Microsporidial Keratitis

Etiology

Microsporidia are spore-forming intracellular organisms found nearly everywhere in nature. Traditionally classified under the kingdom Protista, they have been recently reclassified as fungi based on molecular studies.[4][5] With over 1700 species, the most common species involved in human infection are Enterocytozoon bieneusi and the Encephalitozoon species, primarily Encephalitozoon intestinalis. Microsporidium and Vittaforma corneae are the most common causes of microspordial stromal keratitis, and Encephalitozoon species are the most common cause of microspordial keratoconjunctivitis.[4]  

In addition to primitive genomes and ribosomes similar to protozoa, the absence of mitochondria, centrioles, and peroxisomes gives microsporidia its prokaryotic features. However, the presence of mitochondrial hsp70, mitosome, and alpha-beta tubulin has resulted in their reclassification to fungi.[6][7]

Microsporidia are opportunistic waterborne pathogens commonly transmitted through contaminated water, like lakes, swamps, swimming pools, or spring water.[8][9][10] Among humans, microsporidia affects the gastrointestinal tract, brain, eyes, muscles, bones, and lungs.[11] While microspordial keratoconjunctivitis and microspordial stromal keratitis are the most prevalent ocular presentations, microsporidia also cause scleritis and endophthalmitis.[5][12][13] Historically, individuals who develop microsporidial keratoconjunctivitis were immunocompromised, often associated with HIV infection.[14][15] However, recent literature indicates that even immunocompetent patients can experience keratoconjunctivitis.[16][17][18] Conversely, deep stromal keratitis is primarily observed among immunocompetent individuals. 

Epidemiology

Endemic to Southeast and South Asian countries, ocular microsporidiosis is an infrequent but noteworthy contributor to infectious keratitis, with a prevalence of 0.4% observed in certain regions.[19] Specifically, Thailand, Taiwan, Singapore, and India are identified as endemic areas for microsporidial keratitis, marked by an upsurge in reported cases.[19] Adopting advanced diagnostic techniques, including various staining methods and PCR, has facilitated more accurate and timely diagnoses of this condition.

A notable study reveals that nearly 20% of all clinically diagnosed cases of infective keratitis in these regions are attributed to microsporidia, with incidences peaking during the rainy season. Moreover, a study conducted in South India involving over 10,000 patients with conjunctivitis unveils a significant proportion presenting with microsporidial keratoconjunctivitis among those who underwent corneal scraping. The underreporting of ocular microsporidia infection is likely due to the self-limited nature of microsporidial keratoconjunctivitis and the lack of comprehensive evaluations in patients with conjunctivitis. Clinicians often rely on clinical diagnosis due to these factors.[19] Large-scale epidemiological studies are imperative to clarify further endemic zones, geographical variation, and the mode of disease transmission.[19][20][21] 

Pathophysiology

Microsporidium is an obligate intracellular spore providing enhanced dispersion and protection against adverse environmental conditions. Its cell wall comprises a double envelope, with the outer portion containing glycoproteins and the inner part primarily chitin. The cytoplasm and nuclei form the infectious portion, known as sporoplasm. Changes in pH, exposure to certain cations, or low-dose ultraviolet radiation stimulate the polar filament to inject sporoplasm into the host.[22] Interestingly, spore intake occurs in cell cultures without releasing these polar tubules.

Factors inhibiting germination are magnesium chloride, ammonium chloride, minimal salt levels, sodium fluoride, ultraviolet exposure, temperatures >40 °C, and agents like itraconazole. Once inside the host cell, new spores form and infect neighboring cells. The symptoms and severity of microsporidiosis vary depending on the infecting species and the host's immune response, with severe cases often observed in significantly immunocompromised patients, particularly those with HIV.

Direct inoculation from contaminated water is the most likely cause of infection. Risk factors for acquiring microsporidial keratitis include contact lens usage, particularly inadequate hand hygiene or lens care practices, topical corticosteroid application, insect bites, ocular surgeries, and contact with domestic animals.[10] Immunosuppression remains a notable risk factor. The increased incidence during the rainy season suggests water contaminated with microsporidia or an increased insect population may contribute to transmission. The presence of risk factors among affected individuals varies widely, ranging from as low as 22.2% to as high as 100%.

