Molecular Detection and Genotyping of Enteric Protists in Asymptomatic Schoolchildren and Their Legal Guardians in Madrid, Spain

Asymptomatic carriage of diarrhoea-causing enteric protist parasites in the general population is poorly understood, particularly in medium- to high-income countries. This molecular epidemiological survey investigates the presence, molecular diversity, and household transmission of Giardia duodenalis, Cryptosporidium spp., Blastocystis sp., and Enterocystozoon bieneusi in schoolchildren aged 2–13 years (n = 74) and their legal guardians (n = 6) in Madrid, Spain. Enteroparasite detection and genotyping was conducted in stool samples by molecular (PCR and Sanger sequencing) methods. Potential associations linked to infections were investigated through epidemiological questionnaires. Giardia duodenalis was the most prevalent enteric parasite found (14%, 95% CI: 7.1–23), followed by Blastocystis sp. (10%, 95% CI: 6.2–22) and Cryptosporidium spp. (3.8%, 95% CI: 0.78–11). None of the participants tested positive for E. bieneusi. Sequence analyses revealed the presence of G. duodenalis assemblage B, sub-assemblage BIV in a single child. The three Cryptosporidium isolates obtained were assigned to C. hominis, two of them belonging to the gp60 subtype IbA10G2. Four Blastocystis subtypes were identified including ST2 (38%, 3/8), ST3 (25%, 2/8), ST4 (25%, 2/8), and ST8 (12%, 1/8). All G. duodenalis and Cryptosporidium isolates were detected in children only. Blastocystis ST3 and ST4 were circulating in members of the same household. Blastocystis carriage rates increased with the age of the participants. Presence of diarrhoea-causing enteric protists was common in apparently healthy children.