New Strategy for the Diagnosis of Keratomycosis and Fungal Endophthalmitis

Abstract

 

Purpose:

 

Keratomycosis (KM) and fungal endophthalmitis (FE) may lead to visual impairment,and rapid diagnosis and on time treatments are essential for prognosis improvement. Fungal culture may require several days and previously described PCRs are difficult to be run routinely. The goal of this work was to design and validate a new strategy to detect fungi associated with KM and FE, with no need of any kind of post amplification procedures

 

Methods:

 

Corneal samples were divided in 2 aliquots: the first dipped into Sabouraud’s agar and the second kept dry. Fungi were isolated and characterized according to routine methods. An internal control introduced into each sample was extracted simultaneously with Magnapure after lyticase treatment. The primers for r-t-PCR were chosen by combinatory analysis of conserved sequences of Candida sp. [18S ribosomal RNA gene; internal transcribed spacer 1, 5.8S ribosomal RNA gene, and internal transcribed spacer 2,; and 28S ribosomal RNA gene]; Fusarium sp. and Aspergillus sp. [18S rRNA gene, 5.8S rRNA gene, 28S rRNA internal transcribed spacer 1 (ITS1) and transcribed spacer 2 (ITS2)]

 

Results:

 

The selected sequences presented in Talbe1 detect 1 colony or less of KM and FE associated fungi.

 

Conclusions:

 

The in-silico analysis predicted sequences for an inexpensive FK and FE molecular diagnosis tool. The cost are dramatically reduced because there is no need for fluorophore-labelled TaqMan probes. The High Resolution Melting technology (HRM) may benefit from this approach, allowing in 60 minutes detecting and identifying fungi using one pair of primers. The high sensitivity and specificity for these primers using the SYBR green approach should serve as proof of concept for the development of future HRM assays.  

 

Keywords: keratitis • fungal disease • microbial pathogenesis: clinical studies