Histopathological Evaluation of Corneal Tissues in Microsporidia Stromal Keratitis

Abstract

Purpose: The purpose of this study was to evaluate the histopathological factors in non-resolving cases of microsporidia stromal keratitis (MSK) through the study of corneal buttons obtained during therapeutic penetrating keratoplasty (TPK).

Methods: This was a retrospective noncomparative consecutive case series. This case series included 22 corneal buttons (22 patients) of histologically diagnosed MSK between June 2015 and April 2023. Records of preoperative clinical and microbiological data, and postoperative microbiological and histopathologic data of the corneal buttons were evaluated.

Results: Histologic evaluation was conducted of the buttons for morphologic changes, degree and distribution of inflammatory cells, presence of microsporidial spores, and their degree and distribution within the corneal buttons. This study evaluated 22 patients with MSK, highlighting clinical, microbiological, treatment, and histopathological findings. The mean patient age was 57.1 ± 13.4 years (range = 22–83 years). The median interval from symptom onset to presentation was 4 months, and the mean time from presentation to keratoplasty was 1 month. Microsporidia spores were detected in 59% of cases through smears, with 41% of cases showing no organisms on microbiological tests. Targeted therapy using polyhexamethylene biguanide (PHMB) 0.02% was given in 13 cases, whereas 9 cases were treated empirically. Histopathology showed no significant correlation between the distribution of inflammatory cells and that of microsporidia. Moderate microsporidia severity correlated with longer symptom duration (10.0 ± 6.36 months). These findings underscore the complexity of MSK management and the variable outcomes.

Conclusions: The progression of MSK in advanced stages appears to be influenced by a combination of pathogen-related factors, such as high microsporidial load with deep stromal penetration, and host-related factors, including a pronounced inflammatory response. Additionally, the limited effectiveness of topical PHMB may contribute to disease progression.

Microsporidia are spore-forming, obligate intracellular eukaryotic parasites classified under the phylum Microspora and the kingdom Protista.^1–3 Recent morphological and molecular studies have reclassified microsporidia as fungi. These parasites infect both vertebrates and invertebrates, with their infectious stage being the intracellular spore.⁴ In humans, microsporidia primarily act as opportunistic pathogens, affecting individuals with conditions such as AIDS, organ transplant recipients on immunosuppressive therapy, and, in some cases, immunocompetent individuals. Microsporidiosis can involve multiple organs, most commonly the gastrointestinal tract, followed by the eyes, sinuses, respiratory system, muscles, kidneys, and central nervous system.⁵ 

The eye is the second most frequently affected site, with the cornea being a primary target regardless of immune status. Common ocular manifestations include superficial epithelial keratoconjunctivitis, stromal keratitis, and endophthalmitis.^6–8 Endophthalmitis cases are predominantly reported in immunocompromised individuals.^8,9 Although keratoconjunctivitis was initially observed in immunocompromised patients, it is now recognized in epidemic outbreaks and even in healthy individuals.^10,11 It typically presents as multifocal, coarse granular corneal lesions with punctate epithelial defects and is self-limiting in nature.¹² 

Ocular microsporidiosis varies in clinical presentation depending on the genus involved. For instance, Encephalitozoon primarily affects corneal and conjunctival epithelial cells, causing diffuse punctate epithelial keratoconjunctivitis, whereas Nosema and Microsporidium often invade the corneal stroma, targeting keratocytes.¹³ 

Microsporidial stromal keratitis (MSK) is a distinct clinical entity, presenting with deep stromal infiltration and nonspecific features that can lead to delayed diagnosis or misdiagnosis.¹⁴ These infections are typically slow-growing and lack hallmark signs, resulting in diagnostic challenges.¹⁵ Conventional microbiological tests often fail due to the organism's inability to grow in standard media, necessitating a high index of suspicion during microscopic evaluation of stained samples. 

Management of MSK remains contentious. Systemic albendazole combined with topical chlorhexidine, or oral itraconazole with topical voriconazole, have shown efficacy in treating MSK.^16,17 Penetrating keratoplasty (PK) is widely regarded as the gold standard treatment for advanced MSK. This study primarily aims to determine whether disease progression despite antimicrobial treatment is driven by persistent infection, ongoing inflammation, or a combination of both. We expect an inverse correlation between the inflammatory infiltrates and microsporidial spores as a heavy spore load would inhibit the inflammatory response to perpetuate their growth. We hypothesize that persistent microsporidial infection and host inflammatory responses contribute independently to MSK progression, which can be differentiated through histological analysis, therefore, a histopathological evaluation of corneal buttons has been utilized to assess the role of pathogen factors, such as the degree and distribution of microsporidial spores, and host factors, including inflammatory response, tissue damage, and vascularization, in the progression of MSK. 

