Genotyping and phylogenetic analysis of Pythium insidiosum causing human corneal ulcer

Pythium insidiosum belongs to the Oomycetes, which are known to cause serious life-threatening infectious condition in humans and animals. Corneal infections caused by P. insidiosum are rare and difficult to treat. The molecular-based diagnosis of Pythium is employed for the species identification and to study molecular phylogenetic relationship. Based on Cytochrome oxidase II (cox II) gene, P. insidiosum is categorized into three clades or groups: Clade-I or ATH (American strains), Clade-II or BTH (American, Asian, and Australian strains), and Clade-III or CTH (mostly Thailand strains). This study focused on the molecular identification of Pythium insidiosum from patients with corneal ulcer using ITS regions and clade identification by cox II gene sequencing and correlated with the clinical outcome. The isolates were collected from Aravind Eye Hospital, Madurai, India, from April to December 2018. Through the microbiological laboratory reports, 15 isolates of Pythium sp. from keratitis patient were selected, followed by DNA extraction, ITS, and cox II gene sequencing and phylogenetic analysis using the reference sequences from NCBI database. All 15 P. insidiosum isolates were phylogenetically clustered together as a single group and where also placed distantly from other Pythium species (outgroup). Most ocular isolates fell into either clade BTH or clade CTH, and none of our ocular isolates were in clade ATH. Two of the strains were very distinct and did not match any of the clusters indicating different lineages. There was no significant difference between clinical outcome and genotype of P. insidiosum.