| [Objective] To evaluate the identification methods of Candida parapsilosis complex collected from CHIF-NET 10 and 11. To investigate the molecular epidemiology of national wide, and studied the antifungal susceptibilities as well as the resistance mechanisms using whole genome sequencing.[Methods] The 352 isolates of C. parapsilosis were confirmed by molecular identification. The accurate rate of original identification by each participating hospital was evaluated compared to the results of molecular identification. The performance of Bruker MS and Vitek MS were evaluated for C. parapsilosis identification. The microsatellite typing was used to analyze the 151 isolates from bloodstream. The newly developed antifungal susceptibility testing method YeastOne was used to test the 352 isolates. The azole susceptible and resistant matched isolates with same genemic background were studied to explore the resistance mechanism by whole genome sequencing and RNA expression test.[Results] Most Candida parapsilosis isolates were collected from bloodstream, and the patients suffered invasive Candiasis diseases were old people from general ICU, surgeon ward, surgical ICU and VIP wards, et al.. The average age is 59 years old, the the patients elder than 80years old accounted for 16%. In addition, most patients had catheters with them, which is a risk factor of infections. Compared to molecular identification, the accurate rate of original identification of Candida parapsilosis sensu stricto is 90.8%, and commonly misidentified as Candida glabrata, Pichia guilliermondii, and Candida famata. For Candida metapsilosis and Candida orthopsilosis, they could not be identified by routine identification methods. The MALDI-TOF MS is really a good method to identify Candida species,98.4% and 97.7% of C. parapsilosis sensu stricto could be identified by Bruker MS and Vitek MS, respectively. Vitek MS could not identify C. metapsilosis or C. orthopsilosis because of there is no reference spectrum in its database, while even though Bruker Biotyper has, the identification rate was not high as well. All the 151 isolates of C. parapsilosis sensu stricto were genotyped into 106 genotypes, and the diffused distributed among 22 hospitals. In Hua Xi Hospital and 301 Hospital, there were isolates spreaded in several wards. In Tianjin General Hospital, an outbreak happened in three wars, and 111 isolates from 91 patients were involved, lasting for more than one year. The susceptibilities of C. parapsilosis complex were tested by YeastOne method. The resistant rate to fluconazole was 5.2%, and the MIC50 as well as MIC90 were similar to the global data. The MICs of C. parapsilosis complex to echinocandins were higher than other Candida species. By whole genome sequencing of matched suscptible and resistant isolates, the mutation in MRR1 resulted in MRR1 overexpression which regulated MDR1 overexpression, then leaded to the fluconazole resistance.[Conclusions] By evaluating different identification methods, some hospital which use the routine metnod should pay attention to the misidentified results. Some hospital equiped with MALDI-TOF MS could use it in routine work. The epidemiology study showed that the isolates could spread among hospitals, especially there was an outbreak case in one hospital. The government should pay attention to this issue and take some measures to control outbreak cases. Nowdays, whole genome has not been widely used in fungi, and our study demonstrated that it could be used in Candida species studies, and found the possible resistance mechanisms, and this is the first time to study the fluconazole resistance mechanisms in clinical isolates. However, more research should be done to illustrate the exact resistance mechanism. |
