**3. Recent advances in diagnosis**

### **3.1 Clinical diagnosis**

Fungal keratitis can be diagnosed based on characteristic clinical features. Patients with keratitis usually present with sudden onset of pain, photophobia, watering and discharge and reduced vision. In fungal keratitis, symptoms are much milder than the signs [53].

A fungal keratitis classically presents as a dry, raised lesion with crenate or feathery borders, presence of satellite lesions and a hypopyon. An immune ring of Wesseley may be visible due to deposition of immune complexes and inflammatory cells around the ulcer (**Figures 1**–**5**). However, a study reported that Clinicians could correctly distinguished the microbial kingdom for 54 (73%) of 74 culture-positive infections, including 41 (79%) of 52 bacterial keratitis, 5 (38%) of 13 fungal

#### **Figure 1.**

*Plaque-like ulcer with slightly defined margins, marked conjunctival injection and chemosis; fungal isolatecandida albicans.*

*Fungal Keratitis: Recent Advances in Diagnosis and Treatment DOI: http://dx.doi.org/10.5772/intechopen.98411*

#### **Figure 2.**

*A dry looking lesion with greyish white raised exudate appearing as plaque with hypopyon in a 56-year-old male with fugal keratitis from Aspergillus.*

#### **Figure 3.**

*A greyish white infiltrate with feathery borders and a satellite lesion in a case fungal keratitis caused by Fusarium.*

#### **Figure 4.** *Severe fungal keratitis with feathery edges in case Fusarium Keratitis.*

#### **Figure 5.**

*Corneal thinning and necrosis in severe fungal keratitis caused by Fusarium in a 48-year-old male with history of topical steroid instillation.*

keratitis, and 8 (89%) of 9 amoebic keratitis correctly [54]. In a photographic survey, clinician were able to distinguish between bacterial and fungal aetiologies 66% of the time. In 39 cases of fungal keratitis, the clinicians predicted genus in 27% of cases and species in 7.9% of cases [55].

#### *3.1.1 Confocal microscopy*

In vivo confocal microscopy (IVCM) of the cornea has been emerged as clinically useful non-invasive technique for early diagnosis of FK. It produces images from the cornea with a resolution of one micrometer (μm), which is enough for imaging of microorganisms larger than one μm, such as Acanthamoeba cysts and fungal hyphae [56]. This provides rapid and reliable diagnosis however, a clinical consensus in the interpretation of IVCM images is still lacking.

IVCM can directly visualize filamentous fungi within the whole cornea of patients. Confocal microscopy in vivo uses serial images to create optical sections through the full-thickness of the living cornea. It allows rapid identification of fungi and can be used to differentiate between fungal species.

Brasnu et al. diagnosed all the cases of suspected fungal keratitis (five out of five) caused by different fungal species using IVCM with sensitivity equal to the direct microscopy and culture [56]. They analyzed IVCM images of keratitis obtained using the Heidelberg Retina Tomograph (HRT) II confocal microscope (Heidelberg Engineering, Heidelberg, Germany) in five patients (four patients with *Fusarium soloni* and one patient with *Candida albicans* infection), and three donor corneas with Aspergillus fumigatus, *F. solani*, and *C albicans* infection. *F. soloni* hyphae seen as high contrast lines 3–5 microns (µm) in diameter, 200–300 µm in length, with a branching angle of 90° in IVCM images from patients as well as from the infected donor cornea. *A. fumigatus* hyphae seen as numerous high-contrast lines 200–300 µm in length and 3–5 µm in width, with the branching angle 45° in the infected donor cornea. *C albicans*-infected patient's cornea revealed numerous high-contrast elongated particles measuring 10–40 µm in length and 5–10 µm in width. *C albicans*-contaminated donor cornea revealed numerous characteristic high-contrast elongated particles measuring 10–40 µm in length and 5–10 in µm in width, consistent with Candida pseudofilaments [56].

The hyper-reflective elements seen on IVCM must be differentiated from the basal corneal epithelial nerves, which have a more regular branching pattern. Stromal nerves, on the other hand, are much larger in diameter (25–50 μm). There are now several studies reported the use of IVCM in diagnosis and monitoring of treatment of fungal keratitis with reported sensitivity of 80–94% [57–60].

IVCM is a noninvasive in vivo technique useful for early identification of fungal elements, monitoring and guidance of treatment, and determination of the depth of infection. The limitation of IVCM are that technique is extremely user-dependent, need a skilled operator and experienced viewer. The dense corneal infiltrates or scarring could preclude proper tissue penetration and visualization.

#### **3.2 Laboratory diagnosis**

Conventional methods for the diagnosis of fungal keratitis include staining of tissue scrapings with Gram-stain, 10% potassium hydroxide (KOH) wet mount, lactophenol cotton blue, Giemsa, or calcofluor white. Reported sensitivity of Gram staining is in the range of 36–50% [61]. KOH is a rapid and an inexpensive and one of the most commonly performed procedures for detection of fungi with a sensitivity of 61–94% and specificity of 91–97% for detecting fungus (**Figure 6**).

*Fungal Keratitis: Recent Advances in Diagnosis and Treatment DOI: http://dx.doi.org/10.5772/intechopen.98411*

**Figure 6.** *Fungal hyphae in KOH wet mount counterstained with methylene blue.*

Lactophenol cotton blue mounts had reported sensitivity of 85% and specificity of 90–91% [62]. Sabouraud dextrose agar medium is considered as a culture medium of choice for isolating fungi however it cause delay in diagnosis. Initial growth occurs within 72 hours in 83% of cultures and within 1 week in 97% of cultures [63]. Sometimes it may be necessary to wait for two weeks to confirm no growth in culture. Over the last decade, a number of newer methods have been devised for detection of fungi.
