Simplified Point-of-Care Testing for Human Pythiosis: Development of a Whole-Blood-Based Lateral Flow Immunoassay

<b>Background/Objectives</b>: Human pythiosis, caused by <i>Pythium insidiosum</i>, is associated with severe morbidity and high mortality when diagnosis is delayed. Culture-based diagnosis is time-consuming and may be insensitive in clinical specimens, highlighting the need for rapid point-of-care serodiagnostic tools. Here, we developed and clinically evaluated the Anti-Pin Antibody Test Strip, a whole-blood-compatible lateral flow immunoassay (LFIA) for detecting anti-<i>P. insidiosum</i> antibodies. <b>Methods</b>: Secretory protein antigens of <i>P. insidiosum</i> were prepared and conjugated to gold nanoparticles for LFIA development. Analytical performance was assessed by determining the limit of detection (LOD) using serial dilutions of pythiosis serum and by evaluating cross-reactivity with sera from patients with other infections. Interference testing examined common anticoagulants and adverse sample conditions (hemolysis, lipidemia, and icterus). Clinical performance was evaluated using 258 serum samples, comprising 48 pythiosis-positive and 210 pythiosis-negative specimens confirmed by immunoblotting and/or culture. Test results were read at 5 min. <b>Results</b>: The assay LOD was a serum titer of 1:1000. No cross-reactivity was observed across the tested infectious and immunologic panels, and no interference was detected from anticoagulants or adverse sample conditions. Whole-blood testing showed no red blood cell interference. In clinical evaluation, sensitivity was 100.00% (48/48), specificity was 95.24% (200/210), and accuracy was 96.12%, with a PPV of 82.76% and an NPV of 100.00%. <b>Conclusions</b>: The Anti-Pin Antibody Test Strip provides rapid (5 min), operationally simple serodiagnosis and may support screening/triage of suspected pythiosis, particularly where laboratory methods are limited.