Introduction

Enteral nutritional therapy (ENT) is typically administered to malnourished patients or those at nutritional risk, aiding in the recovery or maintenance of nutritional status for individuals unable to meet their needs through oral feeding [1]. The use of home enteral nutrition (HEN) is encouraged, as it improves patients’ health outcomes, reduces clinical and nutritional complications, and transitions care from hospitals to households while lowering healthcare costs [2, 3].

Various enteral formulations are used in HEN, including food-based or homemade enteral preparations (HEP), made exclusively from fresh foods; commercial enteral formulas (CEF), composed of isolated nutrients and available ready-to-use or in powder form; and blended enteral preparations (BEP), combining natural foods with commercial formulas [4, 5].

Although CEFs are considered safer in terms of nutritional and hygienic standards, HEPs and BEPs are often preferred due to their customizable nutritional composition, adjustable volume, and lower cost [5, 6].

However, the extensive handling and inclusion of fresh produce in HEPs/BEPs increase the risk of pathogen contamination [7], posing a hidden threat to immunocompromised patients.

While bacterial contamination in HEN formulations has been extensively studied [8–13], no data exist on foodborne parasite contamination, representing a critical gap in HEN safety knowledge. Foodborne pathogens cause 600 million illnesses and 420,000 deaths annually [14], with parasitic diseases affecting 407 million people, including 91.1 million foodborne cases and 52,000 deaths [15]. Notably, helminths rank among the most prevalent global foodborne parasitic hazards [16].

Parasites exhibit high resistance to environmental stressors and disinfection, with common contamination sources including fresh juices, fruits, and vegetables [17–19], frequent ingredients in enteral formulations. Beyond acute diarrhea, intestinal parasites can lead to chronic malnutrition, iron-deficiency anemia, and physical/cognitive impairments, further compromising HEN patients’ already fragile health [20, 21].

Despite the benefits of HEN, no standardized methods or International Organization for Standardization (ISO) norms exist for detecting parasites in enteral formulations. Given this gap and the potential health risks, this study aimed to standardize methodologies for the detection of helminth eggs in HEPs containing fresh produce, enabling future monitoring of HEN safety for vulnerable patients.

Materials and methods

Protocols for determining the recovery efficiency of A. suum eggs in enteral food preparations

A known quantity of A. suum eggs (Dose 1 or Dose 2) was added to each HEP sample. To standardize the method, four protocols were tested in triplicate for each dose/HEP combination, totaling 48 artificial contamination trials (Figure 1).

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Figure 1. Protocols and variables were analyzed for the standardization of a methodology for detecting helminth eggs in homemade enteral preparations (HEP) containing vegetables or fruits, artificially contaminated with two doses of Ascaris suum.

The protocols compared recovery efficiency using the following variables: 1) Dissociation solution—use of 1 M glycine (Synth, Brasil), pH 5.5 or 0.1% Alconox (Alconox Inc., USA); 2) Homogenization—by manual stirring or magnetic stirrer. All protocols shared the following steps: sedimentation time, centrifugation, and reading the entire sediment.

Results

The average egg recovery efficiency rates across the four protocols and two doses (48 total trials) ranged from 47% to 66% for HEP 1 (Table 1) and 40% to 66% for HEP 2 (Table 2).

For HEP 1, Protocol 2 demonstrated the highest recovery rates, using glycine (1 M, pH 5.5) with magnetic stirrer homogenization, achieving 66% for Dose 1 and 55% for Dose 2. Although this method produced a more turbid sample (Figure 2A) compared to the clearer Alconox^® solutions (Figure 2B), it was selected as the optimal method for leafy green vegetable HEPs due to its superior recovery performance.

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Figure 2. Recovery of A. suum eggs from home enteral preparation 1 (HEP 1) using distinct dissociation solutions. (A) A. suum egg (red arrow), visualized at 40× magnification in HEP 1 (1 M glycine, pH 5.5). (B) A. suum egg (red arrow), visualized at 40× magnification in HEP 1 (0.1% Alconox). Scale: 50 μm.

