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Disease transmission and maintenance among humans is the result of interactive processes between the agent, the environment and the human host, known as the epidemiological triad 1. An agent is a factor whose presence is essential for the onset of the disease; a host organism is susceptible to infection by an agent, and the environment is the set of factors that interact with the agent. In this context, enteroparasites appear as a serious public health issue, which has a wide geographical distribution 1-3.
Human contamination by intestinal parasites occurs in both rural and urban areas of developing countries, mainly due to poor sanitary, hygiene and food conditions 4-7. Food security has gained growing importance around the world, given the high frequency and severity of Foodborne Diseases (FBD), which represent a major public health concern 8-10.
In general, the transmission of enteroparasites occurs orally, through the ingestion of water or food contaminated by parasitic structures, being more prevalent in areas where hygienic-sanitary conditions are not adequate, especially because of the poor treatment of water and sewage 11,12.
The consumption of fresh vegetables underscores the importance of monitoring these foods, since intestinal parasites are often found in both rural and urban areas of Brazil due to poor sanitary conditions 13. Controlling the presence of enteroparasites in vegetables is, therefore, of great interest to public health, since it provides data for health surveillance on the hygienic condition of these products and allows the retrospective control of the conditions in which they are produced 14.
With this in mind, lettuce (Lactuca sativa), which is grown in all regions of the country and is very accessible to all economic classes 15, and parsley (Petrosolium sativum), a condiment used to flavor various dishes, stand out as some of the most common products of the Brazilian cuisine 16. These vegetables are recommended by doctors and nutritionists as an essential part of a healthy diet due to their vitamin, mineral and dietary fiber content 17.
Many parasites may be present as contaminants in vegetables and, depending on their characteristics, they may or may not pose a threat to the health of consumers of this type of food without proper hygiene. Said parasites include: Giardia lamblia, Entamoeba histolytica/ dispar, Hymenolepis nana, Ascaris lumbricoides, Stron-gyloides stercoralis, Fasciola hepatica, Enterobius ver-micularis, Trichuris trichiura, hookworms and, not least important, commensal parasites, such as Entamoeba coli and Endolimax nana, great indicators of the hygiene conditions of the population 18.
Therefore, considering the importance of monitoring the incidence of enteroparasites in lettuce and parsley sold in São Mateus, the assessment of the quality of these vegetables offered to the population was proposed. Data provided to the Municipal Sanitary Surveillance on the hygienic-sanitary conditions of these products will contribute to improve the quality of the vegetables consumed by the population.
MATERIAL AND METHODS
Study area
This study was conducted in the municipality of São Mateus, northern region of the state of Espírito Santo, 220km from the capital, Vitória 19. According to the Instituto Brasileiro de Geografia e Estatística (IBGE by its acronym in Portuguese) 20, the town has an estimated population of 124 575 people.
Sample Collection
A sample unit was defined as 50g of leaves obtained from a "head" of crisp lettuce (Lactuca sativa)11, and 8g of leaves obtained from a "bunch" of parsley (Petrosolium sativum), regardless of the weight or size of the "head" or "bunch". A total of 120 samples were analyzed.
Each sample unit was selected randomly, as they were accessible to consumers in the stores where they were purchased.
Collection procedure
All samples (120) were collected from six popular commercial establishments in São Mateus, referred to in this study as 1, 2, 3, 4, 5 and 6. Samples 1 and 6 were characterized as street markets and the remaining as supermarkets. 10 lettuce samples and 10 parsley samples were collected from each establishment.
In both supermarkets and street markets, the samples, once selected, were immediately wrapped (individually) in first-use plastic containers and sent to the Clinical Analysis Laboratory of the Federal University of Espírito Santo, São Mateus Campus, for processing and analysis.
Sample Analysis
In the laboratory, about 50g of leaves suitable for consumption were extracted from each lettuce "head" 11, disregarding burnt and damaged leaves, as well as the stem. For parsley, 8g of leaves from each "bunch" were extracted, also disregarding the parts inappropriate for consumption.
After defoliation and weighing, the sample unit was transferred to a plastic tube, where it was washed with a volume of distilled water of 200 mL for parsley and 400 mL for lettuce. During washing, the leaves were rubbed with the fingers, using latex gloves, and then suspended and agitated so that the liquid present on the surface was drained as deep as possible into the tube. After the procedure, the leaves were discarded and, for each new sample, a new pair of gloves was used 5.
Subsequently, the solutions were filtered to a 200-mL conical cup, through surgical gauze folded in four folds. After filtration, the liquid was kept at rest for a period of 24 hours, according to the spontaneous sedimentation method 21. Subsequently, the obtained precipitate was collected and transferred to 50-mL centrifuge tubes and then centrifuged at 4 000 rpm for one minute. After centrifugation, the obtained precipitate was collected by depositing a drop on a glass slide. It was stained with lugol; the use of cover slips was optional. At least three slides of each sample were examined under a light microscope with objective lenses of 10x and/or 40X.
