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Fungal Keratitis Workup

  • Author: Daljit Singh, MBBS, MS, DSc; Chief Editor: Hampton Roy, Sr, MD  more...
Updated: May 20, 2015

Laboratory Studies

The diagnosis of fungal keratitis continues to be problematic. Many clinical characteristics are not specific to fungal ulcers; therefore, antifungal therapy should be withheld until a diagnosis is confirmed by laboratory studies.

The most important step in the initial management of suspected fungal keratitis is to obtain corneal material for directed smears and inoculation of media. Smears are used to obtain rapid information about the pathogen. Gram stain identifies yeast, and Giemsa stain is useful in detecting fungal elements. However, if fluorescein microscopy is available, acridine orange and calcofluor white are the stains of choice. The primary isolation cultures for fungus are Sabouraud and blood agar at room temperature.

In all patients with suspected fungal keratitis, initial corneal smears and cultures should be performed. Culture media for suspected fungal keratitis should include the same media used for a general infectious-keratitis workup.

  • Corneal scrapings are obtained using a platinum spatula, surgical blade, or calcium alginate swab inoculated on Sabouraud agar plates, and then maintained at 25°C to enhance fungal growth.
  • Brain-heart infusion broth is another medium that can be used.
  • Cycloheximide should not be present in the medium because it inhibits fungal growth.
  • Corneal scrapings provide for initial debridement of organisms and epithelium, which may be a barrier to antifungal penetration.
  • Gram and Giemsa stains of corneal scrapings have sensitivities of about 50% in establishing a diagnosis. Calcofluor white stain (requires a fluorescent microscope) also can identify fungal organisms.
  • Initial growth in culture occurs within 72 hours in 83% of cultures and within 1 week in 97% of cultures.
  • A waiting period of 2 weeks is usually necessary for confirmation of no growth in culture.
  • Culture has been used as the criterion standard to aid ophthalmologists in the diagnosis of fungal keratitis; therefore, the true sensitivity of culture techniques is unknown.

When confronted with a likely fungal ulcer of days or weeks duration, a clinician in a developing country must urgently institute an antifungal treatment regimen; however, the choice of treatment is limited based on the antifungal agents available in that particular market.


Imaging Studies

If clinical evidence or suspicion of posterior segment involvement exists, ophthalmic B-scan ultrasound may be necessary to rule out concurrent fungal endophthalmitis.


Other Tests

The laboratory diagnosis of fungal keratitis may be problematic because of the very small sample obtained by scraping the corneal ulcer. Therefore, recent methods for the identification of fungi have been under study and include immunofluorescence staining, electron microscopy, and confocal microscopy. Confocal microscopy may help in correctly diagnosing early stages of fungal keratitis and in monitoring disease progress at the edges and depth. It may also help guide timely decision for keratoplasty and may be helpful in determining when to stop medication.[2]

The polymerase chain reaction (PCR) technique holds promise as an effective method of diagnosing fungal keratitis because it offers increased sensitivity and significant reduction in the time required to establish a diagnosis.



If corneal smears and cultures are negative at 48-72 hours in a patient who is strongly suspected of having a fungal infection and who is not improving on the initial, broad-spectrum antibacterial therapy, the authors recommend proceeding to a corneal biopsy to establish a diagnosis.

The corneal biopsy specimen should be submitted to the laboratory. A substantial portion also should be submitted for histopathologic examination. The pathologist should be alerted regarding the suspected diagnosis and especially that the specimen is a small piece of cornea.

A negative biopsy result indicates that the destructive corneal process is progressing, and hypopyon exists; therefore, anterior chamber paracentesis or excisional biopsy (keratoplasty) should be performed.


Histologic Findings

Histopathologic examination of corneal buttons can reveal the presence of fungal elements in 75% of patients. Fungal hyphae usually lie parallel to the corneal surface and lamellae. The presence of vertical oriented fungal elements in regard to stromal lamellae depicts high virulence of the organism and usually is associated with more aggressive infection. The Descemet membrane may serve as a partial barrier for invasion of fungal organisms. Penetration of the Descemet membrane by the fungal elements depicts an aggressive organism and a higher risk for contamination of the globe.

Contributor Information and Disclosures

Daljit Singh, MBBS, MS, DSc Professor Emeritus, Department of Ophthalmology, Guru Nanak Dev University; Director, Daljit Singh Eye Hospital, India

Daljit Singh, MBBS, MS, DSc is a member of the following medical societies: American Society of Cataract and Refractive Surgery, Indian Medical Association, All India Ophthalmological Society, Intraocular Implant and Refractive Society, India, International Intra-Ocular Implant Club

Disclosure: Nothing to disclose.


Arun Verma, MD Senior Consultant, Department of Ophthalmology, Dr Daljit Singh Eye Hospital, India

Disclosure: Nothing to disclose.

Specialty Editor Board

Francisco Talavera, PharmD, PhD Adjunct Assistant Professor, University of Nebraska Medical Center College of Pharmacy; Editor-in-Chief, Medscape Drug Reference

Disclosure: Received salary from Medscape for employment. for: Medscape.

