Search

Pythiosis is a severe disease in humans and animals globally, caused by the pathogenic oomycete Pythium insidiosum. Early and accurate detection is crucial for effective treatment, but traditional diagnostic methods have limitations. This study presents an alternative approach using Matrix-Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) for lipid profiling to efficiently identify P. insidiosum. The study involved extracting microbial lipid components using optimized chloroform: methanol biphasic method and creating a lipid profile database with samples from 30 P. insidiosum isolates and 50 various fungi. The methodology was validated on 25 blinded samples for assay detection performance. Unique lipid profiles allowed species-specific identification with high efficiency: scores ≥ 2.682 indicated P. insidiosum, scores ≤ 2.512 suggested fungi, and scores in between pointed to other oomycetes. The assay demonstrated sensitivity, specificity, and accuracy of 100%, 80%, and 88%, respectively, for detecting P. insidiosum. The limited detection specificity was due to false positive samples from closely related Pythium species, which are not a significant clinical concern. The findings show that MALDI-TOF MS lipid profiling can efficiently identify P. insidiosum, offering significant advantages in sample preparation, stability, and reproducibility over protein profile-based methods. This study marks the first instance of lipid profiles being reported for P. insidiosum, paving the way for clinical use in improving accurate detection and facilitating timely treatment interventions. [ABSTRACT FROM AUTHOR]

Pythium insidiosum is an important oomycete pathogen of mammals that causes pythiosis, an endemic disease in warm climates that stands out for its unfavorable prognosis, lethality in the affected species, and difficulties in treatment. This study evaluated in vitro anti-P. insidiosum potential of aqueous, hydroethanolic, and ethanolic extracts of indigenous wild mushrooms from southern Brazil. The extracts were prepared from Amanita gemmata, Amanita muscaria, Auricularia auricula, Gymnopilus junonius, Lactarius deliciosus, Laccaria laccata, Psilocybe cubensis, and Russula xerampelina. In vitro susceptibility assays employed the microdilution technique according to the M38-A2 protocol CLSI. The hydroethanolic and ethanolic extracts of R. xerampelina showed anti-P. insidiosum activity at minimum inhibitory concentrations ranging from 1.87--7.50 mg/mL. The other mushroom species extracts showed no inhibitory effects on growth of P. insidiosum. This is the first study to evaluate the antimicrobial activity of mushrooms on oomycetes, evidencing the antimicrobial potential of R. xerampelina on the pathogen P. insidiosum. So, the present study expands new perspectives, since the secondary metabolites produced by mushrooms can be potential targets for the development of new categories of medicines. However, considering the wide biodiversity of Brazilian mushrooms, we suggested that the search for other basidiomycetes species with anti-P. insidiosum action needs to be expanded. [ABSTRACT FROM AUTHOR]

Background: Pythium insidiosum (P. insidiosum) is the causative agent of pythiosis, an infectious disease with a high morbidity and fatality rate. Pythiosis cases have increased dramatically during the past ten years, particularly in tropical and subtropical areas. Sadly, microbiologists and medical professionals know very little about pythiosis, and the disease is frequently challenging to identify. It is frequently misdiagnosed as a fungal infection. Methods: We report two cases of pythiosis, one was Pythium keratitis, the other was cutaneous pythiosis. The patient with corneal infection had no underlying disease, while the patient with cutaneous pythiosis had a history of liver cirrhosis, diabetes, and psoriasis. The corneal sample and subcutaneous pus were sent for metagenomic Next-Generation Sequencing (mNGS). To further diagnose the isolated strain, P. insidiosum zoospores were induced to produce by coincubation with sterile grass leaves in sterile pond water. Their zoospores were used as an inoculum for drug susceptibility testing by disk diffusion and broth microdilution method. Results: The mNGS of two cases were reported as P. insidiosum. Zoospores were produced after incubation 48h. The zoospores were collected for drug susceptibility assay. All antifungal drugs, antibacterial drugs of β-Lactams, vancomycin, levofloxacin, ciprofloxacin, gentamicin, trimethoprim-sulfamethoxazole, clindamycin have no inhibitory activity against P. insidiosum in vitro. Minocycline, tigecycline, linezolid, erythromycin and azithromycin have significant in vitro activity against P. insidiosum. Based on the susceptibility results, the drug was changed from itraconazole to linezolid and minocycline, along with multiple debridements and drainage for cutaneous pythiosis. The patient was discharged after 24 days of treatment. Conclusions: Early and accurate identification, combined with aggressive surgical debridement and appropriate drug therapy, can greatly improve patient managements. Conventional culture and zoospore induction remain gold standard for diagnosis; however, DNA-based method should be performed simultaneously. The drug susceptibility testing provides profound effects on proper drug selection against P. insidiosum. [ABSTRACT FROM AUTHOR]

ABSTRACT: This study evaluated the inhibitory effect of ozone gas (O3) on the mycelia and zoospores of Pythium insidiosum and verified the morphological changes it caused on the hyphae. The effects of O3 were evaluated on the mycelia (n = 21) and zoospores (n = 10) of P. insidiosum. O3 was bubbled into vials with sterile distilled water and mycelium or induction medium and zoospores. After 15 minutes, aliquots of mycelia and zoospores were grown on yeast agar at 37 °C/96 hours. Ozonated flasks were incubated at 37 ºC/72 hours; and every 24 hours, aliquots were subcultured at 37 ºC/96 hours. Transmission (TEM) and scanning (SEM) electron microscopy of O3-exposed hyphae were performed. The results revealed that O3 inhibited microorganism growth in all ozonated suspensions. Conversely, in control suspensions, P. insidiosum growth occurred within 24 hours of incubation. In the SEM, the treated hyphae presented cavitations and cell wall continuity loss; in the TEM, the organelles and cytoplasmic membrane disappeared, and the cell wall was retracted. We demonstrated, for the first time, that O3 causes irreversible damage on P. insidiosum hyphae and prove in vitro oomicidal action on zoospores and hyphae.

