Molecular detection and subtype characterization of Blastocystis in diarrheal outpatients in Shanghai, China: A case-control study

BACKGROUND: Blastocystis is the most prevalent enteric protozoan colonizing the human gastrointestinal tract, yet its clinical relevance and molecular epidemiological profiles in Shanghai, China's largest international megacity, remain unclear. The present study investigated the occurrence, subtype distribution, genetic diversity, and clinical associations of Blastocystis in diarrheal outpatients in Shanghai, China. METHODS: A case-control study was conducted with 370 fecal specimens (220 from diarrheal patients, 150 age- and sex-matched non-diarrheal healthy controls) collected between June 2023 and March 2026. Blastocystis was detected via PCR amplification of the small subunit ribosomal RNA (SSU rRNA) gene, with Sanger sequencing for subtype assignment. Statistical analyses were performed using categorical tests and logistic regression. RESULTS: The overall Blastocystis prevalence was 11.62% (43/370), significantly higher in diarrheal patients (16.8%; 37/220) than in controls (4.0%; 6/150, P < 0.001), with a higher detection rate in chronic (27.3%; 12/44) versus acute (14.2%; 25/176) diarrhea. Only two subtypes were identified: dominant ST3 (76.7%) and ST1 (23.3%). ST1 exhibited higher genetic diversity and showed an exploratory association with chronic diarrhea (P = 0.042, uncorrected for multiple testing). Age 7-18 years showed a borderline positive correlation with detection in diarrheal patients. CONCLUSIONS: This study found that Blastocystis detection was significantly correlated with diarrheal symptoms in the Shanghai outpatient population. ST3 was the dominant circulating subtype, and a potential association between ST1 and chronic diarrhea was identified, which requires confirmation in larger prospective studies. These results contribute to the regional molecular epidemiological database of Blastocystis and may inform clinical decision-making for patients with persistent unexplained diarrhea.