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

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

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.

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.

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 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.

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.

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.

Automated Cell-Free Multiprotein Synthesis Facilitates the Identification of a Secretory, Oligopeptide Elicitor-Like, Immunoreactive Protein of the Oomycete Pythium insidiosum.

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.

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.

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.

Probing the Phylogenomics and Putative Pathogenicity Genes of Pythium insidiosum by Oomycete Genome Analyses.

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

Development of a PCR Assay Based on a Single-Base Pair Substitution for the Detection of Aeromonas caviae by Targeting the gyrB Gene.

Aeromonas caviae is a bacterial pathogen that causes various infectious diseases in both humans and animals. To facilitate its detection, we developed species-specific primer sets targeting polymorphisms in the gyrB gene for use in a PCR assay. The technique was able to detect 100% (29/29) of the A. caviae strains tested using either of two sets of primers (designated ACF1-ACR and ACF3-ACR), which produced 293-bp and 206-bp amplicons, respectively. Another set of primers (designated ACF2-ACR) yielded a 237-bp amplicon and exhibited 90% (26/29) positive results with respect to A. caviae. None of the primer sets exhibited cross-reactivity with 12 non-A. caviae isolates and 52 other non-Aeromonas bacteria. The detection limit using the ACF2-ACR and ACF3-ACR primer sets in pure culture was 1.6 × 10(3) CFU/mL, or 6 CFU per reaction, whereas that of the ACF1-ACR primer set was 1.6 × 10(4) CFU/mL, or 60 CFU per reaction. In the case of spiked Nile Tilapia Oreochromis niloticus, the sensitivity of all primer sets without enrichment was 1.8 × 10(4) CFU/g, or 30 CFU per reaction. Primer set ACF3-ACR was the best for a PCR assay targeting the gyrB gene, and the PCR technique developed was rapid, specific, and sensitive for the identification of A. caviae.

Relationship of body composition and circulatory adiponectin to bone mineral density in young premenopausal women.

OBJECTIVE: Adiponectin is a recently discovered hormone secreted by adipocytes. Adiponectin plays an important role in the regulation of insulin sensitivity as well as the propensity to inflammation and atherosclerosis. In the present study, the authors explore the relationship between adiponectin and bone mass in premenopausal women. The relationship of fat mass compared to lean body mass to bone mass was also investigated MATERIAL AND METHOD: Two hundred premenopausal women aged between 20 and 40 years were studied. Bone mineral density (BMD) was measured at L2-4 and femoral neck by dual-energy X-ray absorptiometry (DEXA). Serum adiponectin concentrations were measured by radioimmunoassay. RESULTS: At the lumbar spines, factors associated with BMD were age (p < 0.01) and lean body mass (p < 0.001). No independent association with fat mass was demonstrated Likewise, at the femoral neck, only lean body mass was related to BMD (p < 0. 01). In terms of the relation of serum adiponectin to BMD, no association of serum adiponectin to BMD at the lumbar spines or femoral neck was found CONCLUSION: Altogether, the present findings do not suggest the independent role of adiponectin in the accrual of bone mass in females, although such a role still cannot be excluded in men or postmenopausal women.

The implication of assessing a polymorphism in estrogen receptor alpha gene in the risk assessment of osteoporosis using a screening tool for osteoporosis in Asians.

Both genetic and environmental factors interact to determine bone mass and the risk for developing postmenopausal osteoporosis. Recently, an Asian-specific tool, the Osteoporosis Self-Assessment Tool for Asians (OSTA), has been developed to assess the risk of osteoporosis in women. An index is calculated by multiplying the difference in body weight in kilograms and age in years by 0.2 and disregarding the decimal digits. The risk of osteoporosis is classified as high, intermediate or low according to the OSTA index less than -4, -4 to -1 and greater than -1. In the present study we examined how a single nucleotide polymorphism (SNP) in exon 8 of the estrogen receptor alpha (ERalpha) gene affected the predictive value of the OSTA index. Subjects consisted of 358 postmenopausal women who were at least 55 years old. BMDs were measured by DXA, and the SNP in the ERalpha gene was assessed by PCR-RFLP. When considering both the OSTA index and ERalpha genotype in a logistic regression model, it was found that both the OSTA index and the ERalpha genotype independently contributed to the risk of osteoporosis. The odds ratios were 1.58 (95% CI 1.26-1.91) and 2.51 (95% CI 1.42-4.44) for one unit decrement in the OSTA index and each copy of the A allele of the ERalpha genotype, respectively. The joint effect conformed more to a multiplicative model of interaction than an additive model. This suggests that persons with the high-risk genotype are at far greater risk of developing osteoporosis with advancing age or decreasing body weight, the two variables from which the OSTA index is derived. Targeting preventive measures for osteoporosis subjects with risk factors and also disease-susceptibility alleles is likely to be more cost effective.