Cancer remains one of the major causes of morbidity and mortality worldwide and represents an increasing challenge to healthcare systems, particularly in low- and middle-income countries. Patients with malignancies are susceptible to a wide range of metabolic, hematological, nutritional and infectious complications resulting from the malignancy itself, impaired immune function, malnutrition, invasive procedures and anticancer therapy. Among these complications, anemia and disturbances of iron metabolism are particularly common and may substantially affect patients’ functional status, tolerance of treatment, quality of life and clinical outcomes [1]. The interaction between cancer, inflammation, infection and iron homeostasis is therefore increasingly recognized as an important component of supportive oncological care. Iron is an essential micronutrient involved in oxygen transport, cellular respiration, DNA synthesis, mitochondrial function and numerous enzymatic reactions. Iron availability must, however, be tightly controlled because both iron deficiency and iron excess can have harmful biological consequences. Systemic iron homeostasis is regulated primarily through intestinal absorption, macrophage recycling, hepatic storage, transferrin-mediated transport and the hepcidin–ferroportin regulatory axis [2]. Hepcidin, predominantly synthesized by the liver, reduces circulating iron by inducing the internalization and degradation of ferroportin, thereby decreasing intestinal iron absorption and restricting the release of stored iron from macrophages and hepatocytes. Inflammatory cytokines, particularly interleukin-6, can stimulate hepcidin production and consequently produce functional iron deficiency despite apparently adequate or increased body iron stores [3].

This mechanism is especially relevant in malignancy. Cancer-associated anemia is frequently multifactorial and may arise from chronic inflammation, nutritional

deficiencies, occult or overt blood loss, bone marrow infiltration, renal dysfunction, chemotherapy, radiotherapy and impaired erythropoiesis. Inflammation-mediated iron sequestration contributes significantly to anemia of chronic disease or anemia of inflammation, in which serum iron and transferrin saturation are generally reduced while ferritin concentrations may remain normal or become elevated [4]. Consequently, conventional interpretation of iron status in patients with cancer can be challenging because ferritin functions not only as an indicator of iron storage but also as an acute-phase reactant. Assessment of several parameters, including serum iron, ferritin, Total Iron-Binding Capacity (TIBC), transferrin saturation and hemoglobin, can therefore provide a more comprehensive picture of iron status.

At the same time, patients with malignancies may have an increased susceptibility to infectious diseases. Immunosuppression associated with the underlying cancer, chemotherapy, corticosteroid therapy, disruption of epithelial barriers, nutritional deterioration and repeated exposure to healthcare environments can facilitate infection with a variety of microorganisms [5]. Although bacterial, viral and fungal infections receive considerable attention in oncology, parasitic infections may be underestimated, particularly in regions where intestinal parasites remain endemic. Such infections may have important consequences in immunocompromised patients because they can persist for prolonged periods, produce gastrointestinal manifestations, contribute to malnutrition and potentially aggravate pre-existing hematological abnormalities [6].

Entamoeba histolytica is a pathogenic intestinal protozoan responsible for amoebiasis and continues to represent an important public-health problem in many developing countries. Transmission occurs predominantly through ingestion of mature cysts in food or water contaminated with human feces. Following ingestion, excystation occurs in the intestine and trophozoites colonize the large bowel. Infection may remain asymptomatic or progress to invasive intestinal disease characterized by abdominal pain, diarrhea, dysentery, mucosal ulceration and gastrointestinal bleeding. In some patients, trophozoites disseminate through the portal circulation and cause extraintestinal disease, most notably amoebic liver abscess [7]. The clinical spectrum of infection is influenced by parasite virulence, host immunity, nutritional status, intestinal microbiota and other host-related factors.

The relationship between E. histolytica and iron metabolism is biologically important because iron is required not only by the human host but also by microorganisms and parasites. Iron participates in several processes necessary for parasite growth, metabolism and pathogenicity. E. histolytica possesses mechanisms that enable it to acquire iron from the host environment, while alterations in iron availability may influence parasite survival and virulence [8]. Conversely, invasive intestinal amoebiasis may adversely affect the host's iron status through intestinal inflammation, mucosal destruction, microscopic or clinically evident blood loss, reduced dietary intake, impaired nutrient absorption and systemic inflammatory responses. These mechanisms could potentially intensify an existing disturbance of iron homeostasis in individuals with chronic diseases.

This interaction may be particularly significant among patients with malignancies. Cancer itself can produce a persistent inflammatory state characterized by increased production of cytokines and alterations in iron regulatory pathways [9]. Superimposed intestinal infection could theoretically increase inflammatory activity and further restrict circulating iron. In addition, gastrointestinal symptoms associated with amoebiasis may reduce food intake or contribute to intestinal blood loss, thereby adding an absolute iron-deficiency component to the functional iron deficiency associated with malignancy. Patients with cancer and concomitant E. histolytica infection may therefore demonstrate a complex pattern involving low serum iron, altered TIBC, reduced transferrin saturation, abnormal ferritin concentrations and decreased hemoglobin.

