Prevalence of intestinal parasites and molecular characterization of Giardia intestinalis, Blastocystis spp. and Entamoeba histolytica in the village of Fortín Mbororé (Puerto Iguazú, Misiones, Argentina)

Background Intestinal parasites (IPs) are widely distributed worldwide and are one of the major contributors to gastrointestinal disease. Their prevalence is associated with poor access to water, sanitation and hygiene (WASH). The objective of this study was to identify the prevalence of IPs, including soil-transmitted helminths (STH), and their relation to socioeconomic characteristics, as well as a first approach to molecularly characterize the types of Giardia intestinalis, Blastocystis spp. and Entamoeba histolytica present in an indigenous community from Puerto Iguazú, Misiones, Argentina. Methods A cross-sectional study was conducted in the rural settlement of Fortin Mbororé between January and March 2018. Socioeconomic variables, household characteristics, and stool and blood samples were collected. Standard coprological techniques were used to analyze stool samples, and a complete hemogram was performed on the blood samples. Giardia intestinalis microscopy-positive samples were genetically typed by the β-giardin (bg) gene. Molecular identification of Blastocystis spp. subtypes and E. histolytica were carried out by amplification and sequencing of a partial fragment of the small subunit ribosomal RNA gene (SSU rDNA). Results The overall prevalence of IPs was 92.7%, with 72.0% specifically for hookworm. IPs were significantly more prevalent in preschool- and school-age children (P < 0.05). No formal education (P = 0.035), the presence of unimproved floors (P = 0.001) and overcrowding (P = 0.005) were significantly associated with IP infection. Hookworm was associated with anemia (P = 0.019). Molecular characterization revealed the presence of E. histolytica sub-assemblages AII (12.5%), AIII (87.5%) and BIV (100%); one case of sub-assemblage D for G. intestinalis; and the presence of subtypes ST1 (14.8%), ST2 (14.8%) and ST3 (70.4%) of Blastocystis spp. Conclusions Protozoans detected in this study are transmitted mainly through water contaminated with fecal matter, evidencing the need to improve the quality of water and sanitation for the inhabitants of Fortín Mbororé. Molecular characterization showed that domestic animals can be implicated in the zoonotic transmission of G. intestinalis and Blastocystis spp. to humans. A hyperendemic area for STH was found, with hookworm prevalence greater than 50%. Therefore, improvements in WASH as well as mass deworming programs need to be implemented in this area to control and decrease the prevalence of IPs in general and STH in particular. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1186/s13071-021-04968-z.


