Corrigendum: Persisting Cryptococcus yeast species Vishniacozyma victoriae and Cryptococcus neoformans elicit unique airway inflammation in mice following repeated exposure.

[This corrects the article DOI: 10.3389/fcimb.2023.1067475.].

Optimization of Aspergillus versicolor Culture and Aerosolization in a Murine Model of Inhalational Fungal Exposure.

Aspergillus versicolor is ubiquitous in the environment and is particularly abundant in damp indoor spaces. Exposure to Aspergillus species, as well as other environmental fungi, has been linked to respiratory health outcomes, including asthma, allergy, and even local or disseminated infection. However, the pulmonary immunological mechanisms associated with repeated exposure to A. versicolor have remained relatively uncharacterized. Here, A. versicolor was cultured and desiccated on rice then placed in an acoustical generator system to achieve aerosolization. Mice were challenged with titrated doses of aerosolized conidia to examine deposition, lymphoproliferative properties, and immunotoxicological response to repeated inhalation exposures. The necessary dose to induce lymphoproliferation was identified, but not infection-like pathology. Further, it was determined that the dose was able to initiate localized immune responses. The data presented in this study demonstrate an optimized and reproducible method for delivering A. versicolor conidia to rodents via nose-only inhalation. Additionally, the feasibility of a long-term repeated exposure study was established. This experimental protocol can be used in future studies to investigate the physiological effects of repeated pulmonary exposure to fungal conidia utilizing a practical and relevant mode of delivery. In total, these data constitute an important foundation for subsequent research in the field.

Fungal diversity in homes and asthma morbidity among school-age children in New York City.

BACKGROUND: Asthma development has been inversely associated with exposure to fungal diversity. However, the influence of fungi on measures of asthma morbidity is not well understood. OBJECTIVES: This study aimed to test the hypothesis that fungal diversity is inversely associated with neighborhood asthma prevalence and identify specific fungal species associated with asthma morbidity. METHODS: Children aged 7-8 years (n = 347) living in higher (11-18%) and lower (3-9%) asthma prevalence neighborhoods were recruited within an asthma case-control study. Fungal communities were analyzed from floor dust using high-throughput DNA sequencing. A subset of asthmatic children (n = 140) was followed to age 10-11 to determine asthma persistence. RESULTS: Neighborhood asthma prevalence was inversely associated with fungal species richness (P = 0.010) and Shannon diversity (P = 0.059). Associations between neighborhood asthma prevalence and diversity indices were driven by differences in building type and presence of bedroom carpet. Among children with asthma at age 7-8 years, Shannon fungal diversity was inversely associated with frequent asthma symptoms at that age (OR 0.57, P = 0.025) and with asthma persistence to age 10-11 (OR 0.48, P = 0.043). Analyses of individual fungal species did not show significant associations with asthma outcomes when adjusted for false discovery rates. DISCUSSION: Lower fungal diversity was associated with asthma symptoms in this urban setting. Individual fungal species associated with asthma morbidity were not detected. Further research is warranted into building type, carpeting, and other environmental characteristics which influence fungal exposures in homes.

Persisting Cryptococcus yeast species Vishniacozyma victoriae and Cryptococcus neoformans elicit unique airway inflammation in mice following repeated exposure.

Background: Allergic airway disease (AAD) is a growing concern in industrialized nations and can be influenced by fungal exposures. Basidiomycota yeast species such as Cryptococcus neoformans are known to exacerbate allergic airway disease; however, recent indoor assessments have identified other Basidiomycota yeasts, including Vishniacozyma victoriae (syn. Cryptococcus victoriae), to be prevalent and potentially associated with asthma. Until now, the murine pulmonary immune response to repeated V. victoriae exposure was previously unexplored. Objective: This study aimed to compare the immunological impact of repeated pulmonary exposure to Cryptococcus yeasts. Methods: Mice were repeatedly exposed to an immunogenic dose of C. neoformans or V. victoriae via oropharyngeal aspiration. Bronchoalveolar lavage fluid (BALF) and lungs were collected to examine airway remodeling, inflammation, mucous production, cellular influx, and cytokine responses at 1 day and 21 days post final exposure. The responses to C. neoformans and V. victoriae were analyzed and compared. Results: Following repeated exposure, both C. neoformans and V. victoriae cells were still detectable in the lungs 21 days post final exposure. Repeated C. neoformans exposure initiated myeloid and lymphoid cellular infiltration into the lung that worsened over time, as well as an IL-4 and IL-5 response compared to PBS-exposed controls. In contrast, repeated V. victoriae exposure induced a strong CD4+ T cell-driven lymphoid response that started to resolve by 21 days post final exposure. Discussion: C. neoformans remained in the lungs and exacerbated the pulmonary immune responses as expected following repeated exposure. The persistence of V. victoriae in the lung and strong lymphoid response following repeated exposure were unexpected given its lack of reported involvement in AAD. Given the abundance in indoor environments and industrial utilization of V. victoriae, these results highlight the importance to investigate the impact of frequently detected fungal organisms on the pulmonary response following inhalational exposure. Moreover, it is important to continue to address the knowledge gap involving Basidiomycota yeasts and their impact on AAD.

