Theerapong Krajaejun

Genome sequence of the necrotrophic plant pathogen Pythium ultimum reveals original pathogenicity mechanisms and effector repertoire

BackgroundPythium ultimum is a ubiquitous oomycete plant pathogen responsible for a variety of diseases on a broad range of crop and ornamental species.ResultsThe P. ultimum genome (42.8 Mb) encodes 15,290 genes and has extensive sequence similarity and synteny with related Phytophthora species, including the potato blight pathogen Phytophthora infestans. Whole transcriptome sequencing revealed expression of 86% of genes, with detectable differential expression of suites of genes under abiotic stress and in the presence of a host. The predicted proteome includes a large repertoire of proteins involved in plant pathogen interactions, although, surprisingly, the P. ultimum genome does not encode any classical RXLR effectors and relatively few Crinkler genes in comparison to related phytopathogenic oomycetes. A lower number of enzymes involved in carbohydrate metabolism were present compared to Phytophthora species, with the notable absence of cutinases, suggesting a significant difference in virulence mechanisms between P. ultimum and more host-specific oomycete species. Although we observed a high degree of orthology with Phytophthora genomes, there were novel features of the P. ultimum proteome, including an expansion of genes involved in proteolysis and genes unique to Pythium. We identified a small gene family of cadherins, proteins involved in cell adhesion, the first report of these in a genome outside the metazoans.ConclusionsAccess to the P. ultimum genome has revealed not only core pathogenic mechanisms within the oomycetes but also lineage-specific genes associated with the alternative virulence and lifestyles found within the pythiaceous lineages compared to the Peronosporaceae.

Clinical and Epidemiological Analyses of Human Pythiosis in Thailand

BACKGROUND Pythiosis is an emerging and life-threatening infectious disease in humans and animals that is caused by the pathogenic oomycete Pythium insidiosum. Human pythiosis is found mostly in Thailand, although disease in animals has been increasingly reported worldwide. Clinical information on human pythiosis is limited, and health care professionals are unfamiliar with the disease, leading to underdiagnosis, delayed treatment, and poor prognosis. METHODS To retrospectively study the clinical and epidemiological features of human pythiosis, we analyzed clinical data from patients with pythiosis diagnosed during the period of January 1985 through June 2003 at 9 tertiary care hospitals throughout Thailand. RESULTS A total of 102 cases of human pythiosis were documented nationwide. A substantial proportion (40%) of cases occurred in the last 4 years of the 18-year study interval. Clinical presentations fell into 4 groups: cutaneous/subcutaneous cases (5% of cases), vascular cases (59%), ocular cases (33%), and disseminated cases (3%). Almost all patients with cutaneous/subcutaneous, vascular, and disseminated pythiosis (85%) had underlying thalassemia-hemoglobinopathy syndrome. Most ocular cases (84%) were associated with no underlying disease. A majority of the patients were male (71%), were aged 20-60 years (86%), and reported an agricultural occupation (75%). Regarding treatment outcomes, all patients with disseminated infection died; 78% of patients with vascular disease required limb amputation, and 40% of these patients died; and 79% of patients with ocular pythiosis required enucleation/evisceration. CONCLUSIONS Here, we report, to our knowledge, the largest case study of human pythiosis. The disease has high rates of morbidity and mortality. Early diagnosis and effective treatment are urgently needed to improve clinical outcomes. Because P. insidiosum is distributed worldwide and can infect healthy individuals, an awareness of human pythiosis should be promoted in Thailand and in other countries.

Development and Evaluation of an In-House Enzyme-Linked Immunosorbent Assay for Early Diagnosis and Monitoring of Human Pythiosis

ABSTRACT Human pythiosis is an emerging, fatal, infectious disease caused by Pythium insidiosum and occurs in both tropical and subtropical countries. Thalassemic patients, farmers, and aquatic-habitat residents are predisposed to this disease. Delayed treatment due to the long time required for isolation and identification of the causative organism, as well as the difficulty in obtaining internal organ specimens, results in high morbidity and mortality. To facilitate rapid diagnosis, an in-house enzyme-linked immunosorbent assay (ELISA) for the detection of immunoglobulin G antibodies against P. insidiosum was developed and evaluated for the diagnosis and monitoring of human pythiosis. Sixteen sera were collected from seven culture-proven human pythiosis cases. A total of 142 sera from thalassemic patients, from patients with other infectious diseases, and from healthy blood donors served as controls. All sera were tested in duplicate. By choosing a suitable cutoff point to maximize sensitivity and specificity, sera from pythiosis cases were all determined to be positive, whereas sera from control groups were all determined to be negative. ELISA signals from serial samples of sera taken from treated patients showed gradually declining levels of antibodies to P. insidiosum. The ELISA test was highly sensitive (100%) and specific (100%) and was useful for early diagnosis and for monitoring the treatment for pythiosis.

