The Role Of Scanning Electron Microscopy (Sem) In The Diagnosis Of Atypical Cases Of Acanthamoeba Negative To Standard Tests

The diagnosis of Acanthamoeba keratitis can be done using corneal scraping, polymerase chain reactions (PCR), with specific culture media or in confocal laser scanning microscopy (CLSM). As early signs may be mild and non-specific, diagenosis is often difficult and delayed. The aim of this study is to evaluate the presence of Acanthamoeba in suspected microbial keratoconjunctivitis with negative culture tests and the effectiveness of the SEM examination on conjunctival scraping in the identification of pathogens not detected with usual microbiological exams.

Multicenter study carried out by the Ophthalmology Unit of the University Hospital of Salerno and by the University of Naples Federico II. The SEM evaluation is carried out by Service Biotech srl.

112 eyes of 93 patients, affected by KC with negative swab, underwent scraping of the superior tarsal conjunctiva and cytological and microbiological evaluation in SEM. Exclusion criteria: age < 18 years, corneal abscess and  ulcer >3 mm, abscess, typical clinical pictures of herpetic infection, allergy or severe dry eye. In cases of Acanthamoeba detection, CLSM and PCR were carried out. The SEM examination was repeated after anti-amoebic therapy and a negative fluotest. At negative result of fluotest, after PHMB 0.02% and Chlorhexidine therapy, SEM exam was repeated; in case of persistence of Acanthamoeba, further therapeutic cycles and follow-up were carried out with subsequent microscopy control.

Pathogens were identified in 103/112 eyes (91.9%). In 23 of them (20.5%) Acanthamoeba was detected. Of the latter, 7 presented only cysts, 11 trophozoites, 5 mixed forms; in 5 of them other pathogens coexisted. CLSM highlighted the parasite in 13 eyes (4 cases of mixed forms, 8 of trophozoites, 1 of cysts); PCR testing for Acanthamoeba was positive in 16/23 cases. The therapy was effective in all cases diagnosed, with negative corneal fluotest and eradication of the germ at SEM examination in 21- 56 days (average 34.2 ± 2.1).

91.9% of KC tested negative on culture tests showed pathogenic germs, with a high prevalence of Acanthamoeba, in an atypical, non-abscess clinical form. CLSM did not allow diagnostic confirmation 10/23 eyes (43.5%), presumably due to the absence of stromal invasion by the parasite; similarly PCR was positive only in 69.6% of cases, probably for reduced microbial load in the sample. SEM applied to conjunctival scraping, minimally invasive and well tolerated, allowed the etiological diagnosis of atypical KC and the resolution of the clinical picture even in cases testing negative by PCR and CLSM in all cases.  Limits of this exam are the need for dedicated equipment and experienced operators in cytological and microbiology evaluation in SEM.