1. Introduction

Intestinal parasites are a major public health problem in developing countries. Many parasites are responsible for these infections. Some, like E. histolytica, E. dispar, Giardia lamblia, Hymenolepis nana, Taenia solium, Ascaris lumbricoides, Trichuris trichiura, Cryptosporidium parvum and Enterobius vermicularis are transmitted by contaminated food or water ^[1]. They are also strongly associated with poor sanitation, hygiene, living and housing conditions, affecting not only the health of the individual but also the workforce thus having an impact in generating the country's wealth.

In literature, reports available on the prevalence of intestinal parasites in Brazil are scarce and very circumstantial because they generally consider specific populations (school, children, elderly or immunocompromised patients) and only reflect the reality of small towns. Thus, one does not know the prevalence of these parasites at the national level ^[2].

In relation to environmental conditions, underdeveloped countries are at higher risk of contamination. However, it is not a sine qua non. An example is Brazil, where, despite favorable conditions for E. histolytica, E. dispar predominates in most states. In a study by ^[3] on the prevalence of intestinal parasites in Fortaleza in two periods: before (1992-1996) and after the implementation of health interventions (2010), it was observed that improvements in health conditions in the area were reflected in a sharp change in the prevalence of intestinal parasites in the assessed community.

Several epidemiological surveys have been conducted to estimate the incidence and prevalence of amebiasis in Brazil. The Northern region stands out with the highest rates, with Belém-PA having the highest prevalence of E. histolytica with 29.5% of the resident population in the metropolitan area being infected ^[4].

Considering the endemicity of parasitic infection in the northern region compared to other regions of Brazil, with similar characteristics: lack of sanitation infrastructure and exposure to poor socioeconomic conditions, would the eating habits of individuals be considered a key risk factor for intestinal parasites? The possible association of dietary habits, environmental exposure and hygienic-sanitary factors to the prevalence of intestinal parasites in the region was investigated.

2. Materials and Methods

The study consisted of 318 patients treated at the Unified Medical Care Sector (SOAMU) of the Evandro Chagas Institute/MS (IEC) and the João de Barros Barreto University Hospital/UFPA (HUJBB), randomly invited to participate in the study. Patients were residents in Belém-PA and/or the metropolitan area, consisting of Belém, Ananindeua, Marituba, Benevides, Santa Barbara and Santa Izabel (Legislative Assembly of Pará 1995 and 2010). The material was collected in the months of March, April and July 2013, in both locations.

This research was approved by the Research Ethics Committee (COEP) of the Federal University of Minas Gerais. Before the collection, an interview was carried out, where the individual was presented with an Informed Term of Consent (TCLE) and a detailed explanation of the research objective.

A questionnaire was applied about the hygiene profile - sanitary and food, blood was collected and stool requested from the participants.

The results of parasitological and serological tests were sent to the participants about 30 days after collection, the positive cases were referred to medical care in the collaborating institution.

A semi structured questionnaire was applied in order to evaluate a possible relation between hygienic-sanitary and dietary factors and seropositivity for invasive amebiasis in the participating study subjects.

The sanitary-dietary investigation presents approaches on the individual's profile regarding living conditions, hygiene and nutrition, which can provide information related to risk factors for incidence of infectious diseases via oral-fecal contamination.

With the sanitary-dietary investigation, information on the following variables was obtained: demographics (sex), eating habits (type of diet, alcohol intake, food hygiene), factors related to housing (type of construction, water quality, access to sanitation, garbage disposal), factors related to water distribution and wastewater treatment.

Afterward, participants were advised on preventive measures to avoid infection by intestinal parasites.

Fecal material was collected in pots made available by the institution and frozen at -20°C on reception, without adding preservatives. The samples were then sent to the Laboratory of amoebiasis and Intestinal Parasites (LAPI) in the Institute of Biological Sciences (ICB) at UFMG.

Upon analysis, the sample was thawed and concentrated by centrifugation in formol-ether for microscopic observation of pellets, and then stained with Lugol for intestinal parasites research.

For the diagnosis of infection by E. histolytica/E. dispar, optical microscopy, has been historically chosen, despite its inability to differentiate the two forms.

In this study, the ELISA technique was used, according to a protocol defined by ^[5] modified and used as a routine method for amebiasis serology in the and in the Laboratory of Amebiasis and Intestinal Parasites (LAPI / ICB / UFMG).

Venous blood (5ml) was collected in vacuum tubes from each individual without an anticoagulant. After clot formation, the serum was separated, aliquotted in plastic tubes and stored in a freezer at -20°C.

