Preliminary Validation of a Colorimetric Loop-Mediated Isothermal Amplification (c-LAMP) Assay for Detection of Pythium insidiosum in Clinical Specimens

Abstract

Pythiosis is an emerging infectious disease caused by the oomycete Pythium insidiosum, affecting humans and animals primarily in subtropical and tropical regions. The pathogen is commonly found in swampy environments, and exposure can lead to diverse clinical manifestations. In humans, ocular and vascular infections predominate, whereas in animals, cutaneous/subcutaneous or gastrointestinal disease is more common. Medical therapy is frequently ineffective, and many patients require extensive surgical intervention. Advanced cases may progress to fatal outcomes. Therefore, early and accurate detection is critical for improving clinical management. This study evaluated a colorimetric loop-mediated isothermal amplification (c-LAMP) assay compared with an established multiplex PCR (m-PCR) assay for the detection of P. insidiosum in clinical specimens from animals with and without pythiosis. When tested on 47 frozen tissue samples, c-LAMP demonstrated superior diagnostic performance, with markedly greater sensitivity (83.9% vs. 41.9%), higher accuracy (78.7% vs. 61.7%), and a shorter turnaround time (65 vs. 180 min). However, c-LAMP yielded five false-positive results, likely due to nonspecific amplification or contamination. Improved sample-handling practices increased the specificity from 68.8% to 93.8%. In contrast, m-PCR showed perfect specificity (100.0%) but substantially lower sensitivity, resulting in a high false-negative rate. In conclusion, these preliminary findings suggest that c-LAMP is a promising rapid screening tool for suspected pythiosis, particularly in resource-limited settings. Nevertheless, confirmatory testing remains necessary for positive or equivocal c-LAMP results.