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。利用Illumina miSeq高通量测序或Sanger测序，对美国亚特兰大（乔治亚州）、本宁顿（佛蒙特州）、里诺（内华达州）和费城（宾夕法尼亚州）等不同地理区域收集的真菌rRNA测序数据进行分析，以表征空气和灰尘样本中的真菌多样性。在2017财年，该项目扩展到包括NIOSH健康危害评估（HHEs）中收集的职业环境样本中的真菌多样性分析。对大麻生产行业收集的区域和个人空气样本的分析表明，真菌的多样性主要由子囊菌门的物种组成，包括植物致病性物种，如引起灰霉的灰霉菌。在另一个最近取样的生产设施中，也列举了其他放置在担子菌门的植物病原体，包括黑孢菌。这些卫生保健机构强调，工人也可能暴露于微生物污染物，包括以前与职业性超敏性肺炎和过敏性致敏有关的物种。ITS rRNA基因测序研究的结果也为以前使用传统评估方法忽略的职业环境中的真菌谱提供了新的见解。作为纽约市邻里哮喘和过敏研究的一部分，其他研究正在检查真菌污染物的分子多样性。初步分析表明，粉尘样本中普鲁兰小孢子虫、光斑青霉菌、色斑青霉菌和交替孢霉因居住类型（单户、多户或公寓）和社区哮喘患病率而异。初步结果表明，包括人为行为改变、住房类型和社区环境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也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。使用Illumina miSeq捕获的真菌多样性正在与使用qPCR获得的结果进行比较，数据分析正在进行中。重组真菌生物标志物抗原抗体的开发工作仍在继续。这些抗体的使用对于真菌生物标志物的定量至关重要，特别是对于那些正在被国家毒理学规划研究的真菌。NIOSH最近从Ulocladium chartarum中开发了一种重组的ala1同源物，暂定名为Ulo c1。已制备了针对重组Ulo c1的多克隆抗体（pAb）。在2017财年，已经确定了pAb与rUlo c1的反应性以及与其他Alt a1同源物的交叉反应性，并开发了夹心和抑制elisa。表位定位实验和免疫测定的优化将在2018财年继续进行。

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. Fungal rRNA sequencing data from samples collected from a variety of geographic regions within the United States including Atlanta (Georgia), Bennington (Vermont), Reno (Nevada) and Philadelphia (Pennsylvania) were analyzed using Illumina miSeq high throughput sequencing or Sanger sequencing to characterize fungal diversity in air and dust samples. In FY17 the project was extended to include the analysis of fungal diversity in occupational environmental samples collected in NIOSH Health Hazard Evaluations (HHEs). Analysis of area and personal air samples collected in the cannabis production industry has demonstrated a diversity of fungi primarily composed of species placed in the phylum Ascomycota including plant pathogenic species such as Botrytis cinerea, which causes grey mold. Other plant pathogens placed in the phylum Basidiomycota, including Wallemia sebi have also been enumerated in another recently sampled production facility. These HHEs have highlighted that workers can also be exposed to microbial contaminants including species that have been previously associated with occupational cases of hypersensitivity pneumonitis and allergic sensitization. The results of ITS rRNA gene sequencing studies have also provided new insights into the spectrum of fungi in occupational environments previously overlooked using traditional methods of assessment. Additional studies are examining the molecular diversity of fungal contaminants as part of the NYC Neighborhood Asthma and Allergy Study. Preliminary analysis of analyzed dust samples indicates that Aureobasidium pullulans, Penicillium glabrum, Wallemia sebi and Alternaria alternata varied by housing type (single, multi-family or apartment) and neighborhood asthma prevalence. Preliminary results suggest that multiple environmental factors including anthropogenic behavior modification, 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 also 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. Fungal diversity captured using Illumina miSeq is being compared to results obtained using qPCR and the data analysis is ongoing. Work has continued in the development of antibodies to recombinant fungal biomarker antigens. The utility of these antibodies is critical for the quantification of fungal biomarkers, particularly to those fungi that are being studied by the NTP. NIOSH has recently developed a recombinant Alt a 1 homologue from Ulocladium chartarum tentatively named Ulo c 1. Polyclonal antibodies (pAb) have been produced toward the recombinant Ulo c 1. In FY17, pAb reactivity with rUlo c 1 as well as cross-reactivity with other Alt a 1 homologs has been determined and sandwich and inhibition ELISAs developed. Epitope mapping experiments and optimization of the immunoassay will continue in FY18.