Identification of risk factors and high-risk areas for transmission of intestinal parasites in model locality in Slovakia

Abstract

Intestinal parasitoses remain a significant health concern, particularly in populations living in areas with poor hygiene and poor living conditions. These infections can cause intestinal problems and pose health risks, especially for children. In Slovakia, such infections are concentrated around Roma settlements. The most effective protection against intestinal parasitoses requires raising public awareness about the disease in general, which is usually lacking. A parasitological analysis using sedimentation methods revealed that 5.95% of the 2,503 stool samples collected from 59 villages in the Rožňava region tested positive for intestinal parasites. The most prevalent species were Ascaris lumbricoides and Giardia duodenalis. Results of the chi-squared test of independence showed a significantly higher infection rate among people from Roma communities and also among residents from rural areas. Correlation analysis identified population density, access to water and sanitation, and education levels as key factors influencing infection rates. Spatial analysis further highlighted areas at higher risk of contracting intestinal parasite diseases within the study area. Ultimately, the insights and information gained from this study can contribute to the development of better-targeted strategies for reducing the occurrence of intestinal parasitoses and improving the overall health situation for the inhabitants of these marginalised areas.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-11547-0.

Introduction

Despite advances in modern healthcare, intestinal parasitoses continue to pose a significant health issue, even in the 21 st century, and in countries where their presence might be unexpected. These parasitic infections, caused by soil-transmitted helminths and intestinal protozoans, are predominantly found in areas with poor hygiene and sanitation¹. Often, they are classified as neglected diseases, as much of the public health attention on these parasitic diseases focuses on the developing countries, or they are sometimes ignored, leaving their presence in high-income countries relatively unexplored. Initially, as the eggs/oocysts are developing, these infections can cause flu-like symptoms, diarrhoea or intestinal discomfort². However, if left untreated, they can progress into more serious health issues, including watery diarrhoea, vomiting, and malnutrition. Children are especially vulnerable, as these diseases can result in serious developmental complications and stunted growth². While Slovakia does not have a high overall prevalence of intestinal parasite infections, there are areas with higher rates of intestinal parasite infections^1,3. These areas are predominately inhabited by marginalised groups of people. In Slovakia, these groups are mainly represented by Roma communities. Their living environments are influenced by poor municipal services and low personal hygiene standards. According to the Atlas of Roma Communities 2019 (ARC)⁴, nearly 36% of Roma residents in these areas lack access to running water, and over 60% live without proper sewage systems. Frequent overcrowding further destabilises living conditions in these areas. On average, there are eight people per dwelling in Roma settlements⁴. This is nearly three times the national average of 2.5 people per household⁵. Additionally, the presence of a higher number of stray dogs further contributes to environmental contamination, as these animals roam freely around the settlements without any care. Their behaviour, such as coprophagy, could contribute to the spread of not-typical dog intestinal parasite eggs and thus pose additional health risks. Moreover, marginalised Roma communities in villages are often pushed to the outskirts of main development. Thus, they are closer to forests and fields, increasing their contact with wild animals, which are also sources of parasitic diseases⁶. This close proximity to wildlife further contaminates their living environment, creating ideal conditions for the cycle of infection to continue and posing a risk for these communities. These aforementioned conditions are particularly common in eastern Slovakia, especially in the Košice Self-Governing region (KSGR). Some parts of this region had experienced significant economic decline in recent decades. This has resulted in higher rates of unemployment and poverty in the region⁷. Also, according to the ARC⁴, the KSGR has the highest proportion of Roma communities in Slovakia. Inhabitants of these areas can move freely within villages. They share communal spaces with the majority, such as public transportation, retail centres or health facilities. This allows for intestinal parasites to be transmitted to the majority population, as they may come into contact with infectious stages of intestinal parasites. Under certain circumstances, this can negatively impact the health of the population and significantly increase the risk of intestinal parasitic infections for the whole population. The combination of various factors (limited access to basic services, overcrowded living conditions, inadequate sanitation, and restricted healthcare access) amplifies potential health risks in these areas. Most research on intestinal endoparasites in the human population in Slovakia focuses solely on the epidemiological situation across various regions, often just highlighting the presence of parasitic stages in human stool samples, dog faeces, or environmental samples^3,8–10. However, only a handful of studies have gone beyond the epidemiological situation to investigate which factors are most responsible for the transmission of endoparasitic diseases and to identify the areas with the highest risk of infection. This leaves significant gaps in our understanding of the underlying causes and risk determinants for the parasite transmission in Slovakia. In recent studies, Ihnacik et al.¹ and Ihnacik et al.¹¹ have begun to address these knowledge gaps. In addition to mapping the occurrence of endoparasites, they are also analysing the influence of key risk factors such as socio-economic conditions, access to sanitation, and environmental influences on the number of parasitoses in certain areas in Slovakia. Thus, the primary aim of this study was to shed light on an epidemiological situation in the chosen study area in Slovakia, providing valuable information about the health of the population. Another key objective was to identify and assess factors that influence the spread of endoparasites in the study area and further improve analysis methods. Additionally, risk map that highlights areas of high- and low-risk of parasitosis transmission within the study area were created.

