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

Abstract

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.

Recent update in diagnosis and treatment of human pythiosis

“…As opposed to the standard stains (i.e., GMS, PAS and H&E), several immunohistological staining assays have been developed to facilitate microscopic detection of P. insidiosum (Inkomlue et al, 2016;Keeratijarut et al, 2009;Triscott, Weedon & Cabana, 1993;Mendoza, Ajello & McGinnis, 1996;Mendoza, Kaufman & Standard, 1987;Brown et al, 1988). These assays rely on the use of rabbit antiserum generated against crude protein extract of P. insidiosum.…”

“…A novel immunohistological method targeting elicitins that are only present in oomycetes, including P. insidiosum (Jiang et al, 2006;Lerksuthirat et al, 2015), has been reported to be highly specific. Recently, Inkomlue et al used a rabbit antiserum against recombinant elicitin (ELI025) of P. insidiosum for the development of an immunohistochemical assay (Inkomlue et al, 2016), and found that all 38 P. insidiosum samples were detected, but not 49 control samples of various fungi, including Fusarium species and Mucorales.…”

“…Resected vessel and soft tissue specimen sent for histological examination should be labelled-proximal or distal-in order to identify the disease-free surgical margin by Grocott-Gömöri methenamine silver (GMS) staining (Sermsathanasawadi et al, 2016). Further immunohistochemistry staining for P. insidiosum is helpful to confirm the diagnosis, especially if the standard microbiological culture is not available or fails to identify the pathogen (Inkomlue et al, 2016;Keeratijarut et al, 2009).…”

Assessment of matrix-assisted laser desorption ionization-time of flight mass spectrometry for identification and biotyping of the pathogenic oomycete Pythium insidiosum

“…Identification of P. insidiosum cannot rely solely on microscopic characteristics because this organism shares filamentous morphologies with true fungi (i.e., Aspergillus species, Fusarium species, and fungal species of the class Zygomycetes). Several immunodiagnostic tests (i.e., immunodiffusion, Western blot, hemagglutination, enzyme-linked immunosorbent assay, immunochromatography, and immunohistostaining assay) have been developed for direct detection of the anti-P. insidiosum antibodies in serum samples or the P. insidiosum hyphae in an infected tissue (Pracharktam et al, 1991;Mendoza et al, 1997;Krajaejun et al, 2002Krajaejun et al, , 2006aKrajaejun et al, , 2009Jindayok et al, 2009;Keeratijarut et al, 2009Keeratijarut et al, , 2013Supabandhu et al, 2009;Chareonsirisuthigul et al, 2013;Inkomlue et al, 2016;Intaramat et al, 2016;Lohnoo et al, 2018). In addition, a number of molecular-based assays (i.e., polymerase chain reaction and sequence homology analysis) can be used to detect the specific DNA sequence of P. insidiosum in clinical samples (Badenoch et al, 2001;Znajda et al, 2002;Vanittanakom et al, 2004;Krajaejun et al, 2011;Keeratijarut et al, 2014Keeratijarut et al, , 2015Rujirawat et al, 2017).…”

Biochemical and genetic analyses of the oomycetePythium insidiosumprovide new insights into clinical identification and urease-based evolution of metabolism-related traits

“…Isolation of the pathogen from infected tissues by the standard microbiological procedure is time-consuming and requires experience ( Chaiprasert et al, 1990 ). A number of detection tools such as serological tests ( Pracharktam et al, 1991 ; Krajaejun et al, 2002 , 2006a , 2009 ; Grooters et al, 2002 ; Jindayok et al, 2009 ; Supabandhu et al, 2009 ; Chareonsirisuthigul et al, 2013 ; Keeratijarut et al, 2013 ; Intaramat et al, 2016 ), immunostaining assays ( Keeratijarut et al, 2009 ; Inkomlue et al, 2016 ), and molecular biology methods ( Grooters & Gee, 2002 ; Botton et al, 2011 ; Keeratijarut et al, 2014 , 2015 ; Rujirawat et al, 2017 ), have been successfully developed for P. insidiosum infection. However, such tools are not generally available in non-reference clinical laboratories, resulting in missed or delayed diagnosis of pythiosis.…”