Case 4 -

Acanthamoeba/Staphylococcus Aureus/Candida Parapsilosis/Aspergillus Fumigatus/Fusarium Species/Herpes Virus Lynn Schoenfield, Cleveland Clinic, Cleveland, OH

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Clinical History Case 4a: 49 year old male with presumed fungal endophthalmitis. Case 4b: 82 year old female with corneal melt syndrome. Case 4c: 74 year old male with HPN and DM with previous corneal transplant and expected endophthalmitis. He subsequently required enucleation, which revealed endophthalmitis and necrotizing keratitis. Case 4d: 79 year old male with perforated ulcer. He subsequently required enucleation. Case 4e: 59 year old female with keratitis and history of contact lens use. Case 4f: 80 year old female with chronic corneal ulcer. Case 4a - Slide 1 Acanthamoeba: H&E and GMS stains showing double- walled cysts measuring approximately 20 microns in size. Click to view with ImageScope Click to view with a Web-Based Viewer Case 4a - Slide 2 Acanthamoeba: H&E and GMS stains showing double- walled cysts measuring approximately 20 microns in size. Click to view with ImageScope Click to view with a Web-Based Viewer Case 4b - Slide 1 Staphylococcus aureus: H&E and gram stains, the latter showing few clusters of gram positive cocci. Click to view with ImageScope Click to view with a Web-Based Viewer Case 4b - Slide 2 Staphylococcus aureus: H&E and gram stains, the latter showing few clusters of gram positive cocci. Click to view with ImageScope Click to view with a Web-Based Viewer Case 4c - Slide 1 Candida parapsilosis: H&E and GMS stains showing yeasts and keratitis. Click to view with ImageScope Click to view with a Web-Based Viewer Case 4c - Slide 2 Candida parapsilosis: H&E and GMS stains showing yeasts and keratitis. Click to view with ImageScope Click to view with a Web-Based Viewer Case 4c - Slide 3 Candida parapsilosis: H&E and GMS stains showing yeasts and keratitis. Click to view with ImageScope Click to view with a Web-Based Viewer Case 4d - Slide 1 Aspergillus fumigatus: H&E and PAS stains showing necrotizing keratitis with ulcer and associated septated fungi. Click to view with ImageScope Click to view with a Web-Based Viewer Case 4d - Slide 2 Aspergillus fumigatus: H&E and PAS stains showing necrotizing keratitis with ulcer and associated septated fungi. Click to view with ImageScope Click to view with a Web-Based Viewer Case 4e - Slide 1 Fusarium species: H&E and PAS stains showing necrotizing keratitis with ulcer and associated large irregular septated fungi. Click to view with ImageScope Click to view with a Web-Based Viewer Case 4e - Slide 2 Fusarium species: H&E and PAS stains showing necrotizing keratitis with ulcer and associated large irregular septated fungi. Click to view with ImageScope Click to view with a Web-Based Viewer Case 4f - Slide 1 Herpes virus: H&E stain and immunoperoxidase stain for Herpes virus showing typical multinucleated giant cells with intranuclear inclusions which are positive for Herpes virus with immunoperoxidase staining. Click to view with ImageScope Click to view with a Web-Based Viewer Case 4f - Slide 2 Herpes virus: H&E stain and immunoperoxidase stain for Herpes virus showing typical multinucleated giant cells with intranuclear inclusions which are positive for Herpes virus with immunoperoxidase staining. Click to view with ImageScope Click to view with a Web-Based Viewer Introduction: Infectious keratitis is a potentially serious disease which can lead to significant ocular morbidity. Critical to salvaging good vision is quick clinical recognition of the entitiy often with laboratory confirmation of the inciting microbe. Pathological/Microscopic Findings and any Immunohistochemical or Other Studies: Acanthamoeba: Double-walled cysts measuring approximately 20 microns in size are sometimes seen with routine H&E staining but can be highlighted with GMS, PAS, and giemsa stains. Culture confirmation can be done by inoculating the specimen on a lawn of E. coli. Case 2 - Staphylococcus aureus: H&E and gram stains, the latter showing few clusters of gram positive cocci, identified on routine culture as Staphylococcus aureus. Case 3 - Candida parapsilosis: H&E and GMS stains showing yeasts and keratitis, which were proven by culture to represent Candida paapsilosis. Case 4 - Aspergillus fumigatus: H&E and PAS stains showing necrotizing keratitis with ulcer and associated septated fungi. The fungal hyphae branch at approximately 45 degrees, which is typical for Aspergillus. Fungal culture confirmed Aspergillus fumigatus. Case 5 - Fusarium species: H&E and PAS stains showing necrotizing keratitis with ulcer and associated large irregular septated fungi. The patient had a history of contact lens use, and culture confirmed Fusarium species. Case 6 - Herpes virus: H&E stain and immunoperoxidase stain for Herpes virus showing typical multinucleated giant cells with intranuclear inclusions which were positive for Herpes virus with immunoperoxidase stain. This was a known clinical case of Herpes keratitis with a chronic corneal ulcer. Differential Diagnoses: The differential diagnosis is non-infectious keratitis. Examples of these include: 1)exposure keratitis — due to dryness of the cornea caused by incomplete or inadequate eye-lid closure. 