For HEP 2, Protocol 4 using 0.1% Alconox^® with magnetic stirrer homogenization showed the best results, with recovery rates of 66% for Dose 1 and 52% for Dose 2. This protocol was consequently chosen for berry fruit-containing HEPs.

Ascaris sp. eggs were not detected in any blank/negative controls analyzed.

The statistical analysis revealed significant differences between protocols for Dose 1 (207 eggs) in both HEP types (HEP 1: p = 0.01; HEP 2: p = 0.00001), while no significant differences were found for Dose 2 (76 eggs) in either HEP 1 (p = 0.74) or HEP 2 (p = 0.70). Comparison of recovery efficiency between doses within each protocol showed no statistically significant differences for either HEP type: HEP 1 showed p = 0.110 for Dose 1 and p = 0.204 for Dose 2, while HEP 2 showed p = 0.103 for Dose 1 and p = 0.502 for Dose 2.

During the standardization of the methodology, natural contamination by Toxocara sp. eggs were detected in HEP 1, and hookworm eggs in HEP 2. The viability test using Trypan Blue dye revealed that both eggs were viable, as they did not take up the dye.

Discussion

The presence of pathogens in fresh produce and ingredients commonly used in blenderized formulations in HEN, can pose a threat to human health since this type of food does not undergo thermal treatment [11, 27]. Among them, parasites remain a great challenge because they are difficult to remove from fresh produce, are highly resistant to sanitization processes, and exhibit a low infective dose, amplifying both the likelihood of transmission and the associated public health risks [28, 29]. This is particularly concerning for immunocompromised patients (e.g., immunodeficient or immunosuppressed individuals), since the use of parasite-exposed HEN may exacerbate their clinical condition.

However, the detection of foodborne parasites is usually underreported [30, 31] and faces challenges due to methodological complexity and, in some cases, high isolation costs [32, 33], which can represent a prohibitive cost for regions more vulnerable and susceptible to intestinal parasitosis, such as Latin America.

Moreover, there are currently no regulations assessing parasitic contaminants in complex food matrices like fresh-food enteral formulations. These nutrient-dense mixtures, designed for medically vulnerable patients, combine multiple ingredients that complicate parasite detection.

To the best of our knowledge, this is the first study to standardize and measure the detection sensitivity of two methodologies for detecting parasites in mixed fresh produce used in enteral formulations intended for patients with special healthcare needs.

The standardized protocols for helminth egg detection presented here offer a practical tool that can be readily implemented by both public and private food safety agencies. The protocols would enhance food safety monitoring and reinforce public health protection. Furthermore, a validated method would enable rapid response during foodborne outbreak investigations involving these products.

Effective helminth parasite detection in food involves critical steps, as it requires optimal elution and concentration from the food matrix [34, 35]. While various solutions exist for this purpose, we selected two specific approaches for testing in HEP: a surfactant-based solution (0.1% Alconox^®); and an amino acid solution (1 M glycine, pH 5.5).

Glycine is synthesized through alkaline hydrolysis of meat/gelatin using potassium hydroxide and chemically produced from monochloroacetic acid and ammonia [36]. Due to its surfactant properties, glycine effectively interacts with cysts, oocysts, and helminth eggs that are tightly bound to plant matrices, enhancing their detachment [23, 24, 32].

The composition of Alconox^® contains a surfactant called sodium dodecylbenzenesulfonate (C₁₂H₂₅C₆H₄SO₃Na) and tetrasodium pyrophosphate (Na₄P₂O₇). The mixture of these two compounds has been shown to effectively remove protozoa from fresh produce [37, 38].