Background
Intestinal parasite infections (IPIs) are a global public health problem due to their high prevalence and worldwide distribution, especially in populations from tropical and subtropical areas of the developing world [1]. Although they can affect any age group, children are the most affected by the consequences of infection [1,2]. Some of the most important causal agents are protozoans (e.g. Entamoeba histolytica, Giardia intestinalis) and helminths, with soil-transmitted helminths (STH, referring to Strongyloides stercoralis, Ascaris lumbricoides, Trichuris trichiura and hookworm) the most prevalent [2], and listed as part of the neglected tropical diseases (NTDs) by the World Health Organization (WHO) [3]. Several studies reported varying prevalence of IPIs in the population, which depends on socioeconomic status, sanitary and environmental conditions and access to water, as well as changes in lifestyle as a result of acculturation and environmental degradation processes [4][5][6][7].
Even though most of these infections are asymptomatic, intestinal parasites (IPs) cause malabsorption, nutritional syndromes, malnutrition, morbidity and deficiencies in children's growth [8,9]. Moreover, STH infections are implicated in the etiology of iron-deficiency anemia (IDA) in developing countries [10]. Specifically, moderate and heavy hookworm infections have been strongly associated with the development of anemia, due to chronic intestinal blood loss [11], as well as with cognitive impairment in school-age children and negative impact on psychomotor and language development of preschool-age children [12,13]. All these factors contribute to the economic impact of the disease and perpetuation of poverty, causing more than 100,000 annual deaths [2] and susceptibility to develop other diseases as adults [14].
Several studies have described the presence of IPs in northern Argentina, showing a high prevalence of protozoans with a predominant presence of G. intestinalis and Blastocystis spp., as well as STH [5,15]. Additionally, Argentina has a heterogeneous prevalence of STH infections, with high prevalence in the north [16]. Anemia is considered a public health problem in the country, particularly in the northwest, where 38% of preschoolage children and 19% of women are anemic [17]. However, the prevalence found could be higher than the one reported due to difficulty in diagnosis if the parasite load is low and given that larval/egg output tends to be irregular in helminths [18]. Moreover, the diagnostic methods commonly used for STH detection have a low sensitivity for S. stercoralis or fail to detect it altogether [19,20].
Resolution WHA62.12 from the WHO World Health Assembly (WHA) urges authorities to "implement interventions against neglected tropical diseases" [21] for STH in endemic areas based on mass drug administration (MDA), normally using a single dose of either albendazole or mebendazole for A. lumbricoides, T. trichiura and hookworm, and ivermectin for S. stercoralis. Currently, Argentina does not have a deworming program at either the national or provincial level.
Although several epidemiological studies have been carried out in northern Argentina, none of them have delved into the molecular aspect of G. intestinalis, Blastocystis spp. and E. histolytica. These enteric protozoans, including Cryptosporidium spp., are regarded as the most common and important causes of protozoan-diarrheal disease in humans globally. Even though the effects caused by Blastocystis spp. are still debatable, several pieces of evidence have emerged pointing to its pathogenic role in intestinal disorders [22,23]. Determining the molecular frequency and subtype of these protozoans is important to ascertain the sources of infection, transmission dynamics and zoonotic potential [23].
Giardia intestinalis, one of the major worldwide contributors to diarrheal disease, is a complex formed of eight genotypes identified to date (A-H), with several sub-assemblages; but only genotype A and B have been associated with human infections [24]. With respect to Blastocystis spp., based on its high level of genetic diversity, it is classified into different global ribosomal subtypes (STs). Currently, 17 subtypes have been described in different areas, with ST1 to ST9 and ST12 colonizing humans. In humans from Europe, ST1, 2, 3 and 4 reportedly occur most commonly, whereas ST1, 2 and 3 commonly occur in South America [22,25].
The aim of this study was to identify the prevalence of intestinal parasites, including STH, due to the presence of living conditions appropriate for their transmission; identify any association between prevalence and socioeconomic characteristics; and perform a first approach to molecularly type G. intestinalis, Blastocystis spp. and E. histolytica present in an indigenous community from Puerto Iguazú, Misiones, Argentina. The results from this study constitute the first published report on G. intestinalis and Blastocystis genotypes circulating in the province of Misiones.

Study area and study population
This study was conducted in the city of Puerto Iguazú, located in the province of Misiones (25° 35′ 52″ S, 54° 34′ 55″ W), a subtropical province of northeastern Argentina ( Fig. 1) and part of the most biodiverse region of the country [26], which houses the Iguazú National Park and Iguazú Falls. Puerto Iguazú is in the tri-border area of Argentina, Brazil and Paraguay; the three countries are naturally divided by the Paraná and Iguazú Rivers. The region is characterized by a subtropical climate with no dry season. The median annual temperature is 21 °C, with annual rainfall of 1883.2 mm [27]. The predominant soil type is lateritic of deep red color [26].
Around the city's periphery, Mbyá Guaraní aboriginal communities have settled into villages, including the village of Fortín Mbororé, which is composed of around 200 families. The primary source of income is from guided tours organized to visit the village, handcrafts and social plans. These communities also all share similar water and sanitation conditions and are homogeneous in their economic status, with low monthly incomes [16]. The living conditions in Fortín Mbororé are characterized by a lack of water and sanitation [16] and houses made of adobe bricks with unimproved roofs and dirt floors, and practically the entire population, both children and adults, walk barefoot (Fig. 2). Most houses have a single room for sleeping, and therefore overcrowding is common.

