Detection of Entamoeba Spp. in Jordanian Clinical Samples by Traditional and Molecular Methods
DOI:
https://doi.org/10.22317/imj.v8i4.1296Keywords:
Amoebiasis, Diarrhea, Entamoeba, Antigen detection, PCR, Jordan.Abstract
Objective: To evaluate the efficacy of polymerase chain reaction (PCR), enzyme-linked immune-sorbent assay (ELISA), and immunochromatography-based methods compared to conventional microscopy for detecting Entamoeba histolytica in diarrheal stool samples, and to identify a reliable diagnostic tool for amoebiasis.
Methods: A total of 68 fresh diarrheal stool samples were collected from three hospitals in Amman. Samples were tested using microscopy, TechLab E. histolytica II ELISA, and nested-PCR. Additionally, Operon Simple immunochromatography test was performed on 29 randomly selected samples. Detection rates of the methods were compared, and the concordance between techniques was analyzed.
Results: Microscopy detected E. histolytica cysts and/or trophozoites in 76.5% (52/68) of the samples, while ELISA and PCR detected the parasite in 10.3% (7/68) and 29.4% (20/68), respectively. Among the 29 samples tested by immunochromatography, 79.3% (23/29) were positive by microscopy, and 48.3% (14/29) were positive for E. histolytica antigen. Discrepancies were noted, as 3 PCR-positive samples were negative by both ELISA and microscopy, and 7 of the microscopically negative samples were PCR-positive. Immunochromatography detected antigens in 2 of the 16 microscopically negative samples.
Conclusion: None of the methods was found superior to microscopy, though microscopy's limitations, such as its inability to distinguish pathogenic from non-pathogenic Entamoeba species, compromise its reliability. Antigen- and DNA-based methods demonstrated higher specificity, with immunochromatography outperforming ELISA and PCR in simplicity and usability. Further studies with larger sample sizes and a standardized reference method are needed to establish the most effective diagnostic approach for E. histolytica.
References
Davis PH, Zhang X, Guo J, Townsend RR, Stanley SL. Comparative proteomic analysis of two Entamoeba histolytica strains with different virulence phenotypes identifies peroxiredoxin as an important component of amoebic virulence. Mol Microbiol. 2006 Sep;61(6):1523–32.
https://doi.org/10.1111/j.1365-2958.2006.05344.x
Fontecha GA, García K, Rueda MM, Sosa-Ochoa W, Sánchez AL, Leiva B. A PCR-RFLP method for the simultaneous differentiation of three Entamoeba species. Exp Parasitol. 2015 Apr 1;151–152:80–3. https://doi.org/10.1016/j.exppara.2015.02.003
Debenham JJ, Tysnes K, Khunger S, Robertson LJ. Occurrence of Giardia, Cryptosporidium, and Entamoeba in wild rhesus macaques (Macaca mulatta) living in urban and semi-rural North-West India. Int J Parasitol Parasites Wildl. 2017 Apr 1;6(1):29–34. https://doi.org/10.1016/j.ijppaw.2016.12.002
Guillén N. Pathogenicity and virulence of Entamoeba histolytica, the agent of amoebiasis. Vol. 14, Virulence. Taylor and Francis Ltd.; 2023. https://doi.org/10.1080/21505594.2022.2158656
Gonin P, Trudel L. Detection and differentiation of Entamoeba histolytica and Entamoeba dispar isolates in clinical samples by PCR and enzyme-linked immunosorbent assay. J Clin Microbiol. 2003 Jan 1;41(1):237–41. https://doi.org/10.1128/jcm.41.1.237-241.2003
Ali IKM, Roy S. A Real-Time PCR Assay for Simultaneous Detection and Differentiation of Four Common Entamoeba Species That Infect Humans. J Clin Microbiol. 2020 Dec 17;59(1). https://doi.org/10.1128/jcm.01986-20
