1. INTRODUCTION
2. EPIDEMIOLOGY
3. PREDISPOSING FACTORS
4. COMMON ETIOLOGICAL AGENTS
5. PRESENTING SYMPTOMS OF THE PATIENT
6. CLINICAL FEATURES
7. LABORATORY DIAGNOSIS
8. DIFFERENTIAL DIAGNOSIS
9. TREATMENT OF FUNGAL KERATITIS
10. COMPLICATIONS OF FUNGAL KERATITIS
11. BACTERIAL CO - INFECTION DURING FUNGAL KERATITIS
12. CONCLUSION
13. REFERENCES

 

Diseases affecting the cornea are a major cause of blindness all over the world, second only to cataract in overall importance. ¹ A range of infectious and inflammatory eye conditions affect the transparency of the cornea and cause corneal blindness. Infectious keratitis is one of the most common causes of ocular morbidity in the developing world. The etiological agents causing infectious keratitis include bacteria, virus and fungi. Fungal infections are not a common cause but still represent a major cause of infectious keratitis because devastating ocular damage can result if the infection is not diagnosed and treated promptly and effectively.

Fungal infection of the cornea (keratomycosis, mycotic keratitis or fungal keratitis) was first described by Leber in Germany in the year 1879.¹ Fungal keratitis remains a diagnostic and therapeutic challenge to the ophthalmologist. Difficulties are related to establishing a clinical diagnosis, isolating the etiologic fungal organism in the laboratory, and treating the keratitis effectively with topical antifungal agents. Unfortunately, delayed diagnosis is common, primarily because of lack of suspicion; even if the diagnosis is made accurately, management remains a challenge because of the poor corneal penetration and the limited commercial availability of antifungal agents.  

Moreover, the incidence of fungal keratitis has increased over the past 30 years.² This increased occurrence of fungal keratitis is a result of the frequent use of topical corticosteroids and antibacterial agents in treating patients with keratitis, the rise in the number of patients who are immunocompromised, and better laboratory diagnostic techniques that aid in its diagnosis.

 

 

Fungal keratitis is a major blinding eye disease in Asia . One report from South India found that 44% of all central corneal ulcers are caused by fungi. This high prevalence of fungal pathogens in South India is significantly greater than that found in similar studies in Nepal (17%), Bangladesh (36%), Ghana (37.6%), and south Florida (35%). In China , the incidence of fungal keratitis has increased during the past decade. In temperate climates, such as Britain and the northern United States , the incidence of fungal keratitis remains very low. ³

In a study done to review the epidemiological characteristics, microbiological profile, and treatment outcome of patients with suspected microbial keratitis, Out of 5897 suspected cases of microbial keratitis 1360 were fungal (38.2%) and 86, cases were of Acanthamoeba (2.4%). ⁴

 

The risk factors include:

 

Filamentous fungi form the major etiologic agents of fungal keratitis. Fusarium and Aspergillus are the main pathogens . The other most frequently isolated agents includ e Curvurlaria, Helminthosporium, Alternaria, Penicillium and Bipolaris species.

Yeast can also cause keratitis. Gopinathan et al. from India have reported Candida as a rare fungal corneal pathogen (0.7%). ⁵

According to the published literature more than 56 genera of fungi, comprising of over 100 species have been incriminated as the causative agents of fungal keratitis. A careful review of the literature shows the role of various fungal isolates in causing keratomycosis throughout the world, with differing predominant risk factors. ¹

 

Invariably, fungi are not able to penetrate the intact corneal epithelium, unless the eye is severely immunocompromised due to long term use of antibiotics or steroids or due to any other predisposing conditions.

 

 

The clinical appearance of fungal keratitis varies greatly depending on the duration and severity of infection.

 

 

 

 

 

 

Sclerotic scatter can be used to highlight the density and scalloped borders of the fungal lesion. Many fungal ulcers demonstrate no striking morphological pattern, and it often is not possible to differentiate clinically between fungal keratitis and bacterial keratitis.

 

Laboratory diagnosis can be made by means of smear, staining, fungal culture, polymerase chain reaction, and Confocal microscopy. The various clinical samples, for laboratory diagnosis, include (a) corneal scraping (b) corneal biopsy and (c) anterior chamber aspirate.

Corneal Biopsy
It is a relatively invasive (trephining) procedure and requires minor OT. The indications of biopsy are (a) strong clinical suspicion of fungal keratitis (b) at least twice negative smear and culture report (c) no clinical improvement on empiric antibiotic therapy. The biopsied material is preferably removed enbloc. It is bisected, half being sent to microbiology laboratory for homogenization and culture and smear examination, and the remaining half put in 10% buffered formalin for histopathological examination.