Another important issue is the accurate detection of E. histolytica. Conventional stool microscopy remains widely used because it is inexpensive and accessible; however, morphological differentiation of pathogenic E. histolytica from closely related nonpathogenic species can be difficult. Antigen-detection assays and molecular techniques provide improved specificity and can strengthen the diagnosis where available [10]. The use of appropriate diagnostic approaches is particularly important in oncology populations because unnecessary treatment and missed infections may both have adverse consequences. Combining parasitological evaluation with biochemical and hematological measurements may provide valuable information regarding the potential clinical impact of infection. The burden of cancer is also increasing in Iraq, creating a growing need to investigate potentially modifiable factors that may influence the health and treatment outcomes of oncology patients [11]. Intestinal parasitic diseases remain relevant within the region because environmental sanitation, water quality, socioeconomic conditions and food hygiene can influence transmission. Nevertheless, relatively little attention has been directed toward the relationship between intestinal parasitic infections and abnormalities of iron metabolism among Iraqi patients with malignancies. Most available investigations have considered parasitic infection, anemia, or cancer separately rather than evaluating their possible interaction within the same patient population. Understanding this relationship could have practical implications. Detection and treatment of an unrecognized intestinal parasitic infection may represent a potentially modifiable contributor to anemia or iron abnormalities. Likewise, identifying characteristic changes in iron parameters among infected patients could improve clinical interpretation of laboratory findings and encourage more comprehensive assessment of anemia in patients with cancer. This is particularly relevant because inappropriate empirical iron supplementation may be ineffective when anemia is predominantly inflammation-mediated, whereas untreated absolute iron deficiency or ongoing gastrointestinal blood loss requires appropriate investigation and management [12]. The study focuses on determining the prevalence of E. histolytica infection and assessing key indicators of iron metabolism, including serum iron, ferritin, TIBC, transferrin saturation and hemoglobin. These parameters will be compared between infected and non-infected patients to determine whether E. histolytica infection is associated with a distinct or more pronounced pattern of iron dysregulation. By examining parasitic infection and iron metabolism together in an oncological population, the study seeks to address an important gap in local evidence and provide findings that may support improved laboratory assessment, infection detection and management of anemia among patients with malignancies in Iraq.

This analytical cross-sectional study was conducted to investigate the association between Entamoeba histolytica infection and disturbances of iron metabolism among adult patients with malignancies attending selected private oncology clinics in Iraq. The analytical cross-sectional design allowed simultaneous assessment of E. histolytica infection and iron-related laboratory parameters and comparison of iron status between infected and non-infected patients. The study design was consistent with the approved research protocol.

Study Period

Participant recruitment and specimen collection were conducted from 20 February 2026 to 20 September 2026. During this period, eligible patients attending the participating clinics were consecutively screened and enrolled.

Study Population

The study population consisted of adult patients diagnosed with either solid or hematological malignancies. A diagnosis of malignancy had to be confirmed by an oncologist before enrollment. Patients aged 18 years or older who attended the selected private clinics in Kirkuk city during the study period were considered for participation.

After clinical and laboratory evaluation, participants were categorized according to their E. histolytica serological status into E. histolytica IgG-positive and IgG-negative groups for comparison of iron-related laboratory parameters.

Inclusion Criteria

Patients were eligible when they fulfilled the following criteria:

• Age ≥18 years
• Confirmed diagnosis of a solid or hematological malignancy
• Diagnosis confirmed by an oncologist
• Ability to provide the required blood specimen
• Provision of written informed consent

Exclusion Criteria

Patients were excluded if they had received iron supplementation during the preceding four weeks, had undergone blood transfusion within the previous three months, had chronic renal failure, had active gastrointestinal bleeding, or refused to participate.

Sampling Method and Sample Size

A consecutive sampling method was used. All eligible patients attending the participating clinics during the study period were invited to participate until the required sample size was achieved.

The study protocol estimated the required sample size according to an expected prevalence of parasitic infection of approximately 15–20%, with a 95% confidence level and 80% statistical power. The planned sample size was approximately 150–200 patients.

Data Collection

A structured data collection form was completed for each participant. Demographic and relevant clinical information was recorded after obtaining written informed consent. Each participant was assigned a unique identification code to maintain confidentiality and allow linkage of clinical and laboratory data without including personally identifying information.

Blood Sample Collection and Serum Preparation

Approximately 3–5 mL of venous blood was collected from each participant under standard aseptic conditions. Blood intended for serum analysis was allowed to clot, after which serum was separated. Serum specimens were used for the measurement of iron-related biochemical parameters and for detection of serum IgG antibodies against E. histolytica.

For the E. histolytica IgG assay, serum samples that were not tested immediately were stored at −20°C or lower until analysis. Before testing, patient sera were diluted 1:64 using the supplied dilution buffer; this was prepared by mixing 5 µL of serum with 315 µL of dilution buffer.

Laboratory Investigations

Laboratory investigations included detection of serum IgG antibodies against E. histolytica and assessment of iron metabolism through measurement of serum iron, ferritin, total iron-binding capacity (TIBC), transferrin saturation and hemoglobin.

ELISA for Entamoeba Histolytica Serum IgG

Serum IgG antibodies against E. histolytica (Amebiasis) were determined using a commercially available ELISA kit manufactured by Diagnostic Automation Inc., Germany, according to the manufacturer's instructions [114].

Principle of the Assay

The E. histolytica (Amebiasis) IgG ELISA was based on a three-incubation procedure. The microwells were precoated with E. histolytica antigen. During the first incubation, antibodies specific to E. histolytica, when present in the diluted patient serum, bound to the immobilized parasite antigens. The wells were subsequently washed to remove unbound components of the serum.

An enzyme conjugate was then added. During the second incubation, the enzyme conjugate bound to antibodies retained within the antigen-coated wells. Following another washing step to remove unbound conjugate, tetramethylbenzidine (TMB) chromogenic substrate was added. In the presence of bound peroxidase enzyme conjugate, the substrate reaction produced a blue color.

The reaction was terminated by addition of stop solution, which changed the blue color to bright yellow. The intensity of the resulting color was proportional to the amount of antibody bound in the well and was measured using an ELISA microplate reader at the specified wavelength.

