The Repurposed Drug Disulfiram Inhibits Urease and Aldehyde Dehydrogenase and Prevents In Vitro Growth of the Oomycete Pythium insidiosum.

Pythium insidiosum is an oomycete microorganism that causes a life-threatening infectious disease, called pythiosis, in humans and animals. The disease has been increasingly reported worldwide. Conventional antifungal drugs are ineffective against P. insidiosum Treatment of pythiosis requires the extensive removal of infected tissue (i.e., eye and leg), but inadequate surgery and recurrent infection often occur. A more effective treatment is needed for pythiosis patients. Drug repurposing is a promising strategy for the identification of a U.S. Food and Drug Administration-approved drug for the control of P. insidiosum Disulfiram has been approved to treat alcoholism, but it exhibits antimicrobial activity against various pathogens. In this study, we explored whether disulfiram possesses an anti-P. insidiosum activity. A total of 27 P. insidiosum strains, isolated from various hosts and geographic areas, were susceptible to disulfiram in a dose-dependent manner. The MIC range of disulfiram against P. insidiosum (8 to 32 mg/liter) was in line with that of other pathogens. Proteogenomic analysis indicated that several potential targets of disulfiram (i.e., aldehyde dehydrogenase and urease) were present in P. insidiosum By homology modeling and molecular docking, disulfiram can bind the putative aldehyde dehydrogenase and urease of P. insidiosum at low energies (i.e., -6.1 and -4.0 Kcal/mol, respectively). Disulfiram diminished the biochemical activities of these enzymes. In conclusion, disulfiram can inhibit the growth of many pathogenic microorganisms, including P. insidiosum The drug can bind and inactivate multiple proteins of P. insidiosum, which may contribute to its broad antimicrobial property. Drug repurposing of disulfiram could be a new treatment option for pythiosis.

Assessment of temperature-dependent proteomes of Pythium insidiosum by using the SWISS-PROT database.

Pythium insidiosum causes the life-threatening disease, called pythiosis. Information on microbial pathogenesis could lead to an effective method of infection control. This study aims at assessing temperature-dependent proteomes, and identifying putative virulence factors of P. insidiosum. Protein extracts from growths at 25°C and 37°C were analyzed by mass spectrometry and SWISS-PROT database. A total of 1052 proteins were identified. Upon exposure to increased temperature, 219 proteins were markedly expressed, eight of which were putative virulence factors of P. insidiosum. These temperature-dependent proteins should be further investigated for their roles in pathogenesis, and some of which could be potential therapeutic targets.

Seroprevalence of anti--Pythium insidiosum antibodies in the Thai population.

Pythiosis is a life-threatening disease of humans and other animals in tropical and subtropical countries. The causative agent is Pythium insidiosum. Diagnosis of pythiosis can be missed due to the lack of awareness in the medical community. Treatment of the disease is difficult and challenging. Most pythiosis patients end up losing an infected organ (i.e., eye or leg), and many die from uncontrolled infection. In 2006, the largest series of human cases of pythiosis (∼100) was reported from Thailand, highlighting the nationwide distribution of this high morbidity and mortality disease. The global distribution of P. insidiosum is demonstrated by its detection in several regions around the world. Epidemiological studies of exposure to the pathogen in the general population are lacking. Here we used a combination of two established diagnostic tools (i.e., ELISA and Western blot) to explore the seroprevalence of anti-P. insidiosum antibodies in 2641 individuals, aged ≥ 15 years, sampled from Thailand. Four individuals were identified with anti-P. insidiosum antibodies in their sera, thus providing a statistically-estimated prevalence of ∼7 in 10000 or ∼32000 in the entire Thai population. The detection of the anti-P. insidiosum antibodies in healthy people with no history of pythiosis suggests that subclinical infections can occur. Taking into account the seroprevalence of anti-P. insidiosum antibodies, the global distribution of the organism, the nationwide distribution of patients, and the high morbidity and mortality of the disease, awareness of pythiosis should be raised as a public health concern in Thailand and other countries.