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. [ABSTRACT FROM AUTHOR]

ABSTRACT: Pythiosis is a life-threatening disease that affects various species and is prevalent in regions with high humidity. The ailment is increasingly reported globally, and in Brazil, it is an important cause of profound economic and health losses in equines. This study aimed to explore the potential of intradermal immunotherapy as both a diagnostic and therapeutic approach for pythiosis in horses from the Pantanal region of Mato Grosso, Brazil. The horses were divided into three groups: those never diagnosed with pythiosis or with lesions that could be mistaken for pythiosis (Group 1); previously infected but successfully treated horses (Group 2); and horses with a positive diagnosis for pythiosis that were under treatment (Group 3). An immunotherapeutic product (PitiumVac®) was administered intradermally. Injection site reaction and response to immunotherapy were assessed. The results indicated that intradermal immunotherapy could be a viable diagnostic and therapeutic tool, particularly in remote areas where traditional laboratory diagnosis methods are not readily accessible. The efficacy of intradermal administration was comparable to that of subcutaneous administration in treating pythiosis in horses, and the combined use of immunotherapy and triamcinolone acetonide yielded promising results for treating pythiosis in horses. However, further research is required to validate these findings. This study contributes to understanding and managing pythiosis more efficiently by providing a simple, cost-effective, and potentially efficient alternative approach to diagnosis and treatment.

Abstract 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).

Abstract 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; N 50: 229.9 Kb; L 50: 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.

ABSTRACTPythiosis 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.

Clinical relevance: Invasive fungal infections (IFIs) and oomycoses (hereafter termed invasive fungal-like infections [IFLIs]) are characterised by penetration of tissues by fungal elements. The environment is the most common reservoir of infection. IFIs and IFLIs can be frustrating to treat because long treatment times are usually required and, even after attaining clinical cure, there may be a risk of relapse. Owner compliance with medication administration and recheck examinations can also decline over time. In addition, some antifungal drugs are expensive, have variable interpatient pharmacokinetic properties, can only be administered parenterally and/or have common adverse effects (AEs). Despite these limitations, treatment can be very rewarding, especially when an otherwise progressive and fatal disease is cured. Aim: In the second of a two-part article series, the spectrum of activity, mechanisms of action, pharmacokinetic and pharmacodynamic properties, and AEs of antifungal drugs are reviewed, and the treatment and prognosis of specific IFIs/IFLIs - dermatophytic pseudomycetoma, cryptococcosis, sino-orbital aspergillosis, coccidioidomycosis, histoplasmosis, sporotrichosis, phaeohyphomycosis, mucormycosis and oomycosis - are discussed. Part 1 reviewed the diagnostic approach to IFIs and IFLIs. Evidence base: Information on antifungal drugs is drawn from pharmacokinetic studies in cats. Where such studies have not been performed, data from 'preclinical' animals (non-human studies) and human studies are reviewed. The review also draws on the wider published evidence and the authors' combined expertise in feline medicine, mycology, dermatology, clinical pathology and anatomical pathology. Abbreviations for antifungal drugs: AMB (amphotericin B); FC (flucytosine); FCZ (fluconazole); ISA (isavuconazole); ITZ (itraconazole); KCZ (ketoconazole); PCZ (posaconazole); TRB (terbinafine); VCZ (voriconazole). [ABSTRACT FROM AUTHOR]

Clinical relevance: In contrast to superficial fungal infections, such as dermatophytosis, invasive fungal infections (IFIs) are characterised by penetration of tissues by fungal elements. Disease can spread locally within a region or can disseminate haematogenously or via the lymphatics. The environment is the most common reservoir of infection. Since fungal spores are airborne, indoor cats are also susceptible to IFIs. Some environmental fungi are ubiquitous and present globally, while others are endemic or hyperendemic within specific geographic regions. Zoonotic pathogens include Microsporum canis, Sporothrix schenckii and Sporothrix brasiliensis. Aim: In the first of a two-part article series, the approach to the investigation of feline IFIs and oomycoses is reviewed. As well as tips for diagnosis, and information on the ecological niche and distribution of fungal pathogens, the review covers clinical presentation of the most common IFIs, including cryptococcosis, histoplasmosis, blastomycosis, coccidioidomycosis, sporotrichosis, phaeohyphomycosis, aspergillosis and dermatophytic pseudomycetoma, as well as the oomycoses pythiosis, lagenidiosis and paralagenidiosis. In Part 2, the spectrum of activity, mechanisms of action, pharmacokinetic and pharmacodynamic properties and adverse effects of antifungal drugs are reviewed, and the treatment and prognosis for specific IFIs and oomycoses are discussed. Evidence base: The review draws on published evidence and the authors' combined expertise in feline medicine, mycology, dermatology, clinical pathology and anatomical pathology. [ABSTRACT FROM AUTHOR]

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). [ABSTRACT FROM AUTHOR]

We report cutaneous pythiosis in 2 dogs in Italy that had recurrent exposure to the same freshwater habitat. Phylogenetic analysis placed the isolates within Pythium insidiosum complex cluster IV, corresponding to P. periculosum. In Italy, pythiosis should be considered in differential diagnoses by human and veterinary health professionals.

