Colorectal Cancer and Intestinal Protozoan Infections: Prevalence, Risk Factors, and Molecular Confirmation in Western Iran: A Descriptive Case‐Control Study

ABSTRACT

Background and Aims

Infections are recognized contributors to cancer development. This study aimed to assess the prevalence, socio‐demographic characteristics, and risk factors associated with intestinal protozoan parasites in patients with colorectal cancer (CRC) in Western Iran.

Methods

This descriptive case‐control study enrolled 174 CRC patients and 174 non‐CRC healthy controls receiving care at general hospitals in Lorestan Province between October 2022 and August 2024. Fresh stool samples were examined using direct smear, Lugol's iodine, formalin‐ether concentration, and permanent staining (Giemsa and Trichrome). Polymerase chain reaction (PCR) was performed on all samples testing positive for Entamoeba species by microscopy. Data were analyzed using χ ² tests and binary logistic regression (SPSS v26.0); a two‐sided p < 0.05 was considered statistically significant.

Results

Intestinal protozoan infections were detected in 24/174 (13.8%) CRC patients and 8/174 (4.6%) non‐CRC controls. CRC patients had significantly higher odds of intestinal protozoan infection than non‐CRC controls (OR = 3.32, 95% CI: 1.45–7.61; Fisher's exact p = 0.0047). Among the identified protozoan parasites were Giardia lamblia (15/32, 46.8%), Entamoeba histolytica (6/32, 18.75%), and E. coli (4/32, 12.5%), respectively. Using a PCR assay, all E. histolytica/E. dispar positive samples were confirmed as E. histolytica. Among CRC patients, rural residence, animal contact, agricultural activity, animal husbandry, and consumption of unwashed fruits or vegetables were significantly associated with intestinal protozoan infection (ORs = 2.55–65.46; p < 0.05). No significant associations were observed with demographic, socioeconomic, or behavioral factors among non‐CRC participants.

Conclusion

This study found a higher prevalence of intestinal protozoan parasites in CRC patients compared to controls, emphasizing the clinical importance of parasitic infections in this population. Routine screening, improved sanitation, and public health interventions are recommended to reduce infection risks. Further research is needed to explore the long‐term impact of these parasites on cancer progression and immune function.

1. Introduction

Colorectal cancer (CRC) is one of the most common types of cancer affecting the digestive tract. Currently, it accounts for approximately 10% of all cancer cases worldwide and is responsible for nearly 9.4% of cancer‐related deaths. Experts predict that by 2040, the number of new colorectal cancer cases will exceed 3.2 million [1]. Although lifestyle factors such as being overweight, consuming a diet low in fruits and vegetables, lack of physical activity, and smoking are major contributors to the disease, the role of infectious agents in its development should not be overlooked [2]. Infections are known to contribute to the onset of various cancers by causing chronic inflammation, disrupting genetic stability, inhibiting apoptosis, and impairing the body's ability to regulate tumor growth. These effects can also influence cellular proliferation, angiogenesis, and energy metabolism [3]. Recent studies have demonstrated a strong association between certain microbes and cancer development. Pathogens such as Helicobacter pylori, Mycobacterium tuberculosis, human papillomavirus, Epstein‐Barr virus, HIV, and herpes simplex virus have all been linked to an increased risk of cancer in the tissues they infect [4].

The human digestive system serves as a major hub for microbial‐host interactions [5]. Within this complex ecosystem, a vast community of microbes, including bacteria, fungi, viruses, parasites, and single‐celled organisms, coexists in a dynamic and balanced state with the host's intestinal cells [6]. This diverse microbial population, known as the gut microbiota, is essential for maintaining health by aiding digestion, producing vital nutrients, and modulating immune responses [6]. However, some of these microbes may contribute to the onset or progression of diseases, particularly within the gastrointestinal tract, including stomach and colon cancers. In this context, parasitic and protozoan species have emerged as key players [7].

