Rapid Identification Of Candida Species Relevant To Fungal Infection And Phenotype Switching Of Candida Parapsilosis

Chapter I Rapid identification of Candida species relevant to deep fungal infection by molecular biology methodsSection I Diagnosis of six medically important Candida species by PCR-RFLP methodObjective To diagnose six Candida species relevant to invasive fungal infections by PCR-RFLP method. Methods To identify 6 standard strains including Candida albicans, Candida krusei, Candida parapsilosis, Candida glabrata, Candida tropicalis, and Candida guilliermondii, the ITS rDNA region was PCR-amplified using primer pairs PR1-PR3, and digested with the enzymes HpyF10VI, Mspl, Hinfl, HhaⅠ, HaeⅢand HhaⅠ+HaeⅢ, then the strains were distinguished by restriction fragment length polymorphism (RFLP) patterns. Results The six medically important Candida species could be distinguished on the basis of the sequence of the 5.8S-ITS rDNA region using MSPI or HhaⅠ+HaeⅢrestriction enzyme reaction. Conclusions This simple and inexpensive procedure can be useful for rapid identification of the six medically important Candida species.Section II Rapid identification of six Candida species relevant to deep fungal infection by multiplex PCRObjective To study the application of multiplex PCR in the identification of six Candida species relevant to invasive fungal infections. Methods 6 type candida strains (C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, C. krusei and C. guilliermondii) were cultured on CHROMagar Candida for preliminary identification, and we also developed a rapid and simple multiplex PCR-based method able to specifically identify the 6 Candida species based on the amplification of particular DNA fragments of the ITS1 and ITS2 regions. Results CHROMagar Candida can identify the most commonly isolated Candida species especially C.albicans, C. tropicalis and C. krusei. The color of C. parapsilosis, C. glabrata and C. guilliermondii, were difficult to define because of the similar pastel tones. Multiplex PCR reactions yielded both universal and species-specific amplicons and can easily identify the six candida species. Conclusions Compared with CHROMagar, this novel multiplex PCR method is more rapid and accurate for identification of the six candida species. ChapterⅡIdentification of Candida spieces from clinical specimens and analysis of the clinical featureSectionⅠIdentification of Candida spieces from clinical specimens by multiplex PCRObjective To identify Candida spieces from clinical specimens by multiplex PCR.Methods 50 Candida isolates from clinical samples were collected and identified by CHROMagar Candida medium and multiplex PCR method. These two identification methods were compared according to the results of ITS region sequencing. Results The accuracy of CHRO Magar Candida medium was 74%, and the accuracy of multiplex PCR was 92%. Significant differences were found between the two methods. Multiplex PCR can rapidly and accurately identify 6 most common clinical pathogenic Candida, including C. albicans, C. parapsilosis, C. krusei, C. glabrata, C. tropicalis and C. guilliermondii. The undiagnosed four isolates were not included in our multiplex diagnostic system. Conclusions The multiplex PCR method can indeed rapidly and accurately identify the 6 most common clinical pathogenic Candida, better than traditional CHROMagar diagnostic method.SectionⅡAnalysis of clinical pathogenic Candida infectionObjective To study the species distribution and clinical feature of Candida isolates obtained from clinical specimens. Methods Species identification were carried out on 50 Candida isolates obtained from clinical specimens, from cases attending our hospital during 2009. The patients'name, sex, age and infected location were recorded, and the species distribution and clinical feature of Candida isolates were studied. Results C. albicans represented 56% of the isolates, C. parapsilosis 18%, C. guilliermondii 8%, C. tropicalis 4%, C. glabrata 4%, C. krusei 2%and other species 8%. C. parapsilosis was the most frequently encountered non-albicans Candida species(18%). There were some other rare candida including C. zeylanoides, C. lipolytica and C. haemulonii.Conclusions C. albicans occurred in an important number of case infections, and there was also a very high frequency of C. parapsilosis infection. It is important to diagnosis Candida isolates to species for appropriate antimicrobial treatment.ChapterⅢPhenotype switching of Candida parapsilosisObjective To investigate the phenotype switching of C. parapsilosis on different medium and the effect of temperature on the white-opaque switch. Methods 9 isolates of C. parapsilosis were collected, mainly from the skin of healthy people and clinic patients. Modified Lee's medium and YPD medium supplemented with phloxine B were used to study phenotype switching and the effect of different temperature. Results C. parapsilosis was capable of high-frequency phenotypic switching and white-opaque switch, on both Lee's medium and YPD medium supplemented with phloxine B. The proportion of opaque colony under 37℃was lower than 25℃on Lee's medium, when evaluating white to opaque switching. Conclusions C. parapsilosis is capable of high-frequency phenotypic switching and white-opaque transition. Opaque cells of C. parapsilosis are unstable at 37℃, switching to more virulent white form.