The exact transmission route of ocular microsporidiosis remains unclear. While fecal-oral transmission is implicated in intestinal microsporidiosis, the viability of microsporidia spores against variable temperatures suggests alternative routes.[23] Certain pathogenic strains have been identified in both surface and groundwater, suggesting that unsanitary water sources, combined with subpar sanitary infrastructures, can be the nidus of infection.

Ocular trauma is another possible transmission route, as it can directly introduce these organisms into the corneal epithelium upon contact with soil or water-containing spores. The United States Environmental Protection Agency (EPA) recognizes microsporidia as a potential microbial waterborne contaminant.[24]

While the literature lacks a precise threshold, a CD4+ T-lymphocyte count below 100 cells/mm³ is commonly associated with increased susceptibility to microsporidial infection.[25][26]  Earlier research indicates a correlation between diminished CD4+ counts, often observed in individuals with AIDS, and the development of microsporidial keratoconjunctivitis. However, findings from Loh et al challenge this immunity-based theory, revealing a significant percentage of cases with normal CD4+ and CD8+ T-lymphocyte counts and negative serology.[27]  

Histopathology

The morphological identification of organisms on corneal tissue scrapings plays a crucial role in diagnosing microsporidial keratitis. The most helpful stain combines 10% potassium hydroxide (KOH) with 0.1% calcofluor white (KOH+CFW). A recent report by Mittal et al demonstrates that microsporidia spores are strongly acidic and heat fast. They stain strongly and most discretely when stained with the conventional Ziehl-Neelsen staining method using 20% sulphuric acid. A Gram stain reveals intraepithelial, gram-positive, oval-shaped spores on transmission electron microscopy. Characteristic periodic acid–Schiff-positive granules are identifiable at one end of mature microsporidia spores. However, the Giemsa stain is not considered useful due to its low sensitivity in detecting microsporidia. 

Corneal button specimens in patients infected with Vittaforma corneae reveal oval-shaped spores measuring 3.5 to 5 µm and 11 to 13 coils in the polar tubule.[28] The spores of the Encephalitozoon species are 2 to 3 µm and contain 4 to 7 coils in the polar tubule.[28] Most notable with the Encephalitozoon species is the presence of a parasitophorous vacuole, a protective bubble made of plasma membrane surrounding microsporidia in the host. The spores are the only form that can survive outside the host cell. Histopathology reveals varying degrees of inflammation with a near absence of inflammatory cells in patients who are immunosuppressed.[29] Organisms commonly confused morphologically with microsporidia are Toxoplasma gondii, Leishmania spp., Histoplasma capsulatum, and Cryptosporidia spp.[30]

History and Physical

Examination for microsporidial keratitis involves a thorough evaluation of the patient's ocular symptoms, medical history, and risk factors for infection. Clinical examination typically includes a detailed assessment of visual acuity and slit-lamp biomicroscopy to inspect the cornea for characteristic findings such as epithelial lesions, stromal infiltrates, and conjunctival inflammation. Additionally, associated symptoms such as redness, tearing, photophobia, and foreign body sensation are noted.

Microsporidial Keratoconjunctivitis

Infection caused by Encephalitozoon species typically remains localized to the epithelial cells of the cornea and conjunctiva. In patients with microsporidial keratoconjunctivitis, symptoms usually appear 1 to 2 weeks after exposure. Patients often report exposure to contaminated water or trauma, mainly in the rainy season. Additionally, a history of exposure to pool water, natural springs, contaminated soil, topical steroid use, insect bites, contact with domestic animals, or participation in water sports may be present. 

Common complaints include redness, decreased visual acuity, tearing, eyelid swelling, eye pain, and a foreign body sensation. Initially, symptoms are typically unilateral but can progress to both eyes.[8] Bilateral involvement is more likely in patients with compromised immune systems, occurring in up to 32% of cases in healthy individuals. Both eyes may be affected simultaneously, or symptoms may develop in 1 eye first, followed by the other within 1 week to 3 months.

Clinical examination features include the following:[31][32]

• Multiple fine to coarse punctate, raised epithelial corneal lesions with a stuck-on appearance
• Lesions stain variably with fluorescein
• Lesions localize peripherally, paracentral, or diffusely
• Significant conjunctival congestion
• Typically not purulent
• Potential anterior chamber inflammation and keratic precipitates

Some cases present with multiple subepithelial punctate infiltrates, making microsporidial keratoconjunctivitis indistinguishable from adenoviral keratoconjunctivitis. Reports exist of a pseudomembrane forming on the conjunctiva.