The material was transported covered in artificial ice, inside sealed coolers for to the Laboratory of amoebiasis and Intestinal Parasites diseases at the ICB Institute of UFMG where the technique was performed.

The presence of anti-E. histolytica antibodies was detected in the sera of the participants by means of the ELISA reaction.

Plates were coated with antigens obtained from axenic strains and cutoff point was determined for the community under study, using negative sera as controls.

The confirmation of E. histolytica infection is an invasive indicator of intestinal amoebiasis and/or extra intestinal amebiasis, with hepatic abscesses being among the most frequent extra-intestinal forms ^[6].

Absolute frequency was calculated individually for the questionnaire results, the Parasitological Stool Examination (EPF) and serology, for a clearer and independent view of the data. The SPSS program (Statistical Package for Social Science - Version 22.0) was then used for association between the results obtained in each section.

Initially, the average, median, quartiles, minimum, maximum and standard deviation were used for the identification of the main characteristics of the participants. For comparison between groups, the comparison test of two independent groups t-Student was used.

In the analysis of normality, the Kolmogorov-Smirnoff test was used, finding that the variable “percentage of ancestry” did not have normal distribution. Thus, when comparing the groups, the continuous variables were compared using the Mann-Whitney U-test and Kruskal-Wallis non-parametric ANOVA.

In analyzing the normality of continuous variables, the Kolmogorov-Smirnoff test was used. Categorical variables were described as proportion and percentages, and continuous variables as mean ± SD, or median with interquartile range (25-75%). When comparing the groups, Student's t-test was used for continuous parametric variables, and U-Mann-Whitney test for the non-parametric.

To Compare the categorical variables, the chi-square test or Fisher's exact test were used. The non-parametric Kruskal-Wallis ANOVA was also used to compare multiple groups, followed by the Dunn test. Differences with p <0.05 were considered statistically significant at the 95% confidence interval.

3. Results

Analyses were performed on the stool of 195 individuals who provided the material. The parasite with the highest frequency was Blastocystis sp (25.6%), followed by E. histolytica / E. dispar (9.7%) and Endolimax nana (8.2%). The table below (Table 1) shows the distribution of parasites in the tests and the relation to water supply and sewage services.

Table 1. Association of different parasites with the water supply and sewage service

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In serology, a high positivity index was observed in the ELISA test, at a dilution of 1:100 in serum samples and 2 standard deviations. Of the 318 participants in the study, 140 patients (44.03%) were positive for anti E. histolytica antibodies, i.e., presented or present amoebic infection, since the detection of IgG may mean a returning or current infection.

The association between positive and negative serology with the results of stool testing are shown in Table 2.

Table 2. Prevalence of parasite species and serology for E. histolytica

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The objective of this survey from the collected data was to identify environmental factors, health and eating habits that could contribute to the incidence and aggravation of infectious diseases with an oral-fecal contamination route.

The survey covered the following topics: sex, quality of water consumed, exposure to environmental risk factors, personal hygiene, food quality, consumption of alcohol.

The survey had 43.7% male individuals and 56.3% females.

Regarding the water supply available to the residences, most have access to the general network (55.7%). 44.7% of participants consumed well water and 0.9% reported that they used water from other sources such as river, streams, etc.

Considering the high percentage of users of non potable water, participants were asked about the care of the water consumed at home. Among the respondents, 29.9% filtered water before consumption, 0.9% do chlorination in their own residence, none of the respondents claimed to use the boiling process (0%) and most (69.5%) reported consuming water just the way it reached the household, without further treatment.

Among the individuals who chose “not do any treatment” to the water consumed in their household, there were many reports of mineral water use, however, in the absence of resources to acquire this, they consumed any water available in the home regardless of the origin.

In 54 (17.0%) households, the general sewage network was the target of sanitary wastewater, while the septic tank was present at the residence of 236 respondents (74.2%), the answer "other" was informed by 1 participant, and 6.9% of households had no sewage at all.

Garbage Destination

The conditions of allocation of the waste produced in the home were assessed. The reported data showed that in 94.7% of households, garbage is collected frequently, in 12 households garbage is burnt/buried (3.8%) and 6 residents leave it out in the open (1.6% ).

Type of Housing Construction

The housing conditions of the participants: type of construction, number of rooms and number of residents were assessed. For the last two questions there were no defined options for answers, the categories were prepared from the data obtained. Thus, the number of rooms mentioned ranged from 1 to 10 and the amount of people in each residence from 1 to 13.

Most respondents (78.9%) lived in brick houses. Two respondents said they live in wattle and daub houses (0.6%), 48 people said they live in wooden houses (15.1%) and 18 (5.7%) on stilts.