Pythiosis is characterized most commonly by ulcerative dermatitis, mainly in the limbs of sheep and occasionally of goats. In sheep, Pythium insidiosum is also responsible for necrotizing rhinitis characterized by marked enlargement and deformity of the nasal region, severe respiratory difficulty, and bloody nasal discharge. A third form of pythiosis in sheep affects the digestive tract, involving ulceration of the esophagus, forestomachs, and abomasum. Pythiosis in sheep and goats has been reported only in Brazil where it occurs mainly in the semiarid region of the country, when animals congregate and stay for longer periods of time within or around water reservoirs. However, it has been reported as well in areas of humid environments, such as the Pantanal of Mato Grosso and in the Brazilian Cerrado. The diagnosis of the different presentations of pythiosis is based on gross and microscopic findings, coupled with detection of the agent by immunohistochemical, molecular, and/or culture-based methods.

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. [ABSTRACT FROM AUTHOR]

The aquatic oomycete Pythium insidiosum is an emerging pathogen highly relevant in human and veterinary medicine and an etiologic agent of pythiosis, a disease of worldwide distribution mainly affecting horses, dogs, and humans, presenting cutaneous, subcutaneous, ocular, gastrointestinal, and systemic forms. The available therapeutic methods to treat this disease and its forms are not entirely effective, thus highlighting the need to investigate the forms of treatments with better efficacy, such as compounds from different pharmacological classes, compounds of natural origin, and new technological alternatives, including nanotechnology. Therefore, this study evaluated scientific publications regarding the use of nanotechnology in P. insidiosum treatment. For this, a systematic literature review, was carried out on articles published from 2010 to 2022 on the LILACS, MEDLINE, Google Scholar, PubMed, and SciELO databases using the descriptors ‘Pythium insidiosum,’ ‘pythiosis,’ ‘nanotechnology,’ ‘nanoparticles,’ ‘nanoemulsion,’ and ‘treatment.’ We reported 162 articles for the researched theme; although, only four studies were included because they met the criteria established herein. A meta-analysis was used for the statistical analysis of the data obtained in vitro studies, and we reported the use of nanotechnology can be a promising alternative in developing antimicrobial compounds with anti-P. insidiosum activity. Nevertheless, additional research is needed to verify the potential use of this technology in clinical therapy against P. insidiosum infections. [ABSTRACT FROM AUTHOR]

Pythium insidiosum, an aquatic oomycete with pathogenic potential in mammals, causes gastrointestinal and cutaneous disease in dogs. Mefenoxam, an agricultural anti-oomycotic compound, has a demonstrated the ability to inhibit P. insidiosum growth in vitro and has been associated with efficacy in treating gastrointestinal pythiosis in several case reports. Electronic medical records of dogs seen at University of Florida Small Animal Hospital and treated with mefenoxam between 2013 and 2020 were searched. Dogs were included in this study upon previous definitive diagnosis with either organism identification using culture, PCR, or antibody ELISA, or a combination of these tests with or without supportive histopathological analysis. Since 2013, mefenoxam had been administered to 25 dogs with cutaneous pythiosis and 16 dogs with gastrointestinal pythiosis. In both gastrointestinal and cutaneous pythiosis groups, the administration of mefenoxam was associated with a survivability rate of approximately 51%. There was a statistically significant difference in the time to death between cutaneous (245 days (52–530)) and gastrointestinal (90 days (21–203)) groups; dogs infected with cutaneous pythiosis survived significantly longer after being diagnosed with the disease (p = 0.035). The dogs in this study experienced increased survival rates and time to death, in the absence of side effects due to mefenoxam, compared with previously published literature. The results of this retrospective study, with some limitations, are promising and should prompt further investigation into the use of mefenoxam in the treatment of both gastrointestinal and cutaneous pythiosis. [ABSTRACT FROM AUTHOR]

A case of meningoencephalitis caused by Pythium insidiosum secondary to rhinitis is reported in a three-year-old crossbred sheep from a herd of 15 animals, raised extensively and with free access to a weir. The animal presented mild dyspnea, blindness, mydriasis, opisthotonos, nystagmus, incoordination, decreased mandibular tone, and spasticity of the pelvic limbs. Macroscopic examination of the nasal cavity showed a blackish-red, irregular, friable mass that bilaterally compromised the nasal septum and the rostral portion of the nasal turbinates. In the brain, there was diffuse thickening of the leptomeninges of the cerebellum and ventral portion of the brainstem characterized by yellowish, granular material associated with vessel hyperemia. On the floor of the fourth ventricle, there was deposition of yellowish, irregular, slightly granular material that protruded towards the obex and displaced the cerebellum dorsolaterally. Microscopically, there were pyogranulomatous, eosinophilic, necrotizing rhinitis and fibrinosuppurative, eosinophilic, necrotizing meningoencephalitis, both associated with thrombosis, vasculitis, and intralesional hyphae. The hyphae were impregnated with silver and presented thin, parallel walls, rarely septate and branched. At immunohistochemistry, the hyphae were immunostained with polyclonal anti-P. insidiosum antibody in fragments of the cerebellum and nasal cavity. The findings showed that P. insidiosum rhinitis can secondarily affect the nervous system of sheep, causing nonspecific neurological clinical signs. [ABSTRACT FROM AUTHOR]

Gastrointestinal (GI) pythiosis is a severe and often fatal disease in dogs that traditionally has been poorly responsive to medical treatment. Although aggressive surgical resection with wide margins is the most consistently effective treatment, lesion location and extent often preclude complete resection. Recently, it has been suggested that the addition of anti-inflammatory doses of corticosteroids may improve outcome in dogs with nonresectable GI pythiosis. This report describes 3 dogs with colonic pythiosis in which complete resolution of clinical signs, regression of colonic masses, and progressive decreases in serological titers were observed after treatment with itraconazole, terbinafine, and corticosteroids. This treatment protocol represents a promising treatment for dogs with GI pythiosis in which surgical intervention is not feasible.