Evolution of the Sterol Biosynthetic Pathway of Pythium insidiosum and Related Oomycetes Contributes to Antifungal Drug Resistance.

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.

Development of an Anti-Elicitin Antibody-Based Immunohistochemical Assay for Diagnosis of Pythiosis.

Pythiosis is an emerging and life-threatening infectious disease of humans and animals living in tropical and subtropical countries and is caused by the fungus-like organism Pythium insidiosum. Antifungals are ineffective against this pathogen. Most patients undergo surgical removal of the infected organ, and many die from advanced infections. Early and accurate diagnosis leads to prompt management and promotes better prognosis for affected patients. Immunohistochemical assays (IHCs) have been developed using rabbit antibodies raised against P. insidiosum crude extract, i.e., culture filtrate antigen (CFA), for the histodiagnosis of pythiosis, but cross-reactivity with pathogenic fungi compromises the diagnostic performance of the IHC. Therefore, there is a need to improve detection specificity. Recently, the elicitin protein, ELI025, was identified in P. insidiosum, but it was not identified in other human pathogens, including true fungi. The ELI025-encoding gene was successfully cloned and expressed as a recombinant protein in Escherichia coli. This study aims to develop a new IHC using the rabbit anti-ELI025 antibody (anti-ELI) and to compare its performance with the previously reported anti-CFA-based IHC. Thirty-eight P. insidiosum histological sections stained positive by anti-ELI-based and anti-CFA-based IHCs indicating 100% detection sensitivity for the two assays. The anti-ELI antibody stained negative for all 49 negative-control sections indicating 100% detection specificity. In contrast, the anti-CFA antibody stained positive for one of the 49 negative controls (a slide prepared from Fusarium-infected tissue) indicating 98% detection specificity. In conclusion, the anti-ELI based IHC is sensitive and specific for the histodiagnosis of pythiosis and is an improvement over the anti-CFA-based assay.

Protein A/G-based immunochromatographic test for serodiagnosis of pythiosis in human and animal subjects from Asia and Americas.

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.

Evaluation of nested pcr technique for detection of Pythium insidiosum in pathological specimens from patients with suspected fungal keratitis.

Diagnosis of Pythium keratitis is problematic due to the difficulty in obtaining a culture report resulting in unnecessarily prolonged usage of antimicrobial medication due to misdiagnosis. This study evaluated and compared nested PCR technique with culture and immunoperoxidase staining assays of Pythium insidiosum in paraffin-embedded corneal tissues from patients with suspected fungal keratitis. Six of 51 pathological reports compatible with fungal infection and 6 of 48 culture-proven fungal keratitis were identified as Pythium. Twenty-seven specimens were PCR-positive for Pythium insidiosum. In comparison with fungal culture for P. insidiosum, PCR had 83% sensitivity and 77% specificity with fair agreement (Kappa score of 0.227, p = 0.001). The mean age of PCR-positive is younger than PCR-negative group and there is a female preponderance in Pythium-infected group (p = 0.002 and p = 0.004, respectively). Nineteen specimens had positive results using immunoperoxidase staining assay with fair agreement to culture method (Kappa 0.340, p < 0.001), and 83% sensitivity, 85% specificity and 85% accuracy (95% CI: 76.7-90.7). PCR-based technique compared with culture and/or immunoperoxidase staining assay had 91.7% sensitivity, 81.8% specificity and 83% accuracy (95% CI: 74.5-89.1) with moderate agreement (Kappa 0.477, p < 0.001). Thus nested PCR detection of P. insidiosum should be employed in preliminary diagnosis of Pythium keratitis in order to initiate proper management.

Efficiency comparison of three methods for extracting genomic DNA of the pathogenic oomycete Pythium insidiosum.

