A direct comparison of STS-PCR and RFLP for detection and subtyping of human Blastocystis in Iran

Blastocystis is a genetically diverse unicellular eukaryotic intestinal parasite with no gold-standard diagnostic method. This study, conducted in Iran, is the first to directly compare Sequence-Tagged Site (STS) PCR and restriction fragment length polymorphism (RFLP) for its diagnosis of human. It builds on our previous work with the same cohort. We aimed to evaluate the diagnostic sensitivity and specificity of these methods using nested PCR as the internal reference. In addition, we assessed subtypes and genetic diversity using STS-PCR. A total of 124 fecal samples were included: 64 previously confirmed positive and 60 negative by nested PCR. These were used to evaluate the diagnostic performance of STS-PCR. RFLP was also applied to the nested PCR-negative samples. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and 95% confidence intervals (CIs) were calculated. STS-PCR detected 39 of the 64 specimens that were positive by nested PCR (sensitivity 60.9%, specificity 100%, NPV 71%, PPV 100%). It failed to detect 25 cases, which may be attributable to primer mismatches or PCR inhibitors. RFLP detected 55 of the 64 positive cases (sensitivity 85.9%, specificity 100%, NPV 87%, PPV 100%). STS-PCR uniquely identified nine samples missed by RFLP. All nested PCR-negative samples remained negative by both methods. Sequencing of two STS-PCR false-negative samples confirmed ST1 and ST3. Differences in detection patterns between STS-PCR and RFLP underscore the value of multi-assay molecular analysis in this dataset. Further validation is required to determine whether similar patterns are observed in other populations or laboratory settings.