{"title":"A Simple Alcohol-based Method of Oocyst Inactivation for Use in the Development of Detection Assays for Cryptosporidium","authors":"Biniam Hagos, Robert E. Molestina","doi":"10.1016/j.fawpar.2022.e00163","DOIUrl":null,"url":null,"abstract":"<div><p><em>Cryptosporidium spp.</em> are obligate, intracellular parasites that cause life-threatening diarrhea among children and immunocompromised adults. Transmission occurs by the fecal-oral route following ingestion of thick-walled oocysts that can contaminate, persist, and resist disinfection in water and food. Sodium hypochlorite, peroxides, ozone, formaldehyde, and ammonia are suitable disinfectants against <em>Cryptosporidium</em> oocysts. Effective concentrations of these chemicals can be toxic and not practical for downstream research use of non-viable oocysts. Oocyst inactivation approaches such as UV light, heat, and treatments with ethanol or methanol are generally more accessible for routine lab use, yet their applicability in <em>Cryptosporidium</em> assay development is limited. The aims of this study were to evaluate methods of inactivation of <em>Cryptosporidium</em> oocysts that can be readily applied in the laboratory and test the utility of whole inactive oocysts in quantitative PCR (qPCR). Experiments were performed on <em>C. parvum</em> oocysts subjected to heat (75 °C/10 min) or treated with increasing concentrations of ethanol and methanol over time. Viability assays based on propidium iodide (PI) staining, <em>in vitro</em> excystation, and infection of the Hct-8 cell line were used to evaluate the efficacies of the treatments. Excystation of sporozoites was not impaired with 24 h exposures of oocysts to 50% ethanol or methanol, even though significant PI incorporation was observed. Concentrations of ≥70% of these chemicals were required to completely inhibit excystation and infection of Hct-8 cells <em>in vitro</em>. Inactivated oocysts stored for up to 30 days at 4 °C retained cyst wall integrity and antigenicity as observed by light microscopy and immunofluorescence. Moreover, non-viable oocysts applied directly in qPCR assays of the COWP gene were useful reference reagents for the identification and quantification of <em>Cryptosporidium</em> in spiked water samples. In summary, we have established a practical approach to inactivate <em>C. parvum</em> oocysts in the laboratory that is suitable for the development of detection or diagnostic assays targeting the parasite.</p></div>","PeriodicalId":37941,"journal":{"name":"Food and Waterborne Parasitology","volume":null,"pages":null},"PeriodicalIF":2.9000,"publicationDate":"2022-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/f5/25/main.PMC9249555.pdf","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Food and Waterborne Parasitology","FirstCategoryId":"1085","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S2405676622000208","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"PARASITOLOGY","Score":null,"Total":0}
引用次数: 0
Abstract
Cryptosporidium spp. are obligate, intracellular parasites that cause life-threatening diarrhea among children and immunocompromised adults. Transmission occurs by the fecal-oral route following ingestion of thick-walled oocysts that can contaminate, persist, and resist disinfection in water and food. Sodium hypochlorite, peroxides, ozone, formaldehyde, and ammonia are suitable disinfectants against Cryptosporidium oocysts. Effective concentrations of these chemicals can be toxic and not practical for downstream research use of non-viable oocysts. Oocyst inactivation approaches such as UV light, heat, and treatments with ethanol or methanol are generally more accessible for routine lab use, yet their applicability in Cryptosporidium assay development is limited. The aims of this study were to evaluate methods of inactivation of Cryptosporidium oocysts that can be readily applied in the laboratory and test the utility of whole inactive oocysts in quantitative PCR (qPCR). Experiments were performed on C. parvum oocysts subjected to heat (75 °C/10 min) or treated with increasing concentrations of ethanol and methanol over time. Viability assays based on propidium iodide (PI) staining, in vitro excystation, and infection of the Hct-8 cell line were used to evaluate the efficacies of the treatments. Excystation of sporozoites was not impaired with 24 h exposures of oocysts to 50% ethanol or methanol, even though significant PI incorporation was observed. Concentrations of ≥70% of these chemicals were required to completely inhibit excystation and infection of Hct-8 cells in vitro. Inactivated oocysts stored for up to 30 days at 4 °C retained cyst wall integrity and antigenicity as observed by light microscopy and immunofluorescence. Moreover, non-viable oocysts applied directly in qPCR assays of the COWP gene were useful reference reagents for the identification and quantification of Cryptosporidium in spiked water samples. In summary, we have established a practical approach to inactivate C. parvum oocysts in the laboratory that is suitable for the development of detection or diagnostic assays targeting the parasite.
期刊介绍:
Food and Waterborne Parasitology publishes high quality papers containing original research findings, investigative reports, and scientific proceedings on parasites which are transmitted to humans via the consumption of food or water. The relevant parasites include protozoa, nematodes, cestodes and trematodes which are transmitted by food or water and capable of infecting humans. Pertinent food includes products of animal or plant origin which are domestic or wild, and consumed by humans. Animals and plants from both terrestrial and aquatic sources are included, as well as studies related to potable and other types of water which serve to harbor, perpetuate or disseminate food and waterborne parasites. Studies dealing with prevalence, transmission, epidemiology, risk assessment and mitigation, including control measures and test methodologies for parasites in food and water are of particular interest. Evidence of the emergence of such parasites and interactions among domestic animals, wildlife and humans are of interest. The impact of parasites on the health and welfare of humans is viewed as very important and within scope of the journal. Manuscripts with scientifically generated information on associations between food and waterborne parasitic diseases and lifestyle, culture and economies are also welcome. Studies involving animal experiments must meet the International Guiding Principles for Biomedical Research Involving Animals as issued by the Council for International Organizations of Medical Sciences.