Materials and methods

Study area

For this study, the Rožňava district, located in the KSGR of Eastern Slovakia (Fig. 1), was selected as the research area. This district consists of 60 villages and 2 towns. It has a population of 59,345 inhabitants, with a population density of 50.57 people per square kilometre⁵. The whole region is located in a mild subterranean zone with an average of 788 mm of precipitation annually¹². Today, it is one of the less developed areas in Slovakia, belonging to the regions with the highest unemployment rate (above 15%) in the country⁷. According to estimates from ARC, the Roma population constitutes 32% of the total population⁴, which is significantly higher than the 5% reported in the census⁵. Along with the regions of Revúca, Rimavská Sobota, and Lučenec, it is colloquially known as part of the “Hungry Valleys,” due to the persistent economic and social challenges faced in these areas.

Fig. 1.

Location of district of Rožňava (Red) in Košice Self-Government region (Green). The map was prepared by Lukáš Ihnacik in OpenStreetMap (Map data from “OpenStreetMap” http://www.openstreetmap.org/copyright, © OpenStreetMap contributors).

Data collection

Information about the Roma population was sourced from ARC⁴. This atlas is one of a kind and focuses on the living conditions of marginalised communities in Slovakia. General demographic and village data at the municipal/village level (LAU 2/level 6 of administrative boundary division) were obtained from the national vide census, which was conducted in Slovakia in 2021⁵. Additionally, the geographical location of settlements and villages and other geographical data were gathered from the Slovakian orthophoto map. For risk factor analysis, 10 different factors were selected from various sources, including population density, Roma population density, location of settlements, access to water in villages and settlements, usage of sewage in villages and settlements, usage of sumps in villages and settlements, and education level of the population in different villages of this region.

Parasitological analysis

A total of 2,503 human stool samples were collected and analysed from 59 locations in the Rožňava district from individuals who showed no apparent symptoms of intestinal parasite infection. Sampling was conducted over a period from September 2021 to January 2024. Samples were obtained either directly from volunteers or through collaboration with paediatricians, general practitioners, or parents interested in their children’s health. Each sample was accompanied by signed informed consent form containing basic demographic information about the volunteer, including age, gender, address, and minority or majority residency status. Samples missing more than one of the above-collected criteria or lacking a signature were excluded from the study. Such personal and sensitive data had to be handled with great care. It was essential to ensure that the information was generalised and anonymised based on the village of origin, guaranteeing that individuals could not be identified or recognised from the published data. All samples were stored without preservation at 4 °C and immediately transported for further parasitological examination to the laboratory at the Institute of Parasitology of the Slovak Academy of Sciences in Košice. Parasitological analyses were performed within 24–48 h. Stool samples were coprologically analysed using either the commercially available Paraprep L sedimentation kit (Mondial, France) or by using a SAF modified concentration (sodium acetate, acetic acid, and formalin) method. Paraprep L is a single-use, disposable kit offering prevention against cross-contamination. Sample preparation and analysis followed the manufacturer’s instructions. Briefly, 0.5 g of stool samples was placed in a mixing tube and mixed in with 2 ml of ethyl acetate and 6 ml of 10% formalin. The mixing tube was connected to a collection tube with a filter in between. After 24 h of incubation at room temperature, it was centrifuged at 500 x g for 1 min. The supernatant discarded, and the sediment examined microscopically with a Leica DM 5000B light microscope (Leica Microsystems; Wetzlar, Germany) at 100x and 400x magnification for the presence especially of helminth eggs. The SAF concentration technique was also used for the detection of helminth eggs and protozoa cysts. Briefly, ~ 1–2 g of faeces was resuspended in SAF solution and strained through a medical gauze into a centrifuge tube. After centrifugation at 500 x g for 1 min, the supernatant was decanted. The sediment was mixed with 7 ml of 0.85% NaCl and 2–3 ml of diethyl ether; the tube was closed, shaken vigorously, and centrifuged again at 500 x g for 5 min. Top layers were discarded, and the resulting sediment was examined microscopically with a Leica DM 5000B light microscope (Leica Microsystems; Wetzlar, Germany) at 100x and 400x magnification for the presence of helminths and at 1000x magnification with the use of immersion oil for protozoa. Intestinal parasitic stages (eggs of helminths A. lumbricoides, T. trichiura, E. vermicularis, and cysts of G. duodenalis) were identified based on morphological and morphometric characteristics by light microscopy¹³.