Study design
A cross-sectional study was conducted in Fortín Mbororé between January and March 2018, as a community-wide intervention. The study included individuals older than 1 year. A total of 61 households were visited, georeferenced and characterized using a house-by-house questionnaire, Fig. 1 Map of the study area. The households from the village of Fortín Mbororé are shown as yellow dots, which are located just outside the urban area of the city of Puerto Iguazú, Misiones, Argentina. South America image was obtained from www. mapas parac olore ar. com. This map was created with QGIS 3.14 (www. qgis. org) collecting data on all members of the household, including socioeconomic variables; these are detailed in the results (Table 4).
Oral bilingual (Spanish/Mbyá-Guaraní) explanations on sample collection were provided along with sample collection containers without any fixative [one per person], and were retrieved on the following day. The fresh samples were transported without fixative in a refrigerated icebox and kept at 4 °C in the lab until analysis within 24 h of collection.
Together with stool samples, blood samples were drawn though venipuncture into H2PP tubes (VITIS ® , Prunus SLR, Argentina) containing EDTA-K3 anticoagulant and transported to a private clinical laboratory (Clínica SAM) located in Puerto Iguazú for hematological analysis. A complete hemogram was performed, including hemoglobin values (Hgb), white blood cell counts, eosinophil relative count and hematocrit. Subjects were classified as anemic or not anemic using the thresholds to define anemia according to sex and age as defined by WHO [28]. All participants were offered anthelminthic treatment according to the WHO preventive chemotherapy for human helminthiasis [29] and national guidelines [30], with the inclusion of ivermectin (200 µg/kg), together with WASH [water, sanitation, and hygiene] education workshops to improve hygiene habits.

Stool examination
To determine the presence of intestinal parasites, samples were processed with four different diagnostic techniques [31][32][33] to optimize the detection of a diverse parasite spectrum. The techniques used included the Ritchie concentration technique for detection of both protozoan and helminth parasites, Baermann concentration for the detection of larvae, Kato-Katz to measure infection intensity of helminth parasites, and modified Ziehl-Neelsen stain for detection of coccidia. The Kato-Katz technique was used only if eggs or larval stages were previously detected by the Ritchie or Baermann method; results were recorded as eggs per gram (EPG) and classified as light, moderate or heavy infection following WHO guidelines [34]. Aliquots of the fresh samples were stored either in 10% formalin for confirmatory techniques or at −20 °C with no fixative for molecular biology techniques. If the sample volume was insufficient to perform all diagnostic methods, the sedimentation technique was prioritized due to its overall higher sensitivity [35]. The findings from each of the different methods were recorded in a database.

DNA extraction
Genomic DNA was extracted from 200 mg of concentrated fecal material using the QIAamp DNA Stool Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions, with slight modification; fecal samples were mixed with stool lysis buffer and incubated for 10 min at 95 °C. The DNA was eluted in 100 μl of elution buffer and stored at −20 °C.

Molecular identification of Giardia spp., Blastocystis spp. and Entamoeba spp.
PCR reactions were performed using an MJ Mini Thermal Cycler PTC-1148 (Bio-Rad Laboratories, Inc.). Sterile water was used as a negative PCR control, and previously tested fecal samples containing G. intestinalis, Blastocystis spp., E. histolytica and E. dispar were used as positive controls. The oligonucleotides used for molecular identification and characterization of G. intestinalis, Blastocystis spp. and Entamoeba histolytica/dispar appear in Additional file 1: Table S1. PCR products from all of the reactions were run on a 1% agarose gel, except for the PCR products for Blastocystis spp., which were run on a 2% agarose gel.

