Field study of parasitic contamination of fruits, vegetables and leafy greens in the Ecuadorian Andes

Introduction

The consumption of fruits and vegetables provides essential nutrients for a healthy diet; however, raw vegetables are among the main vehicles for enteroparasites (FAO/WHO, 2008; Punsawad et al., 2019; Al Nahhas & Aboualchamat, 2020), and contamination can occur at any stage of the food production and marketing chain (Trelis et al., 2022). Various studies have focused on cultivation and harvesting stages as a risk phase, due to inadequate farming hygiene, contact with soil contaminated with human and animal feces (by direct defecation on crops or their fertilization), and the use of contaminated water for irrigation, dilution of pesticides, or washing equipment (Pérez-Cordón et al., 2008; Efstratiou et al., 2017; Ercumen et al., 2017; Luz et al., 2017; Karshima, 2018; Trelis et al., 2022).

Although parasites do not multiply in food, they can survive for months, and resistance to some chemical and physical inactivating agents aggravates the situation (Ramos et al., 2013), so parasites can infect individuals in areas far away from the production site (Dixon et al., 2013; Dixon, 2016; Caradonna et al., 2017; Machado-Moreira et al., 2019; Li et al., 2020).

South American countries are among the most important exporters of fresh vegetables. Ecuador has a tropical climate and soils rich in organic matter that allow it to harvest fruits, vegetables, and grains throughout the year for sale to different countries. According to figures from the Agriculture and Livestock Ministry, during the period of 2014-2018, Ecuador raised more than $3,500 million by exporting 6,000,000 tons of fruits and vegetables, including 212 tons of bananas, 139 tons of baby bananas, 81 tons of pineapples, 74 tons of broccoli and 60 tons of mangoes (Ministerio de Agricultura y Ganadería Ecuador, 2020).

Imports of fresh produce from endemic countries have contributed to the spread of parasites to nonendemic countries; diarrhoea epidemics have been reported from the consumption of raspberries, tomatoes, peppers, onions, carrots and radishes (FAO/WHO, 2008; Dixon, 2016; Machado-Moreira et al., 2019; Li et al., 2020). The WHO highlights the importance of leafy greens (spinach, lettuce, cabbage, watercress, basil, mint, coriander and parsley) as vehicles for food-borne parasites (FAO/WHO, 2008), among which are Cyclospora cayetanensis, Cryptosporidium spp. and Giardia duodenalis, Toxoplasma gondii, Entamoeba histolytica, Blastocystis sp., Cystoisospora belli, Balantidium coli, Dientamoeba fragilis and geohelminths (Ramos et al., 2013; Gamble, 2015; Dixon, 2016; Caradonna et al., 2017; Karshima, 2018; Robertson, 2018; Trelis et al., 2022).

In Ecuador, there is a dramatic health situation that affects all rural Andean regions, especially those located at high altitudes, which are mostly inhabited by indigenous populations that have agriculture, livestock, and other animal husbandry as a means of subsistence (González-Ramírez et al., 2021, 2022). Moreover, it is important to note that farmers need to learn good agricultural practices since they may not have adequate training, confidence, or economic resources to maintain crops and animals with proper hygiene to guarantee food quality production.

A review indicated that there are no studies carried out in other provinces of Ecuador; only one report evaluated the risk factors associated with parasite transmission and described a total of 70.6% contamination of fruits and vegetables grown in six rural communities in the parish of San Andres, Chimborazo province (González-Ramírez et al., 2022). Due to this alarming contamination figure, we proposed evaluating the parasitic contamination of all products (fruits, vegetables and leafy greens) grown in the capital of San Andres, an agricultural zone of the Ecuadorian Andes. The probable causes of produce contamination during the primary production phases are likely due to sanitary control during these stages of the production chain; these need to be analyzed to minimize the risk of infections, which will benefit exports with favourable consequences on the country’s economy.

Methods

Results

When analyzing the different crop products, a total of 898 (63.4%) were contaminated by parasites. A statistically significant difference between the overall contamination rates, the leafy greens (76.9%) were more contaminated than vegetables (67.8%) and fruits (48.4%) (P<0.0001). Also identified were 15 protozoa and 2 helminth nematodes, protozoa also had a higher prevalence (49.6%) than nematodes (15.5%) (P<0.0001). Blastocystis sp. was outstanding among protozoa (33.5%) (P<0.0001), followed by Eimeria spp. (26.3%), Entamoeba spp. (10.3%), Giardia spp. (8.3%) and Cryptosporidium spp. (6.6%). Between the nematodes, Strongylida were more frequent than Ascaris spp. (P<0.0001) (see Table 1).