Materials and Methods

A retrospective review was conducted on the demographic data, clinical features, microbiological findings, treatment details, and outcomes of histopathologically confirmed cases of microsporidial stromal keratitis treated with therapeutic penetrating keratoplasty (TPK) between June 2015 and April 2023 at L. V. Prasad Eye Institute, Bhubaneswar, India. The study received approval from the Institutional Review Board (IEC# 2023-178-BHR-3), and all patient data were handled confidentially. The research adhered to the ethical principles outlined in the Declaration of Helsinki 1964 and its subsequent amendments. 

Baseline and Clinical Characteristics

Data collected included age, gender, comorbidities, duration from symptom onset to hospital presentation, duration from presentation to TPK, total time from symptom onset to TPK, follow-up duration, and treatment outcomes. Visual acuity and clinical features recorded during slit-lamp examinations at the first hospital visit were reviewed. Hospital protocol mandated that patients with microbial keratitis must undergo bilateral syringing, blood sugar testing, slit-lamp photography, posterior segment ultrasonography, and corneal scraping for smear and culture. The treatment was initiated based on smear results. TPK was performed for cases with poor response to therapy, keratitis extending to the limbus/posterior segment, or with perforation. The clinical parameters analyzed included the maximum diameter of corneal infiltration and epithelial defect, presence of keratic precipitates, hypopyon, and perforation. 

Microbiological Examination

Corneal lesions were scraped with the subjects under topical anesthesia using a sterile surgical blade (#15), and specimens were subjected to Gram staining, potassium hydroxide (10% KOH) with calcofluor white (CFW) preparation, and cultured on blood, chocolate, non-nutrient, Sabouraud dextrose, and potato dextrose agars, as well as thioglycolate broth, brain-heart infusion, and Robertson cooked meat medium. Alternative methods, such as specialized media, tissue culture techniques, and serological approaches, were not utilized. For smaller infiltrates, selected media were used based on clinical discretion. Few samples with high degree of clinical suspicion and/or smear negativity for MSK were tested with conventional PCR-based assays. Corneal tissues obtained during TPK were also examined microbiologically for the presence of spores. 

Histopathological Examination

TPK specimens were fixed in 10% neutral buffered formalin and processed for histopathology. Paraffin-embedded tissues were stained with hematoxylin and eosin, periodic acid-Schiff (PAS), and Gram stains, along with a 1% acid-fast stain (Figs. 1A–C). Microsporidial spores were analyzed for their number, distribution within the stroma, and extension beyond Descemet's membrane (DM). Histological changes in corneal layers, including inflammation (type, degree, and distribution) were documented. 

Figure 1.

 

Histopathological section of corneal button showing microsporidial spores stained with: (A) Periodic acid Schiff (PAS) stain (×40), marked in black arrows; (B) Grams chromotrope (×100), marked with a black star, and (C) 1% acid fast stain (×100), marked with a black arrow; this also shows the presence of the polar tube at one end of the spore.

Inflammation severity was graded semi-quantitatively: mild if the keratocyte nuclei and stroma were clearly visible; moderate if keratocyte nuclei were obscured but stroma remained identifiable; and severe if both keratocyte nuclei and stroma were completely obliterated (Figs. 2A–C).¹⁸ The microsporidial load was categorized as mild, moderate, or severe based on the density of spores within two high-power fields (Figs. 2D–F). The DM was assessed for integrity, noting whether it was intact or breached. 

Figure 2.

 

Light microscopic photographs of microsporidial stromal keratitis showing: (A) (stain, hematoxylin–eosin; original magnification, ×10), mild grade of inflammation, that is, the background stroma and keratocyte nuclei were clearly visible; (B) (stain, hematoxylin and eosin; original magnification, ×20), moderate grade of inflammation, that is, the keratocyte nuclei were obliterated, but the stroma was visible; (C) (stain, hematoxylin and eosin; original magnification, ×20), severe grade of inflammation, that is, complete obliteration of keratocyte nuclei and corneal stroma. Histopathological section of corneal button showing 1% acid fast microsporidial spores: (D) mild infection load (100×); (E) moderate infection load (100×); (F) severe infection load (20×).

