Blastocystis is the most commonly found intestinal protist in the world. Accurate detection and differentiation of Blastocystis including its subtypes (arguably species) are essential to understand its epidemiology and role in human health. We compared (i) the sensitivity of conventional PCR (cPCR) and qPCR in a set of 288 DNA samples obtained from stool samples of gut-healthy individuals, and (ii) subtype diversity as detected by next-generation sequencing (NGS) versus Sanger sequencing. Real-time PCR resulted in more positive samples than cPCR, revealing high fecal load of Blastocystis based on the quantification curve in most samples. In subtype detection, NGS was largely in agreement with Sanger sequencing but showed higher sensitivity for mixed subtype colonization within one host. This fact together with use of the combination of qPCR and NGS and obtaining information on the fecal protist load will be beneficial for epidemiological and surveillance studies.

TITLE: Comparaison des approches de diagnostic moléculaire pour la détection et la différenciation du protiste intestinal Blastocystis sp. chez l’homme. ABSTRACT: Blastocystis est le protiste intestinal le plus répandu dans le monde. La détection et la différenciation précises de Blastocystis, y compris ses sous-types (sans doute des espèces), sont essentielles pour comprendre son épidémiologie et son rôle dans la santé humaine. Nous avons comparé (i) la sensibilité de la PCR conventionnelle (cPCR) et de la qPCR dans un ensemble de 288 échantillons d’ADN obtenus à partir d’échantillons de selles d’individus en bonne santé intestinale et (ii) la diversité des sous-types détectée par le séquençage de nouvelle génération (NGS) par rapport au séquençage Sanger. La PCR en temps réel a donné plus d’échantillons positifs que la cPCR, révélant une charge fécale élevée de Blastocystis sur la base de la courbe de quantification dans la plupart des échantillons. Dans la détection des sous-types, le NGS était largement en accord avec le séquençage de Sanger mais a montré une sensibilité plus élevée pour la colonisation de sous-types mixtes au sein d’un hôte. Ce fait, associé à l’utilisation de la combinaison de qPCR et de NGS et à l’obtention d’informations sur la charge fécale de protistes, sera bénéfique pour les études épidémiologiques et de surveillance.

Department of Bacteria Parasites and Fungi Statens Serum Institut Copenhagen DK 2300 Denmark

Department of Pediatrics 2nd Faculty of Medicine Charles University and Motol University Hospital Prague 150 06 Czech Republic

Institute of Parasitology Biology Centre the Czech Academy of Sciences České Budějovice 370 05 Czech Republic

Institute of Parasitology Biology Centre the Czech Academy of Sciences České Budějovice 370 05 Czech Republic Department of Medical Biology Faculty of Science University of South Bohemia České Budějovice 370 05 Czech Republic

Institute of Parasitology Biology Centre the Czech Academy of Sciences České Budějovice 370 05 Czech Republic Institute of Vertebrate Biology Czech Academy of Sciences Květná 8 Brno 603 65 Czech Republic

Zobrazit více v PubMed

Andersen LOB, 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. PubMed PMC

Billy V, Lhotská Z, Jirků M, Kadlecová O, Frgelevocá L, Wegener Parfrey L, Jirků-Pomajbíková K. 2021. Blastocystis colonization alters the gut microbiome and in some cases, promotes faster recovery from induced colitis. Frontiers in Microbiology, 12, 641483. PubMed PMC

Chabé M, Lokmer A, Ségurel L. 2017. Gut protozoa: friends or foes of the human gut microbiota? Trends in Parasitology, 33, 925–934. PubMed

Cinek O, Polačková K, Odeh R, Alassaf A, Kramná L, Ibekwe MAU, Majaliwa ES, Ahmadov G, Elmahi BME, Mekki H, Oikarinen S, Lebl J, Abdullah MA. 2021. Blastocystis in the faeces of children from six distant countries: prevalence, quantity, subtypes and the relation to the gut bacteriome. Parasites & Vectors, 14, 399. PubMed PMC

El Safadi D, Gaayeb L, Meloni D, Cian A, Poirier P, Wawrzyniak I, Delbac F, Dabboussi F, Delhaes L, Seck M, Hamze M, Riveau G, Viscogliosi E. 2014. Children of senegal river basin show the highest prevalence of Blastocystis sp. ever observed worldwide. BMC Infection Diseseas, 14, 399. PubMed PMC

El Safadi D, Cian A, Nourrisson C, Pereira B, Morelle C, Bastien P, Bellanger AP, Botterel F, Candolfi E, Desoubeaux G, Lachaud L, Morio F, Pomares C, Rabodonirina M, Wawrzyniak I, Delbac F, Gantois N, Certad G, Delhaes L, Poirier P, Viscogliosi E. 2016. Prevalence, risk factors for infection and subtype distribution of the intestinal parasite Blastocystis sp. from a large-scale multi-center study in France. BMC Infection Diseases, 16, 451. PubMed PMC

Haghighi L, Talebnia SE, Mikaeili F, Asgari Q, Gholizadeh F, Zomorodian K. 2020. Prevalence and subtype identification of Blastocystis isolated from human in Shiraz city, southern Iran. Clinical Epidemiology and Global Health, 8, 840–844.

