The study protocol, which includes the study design, questionnaire, informed consent process, and animal sampling procedures, was reviewed and approved by the Research Ethics Committee of the Apurímac Regional Health Directorate (Approval Minute No. 003, dated July 30, 2024). Before enrollment, all pig owners were thoroughly informed about the objectives, procedures, potential benefits, and minimal risks of the study. Written informed consent was obtained from each participant prior to the collection of epidemiological data and biological samples.

Participation by pig owners was completely voluntary, with no financial rewards or pressure, and participants could withdraw from the study at any time without consequences. Confidentiality was strictly upheld, and household identifiers were anonymized during data analysis and reporting to prevent the recognition of individuals or communities.

All animal handling procedures followed national and international animal welfare standards. The study complied with the Peruvian Animal Protection and Welfare Law (Law No. 30407), which requires humane treatment of animals and the prevention of unnecessary pain or distress during handling and sampling. Additionally, procedures adhered to the recommendations of the World Organization for Animal Health (WOAH) Terrestrial Animal Health Code regarding the welfare of production animals.

Blood sampling was performed by trained veterinarians using standard, minimally invasive techniques designed to minimize stress and discomfort. Proper physical restraint methods were employed, and sampling duration was kept as short as possible. Personnel wore personal protective equipment (PPE) to ensure biosafety and safety for both animals and handlers. Additionally, the study adhered to the Animal Research: Reporting of In Vivo Experiments 2.0 guidelines, particularly regarding personnel training, procedural refinement, proper animal handling, and transparent reporting of ethical considerations.

The study was conducted from April to December 2024 in the rural district of José María Arguedas, Andahuaylas province, Apurímac department, Perú. The district is situated at an average altitude of 3,590 m above sea level, with a mean annual temperature of 13°C and an average annual rainfall of 608.9 mm. Geographically, it is located at latitude 13°44′03″ South and longitude 73°21′02″ West and has a reported poverty rate of 44.6% [7]. The combination of a cool-temperate climate that supports parasite survival and inadequate sanitation infrastructure that leads to environmental contamination creates conditions favorable for the persistence and transmission of cysticercosis, as T. solium eggs remain viable for long periods under such conditions.

A quantitative, observational, analytical, cross-sectional design was chosen because it enables the simultaneous assessment of multiple exposures and outcomes and is suitable for identifying associations at a single point in time.

According to the Fourth National Agricultural Census [8], an estimated 6,201 pigs were raised in backyard production systems within the José María Arguedas district. A simple random sampling method was used, applying the formula:

n = NZ²PQ / E²(N−1) + Z²PQ

The minimum sample size was determined based on an expected prevalence of 20% (P), a 5% margin of error (E), and a 95% confidence level (Z), resulting in a needed sample of 237 pigs. Pig-owning households were identified using records from the Integrated Animal Health Management System of the National Agrarian Health Service of Perú [9]. Blood samples were collected from about five pigs per household until the target sample size was achieved. In total, 245 pigs were sampled.

Pigs under 4 months old, pregnant females, animals exhibiting obvious illness, and pigs not raised in backyard systems were excluded from the study.

Interviewers, who were trained local veterinarians, visited randomly chosen households to gather data through face-to-face interviews conducted in Spanish or, if needed, in the local Quechua language. If no household member was available, the interviewer moved on to the nearest neighboring household.

The questionnaire was validated through expert judgment by a panel of specialists who assessed clarity, relevance, and content validity. The instrument collected information on pig characteristics, owner demographics, pig origin, housing conditions, sanitation practices, feeding systems, slaughter practices, and knowledge related to cysticercosis transmission. Missing or incomplete responses were handled using the missing-at-random (MAR) approach.

Blood samples (6–8 mL) were drawn from the inferior vena cava of pigs using 21G Vacutainer needles and 6 mL Vacutainer tubes without anticoagulant (Botica Labortech company, Perú). Serum was separated by centrifugation at 1000 × g for 5 min, frozen, and stored until analysis.

Sampling personnel wore appropriate PPE, including goggles, latex gloves, boots, waterproof coveralls, and masks. Animals were restrained using standard equipment such as mouth gags, stingers, and nose rings to reduce stress and ensure safe sample collection. Samples were transported in insulated containers with cold packs to the Public Health Laboratory of the National University Micaela Bastidas of Apurímac and stored at 2°C–8°C. All samples were labeled and tracked through a documented chain-of-custody process.

Serological detection of porcine cysticercosis was conducted using the EITB assay to identify antibodies against T. solium. Samples were considered positive if they showed reactivity against at least one of seven lentil lectin–purified metacestode glycoproteins (GP; GP50, GP42–39, GP24, GP21, GP18, GP14, and GP13 kDa) [10, 11].

Among the positive samples, 26 showed a single reactive band, 7 showed two bands, and 3 showed three bands. All assays were performed at the Parasitic Immunology Laboratory of the Universidad Peruana Cayetano Heredia (Lima, Perú) following standardized protocols [10, 11]. The EITB assay detects exposure to T. solium eggs and both viable or past infections, and it is considered the reference method for population-level surveillance [6, 12].

