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Epidemiology, Species Composition and Genetic Diversity of Tetra- and Octonucleated Entamoeba Spp. in Different Brazilian Biomes

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Epidemiology, Species Composition and Genetic Diversity of Tetra- and Octonucleated Entamoeba Spp. in Different Brazilian Biomes Calegar et al. Parasites Vectors (2021) 14:160 https://doi.org/10.1186/s13071-021-04672-y Parasites & Vectors RESEARCH Open Access Epidemiology, species composition and genetic diversity of tetra- and octonucleated Entamoeba spp. in diferent Brazilian biomes Deiviane Aparecida Calegar1* , Kerla Joeline Lima Monteiro2, Polyanna Araújo Alves Bacelar1,2, Brenda Bulsara Costa Evangelista1,2, Mayron Morais Almeida1, Jéssica Pereira dos Santos1,2, Márcio Neves Boia3, Beatriz Coronato‑Nunes1,4, Lauren Hubert Jaeger1,5† and Filipe Anibal Carvalho‑Costa1† Abstract Background: Entamoeba species harbored by humans have diferent degrees of pathogenicity. The present study explores the intra‑ and interspecifc diversity, phylogenetic relationships, prevalence and distribution of tetra‑ and octonucleated cyst‑producing Entamoeba in diferent Brazilian regions. Methods: Cross‑sectional studies were performed to collect fecal samples (n 1728) and sociodemographic data in communities located in four Brazilian biomes: Atlantic Forest, Caatinga, Cerrado,= and Amazon. Fecal samples were subjected to molecular analysis by partial small subunit ribosomal DNA sequencing (SSU rDNA) and phylogenetic analysis. Results: Light microscopy analysis revealed that tetranucleated cysts were found in all the studied biomes. The highest positivity rates were observed in the age group 6–10 years (23.21%). For octonucleated cysts, positivity rates ranged from 1 to 55.1%. Sixty SSU rDNA Entamoeba sequences were obtained, and four diferent species were identifed: the octonucleated E. coli, and the tetranucleated E. histolytica, E. dispar, and E. hartmanni. Novel haplotypes (n 32) were characterized; however, new ribosomal lineages were not identifed. The Entamoeba coli ST1 subtype predominated= in Atlantic Forest and Caatinga, and the ST2 subtype was predominant in the Amazon biome. E. histol- ytica was detected only in the Amazon biome. In phylogenetic trees, sequences were grouped in two groups, the frst containing uni‑ and tetranucleated and the second containing uni‑ and octonucleated cyst‑producing Entamoeba species. Molecular diversity indexes revealed a high interspecifc diversity for tetra‑ and octonucleated Entamoeba spp. (H SD 0.9625 0.0126). The intraspecifc diversity varied according to species or subtype: E. dispar and E. histo- lytica showed± = lower diversity± than E. coli subtypes ST1 and ST2 and E. hartmanni. Conclusions: Tetra‑ and octonucleated cyst‑producing Entamoeba are endemic in the studied communities; E. histolytica was found in a low proportion and only in the Amazon biome. With regard to E. coli, subtype ST2 was *Correspondence: [email protected] †Lauren Hubert Jaeger and Filipe Anibal Carvalho‑Costa contributed equally to this work 1 Laboratório de Epidemiologia e Sistemática Molecular, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, RJ, Brazil Full list of author information is available at the end of the article © The Author(s) 2021. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Calegar et al. Parasites Vectors (2021) 14:160 Page 2 of 13 predominant in the Amazon biome. The molecular epidemiology of Entamoeba spp. is a feld to be further explored and provides information with important implications for public health. Keywords: Entamoeba genus, Molecular epidemiology, Small subunit ribosomal DNA, Intra‑ and interspecifc diversity Background communities. Similarly, peri-urban and urban communi- Entamoeba species harbored by the human digestive ties in large Brazilian cities in more industrialized states tract have diferent degrees of pathogenicity and impact frequently have poor sanitation infrastructure. In the on public health [1]. Although some species are consid- present study, we explored the species composition, the ered commensal and non-pathogenic, E. histolytica can inter- and intraspecifc genetic diversity and phylogenetic cause serious, life-threatening, and invasive infections relationships, and the prevalence and distribution of such as amoebic dysentery and liver abscess [2]. Enta- Entamoeba species infecting populations living in difer- moeba