Statistical analysis

Following parasitological analysis, samples were categorised into a few distinct groups according to variables such as ethnicity, sex, environment (urban or rural), and age group. This classification allowed for the observation of differences in positivity rates across various demographic and environmental factors. Subsequently, statistical analysis was then performed on obtained parasitological data. Key epidemiological metrics were calculated using RStudio (Posit PBC, USA), including positivity rates to determine the proportion of infected individuals in our sample size, confidence intervals, and the next crude odds ratio to measure the likelihood of higher positivity rates in specific categories. Lastly, p-values and Chi-squared values calculated from the Chi-squared test of independence were used to assess the statistical significance of these associations, allowing us to reject the hypothesis that observed differences occurred by random chance. Additionally, the importance of the selected factors was determined using the correlation analysis function in IBM SPSS Statistics (IBM, USA), comparing positivity rates in individual villages with the data of selected factors. The absolute values of the resulting correlation coefficients were calculated and then converted into percentage values. This was done by dividing the absolute value of each correlation coefficient by the sum of all absolute correlation coefficients, multiplying by 100, and representing these percentages on the graph (Fig. 2). Later, a threshold value of 8% of the correlation coefficient share was determined (based on analysis in our previous research¹), and all factors that exceeded this value were considered important and that they have an impact on the number of positive samples (whether increasing or decreasing the positivity rate). This analysis also provided the basis and inspiration for the subjective weighting of factors (Table 1).

Fig. 2.

Recalculated correlation coefficients (% r) into percentage values of selected factors. The red line is the target line set at 8% of the percentage value of the correlation coefficient.

Table 1.

Factors chosen for risk mapping with their assigned weights.

Factors	density of people	density of Roma	settlement location	water usage in village	water usage in settlement	sewage usage in village	sewage usage in settlement	sumps usage in village	sumps usage in settlement	educational level
Weights (%)	15	10	6	10	6	16	16	5	5	11

Spatial analysis

All of the necessary data were processed in Microsoft Excel (Microsoft, USA) into suitable databases and uploaded into ArcGIS Pro (Version 3.4, ESRI, USA) software. A map of positivity rates for each village was created using point data, with points located at the centre of villages. Graduated colour scales were chosen, and conditional formatting was used to differentiate between different positivity rates. Additionally, indicators of clustering were determined using the spatial autocorrelation (Global Moran’s I) function, which assesses whether data with high or low values are grouped, dispersed, or randomly distributed. For the Conceptualisation of Spatial Relationships parameter, a Fixed Distance Band was used, where each feature will be analysed within the context of neighbouring features. The risk maps were created by using an adjusted suitability approach. In the case of the Rožňava district in Slovakia, this approach requires focusing on socio-economic factors rather than environmental conditions that influence the spread of endoparasitic stages, due to the uniform climate. Values of our chosen factors were reclassified into a common scale from 1 to 5 using the reclassify tool, with natural breaks applied to define category boundaries. Weighted and unweighted risk maps were then created by stacking the reclassified factor layers and combining their values using the raster calculator and weighted overlay functions in ArcGIS Pro.

It’s important to note that risk levels were assigned to whole territorial units, regardless of the specific locations of villages or settlements within them. In some cases, settlements may be located far from a main village but still fall within the same territorial unit as a main village, while being geographically closer to a different village in another territorial unit. Additionally, settlements often have different and worse living conditions compared to villages within the same unit. Therefore, it was decided to distinguish between settlements and villages in the next creation of risk maps. This division was done with the help of ARC⁴ and orthophoto map, which helped identify the precise location of settlements in each territorial unit. Subsequently, only those factors, which could be consistently determined for both villages and individual settlements, were selected. The number of inhabitants for the village and the number of Roma for the settlements, the use of water supply, sewage, and cesspools, in which data could be assigned for both villages and settlements were chosen. It was decided to combine sewage and sump into a single factor called waste management. The methodology for creating risk maps remained the same as before, with the key difference being that analysis was based on point data. This data was later interpolated to produce a continuous surface, allowing risk levels to be extended beyond the points. The interpolation was conducted using the IDW (Inverse Distance Weighting) function to ensure a smooth and accurate representation of risk across the region. This comprehensive analysis enabled us to better understand the distribution of intestinal parasites across the population and identify critical risk factors contributing to the spread of infections as well as high-risk areas where transmission is theoretically the most likely to occur.