Efficacy of Do-It-Yourself air filtration units in reducing exposure to simulated respiratory aerosols.

Many respiratory diseases, including COVID-19, can be spread by aerosols expelled by infected people when they cough, talk, sing, or exhale. Exposure to these aerosols indoors can be reduced by portable air filtration units (air cleaners). Homemade or Do-It-Yourself (DIY) air filtration units are a popular alternative to commercially produced devices, but performance data is limited. Our study used a speaker-audience model to examine the efficacy of two popular types of DIY air filtration units, the Corsi-Rosenthal cube and a modified Ford air filtration unit, in reducing exposure to simulated respiratory aerosols within a mock classroom. Experiments were conducted using four breathing simulators at different locations in the room, one acting as the respiratory aerosol source and three as recipients. Optical particle spectrometers monitored simulated respiratory aerosol particles (0.3-3 µm) as they dispersed throughout the room. Using two DIY cubes (in the front and back of the room) increased the air change rate as much as 12.4 over room ventilation, depending on filter thickness and fan airflow. Using multiple linear regression, each unit increase of air change reduced exposure by 10%. Increasing the number of filters, filter thickness, and fan airflow significantly enhanced the air change rate, which resulted in exposure reductions of up to 73%. Our results show DIY air filtration units can be an effective means of reducing aerosol exposure. However, they also show performance of DIY units can vary considerably depending upon their design, construction, and positioning, and users should be mindful of these limitations.

Mycobiota and the Contribution of Yeasts in Floor Dust of 50 Elementary Schools Characterized with Sequencing Internal Transcribed Spacer Region of Ribosomal DNA.

The assemblage of fungi including unicellular yeasts in schools is understudied. We conducted an environmental study to characterize fungal communities in classroom floor dust. We collected 500 samples from 50 elementary schools in Philadelphia, PA, and evaluated room dampness/mold conditions. Genomic DNA from dust was extracted for internal transcribed spacer 1 Illumina MiSeq sequencing to identify operational taxonomic units (OTUs) organized from DNA sequences. Differential abundance analyses were performed to examine significant differences in abundance among groups. We identified 724 genera from 1490 OTUs. The genus Epicoccum was not diverse but the most abundant (relative abundance = 18.9%). Fungi were less diverse but most dissimilar in composition in the most water-damaged classrooms compared to the least water-damaged, indicating differential effects of individual classroom water-damage on fungal compositions. We identified 62 yeast genera, representing 19.6% of DNA sequences. Cyberlindnera was the most abundant (6.1%), followed by Cryptococcus, Aureobasidium, Rhodotorula, and Candida. The average relative abundance of yeasts tended to increase with increasing dampness and mold score and was significantly (p-value = 0.048) higher in the most water-damaged classrooms (22.4%) than the least water-damaged classrooms (18.2%). Our study suggests the need for further research on the potential health effects associated with exposures to yeasts in schools.

Reduction of exposure to simulated respiratory aerosols using ventilation, physical distancing, and universal masking.

