Investigating Cyclospora cayetanensis in Environmental Waters: Molecular Detection and Genotyping

Abstract

Within the last decade, there has been a continuous increase in Cyclospora cayetanensis cases associated with the consumption of fresh produce grown within the U.S. Environmental sources of contamination beyond fresh produce in the U.S. has not been extensively examined, and this thesis aims to evaluate available molecular detection methods for C. cayetanensis in environmental waters prior to a surveillance study conducted in the Western U.S. California. Currently available genotyping methods for C. cayetanensis utilize a multi-locus sequence typing approach that was evaluated only on human stool samples and need to be evaluated for environmental water matrices to support secondary confirmation and traceback investigations. To assist, our study described in Chapter 2 assessed 10 biomarkers’ performance for genotyping C. cayetanensis in environmental waters, which comprised of 9 from the CDC’s genotyping panel for human stool, consisting of 6 nuclear (CDS-1, CDS-2, CDS-3, CDS-4, HC378, HC360i2) and 3 mitochondrial (MSR, Cmt_534, Cmt_109) biomarkers, as well as one from the FDA mit3PCR for irrigation waters. Successful sequencing and identification of the parasite from secondary sequence confirmation is necessary subsequent to molecular detection with qPCR for environmental samples as the standard 18S rRNA qPCR assay is suspected to cross react with closely related parasites, and the replacement Mit1C qPCR assay targeting the Cox3 gene was yet to be validated for environmental waters. When evaluating results with the Geneious and Bowtie2 mapping algorithms, C. cayetanensis was sufficiently sequenced and confirmed with biomarkers CDS-1 (8/24; 33.3%), CDS-2 (1/24; 4.17%), CDS-3 (7/24; 29.1%), and CDS-4 (7/24; 29.1%) among 14/24 (58.3%) irrigation water samples presumptively positive by 18S rRNA qPCR, and of which none were positive with the Mit1C qPCR assay. Accordingly, we demonstrate the potential of the CDS markers for genotyping C. cayetanensis in environmental waters and potential cross-reactivity with the 18S rRNA qPCR. With the need for a highly specific and sensitive molecular detection tool, the FDA updated the Mit1C qPCR assay as a replacement standard method for molecular detection in environmental waters. As adapting their protocol optimized for the ABI-7500 instrument to more common real-time PCR platforms will broaden research and testing capabilities, in Chapter 3 study, we optimized the Mit1C real-time PCR protocol under ideal clean conditions on our Bio-Rad CFX-96 platform and compared to the ABI-7500 platform. Results indicate that the Mit1C real-time PCR assay performed best with a reduced annealing temperature of 66°C for the Bio-Rad CFX-96, where no optimization and direct adaptation of the Mit1C real-time PCR protocol for the ABI-7500 platform using 67°C could not detect as high as 200 oocysts spiked in irrigation water. With our established tools for molecular detection and sequence confirmation, Chapter 4 describes the study where we then determined the occurrence of C. cayetanensis in environmental water and portable toilet surfaces (fomites) as potential modes of transmission in the Western U.S. California fresh produce growing region. A total of 235 samples including surface water (n=119), reclaimed water (n=41), fomites (n=74), and sludge (n=1) were collected and processed in accordance with the FDA BAM Chapter 19c (July 2020 edition) that describes methods for filtering oocysts, concentrating, and DNA isolation. Molecular detection of C. cayetanensis proceeded with the optimized Mit1C qPCR protocol and a previously developed in-house ITS-2 region qPCR assay. Results indicate no detection of C. cayetanensis in all samples by Mit1C qPCR, where the ITS-2 qPCR assay had very high Cq values for three surface water and one sludge samples. These ITS-2 qPCR positive samples are determined as presumptively positive without supporting sequencing data. C. cayetanensis was not confirmed in reclaimed water and portable toilet surfaces. In summary, this thesis establishes the molecular tools for detecting C. cayetanensis in environmental waters, confirmed its prevalence in Southeast U.S. Florida irrigation waters, and identified presumptive positives in Western U.S. California irrigation water and sludge samples from a fresh produce growing region. Further examination of the parasite in environmental samples is needed to fully determine environmental modes of transmission.