Candidemia:A Clinical Survey And Etiologic Gene Chip Detection Method

Candidemia is bloodstream infection due to Candida spp..In recent years, with the large number of applications of broad-spectrum antimicrobial agents, immune suppression, invasive diagnostic and therapeutic measures, the incidence of candidemia has greatly increased. Candida spp. has been the fourth most common cause of bloodstream infections. Candidemia mainly caused by Candida albican., but in recent years the proportion of non-albicans Candida has gradually increased, and several non-albicans Candida are inherently less susceptible to commonly used antifungal drugs, which add more challenge to clinical treatment. The outcome of candidemia can be affected by a variety of factors, including appropriate antifungal therapy and so on.The clinical diagnosis of candidemia is difficult, traditionally method is blood culture, but it usually costs a long time, and the positive rate is low. The sensitivity and specificity of fungus-specific antigens, antibodies and serological methods metabolites are also unsatisfactory.Gene chip technology (DNA chips, DNA microarrays) is a detection technique based on hybridization, pathogens species can be quickly identified. It is suitable for the analysis of large numbers of samples and practicable and can greatly improve the efficiency of diagnosis. Part 1 Clinical survey of candidemiaIn this part, we retrieved and collected all blood culture Candida spp. positive patients in Huashan Hospital Affiliated to Fudan University from January I.2007 to December 31.2012. Gender, age, department, underlying diseases, species distribution and outcome were described. The results showed that candidemia pathogen species including Candida albicans. Candida tropicalis. Candida parapsilosis, Candida glabrata. Candida krusei. Underlying diseases frequently identified included solid malignancy, gastrointestinal surgery history, diabetes and so on. Presence of central venous catheters and antifungal therapy were factors independently correlated mortality.Part 2 Development of the etiologic gene chip detection method of candidemiaIn this part, we describe a new assay based on gene chip for direct detection and identification of clinical common Candida spp..Universal primers for PCR of Candida spp. DNA is designed according to the conserved 18S rDNA interval of the fungus. Fluorescence is labeled on primers to make the PCR product manifested with fluorescence signal. Based on the variable region of the conserved sequence 18S rDNA, we design 6 specific Candida spp. probes (Candida albicans, Candida tropicalis, Candida parapsilosis, Candida glabrata, Candida krusei and Candida guilliermondii). And at the same time we design of Cryptococcus neoformans specific probe and Aspergillus universal probe to detect blood culture specimens for clinical probable pathogens. The probes are fixed on a same chip, and hybridized with PCR product. The 6 Candida species can be identified. No cross-hybridization was detected with any other bacterial or human DNA. The lower detection limit of the method was 100 fungal cell/PCR reaction. This method has high specificity and good sensitivity.Part 3 Clinical application of the etiologic gene chip detection method of candidemiaIn this part, we collected 440 cases blood samples which alarmed positive in blood culture machine in laboratory of Huashan Hospital Affiliated to Fudan University from 2011 March to 2012 March.22 cases were fungi positive. The method was tested against conventional detection. There were 21 cases of Candida,1 case of Cryptococcus neoformans. It agreed with conventional culture results completely. Therefore, the detection methods of the gene chip used in clinical samples can agree with the conventional culture method. The method only cost a few hours, and can be used for clinical detection Candida spp. alarmed in blood culture machine.