Original Article

Examination of fecal calprotectin level in patients with Entamoeba histolytica from 2020 to 2022

Introduction

Amebiasis is a parasitic infectious disease caused by Entamoeba histolytica in the human body.¹ Abundant biochemical, genetic, and immunological studies have proven that the organism known as E. histolytica consists of two identical morphological species; however, their biological treatment and pathogenesis are different. One of these species is E. histolytica, which is a potentially invasive pathogen. This pathogen can cause amebiasis colitis and some types of extraintestinal amebiasis. Amebiasis can be asymptomatic. Another species, which is called Entamoeba dispar, coexists in the human bowel. This species is non-invasive and does not cause disease.² In Iran, amebiasis is endemic, and its prevalence varies from 8% to 40%.³

During recent decades, many leukocytic products have been discovered that can be secreted into feces. These products are considered as bowel inflammatory factors. Among these products, calprotectin and lactoferrin are the best biomarkers.⁴ These proteins are produced in monocytes, neutrophils, endothelium, and epithelium cells.⁵ Following the activation of neutrophils and monocyte attachment to epithelium or endothelium cells, calprotectin is released, and its level in serum and body fluids is an important inflammatory factor.5⁵ Calprotectin is increased in urine, feces, and synovial fluid during inflammatory conditions and malignancies. In inflammatory bowel disease (IBD), neutrophils migrate into the bowel lumen, which can explain why the calprotectin level rises in this disease.⁶ So, the level of calprotectin in feces is related to the number of neutrophil cells migrated into the bowel lumen.⁷

Studies have shown that calprotectin level measurement can distinguish between IBD and other non-inflammatory diseases.⁸ Also, calprotectin can determine the severity of mucus inflammation, and its levels are correlated to the severity of inflammation and endoscopic indices. As endoscopy procedure is costly and also less intolerable for patients, utilizing an accessible, non-invasive, and inexpensive biomarker like calprotectin is valuable for clinicians.⁹ Because of these roles of calprotectin, we designed this study to determine if calprotectin level can help us distinguish gastroenteritis caused by E. histolytica or not.

Material and Methods

Study design and setting

This is a descriptive-analytic study, conducted in the Sina and Imam Reza Hospital of Tabriz University of Medical Sciences clinics, between 2020–2022. In this study, all the patients with a positive result of E. histolytica in stool exam were involved. To reduce distracting factors, patients with gastrointestinal (GI) malignancies, IBD, immunodeficiency, and who were undergoing radiotherapy and chemotherapy were excluded from the study.

In this study, convenience sampling was done. According to the similar studies, by using sample size calculation formulas (with 90% power and 5% of type 1 error, or alpha) and consideration of 1.4 variances, the sample size was calculated as at least 50 patients.

Data collection and study performing

Patients’ characteristics like age, gender, medical history, and current treatment was obtained by using a checklist. A blood sample was obtained from all the patients to check complete blood count (CBC) and erythrocyte sedimentation rate (ESR). Also, the patients’ stool was examined to measure the level of fecal calprotectin (FCP). Patients’ feces were kept in plastic containers at the temperature of -20 centigrade for 6 hours. FCP level was measured by ELISA method and laboratory kits (Calprest, Dynex Elisa Eurospital, Trieste, Italy). FCP is expressed by milligrams per kilogram of moist feces unit. Its normal range is defined as less than 50 milligrams per kilogram of moist feces.

Statistical analysis

All data were analyzed using IBM SPSS 26, and the Kolmogorov-Smirnov test was used to determine the normality of the collected data. According to the distribution of quantitative data, the mean and standard deviation were reported. We also reported the nominal and ordinal variables with frequencies and percentages.

Results

As mentioned above, 50 participants were involved in this study. Thirty-one participants were male and the rest were female (19 participants). Their mean age was 49.8 years, estimated at 47.5 for females and 50.9 for males. Females’ mean age was less than that of males but the difference was not significant (P = 0.259). Among these participants, 19 participants had more than 50 mg/kg calprotectin in their feces which is considered as positive. The number of males in this group of participants (more than 50 mg calprotectin in a kilogram of feces) was significantly more than that of females (P = 0.035).

Among participants with positive calprotectin in stool, 3 participants had a history of omeprazole consumption, and 3 of them had a history of non-steroidal anti-inflammatory drugs (NSAIDs) use. Among 3 participants with a negative calprotectin test, 2 of them had consumed NSAIDs and 1 of them had consumed omeprazole. However, there was not a significant difference between drug consumption and the result of the calprotectin test (P = 0.369). The results of our study show that there was no significant relation between the calprotectin level and mean age among participants (P = 0.695).

In this study, in terms of the duration of the disease, patients were divided into two groups; more than 7 days and less than 7 days. The results show that the number of patients with positive calprotectin was more in the group of more than 7 days of disease duration, compared to the group of less than 7 days of disease duration (P = 0.018). Meanwhile, there is no significant dispute between females and males in the aspect of the duration of the disease (P = 0.524) (Table 1).