Gastrointestinal (GI) pythiosis is a severe and often fatal disease in dogs that traditionally has been poorly responsive to medical treatment. Although aggressive surgical resection with wide margins is the most consistently effective treatment, lesion location and extent often preclude complete resection. Recently, it has been suggested that the addition of anti-inflammatory doses of corticosteroids may improve outcome in dogs with nonresectable GI pythiosis. This report describes 3 dogs with colonic pythiosis in which complete resolution of clinical signs, regression of colonic masses, and progressive decreases in serological titers were observed after treatment with itraconazole, terbinafine, and corticosteroids. This treatment protocol represents a promising treatment for dogs with GI pythiosis in which surgical intervention is not feasible.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Pythiosis is a serious disease caused by the aquatic oomycete Pythium insidiosum that mainly affects mammals. Unlike fungal and bacterial resistance induced by the indiscriminate use of drugs, P. insidiosum has low susceptibility to antifungal drugs. In this sense, essential oils and their major components emerge as a promising treatment line for this disease. Given the above, this study sought to verify P. insidiosum (n  = 34) susceptibility to the bioactive compounds eugenol, α-terpineol, menthol, and carvacrol and correlate them with the respective essential oils of Eugenia caryophyllata, Melaleuca alternifolia, Mentha piperita , and Origanum vulgare. The essential oils and bioactive compounds were purchased commercially and tested according to the Clinical and Laboratory Standards Institute protocol M38-A2. Our findings showed that eugenol, α-terpineol, and carvacrol had superior anti- P. insidiosum action than their respective essential oils, suggesting that they may be responsible for inhibitory activity against P. insidiosum. Notably, the major compound with the best anti- P. insidiosum activity was α-terpineol; nonetheless, menthol showed less activity than its essential oil. The results imply that essential oils and their major compounds may be important allies in treating pythiosis, expanding the perspectives of developing new drugs with anti- P. insidiosum activity. [ABSTRACT FROM AUTHOR]

This study evaluated in-vitro action of a new molecule, the polypyrrole nanoparticles (Ppy-NP), against Pythium insidiosum isolates using M38-A2/CLSI; the minimal inhibitory (MIC) and minimal oomicidal (MOC) concentrations were also determined. Additionally, changes in the hyphae wall of P. insidiosum CBS 575.85 treated with Ppy-NP were evaluated by scanning electron microscopy (SEM). The MIC100 and MOC for all isolates ranged from 8 to 32 μg mL−1, and the MIC90 and MIC50 were 16 μg mL−1. The SEM showed structural damage to the hyphae of P. insidisoum treated with Ppy-NP, as hyphae surfaces with less turgidity were found, thereby showing scaling and ruptures compared to the control (untreated hyphae). Our findings highlighted the anti- P. insidiosum properties of Ppy-NP proved to be a promising candidate for research using pythiosis experimental models. [ABSTRACT FROM AUTHOR]

The orphan but highly virulent pathogen Pythium insidiosum causes pythiosis in humans and animals. Surgery is a primary treatment aiming to cure but trading off losing affected organs. Antimicrobial drugs show limited efficacy in treating pythiosis. Alternative drugs effective against the pathogen are needed. In-house drug susceptibility tests (i.e., broth dilution, disc diffusion, and radial growth assays) have been established, some of which adapted the standard protocols (i.e., CLSI M38-A2 and CLSI M51) designed for fungi. Hyphal plug, hyphal suspension, and zoospore are inocula commonly used in the drug susceptibility assessment for P. insidiosum. A side-by-side comparison demonstrated that each method had advantages and limitations. Minimum inhibitory and cidal concentrations of a drug varied depending on the selected method. Material availability, user experience, and organism and drug quantities determined which susceptibility assay should be used. We employed the hyphal plug and a combination of broth dilution and radial growth methods to screen and validate the anti-P. insidiosum activities of several previously reported chemicals, including potassium iodide, triamcinolone acetonide, dimethyl sulfoxide, and ethanol, in which data on their anti-P. insidiosum efficacy are limited. We tested each chemical against 29 genetically diverse isolates of P. insidiosum. These chemicals possessed direct antimicrobial effects on the growth of the pathogen in a dose- and time-dependent manner, suggesting their potential application in pythiosis treatment. Future attempts should focus on standardizing these drug susceptibility methods, such as determining susceptibility/resistant breakpoints, so healthcare workers can confidently interpret a result and select an effective drug against P. insidiosum. [ABSTRACT FROM AUTHOR]

Pythiosis is a fatal disease which has high incidence in tropical regions. In contrast with vascular pythiosis, cutaneous and subcutaneous pythiosis are both uncommon. Here, we report a case of subcutaneous pythiosis in a pregnant farmer manifested with a progressively larger and more painful mass at right deltoid. The tissue culture and molecular test were negative for fungi. The diagnosis was supported by the positivity of serum immunochromatographic test (ICT) for pythiosis. Patients responded well to the combination therapy of itraconazole, terbinafine and azithromycin.