The disease can be graded from grade 1 to 4 depending on the presenting features:

• Grade 1: The lesions are greyish-white, <10 in number, and distributed mainly in the peripheral cornea.
• Grade 2: The lesions are central, 10 to 20 in number, elevated epithelial lesions.
• Grade 3: The lesions are 21 to 40 in number.
• Grade 4: The lesions are primarily peripheral and have subepithelial infiltrates.

Disease progression can lead to outcomes like superficial punctate keratitis, which may eventually cause scarring beneath the epithelium. Atypical presentations of microsporidial keratoconjunctivitis can manifest as unique corneal patterns or conditions that mimic other eye diseases, such as Thygeson superficial punctate keratitis. [27]

Microsporidial Stromal Keratitis 

Microsporidial stromal keratitis occurs primarily in immunocompetent individuals and is commonly associated with infections caused by Vittaforma corneae and Microsporidium. This form of infection typically targets the corneal stroma, including the keratocytes. The clinical history and presentation are similar to microsporidial keratoconjunctivitis, but affected patients may have a history of multiple episodes of remission and recurrence.

On average, patients experience symptoms 1 month to 2 years before presentation.[33][34] Clinical manifestations include multifocal, mid to deep stromal infiltrates with the overlying epithelium remaining intact. The stromal infiltrates are nonspecific and associated with edema with or without deep stromal vascularization. The infiltrate can be ulcerative at times.[35]

In cases where patients present with a history of chronic culture-negative stromal keratitis, clinicians should consider including microsporidial stromal keratitis in the differential diagnosis.[34] This form of keratitis can often be overlooked due to its atypical presentation and the challenges associated with its diagnosis.

Evaluation

Scrapings or biopsies of the conjunctiva or cornea typically serve as the primary diagnostic method for confirming microsporidial keratitis. These samples are subjected to various laboratory tests to facilitate the visualization of microsporidia spores.

Transmission Electron Microscopy

Historically, transmission electron microscopy (TEM) played a crucial role in elucidating the classification and comprehension of human microsporidia infections, including those impacting the eye. The distinctive polar tubule, observed through TEM, plays a vital role in identifying and classifying the organism within the Microspora phylum. Despite TEM remaining the gold standard for confirming and identifying microsporidia species, its complex procedure and limited accessibility restrict its practical utility in clinical diagnostics.[36]

Smear Diagnosis

Optical microscopy is a primary method for diagnosing microsporidiosis, as microsporidia are obligate intracellular organisms that cannot be cultured in routine media and require cell cultures for growth. Due to the challenges in consistently culturing microsporidia, direct smear findings become essential. Corneal abnormalities in patients with microsporidial keratoconjunctivitis appear raised and superficial. Clinicians typically scrape off these lesions with a 15-scalpel blade after applying topical anesthetic, and the samples undergo staining to identify microsporidia spores.

Smears initially used for Gram staining can be reused by decolorizing and reapplying a Ziehl-Neelsen stain, thus eliminating the necessity for additional scrapings.[37] Several staining methods are available for verifying the diagnosis of microsporidial keratoconjunctivitis from corneal and conjunctival samples. The combined 10% potassium hydroxide (KOH) with 0.1% calcofluor white (KOH+CFW) staining is the most preferred due to its superior detection rate, closely followed by the modified Ziehl-Neelsen stain, particularly when a fluorescence microscope is not available. The Gram stain follows, with Giemsa staining being the least preferred.

Some researchers have successfully used the modified trichrome staining method when diagnosing microsporidial keratoconjunctivitis.[38] Identification by staining does not allow for identification of the specific species involved. PCR is more commonly used for species-specific identification than electron microscopy.

Due to the presence of deep stromal infiltrates, diagnosing microsporidial stromal keratitis poses greater challenges. Corneal scrapings may not provide sufficient diagnostic information in this condition. A corneal biopsy or therapeutic penetrating keratoplasty in chronic culture-negative keratitis is a valid option to reach the diagnosis. The biopsy or excised corneal button is subjected to histopathological evaluation for a tissue diagnosis. 