Participants were divided into two groups in terms of severity of symptoms: mild and severe. 31 of them were in the severe group, and the rest of them were in the mild group. Calprotectin level was significantly higher in the severe group than the mild one (P = 0.009). Meanwhile, there is no significant dispute between the severity of symptoms and calprotectin levels in terms of gender (P = 0.660) (Table 2).

Discussion

Entamoeba is a monocellular parasite that can cause infection in the bowel. The symptoms of this infection include diarrhea, crampy pain, nausea, and vomiting. Many indices are used to diagnose and follow up on these patients; one of these indices is assessing fecal calprotectin. Fecal calprotectin can also demonstrate the severity of infection.

Recent studies illustrate that the level of fecal calprotectin can be elevated in Entamoeba-infected patients. This protein is produced and secreted by mucosal cells of the bowel and stomach and plays an important role against microbes. In the case of Entamoeba infection, a microbe’s invasion into mucus can lead to an increase in fecal calprotectin secretion.¹⁰

In a recent novel study, scientists found that fecal calprotectin level in patients with severe infection is significantly higher than in patients with mild disease.¹¹ As a result, fecal calprotectin level can predict the severity of the disease in addition to its diagnosis.¹²

The results of a study show that the level of fecal calprotectin in patients with dysentery (bloody diarrhea) is higher in comparison with patients without dysentery.⁸ In another study, calprotectin levels in hospitalized patients with severe symptoms were higher than outpatients with mild symptoms.¹³

In a comparative study, fecal calprotectin levels in IBD patients were measured. It was demonstrated that the level of calprotectin increases with the severity of the disease. Also, this increase was more common among patients with IBD and infectious causes of bowel inflammation.¹⁴

In another study, the results showed that fecal calprotectin levels in other infections like C. difficile were higher than normal ranges. As a result, although calprotectin is a sensitive marker for diagnosis and prediction of severity of disease, it is not a specific marker.¹⁵

Regarding these results, fecal calprotectin levels can be a useful index for the diagnosis and severity prediction of diseases like intestinal amebiasis and other causes of bowel inflammation. This can help clinicians make better decisions for the treatment of patients and provide appropriate follow-ups.¹⁶

In a comparative study, fecal calprotectin level was measured in two groups of patients: ulcerative colitis and amebiasis colitis; and then compared.¹⁷ The result showed that patients with amebiasis colitis had higher levels than patients with ulcerative colitis. It turns out that calprotectin levels can be useful in distinguishing the cause of colitis.¹⁸

In another study, fecal calprotectin level was compared with levels of specific antigens of Entamoeba in infected patients.¹⁹ It showed that the levels of these two parameters are correlated with each other. In other words, as the level of specific antigens rises, calprotectin level increases. This also indicated that this index can be used to predict the severity of the disease.¹³

In a study, the level of fecal calprotectin was measured and compared in two groups of patients: with symptoms and without symptoms.²⁰ The results showed that fecal calprotectin in symptomatic patients was higher than in asymptomatic patients, which illustrates that it can be useful for diagnosis and prediction of the severity of the disease.¹⁴

Although all these studies show that fecal calprotectin level is a sensitive marker for diagnosing infection, other precise tests like microscopic investigation or molecular tests must be done in order to confirm the diagnosis. These tests are used to differentiate the diagnosis and distinguish Entamoeba infection from other causes of bowel infection.²¹

A similar study, in which the calprotectin level was measured in IBD patients, showed that as IBD becomes severe, calprotectin level increases. This is a general pattern for all IBDs. Moreover, it can be used to assess the severity of the disease and its follow-up.²²

According to the results of our study, as the duration of the disease extends, fecal calprotectin level increases, which can show the severity of inflammation and the body’s immune system response to infection.²³ To confront this infection, the immune system becomes activated, and inflammatory cells, along with cytokines and inflammatory proteins like calprotectin, are present in the bowel.²⁴ That can explain why calprotectin levels rise. Extended duration of disease can indicate that inflammation is persistent in the bowel. However, fecal calprotectin is one of the markers used to diagnose and follow up on disease and it is worth mentioning that for confirmation of the diagnosis, other methods and tests are needed.

Conclusion

As a conclusion, fecal calprotectin levels can help clinicians diagnose and predict the severity of disease in patients infected with amebiasis, especially in long-term conditions.

Competing Interests

There is no conflict of interest in this work.

Ethical Approval

The study process was reviewed and approved by the ethics committee of Tabriz University of Medical Sciences, according to the declaration of Helsinki (ethics code: IR.TBZMED.REC.1401.959). Before collecting data, informed consent was obtained from all patients. All methods were carried out following relevant guidelines and regulations.

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