Pythium insidiosum is a rare fungus-like pathogen that is known to cause pythiosis in mammals with high morbidity and mortality. Identification of the pathogen is essential for timely treatment and rational use of antibiotics. However, Pythium insidiosum is difficult to detect via conventional microbiological tests. The current gold standard is polymerase chain reaction, which is lacking in most hospitals since human pythiosis is rare in China. In this study, we used metagenomic Next-Generation Sequencing and identified Pythium insidiosum in a 56-year-old Chinese male who was hospitalized due to severe edema in the right lower limb with scattered darkening indurations. The patient had a history of cirrhosis and occupational exposure to swamp water. Serological level of immune biomarkers indicated immunodeficiency, and Proteinase 3-Anti-Neutrophil Cytoplasmic Antibody was positive. Surgical incision of the lesions revealed radiating and reticular cutaneous ulcers. Microbial infections were suspected but conventional tests failed to discover the etiology. Empirical use of penicillin, vancomycin, and ceftriaxone had no effect. As a result, the peripheral blood and tissue biopsies were sent for metagenomic Next-Generation Sequencing, which reported Pythium insidiosum. This finding was corroborated by pathological staining, wholegenome sequencing, and internal transcribed spacer sequencing. Notably, antifungal treatment was ineffective, but the patient responded well to oral trimethoprim--sulfamethoxazole, which may be due to the folp gene found in Pythium insidiosum genome. Our study prompts future studies to determine the optimal treatment of skin pythiosis. [ABSTRACT FROM AUTHOR]

Abstract Objectives We employed the Illumina NGS platform to sequence genomes of 4 different strains of the pathogenic oomycete Pythium insidiosum, the causative agent of pythiosis. These strains were isolated from humans in Thailand (n = 3) and the United States (n = 1), and phylogenetically classified into clade-I, -II, and -III. Our study augmented the completeness of the P. insidiosum genome database for exploration of the biology, evolution, and pathogenesis of the pathogen. Data description One paired-end library (180-bp insert) was prepared from a gDNA sample of P. insidiosum strains ATCC200269 (clade-I), Pi19 (clade-II), MCC18 (clade-II), and SIMI4763 (clade-III) for whole-genome sequencing by Illumina HiSeq2000/HiSeq2500 NGS platform. A range of 28.4–59.4 million raw reads, accounted for 3.0–7.3 Gb, were obtained and assembled into the genome sizes of 47.1 Mb (15,153 contigs; 85% completeness; 19,329 open reading frames [ORFs]) for strain ATCC200269, 35.4 Mb (14,576 contigs; 83% completeness; 13,895 ORFs) for strain Pi19, 34.5 Mb (11,084 contigs; 84% completeness; 13,249 ORFs) for strain MCC18, and 47.1 Mb (15,162 contigs; 85% completeness; 19,340 ORFs) for strain SIMI4763. The genome data can be downloaded from the NCBI/DDBJ databases under the accessions BCFN00000000.1 (ATCC200269), BCFS00000000.1 (Pi19), BCFT00000000.1 (MCC18), and BCFU00000000.1 (SIMI4763).

Pythium insidiosum, an aquatic oomycete with pathogenic potential in mammals, causes gastrointestinal and cutaneous disease in dogs. Mefenoxam, an agricultural anti-oomycotic compound, has a demonstrated the ability to inhibit P. insidiosum growth in vitro and has been associated with efficacy in treating gastrointestinal pythiosis in several case reports. Electronic medical records of dogs seen at University of Florida Small Animal Hospital and treated with mefenoxam between 2013 and 2020 were searched. Dogs were included in this study upon previous definitive diagnosis with either organism identification using culture, PCR, or antibody ELISA, or a combination of these tests with or without supportive histopathological analysis. Since 2013, mefenoxam had been administered to 25 dogs with cutaneous pythiosis and 16 dogs with gastrointestinal pythiosis. In both gastrointestinal and cutaneous pythiosis groups, the administration of mefenoxam was associated with a survivability rate of approximately 51%. There was a statistically significant difference in the time to death between cutaneous (245 days (52–530)) and gastrointestinal (90 days (21–203)) groups; dogs infected with cutaneous pythiosis survived significantly longer after being diagnosed with the disease (p = 0.035). The dogs in this study experienced increased survival rates and time to death, in the absence of side effects due to mefenoxam, compared with previously published literature. The results of this retrospective study, with some limitations, are promising and should prompt further investigation into the use of mefenoxam in the treatment of both gastrointestinal and cutaneous pythiosis.