Molecular Techniques in Diagnosis

PCR is a powerful tool for diagnosing microsporidial infections and determining the species involved. This molecular technique targets pathogen DNA by binding to a specifically designed set of primers and amplifying it repeatedly in the presence of free nucleotides by a thermostable polymerase enzyme. Utilizing known sequenced pan-microsporidia primers, PCR amplifies a fragment of 16S rRNA, offering a sensitivity of 83% and specificity of 98%.[39] 

Nucleic acids extracted from corneal scrapings or biopsies typically undergo a pan-microsporidian PCR followed by DNA sequencing and a Basic Local Alignment Search Tool (BLAST) search for species-specific identification. Recently, a duplex PCR method has emerged, incorporating pan-microsporidian primers and species-specific ones for Vittaforma corneae.[40] Despite the availability of specific primers for ocular microsporidia species since 1994, they have not gained significant traction in clinical microbiology. Compared to earlier methods, a notable absence of commercial kits specifically designed for diagnosing ocular microsporidiosis exists. 

Introduced in 1996, PCR combined with Southern analysis has successfully distinguished Encephalitozoon species in urine samples. However, its widespread adoption for ocular infections has been limited thus far. Nonetheless, PCR remains a valuable tool for various microsporidia infections across diverse clinical specimens. For example, a 2004 study by Conners et al detailed a PCR method using specific rRNA primers, followed by sequencing to identify microsporidia in corneal samples.

Other studies indicate the utility of real-time PCR assays and multiplex PCR in different contexts, showing potential for broad applications, including the diagnosis of microsporidial keratoconjunctivitis. A newly reported oligonucleotide microarray also seems promising, showcasing the ability to detect multiple species. Its efficacy in ocular sample diagnostics is yet to be determined.[41]

Cell Cultivation Techniques

Due to their intracellular nature, microsporidia can only propagate in cell culture systems. During the early 1990s, researchers successfully adapted 3 microsporidia isolates, specifically Encephalitozoon hellem, from corneal tissue and conjunctival samples of patients with AIDS to Madin-Darby Canine kidney epithelial (MDCK) cells.

Additionally, various other cell types were evaluated for cultivating microsporidia, such as E6, HLF, MRC-5, RK-13, HeLa, Vero, SIRC, and fetal bovine lung fibroblasts. The Vero cell line proved the most effective when comparing the Vero, HeLa, and SIRC cell lines for growing 4 distinct microsporidia species.[42] Despite these advancements, the method of nurturing these organisms in cell lines has not been streamlined for the routine diagnosis of microsporidial keratoconjunctivitis.[42]

Antigen Detection Techniques

In clinical microbiology, antigen detection using various specimens is a common way to identify numerous infectious conditions. This method frequently involves the use of either monoclonal or polyclonal antibodies. Specific antibodies, especially those targeting the spore wall or polar tubule, are available for multiple microsporidia species.[43] Research from the early 20th century indicates the effective application of immunofluorescent antibody techniques to identify ocular microsporidia using species-specific antisera.[40] 

Many researchers feel antigen detection techniques are less sensitive than PCR and, if used, should be used as a supplement to conventional histological methods. Indirect immunofluorescence assay is neither widely available on the market nor regularly used in diagnosing ocular microsporidiosis. In addition, testing for specific antibodies against microsporidia in blood for diagnostic purposes in cases of ocular microsporidiosis is not endorsed and remains inaccessible.[43]

Imaging Techniques

Anterior segment optical coherence tomography (AS-OCT) offers a contact-free, high-definition method to visualize cross-sections of the cornea. A study by Thanathanee et al reveals the following changes in patients with microsporidia keratoconjunctivitis:

• Hyperreflective dots are limited to the epithelial layers of the cornea
• No extension into the stromal layer 
• Hyperreflective dots are slightly raised above the epithelial surface in most cases

These features distinguish microsporidia lesions from the nummular scars caused by adenovirus, which present as more prominent subepithelial anomalies, manifesting milder reflectivity and a smoother epithelial surface. Coupled with microbiological analyses, AS-OCT may offer diagnostic clarity and a means to track treatment progress. Further studies are necessary.[44]

