Scanning Electron Microscopy Of Conjunctival Scrapings: Our Experience In The Diagnosis Of Suspected Infectious Keratitis With Negative Culture Tests

Keratoconjunctivitis can be due to infections, immuno-allergic mechanisms, dry eye or harmful agents. When infection is suspected, broad-spectrum therapy is generally initiated, preferably after conjunctival or corneal sampling for microbiological examinations. The study evaluates 74 eyes affected by keratoconjunctivitis of suspected infectious nature with negative conjunctival swab for bacteria and fungi, which had not shown improvement after topical antibiotic therapy. The aim of the study is to evaluate the effectiveness of a diagnostic method based on the scanning microscope examination of scrapings of the upper tarsal conjunctiva to identify the microbes responsible for the clinical picture. 

Cases of presumed microbial keratoconjunctivitis reported at the Cornea Unit of the University Hospital of Salerno and at the University of Naples Federico II are evaluated. Most of the patients had already performed clinical and microbiological evaluation at other ophthalmic centers and had been sent to a second level unit due to an unsatisfactory response to the prescribed therapies.

74 eyes of 74 consecutive patients (34 males and 40 females, ranging from 18 to 86 years, with an average age of 53.8) presenting signs and symptoms consistent with infectious keratoconjunctivitis, but with recent negative culture tests,were examined. In case of bilateral involvement, the eye with the most severe signs of inflammation and corneal damage was considered. All the patients had undergone broad-spectrum local antibiotic therapy for at least 5 days whitout any clinical improvement.  The exclusion criteria were: corneal ulceration with diameter >3 mm and deeper than 80% of the corneal thickness; age <18 years; pregnant or lactating women; severe dry eye; Vernal or atopic keratoconjunctivitis. 

In 68 eyes out of 74 examined, the presence of pathogenic microorganisms was detected. In 33 (48.53%) of them two or more pathogens were present. In the six patients in whom the presence of pathogenic microorganisms was not detected, microscopic signs of dry eyes or allergic reactions were found. The germs found are, in order of frequency: Candida (23), Acanthamoeba (21), Mycoplasma (19), Chlamydia (7), Mycobacteria (6), Micrococci (4), Pseudomonas (4), Cocci (3), Aspergillus (3), , Cryptococcus (1), Cladosporium (1); in most cases the detection of Candida, Acanthamoeba, Mycoplasma, Chlamydia and Micrococci was associated with other microorganisms. 

The results of the study indicate a high sensitivity of the microbiological diagnostic test based on SEM evaluation. This method allowed to detect the presence of microbial agents in 92% of the eyes with negative results to standard culture tests, as well as to obtain further information deriving from the evaluation of the inflammatory cells present. The therapy developed on the basis of these data proved to be effective in all the examined cases, in a period ranging from 2 to 10 weeks. We also noted a good tolerability, linked to the sampling methods, which do not provide for direct scraping of the corneal lesion. Finally, the study also confirms the validity of the clinical approach in identifying patients with suspected infection.