To limit community spread of SARS-CoV-2, CDC recommends universal masking indoors, maintaining 1.8 m of physical distancing, adequate ventilation, and avoiding crowded indoor spaces. Several studies have examined the independent influence of each control strategy in mitigating transmission in isolation, yet controls are often implemented concomitantly within an indoor environment. To address the influence of physical distancing, universal masking, and ventilation on very fine respiratory droplets and aerosol particle exposure, a simulator that coughed and exhaled aerosols (the source) and a second breathing simulator (the recipient) were placed in an exposure chamber. When controlling for the other two mitigation strategies, universal masking with 3-ply cotton masks reduced exposure to 0.3-3 µm coughed and exhaled aerosol particles by >77% compared to unmasked tests, whereas physical distancing (0.9 or 1.8 m) significantly changed exposure to cough but not exhaled aerosols. The effectiveness of ventilation depended upon the respiratory activity, that is, coughing or breathing, as well as the duration of exposure time. Our results demonstrate that a layered mitigation strategy approach of administrative and engineering controls can reduce personal inhalation exposure to potentially infectious very fine respiratory droplets and aerosol particles within an indoor environment.

Spring is associated with increased total and allergenic fungal concentrations in house dust from a pediatric asthma cohort in New York City.

Introduction: Asthma and allergy symptoms vary seasonally due to exposure to environmental sources of allergen, including fungi. However, we need an improved understanding of seasonal influence on fungal exposures in the indoor environment. We hypothesized that concentrations of total fungi and allergenic species in vacuumed dust vary significantly by season. Objective: Assess seasonal variation of indoor fungi with greater implications related to seasonal asthma control. Methods: We combined next-generation sequencing with quantitative polymerase chain reaction (qPCR) to measure concentrations of fungal DNA in indoor floor dust samples (n = 298) collected from homes participating in the New York City Neighborhood Asthma and Allergy Study (NAAS). Results: Total fungal concentration in spring was significantly higher than the other three seasons (p ≤ 0.005). Mean concentrations for 78% of fungal species were elevated in the spring (26% were significantly highest in spring, p < 0.05). Concentrations of 8 allergenic fungal species were significantly (p < 0.5) higher in spring compared to at least two other seasons. Indoor relative humidity and temperature were significantly highest in spring (p < 0.05) and were associated with total fungal concentration (R2 = 0.049, R2 = 0.11, respectively). Conclusion: There is significant seasonal variation in total fungal concentration and concentration of select allergenic species. Indoor relative humidity and temperature may underlie these associations.

Face mask fit modifications that improve source control performance.

BACKGROUND: During the COVID-19 pandemic, face masks are used as source control devices to reduce the expulsion of respiratory aerosols from infected people. Modifications such as mask braces, earloop straps, knotting and tucking, and double masking have been proposed to improve mask fit however the data on source control are limited. METHODS: The effectiveness of mask fit modifications was determined by conducting fit tests on human subjects and simulator manikins and by performing simulated coughs and exhalations using a source control measurement system. RESULTS: Medical masks without modification blocked ≥56% of cough aerosols and ≥42% of exhaled aerosols. Modifying fit by crossing the earloops or placing a bracket under the mask did not increase performance, while using earloop toggles, an earloop strap, and knotting and tucking the mask increased performance. The most effective modifications for improving source control performance were double masking and using a mask brace. Placing a cloth mask over a medical mask blocked ≥85% of cough aerosols and ≥91% of exhaled aerosols. Placing a brace over a medical mask blocked ≥95% of cough aerosols and ≥99% of exhaled aerosols. CONCLUSIONS: Fit modifications can greatly improve the performance of face masks as source control devices for respiratory aerosols.

Efficacy of Ventilation, HEPA Air Cleaners, Universal Masking, and Physical Distancing for Reducing Exposure to Simulated Exhaled Aerosols in a Meeting Room.

There is strong evidence associating the indoor environment with transmission of SARS-CoV-2, the virus that causes COVID-19. SARS-CoV-2 can spread by exposure to droplets and very fine aerosol particles from respiratory fluids that are released by infected persons. Layered mitigation strategies, including but not limited to maintaining physical distancing, adequate ventilation, universal masking, avoiding overcrowding, and vaccination, have shown to be effective in reducing the spread of SARS-CoV-2 within the indoor environment. Here, we examine the effect of mitigation strategies on reducing the risk of exposure to simulated respiratory aerosol particles within a classroom-style meeting room. To quantify exposure of uninfected individuals (Recipients), surrogate respiratory aerosol particles were generated by a breathing simulator with a headform (Source) that mimicked breath exhalations. Recipients, represented by three breathing simulators with manikin headforms, were placed in a meeting room and affixed with optical particle counters to measure 0.3-3 µm aerosol particles. Universal masking of all breathing simulators with a 3-ply cotton mask reduced aerosol exposure by 50% or more compared to scenarios with simulators unmasked. While evaluating the effect of Source placement, Recipients had the highest exposure at 0.9 m in a face-to-face orientation. Ventilation reduced exposure by approximately 5% per unit increase in air change per hour (ACH), irrespective of whether increases in ACH were by the HVAC system or portable HEPA air cleaners. The results demonstrate that mitigation strategies, such as universal masking and increasing ventilation, reduce personal exposure to respiratory aerosols within a meeting room. While universal masking remains a key component of a layered mitigation strategy of exposure reduction, increasing ventilation via system HVAC or portable HEPA air cleaners further reduces exposure.

