2010-08-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/657772摘要：引起人類隱球菌感染疾病(cryptococcosis)的病因Genus Cryptococcus 可分為2種species, Cryptococcus neoformans及Cryptococcus gattii。研究顯示兩種species的地理分布、環境來源、臨床表現及抗黴菌藥物感受性不同。傳統上隱球菌可以進ㄧ步分為3種亞型：C. neoformans var. grubii (血清A型)，C. neoformans var. neoformans(血清D型)，C. neoformans var. gattii (血清B型及C型)，及hybrid血清AD型。本研究擬探討台灣地區隱球菌的族群結構。計畫由至少三家醫院回溯性及前瞻性收集隱球菌臨床分離菌株。該菌株之臨床資料將同步收集，包括免疫狀態、鴿子等之暴露，抗黴菌藥物之使用及治療反應。所有菌株將以生化方法決定亞型(variety)。基因型之決定及比較擬引用microsatellite特異引子M13進行PCR指紋比對，及URA5基因增幅以限制酶分解片段RFLP分析。臨床菌株預計可分為8個分子分型，5種血清型及3種亞型。4種抗黴菌藥物體外感受性將以肉汁微稀釋法(broth microdilution method) (amphotericinB, flucytosine, fluconazole及voriconazole)，紙錠滲透法(disc diffusion method) (fluconazole, voriconazole)及Etest (amphotericinB) 決定。此外，確定診斷之隱球菌腦膜炎或散播性隱球菌感染病患之血液及腦脊髓液(依例行醫療常規進行診斷及減壓治療時採集的)將收集，並偵測檢體中之菌量及時序變化，擬以real-time PCR偵測檢體中之DNA量，以平板塗抹法決定腦脊髓液中存活的菌量。前述兩種菌量，加上醫療常規檢測之抗原指數，將分別依病人免疫狀態、抗黴菌藥物使用及治療反應、菌株species、體外感受性結果進行分析。預計三年之研究預計可分析100-200株臨床分離菌株，可追蹤分析60-90例病人。<br> Abstract: Cryptococcosis is caused by 2 species in the genus Cryptococcus, Cryptococcus neoformans and Cryptococcus gattii. Global data as well as our previous data suggest that the geographic distribution, environment reservoir, clincial presentation and antifugnal susceptibility of these two species are different. Cryptococcus was classically subdivided into the three varieties: C. neoformans var. grubii (serotype A), C. neoformans var. neoformans (serotype D), C. neoformans var. gattii (serotypes B and C), and the hybrid serotype AD. As local data was limited and disease burden seem increased, this 3-year study plans to determine the population structure of the etiologies of cryptococcosis in Taiwan.To this effect, clinical isolates will be collected both retrospectively and prospectively from at least three hospitals in Taiwan. Clinical data (immune status, pigeon exposure), use of antifungal agents, antifungal response will be reviewed. The variety of these isolates will be determined by biochemical methods and genotypes will be evaluated and compared by using M13 polymerase chain reaction-fingerprinting and orotidine monophosphate pyrophosphorylase (URA5) gene restriction fragment length polymorphism analysis with HhaI and Sau96I in a double digest. Clinical isolates will be grouped into one of the eight molecular types, 5 serotypes and three varieties. In vitro antifungal susceptibility of clinical isolates of C. neoformans will be evaluated and analyzed stratified by varieties. The in vitro susceptibility of nonduplicate isolates to four antifungal agents will be determined by broth microdilution method (amphoterin B, flucytosine, fluconazole and voriconazole), disc diffusion method (fluconazole and voriconazole) and Etest (amphotericin B). Special attentions will be paid for those patients infected due to fluconazole-dose-dependent susceptible or resistant isolates. In addition, C. neoformans in clinical specimens (blood and cerebral spinal fluids) collected according to routine practice from patients with confirmed cryptococcal meningitis and/or disseminated cryptococcosis will be quantitatively by real-time LightCycler PCR assay, colony-forming units, and capsular polysaccharide antigen assay. The fungal kinetic data will be analyzed based on immune status, antifungal therapy and response.It is anticipiated that 100-200 nonduplicate clinical isolates will be collected and evaluated, and clincial specimens (average 10 specimens per patients) collected from 30-90 patients will be monitored for the fungal kinetic during this three-year study.隱球菌族群結構指紋比對血清型抗黴菌藥物抗黴菌藥物感受性antifungal susceptibilityantifungal responseCryptococcus gattiiCneoformans vargrubiiPCR fingerprintingrestriction fragment length polymorphism