When comparing the percentages of contamination of fruits, vegetables and leafy greens by the different parasites, the statistical analysis determined that, in fruits, a higher prevalence of Blastocystis (37.4%) (P=0.0018), Cryptosporidium (7.6%) (P<0.0001), Cyclospora (6%) (P<0.0001) and Endolimax nana (6%) (P=0.0028) was found. Vegetables were mostly contaminated by helminths (24.2%) (P<0.0001), represented mainly Strongylida (23.6%) (P<0.0001). Finally, the leafy greens showed greater contamination by Eimeria (33.5%) (P=0.0002), Entamoeba spp. (16.7%) (P<0.0001), Balantidium (15.0%) (P<0.0001) and Giardia (12.6%) (P=0.0002), which comprised the highest contamination with protozoa (61.4%) (P<0.0001) and a total parasitic contamination of 76.9% (P<0.0001) (see Table 1).

Table 2 summarizes the results obtained according to the type of fruit, the highest number of protozoa was found in strawberries (60.2%) (P<0.0001), with Blastocystis sp. (59.2%) (P<0.0001), E. nana (17.35%) (P<0.0001) and Cyclospora spp. (14.3%) (P=0.0011) in contrast to peaches, which were more often contaminated with helminths (30%) (P<0.0001).

Parasitic contamination in the different types of vegetables is detailed in Table 3, the highest was found in red (84%) and white (82.4%) onions, followed by chili pepper (78%) (P<0.0001). It is important to highlight the level of contamination detected in other vegetables that are eaten raw (carrot 66%, radish 72.1% and pepper 44%). In the analysis to contrast the total parasitic contamination between protozoa (47.1%) and helminths (24.2%), it was possible to verify a higher frequency of protozoa (P<0.0001).

The parasitic contamination in leafy greens was significantly different among types (Table 4); greater percentages of parasites were found in cabbage (100%), alfalfa (90.2%) and parsley (82.4%). It was possible to verify higher contamination of the cabbage with Eimeria (53.8%) (P<0.0001) and with Endolimax nana (13.5%) (P=0.0002), lettuce with Entamoeba spp. (36.2%) (P<0.0001), and parsley with Blastocystis (56.9%) (P=0.0071).

Comparative analysis of parasitic contamination rates detected between fruits and vegetables/leafy greens (Table 5) showed higher parasites percentages in vegetables/leafy greens, with significant differences in the total (72%) (P<0.0001), protozoa (53.7%) (P<0.0001) and helminths (20.9%) (P<0.0001). A higher prevalence of Eimeria (29%) (P=0.0027), Entamoeba spp. (13%) (P<0.0001), Giardia (10.2%) (P=0.0007), and Balantidium (10.2%) (P<0.0001) were found. In contrast, higher percentages of Blastocystis (37.4%) (P=0.0199) and Cyclospora (6%) (P=0.0313) were found in fruits.

When parasitic contamination was compared between leafy greens (76.9%) and vegetables (67.8%), a statistically significant difference was found (P =0.0024) (see Table 6). This result was supported by the highest contamination of leafy greens with Blastocystis (35.9%) (P=0.0064), Eimeria (33.5%) (P=0.0063), Balantidium (15.1%) (P<0.0001), Entamoeba spp. (16.8%) (P=0.0021) and Giardia (12.6%) (P=0.0290). However, vegetables were found to be more contaminated by helminths than leafy greens (24.2%) (P=0.0082), determined by Strongylida (23.6%) (P=0.0150).

Discussion

The results of the present study prove that the fruits, vegetables and green leafy that are cultivated and harvested in the capital of San Andrés, the area with the highest agricultural production in the Ecuadorian Andes, present significant contamination with parasites. Multiparasitism in the samples analyzed reflects inadequate hygiene conditions during agricultural activities and the crops products thus obtained represent a possible vehicle for parasites when consumed without adequate sanitation. It is important to note that agricultural production is marketed locally, regionally, nationally, and internationally; therefore, the risk of contagion to individuals is extrapolated to non-endemic areas.