BACKGROUND: The fungus-like organism Pythium insidiosum is the causative agent of a life-threatening tropical infectious disease, pythiosis, which has high rates of morbidity and mortality. A lack of reliable diagnostic tools and effective treatments for pythiosis presents a major challenge to healthcare professionals. Unfortunately, surgical removal of infected organs remains the default treatment for pythiosis. P. insidiosum is an understudied organism. In-depth study of the pathogen at the molecular level could lead to better means of infection control High quality genomic DNA (gDNA) is needed for molecular biology-based research and application development, such as: PCR-assisted diagnosis, population studies, phylogenetic analysis, and molecular genetics assays. OBJECTIVE: To evaluate quality and quantity of the P. insidiosum gDNA extracted by three separate protocols intended for fungal gDNA preparation. MATERIAL AND METHOD: Seven P. insidiosum isolates were subjected to gDNA extraction by using conventional-extraction, rapid-extraction, and salt-extraction protocols. RESULTS: The conventional protocol offered the best gDNA in terms of quality and quantity, and could be scaled up. The rapid-extraction protocol had a short turnaround time, but the quality and quantity of the gDNA obtained were limited. The salt-extraction protocol was simple, rapid, and efficient, making it appealing for high throughput preparation of small-scale gDNA samples. CONCLUSION: Compared to rapid-extraction protocol, both conventional-extraction and salt-extraction protocols provided a better quality and quantity of gDNA, suitable for molecular studies of P. insidiosum. In contrast to the other two methods, the salt-extraction protocol does not require the use of hazardous and expensive materials such as phenol, chloroform, or liquid nitrogen.

Pins Gene Table v2.0: An Online Genome Database of 37 Pythium insidiosum Strains for Gene Content Exploration and Phylogenomic Analysis.

Unlike most pathogenic oomycetes, Pythium insidiosum infects humans and animals instead of plants. P. insidiosum has three clinically relevant genotypes/clades that cause a severe disease called pythiosis. To develop strategies for infection control, it is necessary to understand the biology and pathogenesis of this pathogen. Investigating the evolutionary mechanisms behind the host-specific adaptation is vital, and comparative genomic analysis can help with this. To facilitate genomic analysis, an online bioinformatics tool called P. insidiosum (Pins) Gene Table v2.0 was developed. This tool includes genomic data from 37 genetically diverse P. insidiosum strains and four related species. The database contains 732,686 genes, grouped into 80,061 unique clusters and further divided into core and variable categories at genus, species, and genotype levels. A high-resolution phylogenomic relationship among P. insidiosum strains and other oomycetes was projected through hierarchical clustering and core gene analyses. 3156 P. insidiosum-specific genes were shared among all genotypes and may be responsible for causing disease in humans and animals. After comparing these species-specific genes to the MvirDB database, 112 had significant matches with 66 known virulence proteins, some of which might be involved in vascular occlusion, which is a pathological feature of pythiosis. The correlation of genotypes, geographic origins, and affected hosts of P. insidiosum suggests that clade-I strains are more specific to animals, while clade-II/III strains are more specific to humans. The clade-specific genes might link to host preference. In summary, Pins Gene Table v2.0 is a comprehensive genome database accessible to users with minimal bioinformatics experience for the analysis of P. insidiosum genomes.

Potential anti-Pythium insidiosum therapeutics identified through screening of agricultural fungicides.