2)photokeratitis — keratitis due to intense ultraviolet radiation exposure (e.g. snow blindness or welder's arc eye.), 3)ulcerative keratitis, 4)ring ulcer (due to a multitude of systemic illnesses, non- infectious as well as infectious)and 5)severe allergic response (i.e. vernal keratoconjunctivitis). A pathologist is less likely to see tissue for these entities. Sometimes there is inflammation associated with graft failure, but it is usually mild. The clinical impression of the ophthalmologist often gives clues to the etiologic microbe. Final Diagnosis: Case 1 - Acanthamoeba Case 2 - Staphylococcus aureus Case 3 - Candida parapsilosis Case 4 - Aspergillus fumigatus Case 5 - Fusarium species Case 6 - Herpes virus Case Discussion: 1. Acanthamoeba Case #1 is actually a research case in which the corneas of pigs were inoculated with Acanthamoba shortly before enucleation. This explains the multiple cysts present and paucity of inflammation. It was given, though, because of the high number of cysts, cut at different angles, so that participants will have an easy reference in the future when confronted with the typical specimen that might have rare cysts for diagnosis. The cysts measure approximately 20 microns in size and are double-walled with a surrounding clear space. The inflammatory infiltrate is usually neutrophilic or histiocytic, sometimes with granuloma formation. Culture may confirm the presence of this amoeba, but often PCR is required. Confocal microscopy is used by some ophthalmologists for a clinical diagnosis. Acanthamoeba keratitis (AK) is a relatively new infectious entity that is difficult to treat. Early diagnosis and appropriate treatment are essential for good outcome. Acanthamoeba is a free-living opportunistic protozoan that is widely distributed in the environment (found in soil, dust, contaminated water, air conditioners, contact lens fluid, sewage, and from the nose and throats of healthy individuals). It can produce serious, if uncommon, human infections, including blinding keratitis and fatal encephalitis. It is one of the four genera of protozoan parasites that cause significant clinical disease in humans: Naegleria (meningoencephalitis), Acanthamoeba and Balamuthia (both causing granulomatous amebic encephalitis and keratitis due to Acanthamoeba) , and Prototheca (cutaneous, musculoskeletal, or disseminated infections). The increase in Acanthamoeba keratitis, especially in contact lens wearers, has generated new interest in this group of amebas. [1] There are ten-fold variations in the reported incidence of the disease in the national populations, from as few as 0.15 per million in the United States to as high as 1.4 per million in the United Kingdom. These differences have been shown, at least in large part, to relate to the prevalence of contact lens (CL) use (incidence rates for CL users vary from 1.65 to 2.01 per million in the US to 17.53 to 19.50 per million in the UK.) [2], the contamination of domestic water and swimming pools by Acanthamoeba, the amoebicidal efficacy of CL care systems, regional variations in the availability of different CL care systems, the use of reusable soft CLs, and the prevalence of the use of diagnostic techniques for AK. [3, 4] In non–lens users AK is usually associated with trauma and exposure to contaminated water or soil, often in agricultural workers. [5] There are pathogenic and nonpathogenic species. Acanthamoeba castellanii and A. polyphaga are the most common species to cause keratitis. [2] The taxonomy and classification of these protozoa are currently under revision, following the successful application of molecular techniques which have led to the identification of 15 different genotypes (T1-T15) based on rRNA gene sequencing [6]. Attempts to correlate pathogenicity with certain genotypes are underway; studies to date have shown that 90 percent of Acanthamoeba isolates that produce human infections belong to the T4 genotype [7] . Acanthamoeba has both a trophozoite and a resistant cyst stage but no flagellate stage. The trophozoite has an amoeboid shape with pseudopodia and feeds on small algae, bacteria, and other protozoans. In the cornea, they are thought to feed on keratocytes. Reproduction is asexual by binary fission. The cyst is the dormant form of the organism. Trophozoites and cysts vary in size from 25 to 50 µm and 15 to 30 µm respectively, which is well within the 2 µ m resolution of modern in vivo confocal microscopes. Trophozoite encystment allows the organism to survive an adverse environment, including the nutrient deficiency and noxious chemicals that it is exposed to in keratitis, and