# Food and Drinking Water as Sources of Pathogenic Protozoans: An Update
## 18 Discussion
Figure 2: Number of waterborne or foodborne acute cases/outbreaks reported for pathogenic protozoans since 2019 [9, 19, 19, 35, 122, 123, 131, 132, 133, 142, 134, 154, 157, 168, 169, 170, 172, 173, 191, 193, 195, 196, 197, 198, 199, 201, 209, 226, 233, 237, 239, 243, 255, 256, 260, 268, 269, 273, 274, 280, 282, 286]. Outbreaks are only shown for _Cryptosporidium_ species. In most cases, links to infection sources were defined by epidemiological investigations since the organisms were not detected from food or water samples causing the illnesses either because the samples were not anymore available at the time of the outbreaks or due to lack of sensitivity of the analytical methods [9, 113]. Indeed, the long incubation period, lasting some weeks before symptom manifestation, and intermittent emissions can confound case recognition and the identification of the infection source in most foodborne outbreaks [9, 129, 131, 154, 203]. In addition, methods available for protozoan detection in food matrices are considered inefficient. A guideline from the International Organization for Standardization (ISO), ISO 18744:2016 [293] is available only for the detection and enumeration of _Cryptosporidium_ and _G. intestinalis_ in vegetables and berry fruits. However, it does not evaluate viability or identify the species and the genotype. Therefore, the development of standard analytical methods is still needed for other food matrices from which the recovery of the parasites is difficult, as in the case of _Cryptosporidium_ from milk and meat [9, 128, 169]. The standardization of reliable detection methods would greatly improve prevention and could represent an essential tool for surveillance. Figure 3 shows the identified sources of protozoan infections in the most recent cases and outbreaks and the origin of contamination if known. Figure 3 shows that in most outbreaks the origin of contamination was not identified. Therefore, future research should be aimed at finding links between protozoan pathogens in food or water and the sources of contamination. In particular, advanced molecular detection methods should be applied to soil, irrigation water, or washing water that comes into contact with fresh produce. To this end, new genotyping methods, still unavailable for some species and subtypes, would be essential for pathogen source tracking. Additionally, metagenomics techniques can be exploited to elucidate the protozoan pathogen transmission chain, as shown by a study that used metataxonomic and metagenomic techniques, coupled with traditional outbreak investigation, to trace a foodborne outbreak caused by _C. parvum_ lidA24G1 in Sweden. In that study, case interviews, sampling of patients, and back-tracing identified romaine lettuce from a specific farm as the outbreak source. The microorganism was found in other samples of lettuce from the farm and the microbiota of the contaminated lettuce and other crops, identified with both 165 rRNA gene V4 hypervariable region amplicon metagenomics and shotgun sequencing, comprised species associated with human and animal fecal environments and untreated sewage. Since the farm used only chemical fertilizers and irrigation water from a deep well that was not contaminated at the time of the outbreak, it was concluded that the most likely source of fecal pollution was human and derived either from wastewater contamination of the irrigation water or water used for washing the lettuce or else from infected people handling the lettuce [133]. The implementation of surveillance programs in countries that still lack plans for protozoan infection monitoring is another critical aspect to be considered since even protozoans with a high pathogenic potential are still not included among microbial pathogens under control [156].