Genotypic Analysis of Enterobius vermicularis (Rhabditida: Oxyuridae, Linnaeus, 1758) Among Infected Individuals in Bulgaria: A First Phylogenetic Study.

Enterobiasis, caused by the nematode Enterobius vermicularis, remains a widespread public health issue, yet data regarding its genetic structure in Southeast Europe are scarce. This study presents the first molecular and phylogenetic characterization of E. vermicularis isolates from Bulgaria. Between 2022 and 2025, perianal tape test samples were collected from 128 individuals (92.2% of whom were children) with enterobiasis from 17 regions of the country. Molecular identification was performed via nested PCR targeting a 324 bp fragment of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene, followed by Sanger sequencing. Phylogenetic relationships were analyzed using Maximum Likelihood (IQ-TREE), and population genetic indices were calculated using DnaSP v6. Phylogenetic analysis revealed that all 128 Bulgarian isolates belong to genotype B, clustering closely with sequences from other European and Asian countries. Genetic diversity analysis showed remarkably low variation, with a haplotype diversity (Hd) of 0.1507 ± 0.0416 and a nucleotide diversity (π) of 0.00082 ± 0.00015. Among the 11 identified haplotypes, a single dominant haplotype (Hap_1) accounted for 92.2% of all samples and was distributed across all sampled geographic regions. Tajima's D was significantly negative (-2.314, < 0.05), suggesting a recent population expansion or purifying selection. The dominance of genotype B and the extremely low genetic diversity suggest a recent introduction or clonal expansion of E. vermicularis in Bulgaria. These findings provide essential baseline data for monitoring transmission dynamics and implementing effective control strategies in the Balkan region.