Molecular Characterisation of Fungi from Mycotic Keratitis and Invasive Infections and Comparison with Conventional Methods

Abstract

Introduction: Fungi can cause systemic or localised infections in human beings, among which mycotic keratitis and Invasive Fungal Infections (IFIs) are very important, which can lead to severe complications and fatal consequences. In some instances, fungal culture isolates may not be identified accurately on the basis of morphology and microscopy. In those cases, molecular characterisation of isolates becomes more helpful in identification, which is essential for providing appropriate antifungal therapy and avoiding bad prognosis. Aim: To identify the fungi collected from patients of mycotic keratitis and IFIs (diagnosed by conventional methods) using molecular methods and to compare the results with the results of conventional methods of identification. Materials and Methods: In the present prospective study, fungal culture isolates were taken from 24 keratomycosis and seven IFIs patients (diagnosed by conventional methods) reporting to the OPD and wards of AIIMS Hospital, Delhi, India (March 2016 to March 2017). Isolates were further subcultured for DNA isolation and amplification for the variable ITS1 and ITS2 regions, and processed for nucleotide sequencing. The obtained sequences for ITS1 and ITS2 regions were searched for homology with NCBI-BLAST program. Results of molecular diagnosis at species level were compared with the conventional methods. Results: Conventional methods could identify Fusarium spp., Alternaria spp., Bipolaris spp., Cladosporium spp., Penicillium spp., Rhizopus spp., up to genus level only, which could be further identified up to species level by molecular methods. Phenotypically identified isolates of Acremonium spp. was identified as Simplicillium spp., Alternaria spp. as Chaetomium globosum, Mucor spp. as Rhizopus oryzae by molecular methods. Conclusion: As prognosis and therapy varies among different genera of fungi and in some cases from species to species, correct species level identification is important; hence, molecular methods may be complemented to traditional methods. Identification of rare fungal isolates is also important in clinical mycology, so that they will not be discarded as contaminants, which is possible using molecular techniques.

Keywords

• fungal keratitis
• invasive fungal infection
• keratomycosis
• nucleotide sequencing