Quality control procedures highlighted the high sensitivity and specificity of the EITB assay. The positive control consistently exhibited the characteristic seven-band pattern associated with confirmed cysticercosis, confirming antigen integrity and reagent performance. Negative control sera showed no band reactivity.

To ensure specificity, sera from individuals with other parasitic infections were used to evaluate potential cross-reactivity. Samples with weak or atypical banding patterns were retested. The assay employs lentil lectin affinity–purified GPs derived from T. solium cyst fluid, originally developed by the U. S. Centers for Disease Control and Prevention, with stable reagents prepared under controlled laboratory conditions [10, 11].

The spatial distribution of porcine cysticercosis was examined using GIS techniques. A total of 49 pig-raising households across 10 localities were georeferenced with a GARMIN eTrex® 10 GPS device, with an accuracy of <10 m. Coordinates were processed in RStudio (R version 4.4.1, R Core Team, Vienna, Austria) utilizing the raster and tidyverse packages.

Inverse Distance Weighting interpolation was used to visualize spatial clustering in accordance with Tobler’s first law of geography. This method gives greater weight to nearby points when estimating values at unsampled locations, creating a continuous raster surface. Infection foci were defined as areas with more than two seropositive households within a 200 m radius.

All collected data were checked for completeness, consistency, and accuracy before analysis. Data cleaning involved identifying and correcting entry errors, resolving inconsistencies, and managing missing values. Missing or incomplete responses were addressed using the MAR assumption to reduce potential bias. Qualitative variables were coded into standardized numerical formats to facilitate statistical processing.

The cleaned dataset was entered into Microsoft Office Excel 365 (Microsoft Corp., Washington, USA) for initial organization and then analyzed using R statistical software version 4.4.1 (R Core Team, Vienna, Austria). Descriptive statistics summarized pig owners’ characteristics, pig demographics, husbandry practices, and sanitation variables. Categorical variables were presented as frequencies and percentages.

Associations between porcine cysticercosis seropositivity and categorical explanatory variables were first evaluated using chi-square tests. Crude odds ratios (ORs) with 95% confidence intervals (CIs) were then calculated to measure the strength of these associations. Variables with p-values ≤ 0.05 in bivariate analyses were considered statistically significant and were included in multivariate modeling.

Univariate logistic regression analyses were performed to identify potential risk factors associated with pig owners’ characteristics (such as sex, education level, knowledge of cysticercosis transmission, pork consumption habits, latrine ownership, pig confinement, access to latrines, number of pigs reared, feed type, and home slaughter practices) and pig-related factors (including sex, reproductive class, origin, and housing location).

A multivariate logistic regression model was then developed to identify independent predictors of porcine cysticercosis while controlling for confounding variables. Backward stepwise elimination based on the Wald statistic was used, starting with a full model that included all candidate variables. Variables with p-values > 0.05 were sequentially removed, and adjusted ORs with 95% CIs were reported for the final model. Statistical significance was set at p ≤ 0.05.

Potential confounding was managed both during the design phase (using standardized inclusion and exclusion criteria) and during the analysis phase (via multivariate adjustment). Model assumptions were checked, and the findings were interpreted in light of the cross-sectional design. It is recognized that the identified associations and spatial risk patterns might not be directly applicable to regions with different environmental conditions or pig production systems.

All pig farmers reported having access to treated water (100%), and the average age of respondents was 40.5 years (standard deviation [σ] = 13.5). As shown in Table 1, most pig owners were women (67.3%) and had completed primary education (40.8%). The majority of respondents reported consuming pork products quarterly (85.1%). Sanitation coverage was high, with 93.9% of households possessing latrines, and 91.8% allowing pigs access to these facilities. Regarding husbandry practices, most farmers raised fewer than 10 pigs (93.9%) and primarily fed them household waste (89.8%).

The demographic characteristics of pigs raised in backyard systems are shown in Table 2. Most pigs were females (56.3%), classified as adults (64.1%), and mainly obtained from livestock fairs (69.4%).

The overall seroprevalence of porcine cysticercosis was 14.7% (36/245; 95% CI = 10.1–19.3). As shown in Table 3, no statistically significant differences were observed in the prevalence of cysticercosis based on the location where pigs were obtained (p > 0.05).

A total of 10 localities within the José María Arguedas district were sampled, with approximately five pigs examined per household. Table 4 shows that the Checche locality had the highest seroprevalence of porcine cysticercosis (60%; 95% CI = 26–87), which was significantly greater than that of other localities (p < 0.05). This spatial clustering is depicted in the serological sample distribution map (Figure 1) and the georeferenced household-level distribution of pigs with and without cysticercosis across the district (Figure 2).