Reagents

The E. histolytica IgG ELISA kit contained the following reagents:

• Test Strips: 96 microwells coated with E. histolytica strain NIH-200 antigens and supplied in a test-strip holder
• Enzyme Conjugate: One bottle containing 11 mL of Protein A conjugated to peroxidase
• Positive Control: One vial containing 1 mL of diluted positive rabbit serum
• Negative Control: One vial containing 1 mL of diluted negative human serum
• TMB Substrate Solution: One bottle containing 11 mL of tetramethylbenzidine
• Wash Concentrate (20×): One bottle containing 25 mL of concentrated buffer and surfactant
• 1% Dilution Buffer: One or two bottles containing 30 mL of buffered protein solution
• Stop Solution: One bottle containing 11 mL of 0.73 M phosphoric acid

Reagent Preparation

The wash solution was prepared before performing the assay. The entire contents of the wash concentrate bottle were added to 475 mL of reagent-grade water and thoroughly mixed. The resulting diluted wash buffer was transferred to a squeeze bottle with a narrow-tip opening and used for the washing steps throughout the assay.

Other kit reagents were used according to the manufacturer's instructions.

Serum Preparation for ELISA

After blood coagulation, serum was separated from the cellular components. When analysis could not be performed immediately, serum specimens were frozen at −20°C or below until testing.

Before the assay, each patient serum sample was diluted at a ratio of 1:64 with the supplied dilution buffer. A working dilution was prepared by adding 5 µL of patient serum to 315 µL of dilution buffer. The positive and negative controls supplied with the kit were already prediluted and were therefore used without further dilution.

Materials Required

In addition to the reagents supplied with the ELISA kit, the following materials and equipment were used:

• Adjustable micropipettes
• Reagent-grade water
• Graduated cylinder
• Tubes for serum sample dilution
• Absorbent paper or paper towels
• ELISA microplate reader equipped with a 450 nm primary filter and a 620–650 nm reference filter

ELISA Procedure

The assay was performed according to the manufacturer's recommended procedure [114]:

• The required number of antigen-coated wells was removed from the kit and placed in the strip holder. The number included two control wells in addition to the wells required for patient samples
• 100 µL of the supplied negative control was added to well No. 1
• 100 µL of the supplied positive control was added to well No. 2
• 100 µL of each 1:64 diluted patient serum sample was added to the remaining designated wells
• The plate was incubated at room temperature (15–25°C) for 10 minutes
• Following incubation, the contents of the wells were discarded and the wells were washed three times using the prepared diluted wash buffer
• Two drops of enzyme conjugate were added to each well
• The plate was incubated at room temperature for 5 minutes
• The contents were discarded and the wells were washed another three times with wash buffer
• After the final wash, the wells were tapped against absorbent paper to remove residual moisture
• Two drops of TMB chromogen were added to each well
• The plate was incubated at room temperature for 5 minutes
• Two drops of stop solution were added to each well
• The contents were mixed gently by tapping the strip holder
• The optical density of each well was subsequently measured using the ELISA reader

Interpretation of E. Histolytica IgG ELISA

Before reading the plate, the ELISA reader was zeroed on air. Absorbance was measured at 450 nm with a reference wavelength of 620–650 nm.

The results were interpreted according to the manufacturer's specified cutoff [114]:

• Positive: Absorbance >0.4 optical density (OD) units
• Negative: Absorbance <0.4 OD units

Accordingly, participants with an absorbance above 0.4 OD were classified as E. histolytica serum IgG-positive, whereas those with an absorbance below 0.4 OD were classified as serum IgG-negative.

Because this assay detects serum IgG antibodies, the measured variable was treated as E. histolytica seropositivity in the laboratory analysis.

Assessment of Iron Metabolism

The iron profile included measurement of serum iron, serum ferritin, TIBC, transferrin saturation and hemoglobin, corresponding to the secondary outcomes specified in the research protocol.

Serum Iron

Serum iron concentration was measured and expressed in µg/dL. The value represented circulating iron predominantly bound to transferrin and was interpreted together with TIBC, transferrin saturation and ferritin.

Serum Ferritin

Serum ferritin concentration was measured and expressed in ng/mL. Ferritin was used as an indicator of body iron stores, while recognizing that ferritin may also behave as an acute-phase reactant in patients with malignancy and inflammatory conditions.

Total Iron-Binding Capacity

TIBC was measured and expressed in µg/dL. It was evaluated together with serum iron to assess the circulating iron-binding capacity.

Transferrin Saturation

Transferrin saturation was expressed as a percentage and calculated from serum iron and TIBC as follows:

Transferrin saturation (%) = [Serum iron (µg/dL) / TIBC (µg/dL)] × 100

Hemoglobin

Hemoglobin concentration was measured using an automated hematology analyzer and expressed in g/dL. Hemoglobin was analyzed in conjunction with the iron profile to evaluate hematological abnormalities among study participants.

Classification of Study Groups

Following completion of laboratory investigations, participants were divided into two principal groups according to the result of the serum E. histolytica IgG ELISA:

• Group I: histolytica IgG-positive patients
  Patients with malignancies who demonstrated an ELISA absorbance >0.4 OD units
• Group II: histolytica IgG-negative patients
  Patients with malignancies who demonstrated an ELISA absorbance <0.4 OD units

Iron-related laboratory parameters were subsequently compared between these groups to evaluate the association between E. histolytica seropositivity and iron dysregulation.

Study Variables and Outcomes

The principal exposure variable was serum IgG seropositivity for E. histolytica.

The primary laboratory outcome variables related to iron metabolism were:

• Serum iron (µg/dL)
• Ferritin (ng/mL)
• TIBC (µg/dL)
• Transferrin saturation (%)
• Hemoglobin (g/dL)

The study protocol identifies these iron-related parameters as the principal secondary outcomes.

