In besides bacterial and viral infections, protozoan parasites may infect the human digestive tract, leading to severe illnesses. Cryptosporidium parvum, Giardia lamblia and Entamoeba histolytica are the principal protozoal infections responsible for diarrhoea and associated illnesses (giardiasis and amoebiasis, respectively), which are characterised as acute and self-limiting dysentery. However, in certain patients, the condition may progress to a chronic state, resulting in long-term consequences such as starvation, growth retardation and cognitive deficits [1,2]. Protozoan parasites are unicellular eukaryotes that inhabit either extracellularly or intracellularly within host cells. They have developed into effective pathogens owing to their exceptional capacity to circumvent immunological responses, enabling them to elude both adaptive humoral and cellular immunity [3]. E. histolytica, as an obligatory parasite in the large intestine, occupies a niche alongside numerous microbial occupants, feeding on bacteria and its pathogenicity is directly associated with exposure to bacterial microbiota. Infection with E. histolytica directly affects the bacterial communities inside the gut microbiota [4]. Giardiasis is an ailment induced by Giardia intestinalis (G. intestinalis) in the small intestine. The infection may be asymptomatic or may manifest symptoms like intermittent diarrhoea, nausea, vomiting, anorexia, epigastric discomfort, weakness and weight loss. Moreover, the parasite's absorbing discs induce mucosal irritation, excessive mucus production and different absorption abnormalities [5].

Oxidative stress significantly influences the progression and outcome of parasitic infections in both the host organism and the parasite attempting to endure. The host employs substantial quantities of Reactive Oxygen Species (ROS), primarily superoxide anion (O2•−) and Hydrogen Peroxide (H2O2), to combat the advancing parasite illness. Conversely, the parasite evolves very powerful defence mechanisms and resistance to ROS, endeavouring to survive within the colonised host organism by exploiting available resources and its living environment, thereby inducing the host's deterioration.[6]. Oxidative damage occurs, resulting in increased lipid peroxidation and elevated MDA levels in patients. The prolonged infection with these parasites and the resultant accumulation of free radicals can significantly deplete both enzymatic and non-enzymatic antioxidants in the body, thereby disrupting oxidative balance and resulting in oxidative damage for patients. This, in turn, contributes to various chronic diseases, including cancer, cardiovascular disease and diabetes [7]. In mammals, the regulation of osmotic pressure and water distribution throughout distinct bodily fluid compartments is predominantly governed by the four principal electrolytes: Na+, K+, Cl- and HCO3-. Besides water homeostasis, these electrolytes are crucial for pH maintenance, regulation of cardiac and muscular function, participation in oxidation-reduction reactions and serving as enzyme cofactors in catalysis [8]. It is evident that aberrant electrolyte levels may be either a cause or a consequence of several illnesses and the assessment of electrolytes is a critical function of the clinical laboratory [9].

The study involved the collection of 110 stool samples (88 from patients with chronic diarrhoea and 22 additional samples) using clean, sealed plastic containers with lids. These samples were obtained from children under 3 years of age suffering from chronic diarrhoea and other intestinal disorders who visited hospitals in Salah al-Din Governorate and certain private laboratories, during the period from early December 2024 to March 2025. The personal information was documented using a designated form that encompassed gender, age, weight, date of visit, area of residence, intestinal parasite infection and sickness symptoms. Upon microscopic examination and ELISA analysis of each sample, the samples were categorised into three groups based on the aetiology of the diarrhoeal infection as follows:

• Group 1: For patients suffering from diarrhea and diagnosed with Giardia lambelia (n:28)

• Group 2: For patients suffering from diarrhea and diagnosed with E. histolytica (n:20)

• Group 3: For patients suffering from non-parasitic diarrhea (n:40)

• Group 4: For healthy individual as control (n:22)

Microscopic Examination 

Stool samples were analysed in the laboratory by assessing consistency, colour, odour and the presence of mucus or blood. Stool specimens were obtained with a sterile wide-mouth screw-cap container and fresh samples were analysed under light microscopy at high power magnification of 40X [10]. A small volume (1-3 ml) of stool specimens was collected in sterile screw-cap containers and stored at -20°C until analysed by ELISA. 