Pythiosis is a life-threatening infectious disease caused by the oomycete Pythium insidiosum. Clinical manifestations of pythiosis include an eye, blood vessel, skin, or gastrointestinal tract infection. Pythiosis has been increasingly reported worldwide, with an overall mortality rate of 28%. Radical surgery is required to save patients' lives due to the limited efficacy of antimicrobial drugs. Effective medical treatments are urgently needed for pythiosis. This study aims to find anti-P. insidiosum agents by screening 17 agricultural fungicides that inhibit plant-pathogenic oomycetes and validating their efficacy and safety. Cyazofamid outperformed other fungicides as it can potently inhibit genetically diverse P. insidiosum isolates while exhibiting minimal cellular toxicities. The calculated therapeutic scores determined that the concentration of cyazofamid causing significant cellular toxicities was eight times greater than the concentration of the drug effectively inhibiting P. insidiosum. Furthermore, other studies showed that cyazofamid exhibits low-to-moderate toxicities in animals. The mechanism of cyazofamid action is likely the inhibition of cytochrome b, an essential component in ATP synthesis. Molecular docking and dynamic analyses depicted a stable binding of cyazofamid to the Qi site of the P. insidiosum's cytochrome b orthologous protein. In conclusion, our search for an effective anti-P. insidiosum drug indicated that cyazofamid is a promising candidate for treating pythiosis. With its high efficacy and low toxicity, cyazofamid is a potential chemical for treating pythiosis, reducing the need for radical surgeries, and improving recovery rates. Our findings could pave the way for the development of new and effective treatments for pythiosis.IMPORTANCEPythiosis is a severe infection caused by Pythium insidiosum. The disease is prevalent in tropical/subtropical regions. This infectious condition is challenging to treat with antifungal drugs and often requires surgical removal of the infected tissue. Pythiosis can be fatal if not treated promptly. There is a need for a new treatment that effectively inhibits P. insidiosum. This study screened 17 agricultural fungicides that target plant-pathogenic oomycetes and found that cyazofamid was the most potent in inhibiting P. insidiosum. Cyazofamid showed low toxicity to mammalian cells and high affinity to the P. insidiosum's cytochrome b, which is involved in energy production. Cyazofamid could be a promising candidate for the treatment of pythiosis, as it could reduce the need for surgery and improve the survival rate of patients. This study provides valuable insights into the biology and drug susceptibility of P. insidiosum and opens new avenues for developing effective therapies for pythiosis.

A peptide ELISA to detect antibodies against Pythium insidiosum based on predicted antigenic determinants of exo-1,3-beta-glucanase.

Human pythiosis is a life-threatening infectious disease caused by the oomycete Pythium insidiosum. Diagnosis of pythiosis relies on culture identification, serodiagnosis, and molecular-based assay. Preparation of a serodiagnostic test requires culture filtrate antigen (CFA) extracted from the live pathogen. A 74-kDa immunoreactive protein of P. insidiosum, is encoded by the exo-1,3-beta-glucanase gene (PinsEXO1). PinsEXO1 protein is recognized by sera from pythiosis patients but not by sera from uninfected patients; therefore, this protein could be used to detect anti-P. insidiosum antibodies. In this study we aimed to: identify, synthesize, and evaluate an antigenic determinant (epitope) of PinsEXO1 to be used to serodiagnose pythiosis based on peptide ELISA, and to compare the diagnostic performance of that test with the current CFA-based ELISA. Two antigenic determinants of PinsEXO1 (Peptide-A and -B) were predicted using the PREDITOP program. The sera from 34 pythiosis patients and 92 control subjects were evaluated. Peptide-A, Peptide-B, and CFA-based ELISAs all had a specificity of 100%. Peptide-B ELISA had a sensitivity of 91% and an accuracy of 98% and both Peptide-A and CFA-based ELISAs had a sensitivity of 100% and an accuracy of 100%. Peptide-A is a more efficient epitope than Peptide-B, and can be used as an alternative antigen to develop a serodiagnostic assay for pythiosis.

Comparative Genomic Analysis Reveals Gene Content Diversity, Phylogenomic Contour, Putative Virulence Determinants, and Potential Diagnostic Markers within Pythium insidiosum Traits.