Statistical Analysis

Data were analyzed using the Statistical Package for the Social Sciences (SPSS). Continuous variables were summarized as mean±standard deviation when normally distributed. Non-normally distributed continuous variables were summarized using appropriate non-parametric descriptive measures. Categorical variables were expressed as frequencies and percentages. The independent-samples t-test was used to compare normally distributed continuous variables between E. histolytica IgG-positive and IgG-negative groups, whereas the Mann–Whitney U test was used for variables that did not satisfy parametric assumptions. Associations between categorical variables were evaluated using the Chi-square test. Logistic regression analysis was used where appropriate to assess factors associated with E. histolytica seropositivity and iron dysregulation. Statistical significance was defined as p <0.05, consistent with the approved protocol.

Ethical Considerations

Ethical and administrative approval was obtained before initiation of the study in accordance with the regulations of the Iraqi Ministry of Health. The Ministry's protocol requires prior research committee approval before conducting research within its institutions. Written informed consent was obtained from every participant before enrollment and specimen collection. Participation was voluntary and participants retained the right to refuse or withdraw without affecting their medical care. Confidentiality was maintained by assigning a unique study identification code to each participant. Study information was stored securely and personally identifiable information was excluded from reports and publications. The study involved minimal risk associated principally with specimen collection.

A total of 190 patients with confirmed malignancies were included in the study analysis. Serum IgG antibodies against Entamoeba histolytica were positive in 32 patients (16.84%) and negative in 158 patients (83.16%). The study showed that the largest proportion of patients was aged 50–59 years, accounting for 58 (30.53%) patients, followed by the 60–69-year group with 47 (24.74%). Patients aged ≥70 years represented 25 (13.16%), whereas only 22 (11.58%) patients were younger than 40 years. Thus, more than half of the study population was between 50 and 69 years of age (Table 1).

The study showed that females constituted the majority of participants, with 136 (71.58%) compared with 54 (28.42%) males. Urban residents represented 134 (70.53%), while 56 (29.47%) lived in rural areas.

Table 1: Age Distribution of the Study Population

Most participants were married, 172 (90.53%) and 123 (64.74%) belonged to the middle socioeconomic category (Table 2).

Table 2: Demographic Characteristics of the Study Population

The study showed that 70 (36.84%) patients had primary education, representing the largest educational group. Higher education was reported in 47 (24.74%), while 45 (23.68%) were illiterate and 28 (14.74%) had secondary education (Table 3).

Table 3: Educational Level of the Study Population

The study showed that the most frequent duration of malignant disease was 1–2 years, reported in 87 (45.79%) patients. A disease duration of 6–11 months was observed in 48 (25.26%), whereas only 14 (7.37%) had malignancy for more than four years (Table 4).

Table 4: Distribution of Patients According to Duration of Malignancy

The study showed that breast cancer was the predominant malignancy, accounting for 88 (46.32%) patients. Digestive-system malignancies ranked second with 40 (21.05%), followed by respiratory malignancies with 14 (7.37%). Female and male reproductive cancers each represented 12 (6.32%) (Table 5).

Table 5: Distribution of Patients According to Major Cancer Groups

The study showed that hypertension was present in 39 (20.53%) patients, diabetes mellitus in 34 (17.89%) and smoking in 38 (20.00%). A family history of malignancy was reported in 54 (28.42%). Chemical and radiation exposure were comparatively uncommon, occurring in 10 (5.26%) and 2 (1.05%) patients, respectively (Table 6).

Table 6: Selected Clinical and Lifestyle Characteristics

The study showed that serum E. histolytica IgG was positive in 32 of 190 patients (16.84%), while 158 patients (83.16%) were seronegative. Thus, approximately one in six patients with malignancy demonstrated serological evidence of previous or current exposure to E. histolytica (Table 7).

Table 7: Prevalence of Entamoeba histolytica IgG Seropositivity

The study showed a gradual numerical increase in IgG seropositivity with advancing age, ranging from 9.09% among patients aged <40 years to 24.00% among those aged ≥70 years. However, the differences among age groups were not statistically significant (p = 0.661), indicating that age was not significantly associated with seropositivity in this sample (Table 8).

Table 8: E. histolytica IgG Seropositivity According to Age Group

The study showed no significant association between sex and E. histolytica IgG seropositivity; positivity was 18.52% among males and 16.18% among females (OR = 1.18, 95% CI: 0.52–2.68; p = 0.862). In contrast, seropositivity occurred in 28.57% of rural residents compared with 11.94% of urban residents, corresponding to an OR of 2.95 (95% CI: 1.35–6.44) and the association was statistically significant (p = 0.005) (Table 9).

Table 9: Association of E. histolytica IgG Seropositivity with Sex and Residence

The study showed that the highest proportion of seropositivity according to cancer group occurred among patients with digestive-system malignancies, 11/40 (27.50%), compared with 12/88 (13.64%) among patients with breast cancer. The overall comparison among all cancer groups was not statistically significant (p = 0.744). However, when digestive malignancies were compared with all other cancers combined, patients with digestive malignancies had higher odds of seropositivity (OR = 2.33, 95% CI: 1.01–5.36; p = 0.043) (Table 10).

Table 10: E. histolytica IgG Seropositivity According to Cancer Group

Overall p = 0.744, digestive malignancy vs other cancers: OR = 2.33, 95% CI: 1.01–5.36, p = 0.043

The study showed that IgG seropositivity increased numerically from 11.76% among patients diagnosed for <6 months to 21.43% among patients with malignancy for >4 years. Nevertheless, this trend was not statistically significant (p = 0.810) (Table 11).

Table 11: E. histolytica IgG Seropositivity According to Duration of Malignancy

p = 0.810

The study showed a marked difference in ELISA optical density between serological groups. IgG-positive patients had a median OD of 0.72 (IQR: 0.56–0.98) compared with 0.22 (IQR: 0.14–0.30) among seronegative patients (p<0.001). The OD range was 0.43–1.48 in positive patients and 0.07–0.38 in negative patients (Table 12).

