Hemagglutination Test for Rapid Serodiagnosis of Human Pythiosis

Jindayok, Thanyasiri; Piromsontikorn, Savittree; Srimuang, S; Khupulsup, Kalayanee; Krajaejun, Theerapong

doi:10.1128/cvi.00113-09

Cited by 45 publications

(41 citation statements)

References 29 publications

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“…Synthetic peptides s74-1 and s74-2 were each used to coat ELISA plates and tested against the serum samples from patients with pythiosis (samples T1 to T5) and controls (samples C1 to C5). Among the pythiosis sera, pythiosis serum samples T1 (hemagglutination [HA] titer [12] ϭ 1:5,120) and T5 (HA titer ϭ 1:40,960) gave remarkably high ELISA signals (1.2 and 0.8, respectively, for s74-1; 1.2 and 1.0, respectively, for s74-2), while pythiosis serum samples T2 (HA titer ϭ 1:2,560), T3 (HA titer ϭ 1:1,280), and T4 (HA titer ϭ 1:1,280) gave weak to moderate ELISA signals (0.4, 0.3, and 0.3, respectively, for s74-1; 0.5, 0.4, and 0.3, respectively, for s74-2). In general, the s74-1 (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…For each new antigen preparation, the assay protocol needs extensive reevaluation in regards to antigen concentration, the optimal dilution of the serum samples, cutoff points, and the effective concentration of other reagents before it is suitable for clinical use. In addition, recent reports of false-positive results for some diagnostic tests (12,15) indicate a problem regarding the specificity of detection of the crude extract antigen. Taken together, these complications indicate an ongoing need for a more reliable and specific antigen source for the development of serological assays.…”

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confidence: 99%

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The 74-Kilodalton Immunodominant Antigen of the Pathogenic Oomycete Pythium insidiosum Is a Putative Exo-1,3-ß-Glucanase

Krajaejun

Keeratijarut

Sriwanichrak

et al. 2010

Clin Vaccine Immunol

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The oomycetous, fungus-like, aquatic organism Pythium insidiosum is the causative agent of pythiosis, a life-threatening infectious disease of humans and animals living in tropical and subtropical areas of the world. Common sites of infection are the arteries, eyes, cutaneous/subcutaneous tissues, and gastrointestinal tract. Diagnosis of pythiosis is time-consuming and difficult. Radical excision of the infected organs is the main treatment for pythiosis because conventional antifungal drugs are ineffective. An immunotherapeutic vaccine prepared from P. insidiosum crude extract showed limited efficacy in the treatment of pythiosis patients. Many pythiosis patients suffer lifelong disabilities or die from an advanced infection. Recently, we identified a 74-kDa major immunodominant antigen of P. insidiosum which could be a target for development of a more effective serodiagnostic test and vaccines. Mass spectrometric analysis identified two peptides of the 74-kDa antigen (s74-1 and s74-2) which perfectly matched a putative exo-1,3-ß-glucanase (EXO1) of Phytophthora infestans. Using degenerate primers derived from these peptides, a 1.1-kb product was produced by PCR, and its sequence was found to be homologous to that of the P. infestans exo-1,3-ß-glucanase gene, EXO1. Enzyme-linked immunosorbent assays targeting the s74-1 and s74-2 synthetic peptides demonstrated that the 74-kDa antigen was highly immunoreactive with pythiosis sera but not with control sera. Phylogenetic analysis using part of the 74-kDa protein-coding sequence divided 22 Thai isolates of P. insidiosum into two clades. Further characterization of the putative P. insidiosum glucanase could lead to new diagnostic tests and to antimicrobial agents and vaccines for the prevention and management of the serious and life-threatening disease of pythiosis.

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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

The 74-Kilodalton Immunodominant Antigen of the Pathogenic Oomycete Pythium insidiosum Is a Putative Exo-1,3-ß-Glucanase

Krajaejun

Keeratijarut

Sriwanichrak

et al. 2010

Clin Vaccine Immunol

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“…The current diagnostic modalities, including culture identification (9)(10)(11), serodiagnosis (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22), and molecular-based detection (22)(23)(24)(25)(26)(27), are fraught with problems. For example, culture identification is time-consuming and often fails to grow and to identify the organism.…”

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confidence: 99%

Development of an Anti-Elicitin Antibody-Based Immunohistochemical Assay for Diagnosis of Pythiosis

et al. 2016

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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.

