Identification and characterization of fungal exposures

为了更全面地评估人类暴露的真菌种类，设计了一项真菌核糖体RNA （rRNA）基因测序研究，以验证美国室内建筑环境中的真菌生物气溶胶比以前使用传统分析方法估计的要多样化得多的假设。在2015财年，使用Illumina miSeq对来自纽约市哮喘儿童和/或霉菌修复工人家中的床、卧室地板和厨房区域的350份灰尘样本进行了真菌多样性评估，并与使用qPCR获得的结果进行了比较。对来自这些个体研究的样本中的真菌多样性的分析提供了新的数据集，这些数据集为在建筑环境中经常恢复的真菌物种提供了更深入的了解。粉尘样本分析显示，普鲁兰小孢子虫、光斑青霉、白孢虫和交替孢虫因居住类型（单户、多户或公寓）和社区哮喘患病率而异。初步结果表明，包括人为行为、住房类型和社区在内的多种环境因素是影响纽约市中等收入家庭真菌多样性的重要变量。在室内灰尘中测量到的a . alternata与呼出的一氧化氮有关，特别是在燃烧副产物暴露较高的儿童中，这表明这两种暴露可能与气道炎症相互作用。作为该项目的另一个组成部分，NIOSH正在将环境相对发霉指数（ERMI）与真菌rRNA测序数据进行比较，这些数据来自于从真菌污染明显的家庭收集的空气和灰尘样本。ERMI已越来越多地被商业部门使用，并识别出通常在与水渗透相关的潮湿和潮湿条件下茁壮成长的真菌。扩展的数据集将改进ERMI面板，使其更充分地代表存在于其他类型环境中的真菌的完整谱。这些研究正在检查来自美国许多不同研究地点的数据，以对受污染和未受污染的居住和职业环境中真菌rRNA的多样性进行分类和排序。研究地点包括佐治亚州亚特兰大的住宅，康涅狄格州哈特福德和佛蒙特州本宁顿的办公楼，以及缅因州威斯卡塞特的一所学校。这项工作的第三个方面是鉴定和表征交叉反应真菌生物标志物。该项目的这一部分的总体目标是确定广泛的交叉反应性真菌过敏原，目的是开发改进的免疫学方法来检测真菌生物气溶胶和过敏致敏。为了实现这一目标，NIOSH正在开发重组真菌抗原的物种特异性单克隆抗体（mab）。这些单克隆抗体对于真菌暴露的物种特异性生物标记物的量化将是重要的。每个单抗将测试对一组密切相关和远相关的职业污染物的反应性。识别相同抗原的单克隆抗体将被选择进行进一步的表征，并用于开发抑制或夹心elisa。土曲霉最初被用作这些研究的模式真菌生物。全球毛藻（chetomium globosum）的基因组现在也已经被测序，允许使用重组技术开发真菌特异性蛋白质。制备了三种与毛囊烯醇化酶反应的单抗，并对其进行了表征。目前正在使用类似的方法来鉴定和表征来自chartarum的其他候选真菌过敏原，并开发可用于跟踪暴露和效果的单克隆抗体。chartarum是一种公认的水分渗透生物标志物，但在污染环境中没有可用的免疫测定方法来检测该物种。本项目开发的试剂将用于表征IgE对致敏真菌物种之间共享的交叉反应性过敏原的反应性。

To more completely assess the fungal species to which people are exposed, a fungal ribosomal RNA (rRNA) gene sequencing study was designed to test the hypothesis that fungal bioaerosols in the United States indoor built environments are much more diverse than previously estimated using traditional methods of analysis. In FY15, fungal diversity was evaluated in 350 dust samples derived from the bed, bedroom floor and kitchen areas from the homes of asthmatic children and/or mold remediation workers in New York City using Illumina miSeq and compared to results obtained using qPCR. The analysis of fungal diversity in samples derived from these individual studies provided new datasets that have provided greater insight into the fungal species that are frequently recovered in built environments. Analysis of the dust samples showed that Aureobasidium pullulans, Penicillium glabrum, Wallemia sebi and Alternaria alternata varied by housing type (single, multi-family or apartment) and neighborhood asthma prevalence. The preliminary results suggest that multiple environmental factors including anthropogenic behavior, housing type, and neighborhood are important variables that influence fungal diversity within middle-income homes in New York City. A. alternata measured in house dust was associated with fractional exhaled nitric oxide, specifically among children with higher combustion byproduct exposure, suggesting a possible interaction between these two exposures on airway inflammation. As an additional component of this project, NIOSH is comparing the Environmental Relative Moldiness Index (ERMI) to fungal rRNA sequencing data derived from air and dust samples collected from homes with well characterized fungal contamination. The ERMI has been increasingly used by the commercial sector and identifies fungi that typically thrive in moist and wet conditions associated with water infiltration. An expanded dataset will improve the ERMI panel so that it more fully represents the complete spectrum of fungi present in other types of environments. These studies are examining data from a number of different study locations in the United States to categorize and sequence the diversity of fungal rRNA in contaminated and non-contaminated residential and occupational environments. The study locations include homes in Atlanta, Georgia, office buildings in Hartford, Connecticut and Bennington, Vermont, and a school in Wiscasset Maine. A third aspect of this work is the identification and characterization of cross-reactive fungal biomarkers. The overall aim of this portion of the project is to identify broadly cross-reactive fungal allergens with the aim of developing improved immunological methods for detecting fungal bioaerosols and allergic sensitization. To accomplish this, NIOSH is developing species-specific monoclonal antibodies (mAbs) to recombinant fungal antigens. These mAbs will be important for the quantification of species-specific biomarkers of fungal exposure. Each mAb will be tested for reactivity against a panel of closely and distantly related occupational contaminants. MAbs that recognize the same antigen will be selected for further characterization and used in the development of either inhibition or sandwich ELISAs. Aspergillus terreus was initially used as a model fungal organism for these studies. The genome of Chaetomium globosum has now also been sequenced, allowing for the development of fungal-specific proteins using recombinant technologies. Three mAbs that react with the Chaetomium globosum enolase have been developed and characterized. A similar approach is currently being used to identify and characterize additional candidate fungal allergens from Ulocladium chartarum and to develop mAbs that can be used to track exposure and effect. Ulocladium chartarum is a recognized biomarker of moisture infiltration but there are no immunoassays available to detect this species in contaminated environments. The reagents developed in this project will be used to characterize IgE reactivity to cross-reactive allergens shared between allergenic fungal species.