A limitation of molecular assays for detection

A limitation of molecular assays for detection of protozoan oocysts in produce is the inability to distinguish live from dead organisms, making it difficult to interpret the results and determine the significance of positive results with respect to the risk posed by the contamination. However, populations of oocysts often consist of both viable and non-viable organisms, and any finding should be considered an indicator of risk. In bacterial detection methods, samples are commonly subjected to an enrichment step prior to detection, so presumably most Teneligliptin hydrobromide Supplier detected are viable or are present in sufficient numbers to permit enumeration and thus represent a food safety risk for consumers. In vitro propagation of protozoan oocysts is generally not possible or practical, so detection methods rely on washing to remove and concentrate minute quantities of oocysts from the food matrix followed by detection by microscopy, immunoassay, or PCR. The recovery rate of oocysts from food matrices varies widely and depends on the produce type and condition, wash method, and wash buffer used. Recovery rates have been demonstrated to be between 3 and 18% for leafy greens and berry fruits (Lalonde and Gajadhar, 2016). Thus, even if there was a reliable method for determining if a very small number of recovered oocysts were indeed non-viable and non-infective, these low recovery rates from the produce would make it very difficult to declare any sample as minimal risk, since viable/infected oocysts could still remain on the sample and the distribution and concentration could be highly variable in the batch of produce from which the sample was taken. The qPCR MCA can provide an estimate of oocyst quantity detected and can reliably detect DNA from as few as 10 oocysts (Lalonde and Gajadhar, 2011). The infectious dose for Cryptosporidium is estimated at 10 or fewer oocysts (Chappell et al., 2006) and is thought to be equally low for Toxoplasma (Dubey et al., 1996; Fayer et al., 2004) and Cyclospora. Even a single oocyst contains 8 infective sporozoites and is capable of establishing an infection. Another factor to consider is that the conditions that favor prolonged shelf life in leafy greens, namely cool, moist conditions, also generally favor oocyst survival in the environment. Therefore, a positive PCR result for detection of protozoan oocysts in produce should be considered an indication of risk to public health. In this survey, T. gondii and C. cayetanensis were detected in the fall months of October and November, and C. parvum during the spring, in May. Outbreaks of cyclosporosis and cryptosporidiosis in endemic regions are commonly seasonal, most typically being reported during the summer months (Hall et al., 2012; Painter et al., 2015). However, the peak seasons can vary depending on the regional climate where contamination occurs, as rainfall, runoff, flooding, drought, temperature, and humidity can impact the survival and/or sporulation of oocysts in the environment (Fletcher et al., 2012; Gajadhar et al., 2015). All of the samples found to contain C. cayetanensis and T. gondii in this study were imported from either the USA or USA and Mexico (package contained a mixture from both countries) where regional climate conditions or pre-/post-harvest practices may have favored oocyst survival and transmission during the fall months. In addition, this survey included 286 samples of domestic leafy greens which were collected and tested during the months they were exclusively available, which was June, July, August, and September due to the relatively short Canadian growing season. Fewer imported samples were tested during these four summer months: 131 (31%) of 417 samples, all of which were from the USA only. Considering the low risk of C. cayetanensis transmission from domestic produce, future surveys for this parasite specifically should be designed to target imported produce during the spring and summer months. The quality of data generated from food safety surveillance studies such as this depends heavily on the selection of a reliable validated method for the detection and the application of appropriate quality assurance measures and controls in the laboratory, including analyst proficiency. The qPCR MCA assay applied here has been demonstrated as fit for use in detecting protozoan oocysts in human feces, leafy greens, and berry fruits (Lalonde and Gajadhar, 2011, 2016; Lalonde et al., 2013). The oocyst isolation method we used was adapted from a previously published method (Cook et al., 2006a,b) which is now being revised as an ISO standard method for detection and enumeration of Cryptosporidium and Giardia in fresh leafy greens and berry fruits; we optimized and validated the method and use it routinely in our laboratory. The assay is limited by relatively low recovery rates of the oocysts from produce (Lalonde and Gajadhar, 2016), and the inability to determine if any recovered oocysts are viable and infective. The broad specificity of the universal primer cocktail, while useful in targeting multiple species of coccidia, may also result in amplification of non-coccidia species in environmental samples (such as yeast) and thus also reduce the assay sensitivity. During routine testing of leafy greens, non-specific amplicons of fungal origin are observed most commonly in samples collected during the summer months (data not shown). This suggests that maintaining cool storage temperatures for samples prior to testing with qPCR MCA is an important measure for ensuring optimal assay sensitivity. In addition, further molecular characterization (ie species-specific PCR, whole genome or targeted sequencing, other single nucleotide polymorphism-based genotyping assays, etc) of positive samples would be necessary to facilitate molecular epidemiology investigations to link contaminated produce to cases of human infection.