Phenotypic And Molecular Characterization Of The Candida Parapsilsis Complex And Candida Dubliniensis

Part I Phenotypic and genotypic characterization of the Candida parapsilosis complexChapter I Phenotypic screening, molecular identification and phylogenetic analysis of the Candida parapsilosis ComplexObjective To explore the species distribution and phylogenetic status of the C. parapsilosis complex in East China. Methods Growth on CHROMagar Candida Medium and carbon-assimilation profiling via API20C AUX system were performed to screen for presumptive C. parapsilosis isolates among a total of 80 isolates collected from 4 cities in East China. Those phenotypically characterized isolates of C. parapsilosis, C. guilliermondii, C. famata and C. lusitaniae were further molecularly identified by sequencing of the ITS region. Phylogenetic trees were constructed based upon the ITS sequence data of the C. parapsilosis complex and phylogenetic relations among isolates were also determined. Results Out of the 80 clinical isolates, sixty (75%) were biochemically characterized as the C. parapsilosis complex, eleven (13.75%) as C. glabrata, four (5%) as C. guilliermondii, and remaining five belonging to other uncommon Candida species. Among those 60 isolates of presumptive C. parapsilosis, three were molecularly identified C. guilliermondii, and 57 belonged to the C. parapsilosis complex, including 41 isolates (71.9%) of C. parapsilosis sensu stricto and 16 C. metapsilosis (28.1%). The identification of presumptive C. guilliermondii, C. famata and C. lusitaniae were also molecularly confirmed. A significant geographic variation with respect to species distribution was observed:out of 37 isolates from Nanjing, Shanghai and Jinan, C. parapsilosis sensu stricto made up the majority (89.2%) and C. metapsilosis represented only 8.8%(4/37); however, C. metapsilosis contributed to 60%(12/20) of all isolates from Nanchang. Phylogenetic trees based on ITS sequence data revealed that C. parapsilosis sensu stricto isolates were evolutionally conserved; C. metapsilosis and C. orthopsilosis showed a closer genetic relationship. There existed two unevenly distributed ITS genotypes, I and II, among C. metapsilosis isolates, with type II being more common. Conclusions (1) There is a geographic variation in terms of species distribution of the C. parapsilosis complex in East China; (2) There are two ITS genotypes within C. metapsilosis.Chapter II Identification and typing of the C. parapsilosis Complex using PCR-RFLP and RAPDObjective To evaluate a novel PCR-RFLP assay and RAPD method for the identification and typing of the C. parapsilosis complex isolates. Methods (1)POM-Stul/Styl assay:A C. parapsilosis complex-specific primer pair POMf/r was designed to amplify a fragment harboring sites of polymorphism in the ITS region which was named as POM. POM was amplified and RFLP analysis was performed using Stul and Styl, respectively. (2)RAPD-PCR using RPO2 was conducted and RAPD fingerprinting patterns were generated. Cluster analysis of RAPD fingerprinting was also performed. Results POMf/r amplified the POM fragment of C. parapsilosis sensu stricto, C. metapsilosis and C. orthopsilosis with high specificity. RFLP analysis using Stul and Styl generated taxon-specific patterns which were in concordance with ITS sequence data. (2) RAPD analysis produced C. parapsilosis sensu stricto-specific and C. metapsilosis-specific fingerprinting. Cluster analysis of the RAPD fingerprinting revealed the presence of two RAPD gentypes which were consistent with ITS genotypes obtained in Chapter I. Conclusions (1)POM-Stul/StyIassay is an effective and accurate molecular method for the differentiation of the C. parapsilosis complex; (2) RAPD offered a rapid and accurate mean to identify C. parapsilosis sensu stricto and C. metapsilosis; (3) There existed two RAPD genotypes which were in agreement with the ITS genotypes.Chapter III Antifungal susceptibility profiling of the Candida parapsilosis complexObjective To profile the susceptibility of the C. parapsilosis complex to five commonly used antifungal agents. Methods The antifungal susceptibility tests were performed according to the CLSI M27-A3 protocol. The tested antifungal agents included amphotericin B, fluconazole, itraconazole, voriconaozle and micafungin. Results All isolates were susceptible to amphotericin B, fluconazole, voriconaozle and micafungin. Six isolates of C. parapsilosis sensu stricto and three C. metapsilosis were found to be dose-dependant-susceptible to itraconazole. No resistant isolate was observed. Conclusions The C. parapsilosis complex isolates resistant to five antifungals were rare in clinical setting. Commonly used systemic antifungals are effective for the management of infections associated with the C. parapsilosis complex. Chapter VI Evaluation of extracellular enzymatic activities of the C. parapsilosis complexObjective To investigate the enzymatic activities of the C. parapsilosis complex, including 21 isolates of C. parapsilosis sensu stricto, one of C, orthopsilosis and 12 of C. metapsilosis. Methods The egg yolk agar, bovine serum albumin agar and tween-80 agar were used to detect phospholipase, proteinase and esterase activities, respectively. Results (1) Phospholipase activities:90.5%(19/21) of C. parapsilosis sensu stricto isolates exhibited phospholipase activity,91.7%(11/12) of tested C. metapsilosis isolates exhibited phospholipase activity. No statistical difference was detected between two species. C. orthopsilosis reference strain showed no activity (2) Proteinase activity: 81.0%(17/21) C. parapsilosis sensu stricto isolates showed proteinase activity. While for C. metapsilosis,83.3%(10/12) were proteinase producers. C. parapsilosis sensu stricto showed stronger proteinase activity than C. metapsilosis. C. orthopsilosis reference strain showed very strong proteinase activity (3)Both species rarely demonstrated esterase activity. Conclusions Candida parapsilosis sensu stricto and C. metapsilosis, just like C. albicans, are strong producers of extracellular phospholipase and proteinase.Part II A study of methods to identify Candida dubliniensisChapter V Evaluation of classic phenotypic markers and a molecular method for the identification of Candida dubliniensisObjective To evaluate six classic phenotypic markers and formulate a novel colony PCR-RFLP method for the differentiation of Candida dubliniensis from C. albicans Methods (1) CHROMagar Candida medium, cornmeal Tween-80 agar,45℃tolerance test, hypertonic tolerance test, tobacco agar test and API20C AUX kit were performed to identify reference strains of C. dubliniensis and C. albicans. A total of 66 clinical isolates of presumptive C. dubliniensislC. albicans were also tested. (2) Colony PCR-Apal/BsiEl: Colony PCR was used to amplify D1-D2 region directly from C. dubliniensislC. albicans and eight other Candida species, and subsequent RFLP analysis using ApaI/BsiEl was performed. Results (1) Hypertonic tolerance test and tobacco agar test reliably identified all reference strains. All 66 isolates were identified as C. albicans; (2) Colony PCR-ApaI/BsiEl successfully identified all reference strains and clinical isolates to the species level. Conclusions A combination of two or more classic phenotypic markers can reliably screen for C. dubliniensis, and colony PCR-Apal/BsiEl can be applied for the rapid and reliable identification of C. dubliniensis.