As for the rooms of the houses, the answers ranged from 1 to 10 rooms, the majority of participants lived in dwellings with four (23.6%) or 5 (22.3%) rooms (Figure 1). The number of residents in each household ranged from 1 to 13, with 4 people per household being more frequent (Figure 1b).

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Figure 1. (a) number of rooms per residence; (B) Number of individuals per residence

Looking to determine the dietary routine of participants, it was found that rice (83.6%), flour (58.9%), beef (38.1%), chicken (28.9%), beans (27.7%), fish (25.5%) and açaí (23.0%) were the most cited and only three participants said they eat shrimp (0.9%).

The frequency in the preferred food consumption revealed that the vast majority (95.3%) of participants consumed their food of “preference” daily.

Seeking to set the frequency of ingestion of raw foods, it was found that 45.3% of participants had this eating habit. Among the individuals who consume raw food, the choice of vegetables was the most frequent (51.9%), followed by legumes (31.8%) and fruits (34.0%). Regarding the intake frequency, 17.3% of respondents said they consume raw foods daily.

Most of the study participants (99.4%) do not grow food at home, just 2 respondents (0.6%) reported the habit of producing vegetable gardens or orchards. These said they lived in coastal communities in municipalities in the metropolitan region of Belém-PA.

Participants who used raw foods were asked about the form of cleaning them. The majority (51.3%) just washed the food in tap water, whereas 46 subjects claimed they sanitized food with vinegar (14.5%), 31 using hypochlorite (9.7%) and 6 cleaned with filtered water (1.9%).

Regarding the intake of high nutritional value protein, the consumption of meat and milk were considered. Most participants responded that they eat meat daily (33.6%), followed by 28% of participants who said to eat meat three times a week and 15.7% of participants who consumed meat two times a day. In a smaller quantity, a once a week (7.9%) consumption was also reported and a weekly consumption of 4 times (5.0%), while participants who said they did not eat meat and those who eat five times per week had the same percentage (4.7%) and finally 0.9% of participants reported eating meat rarely.

With regard to drinking milk, 247 participants (77.7%) reported drinking it every day, followed by 49 participants (15.4%) who do not have the habit of consuming milk. In lower numbers, 2.5% of the participants reported that they rarely consumed milk, while 1.9% consumed three times per week and 1.3% consumed twice or once a week.

Whereas excessive consumption of alcohol leads to intestinal infections, frequency of consumption was investigated. Among the participants, 18.6% said they were consumers, with the majority (11.6%) drinking once a week on average and 6.9% said they consumed twice a week.

Association serology for E. histolytica, with water, sanitation and housing, number of rooms and residents in dwellings (Table 3, Table 4 and Table 5):

Table 3. Relation of E. histolytica serology with water, sanitation and housing

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Table 4. Relation of E. histolytica serology with the number of rooms in housing

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Table 5. Relation of E. histolytica serology with the number of residents per household

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4. Discussion

For contextualization of the reality of Belém and the risks of getting infection by intestinal parasites, positioning becomes necessary. The city of Belém is intersected by Guamá and Acara rivers, which meet in Guajará Bay forming many islands, with high rainfall, which puts the region in a distinct situation of environmental risk and health. One of the main means of transport is the river and the forest is still very present in the landscape of the region.

In feeding, native fruits like açai and peach palm become more scarce every year due to the low sustainability of production and high cost of marketing. Since there is still availability of fish due to geographic site structure, this is still a preferably eaten food with cassava flour and/or açai ^[7].

In the responses to the questionnaire asked during the interview, there were many spontaneous reports on the occurrence of parasitic infections in several episodes throughout the life of individuals. In their childhood, many lived in riverside communities or even used the waters of the rivers and springs for consumption and leisure, and in the report they used painful expressions to express episodes of colitis and diarrhea experienced.

However, we found no association between any of the analyzed factors (gender, quality of water consumed, exposure to environmental risk factors, quality of food and alcohol drinking) with the presence of intestinal parasites. Similar results were found by ^[8], who found high prevalence of intestinal parasites in communities on the outskirts of Belo Horizonte, finding no association with environmental factors and health structure. These results suggest that individual behaviors could be related to higher transmission of intestinal parasites in the two regions.

The poor socioeconomic conditions of the studied region, and the lack of sanitation in most locations, exposes people to greater risk of contracting infections via oral-fecal route. In the northern region of Brazil, the occurrence of intestinal parasites is high, especially in the riverine population, area of greatest deficiency of basic sanitation. The results of ^[9] showed positivity in 94.5% of the Parasitological stool examination (EPF), finding Entamoeba sp. (36.26%) as the most frequent among the protozoa.