Data Management and Statistical Analysis

Patient data were retrieved from electronic medical records (EMRs). Demographic details and clinical features were presented as frequencies and percentages for categorical variables and as mean with standard deviations for continuous variables. Univariate analysis was conducted for each variable. The Fisher's exact test was used to compare categorical data, while the Mann–Whitney U test was applied to continuous data, as the Shapiro-Wilks test indicated a non-normal data distribution. Statistical analyses were performed using IBM SPSS Statistics for Mac, version 27.0.1.0 (IBM Corp., Armonk, NY, USA). A P value of less than 0.05 was considered statistically significant. 

Results

Patient Demography

The mean age of patients was 57.1 ± 13.4 years (range = 22–83 years). The highest incidence was noted between 50 and 80 years (n = 17, 77.2%). There were 13 male (59.1%) and 9 female (40.9%) patients. Type 2 diabetes mellitus, hypertension, and a history of renal transplant was present in 1 case each. A history of trauma with vegetative matter was present in three cases. The mean interval between the onset of symptoms and presentation was 5.6 ± 4.6 months (median = 4 months), between presentation and TPK was 2.8 ± 4.0 months (median = 1 month), and between symptom onset and TPK was 8.3 ± 5.0 months (median = 7 months; Table 1). Keratic precipitates were appreciable in 12 of 22 patients, and hypopyon and perforation in 3 of 22 patients each. 

Table 1.

 

Baseline Characteristics of the Study Population (n = 22)

 

Microbiology

On smear obtained from corneal scraping, microsporidia spores were identified in 13 of 22 (59%) patients of which 9 of 22 patients were positive both on Gram and 10% KOH with 0.1% CFW preparation, 3 of 22 patients on KOH + CFW under fluorescence microscope, and 1 of 22 patients on Gram only. In addition, one patient identified with Gram-positive cocci. On culture, Staphylococcus spp was present in 6 of 22 patients and 1 of 22 patients showed growth of Corynebacterium spp. Conventional polymerase chain reaction (PCR) was performed in 8 out of 22 cases before treatment initiation, with 2 cases testing positive. Both these cases also showed positive results on Gram stain and KOH-Calcofluor stain. A complete microbiologic study of the bisected corneal button obtained at the time of keratoplasty was available in all cases and showed no growth. 

Treatment Outcomes

Medical Management

Thirteen of the 22 cases (59%) were managed as MSK with hourly instillations of topical polyhexamethylene biguanide (PHMB) 0.02%, and oral itraconazole was added in 1 case. The duration of medical treatment was 2.8 ± 4.0 months (median = 1 month). We did not obtain any organism on Gram or KOH + CFW or culture in 9 of 22 (41%) cases, hence, they were kept on empirical combination therapy of fortified cefazolin 5% and ciprofloxacin 0.3% hourly. One case was clinically suspected and treated as herpes simplex virus (HSV) keratitis for 1-year at our institute before proceeding for keratoplasty. 

Surgical Management

The indications for penetrating keratoplasty were: (a) large infiltrate or perforation at the time of presentation, (b) no etiology even on repeated diagnostic workup, (c) no response to medical treatment, and (d) gross thinning or perforation. Three patients underwent TPK on the same day, one of whom had presented with a large perforation. A worsening of infiltrates was noted in 8 out of 22 cases. The remaining 11 cases did not show any signs of improvement over time. Their postoperative medical regimen included topical PHMB 0.02% for a duration of 1 to 3 months. A topical steroid was started on the first postoperative day in 7 cases, on day 7 in 6 cases, on day 14 in 3 cases, on day 21 in 3 cases, and at 1 month in 3 cases. 

After keratoplasty, there was recurrence of microsporidia infection in one case, which warranted evisceration because of corneal melt not salvageable by repeat TPK. The corneal grafts were optically clear in 9 of 22 cases at 1 year. Nine cases were noted to have a failed graft at 1 year, the cause of failure being rejection in 5 cases. A graft infection was noted in three cases (that resolved medically and healed with scarring) and primary graft failure was noted in one case. All the three cases demonstrated Gram positive cocci on smear examination of which two revealed Coagulase negative Staphylococcus species in routine culture. One case was lost-to-follow-up and was categorized as a failed graft. Two were noted to have failing grafts following a rejection episode at 1 year. 

