Background Previous studies have demonstrated that Strongyloides ELISAs frequently exhibit cross-reactivity with infections caused by other helminths. This may result in false-positive results, especially in low-endemic areas, potentially leading to unwarranted treatment. This study aimed to present a new Western blot for the detection of anti-Strongyloides antibodies such as to enhance the accuracy of distinguishing true-positive from false-positive ELISA results.Methods We developed and validated a Western blot to detect anti-S. stercoralis specific antibodies using reference sera from confirmed cases (n=55), non-infected control patients (n=20), and patients with other helminth infections (n=50). Using this sample set, we evaluated the analytical performance of three serologic approaches: an in-house ELISA, the commercially available Bordier-ELISA, and a two-tiered testing procedure combining the in-house ELISA with a confirmatory Western blot. Using receiver operating characteristics (ROC) analysis, setting-specific cut-offs values were determined for both ELISA assays. Further, we conducted a retrospective cohort study using serum samples from solid organ transplant (SOT) candidates (n=310) tested by ELISA during pre-transplant evaluations at Bern University Hospital (2018-2022).Results The Western blot specifically detected human IgG antibodies against S. stercoralis. A two-tier test algorithm (in-house ELISA, followed by confirmatory Western blot) showed a diagnostic sensitivity of 96.4% (95%CI:87.5%-99.6%) and a diagnostic specificity of 98.6% (95%CI:92.3%-100%). Out of 310 SOT candidates' serum samples, 9.3% (n=29) exhibited positive, and 5.2% (n=16) equivocal ELISA results. Testing by Western blot revealed positive S. stercoralis findings for 11.1% (n=5) of these patients. Two of those were initially positive and 3 were equivocal by ELISA.Conclusions The new Western blot is a promising tool for clarifying equivocal S. stercoralis ELISA results, and therefore predisposed to reduce false positive Strongyloides ELISA results, especially in low-endemic regions.
