Identification of Entamoeba Species in Diarrheal Samples Using Sequence Analysis and Nested Multiplex PCR
,
Abstract
Background: Among the many Entamoeba species that infect humans, only Entamoeba histolytica is considered pathogenic, being responsible for amoebiasis or amoebic dysentery.
Methods: Between June and October 2022, a total of 106 stool samples were collected from children under six years of age presenting with diarrhea at Paiji Hospital in the city of Paiji, Iraq. DNA was extracted from all stool specimens to detect the presence of parasitic organisms.
Results: Of the 106 fecal samples, 4 (3.7%) tested positive for Entamoeba spp. using an initial PCR amplification targeting approximately 900 bp of the 18S rRNA gene. Among these, only one sample tested positive for E. histolytica using a nested multiplex PCR assay. In this study, neither E. moshkovskii nor E. dispar was detected. Sequence analysis of the partial 18S rRNA gene revealed that 0.9% of samples were positive for E. histolytica, while 2.8% were positive for E. coli. The sequences were deposited in GenBank under the accession numbers OP868733.1 for E. histolytica and OP868730.1, OP868731.1, and OP868732.1 for E. coli.
Conclusion: Children were infected with different species of Entamoeba. Molecular methods are essential for distinguishing between Entamoeba species due to their significance in accurate diagnosis and appropriate treatment strategies.
1. Fotedar R, Stark D, Beebe N, Marriott D, Ellis J, Harkness J. PCR detection of Entamoeba histolytica, Entamoeba dispar, and Entamoeba moshkovskii in stool samples from Sydney, Australia. J Clin Microbiol. 2007; 45(3):1035-7.
2. Paniker CJ. Paniker's textbook of medical Parasitology. JP Medical Ltd; 2017 Sep 14.
3. Uribe-Querol E, Rosales C. Immune response to the enteric parasite Entamoeba histolytica. Physiology (Bethesda). 2020; 35(4):244-60.
4. Chowdhury RA, Esiobu N, Meeroff DE, Bloetscher F. Different detection and treatment methods for Entamoeba histolytica and Entamoeba dispar in water/wastewater: a review. J Environ Prot. 2022; 13(1):126-49.
5. Alyasari HF, Hashim HO, Altaei AH, Al-Shuhaib MB. Identification of serious clinical amebic dysentery cases in the middle Euphrates region of Iraq. J Biol Sci. 2019; 19 (2):148-154.
6. Feng M, Pandey K, Yanagi T, et al. Prevalence and genotypic diversity of Entamoeba species in inhabitants in Kathmandu, Nepal. Parasitol Res. 2018; 117:2467-72.
7. Rattaprasert P, Nitatsukprasert C, Thima K, et al. Development of nested pcr for identification of Entamoeba coli in human fecal samples. Southeast Asian J Trop Med Public Health. 2022; 53(1):21-36.
8. Begum S, Quach J, Chadee K. Immune evasion mechanisms of Entamoeba histolytica: progression to disease. Front Microbiol. 2015; 6:1394.
9. Liu X, Bao G, Yue M, et al. Prevalence and molecular identification of Entamoeba spp. in non-human primates in a zoological garden in Nanjing, China. Front Vet Sci. 2022; 9:906822.
10. Gomes TD, Garcia MC, Souza Cunha FD, et al. Differential Diagnosis of Entamoeba spp. in Clinical Stool Samples Using SYBR Green Real‐Time Polymerase Chain Reaction. ScientificWorldJournal. 2014; 2014:645084.
11. Puebla LE, Núñez-Fernández FA, Nodarse JF, et al. Diagnosis of intestinal protozoan infections in patients in Cuba by microscopy and molecular methods: advantages and disadvantages. J Microbiol Methods. 2020; 179:106102.
12. Soares NM, Azevedo HC, Pacheco FTF, et al. A Cross-Sectional Study of Entamoeba histolytica/dispar/moshkovskii Complex in Salvador, Bahia, Brazil. Biomed Res Int. 2019; 2019:7523670.
13. Khairnar K, Parija SC. A novel nested multiplex polymerase chain reaction (PCR) assay for differential detection of Entamoeba histolytica, E. moshkovskii and E. dispar DNA in stool samples. BMC Microbiol. 2007; 7:47.
14. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol Biol Evol. 2013; 30(12):2725-9.
15. Carrero JC, Reyes-López M, Serrano-Luna J, et al. Intestinal amoebiasis: 160 years of its first detection and still remains as a health problem in developing countries. Int J Med Microbiol. 2020; 310(1):151358.
