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Analysis of DA Locus of Trna-Linked Short Tandem Repeats Reveals RESEARCH ARTICLE 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 1,2 1 2 3 Urassaya PattanawongID , Chaturong Putaporntip , Azumi Kakino , Naoko Yoshida , 4 1 1 a1111111111 Seiki Kobayashi , Surasuk YanmaneeID , Somchai Jongwutiwes *, 2 a1111111111 Hiroshi TachibanaID * a1111111111 a1111111111 1 Molecular Biology of Malaria and Opportunistic Parasites Research Unit, Department of Parasitology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand, 2 Department of Parasitology, Tokai a1111111111 University School of Medicine, Isehara, Japan, 3 Department of Tropical Medicine and Parasitology, Faculty of Medicine, Juntendo University, Tokyo, Japan, 4 Department of Infectious Diseases, Keio University School of Medicine, Tokyo, Japan * [email protected] (SJ); [email protected] (HT) OPEN ACCESS Citation: Pattanawong U, Putaporntip C, Kakino A, Yoshida N, Kobayashi S, Yanmanee S, et al. (2021) Abstract Analysis of D-A locus of tRNA-linked short tandem repeats reveals transmission of Entamoeba Intestinal parasitic infections, including those caused by Entamoeba species, are a persis- histolytica and E. dispar among students in the tent problem in rural areas of Thailand. The aims of this study were to identify pathogenic Thai-Myanmar border region of northwest Entamoeba species and to analyze their genotypic diversity. Stool samples were collected Thailand. PLoS Negl Trop Dis 15(2): e0009188. https://doi.org/10.1371/journal.pntd.0009188 from 1,233 students of three schools located in the Thai-Myanmar border region of Tak Province, Thailand. The prevalence of Entamoeba infection was measured by polymerase Editor: Kevin SW Tan, National University of Singapore, SINGAPORE chain reaction (PCR) using species-specific primers. Thirty-one (2.5%) positive cases were detected for E. histolytica, 55 (4.5%) for E. dispar, and 271 (22.0%) for E. coli. Positive sam- Received: November 27, 2020 ples for E. histolytica and E. dispar were exclusively obtained from a few school classes, Accepted: February 1, 2021 whereas E. coli was detected in all grades. No infections caused by E. moshkovskii, E. nut- Published: February 18, 2021 talli, E. chattoni, and E. polecki were detected in the students studied. The D-A locus of Copyright: © 2021 Pattanawong et al. This is an tRNA-linked short tandem repeats was analyzed in samples of E. histolytica (n = 13) and E. open access article distributed under the terms of dispar (n = 47) to investigate their diversity and potential modes of transmission. Five geno- the Creative Commons Attribution License, which types of E. histolytica and 13 genotypes of E. dispar were identified. Sequences of the D-A permits unrestricted use, distribution, and reproduction in any medium, provided the original were divergent, but several unique genotypes were significantly prevalent in limited classes, author and source are credited. indicating that intra-classroom transmission has occurred. As it was unlikely that infection Data Availability Statement: Nucleotide would have been limited within school classes if the mode of transmission of E. histolytica sequences have been deposited at the DDBJ/ and E. dispar had been through the intake of contaminated drinking water or food, these EMBL/GenBank database, accession numbers results suggest a direct or indirect person-to-person transmission mode within school clas- LC546869-LC546886. ses. Positive rates for three Entamoeba species were 2-fold higher in students who had sib- Funding: This study was supported by JSPS lings in the schools than in those without siblings, suggesting that transmission occurred (https://www.jsps.go.jp) KAKENHI (grant number even at home due to heavy contacts among siblings. 24406013, JP16H05819, JP17K08811, JP20H03482 to H.T., JP20K07466 to N.Y.), the Thailand Research Fund (https://www.trf.or.th) PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0009188 February 18, 2021 1 / 19 PLOS NEGLECTED TROPICAL DISEASES Transmission of E. histolytica/E. dispar among students (RSA5980054 to C.P.), and the Matsumae International Foundation (https://wwww.mif-japan. Author summary org) (2017 Research Fellowship Program to U.P.). The funders had no role in study design, data Transmissions in endemic areas of the pathogen Entamoeba histolytica and other non-patho- collection and analysis, decision to publish, or genic Entamoeba species such as E. dispar and E. coli are caused by ingestion of drinking preparation of the manuscript. water and foods contaminated with cysts of the parasites. Cases of Entamoeba infections Competing interests: The authors have declared among school-aged children have been reported in