Validation of novel DNA isolation procedures from limited numbers of Cyclospora oocysts

Cyclospora cayetanensis is a highly infectious food- and waterborne parasite that causes gastrointestinal illness in humans and is typically acquired through consumption of fresh produce. Cyclospora infections are characterized by sporadic shedding of oocysts in feces, which, combined with the need to ingest only a few oocysts for full infection, make diagnostics difficult. Since <10 oocysts suffice to cause infection, detection methods must be sensitive and robust. This proposal's main goal is to develop rapid DNA isolation methods from limited numbers of Cyclospora oocysts to support surveillance efforts. We subdivide this problem into two key aims: 1) developing a simple, efficient method for DNA isolation enabling downstream molecular diagnostics, and 2) concentrating oocysts from produce washes. Success would improve the ability to collect, concentrate, and isolate DNA from Cyclospora. The aim is to develop these methods for deployment in production and processing facilities, and to obviate the need for specialized instrumentation or costly reagents.

Cyclosporiasis is a highly infectious gastrointestinal disease caused by the emerging enteric pathogen Cyclosporacayetanensis. The first documented case of cyclosporiasis in the United States is thought to have occurred in 1990 in Chicago and was tentatively linked to a contaminated water source in a physician’s dorm (Huang et al., 1995). In the mid-1990s, several cases were linked to contaminated produce imported from Guatemala, including raspberries and blackberries (Carter et al., 1996; Koumans et al., 1995; Herwaldt et al., 1997). 2018 saw the first outbreak associated with domestic produce, with domestic transmission occurring in every subsequent year. Surveillance efforts in the Southeastern United States indicate that the number of cyclosporiasis outbreaks saw a dramatic increase in prevalence from the 2022 to 2023 outbreak season. Since only a few oocysts are required to cause an infection, rapid and sensitive detection methods are needed. Current molecular methods used to identify and genotype Cyclospora are laborious and equipment-intensive, and the absence of on-site detection impairs outbreak prevention. Rapid detection is hindered via several avenues: Cyclospora cannot be cultured, case reporting is often delayed due to the extensive incubation period before symptoms appear, and clinicians may often omit protozoal infection from the list of differential diagnoses when patients present with diarrheal illness. Despite the increased prevalence of cyclosporiasis in the United States, few methods have been evaluated for rapid and facile DNA isolation from Cyclospora oocysts. This is in stark contrast to other coccidian parasites, on which an extensive body of research exists for rapid molecular detection including thermal, chemical, and physical methods of oocyst rupture. We focus here on novel methods for the concentration and isolation of limited numbers of Cyclospora oocysts, coupled with rapid physicochemical means of oocyst rupture for molecular detection and genotyping. The primary objective of this 1-year proof of concept project is to 1) evaluate an exhaustive array of compounds whose action individually or in combinations is able to rupture the bilayered Cyclospora oocyst wall. In parallel with this primary objective, we will 2) evaluate a method for the magnetic capture of oocysts from produce washes. To meet these objectives, we will rely heavily on robotics- assisted automation to screen test compounds at various concentrations and in combinations. An initial set of test compounds has been chosen based on their action in disrupting polysaccharides, proteins, and lipids, which comprise the coccidian oocyst wall.

Objective 1: Evaluate > 10 methods for efficient DNA extraction from Cyclospora oocysts. 

Objective 2: Validate methods for concentrating oocysts from produce washes.

This project is ongoing. A final report will be provided when the project is finished.