Histopathology

The morphologic changes in the various corneal layers on HPE are as follows: epithelial ulceration in 16 cases, a disrupted Bowman's layer in 7 cases, stromal thinning in 3 cases, stromal necrosis in 11 cases, vascularization in 4 cases, fragmented DM in 13 cases, and perforation in 3 cases. In four cases, vascularization was localized to the deep stroma. 

Inflammation was seen mostly involving all the layers (n = 15, 68.2%), whereas in others it was localized (n = 6, 28.6%), restricted to either the anterior two-thirds or posterior one-third of the stroma. One corneal button did not demonstrate any inflammatory cells. Mixed inflammation was present in 17 corneal buttons and chronic inflammation in 4 buttons. Type, degree, and location of stromal inflammation are shown in Table 2. 

Table 2.

 

Histopathological Characteristics of the Involved Eye

 

Microsporidial spores were either diffuse (n = 12, 54.5%) or localized (n = 10, 45.5%) either to the anterior two-thirds or to the posterior one-third of the stroma. The microsporidial spore load in the corneal buttons was mild in five cases (22.7%), moderate in nine cases (40.9%), and severe in eight cases (36.4%). 

Correlation Between Host Variables and Pathogen Characteristics

There was no significant correlation between inflammation and the distribution of microsporidia on histopathology. In terms of location, 20% of those with localized inflammation had localized microsporidia distribution, whereas 80% had diffuse distribution. Among those with diffuse inflammation, 36.4% had localized and 63.6% had diffuse distribution (P = 0.635). Chronic inflammation had 10% localized and 90% diffuse microsporidia, whereas mixed inflammation showed 27.3% localized and 72.7% diffuse distribution (P = 0.586). The duration of symptoms (P = 0.123), duration from surgery (P = 0.821), and total duration (P = 0.381) also showed no significant difference between localized and diffuse distribution (Table 3). 

Table 3.

 

Correlation of Inflammation With Distribution of Microsporidia on Histopathology Among the Study Population

 

There was no significant association between the severity of microsporidia and inflammation characteristics. Among patients with chronic inflammation, 20% had mild and 80% had moderate microsporidia severity, whereas those with mixed inflammation showed 100% moderate severity and a distribution of 42.9% mild and 57.1% severe microsporidia (P = 0.096). The severity of inflammation (P = 0.401) did not significantly correlate with microsporidia severity. The duration of symptoms (P = 0.430), duration from presentation (P = 0.825), and total duration (P = 0.614) also showed no statistically significant differences across the varying severities of microsporidia (Table 4). 

Table 4.

 

Association of Severity of Microsporidia With Inflammation Characteristics on Histopathology Among the Study Population

 

The analysis showed no significant association between inflammation type and the duration of symptoms, time to surgery, or total duration (P > 0.6). However, symptom duration was significantly longer in the moderate severity group (10.0 ± 6.36 months) compared to the mild and severe groups (P = 0.030). The total duration was also higher in the moderate group, although not statistically significant (P = 0.060). Time to surgery did not differ significantly across severity groups (P = 0.443; Table 5). 

Table 5.

 

Association of Duration of Symptoms, and Time to Surgery With Inflammation Characteristics

 

There was no significant difference between targeted and empirical treatments in terms of inflammation type and microsporidial distribution (Fig. 3). Inflammation was predominantly mixed in both treatment groups (83.3% in targeted versus 77.8% in empirical). For microsporidial distribution, localized cases were more frequent in the empirical group (55.6%) compared to the targeted group (38.5%; Fig. 4). 

Figure 3.

 

(A) Slit lamp photograph of a 50-year-old patient showing infiltrates and epithelial defect measuring 7 × 7 mm. Corneal scraping revealed microsporidial spores. The patient was started on PHMB 0.02% eye drops. The section of the corneal button following keratoplasty shows dense mixed inflammatory cells and refractile spore like structures in interlamellar area involving full thickness on; (B) stain, hematoxylin and eosin (×10); and (C) 1% acid fast stain (×10); (D) Slit lamp photograph of a 69-year-old patient showing infiltrates and epithelial defect measuring 6 × 5 mm. Corneal scraping revealed gram positive bacilli consistent with Corynebacterium species. The patient was started on fortified cefazolin 5% and ciprofloxacin 0.3% eye drops. The section of the corneal button following keratoplasty shows dense mixed inflammatory cells in the posterior stroma and refractile spore like structures in interlamellar area involving the anterior and mid stroma on; (E) stain, hematoxylin and eosin (×20); and (F) 1% acid fast stain (×20).

