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.

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.

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.

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.

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.

Selection of an Appropriate In Vitro Susceptibility Test for Assessing Anti-Pythium insidiosum Activity of Potassium Iodide, Triamcinolone Acetonide, Dimethyl Sulfoxide, and Ethanol.

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.

Genome data of four Pythium insidiosum strains from the phylogenetically-distinct clades I, II, and III.

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).

Identification and Biotyping of Pythium insidiosum Isolated from Urban and Rural Areas of Thailand by Multiplex PCR, DNA Barcode, and Proteomic Analyses.

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.

Immunological Cross-Reactivity of Proteins Extracted from the Oomycete Pythium insidiosum and the Fungus Basidiobolus ranarum Compromises the Detection Specificity of Immunodiagnostic Assays for Pythiosis.

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

Identification, overexpression, purification, and biochemical characterization of a novel hyperthermostable keratinase from Geoglobus acetivorans.

The goal of this study was to identify and biochemically characterize a novel hyperthermostable keratinase from microorganisms for feather waste degradation. Here, a hyperthermophilic Geoglobus acetivorans keratinase (GacK) gene was chosen based on a search of a sequence database. The selected GacK gene was synthesized, cloned, and successfully expressed without a signal peptide in the E. coli system. A monomer of approximately 58 kDa was obtained in a soluble form and purified. The recombinant GacK displayed the highest activity at an optimum temperature of 100 °C and a pH of 10. The hyperthermostable GacK enzymatic performance remained high even after incubation in nonionic surfactants and the chelating agent EDTA. The residual and keratinolytic activities of GacK, as determined with azocasein and keratin azure used as substrates, remained significantly greater than 80% at 130 °C for 7 h. The kinetic parameters Km and Vmax for azure keratin were 0.41 mg/ml and 875.14 unit/mg, respectively, while those for azocasein were 1.51 mg/ml and 505.32 unit/mg, respectively. The results suggest that the enzyme is among the most hyperthermostable keratinases. Because of its enzymatic characteristics to degrade keratin azure at high temperatures, GacK may potentially be utilized in future industrial applications.

Draft genome sequence of the oomycete Pythium destruens strain ATCC 64221 from a horse with pythiosis in Australia.

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), N50 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.

Protein A/G-based enzyme-linked immunosorbent assay for detection of anti-Pythium insidiosum antibodies in human and animal subjects.

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.

Expression, purification, and characterization of the recombinant exo-1,3-ß-glucanase (Exo1) of the pathogenic oomycete Pythium insidiosum.

Pythiosis is a deadly infectious disease of humans and animals living in tropical and subtropical countries. The causative agent is the oomycete Pythium insidiosum. Treatment of pythiosis is challenging. The use of antimicrobial agents usually fails in the treatment of pythiosis. Many patients undergo surgical removal of an infected organ (i.e., eye, arm, and leg). The immunotherapeutic vaccine, prepared from the crude extract of P. insidiosum, shows limited efficacy against pythiosis. The fatal outcome occurs in patients with advanced disease. There are urgent needs for an effective therapeutic modality for pythiosis. Recently, the exo-1,3-ß-glucanase (Exo1) has been identified as a conserve immunoreactive protein of P. insidiosum. Exo1 was predicted to reside at the cell membrane and hydrolyze cell wall ß-glucan during cell growth. An Exo1 ortholog is absent in the human genome, making it an appealing target for drug or vaccine development. We attempted to clone and express the codon-optimized exo1 gene of P. insidiosum in E. coli. To solve the inclusion body formation, expression and purification of Exo1 were achievable in the denaturing condition using SDS- and urea-based buffers. Exo1 lacked hydrolytic activity due to the absence of proper protein folding and post-translational modifications. ELISA and Western blot analyses demonstrated the immunoreactivity of Exo1 against pythiosis sera. In conclusion, we successfully expressed and purified the immunoreactive Exo1 protein of P. insidiosum. The recombinant Exo1 can be produced at an unlimited amount and could serve as an extra protein to enhance the effectiveness of the current form of the vaccine against pythiosis.

Loop-mediated Isothermal Amplification (LAMP) for Identification of Pythium insidiosum.

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.