histolytica produces tetranucleated cysts which ent Brazilian biomes. are indistinguishable from those produced by E. dispar. Te similarity of the cysts led to the adoption of the Methods nomenclature E. histolytica/E. dispar complex, which Description of study area and population, study design also includes Entamoeba moshkovskii, another tetranu- and sampling cleated cyst-producing species. Although E. dispar and Communities from cities located in four Brazilian biomes E. moshkovskii are considered to have less pathogenic were selected: Cachoeiras de Macacu (CAM) in the state potential [3], they have occasionally been associated with of Rio de Janeiro (Atlantic Forest biome), Teresina (TER) invasive disease [4, 5]. Tese fndings have led to the need and São João do Piauí (SJPI) in Piauí (Cerrado and Caat- for further studies to assess the epidemiology of indistin- inga biomes, respectively), and Santa Isabel do Rio Negro guishable tetranucleated amoebas [3]. Entamoeba species (SIRN) in Amazonas and Bagre (BAG) in Pará (Amazon that parasitize other animals can also infect humans [6]. biome) (Fig. 1). In the fve municipalities, the studied Entamoeba coli, which are considered a commensal communities had precarious access to drinking water and harmless organism, produce octonucleated cysts and and poor sanitation systems, to varying degrees and for can be considered a marker of inadequate sanitary con- distinct reasons. Table 1 shows the socio-environmental ditions, denoting greater exposure to other fecal patho- and demographic characteristics of each municipal- gens. Recently, however, potential pathogenicity has been ity and the number of individuals included in each one. attributed to this species. Mexican children infected with Tese regions difer with respect to climate, proportion E. coli have higher levels of stool leucocytes than unin- of the population living in poverty and human develop- fected children, pointing to the possibility of intestinal ment index, among other parameters. Cross-sectional infammatory activity triggered by this organism [7, 8]. studies were carried out in the included areas to collect Additionally, considerable intraspecifc genetic variability fecal samples and sociodemographic data. Entamoeba has been demonstrated for E. coli, which can be divided spp. fecal samples were identifed as positive using para- into two subtypes: E. coli ST1 and E. coli ST2 [9]. sitological methods (Ritchie’s modifed ethyl acetate cen- Typically, the clinical detection of Entamoeba spe- trifugation) [12]. cies in fecal samples has been performed through light microscopy. However, the overlap of morphological char- DNA extraction, polymerase chain reaction (PCR) and DNA acteristics between some species and the morphological sequencing and size variation in structures are limiting factors for Fecal samples positive on light microscopy were sub- microscopic species-specifc diagnosis [10]. Te imple- jected to molecular analyses for species characterization mentation of molecular tools for Entamoeba species and phylogenetic studies. In addition, some Entamoeba characterization has enabled a greater understanding of spp.-negative samples on microscopy were selected, their taxonomy, phylogenetic relationships and epidemi- randomly or when another individual from the same ology [11]. household was positive, to be tested by PCR. Tis was Despite recent advances, the proportion of house- performed in order to improve the number of DNA holds without sanitation systems in many regions of Bra- sequences for genetic analyses. Genomic DNA was zil remains high. Te country has great socioeconomic, extracted from 200 µl of the sedimented fecal material environmental and demographic diversity, and appro- using the ZR Fungal/Bacterial DNA MiniPrep™ extrac- priate access to drinking water is restricted in many tion kit (Zymo Research, Irvine, CA, USA). PCR was Calegar et al. Parasites Vectors (2021) 14:160 Page 3 of 13 Fig. 1 Map of the studied areas in Brazil. The map was created using Google Earth Pro performed using the Platinum Taq DNA Polymerase kit Entamoeba species. Te obtained sequences were depos- (Invitrogen, Waltham, MA, USA), with a fnal volume of ited in GenBank under accession numbers MW026735– 50 μl. Te small subunit rRNA gene locus (SSU rDNA) MW026794. BioEdit version 7.2.5 [15] was used to align of Entamoeba spp. (550 base pairs [bp]) was targeted for
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