Figure 4.

 

Graph illustrating the percentage distribution of corneal buttons categorized by the type of inflammation and microsporidial distribution in cases receiving targeted treatment compared with those undergoing empirical treatment.

Discussion

MSK remains a poorly defined and an underdiagnosed disease, unlike its keratoconjunctivitis variant.^6,19 Despite advancements in diagnostic techniques and medical treatments, MSK generally has a poor prognosis and a high rate of failure with medical therapy, often necessitating keratoplasty in most reported cases. There have been a few case reports demonstrating resolution of MSK with medical management alone in the early stages of MSK.¹⁷ 

MSK is frequently misdiagnosed as bacterial, viral, or fungal keratitis, leading to delays in initiating targeted treatment for microsporidia. The most intriguing aspect of MSK lies in its prolonged pathogenesis and chronic progression, which are believed to result from complex interactions between host and parasite factors within the eye. Huang et al. proposed several theories for this extended course.²⁰ One hypothesis suggests that the cornea's immune tolerance allows microsporidia to persist without triggering a strong immune response.²¹ Additionally, microsporidia are known for their ability to survive within host cells. Another possible explanation is the parasites’ capacity to evade immune detection by residing within keratocytes over extended periods.²⁰ 

Previous histological studies on MSK have included cases with confirmed evidence of microsporidial spores in tissue sections.^21,22 In our study, only histologically confirmed cases of microsporidial infection were included. Of these, 59% were managed as MSK under institutional supervision, with penetrating keratoplasty performed only after monitoring disease progression. This approach allowed for a detailed evaluation of histological and microbiological factors contributing to disease progression, thereby enhancing our understanding of MSK pathogenesis. A retrospective analysis of 34 MSK cases from our institute previously detailed the history, clinical characteristics, and outcomes, emphasizing potential etiological predictors.²² In comparison, another study from the same institute, involving five MSK cases, found all cases to be chronic, with four requiring keratoplasty.⁷ Whereas both studies provided valuable clinical insights, the smaller study particularly underscored the chronic nature of MSK and its strong association with surgical intervention. The present study further builds upon these findings by focusing on the histopathological aspects of MSK progression using corneal buttons obtained during therapeutic keratoplasty. Unlike previous studies, which primarily examined clinical outcomes and epidemiological factors, our study offers a deeper exploration of tissue-level changes that contribute to disease persistence and recurrence. A key similarity across these studies is the significant role of keratoplasty in MSK management, especially in chronic or refractory cases. However, a notable difference lies in the scope of analysis. Although earlier research emphasized clinical progression and outcomes, our study provides a more granular understanding of the underlying histopathological changes. 

The average symptom duration in this series was 5.6 ± 4.6 months, with a median of 4 months. This chronic presentation aligns with literature reporting durations from 4 months to 2 years.^7,22 Diagnostic methods, such as KOH and CFW staining examined under fluorescence microscopy, supplemented by Gram staining, were effective for detecting microsporidial spores. However, culture methods were less useful due to the organism's reliance on tissue culture for growth.²³ 

Histopathological evaluation played a crucial role in diagnosing 10 of 22 cases where microbiological tests were negative, and patients received empirical treatment before keratoplasty. This underscores the importance of preoperative corneal biopsy and/or in vivo confocal microscopy in detecting microsporidial infection when routine smears are inconclusive. Therefore, implementing such techniques can aid early diagnosis and guide appropriate management. 

Interestingly, no correlation was observed between the intensity and distribution of microsporidial spores and inflammatory cell patterns or treatment type. This lack of correlation could be attributed to several potential mechanisms. 

• 1. Immune Evasion by Microsporidia – Microsporidia are known to persist within host cells, particularly keratocytes, potentially avoiding strong immune recognition.
• 2. Localized Immune Privilege of the Cornea – The cornea exhibits immune tolerance, which may allow spores to persist without triggering a proportionate inflammatory response. This could explain why some cases have heavy spore loads without significant inflammation.
• 3. Host-Dependent Variability in Immune Response – Differences in individual immune responses may influence the inflammatory reaction independent of spore burden. Some hosts may mount a robust response leading to diffuse inflammation, whereas others may have a muted reaction despite extensive spore distribution.
• 4. Delayed or Ineffective Immune Activation – The immune system may fail to recognize microsporidial spores immediately due to their intracellular location, leading to an inflammatory response that does not directly correspond to the actual distribution of spores within the cornea.
• 5. Role of Antimicrobial Therapy – Prolonged medical therapy might alter both the spore load and the inflammatory response independently.

