Background: The study aimed to assess the diagnostic and therapeutic management of infectious keratitis at a tertiary referral center, focusing on how microbiological analysis influences clinical outcomes. Methods: A retrospective review was conducted on 220 patients (221 eyes) with infectious keratitis treated between November 2021 and January 2025. Data collected included clinical presentation, microbiological findings, treatment approaches, and outcomes. Statistical analyses examined the relationships between microbiological results, improvements in visual acuity, and the need for rescue surgery. Results: Bacterial keratitis accounted for 64% of cases, followed by viral (20%), fungal (13%), and Acanthamoeba (3%). Microbiological testing was performed in 107 cases, with a culture positivity rate of 75.7%. Positive microbiological findings were significantly associated with better visual acuity (p = 0.019) and a reduced, though not statistically significant, need for rescue surgery. Use of contact lenses and ocular trauma were independent risk factors for culture positivity. Delayed referral (more than 15 days) was linked to longer treatment durations and a higher likelihood of surgical intervention (p < 0.001). Microbiological diagnosis correlates with improved visual outcomes and a decreased need for surgical procedures. Conclusion: Early referral and targeted therapy are essential for optimizing prognosis. The use of contact lenses and cases of ocular trauma should prompt early diagnostic sampling.

Recent research has highlighted the critical role of microbiota in organ transplant outcomes, particularly in the gut. However, the impact of ocular surface microbiota (OSM) on corneal transplantation remains largely unexplored. This piece examines the potential connection between OSM imbalances and corneal graftoutcomes, suggesting that microbial shifts could influence immune responses and transplant success. The OSM, though characterized by low microbial density, plays a critical role in local immune modulation and ocular surface homeostasis. Dysbiosis in this microbiota may compromise the immune privilege of the cornea, potentially increasing the risk of graft rejection. Looking at gut microbiota studies, where dysbiosis has been linked to graft failure, it is reasonable to hypothesize that similar mechanisms might be at play on the ocular surface. Disruptions in cornea’s immune tolerance pathways, such as anterior chamber-associated immune deviation (ACAID), may lead to pro-inflammatory responses that threaten graft survival. In addition, ocular surface diseases such as dry eye disease, microbial keratitis, and allergic conjunctivitis, already associated with OSM dysbiosis, may further exacerbate post-transplant complications. Despite the lack of direct studies linking OSM to corneal transplant outcomes, this opinion piece highlights the necessity for future research. Standardizing microbiota analysis methodologies and exploring therapeutic interventions, such as ocular probiotics, could open new roads for improving corneal transplant success and patient prognosis.

Background/Objectives: Tears are a promising source of biomarkers reflecting both ocular and systemic conditions. However, small sample volumes and low cell yields pose technical challenges in analytical workflows. This study aimed to evaluate the feasibility of quantifying total cell counts and characterizing HLA-DR and CD3 expression in tear-derived cells using an automated cell counter with fluorescence detection (Countess 3 FL). Methods: Tears were collected from 31 patients, centrifuged and the resulting pellet was incubated with HLA-DR and CD3 antibodies, markers of inflammation and T lymphocytes, respectively. Data obtained from Countess 3 FL were compared with conventional flow cytometry and immunofluorescence. For technical performance analysis, precision and reproducibility of cell count and staining were measured. For method validation, an in vitro model of hyperosmolar stress was assessed by culturing conjunctival epithelial cells (CCL20.2) with 350 or 450 mOsm NaCl. Results: The total cell yield in each tear sample correlated with the tear surnatant volume, in a range of 1–40μL (mean total cell number: 1.3 ± 1.1 × 10⁴, correlation analysis with tear volume: r = 0.47, p < 0.05). HLA-DR and CD3 were detected in all samples, with a mean value, respectively, of 43.6% (±21.0) and 25.0% (±15.0) intensity. Data were comparable to those obtained from standard flow cytometry analysis.HLA-DR increase in CCL20.2 exposed to hyperosmolar stress was recorded using Countess 3FL reading, confirming the detection capacity of the proposed method. Conclusions: The automated cell counter can provide HLA-DR and CD3 quantification in tear cell samples, despite the high variability and the low volume availability of tear samples. Method standardization and technical improvements are necessary to strengthen this application in the clinical setting.

Acanthamoeba keratitis (AK) is a rare but sight-threatening corneal infection, caused by free-living protozoa of the genus Acanthamoeba. Although cases are rising in high-income countries, the nonspecific presentation and overlapping features with other keratitis frequently result in misdiagnosis of AK, with poorer outcomes and impaired quality of life. A delayed diagnosis can allow the infection to progress to deeper corneal layers, increasing the risk of severe structural damage and requiring emergency keratoplasty to preserve ocular integrity and regain vision. This retrospective study describes a three-year experience at a Northern Italy tertiary center using an integrated diagnostic workflow combining clinical evaluation, in vivo confocal microscopy, culture, and -since 2024- real-time PCR. Among 39 patients with suspected AK, 11 cases were confirmed. Culture showed 92 % sensitivity in first diagnoses but decreased to 77 % when relapses were included. PCR, introduced as a routine tool in 2024, showed 100 % sensitivity in first diagnoses and proved crucial in detecting low-burden or culture-negative relapses. Confocal microscopy reached a sensitivity and specificity of 82 %. Early targeted anti-amoebic therapy, guided by this integrated approach, contributed to favorable outcomes in most cases, limiting the need for surgical intervention. These findings support the utility of combining methods for timely and accurate diagnosis of AK. Despite ongoing challenges in the clinical management of AK, our findings reinforce the critical value of adopting molecular diagnostics to enable earlier, more accurate identification and targeted treatment.