is the form of the organism responsible for persistent disease. Corneal invasion, at least in animal studies, appears to be due to the release of multiple organisms from cysts and host-derived proteases. Intraocular infection is rare, both in humans and in animal models, despite the organism's ability to penetrate Descemet's membrane and endothelium. This appears to be due to the intense neutrophil response in the anterior chamber. [8] The innate immune response mediated by neutrophils and macrophages seems to be the primary response leading to resolution of the disease. Since in ocular disease control of inflammation without exacerbating infection is so important for visual outcome, understanding the type of immune response is critical. In the hamster model, topical dexamethasone accelerates both trophozoite excystment and proliferation and further, treated trophozoites or cysts induce a significant cytopathic effect on corneal epithelial cells compared with untreated organisms. However if used in conjunction with effective anti-amoeba therapy, excystment might make the organism more susceptible. [2] Cell-mediated immune response has been less studied in animals and thus is not well understood. Necrotic organisms and amoebic cyst walls have been found to remain in corneal tissue for years where they can cause persistent corneal and scleral inflammation in spite of being apparently nonviable. Thus it has been proposed that corneal antigen-presenting cells may result in a T cell response resulting in inflammation, possibly in an autoimmune fashion through molecular mimicry. A provisional diagnosis of AK can be made using the clinical features and confocal microscopy [9] although a definitive diagnosis requires culture, histology, or identification of Acanthamoeba DNA by polymerase chain reaction. Routine use of tissue diagnosis is recommended for making a definitive diagnosis, particularly for patients unresponsive to treatment for AK. Histopathology detection has varied from 21% to 48%, [10, 11] while a culture rate of 55% and a PCR rate of 85% has been reported. [10] Treatment is complicated by the fact that, while the trophozoites are sensitive to many chemotherapeutic agents (antibiotics, antiseptics, antifungals, antiprotozoals including metronidazole, antivirals, and antineoplastic agents), the cyst forms are not. The diamidines (ex. propamidine isethionate) and biguanides (ex. chlorhexidine) are currently the most effective cysticidal agents. [2] Extracorneal complications of AK include cataract, iris atrophy, glaucoma, and peripheral ulcerative keratitis, all possibly due to toxicity from the topical diamidines and/or biguanides. However other potential causes of these complications may be chronic or persistent inflammation, steroid effects, vascular thrombosis or the rare cases of intraocular infection. [2] In conclusion, AK is a painful and serious corneal infection, often associated with contact lens use or following exposure to contaminated water, which presents a challenge in both diagnosis and treatment. 2. Bacteria Case #2 was a culture-proven case of Staphylococcus aureus bacterial keratitis, which was originally treated with both topical and systemic antibiotics. The patient had a history of rheumatoid arthritis and was on steroids. The cornea perforated requiring a penetrating keratoplasty. Due to the prior antibiotic therapy, there are only a few clusters of gram positive cocci present associated with the inflammation seen histologically. In North America, as well as in Europe [12] and Asia, most cases of microbial keratitis arise from a bacterial etiology. In the western US, gram-positive bacteria, especially Staphylococcus and Streptococcus species [13] has been found to be prominent causes. In the southeastern United States although gram-positive bacteria are also prominent, Pseudomonas species are more frequently isolated, [14] attesting to geographic variability. A variety of inciting or risk factors have been recognized in cases of bacterial keratitis. Surface factors such as contact lens wear, trauma, previous corneal surgery or sutures, chronic exposure or irritation, persistent or recurring epithelial defects, tear deficiency or limbal stem cell deficiency states can predispose to the development of bacterial keratitis. Systemic factors such as immunosuppression, atopy, diabetes mellitus, or connective tissue diseases also increase the risk of infection. Geographic location, unusual exposure to animals, contaminated water, or other higher-risk environments also play a role. A study looking at cases from 1950-1988 found an estimated annual incidence of bacterial keratitis in the US of 5.3 per 100,000 people, [15] and was associated with contact lens (CL) wear in over 50% of cases. A study of CL wearers in 1999 in the Netherlands showed an estimated annualized incidence of microbial keratitis of 1.1 per 10,000 users of daily-wear rigid gas-permeable lenses (RGP), 