Overall, these factors suggest that microsporidial keratitis does not follow a simple pathogen-driven inflammatory response but rather a complex interaction among immune tolerance, pathogen survival strategies, and therapeutic modulation. The progression of MSK appears influenced by factors such as high pathogen load, inadequate inflammatory response, and potential limitations of antimicrobial therapy. In the later stages, host factors may also significantly modulate disease progression. 

A notable observation from our study was the shorter interval between the time of presentation and TPK (1 month), compared to the duration from symptom onset to presentation (4 months). This difference could be due to the manipulation of the infected corneal stroma during corneal scraping, which may have exposed the microsporidia spores to the host environment, potentially triggering an immune response and heightened inflammation.²¹ This speculation corroborates with our hypothesis regarding the host modulated response in the later phase of the disease. 

Vemuganti et al. studied the roles of both host and pathogen factors in the progression of fungal keratitis, observing an inverse relationship between the density and spread of inflammatory cells and the extent of fungal filaments.¹⁸ They suggested that, during the early phase, inflammatory cells might actively phagocytose the fungal filaments, or perhaps a high fungal burden could suppress the inflammatory response. In contrast, the chronic phase of fungal keratitis is thought to result from the harmful persistence of inflammation.¹⁸ 

It may be speculated that the inflammation, once initiated following corneal scraping, continues uninhibited. The progression of corneal tissue damage in MSK appears to result from the combined effects of microsporidia and inflammatory mediators. This contrasts with fungal keratitis, where late-stage damage is thought to be primarily inflammation-driven. We propose that incorporating an immunomodulatory drug into targeted therapy could help control the infection. Additionally, monitoring microsporidial spores in vivo using confocal microscopy could provide valuable insights into their persistence or elimination in corneal tissue. 

The detection of both microsporidia and inflammation in corneal buttons following extensive medical therapy, regardless of whether the treatment was targeted or empirical, raises concerns about insufficient drug penetration or potential in vivo resistance. Because microsporidia cannot be cultured in conventional culture media, testing for in vitro drug sensitivity is not feasible. Furthermore, the fact that spores are distributed across the cornea in 54% of cases or are restricted to the anterior two-thirds or posterior one-third in 46% of cases, indicates that the advancement of the disease may be connected to inadequate drug efficacy. Furthermore, the distribution of spores across all corneal layers in 54% of cases or localized to either the anterior two-thirds or posterior one-third in 46%, suggests that disease progression may be linked to suboptimal drug efficacy. Notably, four cases showed mild spore presence with diffuse inflammation and vascularization, supporting the hypothesis that vascularization represents a healing phase of the disease process. 

Only one patient experienced infection recurrence, requiring evisceration, emphasizing that TPK remains the definitive treatment for MSK with a low recurrence rate. In conclusion, the progression of MSK is influenced by pathogen-related factors, such as heavy spore load and deep tissue invasion, as well as host responses like intense inflammation. The limited effectiveness of topical agents like PHMB and voriconazole underscores the need for an optimized treatment protocol to manage MSK effectively. 

Although the retrospective design and small sample size of our study limit the scope for extensive statistical analysis, it remains significant as the largest case series reported to date that evaluates the interaction between host and pathogen factors in this rare condition. Microsporidia-specific microbiological diagnostic techniques such as cell culture and serology were not performed due to resource constraints, which remained another limitation in our study. Further research with larger sample sizes could provide a clearer understanding of these correlations. Moreover, monitoring of such cases with in vivo confocal microscopy was not done in our case series, and neither were the specific microsporidia species identified due to limited resources. There is significant scope for future studies to explore species identification using molecular techniques, investigate host response mechanisms, and conduct multi-center studies with larger, more diverse samples. These efforts will help validate existing findings and improve the reliability and generalizability of MSK research. 

Acknowledgments

Supported by the Hyderabad Eye Research Foundation, Hyderabad, India. 

Disclosure: S. Mandal, None; S. Sucharita, None; V. Deshmukh, None; S.R. Priyadarshini, None; S.K. Sahu, None; S. Das, None