Anterior Chamber Aspirate
Anterior chamber (AC) paracentesis is done when there is strong clinical suspicion of intra ocular infection. In addition, progressive corneal damage and persistent hypopyon are also indicative of this procedure. The aspirate is collected with the help of sterile tuberculin syringe and 22 gauge needle. The AC is tapped via the limbus. The needle should be removed before the specimen is submitted in order to decrease the danger to laboratory personnel. However, the nozzle of the syringe should be sealed with a sterile rubber bung and the whole set should be transported immediately to the laboratory for processing.

Processing Of Samples
The scraped out corneal tissue or the biopsied material after homogenization is divided into three portions, one for Gram staining, one for 10% KOH wet mount and the third for culture.

Direct microscopic evaluation is the most valuable and rapid diagnostic tool for the detection of fungal filaments in corneal scrapings. Giemsa stain and Gram stain are equally sensitive in detecting fungal elements. Thomas et al highlighted the sensitivity of various stains. Gram stain will identify fungal species in 45 to 73% of cases, (Figure 4) and Giemsa will identify fungi in 66%.

10% KOH wet mount is simple, cheap, rapid, and easy to interpret even by ophthalmic technicians. It is an ideal method for practice in tropical and developing countries (Figure 5).

Other staining techniques like periodic acid schiff (PAS) staining, Gomori's methenamine silver staining, calcofluor white, acridine orange, fluorescent stainings have also been recommended.

 

 

Figure 4: Branching fungal hyphae
(Gram stain cytoplasm stained with crystal violet, (×450 magnification)

 

 

Culture and Identification
Corneal material is inoculated onto several agar (solid) plates (blood agar, brain–heart infusion agar, cystine tryptone agar, Sabouraud's glucose–neopeptone agar) in the form of ‘C' streaks; only growth occurring on the ‘C' streaks is considered to be significant. Corneal material is also inoculated into broth (liquid) media (Sabouraud broth, brain–heart infusion broth, thioglycollate broth). These culture media can be made more selective for isolation of fungi by addition of antibacterials (gentamicin, chloramphenicol). The media need to be incubated at 30 and 37 ° C; the use of liquid-shake cultures is also advisable. Although fungal growth usually occurs within 3–4 days (Figure 6 and 7) culture media may require incubation for up to 4–6 weeks.

 

 

 

 

Yeasts can be speciated by looking for chlamydospore formation on corn meal agar and germ tube production as well as various sugar fermentation and assimilation tests, urease test and other biochemical tests.

Interpretation of Culture Report
The causative agents of mycotic keratitis are often saprophytic and also the fungal spores are omnipresent, hence interpretation of fungal growth in the laboratory is sometimes difficult. Therefore, in order to attribute clinical significance to a particular growth, the following criteria need to be considered (1) the laboratory finding should be correlated with clinical presentation, (2) inoculation should be done on ‘C' streak manner and growth occurring only on the ‘C' streak is considered significant, (3) smear results should be consistent with culture, (4) the same fungus should grow in more than one culture medium and (5) the same organism should grow from repeated scrapings.

1. The PCR (Polymerase Chain Reaction): It is universally accepted as a most popular technique for the diagnosis of fungal keratitis. It can yield quick results, confirming the diagnosis of mycotic keratitis within a few hours as compared to culturing which takes at least 5 to 6 days for a positive detection. PCR is of great benefit in rapidly detecting the presence of the organism difficult to culture. The sensitivity of PCR, taking culture as the gold standard, was quite high between 89 to 94%, whereas, specificity ranged between 50% to 88%. ¹ However, the main drawback of PCR is its occasional false positivity, but it can be overcome by application of stringency in laboratory procedures and proper standardization of the techniques.

 Modifications of PCR: PCRSSCP (single stranded conformational polymorphism) and PCR-RFLP (restriction fragment length polymorphism) techniques have also been standardized for fungal identification.

2. Confocal Microscopy is a relatively new, noninvasive technique for imaging the cornea in normal and diseased states.

 

Fungal infections are often misdiagnosed as bacterial, herpetic or amoebic infections, so while evaluating the patient a careful history and a review of all the signs and symptoms of the disease are critical. The below mentioned chart displays the characteristic findings that help determine a specific etiology (Table 2).

