BACKGROUND: Blastocystis is a human gut symbiont of yet undefined clinical significance. In a set of faecal samples collected from asymptomatic children of six distant populations, we first assessed the community profiles of protist 18S rDNA and then characterized Blastocystis subtypes and tested Blastocystis association with the faecal bacteriome community. METHODS: Stool samples were collected from 244 children and young persons (mean age 11.3 years, interquartile range 8.1-13.7) of six countries (Azerbaijan 51 subjects, Czechia 52, Jordan 40, Nigeria 27, Sudan 59 and Tanzania 15). The subjects showed no symptoms of infection. Amplicon profiling of the 18S rDNA was used for verification that Blastocystis was the most frequent protist, whereas specific real-time PCR showed its prevalence and quantity, and massive parallel amplicon sequencing defined the Blastocystis subtypes. The relation between Blastocystis and the stool bacteriome community was characterized using 16S rDNA profiling. RESULTS: Blastocystis was detected by specific PCR in 36% (88/244) stool samples and was the most often observed faecal protist. Children from Czechia and Jordan had significantly lower prevalence than children from the remaining countries. The most frequent subtype was ST3 (49%, 40/81 sequenced samples), followed by ST1 (36%) and ST2 (25%). Co-infection with two different subtypes was noted in 12% samples. The faecal bacteriome had higher richness in Blastocystis-positive samples, and Blastocystis was associated with significantly different community composition regardless of the country (p < 0.001 in constrained redundancy analysis). Several taxa differed with Blastocystis positivity or quantity: two genera of Ruminococcaceae were more abundant, while Bifidobacterium, Veillonella, Lactobacillus and several other genera were undrerrepresented. CONCLUSIONS: Asymptomatic children frequently carry Blastocystis, and co-infection with multiple distinct subtypes is not exceptional. Prevalence and quantity of the organism clearly differ among populations. Blastocystis is linked to both faecal bacteriome diversity and its composition.

Department of Paediatrics and Child Health University of Khartoum Faculty of Medicine Khartoum Sudan

Department of Pediatrics 2nd Faculty of Medicine Charles University Prague and University Hospital Motol 5 Uvalu 84 Prague 5 Czech Republic

Department of Pediatrics Federal Teaching Hospital Abakaliki Ebonyi State University Abakaliki Nigeria

Department of Pediatrics School of Medicine University of Jordan Amman Jordan

Endocrine Centre Baku Str 1 Hashimov 4A AZ1114 Baku Azerbaijan

Faculty of Medicine and Health Technology Tampere University Tampere Finland

Muhimbili National Hospital Dar es Salaam Tanzania

Sudan Childhood Diabetes Center Khartoum Sudan

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Berg G, Rybakova D, Fischer D, Cernava T, Verges MC, Charles T, et al. Microbiome definition re-visited: old concepts and new challenges. Microbiome. 2020;8:103. doi: 10.1186/s40168-020-00875-0. PubMed DOI PMC

Tan KS. New insights on classification, identification, and clinical relevance of Blastocystis spp. Clin Microbiol Rev. 2008;21:639–665. doi: 10.1128/CMR.00022-08. PubMed DOI PMC

Clark CG, van der Giezen M, Alfellani MA, Stensvold CR. Recent developments in Blastocystis research. Adv Parasitol. 2013;82:1–32. doi: 10.1016/B978-0-12-407706-5.00001-0. PubMed DOI

Tito RY, Chaffron S, Caenepeel C, Lima-Mendez G, Wang J, Vieira-Silva S, et al. Population-level analysis of Blastocystis subtype prevalence and variation in the human gut microbiota. Gut. 2019;68:1180–1189. doi: 10.1136/gutjnl-2018-316106. PubMed DOI PMC

Eme L, Gentekaki E, Curtis B, Archibald JM, Roger AJ. Lateral gene transfer in the adaptation of the anaerobic parasite Blastocystis to the Gut. Curr Biol. 2017;27:807–820. doi: 10.1016/j.cub.2017.02.003. PubMed DOI

Gentekaki E, Curtis BA, Stairs CW, Klimes V, Elias M, Salas-Leiva DE, et al. Extreme genome diversity in the hyper-prevalent parasitic eukaryote Blastocystis. PLoS Biol. 2017;15:e2003769. doi: 10.1371/journal.pbio.2003769. PubMed DOI PMC

Beghini F, Pasolli E, Truong TD, Putignani L, Caccio SM, Segata N. Large-scale comparative metagenomics of Blastocystis, a common member of the human gut microbiome. ISME J. 2017;11:2848–2863. doi: 10.1038/ismej.2017.139. PubMed DOI PMC

Maloney JG, Lombard JE, Urie NJ, Shivley CB, Santin M. Zoonotic and genetically diverse subtypes of Blastocystis in US pre-weaned dairy heifer calves. Parasitol Res. 2019;118:575–582. doi: 10.1007/s00436-018-6149-3. PubMed DOI

