PREVALENCE OF ENTEROPARASITES IN RURAL AND URBAN CHILDREN IN THE MUNICIPALITIES OF BOCAIÚVA DO SUL AND COLOMBO

The aim of this study was to diagnose enteroparasites and to relate them to socioeconomic factors in schoolchildren from the public system in the municipalities of Colombo and Bocaiúva do Sul, located in the Metropolitan Region from the city of Curitiba, Brazil. Fecal samples were processed using the modified Ritchie and Ziehl-Neelsen staining methods. Analysis of the risk factors, using Tukey test and Odds ratio (OR), was obtained by connecting questionnaires with parasitological results. Of the 414 schoolchildren analyzed, 32.4% (134) were positive for at least one enteroparasite species. The most prevalent were Blastocystis spp. (21.7%), Entamoeba coli (5.5%), Endolimax nana (5.5%), Giardia duodenalis (3.9%), Trichuris trichiura (3.7%) and Ascaris lumbricoides (2.9%). Schoolchildren from rural areas had a higher occurrence of enteric parasites (p<0.05), showing that is necessary to continue with health education, being careful with the food, basic sanitation and parasitological examination of feces before treatment.


INTRODUCTION
Infections caused by enteroparasites are one of the most prevalent parasitic diseases in tropical and subtropical countries, being an important public health problem and a global socioeconomic concern (WHO, 2017).It is estimated that 3.5 billion people are affected by enteroparasites and, of these, 450 million have clinical manifestations (WHO, 2013).Approximately 5.18 million deaths annually are caused by infections attributed to intestinal parasites, where helminths (Ascaris lumbricoides, Trichuris trichiura, Necator americanus and Ancylostoma duodenale) and protozoa (Cryptosporidium sp. and Entamoeba histolytica/ dispar/ moshkovskii complex) are considered to cause deaths (Lozano et al., 2012;PAHO, 2018).
This group of infectious agents are part of the neglected tropical diseases.There is currently a list of priority diseases (human African trypanosomiasis, onchocerciasis, scabies, schistosomiasis and visceral leishmaniasis) for the WHO, directing research for "Paediatric Drug Optimization" (PADO), aiming to meet the specific needs for babies and children (WHO, 2023).
Among the main risk factors associated with parasitic infections are the lack of basic sanitation, deficient socioeconomic and educational characteristics, crowding, consumption of untreated water and food contamination (Nobre et al., 2013;Rodrigues et al., 2020).The enteroparasites prevalence in the Brazilian nation is underestimated due to the lack of studies in many regions of the country also because it is not compulsory to report them.In the State of Paraná, previous studies have shown parasites infection ranging from 2 to 24.8% (Freckleton et al., 2019;Oishi et al., 2019).Although the Metropolitan Region of Curitiba (MRC) presents a Human Development Index (HDI) of 0.809, greater than other regions of Brazil such as the Metropolitan Region of Porto Alegre (HDI of 0.795), the presence of enteroparasites in the Metropolitan Region of Curitiba (MRC) schoolchildren (Ogliari & Passos 2002;Oishi et al., 2019) have the prevalence ranging from 24.8 to 89.7%, highlighting the presence of A. lumbricoides, T. trichiura, Entamoeba coli and Blastocystis sp.Thus, the purpose of this study was to diagnose enteroparasites in schoolchildren from public schools in the city of Colombo and Bocaiúva do Sul, located in the MRC, and to relate them to their socioeconomic and hygienic characteristics.

Study area
The study was carried out in two municipalities in the MRC in the State of Paraná.The city of Colombo is 17 km away from Curitiba, the State´s capital, with an estimated population of 243,726 inhabitants and the city of Bocaiúva do Sul is 40,6 km away from Curitiba, with an estimated population of 12,944 inhabitants (IBGE, 2017ab) (Figure 1).Two schools from Colombo, one urban (School I) and one rural (School II), and other two schools from Bocaiúva do Sul (School III and IV), have some children from urban, rural and from unknown origin, were included in our study.A total of 414 students were enrolled, 207 from Colombo and 207 from Bocaiúva do Sul.Only students between 1 and 10 years of age participated in our study, with an informed consent signed by their guardians.In addition, guardians answered a closed structured questionnaire with socioeconomic and hygienic aspects to assess risk factors for enteroparasites.

