# Food and Drinking Water as Sources of Pathogenic Protozoans: An Update
## 17 Methods for the Detection/Identification of Protozons in Food and Drinking Water  
From the literature reviewed here, variability in the methods used to detect and identify protozoans in food and drinking water emerged. Since method performance can influence result sensitivity and comparability, standardization is needed. Detection of protozoanocysts from vegetable samples includes elution, concentration, and identification. The elution step is crucial for the efficiency of parasite recovery and is affected by changes in elution buffer composition, volume, incubation times, and agitation methods [37,38,47]. Elution optimization allowed the simultaneous detection of _C. parvum, G. intestinalis, T. gondii_ and _C. cayetanensis_ by multiplex polymerase chain reaction (PCR) with 90% certainty of detection for contamination levels of 1-10 ocysts per g of spinach [37]. The washing solution commonly applied and recommended by standardized detection methods consists of 0.1-1% Alconox [38,127,247,248,291], but suspension solutions with a different composition were also reported [47]. Moreover, spontaneous sedimentation of wash suspension instead of centrifugation is still in use [278,283]. After washing,ocyst concentration was carried out via different multi-step centrifugation methods [38,43,47,83,84] that also required optimization for most genera and species. Comparison of the yield for different elution methods would lead to increased repeatability, reproducibility, and sensitivity of protozoan detection in food and drinking water. Detection by optical microscope examination was preceded by flotation in some experimental procedures [38,237,292]. However, this step is not appropriate to detect _Cryptosporidium_ oocysts that can be present in too-low numbers in fresh produce and are too small. PCR was proven to be 10-fold more sensitive than direct microscope examination with modified Ziehl-Neelsen staining for _Cryptosporidium_ spp. or Lugol's iodine staining for _G. intestinalis_. Staining methods are largely adopted for their low cost [37] but these should be replaced with PCR-based detection. The IFA technique, which is recommended by the ISO 18744:2016 norm for the detection and enumeration of _Cryptosporidium_ spp. and _G. intestinalis_ in vegetables and berry fruits [293], allows oocyte's identification based on immunofluorescence, size, and morphology [37; 43; 83; 84; 293]. It was applied to apple juice, coupled with IMS [132]. However, this detection method is affected by interference from the auto-fluorescing particles in the food matrices [37], while molecular detection methods based on DNA extraction followed by conventional PCR or TaqMan probe-based qPCR are not [37; 230]. These methods can be easily standardized since commercial DNA extraction kits can be used to analyze the pellets derived from vegetable washing, apple juice, and water [4; 38; 47; 122; 123; 133], and commercial multiplex assays are available for PCR detection [47; 294]. For water analysis, in different studies, sampling and analysis were conducted according to the standard ISO 15553:2006 and the NF T90-455 standard method for sampling and enumeration of _Cryptosporidium_ oocysts and _Giardia_ cysts in water samples [41; 42; 123] based on the same phases of concentration by filtration on site of large volumes of water, e.g., 250 L, release of the microorganisms by washing the filters, and detection by IMS and IFA. This procedure does not accomplish species-level identification and gives no information on viability or infectivity. Moreover, it can be used only by experienced analysts who have passed competency tests [122; 154]. Therefore, a simplification and evolution of such methods towards the identification of the parasite is desirable. It must be noted that standardized methods of elution and concentration from water had not yet been developed for some protozoans, such as _Entamoeba_ species [210]. A recent study demonstrated that dead-end ultrafiltration (DEUF) coupled with quantitative PCR allowed consistent detection of _C.