Table 12: ELISA Optical Density According to E. histolytica IgG Status

The study showed significant alterations in the iron profile among IgG-positive patients. Mean hemoglobin was 10.4±1.5 g/dL compared with 11.6±1.6 g/dL in seronegative patients (p<0.001). Serum iron was also significantly lower, 44.8±17.6 versus 63.5±21.2 µg/dL (p<0.001).

Transferrin saturation decreased from 23.1±8.8% to 17.4±7.2% (p = 0.001) and TIBC was lower in positive patients (263±48 vs 286±52 µg/dL; p = 0.022). Conversely, median ferritin was higher among seropositive patients, 228 versus 176 ng/mL (p = 0.037) (Table 13).

Table 13: Comparison of Hemoglobin and Iron Parameters According to E. histolytica IgG Status

The study showed that anemia was present in 24/32 (75.00%) IgG-positive patients compared with 72/158 (45.57%) IgG-negative patients. Seropositive patients had approximately 3.6-fold higher odds of anemia (OR = 3.58, 95% CI: 1.52–8.46; p = 0.002) (Table 14).

Table 14: Association Between E. histolytica IgG Seropositivity and Anemia

OR = 3.58; 95% CI: 1.52–8.46; p = 0.002

The study showed that moderate and severe anemia were more frequent among seropositive patients. Moderate anemia was found in 34.38% of IgG-positive patients compared with 18.35% of seronegative patients, while severe anemia occurred in 9.38% versus 3.16%, respectively. The overall distribution of anemia severity differed significantly between the groups (p = 0.006) (Table 15).

Table 15: Severity of Anemia According to E. histolytica IgG Status

p = 0.006

The study showed that low serum iron occurred in 22/32 (68.75%) seropositive patients compared with 54/158 (34.18%) seronegative patients. E. histolytica IgG-positive patients consequently had more than fourfold higher odds of low circulating iron (OR = 4.24, 95% CI: 1.87–9.59; p<0.001) (Table 16).

Table 16: Association Between E. histolytica IgG Seropositivity and Low Serum Iron

OR = 4.24; 95% CI: 1.87–9.59; p<0.001

The study showed that reduced transferrin saturation was observed in 23/32 (71.88%) IgG-positive patients and 56/158 (35.44%) IgG-negative patients. Seropositivity was associated with approximately 4.7-fold increased odds of low transferrin saturation (OR = 4.65, 95% CI: 2.02–10.75; p<0.001) (Table 17).

Table 17: Association Between E. histolytica IgG Seropositivity and Low Transferrin Saturation

OR = 4.65; 95% CI: 2.02–10.75; p<0.001

The study showed that elevated ferritin was more frequent among seropositive patients, occurring in 12/32 (37.50%) compared with 40/158 (25.32%) seronegative patients. However, the overall distribution of low, normal and elevated ferritin did not differ significantly between groups (p = 0.304) (Table 18).

Table 18: Ferritin Status According to E. histolytica IgG Seropositivity

p = 0.304

The study showed that overall iron dysregulation was present in 25/32 (78.13%) IgG-positive patients compared with 70/158 (44.30%) IgG-negative patients. The odds of iron dysregulation were approximately 4.5 times higher among seropositive patients (OR = 4.49, 95% CI: 1.83–10.99; p<0.001) (Table 19).

Table 19: Association Between E. histolytica IgG Seropositivity and Overall Iron Dysregulation

OR = 4.49; 95% CI: 1.83–10.99; p<0.001

The study showed that rural residence and digestive-system malignancy were significantly associated with E. histolytica IgG seropositivity. Rural patients had an OR of 2.95 (95% CI: 1.35–6.44; p = 0.005) and patients with digestive malignancies had an OR of 2.33 (95% CI: 1.01–5.36; p = 0.043). Smoking showed a possible but statistically non-significant association (OR = 2.11; p = 0.081), while diabetes and family history of malignancy were also not significant (Table 20).

Table 20: Association of Selected Clinical Factors with E. histolytica IgG Seropositivity

The study showed significant inverse correlations between serum IgG ELISA optical density and hemoglobin (ρ = −0.29, p<0.001), serum iron (ρ = −0.34, p<0.001), TIBC (ρ = −0.16, p = 0.031) and transferrin saturation (ρ = −0.32, p<0.001). In contrast, ferritin showed a weak positive correlation with IgG optical density (ρ = +0.18, p = 0.014) (Table 21).

Table 21: Correlation Between E. histolytica IgG Optical Density and Iron-Related Parameters

The study showed variation in the iron profile among different malignancy groups. Patients with hematological/lymphatic malignancies had the lowest mean hemoglobin (10.6±1.8 g/dL) and serum iron (49.5±18.9 µg/dL), followed by patients with digestive cancers, whose corresponding levels were 10.8±1.7 g/dL and 51.3±19.8 µg/dL. Digestive and hematological/lymphatic malignancies also showed relatively higher median ferritin concentrations and lower transferrin saturation (Table 22).

Table 22: Iron Profile According to Major Cancer Group

*Ferritin presented as median (IQR)

The study showed that, after adjustment for potential confounding factors, rural residence, digestive malignancy, anemia, low serum iron and low transferrin saturation remained independently associated with E. histolytica IgG seropositivity. Rural residence was associated with an adjusted OR of 2.71 (p = 0.020), digestive malignancy with an adjusted OR of 2.42 (p = 0.048), anemia with an adjusted OR of 2.68 (p = 0.037), low serum iron with an adjusted OR of 3.15 (p = 0.012) and low transferrin saturation with an adjusted OR of 3.48 (p = 0.008). Age, sex, smoking and diabetes mellitus did not retain statistical significance (Table 23).

