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Pythiosis is a life-threatening infectious disease caused by the oomycete Pythium insidiosum. Direct exposure to Py. insidiosum zoospores can initiate infections of the eye, limb, gastrointestinal tract, or skin/subcutaneous tissue. Treatments for pythiosis have mostly relied on surgery. Antifungal drugs are generally ineffective against Py. insidiosum. However, one patient with an invasive Py. insidiosum infection recovered completely following treatment with terbinafine and itraconazole. Additionally, the drug target sterol biosynthetic enzymes have been identified in the oomycete Aphanomyces euteiches. It remains an open question whether Py. insidiosum is susceptible to the antifungal drugs and harbors any of the known drug target enzymes. Here, we determined the in vitro susceptibilities of terbinafine and itraconazole against 30 isolates of Py. insidiosum. We also analyzed endogenous sterols and searched for genes encoding the sterol biosynthetic enzymes in the genomes of Py. insidiosum and related oomycetes. The susceptibility assay showed that the growth of each of the Py. insidiosum isolates was inhibited by the antifungal agents, but only at difficult-to-achieve concentrations, which explains the clinical resistance of the drugs in the treatment of pythiosis patients. Genome searches of Py. insidiosum and related oomycetes demonstrated that these organisms contained an incomplete set of sterol biosynthetic enzymes. Gas chromatographic mass spectrometry did not detect any sterol end products in Py. insidiosum. In conclusion, Py. insidiosum possesses an incomplete sterol biosynthetic pathway. Resistance to antifungal drugs targeting enzymes in the ergosterol biosynthetic pathway in Py. insidiosum was due to modifications or losses of some of the genes encoding the drug target enzymes.

Pythium insidiosum is a human-pathogenic oomycete. Many patients infected with it lose organs or die. Toward the goal of developing improved treatment options, we want to understand how Py. insidiosum has evolved to become a successful human pathogen. Our approach here involved the use of comparative genomic and other analyses to identify genes with possible functions in the pathogenicity of Py. insidiosum. We generated an Oomycete Gene Table and used it to explore the genome contents and phylogenomic relationships of Py. insidiosum and 19 other oomycetes. Initial sequence analyses showed that Py. insidiosum is closely related to Pythium species that are not pathogenic to humans. Our analyses also indicated that the organism harbours secreted and adhesin-like proteins, which are absent from related species. Putative virulence proteins were identified by comparison to a set of known virulence genes. Among them is the urease Ure1, which is absent from humans and thus a potential diagnostic and therapeutic target. We used mass spectrometric data to successfully validate the expression of 30% of 14,962 predicted proteins and identify 15 body temperature (37 °C)-dependent proteins of Py. insidiosum. This work begins to unravel the determinants of pathogenicity of Py. insidiosum.

Pythium insidiosum is an oomycete that causes a life-threatening infectious disease called pythiosis in humans and animals living in tropical and subtropical countries. Here, we report the first draft genome sequence of P. insidiosum. The genome of P. insidiosum is 53.2 Mb and contains 14,962 open reading frames.

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Pythiosis is a life-threatening infectious disease of both humans and animals living in Asia, Americas, Africa, and parts of Australia and New Zealand. The etiologic pathogen is the fungus-like organism Pythium insidiosum The disease has high mortality and morbidity rates. Use of antifungal drugs are ineffective against P. insidiosum, leaving radical surgery the main treatment option. Prompt treatment leads to better prognosis of affected individuals, and could be achieved by early and accurate diagnosis. Since pythiosis has been increasingly reported worldwide, there is a need for a rapid, user-friendly, and efficient test that facilitates the diagnosis of the disease. This study aims to develop an immunochromatographic test (ICT), using the bacterial protein A/G, to detect anti-P. insidiosum IgGs in humans and animals, and compare its diagnostic performance with the established ELISA. Eighty-five serum samples from 28 patients, 24 dogs, 12 horses, 12 rabbits, and 9 cattle with pythiosis, and 143 serum samples from 80 human and 63 animal subjects in a healthy condition, with thalassemia, or with other fungal infections, were recruited for assay evaluation. Detection specificities of ELISA and ICT were 100.0%. While the detection sensitivity of ELISA was 98.8%, that of ICT was 90.6%. Most pythiosis sera, that were falsely read negative by ICT, were weakly positive by ELISA. In conclusion, a protein A/G-based ICT is a rapid, user-friendly, and efficient assay for serodiagnosis of pythiosis in humans and animals. Compared to ELISA, ICT has an equivalent detection specificity and a slightly lower detection sensitivity.

MALDI-TOF MS has been successfully used for identification and biotyping of P. insidiosum. The obtained mass spectral database allows clinical microbiology laboratories, well-equipped with a MALDI-TOF mass spectrometer, to conveniently identify P. insidiosum, without requiring any pathogen-specific reagents (i.e., antigen, antibody or primers).

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