RESULTS
From a total of 120 analyzed samples, 71 (59 %) had one or more parasitic contaminants, and 49 (41 %) were negative. From this total, 60 samples corresponded to lettuce, and 47 (78.3 %) were positive. Among the 47 positive samples, 26 (43.3 %) had polyparasitism (Figure 1).
Figure 1 Parasitism frequency in lettuce and parsley samples from the São Mateus municipality, Espírito Santo State, Brazil
In relation to the 60 parsley samples, 24 (40 %) showed positive results. From the total positive samples, 6 (10 %) had more than one parasite per sample (Figure 1).
There was no statistically significant difference between the results of the presence of parasites in lettuce and parsley. The contamination rate was higher in lettuce than in parsley (p<0.0001), as well as polyparasitism (p=0.0295).
When collection facilities were evaluated, it was observed that, in street markets, 14 (70 %) of 20 lettuce samples and 8 (40 %) of 20 parsley samples were positive. On the other hand, lettuce and parsley samples from supermarkets showed 33 (82.5 %) and 16 (40 %) positive samples, respectively, from a total of 40 analyzed samples (Figure 2). There was no significant difference between establishments and lettuce and parsley samples (p=0.9726).
Figure 2 Frequency of parasitism in lettuce and parsley samples by type of commercial establishments from the São Mateus municipality, Espírito Santo State, Brazil
When the results obtained in each of the evaluated establishments (Figure 3) were compared, it was observed that establishment 6 (street market) showed the highest positive rates for both parsley and lettuce. The second establishment with the largest number of positive samples for both vegetables was number 3 (supermarket), followed by number 5 which, despite having the same number of contaminated parsley samples as establishment 4, presented a higher total contamination due to the large amount of lettuce samples with positive results. Establishment 2 showed a frequency of positive results four times higher for lettuce when compared to parsley, and establishment 1 (street market) had the lowest contamination rates. Although the number of contaminated parsley samples was the same as in establishment 2; positive results for lettuce were two times lower.
Figure 3 Number of contaminated samples of lettuce and parsley from commercial establishments in the municipality of São Mateus, Espírito Santo State, Brazil
When considering each establishment separately, a statistically significant difference was found in relation to positive or negative samples (p=0.0073). It was observed that establishment 1 had the lowest frequency of contaminated samples when compared to other establishments.
Regarding parasitic contaminants in lettuce samples, it was observed that Ancylostomatidae larvae (61.7 %) were the most frequent, followed by F. hepatica eggs (41.7 %) and mites (11.7 %). Other parasites were also found in lower amounts (Table 1).
Table 1 Relationship between parasites found in lettuce (Lactuca sativa) samples, number of positive samples and frequency of contamination in the city of São Mateus, ES
| Parasites | N° of positive | % |
| samples | ||
| Ancylostomatidae | 37 | 61.7 |
| Ascaris lumbricoides | 5.0 | |
| Dipylidium caninum | 1 | 1.7 |
| Enterobius vermicularis | 1.7 | |
| Fasciola hepatica | 25 | 41.7 |
| Hymenolepis nana | 1.7 | |
| Oxyurida | 3 | 5.0 |
| Strongyloides stercoralis | 3.3 | |
| Toxocara cati | 1 | 3.3 |
| Toxocara vitulorum | 1.7 | |
| Trichuris sp. | 2 | 1.7 |
| Balantidium coli | 1.7 | |
| Entamoeba coli | 3 | 5.0 |
| Entamoeba histolytica/dispar | 1.7 | |
| Giardia lamblia | 1 | 1.7 |
| Mites | 7 | 11.7 |
For parsley samples, nine different parasitic structures were identified and parasites of the family Ancylostomatidae were the most frequent (35 %), followed by mites and F. hepatica eggs, both 5 % positive. The other structures had a frequency of 3.3 % and 1.7 % (Table 2).
DISCUSSION
Parasitic infection rates in a given region are frequently deemed as indicators of socioeconomic development. In order to attempt to identify possible means of transmission and frequency of enteroparasites in vegetables consumed by the population of the municipality of São Mateus, a central city of the northern region of the state, 120 samples were analyzed (60 lettuce "heads" and 60 parsley "bunches"), which yielded 78.3 % and 43.3 % positive results, respectively. These results are similar to those found in studies conducted in supermarkets and street markets of Recife, where the index of positive samples reflects the importance of the transmission of parasitosis to people through food 5.