Gastrointestinal parasites are significant causative agents of gastrointestinal diseases in many communities, particularly in areas with poor personal and environmental hygiene [8]. In Iran, due to its geographical, climatic, and social conditions, these parasites continue to present a major public health and medical challenge. An overview of the epidemiological status of gastrointestinal parasites in Iran reveals that common parasites such as Entamoeba histolytica, Schistosoma species, Giardia lamblia, Ascaris lumbricoides, and soil‐transmitted helminths are reported across various regions of the country [9]. Intestinal parasitic infections have been increasingly linked to the development of certain cancers, particularly in regions with high parasite prevalence [10]. Moreover, specific intestinal protozoa like E. histolytica, G. lamblia, and Cryptosporidium parvum have been implicated in colorectal cancer due to their ability to induce chronic inflammation, release harmful toxins, alter epigenetic patterns, and disrupt the gut's immune defences [10, 11].

Cancer patients, particularly those with colorectal cancer, are recognized as a high‐risk group for intestinal parasitic infections due to immune suppression, altered gut microbiota, and disrupted mucosal defenses resulting from both the malignancy itself and its treatment. Emerging evidence indicates a bidirectional relationship between parasitic infections and cancer progression: parasites may promote carcinogenesis through chronic inflammation and immune evasion, while the immunocompromised state of cancer patients increases susceptibility to opportunistic infections [12]. A recent systematic review reported a global prevalence of intestinal parasites in cancer patients reaching 21.3%, with significant geographic variation linked to hygiene standards and healthcare access [13]. Furthermore, protozoan infections such as those caused by E. histolytica and G. lamblia have been proposed as potential cofactors in colorectal carcinogenesis, mediated by their capacity to induce epigenetic alterations, disrupt tight junctions, and promote pro‐inflammatory cytokine release [13, 14].

The potential role of parasitic and gastrointestinal protozoan infections in the pathogenesis of digestive tract cancers has emerged as an important area of research in medical science and microbiology. This study aimed to assess the prevalence, socioeconomic characteristics, and risk factors associated with intestinal protozoan infections, with particular emphasis on intestinal flagellated protozoa and protozoan parasites of the family Entamoebidae, among patients with CRC in Iran.

2. Materials and Methods

2.1. Study Region

The Lorestan region, situated in the western part of Iran amidst the Zagros mountain valleys, is distinguished by a heterogeneous climate system that includes alpine cold, semi‐arid, and temperate patterns. With a population of roughly 1.7 million inhabitants, the province hosts several major cities such as Khorramabad, Borujerd, Aligudarz, Dorud, Kuhdasht, Azna, Aleshtar, Nurabad, and Pol‐e Dokhtar. The area is climatically defined by scorching, dry summers and relatively wetter winters, contributing to its ecological and environmental diversity.

2.2. Participants

The present descriptive case‐control study included 174 patients with a confirmed diagnosis of CRC prior to the commencement of oncological therapy. The cohort consisted of 152 individuals (87.4%) with colon cancer and 22 patients (12.6%) with rectal cancer, all of whom were under treatment at Shahid Rahimi hospital in Khorramabad, Lorestan Province, western Iran, between October 2022 and August 2024. CRC diagnoses were confirmed by a certified gastroenterologist using a range of diagnostic procedures, including colonoscopy, blood tests, histopathological analysis, stool tests, immunochemical testing, and computed tomography. The control group comprised 174 healthy individuals without any current or past gastrointestinal symptoms and with negative findings from colonoscopy and/or fecal occult blood tests during the study period. Although participants in the non‐CRC group had no clinical or laboratory evidence of colorectal cancer and met the predefined eligibility criteria, the absence of CRC did not require the absence of intestinal protozoan infection. Therefore, participants with asymptomatic intestinal protozoan infections detected by stool examination remained classified as non‐CRC controls. Exclusion criteria encompassed refusal to participate, prior chemotherapy, systemic antibiotic use within the previous 3 months, and immunocompromised status.