Gaseous and Particulate Content of Laser Tattoo Removal Plume.

BACKGROUND: There is increasing awareness of the potential hazards of surgical plumes. The plume associated with laser tattoo removal remains uncharacterized. OBJECTIVE: To determine the gaseous, particulate, and microbiological content of the laser tattoo removal plume. MATERIALS AND METHODS: Air sampling was performed during laser tattoo removal from pig skin and from patients. Measurement of metals, volatile organic compounds (VOCs), carbon monoxide (CO), hydrogen sulfide (HS), and ultrafine particulates (UPs) as well as bacterial 16S ribosomal DNA sequencing were performed. RESULTS: Metals were identified in the plume from both pig and human skin. Volatile organic compounds were found at similar levels within and outside the treatment room. Several bacterial phyla were detected in the treatment room, but not outside. High levels of UPs were measured throughout the treatment room during tattoo removal from pig skin. Ultrafine particulates were detected at low levels in the room periphery during tattoo removal from human skin, but at higher levels in the immediate treatment zone. HS and CO were not detected. CONCLUSION: Metals, VOCs, HS, and CO were found at levels below applicable occupational exposure limits. The presence of bacteria is of uncertain significance, but may be hazardous. High levels of UPs require further investigation.

Aspergillus versicolor Inhalation Triggers Neuroimmune, Glial, and Neuropeptide Transcriptional Changes.

Increasing evidence associates indoor fungal exposure with deleterious central nervous system (CNS) health, such as cognitive and emotional deficits in children and adults, but the specific mechanisms by which it might impact the brain are poorly understood. Mice were exposed to filtered air, heat-inactivated Aspergillus versicolor (3 × 105 spores), or viable A. versicolor (3 × 105 spores) via nose-only inhalation exposure 2 times per week for 1, 2, or 4 weeks. Analysis of cortex, midbrain, olfactory bulb, and cerebellum tissue from mice exposed to viable A. versicolor spores for 1, 2, and 4 weeks revealed significantly elevated pro-inflammatory (Tnf and Il1b) and glial activity (Gdnf and Cxc3r1) gene expression in several brain regions when compared to filtered air control, with the most consistent and pronounced neuroimmune response 48H following the 4-week exposure in the midbrain and frontal lobe. Bulk RNA-seq analysis of the midbrain tissue confirmed that 4 weeks of A. versicolor exposure resulted in significant transcriptional enrichment of several biological pathways compared to the filtered air control, including neuroinflammation, glial cell activation, and regulation of postsynaptic organization. Upregulation of Drd1, Penk, and Pdyn mRNA expression was confirmed in the 4-week A. versicolor exposed midbrain tissue, highlighting that gene expression important for neurotransmission was affected by repeated A. versicolor inhalation exposure. Taken together, these findings indicate that the brain can detect and respond to A. versicolor inhalation exposure with changes in neuroimmune and neurotransmission gene expression, providing much needed insight into how inhaled fungal exposures can affect CNS responses and regulate neuroimmune homeostasis.

Bacterial community assemblages in classroom floor dust of 50 public schools in a large city: characterization using 16S rRNA sequences and associations with environmental factors.