Development of an Immunochromatographic Test for Rapid Serodiagnosis of Human Pythiosis

ABSTRACT Human pythiosis is an emerging and life-threatening infectious disease caused by the fungus-like organism Pythium insidiosum. High rates of morbidity and mortality for patients with pythiosis are exacerbated by the lack of early diagnosis and an effective treatment. Here, we developed and evaluated an immunochromatographic test (ICT) for the diagnosis of human pythiosis, in comparison to a standard serological test of immunodiffusion (ID). Culture filtrate antigen of P. insidiosum was used to detect human anti-P. insidiosum antibody. Sheep anti-human immunoglobulin G-colloidal gold conjugate was used to generate an ICT signal. Thirty-three sera from patients with vascular (n = 27), ocular (n = 4), and cutaneous (n = 2) pythiosis and 181 control sera from healthy blood donors (n = 100), as well as patients with a variety of infectious (n = 56) and noninfectious (n = 25) diseases, were included in the test evaluation. The turnaround time for generating a result by the ICT was less than 30 min, while that for ID was ∼24 h. Based on the results for all sera of pythiosis patients and the control groups, the ICT showed 88% sensitivity and 100% specificity and ID showed 61% sensitivity and 100% specificity. By both tests, false-negative results for sera from all ocular pythiosis patients were obtained. In addition, the ID test yielded false-negative results for sera from eight patients with vascular pythiosis and one patient with cutaneous pythiosis. It was concluded that the ICT is a rapid, user-friendly, and reliable serological test for the early diagnosis of vascular and cutaneous pythiosis.

Identification of a Novel 74-Kilodalton Immunodominant Antigen of Pythium insidiosum Recognized by Sera from Human Patients with Pythiosis

ABSTRACT The oomycetous, fungus-like, aquatic organism Pythium insidiosum is the etiologic agent of pythiosis, a life-threatening infectious disease of humans and animals that has been increasingly reported from tropical, subtropical, and temperate countries. Human pythiosis is endemic in Thailand, and most patients present with arteritis, leading to limb amputation and/or death, or cornea ulcer, leading to enucleation. Diagnosis of pythiosis is time-consuming and difficult. Radical surgery is the main treatment for pythiosis because conventional antifungal drugs are ineffective. The aims of this study were to evaluate the use of Western blotting for diagnosis of human pythiosis, to identify specific immunodominant antigens of P. insidiosum, and to increase understanding of humoral immune responses against the pathogen. We performed Western blot analysis on 16 P. insidiosum isolates using 12 pythiosis serum samples. These specimens were derived from human patients with pythiosis who had different forms of infection and lived in different geographic areas throughout Thailand. We have identified a 74-kDa immunodominant antigen in all P. insidiosum isolates tested. The 74-kDa antigen was also recognized by sera from all patients with pythiosis but not by control sera from healthy individuals, patients with thalassemia, and patients with various infectious diseases, indicating that Western blot analysis could facilitate diagnosis of pythiosis. Therefore, the 74-kDa antigen is a potential target for developing rapid serodiagnostic tests as well as a therapeutic vaccine for pythiosis. These advances could lead to early diagnosis and effective treatment, crucial factors for better prognosis for patients with pythiosis.

Development and Application of a Green Fluorescent Protein Sentinel System for Identification of RNA Interference in Blastomyces dermatitidis Illuminates the Role of Septin in Morphogenesis and Sporulation

ABSTRACT A high-throughput strategy for testing gene function would accelerate progress in our understanding of disease pathogenesis for the dimorphic fungus Blastomyces dermatitidis, whose genome is being completed. We developed a green fluorescent protein (GFP) sentinel system of gene silencing to rapidly study genes of unknown function. Using Gateway technology to efficiently generate RNA interference plasmids, we cloned a target gene, “X,” next to GFP to create one hairpin to knock down the expression of both genes so that diminished GFP reports target gene expression. To test this approach in B. dermatitidis, we first used LACZ and the virulence gene BAD1 as targets. The level of GFP reliably reported interference of their expression, leading to rapid detection of gene-silenced transformants. We next investigated a previously unstudied gene encoding septin and explored its possible role in morphogenesis and sporulation. A CDC11 septin homolog in B. dermatitidis localized to the neck of budding yeast cells. CDC11-silenced transformants identified with the sentinel system grew slowly as flat or rough colonies on agar. Microscopically, they formed ballooned, distorted yeast cells that failed to bud, and they sporulated poorly as mold. Hence, this GFP sentinel system enables rapid detection of gene silencing and has revealed a pronounced role for septin in morphogenesis, budding, and sporulation of B. dermatitidis.