In Vivo Confocal Microscopy 

In vivo confocal microscopy (IVCM) is a ground-breaking tool for diagnosing ocular infections. Using IVCM, the lesions of microsporidial keratoconjunctivitis appear as epithelial clusters accompanied by bright, pinpoint-shaped spores. Clinicians can distinguish from adenovirus keratoconjunctivitis based on the presence of a bright basal epithelium, dendritic cells, and active keratocytes in the superficial stroma at the center of the cornea. Additionally, IVCM can potentially assess treatment efficacy and evaluate for deeper stromal penetration. However, its adoption remains limited due to significant interobserver variability and high cost.[45]

Impression Cytology

Impression cytology involves the application of cellulose acetate filter paper to the eye's surface, allowing for the collection of superficial layers lining the ocular surface. Once collected, clinicians can do histological, immunohistological, or molecular analyses of the cells. This test offers a less invasive method and an alternative to corneal smear tests.

Limited documentation of its use exists in the literature, and its potential accuracy in terms of sensitivity and specificity requires further exploration. Through this technique, intracellular and extracellular spores, without inflammatory cells, have been identified, mirroring the findings of smear tests.[46]

Treatment / Management

No consensus exists regarding the management of microsporidial keratitis. The various treatment options are listed below:

Topical Therapy

Superficial infections are self-limited or treated medically. Deeper stromal involvement often requires corneal transplantation to eradicate the infection and improve vision.[47][35] Lubricants are effective for symptomatic relief. In a randomized controlled trial, lubricants are as effective as biguanides.[48] The frequency of lubricant application depends on the severity of patient symptoms, with more frequent dosing in more symptomatic patients. (A1)

Antimicrobial agents

Topical fumagillin and fluoroquinolones are important antimicrobials frequently used in treating microsporidial ocular infections.[4][49] Fluoroquinolones target the topoisomerase and DNA gyrase enzymes. Among fluoroquinolones, ciprofloxacin 0.3%, gatifloxacin 0.5%, moxifloxacin 0.5%, levofloxacin 0.5%, norfloxacin 0.3%, and ofloxacin 0.5% are effective against microsporidia dosed hourly with or without oral albendazole. However, the results are mainly anecdotal, from in vivo and in vitro studies, and not supported by randomized clinical trials.[16][27][16][49][50](B2)

On the other hand, fumagillin is a mycotoxin produced by Aspergillus and treats microsporidia by inhibiting RNA synthesis.[4] Fumagillin may also act by inhibiting the proteinase, type 2 methionine aminopeptidase. Some reports suggest a potential for relapse after treatment cessation. The concentration of bicyclohexyl ammonium fumagillin is 0.113 mg/ml.[16][51][52](B3)

Biguanides

Polyhexamethylene biguanide (PHMB) 0.02% and chlorhexidine gluconate 0.02% are 2 biguanides effective in treating microsporidial keratitis when combined with debridement.[17] Both PHMB and chlorhexidine are disinfectant antiseptics requiring reconstitution to 0.02% as they are not commercially available in the given concentration. Dosing is as frequent as every 30 to 60 minutes.[48](A1)

Diamidines

Sparse reports have highlighted compounds such as propamidine isethionate and hexamidine isethionate 0.1% as part of combination treatments. Currently, in vitro susceptibility testing for these cationic antiseptics does not exist.[53](B3)

Immune modulation and steroids

Patients with persistent or recurrent disease warrant a short course of topical steroids or steroid-sparing agents like tacrolimus 0.3% and cyclosporine 0.5%.[54][55][56] Tacrolimus and cyclosporine modulate and suppress corneal inflammatory responses. Apart from this, patients who develop endotheliitis, limbitis, and anterior chamber reactions warrant topical steroids.[19][21][19][27] Some studies suggest that the early application of topical steroids can prevent complications when dealing with specific types of keratitis. However, unlike adenovirus, studies have not conclusively shown steroids' impact on microsporidia multiplication.(B2)

Antifungal agents

Microsporidia are related to fungi. Azoles treat fungi by inhibiting ergosterol synthesis in the plasma membrane of fungi. Research published by Agatha and others involving 550 patients reveals that fluconazole 0.3%, dosed 4 times daily until symptom resolution, effectively achieves over 95% visual recovery.[21][57]  