Direct contamination with human and animal excrements is a potential source of contamination of anthroponotic and zoonotic parasites for vegetables, so their consumption constitutes an important risk factor associated with the transmission of infective forms. However, it is possible that free-living parasites (Strongylida), also contaminate these crop products, being considered an insignificant finding, in this population where our research group has detected parasite prevalence’s reaching 97.3% in humans (González-Ramírez et al., 2022) and 90.3% in animals (González-Ramírez et al., 2021).

When contrasting the results of the present investigation carried out in the capital of San Andrés with those obtained in six different communities of the same parish, the total percentage of contamination is lower in agricultural products harvested in the capital (63.4%) compared to the other communities further away, located at higher altitudes and with a larger indigenous population (70.6%); likewise, fruits (48.4 versus 67.1%) and vegetables (67.8 versus 73.6%) (González-Ramírez et al., 2022). These differences may be present because the capital of San Andrés offers better environmental sanitation conditions and the farmers have a higher level of education and better access to the urban area.

In the present study, leafy greens were more contaminated (76.9%) than vegetables (67.8%) and fruits (48.4%), likely because these maintain contact with the soil and organic fertilizers from the beginning as seedlings until they are fully grown, and external leaves allow protection for internal plant parts in contact with contaminated soil. The greater parasitic contamination of leafy greens has been explained by the irregularities of their leaves and the roughness of their surface that allows the adhesion of infectious parasitic forms that persist in the environment (Vuong et al., 2007; Allende et al., 2017).

Vegetables were the second most contaminated products after leafy greens, which is explained by the greater contact they maintain with the soil. The rooted vegetables (tubercle) were found to be more parasitized by nematodes (24.3%), always being less than contamination by protozoa (47.1%), possibly because they grew under the ground. It is important to highlight that, onions, carrots and radishes are frequently consumed raw and can function as efficient vehicles for parasites.

In creeping fruits such as strawberries, a greater number of contaminating parasitic species was found, compared to those that grow on shrubs and trees, perhaps due to direct contact with irrigation water (Esteban et al., 2002; Daniels et al., 2016; González-Ramírez et al., 2020), organic fertilizer and the soil (Dixon, 2016; Rodríguez-Eugenio et al., 2019).

In addition, the roughness of its surface is a condition that can also influence in the contamination of blackberry and peaches (Resendiz-Nava et al., 2020). Although, these rough fruits do not come into contact with the soil, nor with irrigation water, the texture of their surface allows the adhesion of parasites dispersed by the wind, insects or the hands of farmers, as explained by Dixon (2016) and Machado-Moreira et al. (2019).

Animal faeces is a source of nutrients for the soil, fertilizing agro-systems at low cost (Daniels et al., 2016), but if this material does not receive prior treatment, it is highly polluting. Among the inappropriate agricultural practices detected in San Andrés, considered risk factors, the fertilization of crops with fresh excreta from parasitized animals, as well as the contamination derived from their displacement on the crops, dispersing viable parasitic infectious forms persist in the environment (Gutiérrez-Rodríguez and Adhikari, 2018; Julien-Javaux et al., 2019; Resendiz-Nava et al., 2020; González-Ramírez et al., 2021). Additionally, the contamination of soils with human fecal matter contained in septic tanks that overflow or leak is another important source of contamination (Daniels et al., 2016; González-Ramírez et al., 2022).

On the other hand, the irrigation of crops with water bodies conducted by channels or contained in artificial wells that receive runoff from rain should be considered a risk factor for parasitic dispersal, as has been verified by Machado-Moreira et al. (2019) and González-Ramírez et al. (2020). These artificial water resources carry a high risk to human health in relation to the spread and increased transmission of parasites as shown by Esteban et al., (2002). Our results question the rationality of irrigation projects through open channels that carry contaminated water and artificial wells from which the animals drink. Furthermore, this water is used for the dilution of fertilizers and fungicides, machinery and the washing of work equipment and utensils that increase the possibility of contamination of vegetables (Dixon, 2016; Machado-Moreira et al., 2019; Trelis et al., 2022).

In addition, aspects of the field that promote the dissemination of parasitic forms found in the soil include flooding, rain, and sprinkler irrigation that allow the transport of microorganisms from the soil to the plants, as confirmed by Efstratiou et al. (2017), or when water drops splash as explained by Dixon (2016).

Likewise, the wind brings dust particles from the ground that aid adherence of parasitic forms to the vegetables or fruits of trees or shrubs (Dixon, 2016; Machado-Moreira et al., 2019), which explains the finding of Strongylida on the woolly surface of peaches. Additionally, insects, rodents, wild animals and the contaminated hands of farmers can spread parasites, as indicated by Dixon (2016), Machado-Moreira et al. (2019) and González-Ramírez et al. (2021).

