Kahler Amy, Hofstetter Jessica, Rodrigues Camila, Mattioli Mia
Waterborne Disease Prevention Branch, Division of Foodborne, Waterborne, and Environmental Diseases, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, 1600 Clifton Road. MS H23-9, Atlanta 30329, GA, United States of America.
Waterborne Disease Prevention Branch, Division of Foodborne, Waterborne, and Environmental Diseases, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, 1600 Clifton Road. MS H23-9, Atlanta 30329, GA, United States of America; Department of Agriculture, Auburn University, 101 Funchess Hall, Auburn, AL 36849, United States of America; Chenega Systems and Solutions, 609 Independence Parkway, Suite 210, Chesapeake, VA 23320, United States of America.
J Microbiol Methods. 2025 Sep;236:107195. doi: 10.1016/j.mimet.2025.107195. Epub 2025 Jul 14.
The coccidian parasite Cyclospora cayetanensis is the causative agent for foodborne outbreaks of cyclosporiasis and multiple fresh produce recalls annually. In recent years, this organism has been reported in the water near produce growing operations during outbreak investigations, prompting a call for more research on its environmental prevalence in the United States. Currently, there is a lack of performance data available on methods for conducting this research, including the performance of DNA extraction methods for molecular testing. Extraction methods for environmental samples must be efficient due to the often-limited amount of target nucleic acid and the potential for molecular inhibitors present in an environmental sample. This study assessed the performance of C. cayetanensis nucleic acid extraction seeded into surface water, produce wash water, and tap water by two methods designed for use with environmental samples: the PowerViral and UNEX methods. The PowerSoil extraction method (2 g) was assessed for C. cayetanensis extraction from seeded sewage sludge - an environmental sample type used to evaluate parasite carriage within communities. Extraction performance of the PowerViral and UNEX methods were also assessed for the detection of the foodborne bacterial pathogen Salmonella and a surrogate for foodborne viruses, murine norovirus (MNV) seeded into surface water, produce wash water, and tap water. The PowerViral method resulted in consistent detection (83-100 %) of C. cayetanensis, S. enterica, and MNV across all water types. Detection rates for the UNEX method ranged from 56 to 100 % prevalence for tap water and wash water, but there were no detections for any microbe from surface water. The PowerSoil method resulted in poor recovery of C. cayetanensis from sludge (≤1 % recovery), while both the PowerViral and UNEX methods effectively recovered C. cayetanensis from sludge (4-36 % recovery).
球虫寄生虫卡耶塔环孢子虫是每年食源性环孢子虫病暴发和多次新鲜农产品召回事件的病原体。近年来，在疫情调查期间，已在农产品种植作业附近的水体中发现了这种生物体，这促使人们呼吁对其在美国环境中的流行情况开展更多研究。目前，缺乏关于开展此项研究方法的性能数据，包括用于分子检测的DNA提取方法的性能。由于环境样本中目标核酸的量通常有限，且存在分子抑制剂的可能性，因此环境样本的提取方法必须高效。本研究通过两种专为环境样本设计的方法，即PowerViral法和UNEX法，评估了接种于地表水、农产品清洗水和自来水中的卡耶塔环孢子虫核酸提取性能。对PowerSoil提取方法（2克）进行了评估，以确定其从接种的污水污泥（一种用于评估社区内寄生虫携带情况的环境样本类型）中提取卡耶塔环孢子虫的能力。还评估了PowerViral法和UNEX法对食源性病原体沙门氏菌以及接种于地表水、农产品清洗水和自来水中的食源病毒替代品小鼠诺如病毒（MNV）的检测性能。PowerViral法在所有水体类型中均能一致检测到卡耶塔环孢子虫、肠炎沙门氏菌和MNV（检出率为83%-100%）。UNEX法对自来水和清洗水的检出率在56%至100%之间，但未从地表水中检测到任何微生物。PowerSoil法从污泥中回收卡耶塔环孢子虫的效果较差（回收率≤1%），而PowerViral法和UNEX法均能有效地从污泥中回收卡耶塔环孢子虫（回收率为4%-36%）。
