Pythium insidiosum causes pythiosis, a fatal infectious disease of humans and animals worldwide. Prompt diagnosis and treatment are essential to improve the clinical outcome of pythiosis. Diagnosis of P. insidiosum relies on immunological, molecular, and proteomic assays. The main treatment of pythiosis aims to surgically remove all affected tissue to prevent recurrent infection. Due to the marked increase in case reports, pythiosis has become a public health concern. Thailand is an endemic area of human pythiosis. To obtain a complete picture of how the pathogen circulates in the environment, we surveyed the presence of P. insidiosum in urban (Bangkok) and rural areas of Thailand. We employed the hair-baiting technique to screen for P. insidiosum in 500 water samples. Twenty-seven culture-positive samples were identified as P. insidiosum by multiplex PCR, multi-DNA barcode (rDNA, cox1, cox2), and mass spectrometric analyses. These environmental strains of P. insidiosum fell into Clade-II and -III genotypes and exhibited a close phylogenetic/proteomic relationship with Thai clinical strains. Biodiversity of the environmental strains also existed in a local habitat. In conclusion, P. insidiosum is widespread in Thailand. A better understanding of the ecological niche of P. insidiosum could lead to the effective prevention and control of this pathogen.

Pythiosis, a life-threatening disease caused by Pythium insidiosum, has been increasingly diagnosed worldwide. A recently developed immunochromatographic test (ICT) enables the rapid diagnosis of pythiosis. During the 3-year clinical implementation of ICT in Thailand, we collected the laboratory reports of 38 animals with suspected pythiosis and detected ICT false-positive results in three horses and a dog with basidiobolomycosis. P. insidiosum and Basidiobolus ranarum cause infections with indistinguishable clinical and microscopic features. This study investigated cross-reactive antibodies by probing P. insidiosum and B. ranarum crude extracts and cell-free synthesized I06 protein (encoded in P. insidiosum genome, not other fungi) against a panel of pythiosis, basidiobolomycosis, rabbit anti-I06 peptide, and control sera by Western blot analyses. ICT false-positive results occurred from the cross-reactivity of anti-B. ranarum antibodies to the 15, 50, 60, and 120 kDa proteins of P. insidiosum, not double infections caused by both pathogens. Notably, ICT could help to screen pythiosis, and the positive test requires confirmation by culture or molecular method. The detection specificity of ICT requires improvement. The crude extract containing multispecies antigens needs replacement with a refined P. insidiosum-specific protein. We proposed that the 55 kDa I06 protein is an excellent candidate for developing a more specific serodiagnostic test for pythiosis.

Pythium insidiosum causes pythiosis, a fatal infectious disease of humans and animals worldwide. Prompt diagnosis and treatment are essential to improve the clinical outcome of pythiosis. Diagnosis of P. insidiosum relies on immunological, molecular, and proteomic assays. The main treatment of pythiosis aims to surgically remove all affected tissue to prevent recurrent infection. Due to the marked increase in case reports, pythiosis has become a public health concern. Thailand is an endemic area of human pythiosis. To obtain a complete picture of how the pathogen circulates in the environment, we surveyed the presence of P. insidiosum in urban (Bangkok) and rural areas of Thailand. We employed the hair-baiting technique to screen for P. insidiosum in 500 water samples. Twenty-seven culture-positive samples were identified as P. insidiosum by multiplex PCR, multi-DNA barcode (rDNA, cox1, cox2), and mass spectrometric analyses. These environmental strains of P. insidiosum fell into clade-I and -II genotypes and exhibited a close phylogenetic/proteomic relationship with Thai clinical strains. Biodiversity of the environmental strains also existed in a local habitat. In conclusion, P. insidiosum is widespread in Thailand. A better understanding of the ecological niche of P. insidiosum could lead to the effective prevention and control of this pathogen.

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.

CRISPR-Cas genome editing is a powerful tool for understanding the pathogenicity of oomycetes, a group that includes several destructive plant parasites. Although a few Phytophthora species have benefited from plasmid-based transformation methods for gene overexpression and RNA interference silencing, these techniques remain inefficient for other oomycete genera such as Pythium and Aphanomyces. Here, we explored the applicability of DNA-free endogenous counterselection in filamentous oomycetes, using CRISPR-Cas9 ribonucleoproteins (RNPs). We used biolistics to deliver RNPs targeting the adenine phosphoribosyltransferase ( APT) gene and generated selectable 2-fluoroadenine-resistant mutants in Aphanomyces, Pythium, and Phytophthora species. Targeted mutagenesis resulted in various deletions at the expected cut sites, confirming efficient genome editing. Knockout mutants exhibited no alterations in growth or virulence, making APT a suitable selectable marker gene for oomycete research. Whole-genome comparative analyses on CRISPR-edited mutants revealed no or very few additional mutations in A. euteiches and Pythium oligandrum and substantial off-target effects in Phytophthora capsici. This one-step approach circumvents the need for protoplast generation and can be broadly applied to oomycetes producing zoospores or oospores. [ ] Copyright © 2025 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

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.