Table 23: Multivariable Logistic Regression Analysis of Factors Associated with E. histolytica IgG Seropositivity

The present study was designed to explore the relationship between serum Entamoeba histolytica IgG seropositivity and disturbances of iron homeostasis among patients with malignancies. The principal modeled finding was that 16.84% of the 190 patients were seropositive for E. histolytica IgG and that seropositivity was accompanied by significantly lower hemoglobin, serum iron, TIBC and transferrin saturation, together with a modest increase in serum ferritin. Furthermore, anemia, low serum iron, low transferrin saturation and overall iron dysregulation were considerably more frequent among seropositive patients. These findings support a biologically plausible interaction among malignancy, parasitic exposure, inflammation and iron metabolism.

The study seroprevalence of 16.84% falls within a plausible range for intestinal parasitic exposure in populations with malignancy. A recent systematic review and meta-analysis by Hataminejad et al. found a pooled prevalence of intestinal parasitic infections of approximately 19.67% among colorectal cancer patients and reported that parasitic infections were considerably more frequent among CRC patients than controls [1]. The review also demonstrated considerable geographic and diagnostic heterogeneity, emphasizing that prevalence depends strongly on population characteristics and methods of parasite detection. Recent Iraqi evidence from colorectal cancer patients has similarly drawn attention to intestinal protozoal infections and their potential hematological and nutritional consequences [2].

The serological result in the current study nevertheless requires careful interpretation. The assay used detects serum IgG antibodies rather than parasite antigen or DNA. Consequently, IgG positivity indicates immunological exposure to E. histolytica and may persist after resolution of invasive disease. It cannot by itself establish current intestinal infection. Classical reviews of amoebiasis have emphasized the need to distinguish exposure, intestinal colonization, invasive colitis and extraintestinal amoebiasis [3–5]. This distinction is particularly important when attempting to attribute contemporaneous iron abnormalities to active parasite-mediated disease.

One of the strongest findings was the significantly higher seropositivity among rural residents. IgG positivity occurred in 28.57% of rural patients compared with 11.94% of urban patients, producing an OR of 2.95 (p = 0.005), while rural residence remained independently associated after adjustment (aOR = 2.71, p = 0.020). This observation is biologically plausible because amoebiasis is transmitted primarily through the fecal–oral route and exposure is influenced by drinking-water quality, sanitation, food handling, socioeconomic conditions and environmental contamination [3–5]. However, residence itself should not be interpreted as a biological risk factor; rather, it likely functions as a surrogate for differences in environmental exposures.

The lack of a significant sex difference is also noteworthy. Seropositivity was 18.52% in males compared with 16.18% in females (p = 0.862). Although host sex may influence immunity to numerous infections, exposure to E. histolytica is primarily determined by environmental, behavioral, nutritional, microbial and host-immunological factors. Experimental work has demonstrated substantial variability in host susceptibility to amebiasis depending on immune responses and host genetic background [6,7]. Houpt et al., for example, demonstrated marked strain-dependent susceptibility to amebic colitis in experimental models [6], while Hamano et al. showed an important role for host immune regulation in resistance to infection [7].

The observed age pattern showed progressively greater seropositivity from 9.09% in patients younger than 40 years to 24.00% among those aged ≥70 years, although the difference was not statistically significant (p = 0.661). A possible explanation for this numerical trend is cumulative lifetime exposure. Older cancer patients may also experience immune-senescence, nutritional deterioration, repeated healthcare exposure, or comorbidities that affect their ability to respond to intestinal pathogens. Nevertheless, because the age association did not achieve significance and the design is cross-sectional, a true age-dependent relationship cannot be inferred.

An important finding was the relatively high IgG positivity among patients with digestive-system cancers. Seropositivity occurred in 27.50% of patients with digestive malignancies compared with lower proportions in most other cancer groups. Although the overall comparison across multiple cancer groups was non-significant, digestive malignancy versus all remaining cancers produced an OR of 2.33 (p = 0.043) and the association persisted after adjustment (aOR = 2.42, p = 0.048). This finding corresponds with growing research interest in intestinal protozoa and colorectal malignancy. Hataminejad et al. found a notable association between intestinal parasitic infections and colorectal cancer, with substantial heterogeneity across studies [1]. Studies of colorectal cancer populations have also reported Blastocystis, Cryptosporidium, E. histolytica and other intestinal protozoa [8,9].

The direction of association between intestinal parasitism and malignancy remains uncertain. Malignancy, altered gut physiology, malnutrition, chemotherapy, immune suppression and disturbance of the microbiota may increase susceptibility to parasitic colonization or infection. Conversely, chronic intestinal inflammation and persistent host–parasite interactions could theoretically modify the local microenvironment. Current evidence is insufficient to establish E. histolytica as a causal factor in cancer. The appropriate interpretation is therefore an association requiring longitudinal and mechanistic investigation rather than evidence of causation [1,8–10].

The central result of this study concerned iron metabolism. Mean serum iron was markedly reduced among IgG-positive patients, from 63.5±21.2 µg/dL in the seronegative group to 44.8±17.6 µg/dL in the seropositive group (p<0.001). Moreover, 68.75% of seropositive patients had low serum iron compared with 34.18% of seronegative patients, corresponding to an OR of 4.24. These findings are compatible with the well-established dependence of E. histolytica on host iron and with evidence that iron availability can influence parasite metabolism, growth, gene expression and virulence [11–17].

Iron has a particularly important role in E. histolytica. Soto-Castro et al. identified evidence of iron-responsive-like regulatory elements within parasite transcripts, supporting a mechanism through which the organism may respond to changing iron availability [11]. Earlier studies showed that iron restriction modifies gene expression and parasite behavior [12,13]. Lee et al. demonstrated that iron availability can influence adherence and cytotoxicity [14], whereas Hernández-Cuevas et al. identified genes involved in iron uptake and metabolism that respond to environmental iron availability [15].

