Browsing by Autor "Packham, Andrea"

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Quantification of viable protozoan parasites on leafy greens using molecular methods.
(2021) Kim, Minji; Shapiro, Karen; Rajal, Verónica B; Packham, Andrea; Aguilar, Beatriz; Rueda, Lezlie; Wuertz, Stefan
Protozoan contamination in produce is of growing importance due to their capacity to cause illnesses in consumers of fresh leafy greens. Viability assays are essential to accurately estimate health risk caused by viable parasites that contaminate food. We evaluated the efficacy of reverse transcription quantitative PCR (RT-qPCR), propidium monoazide coupled with (q)PCR, and viability staining using propidium iodide through systematic laboratory spiking experiments for selective detection of viable Cryptosporidium parvum, Giardia enterica, and Toxoplasma gondii. In the presence of only viable protozoa, the RT-qPCR assays could accurately detect two to nine (oo)cysts/g spinach (in 10 g processed). When different proportions of viable and inactivated parasite were spiked, mRNA concentrations correlated with increasing proportions of viable (oo)cysts, although low levels of false-positive mRNA signals were detectable in the presence of high amounts of inactivated protozoa. Our study demonstrated that among the methods tested, RT-qPCR performed more effectively to discriminate viable from inactivated C. parvum, G. enterica and T. gondii on spinach. This application of viability methods on leafy greens can be adopted by the produce industry and regulatory agencies charged with protection of human public health to screen leafy greens for the presence of viable protozoan pathogen contamination.
Simultaneous detection of four protozoan parasites on leafy greens using a novel multiplex PCR assay.
(2019) Shapiro, Karen; Kim, Minji; Rajal, Veronica B; Arrowood, Michael J; Packham, Andrea; Aguilar, Beatriz; Wuertz, Stefan
Pathogen contamination of fresh produce presents a health risk for consumers; however, the produce industry still lacks adequate tools for simultaneous detection of protozoan parasites. Here, a simple multiplex PCR (mPCR) assay was developed for detection of protozoan (oo)cysts and compared with previously published real-time PCR assays and microscopy methods. The assay was evaluated for simultaneous detection of Cryptosporidium, Giardia, Cyclospora cayetanensis, and Toxoplasma gondii followed by parasite differentiation via either a nested specific PCR or a restriction fragment length polymorphism (RFLP) assay. Spiking experiments using spinach as a model leafy green were performed for assay validation. Leaf-washing yielded higher recoveries and more consistent detection of parasites as compared with stomacher processing. Lowest limits of detection using the nested mPCR assay were 1-10 (oo)cysts/g spinach (in 10 g samples processed), and this method proved more sensitive than qPCR for parasite detection. Microscopy methods were more reliable for visual detection of parasites in lower spiking concentrations, but are more costly and laborious, require additional expertise, and lack molecular confirmation essential for accurate risk assessment. Overall, the nested mPCR assay provides a rapid (<24 h), inexpensive ($10 USD/sample), and simple approach for simultaneous detection of protozoan pathogens on fresh produce.