Jerez Puebla LE, Núñez-Fernández FA, Fraga Nodarse J, Atencio Millán I, Cruz Rodríguez I, Martínez Silva I, et al. Diagnosis of intestinal protozoan infections in patients in Cuba by microscopy and molecular methods: advantages and disadvantages. J Microbiol Methods. 2020 Dec 1;179. https://doi.org/10.1016/j.mimet.2020.106102
Haapanen S, Parkkila S. Management of Entamoeba histolytica infection-treatment strategies and possible new drug targets. https://doi.org/10.1007/7
Alam MA, Maqbool A, Nazir MM, Lateef M, Khan MS, Ahmed AN, et al. Prevalence of entamoeba histolytica-like cysts compared to E. histolytica antigens detected by ELISA in the stools of 600 patients from three socioeconomic communities in the metropolitan city of Lahore, Pakistan. Journal of Parasitology. 2015 Apr 1;101(2):236–9. https://doi.org/10.1645/14-560.1
Hooshyar H, Rostamkhani P. Accurate laboratory diagnosis of human intestinal and extra-intestinal amoebiasis. Vol. 15, Gastroenterology and Hepatology from Bed to Bench. Research Institute for Gastroenterology and Liver Diseases; 2022. p. 343–59. 10.22037/ghfbb.v15i4.2496
Usuda D, Tsuge S, Sakurai R, Kawai K, Matsubara S, Tanaka R, et al. Amebic liver abscess by Entamoeba histolytica. World J Clin Cases. 2022 Dec 26;10(36):13157–66. 10.12998/wjcc.v10.i36.13157
Singh A, Banerjee T, Khan U, Shukla SK. Epidemiology of clinically relevant entamoeba spp. (E. histolytica/dispar/moshkovskii/bangladeshi): A cross sectional study from North India. PLoS Negl Trop Dis. 2021 Sep 1;15(9). https://doi.org/10.1371/journal.pntd.0009762
Shirley DA, Hung CC, Moonah S. Entamoeba histolytica (Amebiasis). Hunter’s Tropical Medicine and Emerging Infectious Diseases [Internet]. 2020 Jan 1 [cited 2024 Jul 31];699–706. Available from: https://linkinghub.elsevier.com/retrieve/pii/B9780323555128000946
Nath J, Ghosh SK, Singha B, Paul J. Molecular Epidemiology of Amoebiasis: A Cross-Sectional Study among North East Indian Population. PLoS Negl Trop Dis. 2015 Dec 3;9(12). https://doi.org/10.1371/journal.pntd.0004225
Fotedar R, Stark D, Beebe N, Marriott D, Ellis J, Harkness J. Laboratory diagnostic techniques for Entamoeba species. Vol. 20, Clinical Microbiology Reviews. 2007. p. 511–32. https://doi.org/10.1128/cmr.00004-07
Rivera WL, De Jesus FC, De Cadiz AE, Villacorte EA, Tongol-Rivera PN, Kanbara H. Entamoeba histolytica infections in a slum community in Manila, Philippines as detected by stool ELISA. Ann Parasitol. 2021;67(4):757–62. 10.17420/ap6704.393
Al-Hilfi AA, Al-Malak MK, Al-Tomah MA. A Prevalence study of Entamoeba spp. In Basrah Province using Different Detection Methods. Baghdad Science Journal. 2021 Apr 30;18(4):1163–72. http://dx.doi.org/10.21123/bsj.2021.18.4.1163
Blessmann J, Buss H, Nu PAT, Dinh BT, Ngo QTV, Van A Le, et al. Real-time PCR for detection and differentiation of Entamoeba histolytica and Entamoeba dispar in fecal samples. J Clin Microbiol. 2002 Dec 1;40(12):4413–7. https://doi.org/10.1128/jcm.40.12.4413-4417.2002
Zeyrek FY ıldız, Turgay N, Unver A, Ustün S, Akarca U, Töz S. Differentiation of Entamoeba histolytica/Entamoeba dispar by the polymerase chain reaction in stool samples of patients with gastrointestinal symptoms in the Sanliurfa Province. Türkiye parazitolojii dergisi / Türkiye Parazitoloji Derneği = Acta parasitologica Turcica / Turkish Society for Parasitology. 2013;37(3):174–8. 10.5152/tpd.2013.39
Zebardast N, Haghighi A, Yeganeh F, Javad S, Tabaei S, Gharavi MJ, et al. Application of Multiplex PCR for Detection and Differentiation of Entamoeba histolytica, Entamoeba dispar and Entamoeba moshkovskii [Internet]. Vol. 9. Available from: http://ijpa.tums.ac.ir
Flaih MH, Khazaal RM, Kadhim MK, Hussein KR, Alhamadani FAB. 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. 10.4178/epih.e2021034