several countries. However, it has not that no competing interests exist. been demonstrated that transmission of protozoa of the Entamoeba genus occurs within school facilities. In addition, genetic information on E. histolytica and other morphologically indistinguishable species, including E. dispar and E. moshkovskii, in Thailand remains scarce. In the present study, we demonstrated that E. histolytica and/or E. dispar are prevalent among school-aged children, but limited to few classes in three rural schools in the Thai-Myanmar border region of northwest Thailand. Although various genotypes of these Entamoeba species were identified, identical genotypes were significantly more prevalent in certain school classes and also among siblings, suggesting that transmission occurred within the classrooms and at home. The possibility of person-to-person transmission among these students via direct or indirect contact during daily activities in classrooms and home is proposed. Introduction Intestinal parasitic infections are a persistent problem in rural areas of Thailand [1±5]. Chronic intestinal parasitic infections can cause malnutrition, anemia, growth retardation, and intellectual developmental delays in preschoolers and students. As such, cross-sectional studies on intestinal parasitic infections have been conducted in Thai children from various locations [6±9]. Micros- copy studies also demonstrated the prevalence of Entamoeba. However, as Entamoeba histolytica is morphologically indistinguishable from species such as E. dispar and E. moshkovskii, the preva- lence of each species in Thailand remains unknown. E. histolytica is the pathological agent of amebiasis, which is associated with an estimated 50 million cases of colitis and liver abscesses that result in 55,000 deaths per year worldwide. E. dispar has mainly been isolated from cysts in fecal samples from asymptomatic carriers and is described as non-pathogenic. E. moshkovskii is pri- marily free-living, but human infections have also been reported in many countries [10±12]. Recent studies have reported polymorphisms for a serine-rich protein gene, chitinase gene, and tRNA-linked short tandem repeats (tRNA-STR) in E. histolytica and E. dispar [13±15]. The tRNA-STR polymorphism is highly divergent, and several reports have correlated specific genotypes with symptoms [16±18]. Genotyping is a useful tool that can be used to investigate the dynamics of infection in families, communities, and schools [19±21]. However, epidemio- logical studies to identify Entamoeba species and characterize their genotypic diversity have not been previously conducted in schools in Thailand. Here, we report the prevalence of intestinal parasites, including protozoa and helminths, with a special focus on Entamoeba, among students from schools located in the Thai-Myanmar border region in northwest Thailand. Genotypic analyses of E. histolytica and E. dispar sug- gested a mode of person-to-person transmission in classrooms and in the home. Methods Ethics statement This study was conducted in accordance with ethical protocols approved by the Institutional Review Board in Human Research, Faculty of Medicine, Chulalongkorn University, Thailand PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0009188 February 18, 2021 2 / 19 PLOS NEGLECTED TROPICAL DISEASES Transmission of E. histolytica/E. dispar among students (IRB Nos. 236/54 and 246/61). Written informed consent was obtained from all participants or from their parents or guardians prior to stool sample collection. Study area and collection of samples A cross-sectional study was conducted at three schools (A to C) located in the Thai-Myanmar border region in Tha Song Yang, the northwestern-most district of Tak Province, Thailand, in July 2018 (Fig 1). School A has a kindergarten (2 grades), primary school (6 grades), and sec- ondary school (3 grades). School B is located on a hill, and is a branch of school A, comprising a small-scale kindergarten and primary school. School C is a secondary school (6 grades). Clean, wide-mouthed screw-capped plastic containers and spatulas were distributed to the children (or their parents) with instructions for stool sample collection. All 1,788 students from three schools: school A (n = 1,144), school B (n = 82), and school C (n = 562), were requested to submit their stool samples. The next day, stool samples were collected, kept cool on ice, and transported to the laboratory at Chulalongkorn University, Bangkok. A total of 1,233 stool samples were obtained, accounting for 69% of total students from the three schools (70.6%, 53.7%, and 67.8% from schools A, B, and C, respectively). Main reasons for the stu- dents not providing their
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