3.5 per 10,000 users of daily-wear soft lenses, and 20.0 per 10,000 of users of extended-wear soft lenses. [16] A more recent study (2008) from Australia found: in daily wear rigid gas-permeable CL wearers, the annualized incidence per 10,000 wearers was 1.2; in daily wear soft CL wearers 1.9; soft CL wearers (occasional overnight use) 2.2; daily disposable CL wearers 2.0; daily disposable CL wearers (occasional overnight use) 4.2; daily wear silicone hydrogel CL wearers 11.9; silicone hydrogel CL wearers (occasional overnight use) 5.5; overnight wear soft CL wearers 19.5 and in overnight wear of silicone hydrogel 25.4. Their conclusions were that the incidence estimates for soft CL use were similar to those previously reported, new lens types have not reduced the incidence of disease, and importantly, overnight use of any CL is associated with a higher risk than daily use. [17] However, decreased cases of CL-related bacterial keratitis were found in another study looking at cases from 1990-1998. [14] Smoking has frequently been reported to be a risk factor for CIEs (corneal infiltrative events) and microbial keratitis. The mechanisms of this relationship are probably multifactorial and may be partly causal and partly confounding. The adverse effects of smoking may stem from toxins, increased pathogens in the subject's resident microbiota, changes in mucous membranes, or there may be a confounding effect with other unmeasured risk-taking behavior, as there is evidence of clustering of risky health behavior in the primary care setting. Bacterial keratitis can be eradicated in many circumstances; however, visual acuity is frequently diminished as a consequence of the infection. [19] The damage to the visual function is determined by the virulence of the organism, the inoculum, host defenses, adequacy of therapy, and the proximity of the keratitis to the central visual axis. For example, highly virulent gram-negative bacterial keratitis may leave little functional impairment if outside the visual axis, whereas a small central corneal scar from a mildly virulent gram-positive organism will have more severe consequences. The ability of the organisms to form biofilms, defined as functional consortiums of microorganisms organized within an extensive extracellular polymer matrix, may inhibit the host immune response as well as limit the bioavailability of antibiotics. [20] This has become more recently recognized in chronic bacterial keratitis, such as infectious crystalline keratopathy. [20, 21] As an ophthalmologic emergency, bacterial keratitis requires immediate treatment. The initial therapy is usually broad-spectrum empiric antibiotic treatment, followed ideally by more specific treatment based on results of cultures and smears of corneal scrapings. Also, culture negative infectious keratitis often responds to empiric broad-spectrum antimicrobial treatment. Fluoroquinolones are effective agents in bacterial keratitis caused by several pathogens and also convenient and practical to use. [14] However, resistant strains are developing. Topical corticosteroids have an unclear role in the management of bacterial keratitis. The typical objective is to reduce an exaggerated inflammatory response and minimize corneal scarring, while not impairing the healing response. One systematic review found that prior usage of corticosteroids increased the risk of antibiotic treatment failure or other infectious complications. [22] From this review, two recommendations reached "most important" levels. First, topical corticosteroids should be avoided if the causative agent is unknown and, second, topical corticosteroids should be utilized when, after using clinical or laboratory criteria, it is deemed important to aid reepithelialization or minimize stromal alteration and scarring. In practice, before administering topical corticosteroids, the American Academy of Ophthalmology Preferred Practice Pattern on this subject suggests waiting 2 to 3 days after topical antibiotic therapy has been initiated and in which progress is being made in treating the infection. [23] If topical corticosteroids are initiated, it is important to follow up the patient closely in the initial period to insure against recrudescence of the infectious process. As with other infectious keratitides, severe cases may require therapeutic penetrating keratoplasty. In conclusion, bacterial keratitis is the most common type of infectious keratitis. The degree of damage to the eye is dependent on factors such as the virulence of the particular type of bacteria as well as the site of infection on the cornea. Treatment needs to be started urgently, typically with a broad-spectrum antibiotic, followed by more specific treatment once culture and sensitivity results are available. Resistance to some antibiotics is becoming a problem. 