Hostname: page-component-586b7cd67f-r5fsc Total loading time: 0 Render date: 2024-11-26T16:51:52.706Z Has data issue: false hasContentIssue false
  • English
  • Français

Development and evaluation of molecular tools for detecting and differentiating intestinal amoebae in healthy individuals

Published online by Cambridge University Press:  14 January 2019

Amal Chihi ,
Christen R. Stensvold Open the ORCID record for Christen R. Stensvold [Opens in a new window] ,
Imene Ben-abda ,
Rania Ben-Romdhane ,
Karim Aoun ,
Emna Siala  and
Aïda Bouratbine
Amal Chihi
Affiliation:
Laboratoire de recherche ‘Parasitologie Médicale, Biotechnologies et Biomolécules’, LR 16-IPT-06, Université Tunis El-Manar, Institut Pasteur de Tunis, 13 place Pasteur, B.P. 74 1002 Tunis Belvédère, Tunisia Faculté des Sciences de Bizerte, Université de Carthage, Jarzouna, 7021, Bizerte, Tunisia
Christen R. Stensvold
Affiliation:
Department of Bacteria, Unit of Mycology and Parasitology, Parasites & Fungi, Statens Serum Institut, Artillerivej 5, DK-2300 Copenhagen S, Denmark
Imene Ben-abda
Affiliation:
Laboratoire de recherche ‘Parasitologie Médicale, Biotechnologies et Biomolécules’, LR 16-IPT-06, Université Tunis El-Manar, Institut Pasteur de Tunis, 13 place Pasteur, B.P. 74 1002 Tunis Belvédère, Tunisia Laboratoire de Parasitologie-Mycologie, Institut Pasteur de Tunis, 13, place Pasteur, B.P. 74 1002 Tunis Belvédère, Tunisia
Rania Ben-Romdhane
Affiliation:
Laboratoire de recherche ‘Parasitologie Médicale, Biotechnologies et Biomolécules’, LR 16-IPT-06, Université Tunis El-Manar, Institut Pasteur de Tunis, 13 place Pasteur, B.P. 74 1002 Tunis Belvédère, Tunisia
Karim Aoun
Affiliation:
Laboratoire de recherche ‘Parasitologie Médicale, Biotechnologies et Biomolécules’, LR 16-IPT-06, Université Tunis El-Manar, Institut Pasteur de Tunis, 13 place Pasteur, B.P. 74 1002 Tunis Belvédère, Tunisia
Emna Siala
Affiliation:
Laboratoire de Parasitologie-Mycologie, Institut Pasteur de Tunis, 13, place Pasteur, B.P. 74 1002 Tunis Belvédère, Tunisia
Aïda Bouratbine*
Affiliation:
Laboratoire de recherche ‘Parasitologie Médicale, Biotechnologies et Biomolécules’, LR 16-IPT-06, Université Tunis El-Manar, Institut Pasteur de Tunis, 13 place Pasteur, B.P. 74 1002 Tunis Belvédère, Tunisia Laboratoire de Parasitologie-Mycologie, Institut Pasteur de Tunis, 13, place Pasteur, B.P. 74 1002 Tunis Belvédère, Tunisia
*
Author for correspondence: Aïda Bouratbine, E-mail: [email protected]
Get access Rights & Permissions [Opens in a new window]