Pythium insidiosum has successfully evolved into a human/animal filamentous pathogen, causing pythiosis, a life-threatening disease, worldwide. The specific rDNA-based genotype of P. insidiosum (clade I, II, or III) is associated with the different hosts and disease prevalence. Genome evolution of P. insidiosum can be driven by point mutations, pass vertically to the offspring, and diverge into distinct lineages, leading to different virulence, including the ability to be unrecognized by the host. We conducted comprehensive genomic comparisons of 10 P. insidiosum strains and 5 related Pythium species using our online "Gene Table" software to investigate the pathogen's evolutionary history and pathogenicity. In total, 245,378 genes were found in all 15 genomes and grouped into 45,801 homologous gene clusters. Gene contents among P. insidiosum strains varied by as much as 23%. Our results showed a strong agreement between the phylogenetic analysis of 166 core genes (88,017 bp) identified across all genomes and the hierarchical clustering analysis of gene presence/absence profiles, suggesting divergence of P. insidiosum into two groups, clade I/II and clade III strains, and the subsequent segregation of clade I and clade II. A stringent gene content comparison using the Pythium Gene Table provided 3263 core genes exclusively presented in all P. insidiosum strains but no other Pythium species, which could involve host-specific pathogenesis and serve as biomarkers for diagnostic purposes. More studies focusing on characterizing the biological function of the core genes (including the just-identified putative virulence genes encoding hemagglutinin/adhesin and reticulocyte-binding protein) are needed to explore the biology and pathogenicity of this pathogen.

MGI short-read genome assemblies of Pythium insidiosum (reclassified as Pythium periculosum) strains Pi057C3 and Pi050C3.

OBJECTIVES: Pythium insidiosum causes a difficult-to-treat infectious condition called pythiosis, with high morbidity and mortality. So far, genome data of at least 10 strains of P. insidiosum, primarily classified in the phylogenetic clades I and II, have been sequenced using various next-generation sequencing platforms. The MGI short-read platform was employed to obtain genome data of 2 clade-III strains of P. insidiosum (recently reclassified as Pythium periculosum) from patients in Thailand and the United States. This work is a part of our attempt to generate a comprehensive genome database from diverse pathogen strains. DATA DESCRIPTION: A 150-bp paired-end library was prepared from a gDNA sample of P. insidiosum (P. periculosum) strains Pi057C3 and Pi050C3 (also known as ATCC90586) to generate draft genome sequences using an MGISEQ-2000RS sequencer. As a result, for the strain Pi057C3, we obtained a 42.5-Mb assembled genome (164x coverage) comprising 14,134 contigs, L50 of 241, N50 of 45,748, 57.6% CG content, and 12,147 ORFs. For the strain Pi050C3, we received a 43.3-Mb draft genome (230x coverage) containing 14,511 contigs, L50 of 245, N50 of 45,208, 57.7% CG content, and 12,249 ORFs. The genome sequences have been deposited in the NCBI/DDBJ databases under the accession numbers JAKCXM000000000.1 (strain Pi057C3) and JAKCXL000000000.1 (strain Pi050C3).

PacBio long read-assembled draft genome of Pythium insidiosum strain Pi-S isolated from a Thai patient with pythiosis.

OBJECTIVES: Pythium insidiosum is the causative agent of pythiosis, a difficult-to-treat condition, in humans and animals worldwide. Biological information about this filamentous microorganism is sparse. Genomes of several P. insidiosum strains were sequenced using the Illumina short-read NGS platform, producing incomplete genome sequence data. PacBio long-read platform was employed to obtain a better-quality genome of Pythium insidiosum. The obtained genome data could promote basic research on the pathogen's biology and pathogenicity. DATA DESCRIPTION: gDNA sample was extracted from the P. insidiosum strain Pi-S for whole-genome sequencing by PacBio long-read NGS platform. Raw reads were assembled using CANU (v2.1), polished using ARROW (SMRT link version 5.0.1), aligned with the original raw PacBio reads using pbmm2 (v1.2.1), consensus sequence checked using ARROW, and gene predicted using Funannotate pipeline (v1.7.4). The genome completion was assessed using BUSCO (v4.0.2). As a result, 840 contigs (maximum length: 1.3 Mb; N50: 229.9 Kb; L50: 70) were obtained. Sequence assembly showed a genome size of 66.7 Mb (178x coverage; 57.2% G-C content) that contained 20,375 ORFs. A BUSCO-based assessment revealed 85.5% genome completion. All assembled contig sequences have been deposited in the NCBI database under the accession numbers BBXB02000001 - BBXB02000840.