Moreover, various actions carried out by farmers can contaminate crop products, including handling of vegetables without hygienic measures by the personnel in charge of sowing and harvesting, as has been verified in various localities (Dixon, 2016; Machado-Moreira et al., 2019; Li et al., 2020). However, the transfer, storage, washing, packaging, distribution, and marketing activities that are carried out after harvest also contribute to food contamination (Etewa et al., 2017; Trelis et al., 2022).

After becoming aware of the parasitic contamination of food grown in the area, consumers from any part of the world should be warned that they must properly sanitize fruits, vegetables and leafy greens before consuming them, especially those that are eaten raw, if its origin is unknown. Just as this alarming contamination has been detected in this agricultural area, it is likely that it also occurs in other rural areas, mainly in low-income countries where producers do not apply hygienic measures during their agricultural practices, as previously was demonstrated by Pérez-Cordón (2008) in the Andean zone of Peru.

The potential effects of primary production activities on food safety need to be considered. These include identifying any specific points where the probability of contamination may exist and taking specific measures to minimize them. Growers are required to implement measures to prevent contamination of air, soil, water, feed, fertilizers, pesticides, or any other agent used in production and to control animal health so that it does not pose threats. If programs are implemented and executed to guarantee sanitary control in the farms and the objectives of food security are achieved in primary production, exports would increase, translating to an increase in the economic income of the producing countries.

The considerable parasitic contamination in vegetables obtained in the field immediately after harvest in this zone might be one of the causes of the high parasitic prevalence in humans (98.2%), mechanical vectors (52.7%) (González-Ramírez et al., 2022), and animals (90.3%) (González-Ramírez et al., 2021), as well as the 100% contamination of man-made water resources (channels and wells) of these rural communities (González-Ramírez et al., 2020).

These results suggest the need to integrate protozoa and helminths into the list of contaminants that are handled in the microbiological criteria required by the Ecuadorian Technical Standard (INEN, 2016). Monitoring only Escherichia coli in vegetables and fruits is not a good indicator of the absence of faecal contamination, nor does it guarantee food safety. Protozoa, whose high resistance to temperatures and disinfectants (Ramos et al., 2013) and low infectious doses have been demonstrated constitute a significant risk for consumers.

Policy decisions should promote the development of mitigation plans that involve health and hygiene education programs for producers and consumers. In addition, more advanced technological procedures and treatments that contribute to contamination prevention, as well as the inactivation and elimination of infectious forms in contaminated fresh produce to improve the quality and safety of these foods in accordance with the standards of Caradonna et al. (2017), should be promoted.

The sedimentation technique, Ziehl Neelsen staining, and measurement with the ocular micrometer performed for parasitic detection in the present study allowed us to carry out a low-cost analysis, as long as, microscopic visualization is done by trained analysts, fresh plant products can be monitored in other endemics areas of developing countries, where biological analysis cannot be performed by molecular techniques because of its high cost. We are aware of the importance of determining the parasitic species by molecular methods for epidemiological control. However, for surveillance studies on the contamination of these products in poor countries, microscopic diagnosis (though insufficient) is relevant because it is the only thing available; these results provide the basis for food safety guidelines to reduce the risk of contamination and minimize the transmission of food-borne parasitic diseases.

The samples of vegetables and fruits were analyzed by light microscopy alone because of limited resources to perform molecular analysis and the difficulty in obtaining permission to transport the samples to a molecular laboratory, but the overall prevalence detected in this study was one of the highest described thus far. In this Andean region of Ecuador, the global contamination of agricultural products by parasites has a mean prevalence of 63.4 and 70.6% (González-Ramírez et al., 2022), which are higher than those described in Phnom Penh, Cambodia 56.0% (Vuong et al., 2007), Alexandria, Egypt 31.7% (El Said Said, 2012), Koforidua, Ghana 57.5% (Kudah et al., 2018), Arba Minch, Ethiopia 54.4% (Bekele et al., 2017), Nakhon Si Thammarat, Thailand 35.1% (Punsawad et al., 2019), and Damascus, Syria 34.4% (Al Nahhas and Aboualchamat, 2020).