 

Many clinical characteristics are not specific to fungal ulcers; therefore antifungal therapy is usually withheld, until a diagnosis is confirmed by laboratory studies. Ideally in all patients with suspected fungal keratitis, initial corneal smears and cultures for bacteria and fungus should be performed. It is difficult to diagnose the predisposing factors and specific causative organism in corneal ulcer. A waiting period of 2 weeks is usually necessary for confirmation of specific fungal growth in culture and drug sensitivity takes much longer time, so it is not of much help to treating surgeon.

Hyphae can be easily seen in wet mount KOH preparations and antifungal treatment can be started by seeing hyphae in corneal scrapings. ^{7, 11}

 Currently, the therapy of fungal disease of the eye is unsatisfactory. Most of the available agents only inhibit the growth of the fungus necessitating the host defense mechanisms to eradicate the infection.

The currently used antifungal agents belong to:

(1) Polyenes (Nystatin, Natamycin, Amphotericin B)

(2) Azoles, including Newer Azoles (Clotrimazole, Micaconazole, Ketoconazole, Fluconazole, Itraconazole, Voriconazole)

(3) Pyrimidines (Fluorocytosine)

(4) Other Derivatives (Echinocandins, Povidone-iodine (Betadine) and Polyhexamethyl biguanide (PHMB) )

1.       Topical natamycin (5%) or amphotericin B 0.15% is usually selected as first-line therapy for superficial keratitis, whether or not septate hyphae or yeast cells have been seen by direct microscopy. The presence of deep lesions necessitates the addition of some form of systemic therapy, such as subconjunctival or intravenous miconazole, oral ketoconazole, oral itraconazole, or oral fluconazole.

2.       If hyphae are definitely seen by microscopy, topical natamycin (5%) is the drug of choice (0.15% amphotericin B is another option).

3.       If yeasts or pseudohyphae are seen on microscopy, topical 0.15% amphotericin B, fluconazole 2%, natamycin 5%, other azoles at 1% in arachis oil, or 1% Flucytosine eye drops can be used.

4.       Econazole (1%) was recommended by some workers in the 1970s but there are no recent reports about the use of this compound as first-line therapy for mycotic keratitis.

5.       Topical therapy is usually applied hourly around the clock for several days and the frequency of application is then gradually reduced; ‘topical saturation therapy' has also been advocated.

6.       Some form of systemic therapy (oral azoles, intravenous miconazole) is usually included for severe keratitis.

Since mycotic keratitis usually responds slowly over a period of weeks to antifungal therapy, clinical signs of improvement should be carefully noted: these include diminution of pain, decrease in size of infiltrate, disappearance of satellite lesions, rounding out of the feathery margins of the ulcer and hyperplastic masses or fibrous sheets in the region of healing fungal lesions. Negative scrapings during treatment do not always indicate that the fungal infection has been eradicated, since there may be active proliferation of the fungi deep in the stroma; hence, therapy should be continued for at least 6 weeks.

Newer Azoles
Voriconazole is a new azole with broad spectrum efficacy for fungal keratitis and endophthalmitis. In a recent study, 50 invitro susceptibility of various fungal isolates in infectious keratitis towards voriconazole was 100%, Ketoconazole 82.4%, Amphotericin-B 76.5%, Itraconazole 67%, Fluconazole 60% and 5-Fluorocytocin 60%. Voriconazole MIC (90) was lowest for Candida species (0.016 mg/ml). They concluded that voriconazole was a better alternative for the therapeutic management of Candida and Aspergillus ocular infections, as compared to other antifungal. ¹

Echinocandins: These drugs have recently emerged as valuable antifungal agents. These are cell wall acting agents unlike Amphotericin-B which acts on fungal cell membranes. These drugs inhibit B 1- 3 glucan synthesis, and include Caspofungin and Micafungin.

Povidone-iodine (Betadine) and Polyhexamethyl biguanide (PHMB): The effectiveness of Povidone iodine and PHMB as topical antifungals was evaluated by a study group in India in experimentally induced Aspergillus fumigatus keratitis in rabbits. The study concluded that 1% Betadine was not effective in fungal keratitis while PHMB 0.02% was moderately effective .

Surgical Therapy
Surgery also helps in supporting the globe, where integrity of the globe is threatened as in case of thinning or perforation of the cornea. If there is persistent epithelial defect, the usual recommendation is a Superficial Lamellar Keratectomy with removal of necrotic stroma and placing a thin conjunctival flap over the ulcerated site. Blood vessels present on the conjunctival flap help in rapid healing of the ulcer.