Christopher J Rapuano, MD Professor, Department of Ophthalmology, Jefferson Medical College of Thomas Jefferson University; Director of the Cornea Service, Co-Director of Refractive Surgery Department, Wills Eye Hospital

Christopher J Rapuano, MD is a member of the following medical societies: American Academy of Ophthalmology, American Ophthalmological Society, American Society of Cataract and Refractive Surgery, Contact Lens Association of Ophthalmologists, International Society of Refractive Surgery, Cornea Society, Eye Bank Association of America

Disclosure: Serve(d) as a director, officer, partner, employee, advisor, consultant or trustee for: Cornea Society, Allergan, Bausch & Lomb, Bio-Tissue, Shire, TearScience, TearLab<br/>Serve(d) as a speaker or a member of a speakers bureau for: Allergan, Bausch & Lomb, Bio-Tissue, TearScience.

Chief Editor

Hampton Roy, Sr, MD Associate Clinical Professor, Department of Ophthalmology, University of Arkansas for Medical Sciences

Hampton Roy, Sr, MD is a member of the following medical societies: American Academy of Ophthalmology, American College of Surgeons, Pan-American Association of Ophthalmology

Disclosure: Nothing to disclose.


George Alexandrakis, MD Consulting Staff and Surgeon, Department of Ophthalmology, Southern California Permanente Medical Group

George Alexandrakis is a member of the following medical societies: American Academy of Ophthalmology

Disclosure: Nothing to disclose.

Anastasios J Kanellopoulos, MD Assistant Program Director, Clinical Associate Professor, Department of Ophthalmology, Manhattan Eye, Ear, and Throat Hospital, New York University

Anastasios J Kanellopoulos, MD is a member of the following medical societies: American Academy of Ophthalmology, Association for Research in Vision and Ophthalmology, Eye Bank Association of America, and International Society of Refractive Surgery

Disclosure: Nothing to disclose.

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Fungal corneal ulcer.
Perforated fungal ulcer.
Fungal infection under treatment.
Perforated fungal corneal ulcer.
Fungal ulcer in an elderly woman.
Fungal ulcer.
Fungal corneal ulcer, with excessive vascularization.
Marginal ulcer, fungus positive.
Healed fungal ulcer.
Fungal keratitis.
Corneal perforation, blocked by a crystalline lens and being covered by epithelium.
Fungal keratitis, being controlled.
Fungal infection.
Fungal infection.
Fungal abscess.
Fungal corneal abscess/ulcer. A proven case of fungal infection, 5 days' duration. Intense infiltration around the abscess.
Surgical trauma producing edema and striate keratitis. The corneal channels stand out in semiopaque corneal tissue, since they themselves are no-tissue spaces.
Surgical trauma producing edema and striate keratitis. The corneal channels stand out in semiopaque corneal tissue, since they themselves are no-tissue spaces.
A network of channels is visible in a case of megalocornea with faint opacification of stroma. The channels stand out as nonstructures.
This kind of opacification is termed keratitis. Anatomically, it appears to be a microchannel structure.
A network of corneal channels stands out inside the arcus senilis of an old patient. Whatever causes the opacification in the corneal tissue is not able to opacify the emptiness of corneal channels.
Network of corneal channels in a 92-year-old patient.
The corneal channels open in the lucid interval channel of Singh.
Peripheral corneal channel network and canal of Singh in 3 dimensions.
Optical section of corneal channels in a case of arcus senilis.
The lucid interval in optical section clearly shows its triangular configuration and an anterior and posterior wall. The apex continues towards corneal channels in the stroma. The lucid interval channel is connected to limbal lymphatics.
The lucid interval channel is connected to the lymphatics at the limbus and the corneal channels centrally.
A blunt wire of 100 micrometers diameter has been pushed into the canal of Singh.
A 230-micrometer blunt cannula in the canal of Singh.
This networklike pattern of fungal corneal infection is explained only by the preferential path of spread through the corneal channel network.
The fungal infection travels in various directions. Also seen are satellite lesions. Satellite lesions and other appearances are explained by the presence of channels in the cornea.
Notice centrifugal, linear, circular, and satellitelike spread of fungal infection through the corneal channels.
This patient presented with infection of the lucid interval of Singh without any evidence of corneal ulceration as a starting point, suggesting systemic spread. A satellite of infection is seen near the 6-o'clock position.
Same patient with infection of the lucid interval of Singh without any evidence of corneal ulceration as a starting point, suggesting systemic spread. A big and a small satellite at the 6-o'clock position.
The same eye showing the spread of fungal infection on the nasal side of the Singh canal. This patient showed no evidence of corneal injury, thus a systemic origin of infection is a distinct possibility.
The whole of the infected lucid interval canal of Singh was opened. The scraping showed the presence of hyphae. The patient was treated by oral medication, local drops, and intracorneal antifungal voriconazole injections. Final vision was 6/6 uncorrected. There was no recurrence.
Optical coherence tomography scans clearly showing the canal of Singh connected to the Schlemm canal.
Optical coherence tomography scans clearly showing the canal of Singh connected to the Schlemm canal.
Fungal keratitis under treatment. The infection has spread into the nearby lucid interval canal of Singh.
The same case as in the previous photo. Optical coherence tomography scans shows the presence of exudates in the lucid interval canal of Singh and the adjoining trabecular meshwork.
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