Etiological Analysis Of Fungal Keratitis And Rapid Detection Of The Predominant Pathogen

Fungal keratitis is a world-wide, refractory and potentially blinding fungal infection,with corneal ulceration, suppurative infection and visual loss, which seriously affect thehealth and life of patients. In recent years, the incidence of fungal keratitis increasesignificantly in association with previous vegetative corneal injury, chronic ocularsurface disease, corneal surgery or transplantation, inadequate use of topical antibioticsand steroids or wearing contact lenses, host hypoimmunity with AIDS, diabetes andsystemic immunodeficiency diseases and so on.Fungal keratitis is an insidious, rapidly progressive disease that is difficult diagnosis.Treatment remains limite by scarcity of effective antifungals and serious resistance withpathogenic fungi. Therefore, it is significant to understand the common clinicalmanifestations, risk factors, age and season of fungal keratitis; grasp the predominantpathogenic fungi and the characteristics of their drug sensitivity; establish a rapid,specific method to identify the pathogens of fungal keratitis. These would be useful forrapid accurately diagnosis, accelerating early antifungal therapy, control the infection,increasing the cure rate, and saving the diseased eye.The commonly used diagnostic methods of ocular infection are direct smearexamination, fungal culture, in vivo confocal microscopy, serological approaches andhistopathological examination. These methods are difficult to meet needs of patientswith lower isolating rate, time-consuming and easily lead to corneal damage by thelimited corneal scraping size and slow growing of pathogenic fungi.In recent years, the DNA-based PCR and DNA sequencing techniques developedgradually. It has broad prospects for application in clinical detection of fungal keratitisdue to theirs high sensitivity and specificity. PCR techniques for diagnostic andepidemiological studies of fungal keratitis include nested PCR, RFLP and others. Butthe clinical detection is still more dependent on fungal culture owing to the shortage ofeffective extraction method with microamounts of DNA from corneal scrapings andspecific primers of important pathogenic fungi. Recently, multiplex PCR as a novelPCR technique has been wildly used in the diagnosis of microbial and parasitic infections, because it can detect a variety of pathogens at the same time and is a simple,rapid method with high isolating rate. It has rarely been reported using this method todiagnose fungal keratitis currently.In this study, we designed the species-specific primers of the predominant pathogensbased on comprehending the etiological and epidemiological characteristics of fungalkeratitis in Jilin Province, combined with improved DNA extraction method ofinfectious corneal tissue, we used multiplex PCR to rapid identify the pathogens withcorneal scrapings of clinical patients.1. Etiological and epidemiological analysis of fungal keratitisThe present retrospective study of225patients with suspected fungal keratitis wasconducted over a period from October2004to December2011in Jilin Province.Isolation, identification and drug sensitivity test of the pathogens, and analysis ofepidemiological characteristics was performed.208cases were diagnosed with fungalkeratitis by KOH wet mount and fungal culture. Fifteen genera,28species and168strains were isolated. Strains of the genus Fusarium were isolated most frequently,followed by Aspergillus and Candida. And F. solani, A. fumigatus and C. glabrata wasthe predominant causative pathogen caused fungal keratitis.The study showed that the incidence fungal keratitis in Jilin Province was increasedduring autumn and winter. The proportion of male and female patients was1.5:1. Mostof the patients were farmers. The average age was50.1±12.5years. Corneal trauma wasthe most common predisposing factors. More than a half of patients were previouslytreated topically with antibiotics, antifungals and steroids. The drug sensitivity testshowed that28isolates of F. solani were sensitive to Natamycin (sensitivity100%),Voriconazole and Amphotericin B, but resistance to Itraconazole.12isolates of A.fumigatus and10isolates of C. glabrata were sensitive to the four antifungalsmentioned above. This indicated Natamycin might be first choice in prevention andtreatment of fungal keratitis in clinic.2. Multiplex PCR of the predominant pathogenic fungiDNA sequence of pathogenic and environmental fungi was amplified by using theuniversal primers of mitochondrial cytochrome b gene. Homology alignment analysis ofDNA sequence was perfromed to design three pairs of species-specific primers of thepredominant pathogen of fungal keratitis, and established and optimized multiplex PCR.F. solani, A. fumigatus and C. glabrata could be amplified with the specific fragment of ~320bp,270bp and230bp, individually, whereas other fungi, bacteria and normalcornea could not be amplified, which showed the designed primers had goodintraspecific universal and interspecific specificity. And the sensitivity of reactionsystem was50pg. With this method, we reclassified the isolates which were difficult toidentification with morphological characters.3. Rapid detection of patients with fungal keratitisFusarium keratitis in guinea pig model was established by using corneal stromalinjection method, and was confirmed by fungal culture and histopathologicalexamination with the achievement ratio was90%. We established the extraction methodof infectious corneal tissue DNA with fungal keratitis. Using the improved DNAextraction and PCR with the universal fungal primers, corneal scraping specimens of42patients with suspected fungal keratitis was detected. The positive rate of PCR (88%)was higher than the fungal culture method, which had statistically significant. Andmultiplex PCR with species-specific primers was used to rapid and specific identify thepredominant pathogens. Conventional fungal culture often took1~2weeks to obtainthe positive identification. While this culture-independent method could rapidly andspecifically diagnose only in4~8hours, with or without fungal infection, single ormixed infection, and infected by which species of fungus.In summary, we analyzed the etiological and epidemiological characteristics of fungalkeratitis in Jilin Province. These informations could be useful for clinical diagnosis andtreatment. And we established the improved DNA extraction method by using Fusariumkeratitis in guinea pig model, designed three pairs of species-specific primers of thepredominant pathogens in Jilin Province, and used multiplex PCR to rapid identify thepathogens in corneal scrapings of clinical patients. It comfirmed this method was a rapid,culture independent diagnostic technique with high specificity and sensitivity. These canbe the foundation for widespread application to the clinic.