Specific detection of Stachybotrys chartarum in pure culture using quantitative polymerase chain reaction

Cruz-Perez, Patricia; Buttner, Mark P.; Stetzenbach, Linda D.

doi:10.1006/mcpr.2001.0347

Cited by 46 publications

(20 citation statements)

References 16 publications

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“…False-negative PCRs can result from the contaminants being coextracted with DNA that acts as a PCR inhibitor. These PCR inhibitors can be removed by either further DNA purification or a dilution step (1,3,24). In this study, the first and second DNA elutions from the aerosols failed in PCRs, but a 1:100 dilution worked for all the PCR tests.…”

Section: Discussionmentioning

confidence: 76%

Detection and Quantification ofWallemia sebiin Aerosols by Real-Time PCR, Conventional PCR, and Cultivation

Zeng

Westermark²,

Rasmuson-Lestander

et al. 2004

Appl Environ Microbiol

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Wallemia sebi is a deuteromycete fungus commonly found in agricultural environments in many parts of the world and is suspected to be a causative agent of farmer's lung disease. The fungus grows slowly on commonly used culture media and is often obscured by the fast-growing fungi. Thus, its occurrence in different environments has often been underestimated. In this study, we developed two sets of PCR primers specific to W. sebi that can be applied in either conventional PCR or real-time PCR for rapid detection and quantification of the fungus in environmental samples. Both PCR systems proved to be highly specific and sensitive for W. sebi detection even in a high background of other fungal DNAs. These methods were employed to investigate the presence of W. sebi in the aerosols of a farm. The results revealed a high concentration of W. sebi spores, 10 7 m ؊3 by real-time PCR and 10 6 m ؊3 by cultivation, which indicates the prevalence of W. sebi in farms handling hay and grain and in cow barns. The methods developed in this study could serve as rapid, specific, and sensitive means of detecting W. sebi in aerosol and surface samples and could thus facilitate investigations of its distribution, ecology, clinical diagnosis, and exposure risk assessment.Wallemia sebi is a deuteromycete fungus capable of growth over a wide range of water activity from 0.69 to 0.997 (15). It can potentially grow in various environments and on different substances and has been isolated from jam, cake, cereals, salted meat, fish, and dairy products (12,23). Up to now, only one species is described in the genus Wallemia. W. sebi grows slowly on commonly used culture media, such as malt extract agar, and is often obscured by the fast-growing fungi. Thus, its presence in different environments has often been overlooked, which in turn hindered the studies on its distribution and ecology. Recently, with the use of selective media for xerophilic fungi, W. sebi has been found to be very common in the agricultural environments of many parts of the world (4,6,9,16).The conidium of W. sebi has a shape of a rough-surfaced sphere of 2.5 to 3.5 m in diameter (18); thus, it can reach the respiratory bronchioles when inhaled. Airborne W. sebi has been suspected to be a causative agent of human allergies, particularly bronchial asthma (17). Elevated levels of immunoglobulin G (IgG) antibodies were observed among Finnish farmers exposed to W. sebi (9). In eastern France, W. sebi has also been identified as playing a role in farmer's lung disease (16). The fungus produces a toxic metabolite, walleminol A, with a bioinhibitory dose effect similar to those of other mycotoxins such as penicillic acid (23).Conventional methods for the detection and quantification of W. sebi rely on microscopic or culture techniques that are time consuming and laborious. Molecular techniques are promising approaches complementary to the conventional detection methods. PCR-based methods have the advantage of detecting the presence of microorganisms in a sample regardless of the...

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Section: Discussionmentioning

confidence: 76%

Detection and Quantification ofWallemia sebiin Aerosols by Real-Time PCR, Conventional PCR, and Cultivation

Zeng

Westermark²,

Rasmuson-Lestander

et al. 2004

Appl Environ Microbiol

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“…Finally, particle size may play a key role when attempting to quantitate some species; for example, the rapid settling of the large spores of Ulocladium species probably accounts for their being underrepresented in the airborne spore load (144,171). Such culture difficulties may eventually be circumvented using new techniques such as PCR (80,347). A final problem in measuring fungal organisms in the indoor environment relates to selective sampling.…”

Section: Technical Problems In Determining Fungal Exposurementioning

confidence: 99%

Indoor Mold, Toxigenic Fungi, andStachybotrys chartarum: Infectious Disease Perspective

2003

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Damp buildings often have a moldy smell or obvious mold growth; some molds are human pathogens. This has caused concern regarding health effects of moldy indoor environments and has resulted in many studies of moisture- and mold-damaged buildings. Recently, there have been reports of severe illness as a result of indoor mold exposure, particularly due to Stachybotrys chartarum. While many authors describe a direct relationship between fungal contamination and illness, close examination of the literature reveals a confusing picture. Here, we review the evidence regarding indoor mold exposure and mycotoxicosis, with an emphasis on S. chartarum. We also examine possible end-organ effects, including pulmonary, immunologic, neurologic, and oncologic disorders. We discuss the Cleveland infant idiopathic pulmonary hemorrhage reports in detail, since they provided important impetus for concerns about Stachybotrys. Some valid concerns exist regarding the relationship between indoor mold exposure and human disease. Review of the literature reveals certain fungus-disease associations in humans, including ergotism (Claviceps species), alimentary toxic aleukia (Fusarium), and liver disease (Aspergillys). While many papers suggest a similar relationship between Stachybotrys and human disease, the studies nearly uniformly suffer from significant methodological flaws, making their findings inconclusive. As a result, we have not found well-substantiated supportive evidence of serious illness due to Stachybotrys exposure in the contemporary environment. To address issues of indoor mold-related illness, there is an urgent need for studies using objective markers of illness, relevant animal models, proper epidemiologic techniques, and examination of confounding factors

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“…Polymerase chain reaction (PCR) techniques are rapidly developing, and further applications of the new genetic techniques for specific detection of microbes in indoor environments can be expected (Cruz‐Perez et al., 2001; Haugland et al., 1999; Meklin et al., 2004; Rintala et al., 2001). Being based on detection of genetic material from microbes, the techniques are independent of the viability of the microorganisms to be detected.…”

Section: Exposure Assessmentmentioning

confidence: 99%

Of microbes and men

Nevalainen¹,

Seuri

2005

Indoor Air

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Specific detection of Stachybotrys chartarum in pure culture using quantitative polymerase chain reaction

Cited by 46 publications

References 16 publications

Detection and Quantification ofWallemia sebiin Aerosols by Real-Time PCR, Conventional PCR, and Cultivation

Detection and Quantification ofWallemia sebiin Aerosols by Real-Time PCR, Conventional PCR, and Cultivation

Indoor Mold, Toxigenic Fungi, andStachybotrys chartarum: Infectious Disease Perspective

Of microbes and men

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