Hemagglutination Test for Rapid Serodiagnosis of Human Pythiosis

ABSTRACT Human pythiosis is an emerging, life-threatening infectious disease, caused by the oomycete Pythium insidiosum. Thailand is an area where human pythiosis is endemic and the genetic blood disorder thalassemia is a predisposing factor. Patients with pythiosis present with arterial occlusions of the lower extremities, corneal ulcers, or chronic cutaneous infections. Diagnosis relies on time-consuming, relatively insensitive tests such as culture identification and immunodiffusion assay. Most patients undergo surgical removal of infected organs, and many die from the infection. Delayed diagnosis results in a poor prognosis. Here, we describe a hemagglutination (HA) test for rapid diagnosis of human pythiosis. Sheep red blood cells were coated with P. insidiosum protein extract and used in duplicated detection assays using serum samples from 33 patients with vascular (n = 27), cutaneous (n = 2), or ocular (n = 4) pythiosis and serum samples from 289 control patients with other infectious diseases (n = 77), with highly positive antinuclear antibody (n = 5), with thalassemia (n = 21), or with no known disorder (i.e., healthy blood donors) (n = 186). Based on receiver-operating characteristic analysis, a serum titer of 1:160 was selected as the cutoff point for the HA test. Serum samples that generated HA at the cutoff titer were read as positive, while samples that did not were read as negative. Positive results were obtained with the serum samples of all patients with vascular and cutaneous pythiosis and with two serum samples from the control group. Negative results were obtained with serum samples from all ocular pythiosis patients and the 287 remaining serum samples from the control group. Sensitivity and specificity of the HA were 88% and 99%, respectively. In conclusion, the HA test for detection of anti-Pythium antibodies is a simple, rapid, and reliable test for serodiagnosis of vascular and cutaneous pythiosis.

Ocular pythiosis: is it under-diagnosed?

PURPOSE To increase awareness of ocular pythiosis by presenting a typical case and summarizing clinical data of 11 ocular pythiosis cases in Ramathibodi Hospital. DESIGN Interventional case report. METHODS A 48-year-old healthy woman with a history of 3-week painful corneal ulcer of left eye was treated with enucleation. RESULTS The histopathology of enucleated eye revealed endophthalmitis and ulcerative keratitis with numerous hyphae in full-thickness of corneal stroma. The culture identification of the causative organism was Pythium insidiosum. The final diagnosis was ocular pythiosis. CONCLUSIONS Pythium insidiosum is a causative agent of pythiosis and is distributed worldwide. Ocular pythiosis may not be uncommon, as it may be underdiagnosed due to unfamiliarity among clinicians and microbiologists. Diagnosis of pythiosis is difficult. The disease has high morbidity, as evidenced by nearly evisceration or enucleation among all patients at Ramathibodi Hospital. Early detection and effective treatment are needed for possible cure.

Performance comparison of immunodiffusion, enzyme-linked immunosorbent assay, immunochromatography and hemagglutination for serodiagnosis of human pythiosis

Pythiosis is a life-threatening infectious disease caused by the fungus-like organism Pythium insidiosum. Morbidity and mortality rates of pythiosis are high. The treatment of choice for pythiosis is surgical debridement of infected tissue. Early and accurate diagnosis is critical for effective treatment. In-house serodiagnostic tests, including immunodiffusion (ID), enzyme-linked immunosorbent assay (ELISA), immunochromatography (ICT) and hemagglutination (HA) have been developed to detect antibodies against P. insidiosum in sera. This study compares the diagnostic performance of ID, ELISA, ICT, and HA, using sera from 37 pythiosis patients and 248 control subjects. ICT and ELISA showed optimal diagnostic performance (100% sensitivity, specificity, positive predictive value and negative predictive value). ICT was both rapid and user-friendly. ELISA results were readily quantitated. ID is relatively insensitive. HA was rapid, but diagnostic performance was poor. Understanding the advantages offered by each assay facilitates selection of an assay that is circumstance-appropriate. This will promote earlier diagnoses and improved outcomes for patients with pythiosis.

Phylogenetic analysis of Pythium insidiosum Thai strains using cytochrome oxidase II (COX II) DNA coding sequences and internal transcribed spacer regions (ITS)

To investigate the phylogenetic relationship among Pythium insidiosum isolates in Thailand, we investigated the genomic DNA of 31 P. insidiosum strains isolated from humans and environmental sources from Thailand, and two from North and Central America. We used PCR to amplify the partial COX II DNA coding sequences and the ITS regions of these isolates. The nucleotide sequences of both amplicons were analyzed by the Bioedit program. Phylogenetic analysis using genetic distance method with Neighbor Joining (NJ) approach was performed using the MEGA4 software. Additional sequences of three other Pythium species, Phytophthora sojae and Lagenidium giganteum were employed as outgroups. The sizes of the COX II amplicons varied from 558-564 bp, whereas the ITS products varied from approximately 871-898 bp. Corrected sequence divergences with Kimura 2-parameter model calculated for the COX II and the ITS DNA sequences ranged between 0.0000-0.0608 and 0.0000-0.2832, respectively. Phylogenetic analysis using both the COX II and the ITS DNA sequences showed similar trees, where we found three sister groups (A(TH), B(TH), and C(TH)) among P. insidiosum strains. All Thai isolates from clinical cases and environmental sources were placed in two separated sister groups (B(TH) and C(TH)), whereas the Americas isolates were grouped into A(TH.) Although the phylogenetic tree based on both regions showed similar distribution, the COX II phylogenetic tree showed higher resolution than the one using the ITS sequences. Our study indicates that COX II gene is the better of the two alternatives to study the phylogenetic relationships among P. insidiosum strains.