Blastocystis sp. is known to be the most prevalent parasite in fecal samples of humans worldwide. In the present report, a case–control study (1:9.89 (≈10)) was performed, by analyzing data from 3682 patients who attended a public hospital in the northern area of Spain showing gastrointestinal symptoms. Diagnosis was performed in human fecal samples by means of optical microscopy. The prevalence of Blastocystis sp. in patients with gastrointestinal symptoms was 9.18% (338/3682). Most of the Blastocystis sp.-infected patients tested negative for protozoa and helminths, and were underweight and foreign-born (26.4%), mainly from Africa and Central/South America. Gastrointestinal symptoms, such as abdominal pain, anorexia, halitosis, plus relative eosinophilia, as well as co-infections with pathogenic bacteria were associated with Blastocystis sp. infection. Both type 2 diabetes and treatment with immunosuppressive medicines at the time of Blastocystis sp. detection were associated with a higher proportion of infected patients. This is the first case–control study of Blastocystis sp. in humans in northern Spain and may contribute to surveillance and intervention strategies by public health authorities.

Limited molecular data exist on the prevalence and subtype distribution of Blastocystis spp., the most prevalent parasite in human and animal feces worldwide. A total of 44 different subtypes (STs) of Blastocystis are currently recognized based on the sequence of the small subunit ribosomal RNA (SSU-rRNA) gene. This is a molecular study of Blastocystis spp. in hospitalized patients with gastrointestinal symptoms in northern Spain. We analyzed 173 Blastocystis-positive patients with gastrointestinal symptoms by using nested PCR for molecular detection, subtype identification, phylogenetic analyses, and genetic diversity assessment. ST2 (34.1%) and ST3 (34.7%) predominated, followed by ST1 (15.6%) and ST4 (15.6%). Mixed infections with different subtypes were observed in some patients. Sequence analysis revealed for the first time in European humans the allele 88 (a variant of ST1). In other cases, alleles commonly found in animal samples were detected (allele 9 in ST2, allele 34 in ST3, and allele 42 in ST4). Phylogenetic analysis showed high variability in ST1 and ST2, suggesting a polyphyletic origin, while both ST3 and ST4 exhibited higher genetic homogeneity, indicating a possible monophyletic origin and recent transmission to humans. These data confirm Blastocystis spp. subtype diversity and may help in understanding the evolutionary processes and potential zoonotic transmission of this parasite.