Diagnosis using ELISA Technique to Detect Parasite Antigens in Stool Samples

Microscopy-positive samples underwent additional analysis using DRG ELISA for antigen detection of G. lamblia and E. histolytica; positive specimens were subsequently evaluated using TechLab E. histolytica II monoclonal ELISA for antigen detection. 

Collecting Blood Samples

Five milliliters of venous blood were collected using a sterile, dry medical syringe from each individual with diarrheal infection at various hospitals in Salah Al-Din Governorate. The blood samples were placed in gel tubes and allowed to coagulate at room temperature for 15-20 minutes. Subsequently, the tubes were centrifuged at 3000 revolutions per minute for 10 minutes to extract the serum, which was then transferred into Eppendorf tubes for various tests and stored at -20°C until the immunological assay was conducted. All samples were analysed simultaneously to prevent repeated freezing and thawing, which could compromise result quality.

Assessment of GSH, MDA and CAT by Enzyme Linked Immunosorbent Assay ELISA

The ELISA technique was utilised to assess serum concentrations. The levels of GSH, MDA and CAT were measured using the ELISA technique. The plate was pre-coated with antibodies specific to human GSH, MDA and CAT. The sample is augmented with GSH, MDA and CAT concentrations. The levels of human GSH, MDA and CAT demonstrated a positive association with the development of colour in the substrate solution. The process is terminated by the addition of an acidic stop solution, after which absorbance is quantified at a wavelength of 450 nm.

Assessment Biochemical Test

Concentrations of [K], [Na] and [Cl], were assessed utilising various analyses developed by the Japanese firm FUJIFILM (FUJI DRI-CHEM SLIDE).

Statistical Analysis

The statistical outcomes were evaluated employing the t-test and ANOVA, with arithmetic means compared by Duncan's multiple range test at a significance threshold of 0.05 [11].

The current investigation revealed an infection rate of 28 (31.8%) for the G. lamblia parasite and 20 (22.73%) for E. histolytica. In non-parasitic diarrhoea, the incidence was 40 (45.5), as illustrated in Table 1 and Figure 1.
 

Figure 1: Microscopic examination, A,The cyst stage of Giardia intestinalis parasite from a stool sample stained with 1% local iodine under 100X. B, The cyst stage of E,histolytica from a stool sample stained with 1% local iodine under 100X

Table 1: The prevalence of diarrhea depend on parasitic and non-parasitic cause

Table 2: Level of GSH, MDA, CAT in parasitic diarrheal infection and non-parasitic diarrhea

Table 3: Level of electrolyte in parasitic diarrheal infection and non-parasitic diarrhea

The present study demonstrated decrease level of GSH in all three groups G1, G2, G3 as compared with control G4 that were (1.5±0.6, 1.9±0.4, 1.5±0.5, 4.2±1.2) pg/ml respectively. While highly increase MDA in patient in G2, followed by G1 and G3 as compared with control were (7.2±1.1, 5.2± 0.9, 5.1±0.5, 3.1±0.5) pg/ml. CAT highly decrease in G2(287±35.53), followed by G1and G3(335±41.21, 312±29.12) as compared with G4(426±50.16). AS shown in Table 2.
 

The present study demonstrated hyponatremia, Hypochloremia and hypokalemia in both parasitic and non-parastic diarrheal infection (Table 3).
 

Entamoeba histolytica induces tissue invasion and damage to the intestinal mucosa, resulting in ulceration, inflammation and pathological alterations in the epithelial cell villi during the acute phase of infection. This leads to the shortening of intestinal villi and dilation of the crypts, which can activate mesenteric lymph nodes and subsequently stimulate adjacent adipose tissues to secrete leptin. In contrast, Giardia exhibits little to no tissue invasion, resulting in a negligible increase in leptin levels. 

