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Genotypic identification of Fusarium species from ocular sources: comparison to morphologic classification and
1Bascom Palmer Eye Institute, Miller School of Medicine, University of Miami, Miami, Florida, USA.
Purpose:
Ocular infections caused by fungal organisms can cause significant ocular morbidity, particularly when diagnosis and treatment are delayed. Rapid and accurate identification of Fusarium species at the subgenus level using current diagnostic standards is timely and insensitive. The purpose of this study is to examine the usefulness of polymerase chain reaction (PCR) analysis of the internal transcribed spacer (ITS) regions (ITS1, 5.8S, and ITS2) in detecting and differentiating Fusarium species from isolates of ocular infections, and to assess the correlation between the genotypic and morphologic classification.
Methods:
Fifty-eight isolates from 52 patients diagnosed with Fusarium ocular infections were retrieved from storage at the Bascom Palmer Eye Institute's ocular microbiology laboratory. Morphologic classification was determined at both a general and a reference microbiology laboratory. DNA was extracted and purified, and the ITS region was amplified and sequenced. Following DNA sequences, alignment and phylogenetic analysis were done. Susceptibility to antifungal drugs was measured according to the Clinical and Laboratory Standards Institute reference method.
Results:
Sequence analysis demonstrated 15 unique sequences among the 58 isolates. The grouping showed that the 58 isolates were distributed among 4 main species complexes. At the species level, morphologic classification correlated with genotypic classification in 25% and 97% of the isolates in a general microbiology and a reference mycology laboratory, respectively.
Conclusions:
The sequence variation within the ITS provides a sufficient quantitative basis for the development of a molecular diagnostic approach to the Fusarium pathogens isolated from ocular infections. Morphology based on microscopic and macroscopic observations yields inconsistent results, particularly at nonreference laboratories, emphasizing the need for a more reproducible test with less user-dependent variability. Fusarium solani tends to be more resistant to certain antifungals (azoles).
Insights
Polymerase chain reaction (PCR) analysis of internal transcribed spacer (ITS) regions offers a reliable method for identifying Fusarium species in ocular infections. This molecular approach provides more consistent results than traditional morphology, aiding in accurate diagnosis and treatment.
Area of Science:
- Ophthalmology
- Medical Mycology
- Molecular Diagnostics
Background:
- Fungal ocular infections cause significant vision loss, especially with delayed diagnosis.
- Current methods for identifying Fusarium species are slow and inaccurate.
- Accurate identification is crucial for effective antifungal treatment.
Purpose of the Study:
- To evaluate the utility of polymerase chain reaction (PCR) for identifying Fusarium species in ocular infections.
- To compare genotypic identification with traditional morphologic classification.
- To assess the correlation between molecular and morphologic methods.
Main Methods:
- DNA extraction and purification from 58 Fusarium isolates from ocular infections.
- Amplification and sequencing of the internal transcribed spacer (ITS) regions.
- Phylogenetic analysis of DNA sequences and morphologic classification.
Main Results:
- Fifteen unique DNA sequences were identified among the 58 isolates.
- Isolates clustered into 4 main species complexes.
- Morphologic classification correlated with genotypic classification in 25% (general lab) and 97% (reference lab) of isolates.
Conclusions:
- Internal transcribed spacer (ITS) sequencing provides a basis for a molecular diagnostic test for Fusarium ocular pathogens.
- Morphologic identification is inconsistent, highlighting the need for reproducible molecular methods.
- Fusarium solani shows resistance to azole antifungals.
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