| Objective: To discuss the application of multiple PCR and mass spectrometry in the rapid identification of yeast-like fungi,and to analyze the distribution of pathogenic fungi and the sensitivity of antifungal drugs in 149 patients with fungemia confirmed by ICU from 2014 to 2018 in Tianjin Medical University General Hospital.In addition,the risk factors of infection in ICU patients with fungal sepsis were investigated,and the microsatellite typing of candida parapsilosis catheter-related bloodstream infection was conducted,providing a basis for the early diagnosis,treatment and control of nosocomial infection of fungemia.Content: 149 cases of patients with fungemia were diagnosed by blood culture from January 2014 to December 2018 in ICU departments of Tianjin Medical University General Hospital,according to Definitions of Invasive Fungal Disease formulated by EORTC/MSG in 2008.Firstly,the diagnostic efficiency of multiple PCR and MOLDITOF methods in rapid identification of yeast-like fungi was compared,and the sequencing results of general primer amplification products were used as the gold standard for identification.Second,to understand the distribution changes of fungi in ICU departments in the past five years,as well as the resistance characteristics of different fungi to fluconazole,voriconazole,itraconazole,5-fluorouracil and amphotericin B.Thirdly,fungi ranked the third among all pathogenic microorganism isolated from blood culture during 2014-2018.The clinical characteristics,risk factors and epidemiological characteristics of fungemia in ICU were analyzed to provide data support for the early treatment and prevention of the disease.Finally,retrospective statistical analysis found that the highest isolation rate of fungi in ICU of the hospital was candida parapsilosis,which often occurred for the mechanism of biofilm.Therefore,microsatellite typing method was selected to genotype 12 strains of candida parapsilosis causing catheter-related bloodstream infection.Methods:(1)for 149 fungi included in the study,VITEK2 compact was routinely used for chemical identification in clinical practice,and rapid identification methods based on different principles were selected: DNA-based typing method--multiple PCR,protein-based typing method –MOLDI-TOF.The accuracy of the two methods in the identification of fungi were compared,using the results of universal primer amplification product sequencing as the gold standard.(2)WHONET 5.6 was used to analyze the distribution characteristics of 149 fungi and the susceptibility to antifungal drugs.Meanwhile,SPSS17.0 software was used to analyze the clinical data and susceptibility factors of 149 cases of fungemia by Logistic multivariate regression analysis.(3)Three microsatellite loci(CP1,CP4 and CP6)with high resolution reported in the literature were selected.Three fluorescein markers were selected for real-time quantitative PCR amplification,and the products were sent for genotype analysis.Results:(1)For 149 fungi isolated from blood culture,the accuracy of VITEK MS identification was 97.2% and the detection time was short,which was significantly better than VITEK2 compact.The identification accuracy of five common candida species by VITEK MS and multiple PCR system was 100%.For the rapid identification of uncommon yeast-like fungi,the experimental conditions for the specific primers of the multiple PCR method still need to be explored.VITEK MS has more advantages in the identification of rare candida.The general primers PCR method can be used to identify the strains with wrong results.(2)Non-candida albicans were the main pathogen in 149 cases of fungemia,and the separation rate of candida parapsilosis was the highest(64 strains,43.0%),followed by Candida albicans(24 strains,16.1%),Candida tropicalis(18 strains,12.1%),Candida glabrata(13 strains,8.7%),and Rhodotorula(12 strains,8.1%).7 strains of Cryptococcus were isolated,accounting for 4.7% of the total,including 3 strains of cryptococcus neoformans and 4 strains of cryptococcus lorentus.149 fungi showed the highest sensitivity to 5-fluorouracil and amphotericin B,and only one candida albicans showed resistance to 5-fluorouracil.Candida albicans and candida parapsilosis of azole drug sensitivity were higher.Candida glabrata and candida tropical with cross azole drug-resistant appeared.Rhodotorula had low sensitivity to azole drugs and cryptococcus showed no resistance to azole drugs.(3)Candida parapsilosis accounted for the largest proportion of candida in ICU bloodstream infection.The primary infection sites were mainly respiratory system and digestive system,and the proportion of multi-site candida infection reached 34.7%.APACHE ? score,initial treatment control for candida as well as the source of infection inadequate were independent risk factors of death in patients with fungemia.(4)Genotyping analysis was conducted on the microsatellite typing results of the three specific sites by R,and 12 candida parapsilosis could be divided into 5 types.Multiple PCR with different fluorescence labeled primers detected different sites at the same time,which improved the efficiency of genotyping.Conclusion:(1)From 2014 to 2018,fungi separated from ICU in Tianjin Medical University General Hospital from high to low,were candida parapsilosis,candida albicans,candida glabrata,tropical candida.Candida parapsilosis separation rate is highest which should be paid attention in clinic.(2)Candida glabrata and candida tropicalis were significantly more resistant to azoles than candida parapsilosis and candida albicans,and rhodotorula was less sensitive to azoles.(3)MOLDI-TOF was significantly superior to the biological encoding instrument method in the rapid identification of fungi.The multiplex PCR technique required less special instruments and has low cost,which was more suitable for rapid typing of common candida in general laboratories.(4)the APACHE ? score of ICU in the diagnosis and treatment of fungal disease was in great value of early diagnosis and antifungal treatment(5)Microsatellite typing technology was an effective tool for genetic diversity research,with high typing efficiency,and was suitable for clinical microbiology laboratory research on molecular epidemiological characteristics.Few reports on the microsatellite typing of rare candida were seen,and the establishment of microsatellite test system for different species need to be studied. |
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