3. Fungi: Cases #3, #4, #5 represent keratitis caused by Candida parapsilosis, Aspergillus fumigatus, and Fusarium species respectively. The patient with Candida had a history of diabetes mellitus, the patient with Aspergillus had no identifiable risk factor, and the patient with Fusarium had a history of contact lens use. All three cases showed significant keratitis, but the last two also demonstrated marked necrotizing keratitis with ulceration. The organisms, being large, can generally be appreciated on routine H&E, but confirmation with a fungal stain such as GMS or PAS is usually done. Sometimes the fungal organisms are only present in the deep stroma with an overlying intact epithelium. In these cases it is easy to understand why cultures from the surface are negative. The type of fungus was identified with fungal culture in these cases. The inflammation spread in all three cases to cause endophthalmitis, and the Candida and Aspergillus cases eventually required evisceration in one and enucleation in the other. Fungi are ubiquitous organisms that are recognized more frequently as ocular pathogens in agrarian, tropical countries than in the developed world. [24] The incidence of fungal keratitis in the United States is estimated to be approximately 1500 cases per year, [25] with the majority of cases arising in the warmer southern and southwestern states. In these areas (and around the world), septate filamentous fungi, usually Fusarium or Aspergillus, are the most common causative organisms, whereas in the northern states, Candida is the most frequently isolated fungal organism. In warmer regions or the tropics, corneal trauma, which might be trivial, and contamination by plant material are the frequent causes of infection. However, in colder climates, where Candida infections predominate, prior corneal disease with topical corticosteroid use and/or systemic disease states that lower host resistance such as alcoholism or diabetes are associated with these infections. Unlike fungal endophthalmitis, fungal keratitis is not associated with systemic fungemia. [24] For clinical purposes, fungi can be classified on a morphological basis into filamentous, yeast, and diphasic forms. Filamentous organisms are multicellular with branched hyphae. Some, the so-called septate organisms such as Fusarium, Cephalosporium, Aspergillus, Curvularia, and Alternaria species, have hyphae that are divided by cell walls, whereas other filamentous fungi such as Mucor and Rhizopus are nonseptate. Yeasts, such as Candida and Cryptococcus, are unicellular fungi that reproduce by budding, but in tissue, they might develop elongated buds (pseudohyphae) or real hyphae. Dimorphic fungi such as Histoplasma, Coccidioides, and Blastomyces demonstrate both a yeast phase that occurs in tissues and a mycelial phase that appears on culture media and saprophytic surfaces. The keratitis tends to be slowly progressive and insidious. In some cases, the surface corneal epithelium heals over an intrastromal infiltrate with only mild inflammation and little pain. However, inflammation at times is severe enough to lead to hypopyon formation. The clinical findings may simulate a bacterial ulcer. However, certain features suggest a fungal infection, including feathery edges or a dry, gray, elevated infiltrate and satellite lesions. The clinical setting and underlying risk factors, such as topical steroid use are important to consider. Also, there have been cases reported of fungal infections (also infections due to bacteria, mycobacteria or viruses) following LASIK. [26, 27] Prior to treatment, scrapings for culture should always be obtained, recognizing that in some cases the fungi are only present deep in the stroma, necessitating a corneal biopsy. The efficacy of available antifungal agents is limited, and there is a relatively high medical treatment failure rate. These agents fall into three main groups: the polyenes (such as amphotericin B, natamycin, and nystatin), the azoles, and the fluorinated pyrimidines. Corticosteroid use is generally not recommended in fungal keratitis but may be used when excessive inflammation appears to be the cause of progressive damage to the ocular surface. Approximately 20% of cases may require penetrating keratoplasty. [24] A now famous outbreak of Fusarium keratitis was first described in Singapore in March 2005 with cases noted later that year in Hong Kong. [28, 29, 30] At that time, Khor and associates reported 68 eyes of 66 patients which developed Fusarium keratitis in Singapore, 98% of whom wore soft contact lenses. All but 4 of the patients (93.9%) reported using any Renu brand disinfection solution (Bausch & Lomb) with 63.6% specifically using Renu with MoistureLoc, which had recently been introduced worldwide in late 2004. [29] Scattered cases were also reported in the United States during the same time frame after its introduction there in 2005. [31] As of May 18, 2006, the CDC had received reports of 130 confirmed cases of Fusarium keratitis infection, defined as clinically consistent fungal keratitis with symptom onset after June 1, 2005, no history of recent ocular trauma, and a corneal culture yielding a Fusarium species. Cases were reported from 26 states and one territory. Patients had a median age of 41 years (range: 12-83), and 85 of 127 (67%) were female. As a result of this infection, corneal transplantation was required in 37 of 120 (31%) cases. This solution was withdrawn from the US market in April 13, 2006 and from world market on May 15, 2006. [32] In conclusion, fungal keratitis is a serious corneal infection with a relatively high medical treatment failure rate. It may occur in the setting of trauma/plant contamination (usually filamentous fungi) or in patients with immunosuppression (typically Candida). 