Characterizing indoor microbial communities using molecular methods provides insight into bacterial assemblages present in environments that can influence occupants' health. We conducted an environmental assessment as part of an epidemiologic study of 50 elementary schools in a large city in the northeastern USA. We vacuumed dust from the edges of the floor in 500 classrooms accounting for 499 processed dust aliquots for 16S Illumina MiSeq sequencing to characterize bacterial assemblages. DNA sequences were organized into operational taxonomic units (OTUs) and identified using a database derived from the National Center for Biotechnology Information. Bacterial diversity and ecological analyses were performed at the genus level. We identified 29 phyla, 57 classes, 148 orders, 320 families, 1193 genera, and 2045 species in 3073 OTUs. The number of genera per school ranged from 470 to 705. The phylum Proteobacteria was richest of all while Firmicutes was most abundant. The most abundant order included Lactobacillales, Spirulinales, and Clostridiales. Halospirulina was the most abundant genus, which has never been reported from any school studies before. Gram-negative bacteria were more abundant and richer (relative abundance = 0.53; 1632 OTUs) than gram-positive bacteria (0.47; 1441). Outdoor environment-associated genera were identified in greater abundance in the classrooms, in contrast to homes where human-associated bacteria are typically more abundant. Effects of school location, degree of water damage, building condition, number of students, air temperature and humidity, floor material, and classroom's floor level on the bacterial richness or community composition were statistically significant but subtle, indicating relative stability of classroom microbiome from environmental stress. Our study indicates that classroom floor dust had a characteristic bacterial community that is different from typical house dust represented by more gram-positive and human-associated bacteria. Health implications of exposure to the microbiomes in classroom floor dust may be different from those in homes for school staff and students. Video abstract.

Inactivation of the multi-drug resistant pathogen Candida auris using ultraviolet germicidal irradiation (UVGI).

BACKGROUND: Candida auris, often a multi-drug resistant fungal pathogen, has become an emerging threat in healthcare settings around the world. Reliable disinfection protocols specifically designed to inactivate C. auris are essential, as many chemical disinfectants commonly used in healthcare settings have been shown to have variable efficacy at inactivating C. auris. AIM: Ultraviolet germicidal irradiation (UVGI) was investigated as a method to inactivate clinically relevant strains of C. auris. METHODS: Ten C. auris and two C. albicans isolates were exposed to ultraviolet (UV) energy to determine the UV dose required to inactivate each isolate. Using a UV reactor, each isolate (106 cells/mL) was exposed to 11 UV doses ranging from 10-150 mJ/cm2 and then cultured to assess cell viability. FINDINGS: An exponential decay model was applied to each dose-response curve to determine inactivation rate constants for each isolate, which ranged from 0.108-0.176 cm2/mJ for C. auris and 0.239-0.292 cm2/mJ for C. albicans. As the model of exponential decay did not accurately estimate the dose beyond 99.9% inactivation, a logistic regression model was applied to better estimate the doses required for 99.999% inactivation. Using this model, significantly greater UV energy was required to inactivate C. auris (103 to 192 mJ/cm2) when compared to C. albicans (78 to 80 mJ/cm2). CONCLUSION: This study demonstrated UVGI as a feasible approach for inactivating C. auris, although variable susceptibility among isolates must be taken into account. This dose-response data is critical for recommending UVGI dosing strategies to be tested in healthcare settings.

Inhalation of Stachybotrys chartarum Fragments Induces Pulmonary Arterial Remodeling.

Stachybotrys chartarum is a fungal contaminant within the built environment and a respiratory health concern in the United States. The objective of this study was to characterize the mechanisms influencing pulmonary immune responses to repeatedly inhaled S. chartarum. Groups of B6C3F1/N mice repeatedly inhaled viable trichothecene-producing S. chartarum conidia (strain A or strain B), heat-inactivated conidia, or high-efficiency particulate absolute-filtered air twice per week for 4 and 13 weeks. Strain A was found to produce higher amounts of respirable fragments than strain B. Lung tissue, serum, and BAL fluid were collected at 24 and 48 hours after final exposure and processed for histology, flow cytometry, and RNA and proteomic analyses. At 4 weeks after exposure, a T-helper cell type 2-mediated response was observed. After 13 weeks, a mixed T-cell response was observed after exposure to strain A compared with a T-helper cell type 2-mediated response after strain B exposure. After exposure, both strains induced pulmonary arterial remodeling at 13 weeks; however, strain A-exposed mice progressed more quickly than strain B-exposed mice. BAL fluid was composed primarily of eosinophils, neutrophils, and macrophages. Both the immune response and the observed pulmonary arterial remodeling were supported by specific cellular, molecular, and proteomic profiles. The immunopathological responses occurred earlier in mice exposed to high fragment-producing strain A. The rather striking induction of pulmonary remodeling by S. chartarum appears to be related to the presence of fungal fragments during exposure.