Alfellani MA, Stensvold CR, Vidal-Lapiedra A, Onuoha ES, Fagbenro-Beyioku AF, Clark CG. Variable geographic distribution of Blastocystis subtypes and its potential implications. Acta Trop. 2013;126:11–18. doi: 10.1016/j.actatropica.2012.12.011. PubMed DOI

Andersen LO, Bonde I, Nielsen HB, Stensvold CR. A retrospective metagenomics approach to studying Blastocystis. FEMS Microbiol Ecol. 2015;91:fiv072. doi: 10.1093/femsec/fiv072. PubMed DOI

Rostami A, Riahi SM, Haghighi A, Saber V, Armon B, Seyyedtabaei SJ. The role of Blastocystis sp. and Dientamoeba fragilis in irritable bowel syndrome: a systematic review and meta-analysis. Parasitol Res. 2017;116:2361–71. doi: 10.1007/s00436-017-5535-6. PubMed DOI

Cinek O, Kramna L, Mazankova K, Odeh R, Alassaf A, Ugochi Ibekwe M, et al. The bacteriome at the onset of type 1 diabetes: a study from four geographically distant African and Asian countries. Diabetes Res Clin Pract. 2018;144:51–62. doi: 10.1016/j.diabres.2018.08.010. PubMed DOI

Cinek O, Kramna L, Odeh R, Alassaf A, Ugochi Ibekwe MA, Gunduz A, et al. Eukaryotic viruses in the faecal virome at the onset of type 1 diabetes: a study from four geographically distant African and Asian countries. Pediatric Diabetes. 2021;in press. PubMed

Neuman V, Pruhova S, Kulich M, Kolouskova S, Vosahlo J, Romanova M, et al. Gluten-free diet in children with recent-onset type 1 diabetes: a 12-month intervention trial. Diabetes Obes Metab. 2020;22:866–872. doi: 10.1111/dom.13974. PubMed DOI

Stensvold CR, Ahmed UN, Andersen LO, Nielsen HV. Development and evaluation of a genus-specific, probe-based, internal-process-controlled real-time PCR assay for sensitive and specific detection of Blastocystis spp. J Clin Microbiol. 2012;50:1847–1851. doi: 10.1128/JCM.00007-12. PubMed DOI PMC

Maloney JG, Molokin A, Santin M. Next generation amplicon sequencing improves detection of Blastocystis mixed subtype infections. Infect Genet Evol. 2019;73:119–125. doi: 10.1016/j.meegid.2019.04.013. PubMed DOI

Santin M, Gomez-Munoz MT, Solano-Aguilar G, Fayer R. Development of a new PCR protocol to detect and subtype Blastocystis spp. from humans and animals. Parasitol Res. 2011;109:205–12. doi: 10.1007/s00436-010-2244-9. PubMed DOI

Edgar RC. Search and clustering orders of magnitude faster than BLAST. Bioinformatics. 2010;26:2460–2461. doi: 10.1093/bioinformatics/btq461. PubMed DOI

McMurdie PJ, Holmes S. phyloseq: an R package for reproducible interactive analysis and graphics of microbiome census data. PLoS ONE. 2013;8:e61217. doi: 10.1371/journal.pone.0061217. PubMed DOI PMC

Team RC: R: A language and environment for statistical computing. 3.4.2 edn. Vienna, Austria: R Foundation for Statistical Computing; 2017.

Rojas-Velazquez L, Maloney JG, Molokin A, Moran P, Serrano-Vazquez A, Gonzalez E, et al. Use of next-generation amplicon sequencing to study Blastocystis genetic diversity in a rural human population from Mexico. Parasit Vectors. 2019;12:566. doi: 10.1186/s13071-019-3814-z. PubMed DOI PMC

Kramna L, Kolarova K, Oikarinen S, Pursiheimo JP, Ilonen J, Simell O, et al. Gut virome sequencing in children with early islet autoimmunity. Diabetes Care. 2015;38:930–933. doi: 10.2337/dc14-2490. PubMed DOI

Callahan BJ, McMurdie PJ, Rosen MJ, Han AW, Johnson AJ, Holmes SP. DADA2: High-resolution sample inference from Illumina amplicon data. Nat Methods. 2016;13:581–583. doi: 10.1038/nmeth.3869. PubMed DOI PMC

Oksanen J, Guillaume Blanchet F, Friendly M, Kindt R, Legendre P, McGlinn D, et al: vegan: Community Ecology Package. R package version 2.5–6. 2019.

Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15:550. doi: 10.1186/s13059-014-0550-8. PubMed DOI PMC

Horiki N, Maruyama M, Fujita Y, Yonekura T, Minato S, Kaneda Y. Epidemiologic survey of Blastocystis hominis infection in Japan. Am J Trop Med Hyg. 1997;56:370–374. doi: 10.4269/ajtmh.1997.56.370. PubMed DOI

El Safadi D, Gaayeb L, Meloni D, Cian A, Poirier P, Wawrzyniak I, et al. Children of Senegal River Basin show the highest prevalence of Blastocystis sp. ever observed worldwide. BMC Infect Dis. 2014;14:164. doi: 10.1186/1471-2334-14-164. PubMed DOI PMC

Petrasova J, Uzlikova M, Kostka M, Petrzelkova KJ, Huffman MA, Modry D. Diversity and host specificity of Blastocystis in syntopic primates on Rubondo Island, Tanzania. Int J Parasitol. 2011;41:1113–1120. doi: 10.1016/j.ijpara.2011.06.010. PubMed DOI

Lhotska Z, Jirku M, Hlozkova O, Brozova K, Jirsova D, Stensvold CR, et al. A Study on the prevalence and subtype diversity of the intestinal protist Blastocystis sp. in a gut-healthy human population in the Czech Republic. Front Cell Infect Microbiol. 2020;10:544335. doi: 10.3389/fcimb.2020.544335. PubMed DOI PMC

Poulsen CS, Efunshile AM, Nelson JA, Stensvold CR. Epidemiological aspects of Blastocystis colonization in children in Ilero Nigeria. Am J Trop Med Hyg. 2016;95:175–179. doi: 10.4269/ajtmh.16-0074. PubMed DOI PMC

Dominguez-Marquez MV, Guna R, Munoz C, Gomez-Munoz MT, Borras R. High prevalence of subtype 4 among isolates of Blastocystis hominis from symptomatic patients of a health district of Valencia (Spain) Parasitol Res. 2009;105:949–955. doi: 10.1007/s00436-009-1485-y. PubMed DOI

Greige S, El Safadi D, Becu N, Gantois N, Pereira B, Chabe M, et al. Prevalence and subtype distribution of Blastocystis sp. isolates from poultry in Lebanon and evidence of zoonotic potential. Parasit Vectors. 2018;11:389. doi: 10.1186/s13071-018-2975-5. PubMed DOI PMC

Maloney JG, Molokin A, da Cunha MJR, Cury MC, Santin M. Blastocystis subtype distribution in domestic and captive wild bird species from Brazil using next generation amplicon sequencing. Parasite Epidemiol Control. 2020;9:e00138. doi: 10.1016/j.parepi.2020.e00138. PubMed DOI PMC

Meloni D, Poirier P, Mantini C, Noel C, Gantois N, Wawrzyniak I, et al. Mixed human intra- and inter-subtype infections with the parasite Blastocystis sp. Parasitol Int. 2012;61:719–722. doi: 10.1016/j.parint.2012.05.012. PubMed DOI

Audebert C, Even G, Cian A, Loywick A, Merlin S, Viscogliosi E, et al. Colonization with the enteric protozoa Blastocystis is associated with increased diversity of human gut bacterial microbiota. Sci Rep. 2016;6:25255. doi: 10.1038/srep25255. PubMed DOI PMC

Nieves-Ramirez ME, Partida-Rodriguez O, Laforest-Lapointe I, Reynolds LA, Brown EM, Valdez-Salazar A, et al. Asymptomatic intestinal colonization with protist Blastocystis is strongly associated with distinct microbiome ecological patterns. mSystems. 2018;3:e00007. doi: 10.1128/mSystems.00007-18. PubMed DOI PMC

O'Brien Andersen L, Karim AB, Roager HM, Vigsnaes LK, Krogfelt KA, Licht TR, et al. Associations between common intestinal parasites and bacteria in humans as revealed by qPCR. Eur J Clin Microbiol Infect Dis. 2016;35:1427–1431. doi: 10.1007/s10096-016-2680-2. PubMed DOI

Forsell J, Bengtsson-Palme J, Angelin M, Johansson A, Evengard B, Granlund M. The relation between Blastocystis and the intestinal microbiota in Swedish travellers. BMC Microbiol. 2017;17:231. doi: 10.1186/s12866-017-1139-7. PubMed DOI PMC

Krogsgaard LR, Andersen LO, Johannesen TB, Engsbro AL, Stensvold CR, Nielsen HV, et al. Characteristics of the bacterial microbiome in association with common intestinal parasites in irritable bowel syndrome. Clin Transl Gastroenterol. 2018;9:161. doi: 10.1038/s41424-018-0027-2. PubMed DOI PMC

Gabrielli S, Furzi F, Fontanelli Sulekova L, Taliani G, Mattiucci S. Occurrence of Blastocystis-subtypes in patients from Italy revealed association of ST3 with a healthy gut microbiota. Parasite Epidemiol Control. 2020;9:e00134. doi: 10.1016/j.parepi.2020.e00134. PubMed DOI PMC