16. Mahmood SA, Bakr HM. Molecular Identification and Prevalence of and in Erbil City, Northern Iraq. Pol J Microbiol. 2020; 69(3):1-10.
17. Flaih MH, Khazaal RM, Kadhim MK, Hussein KR, Alhamadani FA. The epidemiology of amoebiasis in Thi-Qar Province, Iraq (2015-2020): differentiation of Entamoeba histolytica and Entamoeba dispar using nested and real-time polymerase chain reaction. Epidemiol Health. 2021; 43:e2021034.
18. Ziguer AM, Mahdi DS, Khalaf AK. Molecular detection of Entamoeba spp. By using multiplex PCR in Thi-Qar province. Biochem Cell Arch. 2020; 20(2):6363-7.
19. Bahrami F, Haghighi A, Zamini G, et al. Differential detection of Entamoeba histolytica, Entamoeba dispar and Entamoeba moshkovskii in faecal samples using nested multiplex PCR in west of Iran. Epidemiol Infect. 2019;147: e96.
20. Mulinge E, Mbae C, Ngugi B, et al. Entamoeba species infection in patients seeking treatment for diarrhea and abdominal discomfort in Mukuru informal settlement in Nairobi, Kenya. Food Waterborne Parasitol. 2021; 23: e00122.
21. Hamad N, Ahmed R. Intestinal parasites among patients attending general central public health laboratory in Erbil city-Iraq during 1998-2004. J Educ Sci. 2011;24(4):79-86.
22. Ngui R, Angal L, Fakhrurrazi SA, et al. Differentiating Entamoeba histolytica, Entamoeba dispar and Entamoeba moshkovskii using nested polymerase chain reaction (PCR) in rural communities in Malaysia. Parasit Vectors. 2012; 5:187.
23. Norouzi P, Mohaghegh MA, Ghorbani M, et al. Investigating the prevalence of intestinal parasites with an emphasis on Strongyloides stercoralis infection in hospitalized patients: a regional report from Iran. Ann Parasitol. 2020; 66(3): 365-371.
24. Alsaafin Y, Omer A, Felemban O, et al. Prevalence and Risk Factors of Schistosomiasis in Sudan: A Systematic Review and Meta-Analysis. Cureus. 2024;16(11): e73966.
25. Belchior VLR, Toma HK, Batista KM, et al. Predominance of Blastocystis spp., Entamoeba histolytica /e.dispar and other protozoan evaluated through classical and molecular diagnostic techniques in two communities of Niterói-rj / Brazil. Braz J Health Rev. 2021; 4(3):11741-11754.
26. Lepore CM, Kamau L, Kanduma E. Molecular Prevalence of Entamoeba Species among Diarrheal Patients in Eastern Kenya. Eur J Biol Biotechnol. 2022;3(1):39-46.
2. Paniker CJ. Paniker's textbook of medical Parasitology. JP Medical Ltd; 2017 Sep 14.
3. Uribe-Querol E, Rosales C. Immune response to the enteric parasite Entamoeba histolytica. Physiology (Bethesda). 2020; 35(4):244-60.
4. Chowdhury RA, Esiobu N, Meeroff DE, Bloetscher F. Different detection and treatment methods for Entamoeba histolytica and Entamoeba dispar in water/wastewater: a review. J Environ Prot. 2022; 13(1):126-49.
5. Alyasari HF, Hashim HO, Altaei AH, Al-Shuhaib MB. Identification of serious clinical amebic dysentery cases in the middle Euphrates region of Iraq. J Biol Sci. 2019; 19 (2):148-154.
6. Feng M, Pandey K, Yanagi T, et al. Prevalence and genotypic diversity of Entamoeba species in inhabitants in Kathmandu, Nepal. Parasitol Res. 2018; 117:2467-72.
7. Rattaprasert P, Nitatsukprasert C, Thima K, et al. Development of nested pcr for identification of Entamoeba coli in human fecal samples. Southeast Asian J Trop Med Public Health. 2022; 53(1):21-36.
8. Begum S, Quach J, Chadee K. Immune evasion mechanisms of Entamoeba histolytica: progression to disease. Front Microbiol. 2015; 6:1394.
9. Liu X, Bao G, Yue M, et al. Prevalence and molecular identification of Entamoeba spp. in non-human primates in a zoological garden in Nanjing, China. Front Vet Sci. 2022; 9:906822.
