Genetic Diagnosis Of Clinical Pathogenic Fungi Infection

The incidence of opportunistic fungal infections has increased dramatically in the past few decades. Conventional blood cultures are time-consuming and not very effective in detecting yeast growth, although great progress has been made in recent years. Alternative procedures such as the detection of circulating antigens ( or antibodies) , enzymes, or metabolic products lack sensitivity and specificity. Several non-albicans Candida spp. are inherently less susceptible to commonly used antifungal drugs, and their early identification is essential for rapid initiation of empiric treatment. It is important for clinicians to identify the species responsible for the infection.This paper describes a new assay based on ERG11 PCR amplification and reverse hybridization for direct detection and identification of yeasts in clinical specimens.ERG 11 gene is a fragment of cytochrome P450 lanosterol 14ot-demethylase(P450LlAl) gene. This gene is fungus specific, and encodes a highly conserved region. The pair of primers amplified a 300-bp to 350-bp segment of the this gene from the genomic DNAs of the Candida species and Cryptococcus neoformans, The amplification products have been sequenced and compared with the sequence in Genebank. The sequence similarity between them is more than 99.6%. The forward primer P4501 was biotin labeled at 5' end (P4501 bio) in order to analyze and identify by reverse hybridization. The universal primers (P4501, P4502) did not amplify any other bacterial, viral or human cells DNAs tested, and PCR was able to detect the presence of as few as 100 fg DNA or 10 cells of fungus.Because Candida albicans remains the most common fungal pathogen, accounting for at least 50% of all fungal infections, the multiplex PCR method was developed to identify the Candida albican first from fungal pathogens. Other fungi can be identified to species by reverse hybridization. Candida albican DNA was amplified with universal fungal primers and specific Candida albican primers in one reaction tube. Two amplified products were obtained, 300bp (product of universal primers) and 779bp (product of specific primers).The fungal P450L1A1 gene is multicopy and highly conserved, there are variable regions in it. The sequences of species-specific probe come from variable regions. The PCR product was analyzed in reverse cross blot hybridization with species-specific probes directed to a target region of the ERG11 gene of Candida albicans, C.tropicalis, C.krusei, C.glabrata, C.guilliermondii, C. parapsilosis, Cryptococcus neoformans, and the newly described species C.dubliniensis. No cross-hybridization was detected with any other fungal, bacterial or human DNAs tested.The method was tested against conventional identification on 112 different clinical samples from patients with suspected fungal infections. The results agreed with those of culture and phenotyping for all but 5 specimens (Three of which tested yeasts not included in the PCR panel of probes and two in which PCR positivity-culture negativity was justified from blood specimens). Species identification time was reduced from a mean of 3-6 days with conventional identification to 1-2 days with the molecular method. The PCR- reverse cross blot hybridization assay provides a novel approach for identifying fungi and has potential application value in the early diagnosis.PCR-microplate hybridization method was developed. The aim of this work was to obtain a more simple and semi-automated detection protocol. PCR products were detected with a rapid, ELISA-like colorimetric method. Hybridization in microtitre plates is easy to perform and more readily observed than blot hybridization. It is suitable for the analysis of large numbers of samples and practicable in clinical laboratories.