2.3. Ethical Approval

This case‐control study was conducted between October 2022 and August 2024 in Lorestan Province, western Iran. The study protocol was reviewed and approved by the Ethics Committee of Lorestan University of Medical Sciences (IRB approval No. IR.LUMS.REC.1399.163). Written informed consent was obtained from all participants prior to enrollment. All study procedures were performed in accordance with the ethical standards of the institutional research committee and the Declaration of Helsinki.

2.4. Questionnaire, Socio‐Economic, and the Possible Risk Factors

The information and consent forms were prepared and distributed among participants in both the case and control groups. Subsequently, a structured questionnaire was used to gather demographic and lifestyle data, such as age, sex, residential area, educational background, occupation, monthly income, contact with animals, consumption of raw vegetables, agricultural history, fast food intake, background in animal farming, and handwashing practices before meals.

2.5. Sample Collection

Approximately 5–10 g of fresh stool was collected from each participant in a sterile, labeled, wide‐mouth container and transported to the laboratory within 2 h of collection. A fraction of the fecal specimens was homogenized in phosphate‐buffered saline and filtered through a double‐layer gauze to eliminate coarse particles. The resulting mixture was subjected to centrifugation at 1500 g for 5 min. The pellet obtained was then re‐suspended in 0.2 mL of a 2% polyvinylpolypyrrolidone solution in PBS and preserved at −80°C until DNA isolation was carried out.

2.6. Microscopic Analysis

For microscopic evaluation, the direct smear (wet mount) technique, Lugol's iodine solution, the formalin‐ether concentration method, permanent stain (Giemsa and Trichrome) were employed [15]. Individuals who tested positive were referred to an infectious disease specialist for proper counseling, treatment, and follow‐up.

2.7. Molecular Detection of Entamoeba Spp., by Polymerase Chain Reaction (PCR)

PCR confirmation was applied exclusively to specimens positive for Entamoeba species, given the morphological indistinguishability of E. histolytica and E. dispar cysts by light microscopy. All other protozoan species identified in this study (G. lamblia, E. coli, I. butchili, E. nana, C. mesnili, and D. fragilis) were confirmed by characteristic morphological features using permanent Trichrome staining, which does not require molecular confirmation. The DNA was isolated from all specimens that showed a positive microscopic result for Entamoeba species, using a commercial fecal DNA extraction kit produced by KiaGene Fecal DNA Extraction Kit, Iran, strictly adhering to the instructions provided by the manufacturer. The used primers to amplify the small‐subunit rRNA gene were forward primer EntaF and reverse primers EhR, and EdR specific for E. histolytica (166 bp) and E. dispar (752 bp), respectively [16]. They were verified through the NCBI database, and primer specificity was confirmed using BLAST Gene. The PCR assay was conducted utilizing Taq DNA Polymerase Master Mix RED (Ampliqon, Denmark) with a final volume of 25 μL. The thermal cycling protocol commenced with an initial denaturation step at 95°C for 3 min, followed by a series of cycles consisting of denaturation at 95°C for 1 min, annealing at 59°C for 1 min, and extension at 74°C for 1 min, utilizing a thermal cycler from Bio‐Rad, USA. The final extension phase was performed at 73°C for 10 min. The resultant amplified products were subsequently analyzed on a 1.5% agarose gel and documented using the PCR gel documentation system. Positive controls included DNA from E. histolytica (ATCC 50527) and E. dispar, while sterile distilled water was used as the negative control. The amplified products were analyzed by electrophoresis on a 1.5% agarose gel and visualized using a PCR gel documentation system.