10. Gomes TD, Garcia MC, Souza Cunha FD, et al. Differential Diagnosis of Entamoeba spp. in Clinical Stool Samples Using SYBR Green Real‐Time Polymerase Chain Reaction. ScientificWorldJournal. 2014; 2014:645084.
11. Puebla LE, Núñez-Fernández FA, Nodarse JF, et al. Diagnosis of intestinal protozoan infections in patients in Cuba by microscopy and molecular methods: advantages and disadvantages. J Microbiol Methods. 2020; 179:106102.
12. Soares NM, Azevedo HC, Pacheco FTF, et al. A Cross-Sectional Study of Entamoeba histolytica/dispar/moshkovskii Complex in Salvador, Bahia, Brazil. Biomed Res Int. 2019; 2019:7523670.
13. Khairnar K, Parija SC. A novel nested multiplex polymerase chain reaction (PCR) assay for differential detection of Entamoeba histolytica, E. moshkovskii and E. dispar DNA in stool samples. BMC Microbiol. 2007; 7:47.
14. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol Biol Evol. 2013; 30(12):2725-9.
15. Carrero JC, Reyes-López M, Serrano-Luna J, et al. Intestinal amoebiasis: 160 years of its first detection and still remains as a health problem in developing countries. Int J Med Microbiol. 2020; 310(1):151358.
16. Mahmood SA, Bakr HM. Molecular Identification and Prevalence of and in Erbil City, Northern Iraq. Pol J Microbiol. 2020; 69(3):1-10.
17. Flaih MH, Khazaal RM, Kadhim MK, Hussein KR, Alhamadani FA. The epidemiology of amoebiasis in Thi-Qar Province, Iraq (2015-2020): differentiation of Entamoeba histolytica and Entamoeba dispar using nested and real-time polymerase chain reaction. Epidemiol Health. 2021; 43:e2021034.
18. Ziguer AM, Mahdi DS, Khalaf AK. Molecular detection of Entamoeba spp. By using multiplex PCR in Thi-Qar province. Biochem Cell Arch. 2020; 20(2):6363-7.
19. Bahrami F, Haghighi A, Zamini G, et al. Differential detection of Entamoeba histolytica, Entamoeba dispar and Entamoeba moshkovskii in faecal samples using nested multiplex PCR in west of Iran. Epidemiol Infect. 2019;147: e96.
20. Mulinge E, Mbae C, Ngugi B, et al. Entamoeba species infection in patients seeking treatment for diarrhea and abdominal discomfort in Mukuru informal settlement in Nairobi, Kenya. Food Waterborne Parasitol. 2021; 23: e00122.
21. Hamad N, Ahmed R. Intestinal parasites among patients attending general central public health laboratory in Erbil city-Iraq during 1998-2004. J Educ Sci. 2011;24(4):79-86.
22. Ngui R, Angal L, Fakhrurrazi SA, et al. Differentiating Entamoeba histolytica, Entamoeba dispar and Entamoeba moshkovskii using nested polymerase chain reaction (PCR) in rural communities in Malaysia. Parasit Vectors. 2012; 5:187.
23. Norouzi P, Mohaghegh MA, Ghorbani M, et al. Investigating the prevalence of intestinal parasites with an emphasis on Strongyloides stercoralis infection in hospitalized patients: a regional report from Iran. Ann Parasitol. 2020; 66(3): 365-371.
24. Alsaafin Y, Omer A, Felemban O, et al. Prevalence and Risk Factors of Schistosomiasis in Sudan: A Systematic Review and Meta-Analysis. Cureus. 2024;16(11): e73966.
25. Belchior VLR, Toma HK, Batista KM, et al. Predominance of Blastocystis spp., Entamoeba histolytica /e.dispar and other protozoan evaluated through classical and molecular diagnostic techniques in two communities of Niterói-rj / Brazil. Braz J Health Rev. 2021; 4(3):11741-11754.
26. Lepore CM, Kamau L, Kanduma E. Molecular Prevalence of Entamoeba Species among Diarrheal Patients in Eastern Kenya. Eur J Biol Biotechnol. 2022;3(1):39-46.
| Files | ||
| Issue | Vol 20 No 2 (2025) | |
| Section | Original Article(s) | |
| Keywords | ||
| Entamoeba spp. Diarrhea samples Nested multiplex PCR assay | ||
| Rights and permissions | |
|
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. |
How to Cite
1.
Mohammed Abed Z, Hamad RM. Identification of Entamoeba Species in Diarrheal Samples Using Sequence Analysis and Nested Multiplex PCR. Iran J Parasitol. 2025;20(2):253-260.
