Immunological Cross-Reactivity of Proteins Extracted from the Oomycete Pythium insidiosum and the Fungus Basidiobolus ranarum Compromises the Detection Specificity of Immunodiagnostic Assays for Pythiosis

Abstract

Pythiosis, a life-threatening disease caused by Pythium insidiosum, has been increasingly diagnosed worldwide. A recently developed immunochromatographic test (ICT) enables the rapid diagnosis of pythiosis. During the 3-year clinical implementation of ICT in Thailand, we collected the laboratory reports of 38 animals with suspected pythiosis and detected ICT false-positive results in three horses and a dog with basidiobolomycosis. P. insidiosum and Basidiobolus ranarum cause infections with indistinguishable clinical and microscopic features. This study investigated cross-reactive antibodies by probing P. insidiosum and B. ranarum crude extracts and cell-free synthesized I06 protein (encoded in P. insidiosum genome, not other fungi) against a panel of pythiosis, basidiobolomycosis, rabbit anti-I06 peptide, and control sera by Western blot analyses. ICT false-positive results occurred from the cross-reactivity of anti-B. ranarum antibodies to the 15, 50, 60, and 120 kDa proteins of P. insidiosum, not double infections caused by both pathogens. Notably, ICT could help to screen pythiosis, and the positive test requires confirmation by culture or molecular method. The detection specificity of ICT requires improvement. The crude extract containing multispecies antigens needs replacement with a refined P. insidiosum-specific protein. We proposed that the 55 kDa I06 protein is an excellent candidate for developing a more specific serodiagnostic test for pythiosis.

Pythiosis presenting with chronic swelling and painful subcutaneous lesion at right deltoid

“…This supports the value of serologic tests in the diagnosis of pythiosis. However, false positive results for ICT have been reported in animal with basidiobolomycosis [14] .…”

PacBio long read-assembled draft genome of Pythium insidiosum strain Pi-S isolated from a Thai patient with pythiosis

“…This study aims to obtain a better-quality genome of P. insidiosum using the PacBio long-read. The P. insidiosum strain Pi-S was selected because it has been widely referred to in recent immunological, genomic, transcriptomic, and proteomic studies [8, [14][15][16][17][18][19][20]. The short-read version of the strain Pi-S genome data shows 53.2 Mb in size and comprises 1,192 contigs (average length: 44,664 bases; N 50 : 146,252 bases), 52% G-C content, 10% N composition, and 14,962 open reading frames (ORFs) [8].…”

History and Perspective of Immunotherapy for Pythiosis

“…The P. insidiosum genome data [282][283][284][285][286][287] makes it possible to clone the coding sequence for recombinant protein production or synthesize an immunoreactive peptide of the identified immunogens. Several P. insidiosum proteins, such as exo-1,3-beta-glucanase (Exo1), elicitin (ELI025), and OPEL-like protein (I06), have been successfully cloned, expressed, and purified from bacterium-based, and cell-free, protein biosynthesis systems [275,277,279,288]. These recombinant proteins could serve as unlimited reproducible antigen sources for the future development of a protein subunit for preventing and treating pythiosis.…”