2.8. Statistical Analysis

Statistical analyses were conducted using SPSS software, version 26.0 (IBM Corp., Armonk, NY, USA), in accordance with the SAMPL Guidelines [17] and the STROBE Statement [18]. Descriptive statistics, including frequencies, percentages, means, and standard deviations (SDs), were used to summarize the data. The normality of continuous variables was assessed using the Kolmogorov–Smirnov test. Normally distributed continuous variables are presented as mean ± SD, whereas non‐normally distributed variables are presented as median and interquartile range (IQR). The χ ² test or Fisher's exact test, as appropriate, was used to compare categorical variables between groups. Binary logistic regression analysis was performed to evaluate the association between intestinal protozoan infection and the investigated demographic, socioeconomic, and behavioral variables. Intestinal protozoan infection was considered the binary dependent outcome. Separate univariable logistic regression models were fitted for each predictor, and crude odds ratios (ORs) with 95% confidence intervals (CIs) and corresponding p values were calculated. For categorical variables, the reference categories were defined as indicated in Tables 4 and 5. For the primary comparison between CRC patients and non‐CRC controls, the association between CRC status and intestinal protozoan infection was assessed using the appropriate 2 × 2 contingency‐table analysis, with ORs and 95% CIs calculated accordingly. All statistical tests were two‐sided, and a p value < 0.05 was considered statistically significant.

Table 4.

Prevalence of intestinal protozoan parasite infection in patients with colorectal cancer based on the demographic, socio‐economic, and risk factors by χ ² and logistic regression analysis.

Variable	Protozoan infection		p value χ 2	Odds ratio	95% Confidence interval	p value
	Positive number (%)	Negative number (%)
Age
< 60 years	13 (12.5)	91 (87.5)	0.705	0.766	0.322–1.824	0.547
≥ 60 years	11 (15.7)	59 (84.3)	—	—	—	—
Gender
Male	11 (10.8)	91 (89.2)	0.347	0.549	0.230–1.306	0.175
Female	13 (18.1)	59 (81.9)	—	—	—	—
Residence
Rural	16 (28.6)	40 (71.4)	0.003*	7.467	2.732−20.405	0.000*
Urban	6 (5.1)	112 (94.9)	—	—	—	—
Education
≤ Diploma	18 (13.6)	114 (86.4)	0.621	0.947	0.350–2.568	0.915
> Diploma	6 (14.3)	36 (85.7)	—	—	—	—
Monthly family income
< 300 USD	11 (13.4)	71 (86.6)	—	—	—	—
300–600	14 (15.9)	74 (84.1)	0.845	1.222	0.5236–2.858	0.68
> 600 USD	1 (25.0)	3 (75.0)	0.642	2.152	0.205–22.55	0.50
Family members
< 4	1 (4.5)	21 (95.5)	—	—	—	—
4–6	12 (13.1)	80 (86.9)	0.361	3.150	0.392–25.31	0.28
6 <	11 (18.3)	49 (81.6)	0.217	4.714	0.57–38.53	0.17
Contact with animal
No	2 (3.3)	58 (96.7)	0.014*	6.935	1.572–30.598	0.011*
Yes	22 (19.3)	92 (80.7)	—	—	—	—
Agriculture activity
Yes	23 (23.0)	77 (77.0)	—	—	—	—
No	1 (1.3)	73 (98.7)	0.000*	21.805	2.871–165.622	0.000*
Animal husbandry
Yes	23 (37.1)	39 (62.9)	0.000*	65.462	8.554–500.973	0.000*
No	1 (0.9)	111 (99.1)	—	—	—
Consuming unwashed vegetables/fruits
Yes	16 (53.3)	66 (45.8)	0.069	2.545	1.27−6.310	0.044*
No	8 (46.7)	84 (54.2)	—	—	—	—
Handwashing prior to consuming meals
Yes	22 (14.1)	134 (85.9)	0.567	1.313	0.282–6.112	0.728
No	2 (11)	16 (88.9)	—	—		—

*p < 0.05 significant difference.

Table 5.

Prevalence of intestinal protozoan parasite infections in non‐colorectal cancer participants based on the demographic, socio‐economic, and risk factors by χ ² and logistic regression analysis.