Generation of protoplasts provides a powerful experimental research tool for biological and pathogenicity studies of Pythium insidiosum.

INTRODUCTION: Pythiosis is a high-mortality infectious condition in humans and animals. The etiologic agent is Pythium insidiosum. Patients present with an ocular, vascular, cutaneous/subcutaneous, or gastrointestinal infection. Antifungal medication often fails to fight against P. insidiosum. The effective treatment is limited to radical surgery, resulting in organ loss. Fatal outcomes are observed in advanced cases. Pythiosis needs to be studied to discover novel methods for disease control. Genome data of P. insidiosum is publicly available. However, information on P. insidiosum biology and pathogenicity is still limited due to the lack of a cost-effective animal model and molecular tools. MATERIALS AND METHODS: We aimed to develop a high-efficiency protocol for generating P. insidiosum protoplast, and used it to set up an animal model, in vitro drug susceptibility assay, and DNA transformation for this pathogen. RESULTS: P. insidiosum protoplast was successfully generated to establish a feasible pythiosis model in embryonic chicken eggs and an efficient in vitro drug susceptibility assay. DNA transformation is a critical method for gene manipulation necessary for functional genetic studies in pathogens. Attempts to establish a DNA transformation method for P. insidiosum using protoplast were partly successful. Significant work needs to be done for genetically engineering a more robust selection marker to generate stable transformants at increased efficiency. CONCLUSION: This study is the first to report an efficient P. insidiosum protoplast production for clinical and research applications. Such advances are crucial to speeding up the pathogen's biology and pathogenicity exploration.

In vitro antimicrobial activity of volatile organic compounds from Muscodor crispans against the pathogenic oomycete Pythium insidiosum.

Pythium insidiosum is an oomycete capable of causing a life-threatening disease in humans, called pythiosis. Conventional antifungal drugs are ineffective against P. insidiosum infection. A synthetic mixture of the volatile organic compounds (VOCs) from the endophytic fungus Muscodor crispans strain B23 demonstrates antimicrobial effects against a broad range of human and plant pathogens, including fungi, bacteria, and oomycetes. We studied the in vitro effects of B23 VOCs against 25 human, 1 animal, and 4 environmental isolates of P. insidiosum, compared with a no-drug control. The B23 synthetic mixture, at amounts as low as 2.5 microl, significantly reduced growth of all P. insidiosum isolates by at least 80%. The inhibitory effect of the B23 VOCs was dose-dependent. The growth of all isolates was completely inhibited by a dose of 10.0 microl of B23 VOCs, and all isolates were killed by a dose of 20.0 microl. Synthetic B23 VOCs of M. crispans had inhibitory and lethal effects against all P. insidiosum isolates tested. Further studies are needed to evaluate this mixture for treatment of pythiosis.

Global Distribution and Clinical Features of Pythiosis in Humans and Animals.