All methods used in this study were in accordance with the relevant guidelines and regulations. The study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of the Košice self-governing region (Document no. 5436/2019/ODDZ-25820). Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin. The datasets generated and/or analysed during the current study are not publicly available due to security concerns of participants, and we promised it will be anonymous. All data are available from the corresponding author on reasonable request.

Results

In total, we examined 2,503 stool samples for intestinal parasites, which were detected in 149 (5.95%) samples (Table 3). The most prevalent eggs identified were eggs of Ascaris lumbricoides, detected in 117 cases out of the 149 positive samples. This was followed by Giardia duodenalis cysts, found in 28 samples, and a lower number of helminth eggs of Trichuris trichiura found in 12 samples, and Enterobius vermicularis found in 11 cases out of 149 positive samples (Table 2).

Table 3.

Key epidemiological metrics calculated from obtained parasitological data, categorised by overall sample size, ethnicity, sex, environment and age groups. 95% CI – 95% confidence interval; COR – Crude odds ratio; (ref) – reference group for calculating OR; χ² – chi-squared; ^** – p < 0,01; * - COR, χ² and p-value were not calculated for age groups.

Overall	samples	positive	% positive (95% CI)	COR (95% CI)	χ2	p-value
	2503	149	5.95 (5.02–6.90)	-	-	-
Etnicity
Roma	848	135	15.92 (13.52–18.55)	22.19 (12.71–38.74)	227.56	< 0.01**
Non-Roma	1655	14	0.85 (0.46−0.14)	1 (ref)	-	-
Sex
Men	1318	80	6.7 (4.84–7.48)	1.4 (0.74–1.45)	0.068	0.7
Women	1185	69	5.82 (4.55–7.31)	1 (ref)	-	-
Environment
Rural	1286	93	7.23 (5.87–8.78)	1.7 (1.21–2.41)	7.73	< 0.01**
Urban	1217	56	4.60 (3.49–5.93)	1 (ref)	-	-
Age groups*
Infants (0–1)	141	5	3.55 (1.16–8.08)	-	-	-
Kids (2–6)	700	55	7.86 (5.97–10.10)	-	-	-
Adolescents (7–18)	783	71	9.7 (7.14–11.30)	-	-	-
Productive (19–66)	801	17	2.12 (1.24–3.37)	-	-	-
Post-productive (> 66)	78	1	1.28 (0.03–6.93)	-	-	-

Table 2.

Number of positive human stool samples divided according to parasite species, ethnicity and sex. n – number of samples detected with eggs of given parasite; % - percentage of number of samples detected with eggs of given parasite; * - this includes just the number of positive samples with the given intestinal parasites detected; it does not refer to the number of people infected.

Eggs/cysts	Overall*	Ethnicity				Sex
		Roma*		Non-Roma*		Men*		Women*
		n	%	n	%	n	%	n	%
A. lumbricoides	117	106	90.59	11	9.40	63	53.84	54	46.15
T. trichiura	12	8	66.66	4	33.33	8	66.66	4	33.33
E. vermicularis	11	10	90.90	1	9.09	8	72.72	3	27.27
G. duodenalis	28	26	92.85	2	7.69	16	57.14	12	42.85

Firstly, samples were divided according to ethnicity into two groups: Roma or minority and non-Roma or majority group. The highest prevalence of parasites was observed in the minority of Roma people, where the infection rates reached 15.92%. Roma individuals were significantly more infected, with an odds ratio indicating a 22.19 times higher likelihood of infection compared to the non-Roma population (Table 3), who generally live in better hygiene conditions. Among the non-Roma individuals, parasites were detected in just 0.85% of stool samples. Next, we categorised our samples based on ecosystems, dividing them into rural (villages) and urban (towns) settings. We then analysed the occurrence of endoparasitic infections in the population within these groups. The analysis revealed that individuals living in rural areas (villages) had a significantly higher number of samples with endoparasitic developmental stages than those residing in urban areas (towns). The occurrence of samples with parasitic infections in inhabitants from rural areas was 7.23%, compared to just 4.60% observed in individuals from urban areas. This indicates that individuals in rural areas are nearly twice as likely to be infected as those in urban areas. In contrast, when samples were categorised by gender, no significant differences were found between men and women, with positivity rates remaining the same, around 6% for both sexes (Table 3).

