Stephen Vesper

Reduction in asthma morbidity in children as a result of home remediation aimed at moisture sources.

Objective Home dampness and the presence of mold and allergens have been associated with asthma morbidity. We examined changes in asthma morbidity in children as a result of home remediation aimed at moisture sources. Design In this prospective, randomized controlled trial, symptomatic, asthmatic children (n = 62), 2–17 years of age, living in a home with indoor mold, received an asthma intervention including an action plan, education, and individualized problem solving. The remediation group also received household repairs, including reduction of water infiltration, removal of water-damaged building materials, and heating/ventilation/air-conditioning alterations. The control group received only home cleaning information. We measured children’s total and allergen-specific serum immuno-globulin E, peripheral blood eosinophil counts, and urinary cotinine. Environmental dust samples were analyzed for dust mite, cockroach, rodent urinary protein, endotoxin, and fungi. The follow-up period was 1 year. Results Children in both groups showed improvement in asthma symptomatic days during the preremediation portion of the study. The remediation group had a significant decrease in symptom days (p = 0.003, as randomized; p = 0.004, intent to treat) after remodeling, whereas these parameters in the control group did not significantly change. In the postremediation period, the remediation group had a lower rate of exacerbations compared with control asthmatics (as treated: 1 of 29 vs. 11 of 33, respectively, p = 0. 003; intent to treat: 28.1% and 10.0%, respectively, p = 0.11). Conclusion Construction remediation aimed at the root cause of moisture sources and combined with a medical/behavioral intervention significantly reduces symptom days and health care use for asthmatic children who live in homes with a documented mold problem.

Evaluation of rapid DNA extraction methods for the quantitative detection of fungi using real-time PCR analysis

Three comparatively rapid methods for the extraction of DNA from fungal conidia and yeast cells in environmental (air, water and dust) samples were evaluated for use in real-time PCR (TaqMan) analyses. A simple bead milling method was developed to provide sensitive, accurate and precise quantification of target organisms in air and water (tap and surface) samples. However, quantitative analysis of dust samples required further purification of the extracted DNA by a streamlined silica adsorption procedure.

Evaluation of a rapid, quantitative real-time PCR method for enumeration of pathogenic Candida cells in water.

ABSTRACT Quantitative PCR (QPCR) technology, incorporating fluorigenic 5′ nuclease (TaqMan) chemistry, was utilized for the specific detection and quantification of six pathogenic species of Candida (C. albicans, C. tropicalis, C. krusei, C. parapsilosis, C. glabrata and C. lusitaniae) in water. Known numbers of target cells were added to distilled and tap water samples, filtered, and disrupted directly on the membranes for recovery of DNA for QPCR analysis. The assays sensitivities were between one and three cells per filter. The accuracy of the cell estimates was between 50 and 200% of their true value (95% confidence level). In similar tests with surface water samples, the presence of PCR inhibitory compounds necessitated further purification and/or dilution of the DNA extracts, with resultant reductions in sensitivity but generally not in quantitative accuracy. Analyses of a series of freshwater samples collected from a recreational beach showed positive correlations between the QPCR results and colony counts of the corresponding target species. Positive correlations were also seen between the cell quantities of the target Candida species detected in these analyses and colony counts of Enterococcus organisms. With a combined sample processing and analysis time of less than 4 h, this method shows great promise as a tool for rapidly assessing potential exposures to waterborne pathogenic Candida species from drinking and recreational waters and may have applications in the detection of fecal pollution.

Quantitative PCR analysis of house dust can reveal abnormal mold conditions

Indoor mold concentrations were measured in the dust of moldy homes (MH) and reference homes (RH) by quantitative PCR (QPCR) assays for 82 species or related groups of species (assay groups). About 70% of the species and groups were never or only rarely detected. The ratios (MH geometric mean : RH geometric mean) for 6 commonly detected species (Aspergillus ochraceus, A. penicillioides, A. unguis, A. versicolor, Eurotium group, and Cladosporium sphaerospermum) were >1 (Group I). Logistic regression analysis of the sum of the logs of the concentrations of Group I species resulted in a 95% probability for separating MH from RH. These results suggest that it may be possible to evaluate whether a home has an abnormal mold condition by quantifying a limited number of mold species in a dust sample. Also, four common species of Aspergillus were quantified by standard culturing procedures and their concentrations compared to QPCR results. Culturing underestimated the concentrations of these four species by 2 to 3 orders of magnitude compared to QPCR.