4. Herpes: Case #6 is that of a long standing corneal ulcer clinically due to Herpes simplex. Treatment included a "corneal glue", but in the end a penetrating keratoplasty was required. Histopathologically there was pancorneal acute and chronic inflammation with a perforated ulcer. The surface epithelium showed the typical intranuclear Cowdry type A inclusions of Herpes, and an immunoperoxidase stain confirmed this. This is an example of a stromal keratitis, in particular necrotizing keratitis. Within a month after transplant, the patient suffered transplant failure, requiring a second penetrating keratoplasty. This is not an uncommon occurrence in the setting of Herpes keratitis. Herpes simplex virus type 1 (HSV-1) constitutes the vast majority of herpetic ocular infections of the anterior segment and the most common infective cause of blindness in developed countries [34] . Diagnosis is typically made clinically, although serologic and molecular testing is available. There is considerable variation in the literature regarding incidence, presentation, and recurrences of herpes simplex virus keratitis (HSVK), which may be the result of differing study populations, disease definitions, length of follow-up and/or other factors. In the United States, there are approximately 50,000 cases of new and reactivation cases per year. [35] Approximately 25% of cases are the more serious stromal keratitis type. Bilateral involvement is less common (except in children), usually associated with atopy or other systemic immunosuppression and, depending on the definition used, can range from 3% to 12%. [36, 37] Humans are the only natural reservoir, and an estimated 50% to 80% of the adult population has antibodies to HSV-1. [36] After primary infection by HSV (not clear if this represents primary infection of the cornea itself or of the orolabial area), the virus becomes latent in the trigeminal ganglion allowing for reactivation and recurrent infection at later intervals induced by fever, menses, irradiation, emotional stress, etc. Any structure in the anterior segment can be involved and the infection presents, sometimes simultaneously, in several major forms: blepharoconjunctivitis, infectious epithelial keratitis, neurotrophic keratopathy, stromal keratitis, endotheliitis, iridocyclitis, and trabeculitis. Various studies show varying rates of epithelial vs. stromal keratitis and other ocular manifestations. Liesegang et al found that initial episodes involved the eyelids or conjunctiva in 54% of cases, the superficial cornea in 63%, the deep cornea in 6%, and the uvea in 4%. [34] In susceptible individuals, recurrence of the virus can lead to blinding keratitis or uveitis. Recurrence rates for any form of ocular HSV have been estimated at 9.6% at 1 year, 22.9% to 33% at 2 years, 36% to 40% at 5 years, and 63.2% at 20 years. [34, 38, 39, 40] a) Epithelial keratitis Epithelial keratitis is caused by actively replicating virus on the corneal surface. This initial phase of HSV-1 disease presents as minute corneal vesicles that stain negatively with fluorescein dye. This may progress to dendritic keratitis, geographic keratitis, marginal keratitis with limbitis or trophic ulcer (non-healing). Although at least the first three conditions may resolve spontaneously without therapy, antiviral therapy is generally indicated to accelerate resolution. Wilhelmus KR has done mega-analyses over the years on treatment of Herpetic epithelial disease and published the results in the Cochrane Database Syst Rev. [41] In the most recent one (2010) [34] , he analysized 106 comparative treatment trials involving 5872 eyes with dendritic or geographic epithelial keratitis for corneal healing over two weeks . He found that trifluridine, acyclovir, brivudine and ganciclovir were the most effective agents. While not improving outcome, the combination of interferon and an antiviral agent may speed healing. The effectiveness of corneal epithelial débridement is improved by an antiviral agent. Response to topical therapy usually occurs in 2-5 days, with complete resolution in 2 weeks. The use of systemic acyclovir is increasingly preferred over topical agents in the treatment of HSV keratitis, particularly