In vivo confocal microscopy ineffective for Fusarium and Aspergillus differentiation

It’s very hard to tell the difference between Fusarium and Aspergillus species with in vivo confocal microscopy (IVCM) of fungal keratitis, researchers say.

However, IVCM remains an effective tool to detect fungal filaments, according to Jaya Devi Chidambaram, London School of Hygiene and Tropical Medicine, London, and colleagues. They published their findings in the British Journal of Ophthalmology.

The incidence of fungal keratitis is increasing throughout the world, with filamentous fungi to blame for more than 60% of the resultant ulcers in warm, humid climates such as India, they reported.

The outcomes vary between Fusarium and Aspergillus, the two primary species of these fungi. Compared with Fusarium, which is more vulnerable to natamycin, Aspergillus is associated with slower re-epthelialisation, increased risk of perforation, and worse visual acuity at 3 months after presentation.

The two fungi also have different shapes. For example, in one previous study, donor corneas infected in vitro with A. fumigatus and Fusarium solani, Fusarium  filaments were reported to have a hyphal branching angle of 90° in IVCM images from patients and from the infected donor cornea. By contrast, in A. fumigatus, the branching angle in the infected donor cornea was measured as 45°.

In Aspergillus, but not Fusarium, the apical filament can directly bifurcate instead of generating side branches, a shape known as dichotomous branching. Fusarium can develop spores in the infected tissue.

Chidambaram and colleagues examined 106 patients culture positive for Fusarium or Aspergillus keratitis to see whether they could distinguish between them using IVCM.

They excluded 8 patients from the IVCM analysis (5 Fusarium, 2 Aspergillus flavus, 1 A. fumigatus) due to the absence of any measurable branching hyphae in the IVCM images. In the remaining 98 participants, 68 were culture-positive for Fusarium, 24 for A. flavus, 4 for A. fumigatus, and 2 for Aspergillus terreus.

They applanated the HRT3 laser scanning confocal microscope with Rostock Corneal Module (Heidelberg Engineering) onto corneas anaesthetized with 0.5% proparacaine eye drops. They manually focused and recorded a series of volume scans at the centres and margins of the ulcers.

Chidambaram, who was masked to the microbiological diagnosis, personally measured all the branching hyphae present in each IVCM image within each section of each volume scan. The researchers assessed the presence or absence of dichotomous branching in all sections.

Findings

They found no significant different between the Fusarium and Aspergillus  groups in gender, ulcer stromal infiltrate size, presence or absence of diabetes mellitus, or prior use of topical antifungals or steroids.

They did find that participants culture positive for Aspergillus had a median age of 54 years compared with 45 years for those culture positive for Fusarium. The Aspergillus patients had a median of 7 days of symptoms, versus 5 for the Fusarium patients. Of the Aspergillus patients, 80%  presented with ulcers involving the posterior third of the cornea, compared with 54% of Fusarium patients.

The mean branching angle for filaments of Fusarium  was 59.7° (95% confidence interval (CI) 57.7° to 61.8 °), compared to 63.3° (95% CI 60.8° to 65.8°) for Aspergillus. The difference was not statistically significant at the 5% level in univariate analysis. After adjustment for age and gender, it became significant.

In a multivariate analysis including the depth of the ulcer as well as age and gender, the branching angle for Aspergillus was 4.8° greater than for Fusarium (95% CI 1.0° to 8.5°, P = 0.012). The researcher also found that deeper ulcers had a branching angle of 4.0° smaller than all others (95% CI -0.3° to -7.7°, P = 0.034.)

The branching angles from all species together were not significantly affected by symptom duration, presence of diabetes mellitus, ulcer size, or prior steroid or antifungal use.

The researchers also found prior antifungal use caused a reduction in branch angle among Fusarium  patients of 5.9°. However, there was no significant effect of antifungal use on branch angle in Aspergillus.

The researchers detected dichotomous branching in 7 ulcers in the Aspergillus group, and only 4 in the Fusarium group (25.9% versus 6.1%). They did not find any spores.

The findings were consistent with histopathological reports, the researchers noted. One study found that 17% of tissue biopsy specimens diagnosed as Aspergillus based on histopathological appearance were actually culture positive for a variety of non-Aspergillus organisms including Fusarium.

A study in tissue biopsies from infected burn wounds showed that 35% of those classified by histopathology as having hyphae consistent with Aspergillus were culture positive for other organisms.

“In summary, we have found very little difference between the hyphal branching angle in IVCM images taken from culture positive Fusarium and Aspergillus spp ulcers,” the researchers concluded.