Detection of the oomycete Pythium insidiosum by real-time PCR targeting the gene coding for exo-1,3-β-glucanase

Keeratijarut, Angsana; Lohnoo, Tassanee; Yingyong, Wanta; Rujirawat, Thidarat; Srichunrusami, Chutatip; Onpeaw, Pornpit; Chongtrakool, Piriyaporn; Brandhorst, T. Tristan; Krajaejun, Theerapong

doi:10.1099/jmm.0.000117

Cited by 33 publications

(26 citation statements)

References 25 publications

(47 reference statements)

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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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confidence: 99%

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

et al. 2016

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“…In this case and most previous reports, ITS1, ITS2, and 5.8 S rRNA gene are the main target gene amplified and sequenced by PCR [26–29]. Besides, Exo-1,3-β-Glucanase was promoted as a novel target for Pythium insidiosum detection recently [30]. …”

Section: Discussionmentioning

confidence: 59%

Diagnosis and Treatment of Pythium Insidiosum Corneal Ulcer in a Chinese Child: A Case Report and Literature Review

Liu

Chen

et al. 2016

Am J Case Rep

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Patient: Male, 7Final Diagnosis: Pythium insidiosumSymptoms: Painful vision loss • red eyeMedication:—Clinical Procedure: Application of multiple antifungal medicines followed by the penetrating keratoplastySpecialty: OphthalmologyObjective:Rare diseaseBackground:Pythium insidiosum keratitis is a rare but sight-threatening disease with a high morbidity rate. It can be mis-diagnosed as fungal keratitis in clinic settings. We report a case of severe Pythium insidiosum keratitis in a Chinese child, treated with combined approaches.Case Report:A 7-year-old boy from Hainan province in the south of China developed a suppurative corneal ulcer after being in a forest. A mass of hyphae was detected by confocal imaging in vivo, fungal smear test, and histochemical examination. Treatment with Natamycin, fluconazole, and Voriconazole for 1 month was unsuccessful, and a penetrating keratoplasty with anterior vitrectomy was performed. The infection reappeared 1 day after surgery, immediately after which anterior chamber irrigation with 0.02% Fluconazole and amphotericin B solution was performed. Finally, the globe was saved and there was no further recurrent infection. A cultured mycelial organism, which was hard to identify by biomorphology, was confirmed by PCR to be Pythium insidiosum. The zoospores were observed in water environments. The imaging characteristics of P. insidiosum in confocal microscopy are described herein.Conclusions:This is the first case of Pythium insidiosum keratitis reported in China. It can be misdiagnosed as fungal keratitis in the clinic. Improving the awareness of clinicians, promoting early diagnosis, and a multidisciplinary approach, especially early surgery, improve the prognosis.

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“…Besides, the primers, Dx3 and Dx4, did not amplify non-specific target DNA from a variety of fungal species tested, showing 100% detection specificity. Keeratijarut et al introduced an exo 1-targeting real-time PCR assay using the primers, Pr77 and Pr78, for detection of P. insidiosum (Keeratijarut et al, 2015). The detection sensitivity (100%) and specificity (100%) of the real-time PCR was high (Keeratijarut et al, 2015), but shortened the assay turnaround time by eliminating laborious and toxic steps (i.e., gel electrophoresis and ethidium bromide staining).…”

Section: Molecular Analysismentioning

confidence: 99%

Recent update in diagnosis and treatment of human pythiosis

Chitasombat

Jongkhajornpong

Lekhanont

et al. 2020

PeerJ

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Human pythiosis is an infectious condition with high morbidity and mortality. The causative agent is the oomycete microorganism Pythium insidiosum. The pathogen inhabits ubiquitously in a wet environment, and direct exposure to the pathogen initiates the infection. Most patients with pythiosis require surgical removal of the affected organ, and many patients die from the disease. Awareness of pythiosis among healthcare personnel is increasing. In this review, we summarized and updated information on the diagnosis and treatment of human pythiosis. Vascular and ocular pythiosis are common clinical manifestations. Recognition of the typical clinical features of pythiosis is essential for early diagnosis. The definitive diagnosis of the disease requires laboratory testing, such as microbiological, serological, molecular, and proteomic assays. In vascular pythiosis, surgical intervention to achieve the organism-free margin of the affected tissue, in combination with the use of antifungal drugs and P. insidiosum immunotherapy, remains the recommended treatment. Ocular pythiosis is a serious condition and earliest therapeutic penetrating keratoplasty with wide surgical margin is the mainstay treatment. Thorough clinical assessment is essential in all patients to evaluate the treatment response and detect an early sign of the disease recurrence. In conclusion, early diagnosis and proper management are the keys to an optimal outcome of the patients with pythiosis.

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Detection of the oomycete Pythium insidiosum by real-time PCR targeting the gene coding for exo-1,3-β-glucanase

Cited by 33 publications

References 25 publications

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

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

Diagnosis and Treatment of Pythium Insidiosum Corneal Ulcer in a Chinese Child: A Case Report and Literature Review

Recent update in diagnosis and treatment of human pythiosis

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