Jannati R, Tavakoli Pasand S, Garedaghi Y. Evaluation of Different Techniques in Laboratory Diagnosis of Intestinal Amoebiasis. International Journal of Medical Parasitology and Epidemiology Sciences. 2024 Mar 29;5(1):16–23. 10.34172/ijmpes.3129
Verweij JJ, Blangé RA, Templeton K, Schinkel J, Brienen EAT, Van Rooyen MAA, et al. Simultaneous Detection of Entamoeba histolytica, Giardia lamblia, and Cryptosporidium parvum in Fecal Samples by Using Multiplex Real-Time PCR. J Clin Microbiol. 2004 Mar;42(3):1220–3. https://doi.org/10.1128/jcm.42.3.1220-1223.2004
Solaymani-Mohammadi S, Rezaian M, Babaei Z, Rajabpour A, Meamar AR, Pourbabai AA, et al. Comparison of a stool antigen detection kit and PCR for diagnosis of Entamoeba histolytica and Entamoeba dispar infections in asymptomatic cyst passers in Iran. J Clin Microbiol. 2006 Jun;44(6):2258–61. https://doi.org/10.1128/jcm.00530-06
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. https://doi.org/10.1186/1471-2180-7-47
ElBakri A, Samie A, Ezzedine S, Odeh RA. Differential detection of Entamoeba histolytica, Entamoeba dispar and Entamoeba moshkovskii in fecal samples by nested PCR in the United Arab Emirates (UAE). Acta Parasitol. 2013 Jun;58(2):185–90. https://doi.org/10.2478/s11686-013-0128-8
Das S, Rajkumari N, Gunalan A, Rajavelu D, Olickal JJ. A Comparative Analysis of Microscopy, Coproantigen Serology, and Nested Multiplex PCR in the Laboratory Diagnosis of Entamoeba histolytica Infection. J Lab Physicians. 2022 Jun;14(02):125–31. DOI: 10.1055/s-0041-1732488
Rahman SMM, Haque R, Roy S, Mondal MMH. GENOTYPING OF ENTAMOEBA HISTOLYTICA BY REAL-TIME POLYMERASE CHAIN REACTION WITH SYBR GREEN I AND MELTING CURVE ANALYSIS. Vol. 4, Bangl. J. Vet. Med. 2006. https://doi.org/10.3329/bjvm.v4i1.1526
Zaki M, Reddy SG, Jackson TFHG, Ravdin JI, Clark CG. Genotyping of Entamoeba Species in South Africa: Diversity, Stability, and Transmission Patterns within Families. Vol. 187, The Journal of Infectious Diseases. 2003. https://doi.org/10.1086/375349
Blessmann J, Ali IKM, Ton Nu PA, Dinh BT, Ngo Viet TQ, Le Van A, et al. Longitudinal study of intestinal Entamoeba histolytica infections in asymptomatic adult carriers. J Clin Microbiol. 2003 Oct 1;41(10):4745–50. https://doi.org/10.1128/jcm.41.10.4745-4750.2003
Ngui R, Angal L, Fakhrurrazi SA, Lian YLA, Ling LY, Ibrahim J, 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(1). https://doi.org/10.1186/1756-3305-5-187
Parija SC, Prabhakar PK. Evaluation of Lacto-Phenol Cotton Blue for Wet Mount Preparation of Feces. Vol. 33, JOURNAL OF CLINICAL MICROBIOLOGY. 1995. https://doi.org/10.1128/jcm.33.4.1019-1021.1995
Wu WK, Chen CC, Panyod S, Chen RA, Wu MS, Sheen LY, et al. Optimization of fecal sample processing for microbiome study — The journey from bathroom to bench. Vol. 118, Journal of the Formosan Medical Association. Elsevier B.V.; 2019. p. 545–55. https://doi.org/10.1016/j.jfma.2018.02.005
Mirelman D, Nuchamowitz Y, Stolarsky T. Comparison of Use of Enzyme-Linked Immunosorbent Assay-Based Kits and PCR Amplification of rRNA Genes for Simultaneous Detection of Entamoeba histolytica and E. dispar. Vol. 35, JOURNAL OF CLINICAL MICROBIOLOGY. 1997. https://doi.org/10.1128/jcm.35.9.2405-2407.1997
Beck DL, Tanyuksel M, Mackey AJ, Haque R, Trapaidze N, Pearson WR, et al. Entamoeba histolytica: sequence conservation of the Gal/GalNAc lectin from clinical isolates. Experimental parasitology. 2002; 101(2-3), pp.157-163. https://doi.org/10.1016/S0014-4894(02)00113-3
Gatti S, Swierczynski G, Robinson F, Anselmi M, Corrales J, Moreira J, et al. AMEBIC INFECTIONS DUE TO THE ENTAMOEBA HISTOLYTICA−ENTAMOEBA DISPAR COMPLEX: A STUDY OF THE INCIDENCE IN A REMOTE RURAL AREA OF ECUADOR. 2002.
