Special Issue:

Emerging and Re-emerging Animal Health Challenges in Low and Middle-Income Countries

Molecular Detection of Entamoeba histolytica in Human and Cattle

Mohammed Jawad Kadhim*, Qasim Jawad Amer

Department of Parasitology, College of Veterinary Medicine, Al-Qasim Green University, 51013, Babylon, Iraq.

Received | July 18, 2024; Accepted | October 04, 2024; Published | December 04, 2024

*Correspondence | Mohammed Jawad Kadhim, Department of Parasitology, College of Veterinary Medicine, Al-Qasim Green University,51013 Babylon, Iraq; Email: [email protected]

Citation | Kadhim MJ, Amer QJ (2024). Molecular detection of Entamoeba histolytica in human and cattle. J. Anim. Health Prod. 12(s1): 182-186.

DOI | https://dx.doi.org/10.17582/journal.jahp/2024/12.s1.182.186

ISSN (Online) | 2308-2801

Copyright: 2024 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Introduction

Internal protozoa of the genus Entamoeba can cause an infection known as amoebiasis. Following malaria and schistosomiasis in terms of human mortality, E. histolytica is ranked as the third most dangerous parasite (Lourenço, 2023; Baig et al., 2024). Amoebiasis typically spreads in underdeveloped nations due to a lack of sanitation, low hygiene standards, and overcrowding in the housing market. In contrast, people who have traveled to regions where the disease is common are the principal vector for its spread in developed countries (Fletcher et al., 2012). Many cases arise from persons who are carriers of the illness, referred to as cyst passers, and who excrete the parasite in their feces, either in a fully mature or partially formed state (Cui et al., 2019). Monkeys, dogs, cattle, and potentially pigs are naturally affected by E. histolytica, although the likelihood of human exposure from these species is quite low compared to humans themselves (Hossain et al., 2023). Global frequency of E. histolytica infection is significant, particularly in tropical and subtropical regions. Amoebiasis is expected to impact five hundred million worldwide (Roro et al., 2022). Recently a study has approximated that amoebiasis affects fifty million individuals on a yearly basis, resulting in an estimated 100,000 deaths per year (Flaih et al., 2021; Gupta et al., 2022). Entamoeba spp. is a type of protozoan that lives in the intestines and can cling to and damage the tissue lining the intestines. The life cycle of this organism consists of two forms: Trophozoites and cysts. Infection is acquired through the consumption of water or food that has been contaminated with cysts. The trophozoites in the small intestine undergo excystation to reach maturity and establish themselves in the colonic area, where they then attach to the mucosal layer of the large intestines. E. histolytica is the causative agent of both intestinal and extra-intestinal infections (Al-Yasari et al., 2019; Vaisusuk and Saijuntha, 2021; Hamzah et al., 2020). PCR-based methods often demonstrate higher sensitivity and specificity in comparison to microscopy tests (Markussen et al., 2023). This study was conducted in Babylon City, Iraq to assess the efficacy of a multiplex PCR method for the molecular detection of Entamoeba histolytica in both humans and cattle.

Materials and Method

Sampling

In various parts of Babylon Governorate, Iraq, feces samples were taken from 100 humans and 100 animals. Containers containing the samples were sealed and sent cold to the Parasitology Laboratory at the College of Veterinary Medicine at Al-Qasim Green University in Babylon, Iraq. The collection period for these samples was from October 2023 to April 2024.

Microscopic inspection

The condition of the feces and the presence of any visible blood or mucus were meticulously recorded in each sample. Parasite cysts and their vegetative stages were detected using a wet direct dye using iodine dye.

PCR analysis

The DNA was extracted after each 200 mg fecal sample was washed in 1 ml of sterile Phosphate Buffer saline with a pH of 7.2. The samples were then centrifuged at 14,000 × g for 5 minutes, following the instructions of the QIAGEN kit from Hilden, Germany. Primers used in this investigation were F: GGCCGTTCTTAGTTGGTGGA and R: GTGTGTACAAAGGGCAGGGA for a product of 374bp (Albanese et al., 2019; Mosa et al., 2022). 0.2 microliters of DNA polymerase at 5 units per microliter, 2 microliters of DNA at 100 nanograms, 0.6 microliters of each primer at 40 micromoles, and 16.5 microliters of PCR-water were utilized for the PCR reaction component. Thermocycler settings included 92°C denaturation, 40 cycles of denaturation, annealing, and extension (72°C for 45 seconds followed by 72°C for 5 minutes), and 72°C for 5 minutes of final extension.

Statistical assessment

According to Schiefer (1980), statistical analysis was performed on all samples in this study using chi-square to evaluate if there were significant differences for the analyzed variables. Significant differences were defined at the probability level (P≤0.05).

Results and Discussion

Microscopically findings

The morphology and motility of the trophozoite were examined under the microscope, and the nuclei in both the cyst of the parasite and the trophozoite were stained with iodine Figure 1.

 

 

PCR technique

We used PCR on DNA samples that had a concentration of 400 to 710 ng/μl and a purity level of 1.6 to 1.8. Out of 100 human feces samples, 43% tested positive for the ITS gene, according to the total results of the PCR technique (Figure 2). The correlation between frequency and sex of patient showed that out of 54 male 27 were positive (27/54) 50% of infection, while in female were (16/46) 34.8% as described in Table 1. Moreover, the results shown in Table 2 showed that the highly frequency of E. histolytica were in children less than 5 years old 48.6% (18/37). While in patient from 5-10 years and patient more than 10 years were 13/34(38.2) and 12/29(41.4), respectively.

