Correlative microscopy (CLEM) of tarsal conjunctival scraping: A new opportunity in the diagnosis of microbial keratitis

Abstract

Aims/Purpose: To evaluate the efficacy of correlated Scanning Electron Microscopy (SEM)/Optical Microscopy (OM) analysis of conjunctival scrapings in diagnosing infectious keratitis with prior negative culture tests or ineffective broad‐spectrum antibiotic therapy.Methods: 102 eyes of 87 patients (45M, 42F) affected by keratitis were evaluated by cytological examination of scrapings of the superior tarsal conjunctiva in OM (Nikon Eclipse 50i) and SEM (JEOL), after 72 h of wash‐out. Inclusion criteria: age > 18 years, corneal epithelial defects from suspected infection, ineffectiveness of previous broad‐spectrum antimicrobial therapy or negative conjunctival swab. Exclusion criteria: ocular surgery <3 months, immunosuppression, allergic keratitis, severe dry eye, ocular herpes, ulcer >3 mm, anterior chamber involvement. Identification of inflammatory infiltrate (OM) and microorganisms (SEM) was performed by two independent operators with the same devices and methodology. Weekly clinical control with new conjunctival scraping for OM and SEM after cycles of therapy were carried out.Results: Pathogenic microorganisms were detected in 94/102 eyes: Acanthamoeba (22), Candida (18), Mycoplasma (15), Mycobacteria (13), Chlamydia (12), Micrococci (11), Pseudomonas (6), Cocci (6), Aspergillus (5), Fusarium (2); in 14 cases 2 or more different forms coexisted; in 2 cases a subsequent PCR test revealed the presence of HSV‐1; 3 cases showed signs of severe dry eye; other 3 marked eosinophilia. Operator agreement: 99%. The specific treatment for the germs detected determined in all cases clinical and microbiological resolution in 14–49 days (mean 27.6 ± 2.4).Conclusions: CLEM put in evidence the correlation between phlogistic cells and pathogens, allowing the diagnosis and the treatment of different forms of infectious keratitis and other pathological conditions. The interpretation and repeatability, however, require appropriate training and dedicated equipment.