Lastly, the samples were divided into 5 age groups: infants (0–1 years), kids (2–6 years), adolescents (7–18 years), productive age (19–66), and post-productive age (over 66 years). The highest positivity was observed in adolescents (9.07%), followed by kids (7.86%) and infants (3.55%). In contrast, the productive and post-productive age groups have just 2.12% and 1.28% positivity rates, respectively (Table 3).

We incorporated all 10 factors (population density, Roma population density, location of settlements, access to water in villages and settlements, usage of sewage in villages and settlements, usage of sumps in villages and settlements, and education level of the population in different villages of this region) in Spearman’s correlation analysis. The recalculated values of the correlation coefficient are presented in Fig. 2, illustrating the share of influence of the correlation coefficient on overall positivity. As shown in Fig. 2, population density, usage of sewage in villages and settlements, and educational levels were highly above our threshold line of 8% from correlation coefficient share. Roma population density and access to water in villages are exactly or slightly above the threshold. According to the results, we can conclude these are important and have a notable influence on parasite prevalence in the region. However, while they indicate a strong association with positivity rates, they do not demonstrate causality, only the strength of the relationship.

The map of endoparasite positivity in each village shows that the highest positivity rates were concentrated in villages in the eastern part of the Rožňava region, with additional hotspots in the southwestern tip (Fig. 3). The overall highest positivity, ranging between 17% and 26% was recorded in the southeastern tip of the region. However, it should be noted that in some villages we had relatively low sample sizes, which may affect the accuracy and representativeness of the results.

Fig. 3.

Map of distribution of positive samples in Rožňava district displayed as positivity rates in villages. The map was prepared by Lukáš Ihnacik in ArcGIS Pro (Version, 3.4, ESRI, USA).

The results of the Global Moran’s I analysis showed a z-score exceeding 2.58 and a p-value below 0.01, indicating that the clustering of values of high and low values in the Rožňava district has less than a 1% probability of occurring by random chance. Based on these results, we can conclusively confirm that the positivity values are indeed clustered rather than randomly distributed.

Natural breaks were used to categorise values in the legend, and a suitable colour palette was chosen to enhance visual clarity (Figs. 4 and 5). This approach allows for easy visual identification of high-risk areas, with colour variations quickly highlighting the most concerning regions. Green represents lower risk, while red indicates higher risk, with the intermediate colours reflecting varying levels of risk between these extremes. Firstly, both the unweighted and weighted risk maps, were created based on village territorial unit.

Fig. 4.

Unweighted risk map with positivity in each individual village. (*Circles and shades of blue depict the percentage positivity of intestinal parasites in the human population, and each circle represents the centre of the village; **Unweighted risk values are depicted on the map as colour palette, with numbers in the legend representing summed values ​​of unweighted reclassified factor layers categorized by natural breaks). The map was prepared by Lukáš Ihnacik in ArcGIS Pro (Version, 3.4, ESRI, USA).

Fig. 5.

Weighted risk map with positivity in each individual village. (*Circles and shades of blue depict the percentage positivity of intestinal parasites in the human population, and each circle represents the centre of the village; **Weighted risk values are depicted with colour palette, and numbers represent summed values ​​of weighted reclassified factor layers categorized by natural breaks). The map was prepared by Lukáš Ihnacik in ArcGIS Pro (Version, 3.4, ESRI, USA).

With the unweighted risk map, the overall living conditions can also be reflected, as reclassified values of factors were just added together. Lower resulting numbers indicate better overall living conditions, while higher numbers reflect poorer conditions. The final unweighted risk map shows that the highest values, indicating worse living conditions, are primarily concentrated in the central-western part of the region, with some villages in the eastern tip of the region showing higher values (Fig. 4).

Similarly, weighted maps were generated, but with this map creation, weights were assigned to each factor before combining them (Table 1). As illustrated in Fig. 5, the distribution of risk in the weighted map differs from the concentrated pattern observed in the unweighted risk map (Fig. 4). Throughout the Rožňava district, the weighted map shows a medium risk level across most areas, with some areas ranging from lower risk zones (depicted in green) to higher risk zones (depicted in red). Notably, significant concentrations of high risk are observed in the western part of the district, with a few exceptions in the eastern part of the region.