Oduaran OH, Tamburini FB, Sahibdeen V, Brewster R, Gomez-Olive FX, Kahn K, et al. Gut microbiome profiling of a rural and urban South African cohort reveals biomarkers of a population in lifestyle transition. BMC Microbiol. 2020;20:330. doi: 10.1186/s12866-020-02017-w. PubMed DOI PMC

Nourrisson C, Scanzi J, Pereira B, NkoudMongo C, Wawrzyniak I, Cian A, et al. Blastocystis is associated with decrease of fecal microbiota protective bacteria: comparative analysis between patients with irritable bowel syndrome and control subjects. PLoS ONE. 2014;9:e111868. doi: 10.1371/journal.pone.0111868. PubMed DOI PMC

Insel R, Knip M. Prospects for primary prevention of type 1 diabetes by restoring a disappearing microbe. Pediatr Diabetes. 2018;19(8):1400–6. doi: 10.1111/pedi.12756. PubMed DOI

Kato K, Odamaki T, Mitsuyama E, Sugahara H, Xiao JZ, Osawa R. Age-related changes in the composition of gut bifidobacterium species. Curr Microbiol. 2017;74:987–995. doi: 10.1007/s00284-017-1272-4. PubMed DOI PMC

Arboleya S, Watkins C, Stanton C, Ross RP. Gut Bifidobacteria populations in human health and aging. Front Microbiol. 2016;7:1204. doi: 10.3389/fmicb.2016.01204. PubMed DOI PMC

Deng L, Wojciech L, Gascoigne NRJ, Peng G, Tan KSW. New insights into the interactions between Blastocystis, the gut microbiota, and host immunity. PLoS Pathog. 2021;17:e1009253. doi: 10.1371/journal.ppat.1009253. PubMed DOI PMC

Yason JA, Liang YR, Png CW, Zhang Y, Tan KSW. Interactions between a pathogenic Blastocystis subtype and gut microbiota: in vitro and in vivo studies. Microbiome. 2019;7:30. doi: 10.1186/s40168-019-0644-3. PubMed DOI PMC

Iebba V, Santangelo F, Totino V, Pantanella F, Monsia A, Di Cristanziano V, et al. Gut microbiota related to Giardia duodenalis, Entamoeba spp. and Blastocystis hominis infections in humans from Cote d'Ivoire. J Infect Dev Ctries. 2016;10:1035–41. doi: 10.3855/jidc.8179. PubMed DOI

Alzate JF, Toro-Londono M, Cabarcas F, Garcia-Montoya G, Galvan-Diaz A. Contrasting microbiota profiles observed in children carrying either Blastocystis spp. or the commensal amoebas Entamoeba coli or Endolimax nana. Sci Rep. 2020;10:15354. doi: 10.1038/s41598-020-72286-y. PubMed DOI PMC

Castaneda S, Munoz M, Villamizar X, Hernandez PC, Vasquez LR, Tito RY, et al. Microbiota characterization in Blastocystis-colonized and Blastocystis-free school-age children from Colombia. Parasit Vectors. 2020;13:521. doi: 10.1186/s13071-020-04392-9. PubMed DOI PMC

Kodio A, Coulibaly D, Kone AK, Konate S, Doumbo S, Guindo A, et al. Blastocystis colonization is associated with increased diversity and altered gut bacterial communities in healthy Malian Children. Microorganisms. 2019;7:649. doi: 10.3390/microorganisms7120649. PubMed DOI PMC

Stensvold CR, Alfellani M, Clark CG. Levels of genetic diversity vary dramatically between Blastocystis subtypes. Infect Genet Evol. 2012;12:263–273. doi: 10.1016/j.meegid.2011.11.002. PubMed DOI

Cinek O, Kramna L, Mazankova K, Kunteova K, Chuda K, Eric CJC, et al. Virus genotyping by massive parallel amplicon sequencing: adenovirus and enterovirus in the Norwegian MIDIA study. J Med Virol. 2019;91:606–14. PubMed

Not A, Salvador F, Goterris L, Sulleiro E, Lopez I, Balladares M, et al. Microscopic examination after concentration techniques for Blastocystis sp. detection in serial faecal samples: how many samples are needed? Parasite Epidemiol Control. 2020;9:e00137. doi: 10.1016/j.parepi.2020.e00137. PubMed DOI PMC

Effects of Lactiplantibacillus plantarum and Lacticaseibacillus paracasei supplementation on the single-cell fecal parasitome in children with celiac disease autoimmunity: a randomized, double-blind placebo-controlled clinical trial

Comparison of molecular diagnostic approaches for the detection and differentiation of the intestinal protist Blastocystis sp. in humans