Strongyloides stercoralis is a nematode endemic to subtropical and tropical regions which may cause asymptomatic carriage, peripheral eosinophilia, cutaneous, gastrointestinal, and pulmonary disease, or hyperinfection syndrome. Conventional diagnostic methods for strongyloidiasis include faeces culture and microscopy, with low sensitivity in chronic infection due to the low helminth burden, and serology, which may be prone to false-negative results with immunocompromise and false-positive results with other infections and immunological disorders1 2. We evaluated a laboratory-developed real-time polymerase chain reaction (qPCR), detecting the 18S ribosomal RNA gene, compared to conventional diagnostic methods, using serology via enzyme-linked immunosorbent assay (ELISA) as the gold-standard. The population studied was tertiary hospital inpatients and outpatients residing in a non-endemic area. 750 unfixed stool specimens submitted between 2014 and 2018 were tested for S. stercoralis via microscopy and qPCR. Agar plate culture (APC), Harada-Mori culture (HMC), and ELISA were performed in conjunction with 141, 135, and 177 of the specimens respectively. qPCR yielded 13 positive and 730 negative results whilst inhibition occurred in 7 specimens. ELISA yielded 53 positive, 18 equivocal, and 106 negative results. Results for direct diagnostic methods obtained following treatment with ivermectin were excluded from the performance analysis. Compared with ELISA, qPCR, microscopy, APC, and HMC exhibited sensitivities of 38% (13/34), 6% (2/33), 3% (1/29), and 0% (0/24) respectively and specificities of 100%. Our results conflict a meta-analysis in which molecular methods exhibited a sensitivity and specificity of 56.50% and 95.38% respectively compared with parasitological and serological methods, although only 2 of the 14 included studies were performed in non-endemic areas3. There is a paucity of data from non-endemic settings but our results are comparable to those of one study performed in a non-endemic area where APC, qPCR, and serology exhibited sensitivities of 45%, 57%, and 95% respectively4, most likely reflecting the low helminth burden in chronic strongyloidiasis. We recommend employing a combination of molecular, parasitological, and serological methods for diagnosis and screening of strongyloidiasis.