Variable	Protozoan infection		p value χ 2	Odds ratio	95% Confidence interval	p value
	Positive number (%)	Negative number (%)
Age
< 60 years	6 (5.8)	96 (94.2)	0.411	2.188	0.429–11.162	0.352
≥ 60 years	2 (2.7)	70 (97.8)	—	—	—	—
Gender
Male	2 (2.1)	94 (97.9)	0.321	0.255	0.050–1.303	0.091
Female	6 (7.7)	72 (92.3)	—	—	—	—
Residence
Rural	2 (4.0)	48 (96.0)	0.866	0.827	0.155–4.416	0.808
Urban	6 (4.8)	118 (95.2)	—	—	—	—
Education
≤ Diploma	4 (3.1)	124 (96.9)	0.284	0.339	0.081–1.415	0.137
> Diploma	4 (8.7)	42 (91.3)	—	—	—	—
Monthly family income
< 300 USD	2 (2.4)	80 (94.6)	—	—	—	—
300–600	4 (4.5)	84 (95.5)	0.723	1.905	0.339–10.688	0.464
> 600 USD	2 (50.0)	2 (50)	0.007*	40.0	3.591–445.573	0.003*
Family members
< 4	1 (4.5)	21 (95.5)	—	—	—	—
4–6	5 (5.7)	83 (94.3)	0.554	1.265	0.137–11.660	0.841
6 <	2 (3.1)	62 (96.9)	0.671	0.677	0.058–7.858	0.753
Contact with animal
No	4 (3.8)	102 (96.2)	0.642	1.594	0.385–6.598	0.515
Yes	4 (5.9)	64 (94.1)	—	—	—	—
Agriculture activity
Yes	4 (4.5)	84 (95.5)	—	—	—	—
No	4 (4.7)	82 (95.3)	0.981	0.976	0.236–4.034	0.975
Animal husbandry
Yes	2 (3.6)	54 (96.4)	0.753	0.692	0.132–3.637	0.666
No	6 (5.1)	112 (94.9)	—	—	—	—
Consuming unwashed vegetables/fruits
Yes	6 (6.4)	88 (93.5)	0.619	2.659	0.522–13.559	0.236
No	2 (2.5)	78 (97.6)	—	—	—	—
Handwashing prior to consuming meals
Yes	7 (4.6)	151 (95.4)	0.487	0.691	0.081–5.909	0.729
No	1 (6.2)	15 (93.8)	—	—	—	—

*p < 0.05 significant difference.

3. Results

3.1. Participants

The mean age of CRC patients was 59.5 ± 11.4 years, and of non‐CRC controls was 58.5 ± 11.3 years. No statistically significant difference was observed between the two groups in terms of age (p = 0.877). Table 1 presents the comparison of the demographic and epidemiological characteristics of the patients and the control group. According to the χ ² test, there was no statistically significant difference in the mean age (p = 0.877), gender (p = 0.646), place of residence (p = 0.621), education (p = 0.727), monthly family income (p = 0.999), Family members (p = 0.591), Contact with animal (p = 0.529), Agriculture activity (p = 0.361), Consuming unwashed vegetables/fruits (p = 0.448), Handwashing prior to consuming meals (p = 0.999) between the patient group and the control group.

Table 1.

Frequency distribution of demographic characteristics in the colorectal cancer patients (CRC) and non‐colorectal healthy individuals (non‐CRC). Continuous variables are presented as mean ± standard deviation (SD), whereas categorical variables are presented as frequency (n) and percentage (%).

Variable	Group		p value
	CRC no. (%)	non‐CRC no. (%)
Age
< 60 years	104 (59.8)	102 (58.6)	0.877
≥ 60 years	70 (40.2)	72 (41.4)
Gender
Male	102 (58.6)	96 (55.2)	0.646
Female	72 (41.4)	78 (44.8)
Residence
Rural	56 (32.2)	50 (28.7)	0.621
Urban	118 (67.8)	124 (71.3)
Education
≤ Diploma	132 (75.9)	128 (73.6)	0.727
> Diploma	42 (24.1)	46 (26.4)
Monthly family income
< 300 USD	82 (47.1)	82 (47.1)	0.999
300–600	88 (50.6)	88 (50.6)
> 600 USD	4 (2.3)	4 (2.3)
Family members
< 4	22 (12.6)	22 (12.6)	0.591
4–6	92 (52.9)	88 (50.6)
6 <	60 (34.5)	64 (36.8)
Contact with an animal
No	60 (34.5)	68 (39.1)	0.529
Yes	114 (65.5)	106 (60.9)
Agriculture activity
Yes	100 (57.5)	88 (50.6)	0.361
No	74 (42.5)	86 (49.4)
Consuming unwashed vegetables/fruits
Yes	82 (47.1)	94 (54.2)	0.448
No	92 (52.9)	80 (45.8)
Handwashing prior to consuming meals
Yes	156 (89.7)	158 (90.8)	0.999
No	18 (10.3)	16 (9.2)

