Identification and characterization of fungal exposures

为了更全面地评估人们接触的真菌种类，设计了一项真菌核糖体RNA(RRNA)基因测序研究，以检验这一假设，即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更多样化。使用Illumina MiSeq高通量测序或Sanger测序分析了来自美国不同地理区域的样本的真菌rRNA测序数据，其中包括亚特兰大(佐治亚州)、本宁顿(佛蒙特州)、里诺(内华达州)和费城(宾夕法尼亚州)，以表征空气和粉尘样本中真菌的多样性。在2017财年，该项目扩大到包括在NIOSH健康危害评估(HHES)中收集的职业环境样本中真菌多样性的分析。对大麻生产行业收集的地区和个人空气样本的分析表明，真菌种类繁多，主要由子囊菌门的物种组成，包括引起灰霉病的植物病原物种，如灰霉病。在另一个最近抽样的生产设施中也发现了担子菌门中的其他植物病原体，包括Wallia SEBI。这些高强度健康报告强调，工人也可能接触到微生物污染物，包括以前与职业过敏性肺炎和过敏性敏感症有关的物种。其rRNA基因测序研究的结果也为以前使用传统评估方法忽视的职业环境中的真菌谱提供了新的见解。作为纽约市社区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。对分析的粉尘样本的初步分析表明，出芽短梗霉、光滑青霉、黑斑病和交链格孢菌因房屋类型(单户、多户或公寓)和邻里哮喘患病率而不同。初步结果表明，包括人为行为改变、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。在室内粉尘中测得的交链霉菌也与呼出的部分一氧化氮有关，特别是在燃烧副产物暴露较高的儿童中，这表明这两种暴露之间可能存在对呼吸道炎症的相互作用。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。针对重组真菌生物标记物抗原的抗体的开发工作仍在继续。这些抗体的实用性对于真菌生物标记物的量化至关重要，特别是对于NTP正在研究的那些真菌。NIOSH最近开发了一种从轮状丝孢霉(Ulocladium Chartarum)获得的重组Alt a 1同系物，暂定名为Ulo c 1。针对重组Ulo c 1已产生多克隆抗体(Pab)。在2017财年，已测定了Pab与Rulo c 1的反应性以及与其他Alt a 1同源物的交叉反应，并开发了夹心和抑制ELISA。表位映射实验和免疫分析的优化将在18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.