Molecular Detection of Blastocystis ST2 and ST4 and Exploratory 16S rRNA Gene Community Profiles in a 12-Week Dairy-Intervention Pilot Study

Blastocystis is a common intestinal protist whose clinical and ecological significance remains uncertain. This secondary exploratory analysis examined Blastocystis PCR detection in paired baseline and Week 12 stool samples collected during a randomized pilot intervention comparing daily goat-milk and cow-milk consumption. Nineteen healthy Greek Cypriot adults provided 38 stool samples. Stool DNA was screened by PCR targeting a partial 18S rRNA gene fragment, and positive amplicons were Sanger sequenced for subtype assignment. Blastocystis was detected at both time points in 3 of 19 participants (15.8%); ST2 was identified in two participants and ST4 in one. No Cryptosporidium spp. or Giardia duodenalis PCR-positive samples were identified. In exploratory 16S rRNA gene analyses, median Shannon diversity index was 6.10 versus 4.85 at baseline (p = 0.171) and 5.67 versus 4.17 at Week 12 (p = 0.085) in repeat-detection and no-detection groups, respectively. Bray–Curtis PERMANOVA was non-significant at baseline (R2 = 0.0946, p = 0.0815) and Week 12 (R2 = 0.0597, p = 0.8576); Aitchison-distance sensitivity analyses were likewise non-significant (p = 0.0547 and p = 0.3232, respectively). No selected genus-level comparison met the false-discovery-rate-adjusted threshold. The three-participant repeat-detection subgroup precluded reliable analysis of dairy-arm effects, subtype-specific differences, cardiometabolic outcomes, or bacterial-community associations. In this small pilot cohort, ST2 and ST4 were identified in paired baseline and Week 12 stool samples from three healthy adults, providing molecular evidence of repeat detection over the sampled interval. Larger studies with more frequent sampling, quantitative assays, and adequately powered, prespecified analyses are needed to investigate the temporal dynamics and host-associated correlates of Blastocystis detection.