Pythiosis is a difficult-to-treat infectious disease caused by Pythium insidiosum. The condition is unfamiliar among healthcare workers. Manifestation of pythiosis is similar to other fungal infections, leading to misdiagnosis and delayed treatment. The geographical extent of pythiosis at a global scale is unclear. This study aimed to analyze the clinical information recorded in the scientific literature to comprehensively project epidemiological characteristics, clinical features, and future trends of pythiosis. From 1980 to 2021, 4203 cases of pythiosis in humans (n = 771; 18.3%) and animals (primarily horse, dog, and cow; n = 3432; 81.7%), with an average of 103 cases/year, were recruited. Pythiosis case reports significantly increased in the last decade. Pythiosis spanned 23 tropical, subtropical, and temperate countries worldwide. Some patients acquired pythiosis from a trip to an endemic country. Strikingly, 94.3% of human cases were in India and Thailand, while 79.2% of affected animals were in the U.S.A. and Brazil. Clinical features of pythiosis varied. Vascular and ocular pythiosis were only observed in humans, whereas cutaneous/subcutaneous and gastrointestinal infections were predominant in animals. Mortality depended on host species and clinical forms: for example, none in patients with ocular pythiosis, 0.7% in cows with a cutaneous lesion, 26.8% in humans with vascular disease, 86.4% in dogs with gastrointestinal pathology, and 100% in several animals with disseminated infection. In summary, this study reports up-to-date epidemiological and clinical features of pythiosis in humans and animals. It increases awareness of this life-threatening disease, as the illness or outbreak can exist in any country, not limited to the endemic areas.

Nucleic Acid-Based Detection of Pythium insidiosum: A Systematic Review.

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.

Secretome Profiling by Proteogenomic Analysis Shows Species-Specific, Temperature-Dependent, and Putative Virulence Proteins of Pythium insidiosum.

In contrast to most pathogenic oomycetes, which infect plants, Pythium insidiosum infects both humans and animals, causing a difficult-to-treat condition called pythiosis. Most patients undergo surgical removal of an affected organ, and advanced cases could be fetal. As a successful human/animal pathogen, P. insidiosum must tolerate body temperature and develop some strategies to survive and cause pathology within hosts. One of the general pathogen strategies is virulence factor secretion. Here, we used proteogenomic analysis to profile and validate the secretome of P. insidiosum, in which its genome contains 14,962 predicted proteins. Shotgun LC-MS/MS analysis of P. insidiosum proteins prepared from liquid cultures incubated at 25 and 37 °C mapped 2980 genome-predicted proteins, 9.4% of which had a predicted signal peptide. P. insidiosum might employ an alternative secretory pathway, as 90.6% of the validated secretory/extracellular proteins lacked the signal peptide. A comparison of 20 oomycete genomes showed 69 P. insidiosum-specific secretory/extracellular proteins, and these may be responsible for the host-specific infection. The differential expression analysis revealed 14 markedly upregulated proteins (particularly cyclophilin and elicitin) at body temperature which could contribute to pathogen fitness and thermotolerance. Our search through a microbial virulence database matched 518 secretory/extracellular proteins, such as urease and chaperones (including heat shock proteins), that might play roles in P. insidiosum virulence. In conclusion, the identification of the secretome promoted a better understanding of P. insidiosum biology and pathogenesis. Cyclophilin, elicitin, chaperone, and urease are top-listed secreted/extracellular proteins with putative pathogenicity properties. Such advances could lead to developing measures for the efficient detection and treatment of pythiosis.

Severe skin and soft tissue pythiosis acquired in a hot spring in the southwestern United States, a case report and review of North American cases.

BACKGROUND: Human pythiosis, caused primarily by the aquatic oomycete, Pythium insidiosum, is an emerging but uncommon infection in North America. The infection is frequently life-threatening and is often initially unrecognized due to its rarity and similar presentation to certain fungal infections. METHODS: We report a case of skin and soft tissue pythiosis in a patient without significant underlying comorbidities acquired in a New Mexico hot spring and review its successful treatment. We also review all reported pythiosis cases in North America. RESULTS: Eleven confirmed cases of human pythiosis acquired in North America were identified. The majority of cases occurred in children (64%), ten of eleven cases were acquired in the southern U.S., Mexico, Central America or the Caribbean and four of the eleven individuals succumbed to the infection. CONCLUSIONS: With recognition and aggressive surgical and medical treatment good clinical outcomes can be achieved when treating human pythiosis.