel testing of patients allergic to grass- and birch pollen and by the shift from positive to negative response after removal of serum IgE and by using sham sensitization. RESULTS: The prevalence of most BRS was between 32% and 62%. Positive HRT, showing serum IgE specific to one or more of the moulds, was observed in 37% of the individuals. The highest frequency of positive HRT was found to Penicillium chrysogenum and then to Aspergillus species, Cladosporium sphaerospermum and Stachybotrys chartarum. A significant association was found between most BRS and positive HRT, whereas no association was observed between positive HRT to moulds and self reported hay fever or asthma. CONCLUSION: Positive HRT to indoor moulds, showing the presence in serum of IgE specific to the fungi, was found to be related to BRS in individuals working in damp and mouldy buildings. Whether the association is of causal character is a question for further studies. The test may be useful in the evaluation and study of possible mould induced BRS.

Adult↗
PubMed2006

A simple polymerase chain reaction-sequencing analysis capable of identifying multiple medically relevant filamentous fungal species.

Due to the accumulating evidence that suggests that numerous unhealthy conditions in the indoor environment are the result of abnormal growth of the filamentous fungi (mold) in and on building surfaces it is necessary to accurately determine the organisms responsible for these maladies and to identify them in an accurate and timely manner. Historically, identification of filamentous fungal (mold) species has been based on morphological characteristics, both macroscopic and microscopic. These methods may often be time consuming and inaccurate, necessitating the development of identification protocols that are rapid, sensitive, and precise. To this end, we have devised a simple PAN-PCR approach which when coupled to cloning and sequencing of the clones allows for the unambiguous identification of multiple fungal organisms. Universal primers are used to amplify ribosomal DNA sequences which are then cloned and transformed into Escherichia coli. Individual clones are then sequenced and individual sequences analyzed and organisms identified. Using this method we were capable of identifying Stachybotrys chartarum, Penicillium purpurogenum, Aspergillus sydowii, and Cladosporium cladosporioides from a mixed culture. This method was found to be rapid, highly specific, easy to perform, and cost effective.

Aspergillus↗
PubMed2005

A simple multiplex polymerase chain reaction assay for the identification of four environmentally relevant fungal contaminants.

Historically, identification of filamentous fungal (mold) species has been based on morphological characteristics, both macroscopic and microscopic. These methods may often be time-consuming and inaccurate, necessitating the development of identification protocols that are rapid, sensitive, and precise. The polymerase chain reaction (PCR) has shown great promise in its ability to identify and quantify individual organisms from a mixed culture environment; however, the cost effectiveness of single organism PCR reactions is quickly becoming an issue. Our laboratory has developed a simple method to identify multiple fungal species, Stachybotrys chartarum, Aspergillus versicolor, Penicillium purpurogenum, and Cladosporium spp. by performing multiplex PCR and distinguishing the different reaction products by their mobility during agarose gel electrophoresis. The amplified genes include the beta-Tubulin gene from A. versicolor, the Tri5 gene from S. chartarum, and ribosomal sequences from both P. purpurogenum and Cladosporium spp. This method was found to be both rapid and easy to perform, while maintaining high sensitivity and specificity for characterizing isolates, even from a mixed culture.

Air Pollution, Indoor↗
PubMed2003

Use of gas chromatography-mass spectrometry/solid phase microextraction for the identification of MVOCs from moldy building materials.

Gas chromatography-mass spectrometry/solid phase microextraction (GC-MS/SPME) was applied to identify microbial volatile organic compounds (MVOCs) in water-damaged, mold-infested building materials (gypsum board papers (n=2), mineral wool, and masonite) and in cultivated molds (Aspergillus penicillioides, Stachybotrys chartarum, and Chaetomium globosum). Three SPME fibers (65-microm PDMS-DVB, 75-microm Carboxen-PDMS, and 70-microm Carbowax-stableflex) designed for automated injection were used of which the latter showed best performance. A number of previously reported MVOCs were detected both in the building materials and the cultivated molds. In addition, methyl benzoate was identified both in the S. chartarum and A. penicillioides cultures and in the building materials. SPME combined with GC-MS may be a useful method for the determination of MVOCs emitted from mold-infested building materials.

