Development of an Immunofluorescent Staining Assay for Histodiagnosis of Pythiosis

Abstract

Pythiosis is an infectious disease of humans and animals living in tropical and subtropical countries. The etiologic agent is the pathogenic oomycete Pythium insidiosum. The disease has a high rate of morbidity and mortality. Patients usually presented with symptoms associated with arterial or ocular infection. Most patients underwent surgical removal of an infected organs (i.e., legs and eyes) to control the infection. Early and accurate diagnosis is important because it leads to prompt treatment and better prognosis for patients with pythiosis. Here, we developed and evaluated an immunofluorescent staining assay (IFA) for histodiagnosis of pythiosis. Rabbit anti-P. insidiosum antibodies were generated for direct detection of P. insidiosum in paraffin-embedded samples. Sixteen P. insidiosum isolates and 16 other fungi were used to prepare paraffin-embedded culture blocks for diagnostic performance evaluation of IFA. As a result, all culture blocks prepared from P. insidiosum were stained positive, while those prepared from the other fungi (control) were stained negative. IFA was further evaluated using paraffin-embedded tissue blocks prepared from infected tissues of patients with vascular pythiosis (n=3) and other fungal infections (2 Candida albicans, 1 Aspergillus flavus, and 1 Fusarium sp). All infected-tissue blocks from pythiosis patients were tested positive, while those from patients with other mycoses were tested negative. Taken together, the developed IFA provided high diagnostic performance (100% sensitivity and specificity), and could be used to facilitate diagnosis of pythiosis.

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