The Effectiveness of PCR in Diagnosis of Fungal Keratitis

Abstract

Fungi implicated in mycotic keratitis include different species. Conventional methods for the diagnosis of fungal keratitis include staining of corneal scarpings, culture medium (Sabouraud agar) for isolating fungi.Purpose. To evaluate the effectiveness of polymerase chain reaction (PCR) for the detection of fungal etiology in comparison with the conventional diagnostic methods in cases with suspected fungal corneal ulcer.Patients and methods. Seven patients with severe corneal ulcers with more than 3 weeks duration. Corneal scarpings and corneal buttons from seven patients who had undergone therapeutic keratoplasty were used for microbiological and PCR analysis. PCR diagnostic kits for the differential detection of Candida albicans DNA and total fungi DNA (DNA Fungi), which allows to identify most pathogenic fungi without determining their species were used. Microbiological methods: microscopy of gramstained smears, culture techniques, including selective for fungi agar Saburo with chloramphenicol.Results. PCR: Fragments of all corneas removed from keratoplasty (6 patients) revealed fungal-common DNA (Fungi DNA) and did not detect Candida albicans DNA, which correlated with sowing results on Saburo medium (mold fungi found in 5 of 6 corneas). Fungi DNA was also detected in the corneal scraps taken prior to surgery; however, growth of fungi during sowing on various nutrient media was not found.Conclusion. Corneal fungal ulcers are a serious disease, often leading to visual disability. The rapid determination of etiology and the correct choice of therapy determines the outcomes of the disease. The advantage of PCR over the culture method: the speed of obtaining results (4 hours instead of 3–7 days); high sensitivity, which allows detecting fungi not only in the tissue of the removed cornea, but also in scrapes from the cornea ulcer of patients who previously received antifungal therapy. The presence of commercial kits for differential detection of fungal-common DNA and DNA of Candida albicans extends the possibilities of PCR in the screening diagnosis of fungal keratitis and the selection of drugs before determining the type of pathogen.

Modern Technologies in Diagnosis of Fungal Keratitis (Review)

“…Genus and sometimes species of the causative agent are identified by specific appearance of the colony (color, shape, consistency, spore availability, hypha, pseudohypha). This method is considered the golden standard of the diagnosis, is easy enough, and cost-effective, but still having some limitations: difficulty of species diagnosis, long response time (about a week) [ 80 , 81 ], dependence on the researcher experience, non-typical morphology of some colonies, insufficient knowledge of the suitable conditions of cultivation, inaccessibility of stromal layers for corneal scraping in case of deep fungal penetration [ 82 ], false-negative results of inoculation in case of insufficient scraping volume or progression of corneal destruction, the effect of the previous empirical therapy on the inoculation results [ 83 ]. For example, one of the studies [ 84 ] reported negative results of inoculation and unestablished laboratory diagnoses in 37% of patients with fungal keratitis explaining the reason by antibiotic therapy administered before the admission to the hospital.…”

“…The advantages of PCR are indisputable: the results may be obtained within 4 h instead of 3–7 days in cultural investigations; the method is highly sensible enabling to detect a pathogen in a small scrape from the corneal ulcer or the material from patients receiving previously antifungal therapy [ 81 ]. Like other molecular genetic methods, PCR is designed for species identification.…”

“…Polymerase chain reaction. The PCR diagnosis pertains to the methods of molecular genetic diagnosis and is not inferior to or even exceeds microscopy of the stained preparations and cultural methods detecting fragments of fungal DNA even in cases with negative culturing results [81,97].…”