Abstract
Amoebiasis is an infection caused by enteric protozoa, most commonly Entamoeba histolytica, and is globally considered a potentially severe and life-threatening condition. To understand the impact of the parasite genome on disease outcomes, it is important to study the genomes of infecting strains in areas with high disease prevalence. These studies aim to establish correlations between parasite genotypes and the clinical presentation of amoebiasis. We employ a strain typing approach that utilizes multiple loci, including SREHP and three polymorphic non-coding loci (tRNA-linked array N-K2 and loci 1-2 and 5-6), for high-resolution analysis. Distinct clinical phenotype isolates underwent amplification and sequencing of studied loci. The nucleotide sequences were analysed using Tandem Repeats Finder to detect short tandem repeats (STRs). These patterns were combined to assign a genotype, and the correlation between clinical phenotypes and repetitive patterns was statistically evaluated. This study found significant polymorphism in the size and number of PCR fragments at SREHP and 5-6 locus, while the 1-2 locus and NK2 locus showed variations in PCR product sizes. Out of 41 genotypes, two (I6 and I41) were significantly associated with their respective disease outcomes and were found in multiple isolates. We observed that I6 was linked with a symptomatic outcome, with a statistically significant p-value of 0.0183. Additionally, we found that I41 was associated with ALA disease outcome, with a p-value of 0.0089. Our study revealed new repeat units not previously reported, unveiling the genetic composition of E. histolytica strains in India, associated with distinct disease manifestations.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Data availability
Representative sequences obtained in this study were deposited in GenBank under the accession numbers OQ810098-OQ810337.
References
Ali IK, Clark CG, Petri WA Jr (2008) Molecular epidemiology of amebiasis. Infect Genet Evol 8(5):698–707. https://doi.org/10.1016/j.meegid.2008.05.004
Ali IK, Haque R, Alam F, Kabir M, Siddique A, Petri WA Jr (2012) Evidence for a link between locus R-R sequence type and outcome of infection with Entamoeba histolytica. Clin Microbiol Infect 18(7):E235–E237. https://doi.org/10.1111/j.1469-0691.2012.03826.x
Ali IK, Mondal U, Roy S, Haque R, Petri WA Jr, Clark CG (2007) Evidence for a link between parasite genotype and outcome of infection with Entamoeba histolytica. J Clin Microbiol 45(2):285–289. https://doi.org/10.1128/JCM.01335-06
Ali IK, Zaki M, Clark CG (2005) Use of PCR amplification of tRNA gene-linked short tandem repeats for genotyping Entamoeba histolytica. J Clin Microbiol 43(12):5842–5847. https://doi.org/10.1128/JCM.43.12.5842-5847.2005
Atabati H, Kassiri H, Shamloo E, Akbari M, Atamaleki A, Sahlabadi F, Linh NTT, Rostami A, Fakhri Y, Khaneghah AM (2020) The association between the lack of safe drinking water and sanitation facilities with intestinal Entamoeba spp infection risk: a systematic review and meta-analysis. PLoS One 15(11):e0237102. https://doi.org/10.1371/journal.pone.0237102
Ayeh-Kumi PF, Ali IM, Lockhart LA, Gilchrist CA, Petri WA Jr, Haque R (2001) Entamoeba histolytica: genetic diversity of clinical isolates from Bangladesh as demonstrated by polymorphisms in the serine-rich gene. Exp Parasitol 99(2):80–88. https://doi.org/10.1006/expr.2001.4652
Das K, Ganguly S (2014) Evolutionary genomics and population structure of Entamoeba histolytica. Comput Struct Biotechnol J 12(20-21):26–33. https://doi.org/10.1016/j.csbj.2014.10.001
Das K, Mukherjee AK, Chowdhury P, Sehgal R, Bhattacharya MK, Hashimoto T, Nozaki T, Ganguly S (2014) Multilocus sequence typing system (MLST) reveals a significant association of Entamoeba histolytica genetic patterns with disease outcome. Parasitol Int 63(2):308–314. https://doi.org/10.1016/j.parint.2013.11.014
Deshpande AM, Newlon CS (1996) DNA replication fork pause sites dependent on transcription. Science 272(5264):1030–1033. https://doi.org/10.1126/science.272.5264.1030
Escueta-de Cadiz A, Kobayashi S, Takeuchi T, Tachibana H, Nozaki T (2010) Identification of an avirulent Entamoeba histolytica strain with unique tRNA-linked short tandem repeat markers. Parasitol Int 59(1):75–81. https://doi.org/10.1016/j.parint.2009.10.010
