Multiplex PCR Assay for Screening of Mycotoxin genes from Ocular Isolates of Fusarium species.

Abstract

Purpose: Fumonisin B mycotoxin is a secondary metabolism of Fusarium species. These toxins can cause cellular toxicity leading to keratolysis and perforation. Prevalence among Fusarium species is not well documented. Our purpose was to identify mycotoxin genes among clinical ocular isolates of Fusarium species and correlate with clinical outcomes of Fusarium keratitis.

Methods: Twenty-nine clinical isolates of Fusarium obtained from 29 eyes of patients were retrieved from the BPEI Ocular Microbiology Laboratory data bank and subcultured on Sabouraud dextrose agar. All of the isolates were previously identified based on DNA sequencing as F. solani (N=23), F. oxysporum (N=5) and F.dimeru (N=1). DNA extraction was done by QIAamp DNA Mini Kit. Multiplex PCRs were run to confirm the identification of Fusarium species (internal transcribed spacer sequence (ITS), translation elongation factor 1-α (TEF) and β-tubulin) and to detect the presence of 3 mycotoxins, fumonisin B biosynthetic genes (FUM1, FUM8), Deoxynivalenol and Nivalenol. Presence or absence of mycotoxins was compared with patient outcomes (presenting VA, time to cure and urgent surgical management) as well MICs for voriconazole.

Results: Eighty six percent (25/29) of the isolates were confirmed as Fusarium species by ITS (3/29)10.3% and TEF (25/29)86.2%. All ITS positive isolates were identified as F. oxysporum. 76% (19/25) of the TEF positive isolates were identified as F. solani. The 6 non F. solani isolated were also confirmed as Fusarium with the TEF primer set. FUM1 mycotoxin genes were detected in 86.2% (N=25/29) of the Fusarium isolates. The highest rate was detected for F. solani (13 F. solani, 5 F.oxysporum and 1 F. dimerum). No FUM8, Deoxynivalenol genes and Nivalenol genes were detected amnong this clinical isolates group. Initial best-corrected VA ranged from 20/25-20/80 in the FUM1 gene negative group and from 20/20-LP in the FUM1 gene positive group. There was no difference in time to cure between the 2 groups. The presence of fumonisin B (FUM1) production was correlated with progression to PK 100% (N=5/5). F. solani was recovered from all of patients requiring PK. The FUM1 gene positive group had higher mean voriconazole MIC valves (9.5µg/ml) than the negative group (3.2µg/ml)

Conclusions: Mycotoxin production (FUM1) may be common among clinical Fusarium isolates and contribute to a more fulminant clinical course and adverse outcomes

Keywords: 530 fungal disease • 573 keratitis • 537 gene screening