The current study concurs with [12, 13], which demonstrated an elevation of oxidative stress in E. histolytica patients compared to Giardia lambelia.

E. histolytica are subjected to elevated levels of ROS during tissue invasion, intestinal colonisation and extraintestinal propagation, in contrast to G. lamblia. Additionally, enhanced ROS is produced during the degradation of haemoglobin by E. histolytica [14,15]. 

Entamoeba histolytica is an intestinal protozoan parasite responsible for amebic dysentery and liver abscesses in millions of individuals residing in endemic regions. E. histolytica trophozoites encounter highly hazardous reactive oxygen and nitrogen species during tissue invasion. The ability of E. histolytica trophozoites to endure reactive oxygen and nitrogen species is crucial to its pathogenicity and disease progression. E. histolytica is deficient in the majority of the elements of standard eukaryotic antioxidative defence mechanisms, such as CAT, GSH and its metabolic enzymes, serving as the predominant intracellular low molecular weight thiol. This parasite possesses a functioning thioredoxin system, comprising thioredoxin reductase and thioredoxin crucial for maintaining cellular redox balance and antioxidant function [16,17].

The current study demonstrated a reduction in antioxidants such as glutathione and catalase in diarrhoeal patients, irrespective of the infection's aetiology, whether parasitic or non-parasitic. This depletion in GSH concentrations can be elucidated, as GSH, a critical cellular antioxidant, safeguards cells from oxidative damage by interacting with free radicals generated through lipid peroxidation and peroxidase activity. The current study's findings concur with [18], indicating a reduction in glutathione levels in patients with E. histolytica. Glutathione has been demonstrated to safeguard cells against oxidative stress, inhibiting the oxidation-induced conversion of haemoglobin into methaemoglobin. Moreover, it maintains sulfhydryl (-SH) groups in proteins in a reduced form, safeguarding them from oxidation and preventing the deactivation of functional proteins and enzymes [19].Parasitic illnesses are marked by malnutrition, deficiency in essential micronutrients, compromised digestion, reduced nutritional absorption and chronic inflammation [20]. Gastrointestinal parasitism may lead to electrolyte abnormalities like to those observed in hyperkalaemia and hyponatraemia. This investigation indicates a decrease in the concentrations of Na, K, Cl. This work concurs with [21, 22] that Na and K are essential electrolytes that significantly influence the maintenance of acid-base balance and osmotic pressure. Decreased concentrations of these ions can result in substantial problems and unpredictable disruptions in biological processes.

Disorders of sodium levels in cases of diarrhea-related dehydration can represent a medical emergency necessitating fast and effective diagnosis and care. Various studies have demonstrated differing prevalences of hyponatraemia and hypernatraemia in children experiencing dehydration. Research conducted by Samadi et al. indicated that hyponatraemia and hypernatraemia occurred in 20.8% and 6.4% of cases, respectively [23]. The research conducted by Shah et al. revealed that 56% of patients admitted with diarrhoea and dehydration exhibited hyponatraemia, whereas hypernatraemia was observed in 10% of cases [24]. 
 

Hypochloremia results from the depletion of chloride ions due to gastrointestinal conditions such as diarrhoea, vomiting and the utilisation of a nasogastric tube. Excessive vomiting can lead to a markedly greater depletion of chloride than sodium, as the chloride concentration in the stomach exceeds 100 mEq/L, whilst sodium levels are comparatively lower, ranging from 20 to 30 mEq/L. Diarrhoea induced by many disorders can lead to differing levels of electrolyte depletion in the faeces [25].

The present study concluded Giardia lamblia and Entamoeba histolytica, are most common cause of diarrheal infection. In addition, this study concluded increase MDA in diarrheal infection with both parasitic and non-parasitic, while more increase in Entamoeba histolytica. Furthermore, decrease CAT, GSH and electrolyte in all patients

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