The Study On Rapid Diagnosis Of Invasive Candidiasis Based On Rabbit And Murine Models

Candida albicans is one of the most important human opportunity pathogenic fungi.In resent years,infections and mortality caused by Candida albicans increased rapidly.Due to the lack of specific clinical symptoms,diagnosis of invasive candidiasis still remains a challenge today.Blood culture was considered as a gold standard method in clinic,however,it was time consuming and labor intensive.Therefore,the search on rapid laboratory diagnosis method for invasive candidiasis is essential.Part one Rabbit model of invasive candidiasis and change of physiological and biochemical indexesObjective: To establish rabbit model of invasive candidiasis(IC),and observe the physiological and biochemical indexes to evaluate the infection stage of animals.Methods: The rabbit model was established by injecting Candida albicans through ear-margin vein.Blood was collected at before and after infection 1,2,4,7,10,15,20 and 27 days.The whole blood were used for routine hematology tests and plasma were subjected to biochemical analysis.The blood cell were subjected to blood culture.After infection 3 days,liver,kidney,heart and lung tissues were stained by HE to observe the pathological change.Results: After challenged with SC5314,rabbits showed reduction in daily activity and loss of body weight with higher body temperature.Both white blood cells and neutrophil lymphocyte ratio(NLR)increased,especially neutrophil.Blood culture showed that 4 rabbits were positive at 12 h after Candida albicans challenge,one rabbit was positive at D2 and two rabbits were positive at D1.Compared with pre-infection,UA and CK increased significantly,which suggest that the renal and cardiac tissue cells of the rabbit were injured or damaged.As pathological analysis indicated,acute inflammatory reactions could be observed in liver,kidney,heart and lung,with perivascular cellularedema and tissue structure destruction.Part two Plasma levels of Eno antigen and anti-Eno Ig G in IC rabbits modelObjective: To establish a sandwich ELISAs for quantitative detection of Eno,and investigate dynamic changes of rabbit plasma enolase antigen at different infection time points.To evaluate the diagnostic value of enolase antigen by comparing enolase antigen detection,conventional microbial culture and beta glucan detection.Methods: The developed assay was applied to determine Eno levels in IC rabbits at at before and after infection 1,2,4,7,10,15,20 and 27 days.Anti-Eno Ig G were detected by indirect ELISA method.beta glucan were detected by immunoturbidimetry.Results: On D1 and D2,the level of beta glucan in infected rabbits were significantly increased compared with pre-infection(P<0.05).The concentrations of Eno antigen were also significantly increased at same time points(P<0.05).Notably,Eno antigen could be detected on D4 in all IC rabbits,though there was no statistical difference observed.The antibody titer began to increase around D7,which remained a high level until D30.By comparing the detection of Eno antigen,beta glucan and blood culture,the positive rate of beta glucan and Eno antigen was higher than that in the same period of blood culture.The positive rate of Eno antigen detection was higher than that of the same period(1,3)-beta-D glucan positive rate.Part three Development of real-time fluorescence quantitative PCR assays for the detection of candida albicans in IC murine modelObjective: To establish a murine model of invasive candidiasis and real-time fluorescence quantitative PCR detection system,in order to detect Candida albicans rapidly and accurately.Methods: The murine model was established by intraperitoneal injection of Candida albicans,and blood was collected at after infection 1 h,6 h,1 d,2d,5 d.Real-time fluorescence quantitative PCR was used to detect Candida albicans DNA in murine peripheral blood.Results: Babl/C mice showed typical infection symptoms including inactivity,photophobia,chatter and hold together.Candida albicans DNA was detected at 1 h,6 h and 1 d in murine model peripheral blood by real-time fluorescence quantitative PCR.Conclusion:1.The IC rabbit model was successfully established.Candida infection cause damages in liver,lung,kidney,and heart,especially in kidney and heart.2.Plasma Eno antigen concentrations are significantly increased in a short time after Candida albicans challenge,which indicates that enolase could be used as an early diagnostic markers in Candida infection.3.An IC murine model and real-time fluorescence quantitative PCR detection system were successfully established in the present study,by which Candida albicans could specifically identified in peripheral blood of IC murine.