Among the listed individuals, 195 provided material for the stool test. In 40% of the samples evaluated in the EPF, some kind of parasite was found. The complex E. histolytica / E. dispar showed 9.7% positivity. Exceptional numbers for Brazil, nevertheless considered low compared to the numbers found in literature for this region ^[4].

According to ^[10] the time between the evacuation of feces and the transport to the laboratory may influence the result, the ideal is that research take place in three stool samples on alternate periods of up to ten days, thereby increasing security of operations in enteroparasite research as it contemplates the intermittence in their life cycle.

In Brazil, the geographical distribution of amebiasis is diverse, communities located in the Southeast and Northeast with a reduced number of individuals infected with E. histolytica / dispar in relation to the North.

The reference ^[11] found E. histolytica / E. dispar in 34 samples (2.0%) in a study in Rio de Janeiro. In a research conducted in Alagoas in 68 (3.8%) fecal samples analyzed by optical microscopy, the presence of complex E. histolytica /E. dispar was detected ^[12].

The reference ^[8] evaluated the prevalence of intestinal parasites in communities on the outskirts of Belo Horizonte, and identified 3.5% infected with the complex.

The multiparasitism situation is common among the patients in this study, and other parasites were also found. The data indicate a prevalence of Blastocystis hominis (25.6%) and Endolimax nana (8.2%).

The reference ^[13] showed significant association between E. histolytica / E. dispar and B. hominis, from 34 cases positive for E. histolytica / E. dispar, 20 cases (58.8%) also had B. hominis, indicating a possible influence of one on the pathogenicity of the other, as occurs in relation to the bacteria of the intestinal flora.

Respondents mostly consumed water from the water supply system (55.7%), however, there is a significant percentage of individuals who use well water (44.7%), when asked about the fact, these justified that the quality of water reaching the taps was the main reason for the choice. Given that the water coming out of the central supply is of good quality, possibly the contamination occurs along the path leading to the household tap, this could be by inadequate pipes (old, damaged) and / or the reservoir of the houses.

People who said they use water from rivers and streams for drinking without any treatment were categorized as other (0.9%), this figure is considered low compared to the total sample, this data is attributed to the fact that most respondents reside in urban areas, different profile of coastal communities.

During the interview several people said they use bottled water for drinking, however, as financial resources are often limited, they end up alternating with tap water. This statement was fairly frequent and exposure to the risk factor associated with low health education is observed in this statement, as the interviewee does not know the importance of this practice in getting diseases. These participants were then instructed, at the end of the interview, to consume filtered water, since it would solve the issue of quality of drinking water and make consumption of mineral water unnecessary, saving money for the family, this water treatment method is used by 29.9%, who said they filter water before consumption.

Participants who did chlorination in their own homes lived in riverside communities without access to the water supply network and were instructed to use this method by health workers who visit their homes.

The ingestion of water or food contaminated with waste is an important risk factor for the occurrence of parasitic infections ^[14], most of the subjects in our study (69.5%) did not do any treatment in the water consumed in their homes. Despite this, we found no correlation between water consumption in general and a higher risk of parasitic infection. Perhaps this result relates indifference on water quality from the supply network and artesian wells, which, incidentally, consisted of almost half of the respondents.

We know that it is not the water supply only, but also the sewage network is a base for improving the quality of life and increase human development index in a region. Infectious diseases are the main obstacles for the development of regions that do not have the benefits of Sanitary Engineering. In Belém, the majority of respondents (74.2%) said they use septic tanks at their home. Some respondents reported that despite the use of septic tanks or bathroom, the waste produced by residents is distributed in the vicinity of their homes. Considering the high rainfall in the region, the spread of infected waste becomes reality. This behavior of respondents might relate to indifference in the prevalence of intestinal parasites among the recipients of the sewage network and by those who use the septic tank, and we consider that much of the population under study did not have sanitation.

The type of housing was evaluated to idealize the financial condition of the respondents, since we obtained material from individuals of Belém. The type of housing, number of rooms and people in each housing led us to conclude that we worked with a population of low income.

The food consumption patterns depend on factors such as socioeconomic and demographic, cultural and historical aspects of life of individuals ^[15]. So we try to assess the possibility of differences in the menu of the North relate to higher prevalence of amebiasis.

