Primarily Study On Standardization Of Rapid Method For Detection Of Deep-seated Candidosis By PCR

The progress of medical sciences and universal application of new treatments in recent years such as broad-spectrum antibiotics,systemic use of corticoids, antimalignancy chemotherapy and organ transplantation,have resulted in a sharp increase in the number of immunocompromised patients.Immunosuppression is occasionally accompanied with fungal infections of an insidious onset,and some nosocomial.Invasive mycoses have become a major cause of infectious morbidity and mortality in patients receiving immunosuppressive chemotherapy for cancer or organ transplantation.On the other hand,invasive mycoses are not uncommon in immunocompromised patients,such as individuals with AIDS,cancer,drug abuse. More than 150 species of fungi are known to be responsible for infections in human. All of tissues and organs can be involved.Since opportunistic mycoses are often clinically grave and serious,an early,rapid and accurate identification of the pathogenic fungus is critical to timely and appropriate management.Due to growing application of azoles to prevent mycoses,the fungal infections by non-albicana candida (NAC)has seen a steady rise.C.krusei and C.glabrata are the two most common causes of NAC infections.Nonetheless,C.albicans is the most important in invasive cases.Since C.krusei and C.glabrata are naturally resistant to azoles,they are gaining more and more attention.Fungi are traditionally identified by morphology and metabolic characteristics and they reqiure days even weeks to be isolated on cultures. Identification may sometimes be difficult,which would impede the selection of antimicrobial agents in cases in which species identification of the pathogen is vital. Therefore,we devised a rapid and accurate method of identification,especially to identify C.albicans,C.krusei and C.glabrata.In this study,we developed a universal fungal primer for panfungal PCR and multiplex primers for triplex PCR of C.albicans, C.krusei and C.glabrata.Chapter 1 Development of universal fungal primer PCR and specific primer PCR for detection of common deep-seated pathogenic fungi.First,in order to test the specificity of the PCR assay,the universal fungal primer was used to amplify nucleinic acid of C.albicans,C.krusei,C.glabrata,C.tropicalis,Aspergillus fumigatus,Cryptococcus neogenesis,a 260bp PCR product was successfully obtained from these fungi,while human blood,urine,bronchoalveolar lavage,cerebrospinal fluid samples produced negative results.Second,a 402bp PCR product was successfully amplified from C.albicans,a 475bp one from C.krusei,a 632bp one from C.glabrata,while C.tropicalis,Aspergillus fumigatus,Cryptococcus neogenesis,human blood,urine,bronchoalveolar lavage,cerebrospinal fluid samples were negative.Third,serial dilutions of fungal DNA of C.albicans,C.krusei, C.glabrata were amplified with panfungal primer to detect the sensitivity of the PCR. The sensitivity of PCR was 10~2 CFU/ml.The detection of fungal DNA in simulated clinical specimens with the panfungal PCR was explored with human blood,urine, bronchoalveolar lavage,cerebrospinal fluid.Fourth,we developed multiplex primers for C.albicans,C.krusei and C.glabrata for the triplex PCR.The products of 402bp,475bp and 632bp were successfully amplified from the mixture of C.albicans, C.krusei and C.glabrata and the simulated clinical specimens.Finally,rabbit models of disseminated C.albicans infection were constructed through injecting C.albicans intravenous inoculation.Chapter 2 Detection of the simulated clinical specimens with the triplex PCR. First,we compared the sensitivity of PCR in different fluids and the sensitivity of PCR to C.albicans and Aspergillus fumigatus.The blood simulated specimens of C.albicans were cultured at 37℃.The sensitivity of the specimens were deteceted at the different points of time during the course.Second,according to the random table, we added concentrationnally different suspensions of C.albicans,C.krusei and C.glabrata into human blood,urine,bronchoalveolar lavage,cerebrospinal fluid samples.We detected the simulated clinical specimens with the triplex PCR.Simultaneously we cultured the same specimens on SDA medium.The results showed that the positive rate of triplex PCR was not statistically different from that of fungal culture with respect to detecting deep-seated Candidosis,but the time of triplex PCR requied was much shorter than that of fungal culture.The PCR can be acoomplished in 6 hours.As a result,triplex PCR can serve to efficiently detect the clinical specimens of the deep-seated candidosis.Chapter 3 Construction of rabbit models of disseminated C.albieans infection.Rabbits were challenged with C.albicans through intravenous inoculation.The disseminated infection occurred within 1 hour and 6 hour after challenging.At 24 hour,the rabbits begun to be treated with itraconazole injection 5mg/kg,qd×14d.At every point of time,we collected the venous blood of the rabbits and detected the blood with universal fungal primer and specific primer of C.albicans to evaluate the effect of itraconazole treatment.At the same time,the specimens were cultured on SDA medium.The body temperature and white blood cell count of the rabbits were monitored simultaneously.The fungal culture were positive within 1hour after challenging and continued up to 18 hours.After the experiment,the rabbits were dissected.The positive rate and CFU of organs and tissues culture were higher in treated group than the control.The conclusions can be made that PCR is rapid and sensitive in detecing disseminated candidosis and itraconazole injection is effective in treating the disseminated candidosis,but the clearance rate were not so satisfactory esspecially for the fungi in kidney.Conclusion:In this blinded study,we successfully developed the PCR to detect the deep-seated candidosis and evaluated the sensitivity,specificity and reliability of the PCR on detecting the simulated specimens of deep-seated candidosis.The PCR is highly efficient in detecting deep-seated candidosis.The rabbit models of disseminated candidosis were successfully constructured and the effecacy of itraconazole injection in treating the disseminated candidosis were evaluated by molecular method.