Silva MTN, Santana JV, Bragagnoli G, Marinho AM da N, Malagueño E. Prevalência de Entamoeba histolytica/Entamoeba dispar na cidade de Campina Grande, Nordeste do Brasil. Rev Inst Med Trop Sao Paulo. 2014;56(5):451–4. https://doi.org/10.1590/S0036-46652014000500015
Tüzemen NU, Doğan N. [Comparison of direct microscopy, culture, ELISA and molecular methods for diagnosis of Entamoeba histolytica]. Mikrobiyol Bul. 2014 Jan;48(1):114–22. https://doi.org/10.1136/jcp.2004.017822
Furrows SJ, Moody AH, Chiodini PL. Comparison of PCR and antigen detection methods for diagnosis of Entamoeba histolytica infection. J Clin Pathol. 2004 Dec;57(12):1264–6.
Stark D, Van Hal S, Fotedar R, Butcher A, Marriott D, Ellis J, et al. Comparison of stool antigen detection kits to PCR for diagnosis of amebiasis. J Clin Microbiol. 2008 May;46(5):1678–81. https://doi.org/10.1128/jcm.02261-07
Santos R V., Fontes G, Duarte IAC, Santos-Júnior JA, Rocha EMM. Identification of entamoeba histolytica and E. Dispar infection in Maceió, Alagoas state, northeast Brazil. J Infect Dev Ctries. 2016 Oct 1;10(10):1146–50. https://doi.org/10.3855/jidc.6781
Haque R, Uddin Mollah N, Karim Ali IM, Alam K, Eubanks A, Lyerly D, et al. Diagnosis of Amebic Liver Abscess and Intestinal Infection with the TechLab Entamoeba histolytica II Antigen Detection and Antibody Tests [Internet]. JOURNAL OF CLINICAL MICROBIOLOGY. 2000. Available from: https://journals.asm.org/journal/jcm
Haque R, Petri WA. Diagnosis of Amebiasis in Bangladesh. Arch Med Res. 2006 Feb 1;37(2):272–5. https://doi.org/10.1016/j.arcmed.2005.09.001
Abozahra R, Mokhles M, Baraka K. Prevalence and Molecular Differentiation of Entamoeba histolytica, Entamoeba dispar, Entamoeba moshkovskii, and Entamoeba hartmanni in Egypt. Acta Parasitol. 2020 Dec 1;65(4):929–35. https://doi.org/10.1007/s11686-020-00241-y
Guevara Á, Vicuña Y, Costales D, Vivero S, Anselmi M, Bisoffi Z, et al. Use of real-time polymerase chain reaction to differentiate between pathogenic entamoeba histolytica and the nonpathogenic entamoeba dispar in Ecuador. American Journal of Tropical Medicine and Hygiene. 2019;100(1):81–2. 10.4269/ajtmh.17-1022
Lin FH, Chen BC, Chou YC, Chien WC, Chung CH, Hsieh CJ, et al. The Epidemiology of Entamoeba Histolytica Infection and Its Associated Risk Factors among Domestic and Imported Patients in Taiwan during the 2011–2020 Period. Medicina (Lithuania). 2022 Jun 1;58(6). https://doi.org/10.3390/medicina58060820
Tanyuksel M, Petri WA. Laboratory Diagnosis of Amebiasis. Vol. 16, Clinical Microbiology Reviews. 2003. p. 713–29. https://doi.org/10.1128/cmr.16.4.713-729.2003
López-López P, Martínez-López MC, Boldo-León XM, Hernández-Díaz Y, González-Castro TB, Tovilla-Zárate CA, et al. Detection and differentiation of Entamoeba histolytica and Entamoeba dispar in clinical samples through PCR-denaturing gradient gel electrophoresis. Brazilian Journal of Medical and Biological Research. 2017;50(4). https://doi.org/10.1590/1414-431X20175997