Molecular detection of Giardia intestinalis
Samples positive for Giardia spp. through microscopy were screened by a quantitative PCR (qPCR) method targeting a specific 62-bp region of the small subunit rRNA (SSU rRNA) gene of the parasite [36]. Amplification reactions were conducted in total volumes of 25 µl with 3 µl template DNA, 12.5 pmol of primers and 1× TaqMan Gene Expression Master Mix (Applied Biosystems, CA, USA). Reactions were run using the following protocol: an initial hold step of 2 min at 60 °C, 10 min at 95 °C and 45 cycles of 15 s at 95 °C and 1 min at 60 °C.

Molecular typing of G. intestinalis
Giardia intestinalis isolates that tested positive by qPCR with cycle threshold values less than 37 (Ct < 37) were genotyped to assemblage level using a nested PCR encoding a 753-bp fragment of the β-giardin (bg) gene of the parasite [37,38]. In general, PCR mixtures (25 µl final volume) consisted of 8.5 µl of MyTaq Reaction Buffer, containing 5 mM dNTPs and 15 mM MgCl 2 , 2.5 units (U) of MyTaq DNA polymerase (Bioline GmbH, Luckenwalde, Germany), 1 µl of each 10 µM primer pair and 5 µl of extracted DNA for the first PCR reaction. The amplification condition for the first PCR reaction was as follows: initial denaturation at 95 °C for 7 min, followed by 35 cycles (95 °C for 30 s, 65 °C for 30 s and 72 °C for 60 s), and the final extension was at 72 °C for 7 min. For the second PCR reaction, 3 µl of the product from the first PCR reaction was added, and the reaction was performed under the same conditions as above except for the cycling time, which was instead 95 °C for 30 s, 55 °C for 30 s and 72 °C for 60 s.

Molecular typing of Blastocystis spp.
Characterization of the Blastocystis subtypes from the microscopic-positive samples was achieved by PCR, targeting the SSU rRNA gene of the parasite, amplifying a PCR product of ~ 600 bp [39]. The reaction mixture (25 µl) contained 2.5 U of MyTaq DNA polymerase (Bioline GmbH, Luckenwalde, Germany), 5× MyTaq Reaction Buffer, 5 μl of template DNA and 0.5 μM of each primer. Amplification conditions consisted of one step of 95 °C for 3 min, followed by 30 cycles of 1 min each at 94 °C, 59 °C and 72 °C, with an additional 2 min final extension at 72 °C.

Molecular detection of Entamoeba histolytica/dispar
Entamoeba histolytica/dispar (Entamoeba complex) are morphologically identical species. In this study, specific primers were used to identify either E. histolytica or E. dispar. To identify a 166-bp product for E. histolytica and a 752-bp product E. dispar DNA, samples were screened using a specific PCR based on SSU rRNA [40]. The reaction mixture contained 5 µl of DNA, 1.25 µl of each primer, 2.5 U of Taq polymerase (MyTaq DNA polymerase, Bioline GmbH) and 5× MyTaq Reaction Buffer containing 5 mM dNTPs. PCR amplification started with an initial denaturation at 94 °C for 3 min, followed by 30 cycles of 94 °C 1 min, 58 °C for 1 min and 72 °C for 1 min, with a final extension at 72 °C for 7 min.

Sequencing and phylogenetical analysis
All PCR amplicons obtained from G. intestinalis-positive samples were purified using an mi-PCR Purification Kit (Metabion International AG, Martinsried, Germany) and were sent for sequencing in both direction using the corresponding internal primers to the Central Service for sequencing for Experimental Research (SCSIE, University of Valencia, Spain). β-giardin DNA sequences, both forward and reverse directions, obtained from G. intestinalis-positive samples, were viewed using the Chromas 2.6.6v (Technelysium Pty Ltd.) sequence analysis program. The BLASTn tool (http:// blast. ncbi. nlm. nih. gov/ Blast. cgi) was used to compare β-giardin nucleotide sequences with sequences retrieved from the NCBI GenBank database. Generated DNA consensus sequences from the β-giardin fragment were aligned using the ClustalW algorithm, and a phylogenetical tree was constructed using the neighbor-joining method in MEGA X version 10.1.7 (Molecular Evolutionary Genetics Analysis) software. The reliability of the phylogenetic tree at each branch node was estimated by the bootstrap method using 500 replications. Reference β-giardin sequences chosen for comparison were from isolates of human and animal origin collected throughout the world and from Italian clinical samples, which are described in Additional file 2: Table S2. Giardia muris was used as the outgroup.