Similarly, Vollkopf et al.2 studied the occurrence of enteroparasites in lettuce sold in Porto Murtinho-MS, and obtained positive results for 91.5 % of the samples, which suggests poor sanitary conditions in the trade of that food. The same can be observed with the results obtained in a study conducted in the city of Cuiabá 22, where 66.7 % of crisp lettuce samples had some kind of parasitological contamination.
Furthermore, Guilherme et al.23 and Mesquita et al.24 studied vegetables in a street market from a producer in Maringá, Paraná, and supermarkets, street markets and grocery stores in the cities of Niterói and Rio de Janeiro. They obtained contamination rates in lettuce of 23.2 % and 3.9 %, respectively, which is considerably better when compared to the results observed in São Mateus, probably due to better growing conditions and manipulation.
When comparing rates of vegetable contamination, it was observed that the index of positive samples for parasites and parasitic species in lettuce in São Mateus was higher than for parsley, as reported in studies conducted in the states of Paraná and São Paulo 14,23. Since lettuce has large leaves with a much higher contact surface than parsley, its physical structure can interfere with contamination rates. 14 Another factor to be borne in mind is that parsley is considered a repellent plant and can ward off insects and other animals, including nematode larvae 25.
In relation to contamination rates of samples from supermarkets and street markets, there was no significant difference among establishments (p=0.9726). Nevertheless, studies conducted in supermarkets of Cuiabá, Mato Grosso state 22, reported that the contamination process of vegetables probably occurs due to poor hygienic-sanitary conditions during the production chain and marketing. The same was observed by Takayanagui et al.26, who reported that the contamination of vegetables may occur in the vegetable garden itself, as a result of inadequate irrigation water or fertilizers, during transport or handling at points of sale, since successive handling increases the chances of contamination.
An enteroparasites frequency of 78.3 % in lettuce and 43.3 % in parsley in São Mateus is worrisome, taking into account the observed parasitic species, as well as their amount. Among the parasites observed in this study, four helminth eggs of the genus Ascaris stand out, one of which was identified as A. lumbricoides. It is known that the eggs of this parasite have a great capacity of adherence to surfaces 27 and, therefore, it is possible to admit the possibility that more eggs, which were not removed during the washing process, could be found in the vegetables.
Other enteroparasites found in the study were Ancylostomatidae, whose contamination levels in the samples reached 61.7 %. It is noteworthy that most of the findings were represented by larvae, and that hookworm infection in humans occurs only when L3 larvae (infective or filarioid) penetrate actively through the skin, or passively through the mouth 18. Although the larvae found were not differentiated in relation to their stage, the fact that they are present in large amounts and frequenty still alive suggests that ingestion, or even the simple handling of these vegetables, can lead to contamination of the host and to the development of hookworm disease 18.
It is also important to note that F. hepatica eggs, although not directly infectious to man since the transmission of fascioliasis occurs through the ingestion of water and vegetables contaminated with metacercariae 18, show contamination by ruminant feces, probably used in fertilization 14. Similarly, Toxocara sp. eggs in lettuce samples indicate contamination of vegetables with feces of canids or felids, which can result in manifestations of visceral Larva migrans syndrome, including severe pulmonary, ocular and neurological complications 26.
Other species of helminths and protozoa of clinical importance included G. lamblia, E. histolytica/E. dispar, Tricuris sp. and E. vermicularis, which are the parasites that most frequently cause human contamination. Individuals with immune disorders, children and the elderly are most at risk for parasitic and opportunistic diseases, such as Giardia sp. and Entamoeba sp 28.
The consumption of fresh vegetables enables the exposure of a large portion of the population to transmissible forms of parasites. As a result, to avoid contamination, proper hygiene of vegetables is the first step to reduce the transmission of intestinal parasites 28,29. In consequence, it is recommended to soak the vegetables for 15 minutes in water containing hypochlorite (1 tablespoon for 1 liter of water) before they are cut 30.
From a hygienic-sanitary point of view, the results of this study may indicate lack of knowledge on the subject by the communities 31, as well as inadequate practices in cultivation, transportation, storage or trade of vegetables, as observed by Oliveira & Pinto 32, who reported that food contamination is caused mainly by poor water quality, lack of control of pests and vectors, lack of adequacy and/or precariousness of production facilities, transportation and marketing, in addition to inadequate cleaning of equipment, utensils and food handlers themselves.
Thus, considering that the results of this study showed that the vegetables lettuce (Lactuca sativa) and parsley (Petrosolium sativum) sold in supermarkets and street markets in the city of São Mateus had high levels of contamination by parasites, a more effective monitoring by the competent authorities is expected in order to ensure quality in the food that is offered to the population 