for patients with preexisting ocular surface disease who are at high risk for toxicity from topical medications, for patients who are immunocompromised, and for pediatric patients. Some physicians prescribe both oral and topical antiviral agents together when treating infectious HSV keratitis. b) Stromal/endothelial keratitis This is usually an immune-mediated response to non-replicating viral particles, but more severe forms may be caused by live virus. Stromal keratitis can affect all layers of the cornea and may even involve the trabecular meshwork and iris to varying degrees. It is classified based on the predominant site and type of involvement. a) Endothelitis is the most common form and manifests as overlying stromal edema from endothelial dysfunction. Longstanding stromal edema leads to permanent scarring and is the major cause of decreased vision associated with HSVK. b) Necrotizing keratitis shows greater inflammation in the cornea and is thought to be a reaction to live viral particles in the corneal stroma. It is potentially devastating in the acute phase. It is most commonly seen in patients with multiple recurrences, especially with HSV-2, and it may cause corneal melting and perforation. Frequently, it is associated with uveitis and trabeculitis that may lead to recalcitrant glaucoma. c) Immune stromal keratitis manifests as focal, multifocal, or diffuse stromal opacities or an immune ring. It is often accompanied by stromal edema and a mild anterior chamber reaction. The epithelium and endothelium are relatively spared. It is called interstitial keratitis (IK) if accompanied by vascularization. HSV is now the most common cause of IK, especially unilateral, in the US. Immune stromal keratitis leads to corneal blindness through a chronic relapsing and remitting course. d) Keratouveitis is usually granulomatous with large "mutton-fat" keratic precipitates on the endothelium. It is usually immune-mediated. It can lead to significant morbidity from synechiae, cataracts, and glaucoma. Unilateral uveitis associated with high intraocular pressure is almost always caused by HSV. [42] Herpes simplex virus (HSV) keratitis remains primarily a clinical diagnosis based on characteristic features of the corneal lesion. Laboratory studies may help confirm the clinical suspicion in cases lacking typical findings, but they are not readily available in most clinical settings. Laboratory diagnosis is especially less useful in stromal keratitis since there are usually no live virus particles present. a) Epithelial scrapings (Tzanck smear) with giemsa stain may show multinucleated giant cells, resulting from coalescence of infected corneal epithelial cells and intranuclear viral inclusions. However, negative cytology results do not exclude HSV infection. b) Viral cultures obtained within several days of onset of disease and prior to antiviral therapy have a sensitivity of up to 70% and also allow for identification of the HSV subtypes. Various techniques (eg, conventional tube culture, shell vial assay, suspension infection method) are available. c) HSV antigen detection tests, such as the enzyme-linked virus inducible system (ELVIS), are very specific for detecting herpes infection, but they are limited by their lower sensitivity. Cell culture for confirmation of HSV is recommended when the ELVIS test result is negative. d) Polymerase chain reaction using tear samples, corneal epithelium, anterior chamber tap, or corneal buttons may detect viral DNA in cases of herpetic keratitis or keratouveitis. However, it does not distinguish between latent or active HSV infections. Treatment of HSV is different with epithelial and stromal keratitis . This reflects the fact that epithelial disease is caused by live replicating virus while stromal disease is essentially an immune response to viral antigen. Prompt and appropriate treatment may minimize the risk of scarring, which is the major cause of morbidity with HSVK. Though epithelial keratitis spontaneously resolves in approximately 50% of cases, treatment with antivirals is necessary in ulcers larger than 4 mm, marginal ulcers, and ulcers with underlying stromal inflammation. Stromal keratitis is treated with topical steroids as they decrease inflammation and therefore scarring. However, there is some evidence that HSV reactivation while the patient is on steroids results in severe epithelial disease or necrotizing keratitis. Therefore, simultaneous antiviral prophylaxis is recommended. Oral antivirals decrease the risk of HSV reactivation at the ganglion level while topical antivirals are more effective at treating epithelial recurrences but are toxic. Aggressive topical and systemic antivirals along with steroids are necessary in necrotizing keratitis and focal serous iritis. [42] In conclusion, Herpes keratitis is the most common infectious cause of cornea-derived blindness in developed countries. 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