Statistical analysis
Data were analyzed using Stata 14.2 software (STATA Corp., TX, USA). Measures were evaluated using proportions with 95% confidence intervals (95% CI) and means with standard deviations (SD). The Chi-square test was used to compare significant associations between different variables. Associations were obtained comparing presence or absence of protozoa and helminth (by age group and by parasitic species). A probability (P) value < 0.05 was considered as evidence of statistical significance. Odds ratios (OR) with 95% confidence intervals (CI) were used to measure the strength between dependent and independent variables.

Study population
In total, 218 individuals from 61 households provided stool samples. Population distribution was 47.7% male (n = 104) and 52.3% female (n = 114), with a mean age of 19.9 years and a range from 1 to 87 years. Individuals younger than 15 years represented half of the participants (50.9%). Over half of the underage population were in school (57%), and 76% of adults had gone to school, although not all of them had completed their education; 72.6% had attended primary school and only 24.7% high school. Nonetheless, more than half of the population (64.0%) knew how to read and write.

Prevalence of intestinal parasites
The stool samples from all 218 individuals were analyzed through the Ritchie sedimentation technique. Two hundred and three samples had enough material for the Baermann concentration technique, and only those positive for either one of the previous techniques were processed by the Kato-Katz technique (n = 134) in order to quantify helminth eggs. Additionally, 209 of the samples were processed using the modified Ziehl-Neelsen stain.
Due to the small amount of fecal material received from nine of the 218 samples, only the Ritchie technique was performed.
The intensity of hookworm, measured as EPG of feces using the Kato-Katz technique, was mostly of light (66.1%) and heavy (26.2%) infection, and no statistically significant differences were found either between age groups or by sex. The distribution of hookworm infection intensity by age group is reflected in Fig. 3, which shows the frequency of light, medium or heavy infection within each age group. The intensity of the only T. trichiura infection found was light (48 EPG), while the A. lumbricoides infections detected were of light and heavy intensity (168, 408 and 14,352 EPG, respectively).

Household characteristics
The main characteristics from the houses and their relationship with the presence of intestinal parasites are detailed in Table 4. Most of the inhabitants live in overcrowded houses (68.4%), generally with only one room for the whole family. An association between the presence of intestinal parasites and overcrowding (χ 2 = 7.62, Most of the houses were made of wooden walls (83.3%) or thin wooden panels (55.8%) and dirt floors (50.7%). This type of floor was associated with increased transmission of STH (χ 2 = 15.02, df = 1, P = 0.001), and decreased by 72.2% (0.278, 95% CI 0.1-0.6, P = 0.001) for those living in houses with cement floors. Additionally, 87.6% of the participants walked barefoot and 16.2% practiced open defecation, although most had a latrine with simple ground excavation (79.1%). Nonetheless, no significant associations were observed between these factors and infection with IPs.
The most prevalent protozoan parasites observed were those transmitted by the fecal-oral route and through water. Most of the community (63.7%) obtained water from boreholes, and the rest of the families used tap water. None of the families stated that they treated the water for drinking or cooking by either boiling or use of disinfectants (i.e. bleach). There was no association between infection with protozoans as a group and water source, but families drinking from tap water had a higher prevalence of waterborne parasites. When analyzed by protozoan species, infection with E. histolytica/dispar was significantly associated with the use of tap water (χ 2 = 4.39, df = 1, P = 0.020).