Conventional antimicrobial therapies are less effective against pythiosis. Therefore, the goal of this study was to characterize the effects of two ozone (O3) presentations on the growth of canine Pythium insidiosum. In Experiment 1, the pathogen was treated with O3 gas at different concentrations (32, 52, or 72 μg O3 mL−1 for 30 min) or exposure times (72 μg O3 mL−1 for 5, 15, or 30 min). In Experiment 2, pathogen samples were covered with ozonized sunflower oil with distinct peroxide indices. The growth area of P. insidiosum was measured during the first 14 days after treatment. Samples without growth were recultured to assess the germicidal or germistatic action of the respective therapy. In Experiment 1, treatment for 30 min had germicidal action regardless of the gas concentration, whereas shorter exposures to O3 were not effective. In Experiment 2, only ozonized sunflower oil with a peroxide index > 600 mmol equiv. kg−1 inactivated the pathogen. The findings of the present study suggest that exposure to O3 gas and ozonized sunflower oil with a high peroxide index are promising options for the long-lasting treatment of cutaneous pythiosis in humans and domestic animals. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Early phylogenetic analysis of Pythium insidiosum , the etiologic agent of pythiosis in mammals, showed the presence of a complex comprising three monophyletic clusters. Two included isolates recovered from cases of pythiosis in the Americas (Cluster I) and Asia (Cluster II), whereas the third cluster included four diverged isolates three from humans in Thailand and the USA, and one isolate from a USA spectacled bear (Cluster III). Thereafter, several phylogenetic analyses confirmed the presence of at least three monophyletic clusters, with most isolates placed in clusters I and II. Recent phylogenetic analyses using isolates from environmental sources and from human cases in India, Spain, Thailand, and dogs in the USA, however, showed the presence of two monophyletic groups each holding two sub-clusters. These studies revealed that P. insidiosum possesses different phylogenetic patterns to that described by early investigators. In this study, phylogenetic, population genetic and protein MALDI-TOF analyses of the P. insidiosum isolates in our culture collection, as well as those available in the database, showed members in the proposed cluster III and IV are phylogenetically different from that in clusters I and II. Our analyses of the complex showed a novel group holding two sub-clusters the USA (Cluster III) and the other from different world regions (Cluster IV). The data showed the original P. insidiosum cluster III is a cryptic novel species, now identified as P. periculosum. The finding of a novel species within P. insidiosum complex has direct implications in the epidemiology, diagnosis, and management of pythiosis in mammalian hosts. [ABSTRACT FROM AUTHOR]

Abstract Background Pythium, soil-borne plant pathogens, are in the class Oomycetes. They are not true fungi, but are related to diatom and algae. There are two human pathogens including P. insidiosum and P. aphanidermatum. To date, only one case of pythiosis caused by P. aphanidermatum has been reported. We present herein the first case of P. aphanidermatum vascular pythiosis in Asia. Case presentation A 47-year-old Thai woman, living in North Thailand, with ß thalassemia/hemoglobin E presented with acute recurrent arterial insufficiency of both legs. Emergent embolectomy with clot removal was performed. The pathology of the clot exhibited noncaseous granulomatous inflammation with many fungal hyphal elements. PCR identified P. aphanidermatum with 100% identity. Final diagnosis is vascular pythiosis. Unfortunately, the patient eventually expired after treatment with itraconazole, terbinafine, azithromycin, and doxycycline. Conclusions To date, only one case of pythiosis caused by P. aphanidermatum has been reported. We present herein the first case of P. aphanidermatum vascular pythiosis in Asia.

Objective: Pythium insidiosum causes a life-threatening condition called pythiosis. High morbidity and mortality of pythiosis are consequences of delayed diagnosis. We aimed to develop a loop-mediated isothermal amplification (LAMP) assay for the rapid detection of P. insidiosum for use in remote areas, where pythiosis is prevalent. Methods: We designed four LAMP primers to amplify the rDNA sequence. A side-by-side comparison evaluated performances of LAMP and the previously-established multiplex PCR (M-PCR), using gDNA samples extracted from colonies of P. insidiosum (n = 28) and other fungi (n = 54), and tissues of animals with (n = 16) or without (n = 13) pythiosis. Results: LAMP demonstrated a 50% shorter assay duration (1.5 h) and a 10-fold lower limit of detection (10-4 ng) than did M-PCR. Based on colony-extracted gDNAs, LAMP and M-PCR correctly reported P. insidiosum in all 28 samples, providing 100% sensitivity. While M-PCR did not amplify all fungal controls (100% specificity), LAMP falsely detected one organism (98% specificity). Based on the clinical samples, LAMP and M-PCR provided an equivalently-high specificity (100%). However, LAMP showed a markedly-higher sensitivity than that of M-PCR (88% vs. 56%). Conclusions: LAMP is a simple, useful, efficient assay for the detection of P. insidiosum in clinical specimens and pure cultures in resource-limited laboratories.

ABSTRACT: Pythiosis is an emerging infectious disease affecting captive and free-ranging wild animals. We report granulomatous pneumonia due to Pythium insidiosum in two South American coatis (Nasua nasua), who were found dead without any clinical records. Severe granulomatous pneumonia associated with pleural effusion was revealed in the necropsy. Microscopically, variably sized granulomas and pyogranulomas presented negative hyphae profiles at the periphery of their necrotic cores. Grocott methenamine silver stain highlighted these structures, and immunostain (anti- P. insidiosum) was strongly positive. Molecular analysis by polymerase chain reaction amplified P. insidiosum specific DNA. These findings characterized P. insidiosum as a cause of granulomatous pneumonia in coatis and proved that pythiosis needs to be considered in the differential diagnosis of respiratory diseases affecting this species in endemic areas.

Abstract A 7‐year‐old castrated male French Bulldog was examined for chronic large intestinal enteropathy. A colonic mass and thickened rectal mucosa were identified, and histopathologic examination of endoscopic biopsy specimens disclosed eosinophilic proctitis with large (5‐20 μm), irregularly shaped, pauciseptate hyphae that were Gomori methenamine silver and periodic acid‐Schiff positive. Amplification and sequencing of ribosomal DNA extracted from paraffin‐embedded tissues yielded a sequence with 97% identity to GenBank sequences for Basidiobolus ranarum. After itraconazole, terbinafine, and prednisone administration, clinical signs resolved rapidly, and sonographic lesions were largely absent after 6 weeks. Treatment was discontinued by the owner 15 weeks after diagnosis. Three weeks later, the dog collapsed acutely and was euthanized. Necropsy identified metastatic islet cell carcinoma and grossly unremarkable colorectal tissues. However, histopathology of the rectum disclosed multifocal submucosal granulomas with intralesional hyphae morphologically similar to those previously observed. This report is the first to describe medical treatment of gastrointestinal basidiobolomycosis in a dog.

