| Objective: With the wide application of glucocorticoids,broad-spectrum antibiotics and immunosuppressants in clinical practice,the development of new technologies such as organ transplantation,bone marrow transplantation and interventional diagnosis and treatment,as well as the increase of patients with immunodeficiency diseases,the number of invasive Candida infections dominated by Candida albicans has increased year by year.However,due to the limited types of commonly used antifungal drugs in clinical practice,fluconazole has good safety and bioavailability,it is widely used to treat Candida albicans infection.Due to the increase of patients with Candida albicans infection and the frequency of fluconazole.Candida albicans is resistant to fluconazole based antifungal drugs,and the drug resistance rate is high,which seriously affects the treatment effect of patients.In recent years,the mechanism of fluconazole resistance in Candida albicans has been reported in many literatures,which are the changes of drug targets and the overexpression of drug efflux pump.However,at present,there is no epidemiological analysis of Candida albicans infection in Yan’an area and the study on the mechanism of fluconazole resistance.In this study,a retrospective analysis method was used to analyze the epidemiology of patients with invasive Candida albicans infection mainly isolated from two class III class A hospitals in Yan’an area,and to further explore its mechanism of fluconazole resistance,the aim is to provide some basis for the prevention and treatment of invasive Candida albicans infection in this area.Methods:（1）From January 2012 to December 2021,1042 cases of Candida albicans infection were collected from two class III class A hospitals in Yan’an,including576 males and 466 females,aged from 1 day to 96 years,with an average age of（61.72 ±18.11）years.Retrospective analysis was used to refer to relevant cases and data,and records were made in the form of unified Excel spreadsheets,the identification and drug sensitivity analysis of strains were carried out by using CHROMagar Candida chromogenic culture plate medium and merier ATB fungus 3 yeast like fungus drug sensitivity reagent,and the data were analyzed by SPSS 25.0 software;（2）ten strains of Candida albicans resistant to fluconazole and sensitive to fluconazole were selected from the retrospective analysis cases,the strains were identified by VITEK MS matrix assisted laser desorption ionization time of flight mass spectrometer in merier,France,the mic value was measured by colorimetric microdilution method with Thermo Fisher yeastone plate;the target enzyme coding gene ERG11 was amplified,and its product was analyzed by DNA two-way assay;rhodamine 6G（a fluorescent dye）was used to measure the fluorescence intensity of cells by cytomics 500 flow cytometry,and the efflux of strains from drug-resistant and sensitive groups was analyzed;the expression level of efflux pump gene of Candida albicans was detected by ABI 7300 automatic fluorescent quantitative PCR.Results:（1）Among the clinically isolated Candida species,the isolation rate of Candida albicans was the highest,accounting for 73.48%.The annual distribution trend showed an overall increase first and then decrease.In 2015,the number of isolated strains was the largest,and there was no significant difference in gender distribution.People over 60 years old（62.38%）were significantly more prone to Candida albicans infection than people under 60 years old（37.62%）,mainly aged between 60 and 79 years old,and mainly aged between 60 and 69 years old,with a composition ratio of 24.76%,The clinical samples were mainly sputum,accounting for 58.34%（608/1042）,and the patients’ departments were mainly respiratory medicine（35.51%）.（2）Through the analysis of risk factors（single factor and multi factor）,the main risk factors of Candida albicans infection in Yan’an area were the patients’ age ≥ 60 years old,the use of broad-spectrum antibiotics,invasive operation,various intubations and the history of large-scale surgery.（3）The prophylactic,extensive and frequent use of antifungal drugs in the clinical treatment of Candida albicans infection has led to the increasingly serious drug resistance of Candida albicans,and its resistance to fluconazole is more serious than that of other antifungal drugs.During the decade,the number of fluconazole resistant strains accounted for the highest,accounting for 11.71%,the annual distribution trend chart shows that the drug resistance rate in 2015 was the highest,up to 27.13%.（4）The amplification and sequencing results of the target gene ERG11 showed that the mutation G→A occurred in the 414 th base of drug-resistant strain C3,but did not cause the change of glycine（Gly）codon 118;The mutation C→G occurred in the 518 th base of drug-resistant strain C7 and sensitive strain C12,causing the change of amino acid codon 173 and the change of proline（Pro）to arginine（Arg）.Although the change of amino acid was caused,it existed in both drug-resistant and sensitive strains,and it was not a base mutation related to drug resistance reported in the previous literature.It was speculated that the mutation at this site was a meaningless mutation in the mechanism of fluconazole resistance of Candida albicans.（5）Rhodamine 6G,a fluorescent dye,was used to detect the efflux of Candida albicans.The results showed that there were significant differences in the efflux of drug-resistant and sensitive bacteria;RT-PCR detection of efflux pump gene showed that there was significant difference in the expression levels of CDR1 and CDR2 efflux pump genes between drug-resistant and sensitive bacteria（P<0.05）.Conclusions:（1）Candida albicans is the most common Candida isolated in this study,in the past decade,the overall infection rate has increased first and then decreased,the patients are mainly distributed in the age group of 60-79 years old.Most of the clinical cases are in respiratory medicine,and the type of specimen is mainly sputum;（2）The main risk factors for Candida albicans infection are the patient’s age ≥ 60 years old,the use of broad-spectrum antibiotics,invasive operation,various intubation operations and the history of large-scale surgical treatment,and the supervision of such patients should be strengthened in clinical practice;（3）Candida albicans showed different degrees of resistance to antifungal drugs,and the resistance rate to fluconazole was the highest,and the resistance situation was severe;（4）The resistance mechanism of Candida albicans to fluconazole in this region is mainly the overexpression of efflux pump coding genes CDR1 and CDR2,and no significant mutation of target enzyme coding gene was found. |
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