Clinical Application Research Of Diagnosis Of Fungal Keratitis Using Multi-polymerase Chain Reaction

Fungal corneal infection causes severe visual impairment. The number of patients who are induced corneal blindness by Fungal Keratitis has been gradually increasing, particularly among agricultural workers in the developing world. It is important for ophthalmologist to assume correspondent therapeutic schedules according to various fungal strains. Early diagnosis is the key to appropriate therapy. Conventional microbiological assay techniques for diagnosing microbial keratitis rely on culturing and specimen microscopy. But both have apparent deficiencies such as long time period and no Genus Identification of Fungal Pathogens.Two multi-PCR systems were established. System 1 contains Fusarium-specific and Aspergillus, Penicillium and Curvularia-specific primers. System 2 contains Penicillium-specific and Curvularia-specific primers. PCR system specificity was also tested.The PCR assay was optimized using dilution of DNA template in sterile deionized water. We simultaneously detected reaction system sensitivity. Clinical fungus strains were used to research the possibility of PCR systems clinical genus identification; To establish a rapid and simple extracting system of fungal DNA from clinical samples with fungal keratitis and evaluate the usefulness of the multi-PCR systems in comparison with the conventional mycologic methods in the diagnosis of fungal keratitis.Material and methodsThe two systems were tested by Fusarium solani, Fusarium moniliform, Fusarium equiseti, Fusarium graminum, Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Penicillium implicatum and Curvularia lunata. And Human DNA and DNAs of other microorganism in human ocular infections also tested them.The multi-PCR systems were optimized using laddered dilution of Fusarium and Penicillum DNA template in sterile deionized water. The appropriate concentrations of every component in reagent mixtures and rounds parameter were defined. Making use of ultraviolet spectrophotometer to measure the mass concentration of purification DNA template, PCR system sensitivity was detected.Standard fungal filament sterile deionized water dilutions simulated clinical scraping specimens to extract DNA template by Guanidine Thiocyanate lysis, SDS (sodium dodecyl sulfate) lysis, CATB (hexadecyltrimethylammonium bromide) lysis, Triton-xlOO lysing and NaOH. Different DNA templates were added in optimized PCR reaction systems for PCR amplification. Visualization of the PCR product was done by electrophoresis in Tris-borate buffer on a 2% agarose gel incorporating 0.5μg/mL EB. Different methods for DNA lysing were compared according to DNA band brightness on an ultraviolet transilluminator.To test the genus identification accuracy, 72 clinical fungus strains DNA extracted by Guanidine Thiocyanate lysis were amplified in the multi-PCR systems. Two multi-PCR systems were used to diagnose and identify fungal pathogens in fungal keratitis. Corneal scrapings from 129 patients with presumed infectious keratitis were evaluated using this assay, as well as by standard microbiological determination techniques and by smear microscopy, and the results were compared. ResultThe fungi could be identified according to the amplification products by the two multi-PCR systems: In system 1, Fusarium species amplify products about 370 bp; Aspergillus, Penicillium and Curvularia species amplify products about 470 bp. In system 2, Penicillium implicatum amplifys product about 360 bp; Curvularia lunata amplifys product about 300 bp; Fusarium and Aspergillus species amplify no product.None of the bacterial and human DNA was amplified using these primers.In system 1, multi-PCR was sensitive enough to detect the presence of up to lOfg of Fusarium and Aspergillus DNA In system 2,the sensitivity of the multi-PCR was sufficient to amplify lOfg of DNA from Penicillium and lOOfg of DNA from Curvularia.All 9 kinds of fungal DNA template extracted by Guanidine Thiocyanate lysate could be amplified in PCR system, and its product band is the brightest one among the 5 fungi lysate methods. SDS, CATB methods had weak product bands for several kinds of fungi, and Triton-x100 lysing showed weak bands for all the fungi. NaOH is the weakest.68 strains (including Fusarium, Aspergillus, Penicillium, Curvularia) of 72 fungus clinical strains should be tested in the PCR identification theoretically. Among the 68 strains, results of 66 were same as culture. It account for 96.5%. No cross-reaction with other genus was observed.Of the 129 clinical specimens analysed, fungal keratitis was definitively diagnosed by PCR in 102(79%). Ninety -nine (76.7%) specimens were culture positive. All 129 specimens were examined by light microscopy with fungal staining, and 89 (69%) showed fungi. The results of genus identification by PCR were same as that by culture. As to the diagnostic period, PCR is far shorter than culture. ConclusionThe combination of two multi-PCR systems is a rapaid, simple, reliable genus identification method. Guanidine Thiocyanate lysate is potential for fungal identification from cultures and clinical specimens. It provides a rapid and reliable approach to DNA purification protocols in laboratories. The system is promising as a means to diagnose fungal keratitis and offers some advantages over culture methods and smear microscopy, including rapid analysis and the ability to analyse specimens. It has a good clinical application prospect.