Guillén N (2023) Pathogenicity and virulence of Entamoeba histolytica, the agent of amoebiasis. Virulence 14(1):2158656. https://doi.org/10.1080/21505594.2022.2158656
Haghighi A, Kobayashi S, Takeuchi T, Masuda G, Nozaki T (2002) Remarkable genetic polymorphism among Entamoeba histolytica isolates from a limited geographic area. J Clin Microbiol 40(11):4081–4090. https://doi.org/10.1128/JCM.40.11.4081-4090.2002
Haghighi A, Kobayashi S, Takeuchi T, Thammapalerd N, Nozaki T (2003) Geographic diversity among genotypes of Entamoeba histolytica field isolates. J Clin Microbiol 41(8):3748–3756. https://doi.org/10.1128/JCM.41.8.3748-3756.2003
Jaiswal V, Ghoshal U, Mittal B, Dhole TN, Ghoshal UC (2014) Association between allelic variation due to short tandem repeats in tRNA gene of Entamoeba histolytica and clinical phenotypes of amoebiasis. Acta Trop 133:1–7. https://doi.org/10.1016/j.actatropica.2014.01.009
Köhler S, Tannich E (1993) A family of transcripts (K2) of Entamoeba histolytica contains polymorphic repetitive regions with highly conserved elements. Mol Biochem Parasitol. 59(1):49–58. https://doi.org/10.1016/0166-6851(93)90006-j
Labib K, Hodgson B (2007) Replication fork barriers: pausing for a break or stalling for time? EMBO Rep 8(4):346–353. https://doi.org/10.1038/sj.embor.7400940
Mukherjee AK, Chowdhury P, Bhattacharya MK, Ghosh M, Rajendran K, Ganguly S (2009) Hospital-based surveillance of enteric parasites in Kolkata. BMC Res Notes 2:110. https://doi.org/10.1186/1756-0500-2-110
Mukherjee AK, Das K, Bhattacharya MK, Nozaki T, Ganguly S (2010) Trend of Entamoeba histolytica infestation in Kolkata. Gut Pathog 2(1):12. https://doi.org/10.1186/1757-4749-2-12
Nishise S, Fujishima T, Kobayashi S, Otani K, Nishise Y, Takeda H, Kawata S (2010) Mass infection with Entamoeba histolytica in a Japanese institution for individuals with mental retardation: epidemiology and control measures. Ann Trop Med Parasitol 104(5):383–390. https://doi.org/10.1179/136485910X12743554760388
Pattanawong U, Putaporntip C, Kakino A, Yoshida N, Kobayashi S, Yanmanee S, Jongwutiwes S, Tachibana H (2021) Analysis of D-A locus of tRNA-linked short tandem repeats reveals transmission of Entamoeba histolytica and E. dispar among students in the Thai-Myanmar border region of northwest Thailand. PLoS Negl Trop Dis 15(2):e0009188. https://doi.org/10.1371/journal.pntd.0009188
Preet S, Bharati S, Shukla G, Koul A, Rishi P (2011) Evaluation of amoebicidal potential of Paneth cell cryptdin-2 against Entamoeba histolytica. PLoS Negl Trop Dis 5(12):e1386. https://doi.org/10.1371/journal.pntd.0001386
Samie A, Obi CL, Bessong PO, Houpt E, Stroup S, Njayou M, Sabeta C, Mduluza T, Guerrant RL (2008) Entamoeba histolytica: genetic diversity of African strains based on the polymorphism of the serine-rich protein gene. Exp Parasitol 118(3):354–361. https://doi.org/10.1016/j.exppara.2007.09.008
Simonishvili S, Tsanava S, Sanadze K, Chlikadze R, Miskalishvili A, Lomkatsi N, Imnadze P, Petri WA Jr, Trapaidze N (2005) Entamoeba histolytica: the serine-rich gene polymorphism-based genetic variability of clinical isolates from Georgia. Exp Parasitol 110(3):313–317. https://doi.org/10.1016/j.exppara.2005.02.015
Singh PP, Galhotra A (2014) Water, amoebiasis and public health (eds). Springer, New Delhi, pp 169–177. https://doi.org/10.1007/978-81-322-1029-0_11
Stanley SL Jr, Becker A, Kunz-Jenkins C, Foster L, Li E (1990) Cloning and expression of a membrane antigen of Entamoeba histolytica possessing multiple tandem repeats. Proc Natl Acad Sci U S A 87(13):4976–4980. https://doi.org/10.1073/pnas.87.13.4976
Tawari B, Ali IK, Scott C, Quail MA, Berriman M, Hall N, Clark CG (2008) Patterns of evolution in the unique tRNA gene arrays of the genus Entamoeba. Mol Biol Evol:187–198. https://doi.org/10.1093/molbev/msm238
Yanagawa Y, Nagashima M, Gatanaga H, Kikuchi Y, Oka S, Yokoyama K, Shinkai T, Sadamasu K, Watanabe K (2020) Seroprevalence of Entamoeba histolytica at a voluntary counselling and testing centre in Tokyo: a cross-sectional study. BMJ Open 10(2):e031605. https://doi.org/10.1136/bmjopen-2019-031605