Cultivation and aerosolization of Stachybotrys chartarum for modeling pulmonary inhalation exposure.

Objective:Stachybotrys chartarum is a hydrophilic fungal species commonly found as a contaminant in water-damaged building materials. Although several studies have suggested that S. chartarum exposure elicits a variety of adverse health effects, the ability to characterize the pulmonary immune responses to exposure is limited by delivery methods that do not replicate environmental exposure. This study aimed to develop a method of S. chartarum aerosolization to better model inhalation exposures. Materials and methods: An acoustical generator system (AGS) was previously developed and utilized to aerosolize and deliver fungal spores to mice housed in a multi-animal nose-only exposure chamber. In this study, methods for cultivating, heat-inactivating, and aerosolizing two macrocyclic trichothecene-producing strains of S. chartartum using the AGS are described. Results and discussion: In addition to conidia, acoustical generation of one strain of S. chartarum resulted in the aerosolization of fungal fragments (<2 µm aerodynamic diameter) derived from conidia, phialides, and hyphae that initially comprised 50% of the total fungal particle count but was reduced to less than 10% over the duration of aerosolization. Acoustical generation of heat-inactivated S. chartarum did not result in a similar level of fragmentation. Delivery of dry, unextracted S. chartarum using these aerosolization methods resulted in pulmonary inflammation and immune cell infiltration in mice inhaling viable, but not heat-inactivated S. chartarum. Conclusions: These methods of S. chartarum growth and aerosolization allow for the delivery of fungal bioaerosols to rodents that may better simulate natural exposure within water-damaged indoor environments.

Potential occupational and respiratory hazards in a Minnesota cannabis cultivation and processing facility.

BACKGROUND: Cannabis has been legalized in some form for much of the United States. The National Institute for Occupational Safety and Health (NIOSH) received a health hazard evaluation request from a Minnesota cannabis facility and their union to undertake an evaluation. METHODS: NIOSH representatives visited the facility in August 2016 and April 2017. Surface wipe samples were collected for analysis of delta-9 tetrahydrocannabinol (Δ9-THC), delta-9 tetrahydrocannabinol acid (Δ9-THCA), cannabidiol, and cannabinol. Environmental air samples were collected for volatile organic compounds (VOCs), endotoxins (limulus amebocyte lysate assay), and fungal diversity (NIOSH two-stage BC251 bioaerosol sampler with internal transcribed spacer region sequencing analysis). RESULTS: Surface wipe samples identified Δ9-THC throughout the facility. Diacetyl and 2,3-pentanedione were measured in initial VOC screening and subsequent sampling during tasks where heat transference was greatest, though levels were well below the NIOSH recommended exposure limits. Endotoxin concentrations were highest during processing activities, while internal transcribed spacer region sequencing revealed that the Basidiomycota genus, Wallemia, had the highest relative abundance. CONCLUSIONS: To the authors' knowledge, this is the first published report of potential diacetyl and 2,3-pentanedione exposure in the cannabis industry, most notably during cannabis decarboxylation. Endotoxin exposure was elevated during grinding, indicating that this is a potentially high-risk task. The findings indicate that potential health hazards of significance are present during cannabis processing, and employers should be aware of potential exposures to VOCs, endotoxin, and fungi. Further research into the degree of respiratory and dermal hazards and resulting health effects in this industry is recommended.

Collection and Extraction of Occupational Air Samples for Analysis of Fungal DNA.

Traditional methods of identifying fungal exposures in occupational environments, such as culture and microscopy-based approaches, have several limitations that have resulted in the exclusion of many species. Advances in the field over the last two decades have led occupational health researchers to turn to molecular-based approaches for identifying fungal hazards. These methods have resulted in the detection of many species within indoor and occupational environments that have not been detected using traditional methods. This protocol details an approach for determining fungal diversity within air samples through genomic DNA extraction, amplification, sequencing, and taxonomic identification of fungal internal transcribed spacer (ITS) regions. ITS sequencing results in the detection of many fungal species that are either not detected or difficult to identify to species level using culture or microscopy. While these methods do not provide quantitative measures of fungal burden, they offer a new approach to hazard identification and can be used to determine overall species richness and diversity within an occupational environment.