Next, by creating risk maps that considered the locations of settlements and villages separately, we achieved a finer resolution in both the weighted and unweighted maps. Resulting risk maps better reflect the specific circumstances and vulnerabilities of each area, facilitating more information.

Similarly to the unweighted risk maps created earlier (Fig. 4), the highest risk levels can also be interpreted as indicators of worse living conditions. After the interpolation, a continuous surface was generated, allowing us to observe risk values of study areas. The highest unweighted risk values are primarily concentrated in the central part and the western tip of the district, with small pockets of higher risk in the eastern part (Fig. 6).

Fig. 6.

Unweighted risk map, settlements are split from villages, and points are interpolated. (*Unweighted risk values are depicted as colour palette, with numbers representing summed values of unweighted reclassified factor layers categorized by natural breaks). The map was prepared by Lukáš Ihnacik in ArcGIS Pro (Version, 3.4, ESRI, USA).

With the weighted interpolated risk maps, the highest risk areas are no longer as concentrated as in the unweighted interpolated risk maps (Fig. 6). Instead, the high-risk areas now extend across a larger portion of the central part of the district, with notably higher risk in the western tip (Fig. 7). Additionally, smaller pockets of elevated risk are also more easily distinguishable in other parts of the regions.

Fig. 7.

Weighted risk map, settlements are split from villages, and point are interpolated. (*Weighted risk values are depicted as colour palette, with numbers representing summed values of weighted reclassified factor layers categorized by natural breaks). The map was prepared by Lukáš Ihnacik in ArcGIS Pro (Version, 3.4, ESRI, USA).

This approach to risk mapping, with separate assessment of villages and settlements, offers greater accuracy, producing a more precise and granular risk surface. It enables us to identify high- and low-risk areas also in one territorial unit that might have been otherwise overlooked or assigned another risk level, thus providing more information.

Discussion

The first part of the research was focused on assessing the epidemiological situation in the Rožňava district. We found that the overall prevalence of intestinal parasites in the population was 5.95%. Infections with A. lumbricoides were the most common, accounting for nearly 79% of all positive cases. This was followed by G. duodenalis, whose cysts were present in 19% of positive human stool samples. Higher presence of A. lumbricoides may be attributed to the resilience of its eggs, which can embryonate more rapidly than T. trichiura^13,14. In the environment, they can survive for years, thus increasing the likelihood of infection. These findings are consistent with Papajová et al.¹⁰. They reported, in their study on children from 32 segregated Roma settlements from the Košice and Prešov regions, a high occurrence of A. lumbricoides, followed by G. duodenalis. This was probably due to the fact that they used the same detection methods as in our study (kit Paraprep L and SAF). Although we did not perform the standard diagnostic methods for detecting E. vermicularis, which usually requires a transparent gluing plastic strip to be touched on the perineal region^13,15, we still identified eggs of this parasite in 11 of our samples. Detection of E. vermicularis eggs through traditional concentration methods is uncommon and seldom. The female parasite generally gluing their fertilised eggs on perineal skin during the night, rather than producing it inside intestines¹³. This suggests particularly heavy infection, where a significant number of eggs were passed into stool during defecation. It also indicates that individuals with E. vermicularis infection may be undiagnosed in the study area. These individuals can contribute to further transmission.

Samples were divided according to the ethnicity on 848 samples from Roma and 1655 faecal samples from non-Roma inhabitants. It reveals that the risk of intestinal parasites infection is 22.19 times higher among the Roma inhabitants compared to the non-Roma population. It is important to note that the relatively low number of samples from Roma residents in some villages was influenced by mistrust and reluctance to engage with our team. Some residents were concerned about the potential impact on their families and property, despite assurances that our institution has no authority in such matters. Similarly, Štrkolcová et al.¹⁶ reported a higher prevalence of intestinal parasite eggs primarily among Roma children, where 64.4% out of 340 samples were tested positive. In contrast, only four positive cases (3.25%) were observed in 123 samples from non-Roma children. However, unlike our study, this one focused on a single village in eastern Slovakia. The most prevalent parasites were A. lumbricoides and G. duodenalis. These findings align with our study, as they also used two concentration methods for detecting helminth eggs (the flotation method according to Kozák/Magrova) and parazoan cysts (the flotation method according to Faust). Likewise, Pipíková et al.⁸ observed a higher incidence of parasitic infections in Roma than in non-Roma children. However, this study, similarly to Papajova et al.¹⁰, focused on the larger area of ​​the Košice and Prešov self-government regions. Out of 426 stool samples, 72 samples contained eggs of intestinal parasites. Nearly all positive samples were from Roma children (71), with just one positive case in the non-Roma group. Unlike our study, which identified A. lumbricoides and G. intestinalis as the most common parasites, Pipíková et al.⁸ reported in their study A. lumbricoides and T. trichiura as the most prevalent species. This was probably due to the use of the Paraprep L concentration kit for sample analysis. Similarly, as in our study and Papajová et al.¹⁰, this kit primarily detected helminth eggs. The consistent finding of A. lumbricoides as a predominant parasite across studies underscores the environmental persistence of this species. These studies also highlight that the occurrence of intestinal endoparasites was more typical in marginalised Roma population, than in the majority of the population.