Given that enteral formulations can be made up of different ingredients, and that caregivers of bedridden patients add fresh produce, standardizing and measuring the sensitivity of the methodology by applying different eluent solutions demonstrates the need for adaptation and choice depending on the type of food used in the enteral formulation (whether composed of fruits or vegetables). Our results from artificial contamination experiments show that for HEPs composed mostly of vegetables, the 1 M glycine (pH 5.5) solution should be used for helminth egg identification, as it achieved the highest recovery rates with both doses of A. suum eggs. Similarly, for HEPs composed of berry fruits, the best protocol performance was observed when using the 0.1% Alconox^® dissociation solution, regardless of the dose applied.

Previous studies evaluating helminth egg recovery from leafy vegetables have typically employed only one homogenization method [23, 24]. In contrast, our study directly compared two approaches: manual agitation versus magnetic stirring. For both HEP formulations, magnetic stirring yielded superior recovery rates across all doses and dissociation solutions. This advantage likely stems from reduced sample handling and the use of glass beakers, which minimize egg adhesion compared to other materials [39].

In our standardization using HEP 1 (leafy vegetables) and HEP 2 (fruits), we evaluated two contamination levels: a higher dose (207 eggs) and a lower dose (76 eggs). This dual-dose approach strengthens the methodology by demonstrating consistent egg recovery efficiency across different potential contamination scenarios.

The two optimal protocols demonstrated robustness through both high recovery rates and their ability to detect naturally occurring helminth contaminants. During artificial contamination trials using a glycine solution (1 M, pH 5.5) with HEP-containing lettuce, watercress, cabbage, and orange, we identified natural contamination by Toxocara sp. eggs.

Toxocara sp. ranks 20th in the FAO/WHO risk assessment of foodborne parasites [16]. Despite this classification, toxocariasis remains significantly underreported while posing substantial public health challenges. The highest disease burden occurs in Brazil, China, the United States, Korea, Japan, India, Austria, and France [40].

Also, viability tests applied to naturally detected eggs in this study, including Trypan Blue staining, confirmed their viability. This remains the most common viability assessment method due to its cost-effectiveness and simplicity for evaluating Toxocara sp., Ascaris sp., and Trichuris sp. [25, 41]. As a colorimetric reagent, Trypan Blue stains only dead cells (visible under low-resolution microscopy) by penetrating compromised membranes, while viable cells exclude dye [41]. Therefore, the finding of viable eggs, with the potential to infect humans, adds relevance to public health, especially for the vulnerable group that receives this type of food.

Further, the protocol using Alconox^® enabled the detection of hookworm eggs in strawberry preparation. While these eggs are non-infective in this stage, their presence indicates fecal contamination [42].

Due to the growing reliance on outpatient nutritional care, HEN has become an increasingly important intervention. Recognized for its cost-effectiveness, HEN optimizes dietary transitions and improves patients’ quality of life [2]. However, epidemiological data from Europe, North America, Australia, and New Zealand reveal that a significant proportion of foodborne illnesses stem from inadequate food preparation practices in household settings [43, 44].

The detection of other genera of helminth eggs during standardization reinforces that washing with running water alone is not sufficient, demonstrating that parasite removal efficiency depends on both processing methods (washing and elution) and is closely related to food matrix characteristics. Fruits and leafy vegetables have irregular, porous, and rough surfaces, which significantly hinder the effective removal of attached parasites [28].

Moreover, the development of methods for detecting and determining the viability of infectious stages of pathogens in food and other matrices provides important tools for identifying and understanding transmission routes and estimating the risk of infection [29]. While our study established standardized methodologies, it would be relevant for future work to perform its validation and evaluate home-prepared formulations, as we could not control ingredient selection by caregivers. However, between main meals, it is common for individuals who require HEN to receive juices containing vegetables and fruits.

This finding highlights the need to establish stricter guidelines regarding the quality of home-prepared enteral formulations, including the specific consideration of the presence of parasites in enteral nutrition (EN), as well as the implementation of rigorous sanitary control measures, to reduce the risk of a wide variety of foodborne diseases.

These guidelines can support decision-making by healthcare professionals, regulatory agencies, caregivers, and public health managers involved in HEN, promoting the improvement of protocols, standards, safe practices, and policies focused on contamination control and risk reduction to health.