Association of Enterocytozoon bieneusi Infection with chronic/persistent diarrhea and ITS genotypic diversity: a hospital-based case-control study in Suburban Shanghai, China.
Enterocytozoon bieneusi is a globally distributed zoonotic enteric pathogen that remains largely overlooked in routine diarrheal disease surveillance. Although previous studies in Shanghai, China, have reported elevated prevalence in diarrheal populations, case-control data from suburban areas at the peri‑urban interface and the strength of the association between E. bieneusi infection and chronic diarrhea in non-immunocompromised individuals remain poorly characterized. We performed a hospital-based case-control study in suburban Shanghai, enrolling 286 diarrheal outpatients without documented immunodeficiency and 138 asymptomatic controls frequency-matched for age and sex. Fecal specimens were collected and subjected to genomic DNA extraction. E. bieneusi was detected via nested PCR amplification of the ribosomal internal transcribed spacer (ITS) region. Factors associated with infection were identified using multivariate logistic regression. Genotypic diversity and zoonotic potential were assessed by Sanger sequencing and phylogenetic analysis. The overall prevalence of E. bieneusi was 12.2% (35/286) in diarrheal patients, significantly higher than the 2.2% (3/138) observed in asymptomatic controls (P < 0.001). E. bieneusi positivity was independently associated with chronic/persistent diarrhea (adjusted odds ratio = 2.63, 95% confidence interval: 1.25-5.54, P = 0.011). Fourteen distinct ITS genotypes were identified, comprising five known genotypes (D, EbpD, SHW7, Henan-III, and CHG5) and nine novel genotypes (designated SHH2 to SHH10). Thirteen genotypes clustered within Group 1, and one genotype (CHG5) fell within Group 2, two phylogenetic groups that contain genotypes with documented zoonotic potential in global surveillance. E. bieneusi was detected at a relatively high prevalence among diarrheal patients in suburban Shanghai, and its detection was associated with chronic/persistent diarrhea. The predominance of zoonotic genotypes and the identification of nine novel Group 1 genotypes indicate phylogenetic similarity to known zoonotic lineages and warrant further investigation of local zoonotic transmission; no animal or environmental samples were analyzed in this study. These findings suggest that E. bieneusi testing may be considered as part of the differential diagnosis for patients with unexplained chronic/persistent diarrhea and highlight the need for One Health surveillance in the surveyed area.