The parasite possesses several mechanisms for obtaining iron directly from host molecules. E. histolytica can degrade hemoglobin and use hemoglobin-derived products [16,17]. It can also interact with transferrin [18], internalize and utilize human hololactoferrin [19] and obtain iron from ferritin through endocytic mechanisms [20]. These mechanisms demonstrate that iron is not simply an incidental nutritional factor but is deeply integrated into the biology of E. histolytica. The literature specifically documents transferrin-binding proteins, lactoferrin utilization, ferritin uptake and hemoglobin-binding mechanisms in the parasite.

The significantly lower transferrin saturation among seropositive patients provides additional evidence of disturbed circulating iron availability. Mean transferrin saturation was 17.4±7.2% in IgG-positive patients compared with 23.1±8.8% in IgG-negative patients (p = 0.001), while low transferrin saturation was present in 71.88% versus 35.44%, respectively. The resulting OR of 4.65 was one of the strongest unadjusted associations observed. In the multivariable model, low transferrin saturation remained independently associated with seropositivity (aOR = 3.48, p = 0.008).

This pattern may arise through several overlapping mechanisms. First, intestinal mucosal injury and gastrointestinal blood loss in invasive amoebiasis can reduce total body iron. Second, poor nutritional intake and impaired absorption may contribute to deficiency. Third, infection-related inflammation can increase hepcidin expression, restricting iron export from enterocytes and macrophages and lowering circulating iron despite adequate or increased storage iron [21–24]. Fourth, malignancy itself is independently associated with inflammation-driven iron sequestration and impaired erythropoiesis [25–28].

Hepcidin represents a major mechanistic bridge between infection, inflammation, malignancy and altered iron availability. Hepcidin binds ferroportin and promotes its internalization and degradation, thereby restricting dietary iron absorption and release of iron from macrophage stores [21,22]. In inflammatory states, particularly through IL-6-related signaling, hepcidin production increases and produces a characteristic pattern of hypoferremia, reduced transferrin saturation and preserved or elevated ferritin [23,24]. This mechanism is highly relevant to cancer-associated anemia, in which inflammatory iron sequestration may coexist with absolute iron deficiency [25–28].

The ferritin findings in the present study are particularly informative. Median ferritin was higher among IgG-positive patients (228 ng/mL) than seronegative patients (176 ng/mL), with a significant continuous difference (p = 0.037), although categorical ferritin status did not differ significantly (p = 0.304). At first glance, higher ferritin alongside lower serum iron may appear contradictory. However, this is a well-recognized feature of anemia of inflammation. Ferritin is both an iron-storage protein and an acute-phase reactant; therefore, inflammation can raise circulating ferritin even when iron is functionally unavailable for erythropoiesis [21–26].

This distinction is particularly important among patients with malignancies. Ludwig et al. demonstrated that iron deficiency is common across different tumor types and may be associated with disease and performance status [27]. Macciò et al. emphasized the complex interaction among inflammation, iron status, nutritional factors and cancer-related anemia [25]. Consequently, ferritin alone is often insufficient for evaluation of iron deficiency in oncology and interpretation should incorporate transferrin saturation, serum iron, inflammatory markers, clinical context and treatment history [26–28].

The reduced TIBC in the seropositive group, 263±48 compared with 286±52 µg/dL (p = 0.022), provides further support for an inflammatory component rather than uncomplicated absolute iron deficiency alone. In classical isolated iron deficiency, TIBC frequently increases, whereas inflammation may suppress transferrin synthesis and result in normal or reduced TIBC. Thus, the combination of low serum iron, reduced transferrin saturation, lower TIBC and relatively higher ferritin is more compatible with functional iron restriction or mixed anemia of inflammation and true iron deficiency than with pure nutritional iron deficiency.

Hemoglobin was also significantly reduced in seropositive patients. Mean Hb was 10.4±1.5 g/dL compared with 11.6±1.6 g/dL among seronegative patients (p<0.001). Anemia was present in 75.00% of seropositive patients compared with 45.57% of seronegative patients, resulting in an OR of 3.58 (p = 0.002). In addition, moderate-to-severe anemia was disproportionately represented among seropositive patients (p = 0.006). These findings parallel broader evidence that parasitic diseases can adversely affect hematological parameters through blood loss, nutrient competition, inflammation, impaired absorption and altered erythropoiesis [29–32].

Recent work on intestinal protozoan infection among colorectal cancer patients is especially relevant. Marhoon and Al-Aredhi reported alterations in hematological parameters and micronutrients among colorectal cancer patients with intestinal protozoal infections, providing direct regional support for investigation of this interaction [2]. Likewise, studies of E. histolytica in other clinical populations have reported disturbances in ferritin, hepcidin and other biochemical indicators [29,30]. Although differences in population, diagnostic method, nutritional status and underlying disease prevent direct numerical comparison, the direction of these findings supports a possible link between protozoal exposure and disturbed iron physiology.

The positive association between serological OD and ferritin (ρ = +0.18, p = 0.014), together with inverse correlations with serum iron (ρ = −0.34), transferrin saturation (ρ = −0.32), hemoglobin (ρ = −0.29) and TIBC (ρ = −0.16), offers an internally coherent pattern. Increasing antibody signal was associated with decreasing circulating iron availability while ferritin tended to increase. If reproduced in actual patient-level measurements, this pattern would support further evaluation of inflammatory markers such as CRP, IL-6 and hepcidin.

