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.

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.