耐两性霉素B的近平滑与热带假丝酵母菌的体外诱导及耐药分子机制研究

目的：体外诱导近平滑假丝酵母菌（ CP）、热带假丝酵母菌（ CT）对两性霉素B（ AMB）的耐药株,检测其中的耐药基因表达情况。方法 AMB体外构建耐药株,用实时荧光定量PCR法检测耐药基因的表达水平。结果 CP、CT 野生株经体外 AMB 浓度递增法诱导后,形成耐药株最低抑菌浓度（ MIC,≥8μg? mL－1）;CYP51A、CYP51B、CDR1、CDR2、MDR1及MDR2基因在野生株中低水平表达,在耐药株中表达水平明显升高,差别有统计学意义（ P ＜0．05）;CYP51A、CYP51B、CDR1、CDR2、MDR1及MDR2的表达量在CPAMB耐药株中分别是其野生株的136．92,16．33,2．07,14．84,4．07,2．38倍;在CT AMB耐药株中分别是其野生株的8．83,2．98,3．00,2．40,2．30,3．57倍。结论 CP、CT对AMB耐药与CYP51A、CYP51B、CDR1、CDR2、MDR1及MDR2基因过度表达有关。     

18例早产儿真菌败血症临床特点

目的对某院早产儿真菌败血症的临床特点进行分析,为临床诊治提供参考。方法对该院2011年1月—2013年12月18例早产儿真菌败血症的临床资料进行回顾性分析。结果 18例早产儿胎龄为27~36周,出生体重为1 050~3 100 g,其中极低出生体重儿(VLBWI)8例;均有广谱抗菌药物用药史,感染前均长时间静脉营养,10例机械通气,2例经外周静脉穿刺中心静脉置管(PICC)。临床表现以呼吸暂停、抽搐、喂养困难、反应差等为主;出现症状时间为出生后3 h~52 d。13例(72.22%)早产儿血白细胞(WBC)计数异常,12例(66.67%)血小板(PLT)100×109/L,18例(100.00%)C反应蛋白(CRP)均增高,平均CRP浓度为(41.90±26.77)mg/L。感染病原菌以假丝酵母菌属为主,共17例(94.44%),其中包括近平滑假丝酵母菌7例,白假丝酵母菌5例,白假丝酵母菌生物变种4例,无名假丝酵母菌1例。用氟康唑及两性霉素B治疗,15例治愈(83.33%),2例好转(11.11%),1例死亡(5.56%)。结论早产儿真菌败血症以假丝酵母菌感染为主,临床缺乏特异性表现,应严密观察具有高危因素的早产儿临床症状,定期检测血常规及CRP等指标,及时给予抗真菌药物治疗,有助于取得良好的治疗效果。     

系统性念珠菌感染小鼠模型的制备

目的 建立稳定的系统性念珠菌感染小鼠模型,规范其操作方法。方法 采用环磷酰胺对小鼠进行免疫抑制后,选择白色念珠菌（C.albicans）和非白念珠菌（C.parapsilosis）分别接种ICR小鼠,从免疫抑制、菌株制备、接种剂量和接种途径等方面对建模过程进行质量控制,通过生存分析、组织载菌量和病理学检查对模型进行评价。结果 我们建立的系统性念珠菌感染小鼠模型显示肾脏为靶器官,多器官弥散性真菌感染的典型病理组织学改变。结论 通过对建模过程各环节进行规范,可得到稳定的系统性念珠菌感染小鼠模型,该模型可应用于系统性念珠菌感染的致病机理,免疫防御及抗真菌药物筛选等研究领域。     

黄根醇提物体外抗白念珠菌生物膜作用的研究

目的观察黄根醇提取物体外对白色念珠菌生物膜及相关基因的影响。方法 M27-A2测定黄根醇提取物对白色念珠菌的最小抑菌浓度(MIC);XTT法评价黄根醇提取物对白色念珠菌生物膜形成的影响;实时荧光定量RT-PCR(q RT-PCR)检测黄根醇提取物作用后,ALS基因的表达情况。结果黄根醇提取物对白色念珠菌的MIC为8μg/m L;随着浓度的增加,黄根醇提取物对白色念珠菌生物膜的抑制作用增强,呈正相关性;16μg/m L黄根醇提取物对不同生长阶段的白色念珠菌生物膜均有抑制作用,抑制率随生物膜成熟逐渐降低;q RT-PCR结果显示,药物作用后ALS2、ALS3基因表达降低(7.87±0.27比5.15±0.34;6.24±0.51比2.13±0.23,P<0.05),ALS1基因表达无明显变化(6.11±0.14比5.95±0.31,P>0.05)。结论黄根醇提取物对体外白色念珠菌生物膜有较明显的抑制作用,可能通过抑制ALS2、ALS3基因的表达而实现。     