Molecular characterization of G. intestinalis isolates
Through the standard coprological techniques used, 54 samples were found to be positive for Giardia spp. Of these, only those with a high number of cysts/slide were selected for molecular analysis (n = 32), and 29 of the 32 DNA isolates tested positive for G. intestinalis by qPCR. Generated Ct values ranged from 28.3 to 39.1 (median: 32.2; 25th centile: 30.5; 75th centile: 34.0). Only DNA isolates with Ct values ≤ 37 (n = 28) were used for genotyping.
Of the 28 DNA isolates selected, 92.9% (26/28) were successfully amplified at the β-giardin locus and are described in Additional file 3: Table S3. Sequence analyses revealed the presence of assemblages A (30.8%; 8/26) and B (65.4%; 17/26); one canine assemblage (D) was also detected. Type A assemblage sequences were assigned to either sub-assemblage AII (12.5%; 1/8) or AIII (87.5%; 7/8), while all type B sequences were assigned to the BIV sub-assemblage. The phylogenetic analysis performed with the sequences obtained revealed that the sample sequences found herein clustered with the corresponding assemblage and sub-assemblage A-D sequences used as references, although some samples tended to group into independent subgroups, reflecting noticeable changes at the nucleotide level (Fig. 4).

Molecular characterization of E. histolytica/dispar
Through the standard coprological techniques used, 22 samples were found to be positive for the "Entamoeba complex" and processed by PCR. Molecular characterization of the isolated samples showed that 45.5% of them were positive for E. histolytica, and E. dispar was not identified in any of the samples.