Air Pollution, Indoor↗
PubMed2000

Kurstakins: a new class of lipopeptides isolated from Bacillus thuringiensis.

A novel class of lipopeptides was isolated from Bacillus thuringiensis kurstaki HD-1. Four compounds (1-4) were separated by high-performance liquid chromatography and their primary structures determined using a combination of chemical reactions and mass spectrometry. The four lipopeptides were found to have the same amino acid sequence, Thr-Gly-Ala-Ser-His-Gln-Gln, but different fatty acids. The fatty acyl chain is linked to the N-terminal amino acid residue via an amide bond. Each lipopeptide has a lactone linkage between the carboxyl terminal amino acid and the hydroxyl group in the side chain of the serine residue. Antifungal activity was demonstrated against Stachybotrys charatum.

Amino Acids↗
PubMed2002

Indoor air microbes and respiratory symptoms of children in moisture damaged and reference schools.

Microbial indoor air quality and respiratory symptoms of children were studied in 24 schools with visible moisture and mold problems, and in eight non-damaged schools. School buildings of concrete/brick and wooden construction were included. The indoor environment investigations included technical building inspections for visible moisture signs and microbial sampling using six-stage impactor for viable airborne microbes. Children's health information was collected by questionnaires. The effect of moisture damage on concentrations of fungi was clearly seen in buildings of concrete/brick construction, but not in wooden school buildings. Occurrence of Cladosporium, Aspergillus versicolor, Stachybotrys, and actinobacteria showed some indicator value for moisture damage. Presence of moisture damage in school buildings was a significant risk factor for respiratory symptoms in schoolchildren. Association between moisture damage and respiratory symptoms of children was significant for buildings of concrete/brick construction but not for wooden school buildings. The highest symptom prevalence was found during spring seasons, after a long exposure period in damaged schools. The results emphasize the importance of the building frame as a determinant of exposure and symptoms.

Actinobacteria↗
PubMed2003

The relation between growth of four microbes on six different plasterboards and biological activity of spores.

Microbial growth on water-damaged building materials is commonly associated with adverse health effects in the occupants. We examined the growth of Stachybotrys chartarum, Aspergillus versicolor, Penicillium spinulosum, and Streptomyces californicus, isolated from water-damaged buildings, on six different brands of plasterboards. The microbial growth was compared with the biological activity of the spores, that is the potential to induce cytotoxicity and proinflammatory mediators in RAW264.7 macrophages. These results showed that the microbial growth on plasterboard depended on both the microbial strain and the brand of plasterboard used. The biological activity of spores appeared to be regulated by different growth conditions on plasterboards so that good microbial growth was associated with a low bioactivity of the spores, whereas the spores collected from plasterboard supporting only weak growth usually were biologically active. Cytotoxicity of either S. chartarum or A. versicolor did not correlate with any particular growth conditions or induced inflammatory responses. Instead, there were positive correlations between cytotoxicity and levels of induced proinflammatory cytokines for P. spinulosum and S. californicus. These data suggest that both the microbial growth on plasterboard and the resulting bioactivity of spores vary and might be affected by changing the growth conditions provided by the plasterboards.

Air Pollution, Indoor↗
PubMed2005

Evaluation of interference to conventional and real-time PCR for detection and quantification of fungi in dust.

Advances in polymerase chain reaction (PCR) have permitted accurate, rapid and quantitative identification of microorganisms in pure cultures regardless of viability or culturability. In this study, a simple sample processing method was investigated for rapid identification and quantification of fungal spores from dust samples using both conventional and real-time PCR. The proposed method was evaluated for susceptibility to interference from environmental dust samples. Stachybotrys chartarum and Aspergillus fumigatus were used as test organisms. The sensitivity of detection in pure culture was 0.1 spore DNA equivalents per PCR reaction corresponding to 20 spores ml(-1) in the sample. However, 1 spore DNA equivalent per PCR reaction corresponding to 200 spores ml(-1) in the sample was the lowest amount of spores tested without interference in dust samples spiked with spores of either fungal species. The extent of inhibition was calculated using conventional and real-time PCR reactions containing fungal spores, specific primers, specific probes (for real-time PCR) and various amounts of dust. The results indicate that the extent of inhibition by dust on PCR varies with the type and amount of dust, and number of spores. No interference in the analysis of spiked samples was detected from 0.2 mg ml(-1) of four real-life dust samples at p-value >0.05 using 2 x 10(4) spores for conventional PCR and 2 x 10(5) spores for real-time PCR. However, samples containing >0.2 mg ml(-1) real-life dust compromised the PCR assay. These results suggest the potential usefulness of a simple sample processing method in conjunction with PCR for monitoring the fungal content of aerosols collected from indoor environments.