High environmental relative moldiness index during infancy as a predictor of asthma at 7 years of age

BACKGROUND Mold exposures may contribute to the development of asthma, but previous studies have lacked a standardized approach to quantifying exposures. OBJECTIVE To determine whether mold exposures at the ages of 1 and/or 7 years were associated with asthma at the age of 7 years. METHODS This study followed up a high-risk birth cohort from infancy to 7 years of age. Mold was assessed by a DNA-based analysis for the 36 molds that make up the Environmental Relative Moldiness Index (ERMI) at the ages of 1 and 7 years. At the age of 7 years, children were evaluated for allergic sensitization and asthma based on symptom history, spirometry, exhaled nitric oxide, and airway reversibility. A questionnaire was administered to the parent regarding the childs asthma symptoms and other potential cofactors. RESULTS At the age of 7 years, 31 of 176 children (18%) were found to be asthmatic. Children living in a high ERMI value (≥5.2) home at 1 year of age had more than twice the risk of developing asthma than those in low ERMI value homes (<5.2) (adjusted odds ratio [aOR], 2.6; 95% confidence interval [CI], 1.10-6.26). Of the other covariates, only parental asthma (aOR, 4.0; 95% CI, 1.69-9.62) and allergic sensitization to house dust mite (aOR, 4.1; 95% CI, 1.55-11.07) were risk factors for asthma development. In contrast, air-conditioning at home reduced the risk of asthma development (aOR, 0.3; 95% CI, 0.14-0.83). A high ERMI value at 7 years of age was not associated with asthma at 7 years of age. CONCLUSIONS Early exposure to molds as measured by ERMI at 1 year of age, but not 7 years of age, significantly increased the risk for asthma at 7 years of age.

Evaluation of Stachybotrys chartarum in the house of an infant with pulmonary hemorrhage: quantitative assessment before, during, and after remediation.

Stachybotrys chartarum is an indoor mold that has been associated with pulmonary hemorrhage cases in the Cleveland, Ohio, area. This study applied two new quantitative measurements to air samples from a home in which an infant developed PH. Quantitative polymerase chain reaction and a protein synthesis inhibition assay were used to determine the level ofS. chartarum spores and their toxicity in air samples taken before, during, and after a remediation program was implemented to remove the fungus. Initial spore concentrations were between 0.1 and 9.3 spores/m3 of air, and the toxicity of air particulates was correspondingly low. However, the dust in the house contained between 0.4 and 2.1×103 spores/mg (as determined by hemocytometer counts). The remediation program removed all contaminated wallboard, paneling, and carpeting in the water-damaged areas of the home. In addition, a sodium hypochlorite solution was used to spray all surfaces during remediation. Although spore counts and toxicity were high during remediation, air samples taken postremediation showed no detectable levels ofS. chartarum or related toxicity. Nine isolates ofS. chartarum obtained from the home were analyzed for spore toxicity, hemolytic activity, and random amplified polymorphic DNA banding patterns. None of the isolates produced highly toxic spores (>90 μg T2 toxin equivalents per gram wet weight spores) after growth for 10 and 30 days on wet wallboard, but three isolates were hemolytic consistently. DNA banding patterns suggested that at least one of these isolates was related to isolates from homes of infants with previously investigated cases.