Zaki M, Clark CG (2000) Polymorphic DNA markers in Entamoeba histolytica. Arch Med Res 31(4 Suppl):S255–S257. https://doi.org/10.1016/s0188-4409(00)00210-1
Zaki M, Clark CG (2001) Isolation and characterization of polymorphic DNA from Entamoeba histolytica. J Clin Microbiol 39(3):897–905. https://doi.org/10.1128/JCM.39.3.897-905.2001
Zaki M, Meelu P, Sun W, Clark CG (2002) Simultaneous differentiation and typing of Entamoeba histolytica and Entamoeba dispar. J Clin Microbiol 40(4):1271–1276. https://doi.org/10.1128/JCM.40.4.1271-1276.2002
Zermeño V, Ximénez C, Morán P, Valadez A, Valenzuela O, Rascón E, Diaz D, Cerritos R (2013) Worldwide genealogy of Entamoeba histolytica: an overview to understand haplotype distribution and infection outcome. Infect Genet Evol 17:243–252. https://doi.org/10.1016/j.meegid.2013.04.021
Acknowledgements
The authors would like to express their appreciation to all the patients who participated in the study. We are also grateful for the assistance provided by the hospital staff and sample collectors during the sample collection process.
Funding
This study was funded by the Indian Council of Medical Research (ICMR), the Government of India, and Japan Agency for Medical Research and Development (AMED, grant number: JP23fk0108683), Japan. The authors extend their appreciation to all patients who took part in the study.
Author information
Authors and Affiliations
Contributions
The authors confirm their contribution to the article as follows:
Sanjib K. Sardar: conceptualization, methodology, data curation, writing original draft, and formal analysis. Ajanta Ghosal: methodology. Tapas Haldar: methodology. Koushik Das: formal analysis. Yumiko Saito Nakano: validation and investigation. Seiki Kobayashi: visualization. Shanta Dutta: project administration. Tomoyoshi Nozaki: validation. Sandipan Ganguly: conceptualization, visualization, validation, funding acquisition, review and editing, and supervision.
Corresponding author
Ethics declarations
Ethics approval and informed consent
The ethical clearance of this study was reviewed and approved by the Institutional Human Ethics Committee of the ICMR-NICED. Informed consent statements were obtained from the participants.
Competing interests
The authors declare no competing interests.
Additional information
Section Editor: Dietmar Steverding
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary information
ESM 1
Supporting Information (SI) 1: The figure shows the representative amplified PCR products of several studied STR loci of E. histolytica. A and B display the PCR products for the N-K2 locus, while C and D show the products for the locus 5-6. E displays the product for locus 1-2, and F shows the product for the SREHP locus (PDF 313 kb)
ESM 2
Supporting Information (SI) 2: This figure illustrates the distribution of N-K2 genotypes obtained from E. histolytica isolates of different disease outcome groups where each genotype is depicted by a unique colour. K1-K20 are the abbreviation for K1Ind-K20Ind (PDF 45 kb)
ESM 3
Supporting Information (SI) 3: The presented figure displays the distribution of various haplotypes of E. histolytica isolates in different disease outcome groups based on locus 1-2. Each haplotype is distinguished using a unique colour. L1-L6 are the abbreviation for L1Ind-L6Ind (PDF 45 kb)
ESM 4
Supporting Information (SI) 4: This figure displays the distribution of different haplotypes of E. histolytica in various disease outcome groups based on locus 5-6. Each genotype is distinguished using a unique colour. Q1-Q7 are the abbreviation for Q1Ind-Q7Ind (PDF 41 kb)
ESM 5
Supporting Information (SI) 5: This figure displays the distribution of different haplotypes of E. histolytica in various disease outcome groups based on SREHP. Each genotype is distinguished using a unique colour. S1-S15 are the abbreviation for S1Ind-S15Ind (PDF 42 kb)
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Sardar, S.K., Ghosal, A., Haldar, T. et al. Investigating genetic polymorphism in E. histolytica isolates with distinct clinical phenotypes. Parasitol Res 122, 2525–2537 (2023). https://doi.org/10.1007/s00436-023-07952-x
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00436-023-07952-x