Oral Therapy

Antifungals

Itraconazole, an oral antifungal medication, has been administered to treat specific infections without any relapses. However, its definitive efficacy is still under scrutiny.[58] Oral Itraconazole 200 mg/d is effective for disseminated diseases; however, the effectiveness of an oral antifungal in treating microsporidial keratitis has not been established.[59](B3)

Antiprotozoal and antihelminthic medications

Albendazole is an antiprotozoal effective in treating microsporidial keratoconjunctivitis.[60] It exerts its action by inhibiting microtubules, thus impeding cell division. Albendazole is most effective against Encephalitozoon species, with a recommended dose of 400 mg twice daily.[49] (B3)

Corneal Swabbing and Surgical Management

In microsporidial keratoconjunctivitis, the debridement of the corneal epithelium serves dual purposes: collecting samples for microbiological evaluation and reducing the microbial load. This procedure typically involves using a 15-blade or 26-gauge needle, and the affected area normally heals without scarring.[17] Debridement can be utilized alone or with antimicrobial and lubricating agents. However, debridement is not superior to conservative management and increases the risk of superimposed infection. Therefore, it is advisable to reserve debridement for severe cases.[61] (A1)

Repeated corneal swabbing using cotton swabs is another effective, relatively less invasive procedure proposed by Fan and colleagues. This method involves repetitive swabbing of the cornea using cotton swabs, which is less intrusive, causes minimal discomfort, is generally well-received by patients. Moreover, results are typically evident within a week of the procedure.[19] (B2)

The outlook for stromal disease is less optimistic. While some case reports describe successful medical treatment, the efficacy varies. Medications used alone or in combination include topical polyhexamethylene biguanide, chlorhexidine, and voriconazole with or without oral itraconazole or albendazole. However, one study revealed that less than half of the patients responded to medical therapy consisting of topical polyhexamethylene biguanide and chlorhexidine, with or without oral albendazole. Ultimately, 25 of the 34 patients required keratoplasty. Therapeutic penetrating keratoplasty often emerges as the only viable option for patients with corneal stromal involvement.[34][62](B3)

Differential Diagnosis

The differential diagnoses for microsporidial keratoconjunctivitis include:[55]

• Adenoviral keratoconjunctivitis
• Thygeson superficial punctate keratitis
• Dry eye disease
• Filamentary keratitis
• Vesicular stage of herpes simplex virus epithelial keratitis
• Acanthamoeba keratitis
• Atypical mycobacterial keratitis
• Viral keratoconjunctivitis 
• Bacterial conjunctivitis

Thygeson superficial punctate keratitis typically presents bilaterally, featuring coarse raised epithelial lesions predominantly located centrally. This condition responds favorably to treatment with topical steroids.

The differential diagnoses for microsporidial stromal keratitis include:

• Herpetic stromal keratitis
• Fungal corneal ulcer
• Bacterial corneal ulcer

Prognosis

Microsporidial stromal keratitis often follows a pattern of remission and recurrence until surgically removed via penetrating keratoplasty. Conversely, microsporidial keratoconjunctivitis typically resolves without any long-term sequelae or residual effects. In a study involving 332 patients treated for microsporidial keratoconjunctivitis, 49 returned for reevaluation at 6 weeks. Among the returning patients, 16 individuals (4.8%) returned for symptoms of microsporidial keratoconjunctivitis, and the remaining patients had non-microsporidial keratoconjunctivitis eye complaints like refractive error and dacrocystitis. The 3 manifestations of persistence were superficial punctate keratitis, sub-epithelial infiltrates, and uveitis. The 16 patients with symptoms of microsporidial keratoconjunctivitis underwent further treatment and achieved disease resolution. Some of them experienced recurrences at a later date. Visual acuity typically returns following successful treatment, with recovery rates ranging from 70% to 100%. However, unresolved subepithelial infiltrates may lead to worsening vision.[19]

Complications

Nummular scars can persist in cases of inadequately treated microsporidial keratoconjunctivitis. In cases of stromal keratitis, corneal scars and edema usually increase with each recurring episode of inflammation.[55] Increased intraocular pressure due to topical corticosteroid use is an additional medication-related complication. Other potential sequelae are limbitis, endotheliitis, and keratic precipitates.[55] These can be treated with topical steroids.[54]