# Protozoa as the \"Underdogs\" for Microbiological Quality Evaluation of Fresh Vegetables
## 3 Results and Discussion  
Between one and four _G. duodenalis_ fluorescent cysts (Figure 1A) were observed in microscopic slides of four of the 16 samples (25.0%; 95% CI: 9.7 to 49.9%) (Table 2). The same percentage (25.0%) was obtained for the number of _Cryptosporidium_ spp. (Figure 1B) oocysts (between one and eight oocysts) (Table 2); however, none of the _Cryptosporidium_-positive slides were confirmed by PCR. On the contrary, three of the four _Giardia_-positive slides were also confirmed by PCR. \(\beta\)-giardin fragment sequencing analysis revealed the presence of _G. duodenalis_ assemblage A having more than 98% homology with _G. duodenalis_ assemblage A isolates available in the Genebank database (KJ668152.1, KP687765.1, KP63547.1, EU642897.1, EU200934.1). The determination of genotypes is important for the identification of risk to human health, and potentially, the source of contamination. Assemblage A is one of the genotypes that undeniably causes human infections [24]. Globally, significant variations in the prevalence of these parasites have been observed in vegetables, ranging between 0% and 53% for _G. duodenalis_ and between 0% and 63% for _Cryptosporidium_ spp. (reviewed by [PERSON] et al. [60]). Several factors can easily explain this discrepancy, such as different sampling procedures and detection methods having different recovery efficiencies and detection limits, distinct geographic locations having variable seasonal climatic conditions, and the degree of local development. The type of vegetable, in addition to the quality of water used for irrigation and washing, the proximity of livestock, and the presence of organic fertilizers, are also extremely relevant [60]. Unexpectedly, positive samples were only observed for the RTE type of produce. Thus, it is acceptable to discuss the management of the washing systems used in the food industry, even based on these preliminary data. Indeed, [PERSON] et al. [61] noted that the use of treated wastewater for irrigation increases the chance of contamination of RTE/packaged vegetables. Other studies mention that contamination of RTE products by food handlers \begin{table}
\begin{tabular}{c c c c c c c c c} \hline \hline
**Sample** & **Product** & **Product** & **No.** & **PCR** & **No. (ooclysts)** & **PCR** & **Genotyping** & \\
**Presentation** & **Presentation** & **_Giaardia_ & _Giaardia_ & _Cryptosporidium_ & _Cryptosporidium_ & **(Assemblage)** & \\ \hline
2 & Coriander & bulk & 2 & Negative & 0 & n.a. & n.a. & \\
8 & Watercross & RTE & 1 & **Positive** & 0 & n.a. & A & \\
9 & Mixed & RTE & 1 & **Positive** & 0 & n.a. & A & \\
12 & Mixed & RTE & 4 & **Positive** & 8 & Negative & A & \\
13 & Mixed & RTE & 0 & n.a. & 1 & Negative & n.a. & \\
15 & Arugula & RTE & 0 & n.a. & 1 & Negative & n.a. & **LMO** & **LMO** & **detection** \\
16 & Lettuce & bulk & 0 & n.a. & 2 & Negative & n.a. & \
\begin{tabular}{c} Present \\ \(<4.0\times 10^{1}\) \\ \end{tabular} &
\begin{tabular}{c} Positive in \\ 25 g \\ \end{tabular} \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Protozoa and bacteria positive samples. Figure 1: Epifluorescence microscopy images (400\(\times\) magnification) of _Giardia_ cysts (**A**) and _Cryptosporidium_ oocytes (**B**) found in sample 12.  
during packing is also a transmission route of _Cryptosporidium_ and _Giardia_[61, 62, 63]. Therefore, it is clear that current RTE production treatment does not guarantee that fresh produce will be free from protozoa, and that alternative water treatments and increased hygienic measures for food handlers are necessary to ensure the safety of RTE vegetables. Fresh produce has repeatedly been implicated in foodborne outbreaks and recalls, with _Salmonella_ spp., _E. coli_, and _L. monocytogenes_ the most implicated bacteria [64]. However, with the exception of a lettuce sample (sample no. 16) in which _L.