Karen C. Dannemiller

Fungal High-throughput Taxonomic Identification tool for use with Next-Generation Sequencing (FHiTINGS)

Improvements in DNA sequencing technology provide unprecedented opportunities to explore fungal diversity, but also present challenges in data analysis due to the large number of sequences generated. Here, we describe an open source software program “FHiTINGS” that utilizes the output of a BLASTn (blastall) search to rapidly identify, classify, and parse internal transcribed spacer (ITS) DNA sequences produced in fungal ecology studies that utilize next‐generation DNA sequencing. This tool was designed for use with 454 pyrosequencing and is also appropriate for use with any sequencing platform that allows for BLAST searches against the indicated ITS database. For each sequence, FHiTINGS uses the lowest common ancestor method (LCA) to produce a single identification from BLAST output results, and then assigns taxonomic ranks from species through kingdom when possible for each sequence based on the Index Fungorum database. The program then sums and sorts this data into tabular form to enable rapid analysis of the sample, including α‐diversity measures or richness. In silico testing demonstrates the time required to analyze and classify 1000 sequences is reduced from over 2 h by manual sorting to <1 min of computational time when using FHiTINGS, and that the classification output from the software is consistent with that derived from manual sorting of the data.

Indoor emissions as a primary source of airborne allergenic fungal particles in classrooms.

This study quantifies the influence of ventilation and indoor emissions on concentrations and particle sizes of airborne indoor allergenic fungal taxa and further examines geographical variability, each of which may affect personal exposures to allergenic fungi. Quantitative PCR and multiplexed DNA sequencing were employed to count and identify allergenic fungal aerosol particles indoors and outdoors in seven school classrooms in four different countries. Quantitative diversity analysis was combined with building characterization and mass balance modeling to apportion source contributions of indoor allergenic airborne fungal particles. Mass balance calculations indicate that 70% of indoor fungal aerosol particles and 80% of airborne allergenic fungal taxa were associated with indoor emissions; on average, 81% of allergenic fungi from indoor sources originated from occupant-generated emissions. Principal coordinate analysis revealed geographical variations in fungal communities among sites in China, Europe, and North America (p < 0.05, analysis of similarity), demonstrating that geography may also affect personal exposures to allergenic fungi. Indoor emissions including those released with occupancy contribute more substantially to allergenic fungal exposures in classrooms sampled than do outdoor contributions from ventilation. The results suggest that design and maintenance of buildings to control indoor emissions may enable reduced indoor inhalation exposures to fungal allergens.

Identification accuracy and diversity reproducibility associated with internal transcribed spacer-based fungal taxonomic library preparation

This study investigated analytical parameters that are inherently relevant to identifying and quantifying fungal communities based on polymerase chain reaction amplicons. Specifically, we evaluated the accuracy of the BLASTn-based identification for internal transcribed spacer (ITS) sequences generated from pure cultures, and quantified the reproducibility of relative abundances as well as α and β diversity measurements using duplicated environmental samples. The BLASTn-based method produced accurate fungal identification for the pure culture sequences at the genus rank. Percentages of the sequences assigned to correct genera were 99.8%, 99.8% and 99.9% for Alternaria alternata, Cladosporium cladosporioides and Epicoccum nigrum respectively. These fractions were smaller for Aspergillus fumigatus and Penicillium chrysogenum, which have dual nomenclatures or sibling species that are indistinguishable by ITS sequences. Our duplicate environmental analyses demonstrated that α diversity and relative abundance levels were reproducible (r(2)  > 0.9), that variability decreases with increased sequence quantity, and that the differences in distinct environmental samples were larger than differences in replicate samples (β diversity). These results serve to better characterize the identification and quantification limits of ITS-based fungal taxonomic studies, and demonstrate that while diversity quantification is reproducible, limitations in ITS-based taxonomic identification and dual nomenclature conventions are current barriers to identification accuracy.

Formaldehyde concentrations in household air of asthma patients determined using colorimetric detector tubes

Formaldehyde is a colorless, pungent gas commonly found in homes and is a respiratory irritant, sensitizer, carcinogen, and asthma trigger. Typical household sources include plywood and particleboard, cleaners, cosmetics, pesticides, and others. Development of a fast and simple measurement technique could facilitate continued research on this important chemical. The goal of this research is to apply an inexpensive short-term measurement method to find correlations between formaldehyde sources and concentration, and formaldehyde concentration and asthma control. Formaldehyde was measured using 30-min grab samples in length-of-stain detector tubes in homes (n = 70) of asthmatics in the Boston, MA area. Clinical status and potential formaldehyde sources were determined. The geometric mean formaldehyde level was 35.1 ppb and ranged from 5 to 132 ppb. Based on one-way ANOVA, t-tests, and linear regression, predictors of log-transformed formaldehyde concentration included absolute humidity, season, and the presence of decorative laminates, fiberglass, or permanent press fabrics (P < 0.05), as well as temperature and household cleaner use (P < 0.10). The geometric mean formaldehyde concentration was 57% higher in homes of children with very poorly controlled asthma compared to homes of other asthmatic children (P = 0.078). This study provides a simple method for measuring household formaldehyde and suggests that exposure is related to poorly controlled asthma.

Gene expression of indoor fungal communities under damp building conditions: Implications for human health

Dampness and visible mold growth in homes are associated with negative human health outcomes, but causal relationships between fungal exposure and health are not well established. The purpose of this study was to determine whether dampness in buildings impacts fungal community gene expression and how, in turn, gene expression may modulate human health impacts. A metatranscriptomic study was performed on house dust fungal communities to investigate the expression of genes and metabolic processes in chamber experiments at water activity levels of 0.5, 0.85, and 1.0. Fungi at water activities as low as 0.5 were metabolically active, focusing their transcriptional resources on primary processes essential for cell maintenance. Metabolic complexity increased with water activity where communities at 1.0 displayed more diverse secondary metabolic processes. Greater gene expression at increasing water activity has important implications for human health: Fungal communities at 1.0 aw upregulated a greater number of allergen-, mycotoxin-, and pathogenicity-encoding genes versus communities at 0.85 and 0.5 aw . In damp buildings, fungi may display increases in secondary metabolic processes with the potential for greater per-cell production of allergens, toxins, and pathogenicity. Assessments in wet versus dry buildings that do not account for this elevated health impact may not accurately reflect exposure.

Translating research to policy at the NCSE 2017 symposium “Microbiology of the Built Environment: Implications for Health and Design”

Here, we summarize a symposium entitled “Microbiology of the Built Environment: Implications for Health and Design” that was presented at the National Council for Science and the Environment (NCSE) 17th National Conference and Global Forum in January 2017. We covered topics including indoor microbial exposures and childhood asthma, the influence of hospital design on neonatal development, the role of the microbiome in our premise (i.e., building) plumbing systems, antibiotic resistance, and quantitative microbial risk assessment. This symposium engaged the broader scientific and policy communities in a discussion to increase awareness of this critical research area and translate findings to practice.