黄连解毒汤对白色念珠菌的抗真菌作用研究

目的 观察黄连解毒汤对白色念珠菌的体外抗真菌效果.方法 采用微量稀释法药物敏感性试验测定黄连解毒汤对标准菌株、氟康唑敏感株、氟康唑中度敏感株、氟康唑耐药株4种白色念珠菌的最低抑菌浓度.结果 黄连解毒汤对4种白色念珠菌均具有较好的抑制作用,其对白色念珠菌标准菌株的最低抑菌浓度为4mg/ml,对氟康唑敏感型白色念珠菌最低抑菌浓度为8mg/ml,对中度敏感型白色念珠菌最低抑菌浓度为16mg/ml,对耐药型白色念珠菌最低抑菌浓度为 64mg/ml.结论 黄连解毒汤对白色念珠菌有明显的抑制作用,可作为临床用药的参考.     

黄连解毒汤联合氟康唑对耐药白念珠菌麦角甾醇的影响

目的:探讨黄连解毒汤乙酸乙酯提取物(ethyl acetate extract of Huanglian Jiedu decoction,EAHD)联合氟康唑(fluconazole,FLZ)对耐药白念珠菌生物膜麦角甾醇的影响。方法:采用CLSI M27-A3的微量稀释法测定EAHD与FLZ对白念珠菌FLZ耐药株的MIC与SMIC80;棋盘稀释法测定EAHD与FLZ联用对白念珠菌的协同效应;稀释涂布平板法考察EAHD与FLZ联用不同时间对菌体的杀伤作用;分别用HPLC和qRT-PCR法检测耐药白念珠菌生物膜麦角甾醇含量及相关基因的变化。结果:对于9株白念珠菌FLZ耐药株,EAHD对其MIC介于156~1 250 mg·L-1,FLZ对其MIC介于256~2 048 mg·L-1以上,二者联用后MIC显著降低,FICI(部分抑菌浓度指数)最低达0.066;EAHD单用对其SMIC80均高于1 250 mg·L-1,FLZ单用对其SMIC80均高于512 mg·L-1,最高可达2 048 mg·L-1以上,联用组除个别组具有相加作用外也均有协同效应。时间杀菌(time kill,TK)实验显示当作用12 h,二者联用杀菌效应最强。HPLC结果显示联用组、EAHD组与空白组麦角甾醇量相比,分别降低了近85%,50%。qRT-PCR检测FLZ组ERG1,ERG2,ERG6,ERG7,ERG11均上调,ACS1,ACS2,MET6均下调;EAHD组所有基因均下调;联用组ERG2,ERG6,ERG11均显著上调,ERG1,ERG7,ACS1,ACS2,MET6均显著下调。结论:EAHD协同FLZ抗白念珠菌耐药株,可能与抑制麦角甾醇合成相关基因的表达来降低麦角甾醇的合成有关,造成细胞膜损伤,抑制菌体生长。     

The interplay between inflammasome activation and antifungal host defense

Fungal infections cause significant morbidity and mortality in humans, and they are a growing problem due to the increased usage of broad-spectrum antibiotics and immunosuppressive therapies. The equilibrium between the commensal microbial flora and the immune system that protects the host against invasive fungal infection is disturbed during disease, and understanding this disturbed balance is important to develop new therapeutic interventions for the treatment of fungal infection. In the context of tolerating fungi during colonization and eliciting a vigorous immune response to eliminate invading fungal pathogens when needed, the inflammasome has been identified as an integral component of antifungal host defense. It contributes to mucosal host defense by regulating T-helper 17 (Th17) cell responses, and contributes to protective responses such as neutrophil influx during fungal sepsis. Several aspects are important for understanding the role of the inflammasome for antifungal host defense, such as the role of fungal cell wall morphology and its components in triggering the inflammasome, the pattern recognition pathways and downstream signaling cascades involved in the activation of the inflammasome, and the effects of inflammasome activation during fungal infection. The future perspectives of inflammasome research in fungal immunology, with emphasis on targeting the inflammasome for the design of future immunotherapies, is also discussed.     