Initial Characterization of the Hemolysin Stachylysin from Stachybotrys chartarum

ABSTRACT Stachybotrys chartarum is a toxigenic fungus that has been associated with human health concerns, including pulmonary hemorrhage and hemosiderosis. This fungus produces a hemolysin, stachylysin, which in its apparent monomeric form has a molecular mass of 11,920 Da as determined by matrix-assisted laser desorption ionization–time of flight mass spectrometry. However, it appears to form polydispersed aggregates, which confounds understanding of the actual hemolytically active form. Exhaustive dialysis or heat treatment at 60°C for 30 min inactivated stachylysin. Stachylysin is composed of about 40% nonpolar amino acids and contains two cysteine residues. Purified stachylysin required more than 6 h to begin lysing sheep erythrocytes, but by 48 h, lysis was complete. Stachylysin also formed pores in sheep erythrocyte membranes.

Quantitative polymerase chain reaction analysis of fungi in dust from homes of infants who developed idiopathic pulmonary hemorrhaging

Fungal concentrations were measured in the dust of 6 homes in Cleveland, Ohio, where an infant developed pulmonary hemorrhage (pulmonary hemorrhage homes [PHH]) and 26 reference homes (RH) with no known fungal contamination. Quantitative polymerase chain reaction assays for 82 species (or assay groups) were used to identify and quantify fungal concentrations. The ratios of the geometric means of PHH to RH were >1 for 26 species (group I). However, the same ratios were <1 for 10 species (group II). Probit analysis of the sum of the logs of the concentrations of these 2 groups resulted in a 95% probability range for separating PHH from RH homes. The same 82 fungal species were also tested for hemolysin production on sheep’s blood agar (incubated at 37°C). Hemolysins were more commonly produced by group I species (42%) compared with group II species (10%).

Quantification of Stachybotrys chartarum conidia in indoor dust using real time, fluorescent probe-based detection of PCR products

Analyses of fungal spores or conidia in indoor dust samples can be useful for determining the contamination status of building interiors and in signaling instances where potentially harmful exposures of building occupants to these organisms may exist. A recently developed method for the quantification of Stachybotrys chartarum conidia, using real-time, fluorescence probe–based detection of PCR products (TaqMan™ system) was employed to analyze indoor dust samples for this toxigenic fungal species. Dust samples of up to 10 mg were found to be amenable to DNA extraction and analysis. Quantitative estimates of S. chartarum conidia in composite dust samples, containing a four-log range of these cells, were within 25–104% of the expected quantities in 95% of analyses performed by the method. Calibrator samples containing known numbers of S. chartarum conidia were used as standards for quantification. Conidia of an arbitrarily selected strain of Geotrichum candidum were added in equal numbers to both dust and calibrator samples before DNA extraction. Partial corrections for reductions in overall DNA yields from the dust samples compared to the calibrator samples were obtained by comparative analyses of rDNA sequence yields from these reference conidia in the two types of samples. Dust samples from two contaminated homes were determined to contain greater than 103 S. chartarum conidia per milligram in collection areas near the sites of contamination and greater than 102 conidia per milligram in several areas removed from these sites in analyses performed by the method. These measurements were within the predicted range of agreement with results obtained by direct microscopic enumeration of presumptive Stachybotrys conidia in the same samples.

Stachylysin May Be a Cause of Hemorrhaging in Humans Exposed to Stachybotrys chartarum

ABSTRACT Stachybotrys chartarum is a toxigenic fungus that has been associated with human health concerns such as nasal bleeding in adults and pulmonary hemosiderosis (PH) in infants. Seven of eight strains of S. chartarum isolated from homes of infants with PH in Cleveland, Ohio, and the strain from the lung of an infant with PH in Texas produced stachylysin in tryptic soy broth (TSB), whereas only one out of eight strains isolated from control homes produced stachylysin. However, all strains produced stachylysin when grown on TSB with 0.7% sheeps blood. When stachylysin was injected into Lumbricus terrestis, the erythrocruorin hemoglobin (absorbance peaks at 280 and 415 nm) was released, resulting in a lethal effect. These results support the hypothesis that stachylysin may be one agent responsible for hemorrhaging in humans.