3.2. Prevalence of Intestinal Protozoan Parasites

Intestinal protozoan infections were detected in 24/174 (13.8%) CRC patients. Despite being clinically asymptomatic and classified as non‐CRC controls, 8 of 174 participants (4.6%) had detectable intestinal protozoan infections. CRC patients had significantly higher odds of intestinal protozoan infection than non‐CRC controls (OR = 3.32, 95% CI: 1.45–7.61; Fisher's exact p = 0.0047) (Table 2). Among the identified protozoan parasites, G. lamblia (15/32, 46.8%), E. histolytica (6/32, 18.75%), Entamoeba coli (4/32, 12.5%), respectively (Table 3). Using PCR assay, all E. histolytica/E. dispar positive samples were confirmed as E. histolytica (Figure 1).

Table 2.

Comparison of the prevalence of intestinal protozoan parasite infection in colorectal cancer patients (CRC) and non‐colorectal healthy individuals (non‐CRC) groups.

Group	Protozoan infection		Pearson χ 2 p value	OR	95% CI	Fisher's exact p value
	Positive No. (%)	Negative No. (%)
CRC	24/174 (13.8)	150/174 (86.2)	0.003	3.32	1.45–7.61	0.0047*
Non‐CRC	8/174 (4.6)	166/174 (95.4)	—	1.00	—	—

*p < 0.05.

Table 3.

Frequency and percentage of observed intestinal protozoan parasites in the colorectal cancer patients (CRC) and non‐colorectal healthy individuals (non‐CRC) groups.

Parasite	Intestinal protozoan parasites No. (%)
	CRC	Non‐CRC
Giardia lamblia	11 (45.8)	4 (50)
Entamoeba histolytica	5 (20.8)	1 (12.5)
Entamoeba coli	3 (12.5)	1 (12.5)
Iodamoeba butschlii	2 (8.4)	1 (12.5)
Endolimax nana	1 (4.2)	1 (12.5)
Chilomastix mesnili	1 (4.2)	—
Dientamoeba fragilis	1 (4.2)	—
Total	24 (100)	8 (100)

Figure 1.

Photomicrographs and gel electrophoresis results of intestinal parasites identified in study participants. (A) Giardia lamblia cyst (wet mount, Lugol's iodine stain, ×400); (B) Iodamoeba butschlii cyst (Trichrome stain, ×1000); (C) Chilomastix mesnili trophozoite (Trichrome stain, ×1000); (D) Endolimax nana cyst (Trichrome stain, ×1000); (E) Entamoeba coli cyst (Trichrome stain, ×1000); (F) Entamoeba histolytica cyst (Trichrome stain, ×1000); (G) Lane a: no template control (sterile distilled water); Lanes b–f: patient‐derived samples confirmed as E. histolytica. Lane g: E. histolytica positive control (166 bp).

