Pythiosis, a life-threatening infectious condition caused by Pythium insidiosum, has been increasingly reported in humans and animals worldwide. Antifungal drugs usually fail to control the pathogen. The surgical removal of an infected organ is the treatment of choice. Many affected patients die due to advanced infection. A timely and accurate diagnosis could lead to a better prognosis in pythiosis patients and save their lives. Although a standard culture method is available in microbiological laboratories, it is time-consuming, laborious, and insensitive for P. insidiosum identification. Immunological assays have been developed to improve the diagnosis of pythiosis. However, immunological methods are commercially unavailable and primarily detect anti-P. insidiosum antibodies, which constitute indirect evidence of pythiosis, making it challenging to differentiate a past from a recent infection. Moreover, such immunological tests cannot diagnose patients with a local infection, such as in the eye. Nucleic acid-based tests (NATs) are efficient for the direct and rapid detection of P. insidiosum DNA in trace-amount or culture-negative specimens. The reagents and equipment required for NATs are usually available in molecular diagnostic laboratories. Herein, we provide a systematic review to comprehensively present the principal and clinical usages, advantages, and limitations of such NATs in the detection of P. insidiosum. Various NATs have been established to detect P. insidiosum, which can be classified into amplification-based (i.e., PCR assays, isothermal tests, and next-generation sequencing methods) and non-amplification-based (i.e., DNA hybridization) techniques. This concise review on NATs constitutes an up-to-date reference with which healthcare professionals can learn about and decide upon which detection method is suitable for their respective laboratory environments.

Pythiosis, caused by Pythium insidiosum, is a severe infectious disease affecting humans and animals worldwide. There is an urgent need for a simple and rapid detection method for pythiosis, especially in remote areas where this disease is prevalent. To address this, a colorimetric loop-mediated isothermal amplification assay (c-LAMP) using hydroxynaphthol blue dye as a color indicator has been developed. This method utilized a one-step closed-tube system under a single temperature reaction to detect P. insidiosum, minimizing DNA carry-over contamination and eliminating the need for expensive tools. The test result can be easily read through the color change from violet (negative) to sky blue (positive). When tested with DNA samples from P. insidiosum (n = 51) and other fungi (n = 70), c-LAMP showed a detection sensitivity, specificity, and accuracy of 100.0 %, 95.7 %, and 97.5 %, respectively. The assay detection limit was 1 x 10⁻⁵ ng of DNA template, 10,000 times lower than the reference multiplex PCR assay (m-PCR). c-LAMP also showed a faster assay turnaround time, taking only 65 min, as opposed to the 180 min required for m-PCR. This newly established c-LAMP is rapid, cost-effective, and efficient, making it a promising tool for detecting P. insidiosum in resource-limited laboratories.

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.

Pythium insidiosum, the causative agent of pythiosis, remains an important pathogen in tropical and subtropical regions, where delayed diagnosis is associated with severe disease and poor clinical outcomes. We developed a loop-mediated isothermal amplification coupled with lateral flow dipstick (LAMP-LFD) assay for the rapid detection of P. insidiosum. The assay employs dual-labeled LAMP amplicons that are directly detected by a lateral flow dipstick, enabling simple visual interpretation without agarose gel electrophoresis. Analytical performance was evaluated using genomic DNA from 102 clinically relevant isolates, comprising 51 P. insidiosum isolates and 51 non-target organisms. The LAMP-LFD assay correctly detected all P. insidiosum isolates, achieving an inclusivity of 100.0% (95% CI: 93.0–100.0%), and correctly excluded 48 of 51 non-target organisms, yielding an exclusivity of 94.1% (95% CI: 83.8–98.8%). The assay demonstrated a detection limit of 10 fg of genomic DNA per reaction, representing at least 1000-fold greater analytical sensitivity than the reference multiplex PCR (m-PCR) assay. Clinical validation using 47 blinded animal tissue specimens showed a sensitivity of 90.3%, specificity of 87.5%, accuracy of 89.4%, and good agreement with the reference standard (culture and ITS sequence analysis) (κ = 0.76). In comparison, the m-PCR assay achieved a sensitivity of only 41.9%, indicating substantially lower clinical detection performance. In conclusion, direct LFD-based detection preserves the analytical performance of the established P. insidiosum LAMP assay while providing a clear and easily interpretable visual endpoint. The LAMP-LFD assay represents a rapid, sensitive, and practical tool for screening pythiosis, offering improved diagnostic performance and more reliable result interpretation.