Abstract

Amoebae are single-celled parasites frequently colonizing human gut. However, few molecular tools are available for accurate identification. Here, we evaluated a panel of polymerase chain reactions (PCRs) targeting Entamoeba histolytica, Entamoeba dispar, Entamoeba coli, Entamoeba hartmanni, Entamoeba polecki, Endolimax nana and Iodamoeba bütschlii. Thirty-six faecal samples (18 containing at least one amoeba species by microscopy and 18 microscopy negative for amoebae) were tested. Real-time PCRs were used for detection and differentiation of E. histolytica and E. dispar. Conventional PCR with Sanger sequencing were applied for detection and differentiation of E. coli, E. hartmanni, E. polecki, E. nana and I. bütschlii. All microscopy results were confirmed by DNA-based methods. However, more samples were positive for single and mixed amoebic species by DNA-based assays than by microscopy (22 vs 18 and 7 vs 1, respectively). DNA sequencing allowed identification of E. coli subtypes (ST1 and ST2), showed low intra-specific variation within E. hartmanni, identified two phylogenetically distinct groups within E. nana, and identified Iodamoeba at the ribosomal lineage level. Taking into account the high intra-genetic diversity within some of the species at the small subunit (SSU) rRNA gene level, amplification of SSU rRNA genes with subsequent sequencing represents a useful method for detecting, differentiating and subtyping intestinal amoebae.

Keywords

AmoebadiagnosisEndolimaxEntamoebahuman gutIodamoebaparasitePCR
Type
Research Article
Information
Parasitology , Volume 146 , Issue 6 , May 2019 , pp. 821 - 827
Check for updates
Copyright
Copyright © Cambridge University Press 2019 

