Abstract
It is known that even after appropriate treatment, the prognosis of candidemia is poorand candidemia has high mortality rates. The blood culture which is accepted as the goldstandard for the diagnosis, can remain negative in %42 of the cases. Therefore rapid andreliable diagnosis of candidemia is very important to start the antifungal treatment. The aim ofour study was to standardize the polymerase chain reaction (PCR) method by using simulatedsamples in order to detect Candida species in blood samples of candidemia suspected cases.In this study, simulated samples were prepared by using blood samples of healthyvolunteers which were inoculated into tubes with EDTA and BACTEC 9240 blood culturebottles in which no growth was detected and standard strains as C. albicans ATCC 90028, C.tropicalis ATCC 1021, C. parapsilosis ATCC 90018, C. krusei ATCC 6258, Escherichia coliATCC 35218, Staphylococcus aureus ATCC 25923 and clinical isolates of Aspergillusfumigatus, C. kefyr, C. glabrata, C. lusitaniae, C. guilliermondii, Rhodotorula sp.Furthermore, blood culture samples of 23 patients whose blood culture bottles signaled aspositive and showed growth of Candida in agar plates were examined.DNA extraction of all samples were performed according to the standard procedureproposed by the MN Nucleospin Tissue Kit (Macherey-Nagel, Germany) for tissue samples;following the pre-treatment with erythrocyte, leukocyte and fungus cell wall lysis buffers.DNAs were amplified with the PCR method, using synthetic oligonucleotide primers selectedfrom the 5S rDNA region and PCR products were ?electrophoresed? in 2% agarose gel.Presence of a 105 base pair product was considered as positive.The lowest detection limit of PCR has been determined as 100-1000 CFU/ml Candidafor our simulated samples. The presence of a 105 bp band has been observed in samplesprepared with all Candida strains included in the study. Blood samples spiked with E. coli, S.aureus, A. fumigatus and Rhodotorula sp. and negative blood samples has been found asnegative in terms of Candida DNA. The same 105 bp product has been observed in the PCRproducts of blood culture samples with Candida growth.The PCR method applied in this study takes approximately 7 hours, whereas thedetection of Candida growth takes at least 24-48 hours by routine blood culture method. Alsothe application of this method to both blood samples and samples obtained from BACTEC9240 blood culture bottles seems to be an advantage. The cost of this method per patient isdetermined to be approximately 8 YTL and this amount is very small when compared with thecost of wrong or unnecessary antifungal therapy and hospital expenses.As a result, it has been determined that Candida DNA from either blood or BACTECblood culture samples, taken from candidemia suspected cases, can be detected in a shortertime by PCR using PCon 1 and PCon 2 primers which are spesific for 5S rDNA gene.Key words: Candida, blood, PCR