Approval of the ethics committee
The research was approved by the Research Ethics Committee from the Federal University of Paraná (CAAE 91542618.0.0000.0102).

Sample collection and processing methods
Fresh morning fecal samples were transported in a refrigerated Styrofoam box to the Laboratory of Parasitology at the Federal University of Paraná and classified according to the Bristol scale.For the fecal analysis the modified Ritchie technique (Knight et al., 1976) was performed, changing the use of ether to ethyl acetate.The observation was carried out in triplicate.To detect Cryptosporidium spp.and other coccidian protozoa, the modified Ziehl-Neelsen method was applied.A Leitz Dialux 22 optical microscope was used.Measurements were carried out in micrometers (μm) and were taken with a Zeiss ocular micrometer calibrated using a standard Zeiss scale.The conversion of the micrometers measurements was as follows: objective (6.3×)= 18.5 µm, (16×)= 7.3 µm, (25×)= 4.65 µm and (40×)= 2.9 µm to access the structure's size.All samples were analyzed in triplicate.

Statistical analysis
The data obtained were tabulated in the Excel ® 2016 program and analyzed both by descriptive statistics and by inferential statistics using the R. Studio ® version 3.6.2.The information provided through prevalence calculations made it possible to establish population risk statistics (Odds-Ratio) between children who tested positive for enteroparasites and children who tested negative, describing the relationships between a set of variables making it possible to characterize a group based on its positive or negative sample, using the chi-square test or Fisher's test when necessary.All inferences were accompanied by a 95% confidence interval.The tabulated data were transformed into logistic regression and a multivariate analysis (ANOVA) was performed, to verify multifactorial relationships.
In the Table 3 results from urban and rural environments infection is shown.In general, enteroparasites infection resulted more frequent between children from rural environments (50.7%) (68/134) rather than those from urban ones (40.3%) (54/134) with statistically significant differences (Tukey's test, p< 0.05).Blastocystis spp. was most observed in children from rural environments (38.1%) rather than in those from urban ones (23.1%) with significant statistical differences (Tukey's test, p=0.002).
Table 4 shows the relationships between infection results and different variables recorded, allowing the Odds-Ratio calculations.Although no statistically significant differences were observed in the overall infection among both municipalities studied, schoolchildren from rural environments result statistically 2.6 times more likely to became infected.Moreover, given the different schools studied, schoolchildren from school II (Rural-Colombo) present statistically 12.6 times more likely to present infection.The most affected age group was from 6 to 10 years old, with a statistical difference, showing a 1.8% chance of being affected by at least one parasite (Odds-Ratio and Tukey Test, p< 0.05).Amidst all the variables analyzed (medication, water source, place of evacuation, waste destination, sewage treatment and presence of animals) for those children who were positive for some form of parasite, no significant statistical differences were observed (Odds-Ratio and Tukey test, p< 0.05) (Table 5).When observing the socioeconomic data of both municipalities, children who had already taken some medication (Albendazole or Nitazoxanide) and/or natural treatment (mint tea) were 30% less likely to have a parasite (Tukey, p<0.05).It was noted that in Colombo 25.0% of the samples (5/20) that had a positive result had already taken Albendazole, 50.0%(1/2) had taken Nitazoxanide and 22.2% (4/18) mint tea.For Bocaiúva do Sul, mint tea was most used by the individuals sampled and parasitized, 8.7% (14/67), followed by Albendazole with 8.2 (13/48) and Nitazoxanide with 5.1 (8/ 31).