However, in situations where there is keratitis with deep stromal lesions or progressive keratitis with corneal perforation not responding to antifungal treatment, a penetrating keratoplasty is recommended. In this procedure, at least 0.5mm of clear corneal tissue is excised all around the infected area in order to decrease the chances of recurrence . The advantage of this procedure includes the elimination of fungi simultaneously with the secondary inflammatory reaction. Topical treatment with antimycotics is recommended for several days post- operatively. An alternative therapy may be the use of excimer laser at 193 nm.

 

The problems that are frequently encountered include:

1.       Pain - sudden increase and decrease

2.       Severe anterior chamber reaction

3.       Sudden increase in the ocular surface reaction despite medications

1. Pain : This is a normal protective mechanism associated with any lesion anywhere in the body. However, if a patient complains of a sudden or severe increase in pain, one needs to consider secondary glaucoma. This is usually due to the secondary changes in the anterior chamber. They include hypopyon and exudates on the endothelial surfaces causing a block of the trabecular meshwork.

1.1 Perforation
Sudden reduction of pain in a patient with an active uncontrolled keratitis should be looked upon with suspicion rather than relief. Many a times due to the extensive exudation and/or accumulation of the medications (especially antifungal), the underlying wound is hidden. This can lead to a false sense of security and the underlying thinning may be missed.

2 . Severe anterior chamber reaction: In severe fungal infection, the Descemet's membrane can fail to limit the infection to the corneal stroma. Alternatively, the severe anterior chamber reaction secondary to the toxic effects of the microbe can lead to a complete organized hypopyon formation in the anterior chamber.

 3 . Sudden increase in the ocular surface reaction despite medications: Many a times one sees that the corneal ulcer is responding to the medications for first 24-48 h. After this period, the eye becomes significantly redder, there is lid edema and the conjunctival surface too starts to stain with fluorescein. This may be a sign of spreading infection. However, it is more likely to either be an allergic reaction to one of the medications being used or the occurrence due to keratitis medicamentosa.

Other severe complications include:

·         Loss of the eye

·         Severe vision loss

·         Scars on the cornea

 

According to a study, Yeast keratitis can increase the risk of a bacterial co-infection. The risk is more than threefold compared with filamentous fungal keratitis.

Kirk R. Wilhelmus, MD, and colleagues looked at the propensity of keratomycosis for parallel or secondary bacterial infection and the relationship between fungal and bacterial co-isolates. They retrospectively reviewed laboratory records covering 24 years, and found 152 episodes of culture positive fungal keratitis.

The researchers found that 30 keratomycoses cases (20%) met laboratory requirements for polymicrobial infection. Staphylococci were relatively prevalent during fungal keratitis, comprising half of the patients' bacterial co-isolates. It was also observed that some bacterial groups selectively paired with particular fungal infections suggesting that co-pathogens may form a mutual alliance.

The study concluded that Bacterial co-infection can “sometimes complicate fungal keratitis, especially candidiasis.

 

Fungal keratitis is responsible for a significant burden of blinding disease in the developing world. Current treatment methods frequently fail to preserve or restore vision after fungal keratitis. Although emerging antifungal agents show promise, therapeutic gaps will probably persist, and further development is necessary. Priorities should be given to develop and undertake drug trials against filamentous fungal keratitis.

 

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2.       http://emedicine.medscape.com/article/1194167-overview, Last accessed on 6 th July 2010

3.       M. Srinivasan. Fungal keratitis. Curr Opin Ophthalmol 2004; 15:321–327

4.       Usha Gopinathan, Savitri Sharma, Prashant Garg, Gullapalli N Rao. Review of Epidemiological features, microbiological diagnosis and treatment outcome of microbial keratitis: Experience of over a decade. Indian J Ophthalmol:2009;57:273-279

5.       Gopinathan U, Garg P, Fernandes M, et al. The epidemiological features and laboratory results of fungal keratitis: a 10-year review at a referral eye care center in South India. Cornea 2002, 21:555–559

7.       Yashika inderjeet. Review of fungal keratitis. Continuing Professional Development article. Page 1-4

8.       PA Thomas. Fungal infections of the cornea. Eye (2003) 17, 852–862

9.       Vinaykumar Agrawal, MS. First aid for complications of infectious keratitis. Indian J Ophthalmol. 2008 May–Jun; 56 (3) : 221–222

10.    J C Pate, D B Jones, K R Wilhelmus. Prevalence and spectrum of bacterial co-infection during fungal keratitis. Br J Ophthalmol 2006; 90:289–292

11.    V. K. Sharma, M. Purohit & S. Vaidya. Management of Mycotic Keratitis. The Internet Journal of Ophthalmology and Visual Science. 2009; Volume 6: Number 2