Lau YL, Anthony C, Fakhrurrazi SA, Ibrahim J, Ithoi I, Mahmud R. Real-time PCR assay in differentiating Entamoeba histolytica, Entamoeba dispar, and Entamoeba moshkovskii infections in Orang Asli settlements in Malaysia. Parasit Vectors. 2013;6(1). https://doi.org/10.1186/1756-3305-6-250
Lebbad M, Svärd SG. PCR differentiation of Entamoeba histolytica and Entamoeba dispar from patients with amoeba infection initially diagnosed by microscopy. Scand J Infect Dis. 2005;37(9):680–5. https://doi.org/10.1080/00365540510037812
Luciano F, Santos N, De Souza Gonçalves M, Soares NM, Moniz G, Cruz O, et al. Validation and utilization of PCR for differential diagnosis and prevalence determination of Entamoeba histolytica/Entamoeba dispar in Salvador City, Brazil. Vol. 15, Braz J Infect Dis. 2011. https://doi.org/10.1590/S1413-86702011000200005
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 Mar;45(3):1035–7. https://doi.org/10.1128/jcm.02144-06
Johnston SP, Pieniazek NJ, Xayavong M V., Slemenda SB, Wilkins PP, Da Silva AJ. PCR as a confirmatory technique for laboratory diagnosis of malaria. J Clin Microbiol. 2006 Mar;44(3):1087–9. https://doi.org/10.1128/jcm.44.3.1087-1089.2006
Mosleh IM, Shönian G, Geith E, Al-Jawabreh A, Natsheh L. The Jordanian Mid Jordan Valley is a classic focus of Leishmania major as revealed by RFLP of 56 isolates and 173 ITS-1-PCR-positive clinical samples. Exp Parasitol. 2015 Jan 1;148:81–5. https://doi.org/10.1016/j.exppara.2014.11.006
Saidin S, Othman N, Noordin R. Update on laboratory diagnosis of amoebiasis. Vol. 38, European Journal of Clinical Microbiology and Infectious Diseases. Springer Verlag; 2019. p. 15–38. https://doi.org/10.1007/s10096-018-3379-3
Otieno BIA, Matey EJ, Bi X, Tokoro M, Mizuno T, Panikulam A, et al. Intestinal parasitic infections and risk factors for infection in Kenyan children with and without HIV infection. Parasitol Int. 2023 Jun 1;94. https://doi.org/10.1016/j.parint.2022.102717
Monteiro L, Bonnemaison D, Vekris A, Petry KG, Bonnet J, Vidal R, et al. Complex Polysaccharides as PCR Inhibitors in Feces: Helicobacter pylori Model. Vol. 35, JOURNAL OF CLINICAL MICROBIOLOGY. 1997. https://doi.org/10.1128/jcm.35.4.995-998.1997
Holland JL, Louie L, Simor AE, Louie M. PCR Detection of Escherichia coli O157:H7 Directly from Stools: Evaluation of Commercial Extraction Methods for Purifying Fecal DNA. Vol. 38, JOURNAL OF CLINICAL MICROBIOLOGY. 2000. https://doi.org/10.1128/jcm.38.11.4108-4113.2000
Ayana M, Cools P, Mekonnen Z, Biruksew A, Dana D, Rashwan N, et al. Comparison of four DNA extraction and three preservation protocols for the molecular detection and quantification of soil-transmitted helminths in stool. PLoS Negl Trop Dis. 2019;13(10). https://doi.org/10.1371/journal.pntd.0007778
Downloads
Published
How to Cite
Issue
Section
License
Copyright (c) 2024 Iraq Medical Journal
This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.