Nevertheless, our results are similar to those found in Trujillo, Peru, where Pérez-Cordón et al. (2008) reported the presence of Giardia, Cyclospora, E. nana, Iodamoeba buetschlii, Blastocystis and Ascaris lumbricoides. Moreover, the prevalence values are similar to those reported in Mina Gerais, Brazil, by Luz et al. (2017), with 50.9% of vegetables contaminated, with a predominance of nematode larvae (36.5%), Entamoeba coli (26.0%) and eggs of hookworms/Strongyloides spp. (12.9%).

Our results also differ from those obtained by Honório Santos et al. (2019) in Bahia, Brazil, with prevalence’s of 70% in fruits: guava (90%), lemon and apple (70%) and grape (50%). The highest prevalence in this study was of the helminths A. lumbricoides, Ancylostomids, Taenia spp., and Enterobius vermicularis, followed by the protozoa Balantidium coli and Entamoeba coli. These differences might be due to the high altitude of San Andrés, where the evolution of soil-transmitted helminths is limited.

Interestingly, in San Andrés, there were significant differences between contamination in leafy green types, which is consistent with the results of other studies that indicate that the highest-contaminated vegetable is lettuce, reaching rates of 29.5% in Damascus (Al Nahhas and Aboualchamat, 2020), 54.2% in Ghana (Kudah et al., 2018), and 61.1% in Mina Gerais, Brazil (Luz et al., 2017).

Food-borne transmission of protozoan parasites is an emerging issue in developed countries around the world. Giardia, Cryptosporidium and Cyclospora have been implicated in both human and animal illness: unpasteurized apple juice, unwashed onions, salad, mixed baby lettuce, basil, sandwiches, fruit salad and raspberries (Dixon, 2016). Rzezutka et al. (2010), in Lublin, Poland, detected Cryptosporidium sp. in 4.7% of fresh vegetables; in packaged salads, Italy revealed 4.2% contamination of the samples, and the prevalence of each species was for G. duodenalis 0.6%, T. gondii 0.8%, Cryptosporidium spp. 0.9%, C. cayetanensis 1.3%, B. hominis 0.5% and D. fragilis 0.2% (Caradonna et al., 2017). In contrast, Trelis et al. (2022) were able to prove higher contamination with G. duodenalis 23.3% and Cryptosporidium spp. 7.8% in green leafy vegetables marketed in the city of Valencia, Spain.

Information collected at each sampling point checked to field cultivation as the critical step for contamination (Luz et al., 2017). The high parasitic frequency is associated with the inadequate handling of crop products, as well as, to the inefficient sanitary conditions of the places where they are marketed. It is recommended to teach hygienic measures through sanitary education for farmers, merchants, and consumers (Honório Santos et al., 2019).

In these tropical countries, the highest records of parasitic contamination are in vegetables, so they are described as endemic for enteric parasites. From there, they are spread to other countries through fresh vegetables. Developing countries have not been able to control their enteric-parasitosis because of the low socioeconomic and hygienic-sanitary levels, inability to offer adequate sanitary infrastructures and the education that could change of habits in people and prevent soil, water, and food contamination.

The implementation of control measures in fresh produce preharvest and postharvest, as well as an adequate sanitary hygienic level of the producer, handler, and consumer, will be crucial to minimize the food transmission of protozoa and helminths. To control parasites at the time of cultivation and harvest, irrigation with properly treated water, monitoring the health and hygiene of agricultural workers, improving agricultural sanitation, and restricting access of livestock and other animals to crops and surface water bodies (building adequate drinking troughs) are needed. Additionally, proper construction and maintenance of septic tanks is important to prevent contamination by overflow.

Unsafe agricultural practices, such as irrigation with untreated contaminated water and fertilization of the soil with improperly treated animal manure, are used very commonly by small farmers; mainly in developing countries, due to the export of agricultural products. To mitigate this problem, it is necessary to use treated water for irrigation, washing fresh produce, washing hands and equipment. Good hygienic practices by farm workers involved in the cultivation, harvesting and handling of fresh produce are another important means of reducing the likelihood of contamination at the farm level in endemic regions.

Conclusion

This research demonstrated the important parasitic contamination of fruits, vegetables, and leafy greens. Warns about the risk of consuming raw products from these crops, without proper hygiene can be infection source of enteroparasites to humans and animals in this area or in nonendemic areas where these products are marketed. This study establishes the need for strict hygienic measures in growing and harvest areas, which can be achieved by the treatment of soil, manure, and water used for the cultivation of vegetables and fruits, as well as proper disinfection before consumption.