Detection of phospholipase activity of Candida albicans and non albicans isolated from women of reproductive age with vulvovaginal candidiasis in rural area

Background: Vulvovaginal candidiasis (VVC) is most common accounting for 17 to 39% of symptomatic women. Both Candida albicans and non albicans Candida species are involved in VVC. Amongst various virulence factors proposed for Candida, extracellular phospholipases is one of the virulence factor implicated in its pathogenicity. With this background the present study was carried out to find the prevalence of different Candida species and to detect phospholipase producing strains isolated from symptomatic women with VVC. Materials and Methods: At least two vaginal swabs from 156 women of reproductive age with abnormal vaginal discharge were collected. Direct microscopy and Gram’s stained smear examined for presence of budding yeast and pseudo mycelia followed by isolation and identification of Candida species. Extracellular phospholipase activity was studied by inoculating all isolates on Sabouraud’s dextrose egg yolk agar (SDA) medium. Results: Of the 156 women with curdy white discharge alone or in combination with other signs, 59 (37.82%) women showed laboratory evidence of VVC. A total of 31 (52.54%) women had curdy white discharge followed by 12 (20.33%) with other signs and symptoms. C. albicans (62.59%) and non albicans Candida (37.28%) in a ratio of 1.68:1 were isolated. Of the 37 strains of C. albians 30 (81.08%) showed the enzyme activity. Seventeen (56.66%) strains showed higher Pz value of < 0.70 (++++). Conclusion: Although there may be typical clinical presentation of Candidiasis. all the patients did not show laboratory evidence of infection. Pregnancy was found to be major risk factor for development of VVC. C. albicans was prevalent species but non albicans species were also frequently isolated. Extracellular phospholipase activity was seen in C. albicans and not in non albicans Candida isolates.     

Evaluation of the anti-candidal activity of methanolic leaf extract of cleistopholis patens (fam. Annonaceae) on candida species isolated from stage II HIV patients

BackgroundCandida species (sp) is implicated in causing opportunistic disseminated mycotic complications in stage II HIV patients. Cleistopholis patens is a West African medicinal tree reported to have significant antifungal activity against C. albicans.ObjectivesThis study aimed to determine the anti-candidal activity of methanolic leaf extract of Cleistopholis patens against Candida species isolated from stage II HIV patients.MethodsThe minimum inhibitory concentration (MIC) of the extract and Nystatin®® was determined by agar dilution method. The killing rate studies of the plant extract and Nystatin® were also determined.ResultsThe extract had activity against all Candida isolates, with the MIC against the five isolates ranging from 6.0 – 9.8 mg/ml. Nystatin® also demonstrated plausible activity against the isolates with MICs ranging from 0.3125 — 25 mg/ml. Candida albicans strain 2 was the most sensitive to both extract and Nystatin® with MIC values of 6 and 0.3125 mg/ml respectively. Candida krusei was the least sensitive with MIC values of 9.8 and 25 mg/ml for the extract and Nystatin® respectively. The killing rate values for the extract ranged from −0.029 to −0.091 min⁻¹ and that of Nystatin® ranged from −0.076 to −0.112¹⁶ min⁻¹.ConclusionsThe results indicate that the methanolic extract of Cleistopholis patens is a promising clinical alternative besides Nystatin® in the treatment of infections caused by Candida species in stage II HIV patients.     

阿米巴活体微培养法

目的 建立一种既可做阿米巴与病原体共培养实验，又可连续观察阿米巴活体形态细微变化的培养技术。 方法 以24孔细胞培养板覆盖玻片为容器，37 ℃下微量悬滴将阿米巴与白色念珠菌共同培养, 普通光学显微镜（×1000）下连续观察阿米巴的形态、活动能力、吞噬的细菌等情况。 结果 经微量悬滴培养在普通光学显微镜油镜下可连续观察阿米巴各种形态，滋养体呈T、K、Y字形态，并可连续观察吞噬白色念珠菌的过程。 结论 本法可观察到常规固定染色标本无法见到的阿米巴滋养体T、K、Y字形态及其吞噬白色念珠菌过程。