There was a variety of dishes mentioned (31 kinds), with the most common being rice (83.6%), flour (58.8%), beef (38.1%), chicken (28.9%), fish (25.5%), bean (27.7%) and acai (23.0%). We did not identify any different food consumed in other areas that could be related to greater predisposition to amebiasis. We actually did not verify differences in the consumption of raw foods (about 50% of respondents consume them), which can enhance the spread of intestinal parasites, mainly due to poor hygiene of the same.

Most respondents (51.3%) used only tap water for cleaning, similar to what is observed in other regions ^{[8, 16]}.

Most cited food eaten raw were tomato and lettuce. A report by ^[9] shows the presence of parasites in all evaluated lettuce samples. The most frequent were Entamoeba coli (67%), E. histolytica (20%), Giardia sp (13%) and Ascaris lumbricoides (7%).

Protein malnutrition can be an important predisposing factor for acquiring infections ^[17]. We evaluated the intake of protein of high nutritional value by participants through milk and meat consumption. We also assessed alcohol intake as predisposition factor for malnutrition.

Most people said they consumed meat and milk with great frequency. Alcohol consumption remained reasonable (18.6%) with no exacerbations that culminated in danger to health. So our study population did not present nutritional deficiencies.

Our results corroborate a study by ^[15] who found a reduced frequency of consumption of vegetables, fruits and vegetables and high intake of meat, sausages, milk and dairy products in more than 1/3 of the population studied.

Serology for amebiasis was an important analysis in this work. The amebiasis, infection by E. histolytica, can happen across the globe, but symptomatic infections are more common in certain regions. The asymptomatic amoebiasis produces no detectable antibody response in conventional immunoassay. The opposite is observed for symptomatic cases, where there is invasion of intestinal and extra-intestinal tissues by the amoebae with positive serology in almost all cases in unknown time, even after treatment [18-23]^[18]. Considering the above, we can conclude that the positive serology is a marker of severity and prevalence of infection in the region.

The Northern region of Brazil was chosen to carry out the work to maximize the possibility of finding people with the disease. However, diagnosis in this region is usually presumptive. Individuals showing symptoms of severe hepatomegaly and dysenteric colitis are treated for amoebiasis. So it was hard to find anyone with the disease currently. Many of our respondents reported being treated for amoebiasis (the disease is well known in the region). In this clash, serology has become a tool to identify amebiasis whether invasive, active or returning.

There is a small number of research in the region studied using the ELISA method for serology in the amoeba research, the data in the literature only shows values in the EPF, coproantigen and PCR. The method used in an investigation may underestimate or overestimate the prevalence of the disease. The ELISA has high sensitivity and specificity in the diagnosis of amebiasis ^[24].

Immunological and molecular methods have better sensitivity and specificity for optical microscopy. The detection of antibodies by the ELISA test is one of the most used tools in the diagnosis of invasive extra intestinal amebiasis, such as amebic liver abscess, with 90% sensitivity. In the invasive intestinal forms, the sensitivity is around 70% for anti E. histolytica antibodies in serum ^{[25, 26]}.

The positive serology for amebiasis may indicate previous infection, since, the immunoglobulin G (IgG) for E. histolytica detected by the method can be found in the individual years after being infected (Santos and Smith 2008). However, high titers of IgG, are observed only at the beginning of the infection a few weeks of the onset of symptoms of amoebic colitis and amebic liver abscess ^{[25, 27]}.

The results found in the serology of the 318 participants in this study indicated a high frequency (44.03%) of amebiasis in the studied community. However, this method is not routinely used in the diagnosis because of the cost associated with under-reporting of the disease in the region, resulting in no documented evidence which may be used as a parameter for controlling the strategic management of amoebiasis.

To identify E. histolytica in positive samples for the complex in the stool test, we carried out the specific test for E. histolytica coproantigen.

Participants who tested positive also had their stool processed for the specific enzyme immunoassay for identifying E. histolytica. None of the 174 samples analyzed was positive. Possibly due to low levels of antigens present in the samples. According to ^[28], high levels of E. histolytica antigen are required for detection, suggesting an inability of this kit to detect low numbers of parasites in samples, especially in asymptomatic individuals in endemic areas, which may be infected with both types of amoebae.

5. Conclusion

Historically it is considered that eating habits can be considered a determining risk factor for intestinal parasites, the data obtained in this study indicate that this association was not significant.

High prevalence of intestinal parasites and the complex E. histolytica / E. dispar is observed in this population compared to other regions of Brazil.

By identifying symptomatic amoebiasis (active or not), detected by serology, this proved to be very prevalent in the population sample, corroborating descriptions of respondents and the expectation for the region.

Dietary factors, environmental and health were not associated with parasitic infections and even serology for amebiasis, signaling a re-reading of this relationship.

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