Comparative Study of Wheatley’S Trichrome Stain and In-Vitro Culture Against Pcr Assay For the Diagnosis of Blastocystis Sp. in Stool Samples

Background: this study evaluated the performance of routine permanent stain and cultivation method in comparison with polymerase chain reaction assay as the reference technique to detect blastocystis sp. methods: a cross-sectional study was conducted among aboriginal populations that reside in pahang, peninsular malaysia in feb to mar 2015. a total of 359 stool samples were examined using wheatley’s trichrome stain, in-vitro cultivation in jones’ medium and pcr assay. positive amplicons were subjected to sequencing and phylogenetic analysis. results: fifty-six (15.6%) samples were detected positive with blastocystis sp. by wheatley’s trichrome stain and 73 (20.3%) by in-vitro culture, while pcr assay detected 71 (19.8%) positive samples. detection rate of blastocystis sp. was highest in combination of microscopic techniques (27.9%). the sensitivity and specificity of wheatley’s trichrome staining and in-vitro culture techniques compared to pcr assay were 49.3% (95% ci: 37.2-61.4) and 92.7% (95% ci: 89.1-95.4) and 39.4% (95% ci: 28.0-51.8) and 84.4% (95% ci: 79.7-88.4), respectively. however, the sensitivity [60.6% (95% ci: 48.3-71.9)] of the method increased when both microscopic techniques were performed together. false negative results produced by microscopic techniques were associated with subtype 3. the agreement between wheatley’s trichrome stain, in-vitro culture and combination of microscopic techniques with pcr assay were statistically significant by kappa statistics (wheatley’s trichrome stain: k = 0.456, p<0.001; in-vitro culture: k = 0.236, p<0.001 and combination techniques: k = 0.353, p<0.001). conclusion: the combination of microscopic technique is highly recommended to be used as a screening method for the diagnosis of blastocystis infection either for clinical or epidemiological study to ensure better and accurate diagnosis.