DISCUSSION
Overall enteroparasites prevalence detected (32.4%) was lower than that found in the coast of the State of Paraná (46.1%) (Seguí et al., 2018).However, results from Colombo (18.4%) are close to those from Campo do Tenente (24.8%) (Oishi et al., 2019).Protozoa prevalence resulted higher than that of helminths, similar to previous Brazilian study (Oishi et al., 2019).Blastocystis, considered as the most observed intestinal protozoan in humans (Coco, 2017), was the most prevalent species in both municipalities.
However, although the decrease in helminths prevalence detected may be due to health education and mass treatment programs (Guilherme et al., 2004;Oishi et al., 2019).some protozoa considered commensals for humans were detected in this study, such as E. coli, E. nana, E. hartmanni and I. bütschlii and they are indicators of failure in personal hygiene.Most infection was detected in those not symptomatic schoolchildren, with 68% having formed feces, according to Belloto et al., 2011.In the State of Amazonas, with indigenous populations, a parasitic association of 14 species was observed, with emphasis on A. lumbricoides, G. duodenalis and E. coli (Oliveira et al., 2016).However, we have only found single counts of four different parasite species associations, while the most prevalent way of infection was monoparasitism in both municipalities of MRC.Different enteroparasites species detected resulted similar in both municipalities studied.However, children from rural environments were more frequently infected than those from urban ones.A contradictory fact to highlight is the higher A. lumbricoides infection in urban rather than in rural environments.
No differences of infection between males and females were observed, other authors result detected a higher prevalence in females (Coco et al., 2017;Oishi et al., 2019).Children aged 6 to10 years, presented the highest prevalence and higher risk of infection, which corroborates previous research in the State of Minas Gerais, Brazil, where they observed protozoa, such as E. histolytica complex and E. coli, in children of that age-group (Belo et al., 2012).Previous studies showed that mass treatment with anthelmintic (Albendazole) reduced the prevalence of parasites (Machado et al., 1996) and the use of mint has a positive effect in the treatment of the protozoan G. duodenalis (Vidal et al., 2007).In that sense, the use of antiparasitic treatment is a habitual consumption, drugs mainly in Colombo and natural treatment mainly in Bocaiúva do Sul.However, many people take medicine without undergoing a previous laboratory diagnosis test (Cavagnolli et al., 2015).
Water consumption from the public network is responsible for the decline in the prevalence of parasitic species (Cavagnolli et al., 2015;Viana et al., 2017).The largest water supplier for the children's homes in both municipalities is the "Companhia de Saneamento do Paraná" (Sanepar) who supplies treated drinking water.Although the presence of animals such as pigs, chickens and cows in children's homes is considered as a risk factor for infection or reinfection of zoonoses (Zanetti et al., 2021), in the present study, it cannot be considered as a risk factor of infection in neither of the two municipalities studied.
In conclusion, this research is the first parasite prevalence study in schoolchildren from the municipalities of Colombo and Bocaiúva do Sul, where transmission of parasitic species was exposed, with emphasis on Blastocystis spp.Knowing the importance of the fecal-oral route of transmission, there is an urgent need to improve the public health policies for parasite control, prevention methods and improvement of social awareness.

Figure 1 .
Figure 1.Map of Brazil, the location of the State of Paraná and the municipalities of Colombo and Bocaiúva do Sul.

Table 1 .
Prevalence of enteroparasites in schoolchildren from Colombo and Bocaiúva do Sul municipalities (August 2017 to July 2019).

Table 2 .
Frequency of monoparasitism and multiparasitism detected in Colombo and Bocaiúva do Sul municipalities (August 2017 to July 2019).

Table 3 .
Frequencies of enteroparasites infection, in rural and urban environments from Colombo and Bocaiúva do Sul municipalities (August 2017 to July 2019).

Table 4 .
Infection results and characteristics of schoolchildren from Colombo and Bocaiúva do Sul municipalities, in relation to the frequencies of enteroparasites (August 2017 to July 2019).

Table 5 .
Odds-Ratio calculation between infection results and socio-economic and hygienic characteristics of schoolchildren from Colombo and Bocaiúva do Sul municipalities, Paraná (August 2017 to July 2019).