Molecular epidemiology of Dientamoeba fragilis coinfections with Blastocystis sp. and Giardia duodenalis in symptomatic patients from Valencia, Spain

Abstract

PURPOSE: Dientamoeba fragilis is a cosmopolitan intestinal protist with an insufficiently understood life cycle and pathogenicity. Diagnosis by light microscopy is challenging, leading to significant underdiagnosis. Two genotypes (1 and 2) have been described, with genotype 1 being the most frequently detected worldwide. As no molecularly characterized isolates from Spain have been reported, we performed a molecular characterization and evaluated sociodemographic factors of D. fragilis in patients coinfected with Blastocystis sp. and/or Giardia duodenalis. METHODS: Using a multiplex qPCR assay (Allplex? GI-Parasite Assay, Seegene), we analyzed 354 stool samples previously identified as positive for Blastocystis sp. (n = 276), G. duodenalis (n = 63), or both (n = 15) by light microscopy. Eligible positive samples were selected for conventional PCR targeting the SSU rRNA gene and subsequent Sanger sequencing. RESULTS: D. fragilis was detected in 34.5% of samples by qPCR; 32.3% of Blastocystis-positive, 44.9% of Giardia-positive and 46.7% of coinfected samples. We successfully sequenced 22 isolates, all corresponding to genotype 1, identifying two distinct intra-genotypic sequence variants. Younger age was positively associated with D. fragilis, with individuals coinfected with Blastocystis sp. being markedly younger than those monoinfected. Patients with G. duodenalis and D. fragilis were mainly under 15 years of age. CONCLUSION: This study provides the first genotyping of D. fragilis in Spain, demonstrating the circulation of two genotype 1 variants. Findings highlight the underdiagnosis of D. fragilis and its high prevalence in young patients with other fecal-orally transmitted protists. Evaluating coinfections is crucial to understanding parasite relationships and should be considered for clinical management, especially when symptoms persist.