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Evaluation of molecular identification of Aspergillus species causing fungal keratitis
Bader Alshehri1, Manikandan Palanisamy1,2
1Department of Medical Laboratory Sciences, College of Applied Medical Sciences, Majmaah University, Al Majmaah 11952, Saudi Arabia.
Abstract:
Fungal keratitis caused by the species of Aspergillus is a common and leading problem in developing countries like India. In this study, a total of 135 isolates from Aspergillus keratitis were studied by sequence analyses of the internal transcribed spacer (ITS) region performed by nucleotide-nucleotide BLAST analysis followed by the initial identification of the isolates based on conidial and colony morphology. The sequence analysis revealed several unusual species which were never reported in eye infections such as A. tamrii, A. tubingensis, A. braslliensis, A. nomius, A. pseudonomius, A. sydowii, Eurotium amstelodami. The sequence analysis of the ITS region; the β-tubulin and calmodulin genes brought out the genetic diversity among the isolates as the study intended to locate a more sensitive target sequence to study genetic diversity among a set of test fungal isolates. The PCR amplified sequences of the test isolates of the study as well as sequences belonging to section Flavi obtained from Genbank database were compared and analyzed along with three standard isolates by phylogenetic tree (Neighbor-joining) as to find out a target region/gene that could produce a better resolution to differentiate the isolates. Accordingly, the calmodulin gene had provided better resolution compared to ITS and β-tubulin to study the diversity among the test Aspergillus species isolated from fungal corneal ulcer.
Insights
This study identified diverse Aspergillus species causing fungal keratitis in India, including novel species not previously reported in eye infections. Calmodulin gene sequencing proved most effective for differentiating these fungal isolates.
Area of Science:
- Ophthalmology
- Mycology
- Molecular Biology
Background:
- Fungal keratitis, particularly by Aspergillus species, is a significant cause of blindness in developing nations like India.
- Accurate identification of fungal species is crucial for effective treatment and understanding disease epidemiology.
Purpose of the Study:
- To identify and genetically characterize Aspergillus isolates from fungal keratitis cases.
- To evaluate the efficacy of different genetic markers (ITS, β-tubulin, calmodulin) for differentiating Aspergillus species.
Main Methods:
- Morphological identification of 135 Aspergillus isolates.
- Sequence analysis of the internal transcribed spacer (ITS) region, β-tubulin, and calmodulin genes.
- Phylogenetic analysis using Neighbor-joining to assess genetic diversity.
Main Results:
- Identification of common and rare Aspergillus species, including previously unreported species in ocular infections (e.g., A. tamrii, A. tubingensis, A. brasiliensis).
- Calmodulin gene sequencing demonstrated superior resolution in differentiating Aspergillus species compared to ITS and β-tubulin.
- Genetic diversity was observed among the clinical isolates.
Conclusions:
- The study highlights the diverse range of Aspergillus species involved in fungal keratitis.
- The calmodulin gene is recommended as a sensitive molecular marker for studying genetic diversity in Aspergillus keratitis isolates.