The prevalence and spread of endoparasites are influenced by a range of factors. Mostly demographic, climatic, edaphic, and WASH conditions, which can significantly impact the transmission and distribution of intestinal parasites propagational stages. Therefore, understanding the relationship between risk factors and spatial patterns of intestinal parasite infections is essential. In the next part of this study, we examined the influence of ten selected factors on the prevalence of intestinal parasites in the Rožňava district through correlation analysis. Focusing on factors above the threshold of 8% of the correlation coefficient, we identified the key factors affecting intestinal parasite prevalence in this region. Among the analysed factors, just hygiene factors (particularly usage of water in settlements and usage of sewage in both settlements and villages), demographic composition (population density and Roma population density), and educational levels of inhabitants were above threshold and thus were considered important for the spread of intestinal parasites. Our findings are similar to those of Scholte et al.¹⁷, who reported in their study that apart from environmental factors, the poverty index, which is linked with hygiene levels, is closely associated with the transmission of endoparasitic diseases, such as ascariasis and trichuriasis. Massara and Enk¹⁸ underscored that improvements in basic hygiene and health education are critical measures to reduce the risk of A. lumbricoides infection. According to research by Da Silva et al.¹⁹, Wardell et al.²⁰, and Riaz et al.²¹, endoparasite distribution in low-hygiene areas is strongly linked to environmental sanitation (including wastewater treatment and personal hygiene), availability of defecation facilities (toilets, latrines, or open defecation), population density, drinking water sources, and food contamination by endoparasite pathogens. It is important to note that these studies were focused on the countries of the southern hemisphere. In Slovakia, one of the earliest study to explore risk factors influencing the spread and transmission of intestinal parasites in humans was conducted by Ihnacik et al.¹¹. Similarly, as our study, they utilised correlation analysis to identify key factors contributing to infection risk. Their findings identified the proportion of Roma inhabitants and the usage of water and public sewerage as the most significant risk factors. This study focused on selected municipalities in eastern Slovakia, providing valuable insights into the environmental and social determinants of parasitic infections. On the other hand, Wardell et al.²⁰ concluded in their study that average annual temperature and rainfall are among the most influential factors affecting the environmental spread of helminth eggs and protozoan cysts. For our research, temperature and rainfall are less applicable. Due to Slovakia’s relatively uniform climatic zone, variations between localities are not drastic. This homogeneity reduces the impact of climatic differences on regional studies or analyses.

Risk mapping is a useful tool for studying the transmission and potential spread of endoparasites. It is useful in high-risk regions where environmental and socio-demographic factors significantly influence prevalence. By integrating spatial analysis with parasitological data, risk maps provide visual representations of areas with the most likely chance for future endoparasite transmission. In this study, mapping the spatial distribution of intestinal parasite infections across the Rožňava region allowed us to identify high-risk and low-risk areas. For instance, settlements with poor sanitation infrastructure, high population density, and limited access to clean water tend to have a higher prevalence of intestinal parasites than villages. This was apparent in risk mapping based on settlements and villages separately. Earlier efforts by Papajová et al.²² and Blišťan et al.²³ presented pioneering work in the field of risk mapping in Slovakia, where they compared various methods for weighting risk factors in order to create risk maps. In their study, they included water, sanitation and hygiene conditions, educational level, population density, and additionally, age distribution. Use of different spatial methods was described in the study of Lin and Wen²⁴. They highlighted that the use of these methods will provide deeper additional information compared to traditional epidemiological analysis. The importance of spatial analysis in parasitology was stressed already a few years ago, where it was highlighted by Brooker et al.²⁵. They demonstrated the use of spatial methods in neglected tropical disease control. Apart from that, they emphasised how spatial data combined with socio-economic variables and ecological factors could be used to target a mass drug administration program effectively. In the end, they created an atlas, which helps created an estimate of infection intensity by showing where high-risk areas are. In this study, a novel combination of statistical methods and spatial analyses was applied for the first time to assess the risk of intestinal parasite transmission within the human population in Slovakia.