Lhotská Z, Jirků M, Hložková O, Brožová K, Jirsová D, Stensvold CR, Kolísko M, Jirků-Pomajbíková K. 2020. A study on the prevalence and subtype diversity of the intestinal protist Blastocystis sp. in a gut-healthy human population in the Czech republic. Frontiers in Cellular and Infectious Microbiology, 10, 544335. PubMed PMC

Lukeš J, Stensvold CR, Jirků-Pomajbíková K, Wegener Parfrey L. 2015. Are human intestinal eukaryotes beneficial or commensals? PLoS Pathogens, 11, e1005039. PubMed PMC

MacConaill LE, Burns RT, Nag A, Coleman HA, Slevin MK, Giorda K, Light M, Lai K, Jarosz M, McNeil MS, Ducar MD, Meyerson M, Thorner AR. 2018. Unique, dual-indexed sequencing adapters with UMIs effectively eliminate index cross-talk and significantly improve sensitivity of massively parallel sequencing. BMC Genomics, 19, 30. PubMed PMC

Maloney JG, Molokin A, Santin M. 2019. Next generation amplicon sequencing improves detection of Blastocystis mixed subtype infections. Infection, Genetics and Evolution, 73, 119–125. PubMed

McHardy IH, Wu M, Shimizu-Cohen R, Roger Couturier M, Humphries RM (2014) Detection of intestinal protozoa in the clinical laboratory. Journal of Clinical Microbiology, 52, 712–720. PubMed PMC

Nourrisson C, Scanzi J, Pereira B, NkoudMongo C, Wawrzyniak I, Cian A, Viscogliosi E, Livrelli V, Delbac F, Dapoigny M, Poirier P. 2014. Blastocystis is associated with decrease of fecal microbiotaprotective bacteria: comparative analysis between patients with irritable bowel syndrome and control subjects. PLoS One, 9, e111868. PubMed PMC

Nourrisson C, Brunet J, Flori P, Moniot M, Bonnin V, Delbac F, Poirier P. 2020. Comparison of DNA extraction methods and real-time PCR assays for the detection of Blastocystis sp. in stool specimens. Microorganisms, 8, 1768. PubMed PMC

Poirier P, Wawrzyniak I, Albert A, El Alaoui H, Delbac F, Livrelli V. 2011. Development and evaluation of a real-time PCR assay for detection and quantification of Blastocystis parasites in human stool samples: prospective study of patients with hematological malignancies. Journal of Clinical Microbiology, 49, 975–983. PubMed PMC

Popruk S, Adao DEV, Rivera WL. 2021. Epidemiology and subtype distribution of Blastocystis in humans: a review. Infection, Genetics and Evolution, 95, 105085. PubMed

Rojas-Velázquez L, Maloney JG, Molokin A, Morán P, Serrano-Vázquez A, González E, Pérez-Juárez H, Ximénez C, Santin M. 2019. Use of next-generation amplicon sequencing to study Blastocystis genetic diversity in a rural human population from Mexico. Parasites & Vectors, 12, 566. PubMed PMC

Scanlan PD, Stensvold CR, Cotter PD. 2015. Development and application of a Blastocystis subtype-specific PCR assay reveals that mixed-subtype infections are common in a healthy human population. Applied and Environmental Microbiology, 81, 4071–4076. PubMed PMC

Scanlan PD, Knight R, Song SJ, Ackermann G, Cotter PD. 2016. Prevalence and genetic diversity of Blastocystis in family units living in the United States. Infection, Genetics and Evolution, 45, 95–97. PubMed

Scicluna SM, Tawari B, Clark CG. 2006. DNA barcoding of Blastocystis. Protist, 157, 77–85. PubMed

Seyer A, Karasartova D, Ruh E, Güreser AS, Turgal E, Imir T, Taylan-Ozkan A. 2017. Epidemiology and prevalence of Blastocystis spp. in North Cyprus. American Journal of Tropical Medicine and Hygiene, 96, 1164–1170. PubMed PMC

Skotarczak B. 2018. Genetic diversity and pathogenicity of Blastocystis. Annals of Agricultural and Environmental Medicine, 25, 411–416. PubMed

Stensvold CR, Clark CG. 2020. Pre-empting pandora’s box: Blastocystis subtypes revisited. Trends in Parasitology, 36, 229–232. PubMed

Stensvold CR, Arendrup MC, Jespersgaard C, Mølbak K, Nielsen HV. 2007. Detecting Blastocystis using parasitologic and DNA-based methods: a comparative study. Diagnotic Microbiology and Infectious Disease, 59, 303–307. PubMed

Stensvold CR, Ahmed UN, Andersen LOB, Nielsen HV. 2012. Development and evaluation of a genus-specific, probe-based, internal-process-controlled real-time PCR assay for sensitive and specific detection of Blastocystis spp. Journal of Clinical Microbiology, 50, 1847–1851. PubMed PMC

Stensvold CR, Tan KSW, Clark CG. 2020. Blastocystis. Trends in Parasitology, 36, 315–316. PubMed

Stensvold CR, Jirků Pomajbíková K, Wegener Tams K, Jokelainen P, Berg RPKD, Marving E, Petersen RF, Andersen LOB, Angen O, Nielsen HV. 2021. Parasitic intestinal protists of zoonotic relevance detected in pigs by metabarcoding and real-time PCR. Microorganisms, 9, 1189. PubMed PMC

Tan KSW. 2008. New insights on classification, identification, and clinical relevance of Blastocystis spp. Clinical Microbiology Reviews, 21, 639–665. PubMed PMC

Vega L, Herrera G, Munoz M, Patarroyo MA, Maloney JG, Santin M, Ramírez JD. 2021. Gut microbiota profiles in diarrheic patients with co-occurrence of Clostridioides difficile and Blastocystis. PLoS One, 16, e0248185. PubMed PMC

Shaping the human gut microbiota: The role of canine companionship, lifestyle choices, and Blastocystis sp

A cross-sectional survey of Blastocystis sp. and Dientamoeba fragilis in non-human primates and their caregivers in Czech zoos

The opportunistic protist, Giardia intestinalis, occurs in gut-healthy humans in a high-income country

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

A Cross-Sectional Study on the Occurrence of the Intestinal Protist, Dientamoeba fragilis, in the Gut-Healthy Volunteers and Their Animals