Univariate logistic regression analysis showed that the sex of the pig owner (18.2%; 95% CI = 11.9–24.4; p < 0.05) and awareness that humans can be infected with cysticercosis (p = 0.021; 95% CI = 12.5–29.5) were significantly linked to porcine cysticercosis (Table 5).

No statistically significant links were found between pig-related traits and the presence of porcine cysticercosis (p > 0.05) (Table 6).

In the multivariate logistic regression analysis, only the sex of the pig owner remained independently associated with porcine cysticercosis. Pigs raised by female owners had a significantly higher likelihood of infection (OR = 3.2; 95% CI = 1.3–8.2; p = 0.015) (Table 7).

The overall seroprevalence of porcine cysticercosis observed in this study was 14.7%, confirming that this parasitic infection remains endemic in the José María Arguedas district. The persistence of porcine cysticercosis poses a major public health concern and causes economic losses for rural communities, mainly through reduced market value or condemnation of pig carcasses [13]. The prevalence reported here is similar to figures documented in Callao, Perú [14], and in several endemic countries, including Gambia and Senegal [1], Burma [15], Ivory Coast [16], and Tanzania [17–19]. However, higher prevalences have been reported in nearby Andean localities such as Nueva Esperanza, Turpo, and Matapuquio [3], as well as in other Peruvian regions, including Tumbes [13, 20, 21], Amazonas [22], and Tarapoto [23]. Likewise, elevated prevalences have been recorded in Ecuador [24], Colombia [25], Guatemala [26], South Africa [27], Zambia [28], Tanzania [29], Kenya [30], Cameroon [31], Ghana [32], and Indonesia [33], indicating that the level observed in this study reflects moderate endemicity.

Unlike tropical lowland settings, high-altitude Andean backyard production systems are characterized by distinctive ecological and management conditions, including limited waste-disposal infrastructure and climatic factors that may prolong the survival of T. solium eggs in the environment, thereby affecting transmission dynamics.

Access to human feces, latrine availability, and pig confinement are well-known risk factors for porcine cysticercosis [22, 34–36]. In this study, no significant links were found between porcine cysticercosis and pig confinement or latrine ownership. This may be due to the poor quality of most latrines, which were often basic and lacked secure barriers, allowing pigs to access them. Given pigs’ coprophagous behavior and the dominance of free-ranging management systems in the district, exposure to human feces is likely to persist despite limited sanitation coverage.

Furthermore, the widespread practice of feeding pigs with household waste (89.8%) may increase exposure to T. solium eggs [14]. Environmental contamination can be worsened by poor personal hygiene and the presence of untreated human tapeworm carriers. Additionally, water sources may become contaminated through open defecation, which can help spread the eggs, as T. solium eggs can survive in both stagnant and flowing water for long periods [29].

Although no differences were observed in seroprevalence based on the locality where pigs were acquired, significant spatial heterogeneity was evident at the household-level. The Checche locality showed a notably high seroprevalence (60%), indicating a hyperendemic focus. This high prevalence is likely related to the remoteness of Checche and its limited sanitation infrastructure, factors known to hinder livestock commercialization and increase the risk of animal neglect [3, 37].

Cold and humid Andean soils may further support egg survival, maintaining environmental reservoirs even in areas with relatively low human infection pressure. Additionally, the practice of home slaughter, reported by nearly one-fifth of pig owners, increases the risk of human exposure to infected meat, emphasizing the importance of pre-slaughter inspection [29]. The observed spatial clustering underscores the value of GIS–based analyses for identifying micro-endemic foci and prioritizing targeted interventions.

Knowledge that humans can become infected with cysticercosis was significantly linked to porcine cysticercosis, consistent with findings from Tanzania, where limited awareness among household members was associated with increased pig seropositivity [18, 38, 39]. Previous studies have also indicated connections between prior herd infection and ongoing transmission [29]. Notably, Puerta et al. [34] reported that only 15% of pig farmers knew the routes of human infection.

Limited understanding of cysticercosis and its transmission cycle can lead to practices that sustain the parasite’s life cycle [40–42]. These results highlight the importance of ongoing health education programs focused on the life cycle of T. solium, zoonotic risks, and related economic impacts, as lack of knowledge can hinder control and eradication efforts [25, 32, 43].

A key finding of this study was the link between the sex of pig owners and porcine cysticercosis, with pigs raised by women showing a threefold higher risk of infection (OR = 3.2). This aligns with prior reports indicating that women have less cysticercosis-related knowledge compared to men [43]. In rural areas, men often travel to urban centers for work, gaining more exposure to health information, while women, mainly involved in household and animal care, may have fewer opportunities to access such knowledge.

These results highlight the need to include a gender-sensitive approach in cysticercosis control programs. Intersectoral and geotargeted strategies are recommended, using GIS-based hotspot identification to inform surveillance and quick response [3]. Control efforts should focus on targeted health education for women pig farmers, improving sanitation infrastructure and waste management in high-risk areas, and enhancing animal health interventions, such as deworming and meat inspection, all within a comprehensive One Health framework.