Discussion
The overall prevalence of IPs found in individuals from Fortín Mbororé Village, located in Puerto Iguazú, Misiones, Argentina was 92.7%, with a high prevalence of both protozoa and helminths. The most prevalent pathogenic protozoans were G. intestinalis (24.7%) and Blastocystis spp. (57.3%), whose pathogenic capacity is still a debatable issue. The most prevalent STH was hookworm (72%), followed by S. stercoralis with a prevalence of 11.5%. The percentage of individuals with polyparasitism was higher than 88%, evidencing the need for an urgent improvement of the health and living conditions of the population. These results are similar to previous studies carried out in other areas from northern Argentina, in populations who presented higher socio-environmental vulnerability [5,15].
In the multivariate analysis of the data, a higher presence of IPs was observed in preschool-and school-age children. Also, a significant relation between no formal education and a higher presence of IPs was observed, as previously reported [41]. In contrast, parasitism was not associated with gender. The high prevalence of parasites transmitted by the oral-fecal route suggests that water quality is not adequate. Furthermore, the population that obtained water from the public water system was positively associated with infection by E. histolytica/dispar and had a higher probability of harboring a parasitic infection (OR = 1.77). Some of the household characteristics of the study area, such as dirt floor or overcrowding, were associated with a higher prevalence of STH or IPs, as observed in other studies [7,42]. Unimproved sanitation, walking barefoot or practicing open defecation are risk factors that may contribute to an increase in skin-penetrating parasite infections like hookworm. In the present study, households with cement floors showed lower incidence of STH, so an initial improvement of the material conditions of the houses might have an impact on the prevalence of this type of parasite, which would theoretically decrease even more with the use of footwear.
The high prevalence of hookworm and S. stercoralis detected in Iguazú coincides with rates reported previously [4,15], although a large difference in prevalence was observed for S. stercoralis (41.9% vs. 11.5%). These differences may be due to the variability in parasite expulsion in the stool and the low sensitivity and detection limitations of the nonmolecular techniques used [19,43]. There were practically no cases of A. lumbricoides or T. trichiura; these parasites are more influenced by fecal-oral hygiene habits, and tap water and hand washing could contribute to a lower prevalence detected in our study [6]. Although studies investigating risk factors for S. stercoralis infection have mostly reported a higher risk among men, generally attributed to men's extensive exposure to soil during farming activities [44], in our study this parasite was associated more with females. We can only hypothesize on the reason for this, although it is important to note that there was no bias in sampling, since the participation between males and females was not statistically different. One plausible explanation is that men in this area do not perform farming activities; they usually travel outside the village to tourist areas to sell their crafts, while women mostly stay at home to care for the children.
Additionally, 72.4% of the population studied was anemic. The relationship between STH infection, malnutrition and anemia has been extensively shown [15]. In this study, a significant association was observed between the presence of hookworm and anemia in men. Furthermore, the intensity of hookworm infection is related to anemia and morbidity and is a key indicator for measuring the success of large-scale deworming programs [45,46]. Also, deleterious nutritional conditions and protein malabsorption have been associated with elevated parasite loads [15]. However, no association was found between heavy intensity (26.2%) and a greater presence of anemia. This is probably because most of the population studied had light-intensity hookworm infections (66.1%). The low nutritional status of the population previously reported [15] could be influenced, among other factors, by parasite incidence, since, as the study points out, these two factors are associated. Guidelines for anemia and STH recommend implementing deworming programs to improve the public health situation of communities with high prevalence of anemia [10,47].
Infection with E. histolytica can result in invasion of the colon wall and damage to other host tissues (amebiasis), and remains a cause of morbidity and mortality in developing countries [48]. The clinical diagnosis of amebiasis Fig. 4 Phylogenetic relationships of Giardia intestinalis inferred by neighbor-joining analysis of the β-giardin nucleotide sequences. Filled triangles represent reference sequences obtained from GenBank, described in Additional file 2: Table S2. A sequence from Giardia muris was used as the outgroup. Bootstrap values are based on 500 replicates, and only bootstraps > 50% are indicated is usually confirmed by visualization of the parasite by light microscopy, but this has the limitation of being unable to distinguish E. histolytica from E. dispar and E. moshkovskii cysts. Furthermore, the presence of other Entamoeba spp., Iodamoeba spp. or Endolimax spp. can make the diagnosis even more difficult [40]. With the aid of molecular techniques, it was possible to identify 10 E. histolytica-infected individuals in 22 microscopy-positive samples. The remaining 12 negative samples by PCR suggest they are cysts that may belong to another Entamoeba spp. The low prevalence of this protozoan in the current study agrees with previous studies from northern Argentina [49,50].
On the other hand, Blastocystis spp. was the most prevalent species detected in this study, which is a zoonotic parasite that colonizes humans and multiple domestic and wild animals. The high prevalence contrasts greatly with data obtained in another study on the same population (57.3% vs. 5.9%) [15], although another study conducted in the Mbyá-Guaraní communities (Misiones, Argentina) has reported a higher prevalence [4]. The genetic diversity of Blastocystis spp. can be classified based on their polymorphic regions of its small subunit of the ribosomal RNA gene [51]. Some studies have reported that the most frequent subtypes in humans in Latin America are ST1 and ST3 [22,25]. Although its immunopathogenesis is a matter of study and controversy, the ST3 subtype has been associated with a higher frequency in symptomatic patients [52]. This first molecular approximation of Blastocystis spp. in Iguazú indicates that the majority of the population was infected with the ST3 (87%) subtype, although gastrointestinal symptoms were not the subject of an associative analysis in the current study. Considering the zoonotic nature of the parasite, found in almost 60% of the population, and the fact that practically all families live with domestic or farm animals, these may represent a significant zoonotic source of this parasite for the community of Fortín Mbororé.
With respect to G. intestinalis, assemblage B showed a higher prevalence (65.4%) compared to assemblage A (30.8%); sub-assemblages AIII and BIV were the most frequent. These results coincide with the prevalence reported in other studies [24,53], and it is commonly believed that humans are infected only with assemblages A and B. Surprisingly, a case of infection by assemblage D (identified from dogs) was found in this study, coinciding with the recent identification of unusual G. intestinalis genotypes, such as assemblages C, D, E and F in humans [54][55][56]. These results indicate the need to carry out greater genotyping of G. intestinalis infections in order to increase our knowledge on the transmission routes of this parasite.

Conclusion
This study represents the first molecular approach in Puerto Iguazú, describing the genotype of G. intestinalis, Blastocystis spp. and molecular detection of E. histolytica. There was a significant inverse association between age and parasitism, and a higher prevalence