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

Chareonsirisuthigul, Takol; Khositnithikul, Rommanee; Intaramat, Akarin; Inkomlue, Ruchuros; Sriwanichrak, Kanchana; Piromsontikorn, Savittree; Kitiwanwanich, Sureewan; Lowhnoo, Tassanee; Yingyong, Wanta; Chaiprasert, Angkana; Banyong, Ramrada; Ratanabanangkoon, Kavi; Brandhorst, T. Tristan; Krajaejun, Theerapong

doi:10.1016/j.diagmicrobio.2013.02.025

Cited by 45 publications

(52 citation statements)

References 14 publications

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“…Statistical analysis. Detection sensitivity, detection specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy were calculated using Microsoft Excel 2013 software (12).…”

Section: Methodsmentioning

confidence: 99%

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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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Section: Methodsmentioning

confidence: 99%

“…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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“…The serum sample were tested (in duplicate) to detect the anti-P. insidiosum antibodies using an established protein A/G-based ELISA [11] with some modi cations. Brie y, a 96-well polystyrene plate (Corning) was coated with 100 µl of 5 µg/ml culture ltrate antigen (prepared from the human-isolated P. insidiosum strain Pi-S [24]) in 0.1 M carbonate buffer (pH 9.6) and 1.5% NaCl (4 ºC overnight), washed 4 times with PBS and 0.05% tween-20 (PBS-T), blocked with 250 µl of 0.5% bovine serum albumin (Merck) in PBS (37 ºC for 80 min), and washed 4 times again with PBS-T. A diluted serum sample (1:1,600 in PBS; 100 µl) was incubated in each well (37˚C for 1 hr) and was washed 4 times with PBS-T. The peroxidase-conjugated protein A/G (Bio-Rad) (1:100,000 in PBS; 100 µl) was added to each well and incubated at 37˚C for 1 hr.…”

Section: Methodsmentioning

confidence: 99%

“…The appearance of both test and control lines indicated an ICT-positive result, while the presence of only the control line indicated an ICT-negative result. For Western blot analysis, CFA of P. insidiosum [24] was separated by SDS-PAGE (4% stacking and 12% resolving gels) using a Bio-Rad MiniProteon II apparatus (setting: 100v for 90 min) and blotted onto anitrocellulose membrane (Bio-Rad) using a Bio-Rad Mini Trans-Blot apparatus (setting: 100v for 60 min). The blotted membrane was blocked with 5% skimmed milk in PBS with 0.1% Tween-20 (PBS-T) at room temperature for 60 min, and incubated with a serum sample, diluted 1:2,000 in 1% skimmed milk in PBS-T, at room temperature for 3 hr.…”

Section: Methodsmentioning

confidence: 99%

An initial survey of 150 horses from Thailand for anti-Pythium insidiosum antibodies

Htun

Laikul

Pathomsakulwong

et al. 2020

Preprint

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Objectives: Pythium insidiosum causes a deadly condition, called pythiosis, in humans and other animals. The organism has been identified in tropical and subtropical environments worldwide. Since 1985, human pythiosis has been increasingly reported from Thailand. Seroprevalence studies estimated that ~32,000 Thai people have been exposed to the pathogen. In 2018, the first animal pythiosis case in Thailand was diagnosed in a horse. In this study, we surveyed anti-P. insidiosum antibodies in a sample of the Thai equine population. Results: Serum samples were available from 150 out of 6,353 registered horses distributed across Thailand. ELISA detected the anti-P. insidiosum antibodies in three horses. Immunochromatography and Western blot confirmed the presence of the antibodies in one of the ELISA-positive horses. Based on one positive out of 150 tested, the Pythium seroprevalence in the Thai equine population was 0.7%, which is 10 times higher than that of the Thai human population. The seroprevalence of the anti-P. insidiosum antibodies in a small sample of horses suggests a higher incidence of pythiosis in horses than in humans. Larger studies will be necessary to obtain the full picture of the epidemiology of animal pythiosis in Thailand.

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“…A number of assays are available for facilitating diagnosis of pythiosis, such as culture identification and immunodiagnostic tests (Chareonsirisuthigul et al, 2013;Keeratijarut et al, 2009;Vanittanakom et al, 2004). Culture identification is a time-consuming and relatively insensitive technique.…”

Section: Introductionmentioning

confidence: 99%

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

Keeratijarut

Lohnoo

Yingyong

et al. 2015

Journal of Medical Microbiology

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Pythiosis is a life-threatening infectious disease caused by Pythium insidiosum. Early and accurate diagnosis is the key to prompt treatment and an improved prognosis for patients with pythiosis. An alternative to microbiological and immunological approaches for facilitating diagnosis of pythiosis is the PCR-based assay. Until recently, the ribosomal DNA (rDNA) region was the only target available for PCR-based detection of P. insidiosum. Failure to detect P. insidiosum by PCR amplification using the rDNA-specific primers has been reported. PinsEXO1, encoding an exo-1,3-b-glucanase, is an alternative, novel and efficient target for identification of P. insidiosum by conventional PCR. In this study, we aimed to develop a realtime (RT)-PCR approach targeting PinsEXO1 and compare its performance with conventional PCR for the detection of P. insidiosum. Both conventional and RT-PCR assays were positive for all 35 P. insidiosum strains tested, whilst all 58 control fungi were negative. The turnaround time for conventional PCR was 10 h, whilst that for RT-PCR was 7.5 h. The lowest amounts of genomic DNA template required for successful amplification by conventional and RT-PCR were 1 and 1610 24 ng, respectively. In conclusion, the RT-PCR assay retained 100 % sensitivity and 100 % specificity for detection of P. insidiosum. It showed a substantially improved analytical sensitivity and turnaround time that could improve diagnosis of pythiosis. The assay could also facilitate quantitative DNA analysis and epidemiological studies of P. insidiosum.

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

Cited by 45 publications

References 14 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

An initial survey of 150 horses from Thailand for anti-Pythium insidiosum antibodies

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

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