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

Abu-Madi, M, Boughattas, S, Behnke, JM, Sharma, A and Ismail, A (2017) Coproscopy and molecular screening for detection of intestinal protozoa. Parasites & Vectors 10, 414.Google Scholar
Andersen, LO and Stensvold, CR (2016) Blastocystis in health and disease: are we moving from a clinical to a public health perspective? Journal of Clinical Microbiology 54, 524–528.Google Scholar
Andersen, LO, Vedel Nielsen, H and Stensvold, CR (2013) Waiting for the human intestinal Eukaryotome. The ISME Journal 7, 1253–1255.Google Scholar
Beghini, F, Pasolli, E, Truong, TD, Putignani, L, Caccio, SM and Segata, N (2017) Large-scale comparative metagenomics of Blastocystis, a common member of the human gut microbiome. The ISME Journal 11, 2848–2863.Google Scholar
Ben Ayed, S, Aoun, K, Maamouri, N, Ben Abdallah, R and Bouratbine, A (2008 a) First molecular identification of Entamoeba moshkovskii in human stool samples in Tunisia. The American Journal of Tropical Medicine and Hygiene 79, 706–707.Google Scholar
Ben Ayed, S, Ben, Abdallah R, Mousli, M, Aoun, K, Thellier, M and Bouratbine, A (2008 b) Molecular differentiation of Entamoeba histolytica and Entamoeba dispar from Tunisian food handlers with amoeba infection initially diagnosed by microscopy. Parasite 15, 65–68.Google Scholar
Berrilli, F, Di Cave, D, Cavallero, S and D'Amelio, S (2012) Interactions between parasites and microbial communities in the human gut. Frontiers in Cellular and Infection Microbiology 2, 141.Google Scholar
Bouratbine, A, Aoun, K, Siala, E, Chahed, MK, Ben Hassine, L and Meherzi, A (2000) For a better estimation of the prevalence of intestinal parasitism in the Tunis region. Bulletin de la Société de Pathologie Exotique 93, 353–355.Google Scholar
Bouratbine, A, Maamouri, N, Ben Abdallah, RR, Aoun, K, Haouet, S, Boubaker, J, Ben Jilani, S, Ghorbel, AJ, Najjar, T and Ben Ammar, A (2003) Epidemiological, clinical and parasitological data concerning intestinal amebiasis in northern Tunisia. Tunisie Medicale 81, 318–322.Google Scholar
Bruijnesteijn van Coppenraet, LE, Wallinga, JA, Ruijs, GJ, Bruins, MJ and Verweij, JJ (2009) Parasitological diagnosis combining an internally controlled real-time PCR assay for the detection of four protozoa in stool samples with a testing algorithm for microscopy. Clinical Microbiology and Infection 15, 869–874.Google Scholar
Burrows, RB (1959) Morphological differentiation of Enta moeba hartmanni and E. polecki from E. histolytica. American Journal of Tropical Medicine and Hygiene 8, 583–589.Google Scholar
Cimino, RO, Jeun, R, Juarez, M, Cajal, PS, Vargas, P, Echazu, A, Bryan, PE, Nasser, J, Krolewiecki, A and Mejia, R (2015) Identification of human intestinal parasites affecting an asymptomatic peri-urban Argentinian population using multi-parallel quantitative real-time polymerase chain reaction. Parasites & Vectors 8, 380.Google Scholar
Clark, CG and Diamond, LS (1997) Intraspecific variation and phylogenetic relationships in the genus Entamoeba as revealed by riboprinting. Journal of Eukaryotic Microbiology 44, 142–154.Google Scholar
Efunshile, MA, Ngwu, BA, Kurtzhals, JA, Sahar, S, Konig, B and Stensvold, CR (2015) Molecular detection of the carriage rate of four intestinal protozoa with real-time polymerase chain reaction: possible overdiagnosis of Ent amoeba histolytica in Nigeria. American Journal of Tropical Medicine and Hygiene 93, 257–262.Google Scholar
Fotedar, R, Stark, D, Beebe, N, Marriott, D, Ellis, J and Harkness, J (2007) Laboratory diagnostic techniques for Entamoeba species. Clinical Microbiology Reviews 20, 511–532, table of contents.Google Scholar
Gomes Tdos, S, Garcia, MC, de Souza Cunha, F, Werneck de Macedo, H, Peralta, JM and Peralta, RH (2014) Differential diagnosis of Entamoeba spp. in clinical stool samples using SYBR green real-time polymerase chain reaction. The Scientific World Journal 2014, 645084.Google Scholar
Iebba, V, Santangelo, F, Totino, V, Pantanella, F, Monsia, A, Di Cristanziano, V, Di Cave, D, Schippa, S, Berrilli, F and D'Alfonso, R (2016) Gut microbiota related to Giardia duodenalis, Entamoeba spp. and Blastocystis hominis infections in humans from Cote d'Ivoire. The Journal of Infection in Developing Countries 10, 1035–1041.Google Scholar
Incani, RN, Ferrer, E, Hoek, D, Ramak, R, Roelfsema, J, Mughini-Gras, L, Kortbeek, T and Pinelli, E (2017) Diagnosis of intestinal parasites in a rural community of Venezuela: advantages and disadvantages of using microscopy or RT-PCR. Acta Tropica 167, 64–70.Google Scholar
