Rapid Diagnosis Of Fungal Keratitis Using Seminested Polymerase Chain Reaction

Fungal keratitis is a worldwide major blinding eye disease, especiallyin the developing country. In China, the incidence of fungal keratitis hasincreased dramatically during the past decade. Early and accurate diagnosisand reasonable intervention is a critical element for an effective treatmentof fungal keratitis. Today, the decision to diagnosis fungal keratitis is stillbased mainly on clinical information and traditional laboratory techniques.However, conventional laboratory approaches (light microscopy and culture)for the diagnosis of fungal keratitis have many limitations, including lowersensitivity and specifity, and easily affected by subjective factors andantifungal treatment. This has led to the development of rapid,species-specific and effective diagnostic methods such as PCR. Although itis well recognised that amplification of target DNA through PCR withfungal sequence-specific primers is potentially more sensitive, rapid andspecific than traditional laboratory techniques, it is not widely used todiagnose the clinical fungal ketatitis. The amplified sequence you selectedis the key to decide the specifity of PCR. This is in agreement with thefactthat rRNA genes are multiple-copy and highly conserved genes and arepresent in all fungal genera, but the ITS regions(intergenic transcribedspacer regions )which exsist between the 18S and 5.8S and between the5.8S and 28SrDNA gene subunits contain enough internal variation in itssequence, so ITS/5.8SrDNA regions are the ideal amplified sequences.Seminested PCR includs two rounds amplification, so it can maximize thesensitivity and specifity of the PCR method. This study aims to (1)Amplify fungal ITS2/5.8SrDNA by PCR and seminested PCR to detectfungal DNA in corneal scrapings from patients clinically suspected to havefungal keratitis; (2) compare the diagnostic accuracy of seminested PCRanalysis and standard microbiological techniques(light microscopy andculture) for diagnosing keratomycosis; (3) evaluate the practicality of PCRas a diagnostic method of clinically fungal keratitis. 8ethods: ITS2 and 5.8SrDNA regions were amplified by seminestedPCR to detect the DNA of fungi from standard strains and cornealscrapingsfrom patients suspicious of fungal infection(20 eyes,20 cases). The resultswere compared with light microscopy and culture. In addition, MPCR wasperformed to detect the DNA fragment of bacteria and fungi at the sametime in the first-round amplification of seminested PCR. Results: We successfully detected fungi from standard fungal strainsand all 20 corneal scrapings from patients suspicious of fungal infectionusing this seminested PCR method , but not from bacteria and humancorneal cells, the amplified segment is about 290 bp long, the totalidentification time of the two rounds amplification was 5 h. However, inthe first-round amplification, we successfully detected fungi from standardfungal strains and only 13 samples from patients with fungal keratitis anddetected bacteria only from standard bacterial strains using this MultiplexPCR method, the amplified fungal segment is about 550bp long, and theamplified bacterial segment is about 996bp long. The positive rate ofsamples from patients using single MPCR was 65%, which was indifferentas light microscopy (X2=0.107,P>0.05) or culture (X2=2.506,P>0.05), andthe positive rate of samples from patients using seminested PCR was 100%,which was higher than light microscopy (X2=10,P<0.01) or culture(X2=16,P<0.01). The sensitivity of the MPCR and seminested PCR by celldilutions was 5 organisms and 1 organisms per PCR respectively. Conclusions: The sensitivity and specifity of seminested PCR arenotable higher than single PCR. Amplification of ITS2/5.8SrDNA byseminested PCR shows potential as a rapid, sensitive, specific and reliabletechnique for diagnosing clinical fungal keratitis.