Abstract Objectives Genome sequences are a vital resource for accelerating the biological exploration of an organism of interest. Pythium destruens (a synonym of Pythium insidiosum) causes a difficult-to-treat infectious disease called pythiosis worldwide. Detection and management of pythiosis are challenging. Basic knowledge of the disease is lacking. Genomes of this organism isolated from different continents (i.e., Asia and the Americas) have been sequenced and publicly available. Here, we sequenced the genome of an Australian isolate of P. destruens. Genome data will facilitate the comparative analysis of this and related species at the molecular level. Data description Genomic DNA of the P. destruens strain ATCC 64221, isolated from a horse with pythiosis in Australia, was used to prepare one paired-end library (with 180-bp insert) for next-generation sequencing, using the Illumina HiSeq 2500 short-read platform. Raw reads were cleaned and assembled by several bioinformatics tools. A total of 20,860,454 processed reads, accounted for 2,614,890,553 total bases, can be assembled into a 37.8-Mb genome, consisting 13,060 contigs (average length: 2896 bases; range: 300–142,967), N 50 of 11,370 bases, and 2.9% ‘N’ composition. The genome was determined 85.9% completeness, contained 14,424 predicted genes, and can be retrieved online at the NCBI/DDBJ databases under the accession number BCFQ01000000.1.

Abstract Objectives Pythiosis is a deadly infectious disease caused by Pythium insidiosum. Reports of both human and animal pythiosis are on the rise worldwide. Prognosis of the pythiosis patients relies on early diagnosis and prompt treatment. There are needs for an immunodiagnostic test that can detect the disease in both humans and animals. This study aims at reporting an optimized protocol for the development of a protein A/G-based enzyme-linked immunosorbent assay (ELISA) for the detection of anti-P. insidiosum antibody in multiple host species. Results A total of 25 pythiosis and 50 control sera, obtained from humans, horses, dogs, cats, and cows, were recruited for the assay development. With a proper ELISA cutoff point, all pythiosis sera can ultimately be distinguished from the control sera. The successfully-developed protein A/G-based ELISA can detect the anti-P. insidiosum antibodies in serum samples of both humans and animals. It is a versatile, feasible-to-develop, and functional immunodiagnostic assay for pythiosis.

Pythiosis, whose etiological agent is the oomycete Pythium insidiosum, is a life-threatening disease that occurs mainly in tropical and subtropical countries, affecting several animal species. It is frequently found in horses in Brazil and humans in Thailand. The disease is difficult to diagnose because the pathogen’s hyphae are often misdiagnosed as mucoromycete fungi in histological sections. Additionally, there is no specific antigen to use for rapid diagnosis, the availability of which could improve the prognosis in different animal species. In this scenario, we investigated which P. insidiosum antigens are recognized by circulating antibodies in horses and humans with pythiosis from Brazil and Thailand, respectively, using 2D immunoblotting followed by mass spectrometry for the identification of antigens. We identified 23 protein spots, 14 recognized by pooled serum from horses and humans. Seven antigens were commonly recognized by both species, such as the heat-shock cognate 70 KDa protein, the heat-shock 70 KDa protein, glucan 1,3-beta-glucosidase, fructose-bisphosphate aldolase, serine/threonine-protein phosphatase, aconitate hydratase, and 14-3-3 protein epsilon. These results demonstrate that there are common antigens recognized by the immune responses of horses and humans, and these antigens may be studied as biomarkers for improving diagnosis and treatment. [ABSTRACT FROM AUTHOR]

Objectives: We employed the Illumina NGS platform to sequence genomes of 4 different strains of the pathogenic oomycete Pythium insidiosum, the causative agent of pythiosis. These strains were isolated from humans in Thailand (n = 3) and the United States (n = 1), and phylogenetically classified into clade-I, -II, and -III. Our study augmented the completeness of the P. insidiosum genome database for exploration of the biology, evolution, and pathogenesis of the pathogen. Data description: One paired-end library (180-bp insert) was prepared from a gDNA sample of P. insidiosum strains ATCC200269 (clade-I), Pi19 (clade-II), MCC18 (clade-II), and SIMI4763 (clade-III) for whole-genome sequencing by Illumina HiSeq2000/HiSeq2500 NGS platform. A range of 28.4–59.4 million raw reads, accounted for 3.0–7.3 Gb, were obtained and assembled into the genome sizes of 47.1 Mb (15,153 contigs; 85% completeness; 19,329 open reading frames [ORFs]) for strain ATCC200269, 35.4 Mb (14,576 contigs; 83% completeness; 13,895 ORFs) for strain Pi19, 34.5 Mb (11,084 contigs; 84% completeness; 13,249 ORFs) for strain MCC18, and 47.1 Mb (15,162 contigs; 85% completeness; 19,340 ORFs) for strain SIMI4763. The genome data can be downloaded from the NCBI/DDBJ databases under the accessions BCFN00000000.1 (ATCC200269), BCFS00000000.1 (Pi19), BCFT00000000.1 (MCC18), and BCFU00000000.1 (SIMI4763). [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