Air Pollution, Indoor↗
PubMed2000

Acute pulmonary haemorrhage in an infant during induction of general anaesthesia.

Pulmonary haemorrhage is a rare, life-threatening complication of anaesthesia. This report describes the anaesthetic management of an infant who developed laryngospasm and pulmonary haemorrhage during general anaesthesia. The infant was subsequently found to have prior exposure to a fungus, Stachybotrys chartarum, which produces mycotoxins that may have produced capillary fragility in the infant's rapidly growing lungs.

Acute Disease↗
PubMed2005

Effects of microbial cocultivation on inflammatory and cytotoxic potential of spores.

Microbial growth on moisture-damaged building materials is commonly associated with adverse health effects in the occupants. In moisture damage situations, the environmental conditions as well as the dominant microbial species will vary, leading to a diversity of microbes and continual changes in the different microbial populations. Currently, very little is known about the effects of microbial cocultures on the potential harmfulness of the microbial population. In this study we have investigated the effects of cocultivation of certain indoor air microbes on the inflammatory and cytotoxic potential of their spores. We grew various microbial combinations made from strains of Streptomyces californicus, Stachybotrys chartarum, Aspergillus versicolor, and Penicillium spinulosum on wetted plasterboard. After 5 or 10 wk of growth, the spores were collected from the plasterboards, mouse RAW264.7 macrophages were exposed to the spores, and after 24 h the induced inflammatory and cytotoxic responses were analyzed. Among all the tested microbes and their combinations, the spores of Str. californicus proved to be the most potent inducer of cytotoxicity and inflammatory responses. These results indicate also that microbial coculture may support the growth of certain microbes with high immunotoxic potency such as Str.californicus. Furthermore, coculture containing S. chartarum and A. versicolor caused a synergistic increase in cytotoxicity compared to the sum response induced by the pure cultures, but no effect on inflammatory responses was detected. Generally, spore-induced cytotoxicity and production of inflammatory markers increased during the growth period from 5 to 10 wk, suggesting that the immunotoxic potency of spores increases with time.

Air Pollution, Indoor↗
PubMed2004

Culturability and toxicity of sick building syndrome-related fungi over time.

Two experiments were conducted regarding the culturability and toxicity of fungi located on building materials over time and the efficacy of seven laboratory techniques in recovering culturable fungi from sample swabs. In the first experiment, eight sections of drywall were inoculated with Stachybotrys chartarum and stored at 25 +/- 5 degrees Celsius and 20-60% relative humidity (RH) for up to two years. Another eight sections of ceiling tile were stored at 100% RH for 1 year. Six sections of ceiling tile and 15 swabs were also inoculated with Penicillium chrysogenum and S. chartarum respectively and stored under the same conditions for 8 months and 3.3 years. All materials were tested for culturability at the end of the storage period. S. chartarum-inoculated samples were also tested for toxicity. In the second experiment (replicated twice), S. chartarum and Chaetomium globosum were inoculated onto 84 swabs each. Storage was up to 266 days at 25 +/- 5 degrees Celsius and 20-60% RH. Seven techniques were compared regarding the recovery of culturable fungi from the swabs over different time points. Results for Experiment 1 showed that all samples were culturable after the storage period and that the S. chartarum-inoculated drywall samples were toxic. In Experiment 2, all techniques showed high rates of recovery. These data show that despite being without a water source, these organisms can be culturable and toxic after long periods of time under conditions similar to human-occupied dwellings and that a number of preparation techniques are suitable for the recovery of these fungi from inoculated swabs.

Construction Materials↗