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“…Even isolating the organism in pure culture does not guarantee an accurate identification: the procedure is both time-consuming and technically challenging, and the organism may fail to generate characteristic zoospores when cultured in vitro (3,6). Serological methods have been described, including complement fixation, immunodiffusion, fluorescent antibody tests, immunoblots, enzyme-linked immunosorbent assays (ELISA), and hemagglutinin tests (8). However, these techniques display variable sensitivity and specificity, especially during early infection when the immune response is not fully mounted (8).…”

Section: Case Reportmentioning

confidence: 99%

“…Serological methods have been described, including complement fixation, immunodiffusion, fluorescent antibody tests, immunoblots, enzyme-linked immunosorbent assays (ELISA), and hemagglutinin tests (8). However, these techniques display variable sensitivity and specificity, especially during early infection when the immune response is not fully mounted (8). Definitive diagnosis can be achieved using molecular methods, including species-specific nested PCR (3) or DNA sequencing of variable regions (1,2).…”

Section: Case Reportmentioning

confidence: 99%

Molecular Diagnosis of Subcutaneous Pythium insidiosum Infection by Use of PCR Screening and DNA Sequencing

et al. 2012

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h Pythium insidiosum is an emerging human pathogen classified among brown algae and diatoms that can cause significant morbidity and mortality in otherwise healthy individuals. Here we describe a pediatric patient with pythiosis acquired in the southern United States, diagnosed by molecular screening and DNA sequencing of internal transcribed spacer region 1. CASE REPORTA n otherwise healthy 14-year-old girl living in urban north Texas presented to an urgent care facility with a 2-week history of a progressively enlarging, erythematous bump on the medial aspect of her left lower leg. She denied recent travel but was noted to have gone swimming in a swimming pool with signs of algal overgrowth. She was treated with incision and drainage and given a course of oral cephalexin. Although initially responding to antibiotics, over 2 weeks she again developed worsening local edema and erythema. The patient was switched to oral clindamycin but did not show improvement, prompting hospital admission about 3 weeks into her illness. She was administered intravenous clindamycin, and an ultrasound of the area revealed soft-tissue swelling with some patchy areas suggestive of fluid accumulation but without clear signs of an abscess. Incision and drainage was performed, and a drain was placed. Wound cultures were sent and returned positive for Staphylococcus intermedius and Pseudomonas aeruginosa. Based on the culture sensitivities of those organisms, antibiotic therapy was tailored to cefepime, tobramycin, and clindamycin. The patient showed clinical improvement and was discharged on oral ciprofloxacin 1 week after admission.After approximately 5 days, erythema and edema around the wound had visibly increased and the patient was readmitted approximately 1 month after onset of her initial symptoms. She was administered intravenous meropenem and amikacin and incision and drainage was repeated, revealing an abscess measuring 10 cm by 15 cm. A drain was again placed, and wound cultures were sent for growth of bacteria, fungi, and acid-fast bacilli. Histologic sections from debridement material demonstrated an acute and chronic inflammatory infiltrate, interrupted by eosinophilic pools comprised of poorly formed granulomas with central necrosis admixed with neutrophils, eosinophils, and mineralized debris (Fig. 1A). Rare hyphal elements on Gomori methenamine silver stain were identified (Fig. 1B and C). Consequently, amikacin was discontinued, and the patient was started on liposomal amphotericin B (250 mg intravenously [i.v.]

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Hemagglutination Test for Rapid Serodiagnosis of Human Pythiosis

Cited by 45 publications

References 29 publications

The 74-Kilodalton Immunodominant Antigen of the Pathogenic Oomycete Pythium insidiosum Is a Putative Exo-1,3-ß-Glucanase

The 74-Kilodalton Immunodominant Antigen of the Pathogenic Oomycete Pythium insidiosum Is a Putative Exo-1,3-ß-Glucanase

Development of an Anti-Elicitin Antibody-Based Immunohistochemical Assay for Diagnosis of Pythiosis

Molecular Diagnosis of Subcutaneous Pythium insidiosum Infection by Use of PCR Screening and DNA Sequencing

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