ABSTRACT Protein production relies on time-consuming genetic engineering and in vivo expression, which is a bottleneck for functional studies in the postgenomic era. Cell-free protein synthesis (CFPS) overcomes the limitation of in vivo protein biosynthesis by processing in vitro transcription and translation of multiple genes to proteins within hours. We employed an automated CFPS to simultaneously synthesize proteins from 24 genes of the oomycete Pythium insidiosum (which causes the life-threatening disease pythiosis) and screen for a diagnostic and therapeutic target. CFPS successfully synthesized 18 proteins (∼75% success rate). One protein, namely, I06, was explicitly recognized by all pythiosis sera, but not control sera, tested. Py. insidiosum secreted a significant amount of I06. The protein architecture of I06 is compatible with the oligopeptide elicitor (OPEL) of the phylogenetically related plant-pathogenic oomycete Phytophthora parasitica. The OPEL-like I06 protein of Py. insidiosum can stimulate host antibody responses, similar to the P. parasitica OPEL that triggers plant defense mechanisms. OPEL-like I06 homologs are present only in the oomycetes. Py. insidiosum contains two OPEL-like I06 homologs, but only one of the two homologs was expressed during hyphal growth. Twenty-nine homologs derived from 15 oomycetes can be phylogenetically divided into two groups. The OPEL-like genes might occur in the common ancestor, before independently undergoing gene gain and loss during the oomycete speciation. In conclusion, CFPS offers a fast in vitro protein synthesis. CFPS simultaneously generated multiple proteins of Py. insidiosum and facilitated the identification of the secretory OPEL-like I06 protein, a potential target for the development of a control measure against the pathogen. IMPORTANCE Technical limitations of conventional biotechnological methods (i.e., genetic engineering and protein synthesis) prevent extensive functional studies of the massive amounts of genetic information available today. We employed a cell-free protein synthesis system to rapidly and simultaneously generate multiple proteins from genetic codes of the oomycete Pythium insidiosum, which causes the life-threatening disease called pythiosis, in humans and animals worldwide. We aimed to screen for potential diagnostic and therapeutic protein targets of this pathogen. Eighteen proteins were synthesized. Of the 18 proteins, one was a secreted immunoreactive protein, called I06, that triggered host immunity and was recognized explicitly by all tested sera from pythiosis patients. It is one of the OPEL proteins; these proteins are present only in the unique group of microorganisms called oomycetes. Here, we demonstrated that cell-free protein synthesis was useful for the production of multiple proteins to facilitate functional studies and identify a potential target for diagnosis and treatment of pythiosis.

Pythiosis is a life-threatening infectious disease of humans and animals caused by the oomycete microorganism Pythium insidiosum. The disease has been increasingly diagnosed worldwide. P. insidiosum inhabits freshwater and presents in two forms: mycelium and zoospore. Clinical manifestations of pythiosis include an infection of the artery, eye, skin, or gastrointestinal tract. The management of pythiosis is problematic due to the lack of effective treatment. Many patients die from an uncontrolled infection. The drug susceptibility testing provides clinically-useful information that could lead to proper drug selection against P. insidiosum. Currently, no standard CLSI protocol for the drug susceptibility of P. insidiosum is available. This review aims at describing methods and antimicrobial agents for susceptibility testing against P. insidiosum. Several in-house in vitro susceptibility methods (i.e., broth microdilution method, radial growth method, and agar diffusion method) have been established for P. insidiosum. Either mycelium or zoospore can be an inoculum. Rabbit is the commonly-used model of pythiosis for in vivo drug susceptibility testing. Based on the susceptibility results (i.e., minimal inhibitory concentration and inhibition zone), several antibacterial and antifungal drugs, alone or combination, exhibited an in vitro or in vivo effect against P. insidiosum. Some distinct compounds, antiseptic agents, essential oils, and plant extracts, also show anti-P. insidiosum activities. Successfully medical treatment, guided by the drug susceptibility data, has been reported in some pythiosis patients. Future studies should emphasize finding a novel and effective anti-P. insidiosum drug, standardizing in vitro susceptibility method and correlating drug susceptibility data and clinical outcome of pythiosis patients for a better interpretation of the susceptibility results.

Human pythiosis is an infectious condition with high morbidity and mortality. The causative agent is the oomycete microorganism Pythium insidiosum. The pathogen inhabits ubiquitously in a wet environment, and direct exposure to the pathogen initiates the infection. Most patients with pythiosis require surgical removal of the affected organ, and many patients die from the disease. Awareness of pythiosis among healthcare personnel is increasing. In this review, we summarized and updated information on the diagnosis and treatment of human pythiosis. Vascular and ocular pythiosis are common clinical manifestations. Recognition of the typical clinical features of pythiosis is essential for early diagnosis. The definitive diagnosis of the disease requires laboratory testing, such as microbiological, serological, molecular, and proteomic assays. In vascular pythiosis, surgical intervention to achieve the organism-free margin of the affected tissue, in combination with the use of antifungal drugs and P. insidiosum immunotherapy, remains the recommended treatment. Ocular pythiosis is a serious condition and earliest therapeutic penetrating keratoplasty with wide surgical margin is the mainstay treatment. Thorough clinical assessment is essential in all patients to evaluate the treatment response and detect an early sign of the disease recurrence. In conclusion, early diagnosis and proper management are the keys to an optimal outcome of the patients with pythiosis.

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.