During this study, several challenges were encountered, such as the above-mentioned insufficient number of samples in some areas. Next, it was a fact that we worked with several years-old data. However, this is not necessarily a problem for data about municipalities but for information regarding Roma settlements. Roma communities often exhibit significant mobility and fluidity. Due to this population movement or seasonal changes in these areas, the living conditions can change multiple times. Another significant limitation of this study was its reliance on one-time sampling, where each volunteer provided a sample only once. While multiple sampling strategies could bring more information and enhance data reliability, they are very problematic. They include both ethical concerns and the potential distrust towards scientific institutions, particularly in marginalised communities. Addressing these issues in future requires careful research planning and engagement of Roma communities in the future studies. Both of these solutions also bring disadvantages, mainly from a time point of view, since working with Roma communities requires lengthy bureaucratic and ethically right processes. Another important limitation of this study is the reliance on microscopic concentration methods for parasite detection without the use of molecular techniques such as PCR. While microscopy is a well-established and cost-effective approach, especially suitable for detecting soil-transmitted helminth eggs, it is known to underestimate the true prevalence of mainly protozoan parasites. PCR methods, although more sensitive and specific, were not feasible due to the large sample size and resource constraints. Nonetheless, our chosen methods were sufficiently sensitive to detect protozoan infections such as Giardia cysts in some cases.

This study is nevertheless able to bring new information and insights into the spread and transmission of intestinal parasites in the studied localities. It offers essential insights into the epidemiological situation, which would be otherwise overlooked, by various health authorities. It enables the creation of better-targeted interventions and preventive measures to reduce the risk of the spread of intestinal parasite infections. By addressing the root causes of intestinal parasite transmission in high-risk areas, we can bring long-term disease prevention. This would require increased educational efforts, such as presentations in schools for younger generations. Another way to spread awareness is to distribute informative leaflets about intestinal parasites, the diseases they can cause, the method of transmission, and especially prevention. Additionally, the acquisition of essential sanitary equipment, construction of latrines, or installation of sewage systems by municipalities could thereby improve public health protection. Lastly, expanding veterinary services for stray dogs and household animals or regular deworming should also be one of the steps in preventing the spread of intestinal parasitoses. Realistically, presentations and perhaps leaflet distribution (email or paper form) are the most feasible, as these are not financially demanding activities. However, their impact may not be as significant as completing infrastructure or mandatory deworming, which are, on the other hand, quite expensive.

Conclusions

The analysis of parasitological data and factors regarding living conditions in the model Rožňava region revealed significant health risks associated with intestinal parasites, particularly in marginalised communities. The study revealed the highest percentage positivity rates were among Roma inhabitants, especially children, and in rural areas. Spatial analysis and risk mapping identified central and western parts of the district as high-risk areas. This study also highlights the need for improved data collection, better, more up-to-date data sources, and targeted public health interventions to lower the existing risk in this district. Despite the challenges, this research provides valuable insights for authorities, helping them identify areas with the worst living conditions and the highest risk of endoparasite disease transmission. Expanding such studies to other regions of Slovakia, with a larger sample size and inclusion of more relevant socio-economic and environmental factors, could further refine risk assessments and intervention strategies. In conclusion, we see that in general, the importance of spatial analysis for the prediction of the occurrence of diseases is very important, which we observed, for example, during the recent epidemic of Covid-19. By integrating epidemiological, spatial, and temporal data, public health responses can be more precise and impactful in addressing both infectious and parasitic diseases.

Electronic supplementary material

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Author contributions

All authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication.

Funding

This research was supported by the Scientific Grant Agency of the Ministry of Education of the Slovak Republic, by support from the Slovak Academy of Sciences, grant number VEGA 2/0069/25, and by the Slovak Research and Development Agency under contract no. APVV-18-0351.

Data availability

Dataset used in this publication are not publicly available because of safety concern for our participants but they are available from the first author and the corresponding author upon reasonable request.

Declarations

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Ethics statement

The study was conducted in accordance with the Declaration of Helsinki, and approved by the Ethics Committee of Košice self-governing region (Document no. 5436/2019/ODDZ-25820). Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.