 

Table 1: The frequency of E. histolytica in feces of human diagnosed by PCR.

Percentage of total (٪)	Positive	Total cases	Sex
50	27	54	Male
34.8	16	46	female
43	43	100	Total
2.346868			X2
0.125535NS			P value

 

Table 2: The age correlation with the frequency of E. histolytica of fecal human samples.

Percentage of total (٪)	Positive	Total cases	Age
48.6	18	37	Less than 5 years
38.2	13	34	From 5 years to 10 years
41.4	12	29	More than 10 years
43	43	100	Total
0.827671			X2
0.661110 NS			P value

 

For cattle, the total results of PCR technique showed that, out of 100 cow stool samples (59%) were positive for (ITS) gene as depicted in Figure 3. The correlation between frequency of E. histolytica and sex of animal showed that out of 25 male 13 were positive (13/25) 52% of infection, while in female were (46/75) 61% as described in Table 3. On the other hand, the results in Table 4 showed that the highly frequency of E. histolytica were in animals from 2 years old and above 63.2% (24/38). While the animals aged less than 2 years the frequency were 35/62(56.5).

 

Table 3: The frequency of E. histolytica in cattle that diagnosed by PCR.

Percentage of total (٪)	Positive	Total cases	Sex
52	13	25	Male
61	46	75	Female
59	59	100	Total
0.675210			X2
0.411241 NS			P value

 

Table 4: Age correlation with frequency of E. histolytica in cattle fecal samples.

Percentage of total (٪)	Positive	Total cases	Age
56.5	35	62	Less than 2 years
63.2	24	38	From 2 years and above
59	59	100	Total
0.438029			X2
0.508075 NS			P value

 

 

The findings of this study indicate that PCR is a highly efficient method for detecting E. histolytica. Therefore, employing PCR is a feasible method for distinguishing between distinct species. This approach has superior sensitivity and specificity when compared to microscopy (Hamzah et al., 2019). The ITS1 region of the parasite genome is essential for molecular diagnostics, as it is used for both species’ identification and overall diagnosis (Paul and Kannan, 2019). López-López et al. (2017) have utilized a segment of the ITS1 gene to accurately distinguish and categorize E. histolytica and E. dispar in their investigation. Five single nucleotide changes were identified in this gene between the two species. The precise detection of E. histolytica and E. dispar was achieved by various approaches, including real-time PCR. Regrettably, the excessively high cost limits their usage in developing nations, and specific models may generate inaccurately pessimistic results (Calderaro et al., 2015; Van Den Broucke et al., 2018). In addition, the PCR method based on the ITS1 gene was shown to be extremely efficient in the current study. The user’s text is a single period. The findings align with the study conducted by Saba and Meki (2017), which determined that the ITSI gene is the suitable DNA region for identifying E. histolytica in the middle Euphrates region of Iraq. The findings of this experiment were like those seen by (Zebardast et al., 2014; Mohmood et al., 2020). The infection rate was 62.96%, while Iran’s infection rate stood at 67.7%. A study conducted by Sajid et al. (2024) revealed that 3.2% of Malaysians were exclusively infected with E. histolytica, whereas 13.4% had concurrent infections of both E. histolytica and E. dispar. According to the studies conducted by Abdel-Naim (2012) and Al-Aarif and Al-Jarjary (2023), the infection rate of E. histolytica among patients in the outpatient clinic at Al-Zaqaziq University Hospital was 50%, while the infection rate of E. dispar was 35%. Emphasizing the efficacy of the primers developed for diagnosing E. histolytica is crucial. The primers successfully amplified the target section in the ITS1 region, leading to the presence of migration products at the target test. The size of these products was 374 base pairs, as depicted in Figures 2 and 3. This confirms and validates the accuracy of the diagnosis. The discrepancies in the outcomes of the PCR approach can be ascribed to variations in the methodologies employed for DNA extraction from fecal samples and the implementation of PCR, together with potential disparities in the quantity of parasites detected in the feces. Alternatively, the variations in infection rates of these parasites may be attributed to their random spread across numerous countries worldwide, influenced by diverse climatic conditions, cultural practices, and traditional beliefs.

Conclusions and Recommendations

Microscopic examination continues to be a dependable technique for verifying the existence of the parasite. Nevertheless, it lacks efficacy in assessing the parasite’s pathogenicity or discerning its precise classifications. Hence, it is advisable to employ serological or molecular diagnostic techniques in addition to the findings from microscopic inspection. The molecular and serological methods used in this study demonstrated a high level of sensitivity and specificity.

Acknowledgment

Warmest our-thanks for the head of Department of parasitology for their supporting me to completion of the study.

Novelty Statement

Evaluate the potential of Molecular Detection of Entamoeba histolytica in Human and Cattle.

Author’s Contribution

Qasim Jawad Amer and Mohammed Jawad Kadhim are participatred in research desing, proposal writing, experimental and clinical works and also drafting the manuscript.

Ethical approval

Ethical approval was obtained from the ethical committee (127/2023) in the Al-Qasim Green University, College of Veterinary Medicine.

Conflict of interest

The authors have declared no conflict of interest.

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