McHardy, IH, Wu, M, Shimizu-Cohen, R, Couturier, MR and Humphries, RM (2014) Detection of intestinal protozoa in the clinical laboratory. Journal of Clinical Microbiology 52, 712–720.Google Scholar
Morton, ER, Lynch, J, Froment, A, Lafosse, S, Heyer, E, Przeworski, M, Blekhman, R and Segurel, L (2015) Variation in Rural African Gut Microbiota is strongly correlated with colonization by Entamoeba and subsistence. PLoS Genetics 11, e1005658.Google Scholar
Nazer, H, Greer, W, Donnelly, K, Mohamed, AE, Yaish, H, Kagalwalla, A and Pavillard, R (1993) The need for three stool specimens in routine laboratory examinations for intestinal parasites. British Journal of Clinical Practice 47, 76–78.Google Scholar
O'Brien Andersen, L, Karim, AB, Roager, HM, Vigsnaes, LK, Krogfelt, KA, Licht, TR and Stensvold, CR (2016) Associations between common intestinal parasites and bacteria in humans as revealed by qPCR. European Journal of Clinical Microbiology & Infectious Diseases 35, 1427–1431.Google Scholar
Poulsen, CS and Stensvold, CR (2016) Systematic review on Endolimax nana: a less well studied intestinal ameba. Tropical Parasitology 6, 8–29.Google Scholar
Santos, HL, Bandea, R, Martins, LA, de Macedo, HW, Peralta, RH, Peralta, JM, Ndubuisi, MI and da Silva, AJ (2010) Differential identification of Entamoeb a spp. based on the analysis of 18S rRNA. Parasitology Research 106, 883–888.Google Scholar
Siala, E, Toumi, I, Bettaieb, J, Boulehmi, N, Zallega, N, Aoun, K and Bouratbine, A (2015) Evolution of the prevalence of intestinal parasitosis in the region of tunis from 1996 at 2012. Tunisie Medicale 93, 687–691.Google Scholar
Silberman, JD, Clark, CG, Diamond, LS and Sogin, ML (1999) Phylogeny of the genera Entamoeba and Endolimax as deduced from small-subunit ribosomal RNA sequences. Molecular Biology and Evolution 16, 1740–1751.Google Scholar
Stensvold, CR and Nielsen, HV (2012) Comparison of microscopy and PCR for detection of intestinal parasites in Danish patients supports an incentive for molecular screening platforms. Journal of Clinical Microbiology 50, 540–541.Google Scholar
Stensvold, CR and van der Giezen, M (2018) Associations between gut microbiota and common luminal intestinal parasites. Trends in Parasitology 34, 369–377.Google Scholar
Stensvold, CR, Lebbad, M and Verweij, JJ (2011a) The impact of genetic diversity in protozoa on molecular diagnostics. Trends in Parasitology 27, 53–58.Google Scholar
Stensvold, CR, Lebbad, M, Victory, EL, Verweij, JJ, Tannich, E, Alfellani, M, Legarraga, P and Clark, CG (2011b) Increased sampling reveals novel lineages of Entamoeba: consequences of genetic diversity and host specificity for taxonomy and molecular detection. Protist 162, 525–541.Google Scholar
Stensvold, CR, Lebbad, M and Clark, CG (2012) Last of the human protists: the phylogeny and genetic diversity of Iodamoeba. Molecular Biology and Evolution 29, 39–42.Google Scholar
Stensvold, CR, Winiecka-Krusnell, J, Lier, T and Lebbad, M (2018) Evaluation of a PCR method for detection of Entamoeba polecki, with an overview of its molecular epidemiology. Journal of Clinical Microbiology 56, e00154-18.Google Scholar
Suzuki, J, Kobayashi, S, Murata, R, Tajima, H, Hashizaki, F, Yanagawa, Y and Takeuchi, T (2008) A survey of amoebic infections and differentiation of an Entamoeba histolytica-like variant (JSK2004) in nonhuman primates by a multiplex polymerase chain reaction. Journal of Zoo and Wildlife Medicine 39, 370–379.Google Scholar
Tanyuksel, M and Petri, WA Jr (2003) Laboratory diagnosis of amebiasis. Clinical Microbiology Reviews 16, 713–729.Google Scholar
ten Hove, RJ, van Esbroeck, M, Vervoort, T, van den Ende, J, van Lieshout, L and Verweij, JJ (2009) Molecular diagnostics of intestinal parasites in returning travellers. European Journal of Clinical Microbiology & Infectious Diseases 28, 1045–1053.Google Scholar
Verweij, JJ and Stensvold, CR (2014) Molecular testing for clinical diagnosis and epidemiological investigations of intestinal parasitic infections. Clinical Microbiology Reviews 27, 371–418.Google Scholar
Won, EJ, Kim, SH, Kee, SJ, Shin, JH, Suh, SP, Chai, JY, Ryang, DW and Shin, MG (2016) Multiplex real-time PCR assay targeting eight parasites customized to the Korean population: potential Use for detection in diarrheal stool samples from gastroenteritis patients. PLoS ONE 11, e0166957.Google Scholar
Zadrobilkova, E, Walker, G and Cepicka, I (2015) Morphological and molecular evidence support a close relationship between the free-living archamoebae Mastigella and Pelomyxa. Protist 166, 14–41.Google Scholar
Supplementary material: File

Chihi et al. supplementary material

Table S1

Download Chihi et al. supplementary material(File)
File 13.8 KB