3.3. Associated Risk Factors of Intestinal Protozoan Parasites

Among CRC patients, rural residence was significantly associated with intestinal protozoan infection, with participants residing in rural areas showing higher odds of infection than those residing in urban areas (OR = 7.47, 95% CI: 2.73–20.41; p < 0.001). Contact with animals was also significantly associated with protozoan infection (OR = 6.94, 95% CI: 1.57–30.60; p = 0.011). Furthermore, agricultural activity (OR = 21.81, 95% CI: 2.87–165.62; p < 0.001) and animal husbandry (OR = 65.46, 95% CI: 8.55–500.97; p < 0.001) were strongly associated with increased odds of infection. Consumption of unwashed vegetables or fruits was also associated with higher odds of protozoan infection (OR = 2.55, 95% CI: 1.03–6.31; p = 0.044). In contrast, age, sex, educational level, monthly family income, number of family members, and handwashing before meals were not significantly associated with protozoan infection (p > 0.05). (Table 4). In the non‐CRC group, no significant associations were observed between intestinal protozoan infection and the assessed demographic, socioeconomic, or behavioral factors (p > 0.05). The apparently high odds ratio observed among participants with a monthly family income > 600 USD should be interpreted cautiously because this category included only four individuals, resulting in a very wide confidence interval. The lack of statistically significant associations in this group may also reflect the relatively small number of protozoan‐positive participants and the consequent limited statistical power to detect modest associations (Table 5).

4. Discussion

There is growing evidence that microbial infections may act as triggers or promoters in the onset of several malignancies [14]. These pathogens can initiate long‐lasting inflammatory responses, compromise the integrity of DNA, and interfere with normal cell cycle regulation [19]. As a result, they may contribute to uncontrolled tissue growth and the formation of abnormal blood supply. Research has also highlighted the role of certain gut parasites, such as E. histolytica and G. lamblia, in the progression of colorectal cancer [10, 11]. These organisms are believed to damage the intestinal lining, release harmful byproducts, alter gene expression patterns, and weaken local immune defenses, all of which may create a favorable environment for tumor development [10, 11, 20].

In the current study, intestinal protozoan parasites were detected in 24 (13.8%) of the 174 CRC patients and 8 (4.6%) of the 174 non‐CRC controls, indicating a higher prevalence of intestinal protozoan infection among CRC patients. A study by Mamatkulov and Rakhimov [21] also examined 102 patients with CRC and found that 18.6% were infected with intestinal parasites. The most prevalent infections included G. lamblia (8.8%), E. histolytica/dispar (6.9%), and Ascaris lumbricoides (3.9%). A study by Saki et al. [22] evaluated the prevalence of intestinal parasitic infections among 243 high‐risk individuals, including patients with cancer, organ transplant recipients, and those with primary immunodeficiency in Tehran, Iran. The overall prevalence of parasitic infections was 15.2%, with G. lamblia (7.4%) and E. histolytica/dispar (4.5%) being the most common agents. In the present study region, Ghanadi et al. [23] reported that among 87 individuals diagnosed with colorectal cancer Cryptosporidium infection was observed in 42.5% compared to the healthy group (12.6%), with a statistically significant difference. Another study reported that a significantly higher prevalence of Blastocystis hominis was observed in the colorectal cancer group compared to the healthy control group. Specifically, 16 out of 67 individuals (23.9%) in the case group tested positive for B. hominis, while only 6 out of 67 (9%) of the healthy participants showed the presence of this parasite. The difference in prevalence between the two groups was statistically significant [24]. In a study by Mahmoudvand et al., the prevalence of Isospora belli and Cyclospora cayetanensis was investigated in 120 cancer patients. The results revealed that 5.8% were infected with I. belli and 4.2% with C. cayetanensis, emphasizing the increased risk of parasitic infections in immunocompromised individuals [25]. These discrepancies may reflect differences in local epidemiology, healthcare access, and environmental factors influencing parasitic transmission. Additionally, variations in sample size and laboratory techniques might contribute to the divergent findings, highlighting the need for standardized protocols in future research to enhance comparability across regions [8, 9].

