Tehran University of Medical Sciences Iranian Journal of Parasitology 1735-7020 13 1 2018 03 14 127 136 Nabilah Amelia MOHAMMAD Center of Medical Laboratory Technology, Faculty of Health Sciences, Universiti Teknologi MARA, Puncak Alam Campus, Se-langor, Malaysia Mohd Fahmi MASTUKI Center of Medical Laboratory Technology, Faculty of Health Sciences, Universiti Teknologi MARA, Puncak Alam Campus, Se-langor, Malaysia Hesham M. AL-MEKHLAFI Endemic and Tropical Diseases Unit, Medical Research Center, Jazan University, Jazan, Kingdom of Saudi Arabia AND Dept. of Parasitology, Faculty of Medicine and Health Sciences, Sana’a University, Sana’a, Yemen Norhayati MOKTAR Dept. of Pre-Clinical Sciences, Faculty of Medicine and Health Sciences, Universiti Tunku Abdul Rahman, Sungai Long Campus, Selangor, Malaysia Tengku Shahrul ANUAR Center of Medical Laboratory Technology, Faculty of Health Sciences, Universiti Teknologi MARA, Puncak Alam Campus, Se-langor, Malaysia AND Integrative Pharmacogenomics Institute, Universiti Teknologi MARA, Puncak Alam Campus, Selangor, Malaysia 2018 03 14 2018 03 14 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. https://ijpa.tums.ac.ir/index.php/ijpa/article/view/2054 https://ijpa.tums.ac.ir/index.php/ijpa/article/download/2054/816