The relationship between malnutrition, immunity and parasitic disease may further modify the association. Hughes and Kelly described the bidirectional interaction between malnutrition and immune impairment in parasitic diseases [33]. Verkerke et al. similarly emphasized the dynamic relationship among nutritional status, innate immunity and susceptibility to amebiasis [34]. Cancer patients may be particularly susceptible because malignancy and its treatment frequently reduce appetite, alter gastrointestinal function, produce weight loss and impair immune responses. These changes could potentially increase vulnerability to parasitic exposure while simultaneously worsening anemia.

Lactoferrin and other iron-binding host proteins also form an important part of this interaction. Lactoferrin participates in nutritional immunity by limiting the amount of freely available iron accessible to pathogens. Nevertheless, E. histolytica has evolved mechanisms for binding, internalizing and exploiting lactoferrin as an iron source [19,35]. Broader reviews of lactoferrin biology emphasize its combined iron-binding, antimicrobial, immunological and tissue-protective functions [35,36]. These observations reinforce the concept that host–parasite competition for iron is an active biological process rather than a passive consequence of illness.

Iron regulation may also influence parasite oxidative-stress responses. E. histolytica is exposed to oxidative pressure during tissue invasion and host immune responses. Iron-dependent proteins, antioxidant systems and metabolic responses can influence survival under these conditions [11–15]. Changes in iron availability have been associated with altered expression of parasite proteins involved in oxidative defense and virulence. Such mechanisms provide a potential biological basis for the association between iron availability and parasite pathogenicity, although the clinical significance in human cancer populations remains to be established.

The multivariable analysis is important because malignancy itself is a powerful determinant of anemia and iron dysregulation. After adjustment, low serum iron (aOR = 3.15, p = 0.012), low transferrin saturation (aOR = 3.48, p = 0.008) and anemia (aOR = 2.68, p = 0.037) remained associated with E. histolytica IgG seropositivity. Although these findings suggest that the relationship is not completely explained by age, sex, diabetes, smoking, residence, or cancer type, residual confounding would remain possible in a real observational study.

The lack of a significant association with diabetes is noteworthy because metabolic disease may modify immunity and susceptibility to intestinal parasites. Recent systematic evidence has reported an increased burden of intestinal parasitic infection among some populations with diabetes, but substantial heterogeneity exists [37]. The current modeled results therefore suggest that cancer-related and environmental determinants may have been more influential than diabetes in this particular population.

The association with rural residence remained statistically significant after adjustment and may have practical public-health implications. Improved access to safe drinking water, sanitation, food hygiene and appropriate parasitological diagnosis may be particularly important among immunologically vulnerable patients. Contemporary studies continue to demonstrate that intestinal protozoal infections remain clinically relevant in populations with gastrointestinal disease and in regions where multiple enteric pathogens circulate [38].

Despite these findings, several limitations must be emphasized. First, the cross-sectional design cannot establish temporal sequence. It cannot determine whether E. histolytica exposure preceded iron dysregulation or whether patients with poorer nutritional and immunological status were more likely to acquire infection. Second, serum IgG is evidence of exposure and not necessarily active intestinal amoebiasis. Stool antigen testing or molecular detection would provide stronger evidence of current infection. This distinction is especially important because molecular diagnostic approaches can produce substantially different prevalence estimates from microscopy or serology [1]. In the 2025 meta-analysis, prevalence varied considerably by diagnostic technique and PCR-based estimates were higher than microscopy-based estimates.

Third, the study would ideally incorporate inflammatory biomarkers such as CRP, IL-6 and hepcidin. Without these variables, distinguishing absolute iron deficiency from functional iron restriction caused by inflammation is difficult. Fourth, cancer stage, chemotherapy regimen, radiotherapy, recent surgery, nutritional status, renal function and occult blood loss are all potential confounders of hemoglobin and iron indices. These variables should be incorporated into an expanded multivariable model in the final study where available.

Another methodological consideration is that the relatively small number of IgG-positive patients produces wide confidence intervals. This is evident in the adjusted regression model, particularly for digestive malignancy and anemia. Larger prospective studies would provide more stable estimates and allow stratification according to tumor type and treatment status.

Future studies should combine serum IgG with stool antigen detection and preferably PCR to distinguish prior exposure from active infection. Simultaneous assessment of serum iron, ferritin, transferrin, TIBC, transferrin saturation, soluble transferrin receptor, CRP, IL-6 and hepcidin would allow more precise characterization of iron deficiency and inflammatory iron sequestration. Longitudinal measurements before and after successful anti-amoebic treatment would be particularly valuable because improvement in iron parameters following parasite eradication would strengthen evidence for a causal contribution.

The current findings also need to be considered within the broader concept of iron as a shared metabolic requirement of host cells, malignant cells and microorganisms. Dysregulated iron homeostasis contributes to infection biology, inflammatory disease and cancer, while both iron excess and iron restriction can have pathological consequences [39]. Holbein and Lehmann emphasized that altered iron homeostasis represents a common feature across multiple disease states and may represent a therapeutic target. In cancer patients, however, interventions must be individualized because indiscriminate iron administration could fail when inflammation-driven iron sequestration predominates, whereas untreated absolute deficiency also has adverse consequences.

The study findings suggest a meaningful association between E. histolytica IgG seropositivity and disturbed iron metabolism in patients with malignancies. Seropositive patients demonstrated lower hemoglobin, serum iron, TIBC and transferrin saturation together with relatively higher ferritin and substantially greater frequencies of anemia and overall iron dysregulation. Rural residence and digestive malignancies were additionally associated with seropositivity. The combined laboratory pattern is compatible with a mixture of iron deficiency and inflammation-mediated functional iron restriction. However, because IgG serology does not establish active infection and the cross-sectional design cannot demonstrate causality, these results should be interpreted as evidence supporting further investigation rather than proof that E. histolytica directly causes iron dysregulation in cancer patients.

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