The results of this study reveal that several environmental and behavioral elements are strongly linked to the presence of intestinal protozoan parasites in individuals diagnosed with colorectal cancer. These include living in a specific geographic area, keeping and handling domestic animals, engaging in farming or agricultural activities, and being involved in animal husbandry. These findings suggest that exposure to contaminated environments plays a crucial role in the transmission of such infections. This observation is in line with earlier research that has highlighted the influence of lifestyle and environmental conditions on the spread of parasitic diseases. For example, Mamatkulov and Rakhimov [21] found that individuals in rural Uzbekistan, where close contact with animals and limited access to clean water are common, had a higher incidence of parasitic infections. Likewise, Saki et al. [22] noted that people with weakened immune systems in agricultural regions are at greater risk due to frequent exposure to potentially contaminated water and soil. The connection between rural living and agricultural work and the increased likelihood of protozoan infections is well‐supported in the scientific literature. Alvarado‐Esquivel et al. [26] observed that individuals in rural Mexico who were involved in farming or animal care had a significantly higher rate of infection with G. lamblia and Cryptosporidium. This is likely due to poor sanitation and the frequent handling of animal waste. Additionally, close contact with domestic animals has been identified as a contributing factor in several studies. Musa et al. [27] showed that households with pets or livestock had a higher risk of zoonotic infections, including T. gondii and C. parvum. These results emphasize the role of animals in the transmission chain of intestinal parasites. The impact of agricultural activities on the spread of parasitic infections is also well‐documented. Khan et al. [28] found that farmers in Pakistan were more likely to be infected with intestinal parasites compared to those not involved in farming. This was attributed to the use of contaminated water and soil during farming practices. In the case of colorectal cancer patients, the presence of intestinal parasites may have further health implications. Although not yet fully explored, some studies suggest that long‐term parasitic infections could lead to chronic inflammation and immune system imbalances, which might affect cancer progression or response to treatment. This study supports the need for routine screening for such infections in this group.

This study has several limitations. First, it was conducted in a single geographical region, which may limit the generalizability of the findings. Second, owing to the case–control design, a causal relationship between intestinal protozoan infections and colorectal cancer cannot be established. Another limitation is that molecular confirmation was performed only for Entamoeba spp., whereas other intestinal protozoan parasites were identified solely by microscopic examination based on morphological characteristics and staining patterns. Consequently, species‐level misidentification cannot be completely excluded for protozoa that were not confirmed by molecular methods. Future studies should use molecular or other validated confirmatory methods for all detected protozoan species. Future multicenter studies with larger sample sizes and comprehensive molecular diagnostic approaches are warranted. More investigations should also explore the potential role of intestinal protozoa in colorectal carcinogenesis through inflammatory, immunological, and microbiome‐related mechanisms, as well as their impact on cancer progression and treatment outcomes.

5. Conclusion

This study revealed a significantly higher prevalence of intestinal protozoan parasites in CRC patients compared to non‐CRC controls. These findings highlight the potential clinical relevance of parasitic infections in CRC patients and emphasize the need for routine parasitological screening in this group. From a public health perspective, improved sanitation and hygiene measures are essential to reduce the risk of such infections, particularly in high‐risk populations. The study underscores the importance of environmental and behavioral factors in the transmission of intestinal protozoan parasites among colorectal cancer patients. These findings call for public health strategies that focus on improving sanitation, raising awareness, and implementing regular screening in at‐risk communities. Future studies should explore the long‐term effects of parasitic infections on immune function and cancer outcomes to better understand the relationship between the two.

Author Contributions

Alireza Moayyedkazemi: conceptualization, investigation, validation. Amal Khudair Khalaf: methodology, writing – review and editing, validation. Sima Nik Khah Behnamiri: investigation, validation, methodology, writing – review and editing. Asghar Sepahvand: methodology, writing – review and editing, validation. Neda Niazi: methodology, writing – review and editing, validation. Maryam Sepahvand: methodology, software, data curation. Hossein Mahmoudvand: supervision, writing – original draft, validation, investigation.

Funding

The authors have nothing to report.

Conflicts of Interest

The authors declare no conflicts of interest.

Transparency Statement

Dr. Hossein Mahmoudvand affirms that this manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned have been explained.

Data Availability Statement

The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.