老年人口腔白色念珠菌的分布与基因分型研究

目的研究健康老年人口腔白色念珠菌的分布、基因型特点及基因型特点与白色念珠菌分布的相关性。方法来自成都地区212例老年人分为4组：A1（男性，有义齿）；A2（男性，无义齿）；B1（女性，有义齿）和B2（女性，无义齿）。CHROMagar Candida^TM鉴定培养基、碳水化合物同化反应鉴定体系（API 20C AUX）和随机扩增片段多态性分析（randomly amplified polymorphic DNA assays，RAPD）鉴定白色念珠菌。RAPD分析健康老年人口腔白色念珠菌分离株的基因型特征。统计学分析比较不同分组健康老年人口腔白色念珠菌基因型之间的差异。结果212例受检个体中检出89株白色念珠菌（42％），A1、A2、B1、B2白色念珠菌检出率分别为56．2％、21．3％、56．6％、38％。A1组与A2组白色念珠菌检出率差异有统计学意义，P〈0．01。以JWFP和NA为随机引物的RAPD分析将白色念珠菌分为5型。RAPD分析基因型构成在A1组与A2组之间比较差异有统计学意义，P〈0．05，A1组主要以A型为主。结论健康老年人口腔白色念珠菌的检出率与义齿修复相关。义齿修复可能促进具有特定基因型特点的白色念珠菌检出率增高。     

正常儿童口腔中白色念珠菌的分布及基因分组

目的 研究以白色念珠菌为代表的念珠菌，以及白色念珠菌基因分组在不同年龄段儿童口腔中的分布情况。方法 4组不同年龄儿童和1组成人作为研究对象，粘膜拭子法取样，CHROMagar Candida鉴定培养基分离鉴定，PCR方法一组引物确认白色念珠菌，一组引物基因分组。结果 不同年龄组儿童口腔中念珠菌的检出率有所不同，除健康青年外，白色念珠菌在检出的念珠菌中均占多数。白色念珠菌3种基因分组的分布也存在差异，以A组为主。结论 不同年龄阶段健康儿童口腔中，A组白色念珠菌组占主导地位。     

52株生殖道念珠菌病病原菌的鉴定及药敏分析

目的分析女性生殖道念珠菌感染状况以及耐药性情况。方法临床标本接种到沙保弱培养基,分离培养出的真菌经革兰染色、芽管形成试验、CHROMagar显色培养进行鉴定,并进行氟康唑、两性霉素B药敏分析。结果120份临床标本分离培养出念珠菌52株。白色念珠菌占69.2%,热带念珠菌占7.7%,克柔念珠菌占5.8%,其他念珠菌占17.3%;分离出的52株念珠菌对氟康唑、两性霉素B敏感率分别为86.5%、94.2%。结论女性生殖道念珠菌感染仍以白色念珠菌为主。念珠菌对氟康唑和两性霉素B的敏感性有差异,应重视念珠菌的培养鉴定和药敏试验,以指导临床合理选择抗真菌药物。     

白色念珠菌基因分型研究进展

白色念珠菌（Ｃ．ａｌｂｉｃａｎｓ，简称白念）基因分型是近年来白念分子生物学研究中的一个热点。本文综述了脉冲电泳、限制性酶切、Ｓｏｕｔｈｅｒｎｂｌｏｔ杂交、ＲＡＰＤ分析等分子生物学技术在白念基因分型中的应用进展。讨论了计算机辅助分析、核酸序列分析、构建基因图谱等发展趋势     

白色念珠菌诱导小鼠胸腺细胞凋亡的基因调控

目的　探讨白色念珠菌诱导小鼠胸腺细胞凋亡过程中的基因调控作用。方法　经小鼠尾静脉注射白色念珠菌后 ,采用FCM技术测定胸腺凋亡细胞上 5个凋亡相关基因 ( p5 3,bax ,bcl 2 ,fas和c myc)产物的水平在不同时间组的变化。结果　p5 3和bax两个基因产物的水平明显增加 ;而bcl 2 ,fas和c myc基因产物的水平无明显改变。 结论　白色念珠菌诱导小鼠胸腺细胞凋亡 ,可能是经由基因调控的转录途径机制而发生 ,p5 3和bax基因在此过程起重要作用     

312株念珠菌感染分布与药敏分析

随着广谱抗生素，激素和免疫抑制剂的广泛应用，念珠菌引起的病人院内感染逐年增多，随着感染率的升高，以及抗真菌药物在临床上广泛使用，又导致耐药菌株的产生，给临床抗真菌感染的治疗带来一定的困难，尤其病原性真菌在临床上诊断、治疗不易，已经引起临床高度重视。本文将我院近期检出的312株念珠菌感染分布及药敏试验结果报道如下。     

桔梗皂苷D防御口腔黏膜上皮细胞感染白色念珠菌的作用

目的:探讨桔梗皂苷D对白色念珠菌黏附口腔黏膜上皮细胞的影响。方法:MTT法检测不同桔梗皂苷D对口腔上皮KB细胞存活率的影响;将白色念珠菌、KB细胞以及不同浓度的桔梗皂苷D共同培养,革兰染色检测白色念珠菌黏附数,台盼蓝排斥实验检测念珠菌活力和菌丝转换率;ELISA法检测上清液中IL-18与人β-防御素2(HBD-2)的蛋白含量;实时荧光定量PCR和Western blot法分别检测KB细胞中HBD-2 mRNA与蛋白表达的变化。结果:桔梗皂苷D对KB细胞存活率无影响;随着桔梗皂苷D浓度的增加,白色念珠菌的黏附数、菌活力和菌丝转换率逐渐下降,上清液中IL-18与HBD-2的蛋白含量以及KB细胞中HBD-2 mRNA表达与蛋白水平逐渐降低。结论:桔梗皂苷D具有抑菌作用,并参与了口腔黏膜上皮细胞防御白色念珠菌感染的免疫反应。     

硝酸铈对白色念珠菌感染小鼠免疫功能的影响

<正>据报道用硝酸铈作为烧伤的局部治疗药物对革兰阴性肠杆菌效果很好。硝酸铈还可用以治疗湿疹皮肤炎、过敏性皮炎、龈炎等,并取得满意效果。我们发现硝酸铈在体外对白色念珠菌（CA）有明显的抑菌作用,在体内可降低CA感染小鼠的病死率,延长平均存活期,脏器病变好转（另文发表）。本文进而研究硝酸铈对CA感染小鼠的腹腔巨噬细胞和红细胞免疫功能的影响。1 材料和方法1.1 动物分组处理 按随机抽样法将18～22g重的昆明系小鼠（陕西省药品检验所提供）分为4组:A组（正常对照）,对小鼠不作任何处理;B组（感染对照）,尾静脉注射CA悬液0.2ml;C组（硝酸铈对照）,腹腔注射治疗剂量硝酸铈0.2ml,每天1次,连续3d;D组（感染治疗）,注射CA后即用硝酸铈治疗,每天1次,连续 3d。1.2 免疫学指标和检测方法 硝酸铈对感染小鼠外周血白细胞总数的影响用常规法;硝酸铈对感染     

白色念珠菌感染免疫应答的研究进展

白色念珠菌的致病是其与机体免疫系统相互作用的结果.研究发现,白色念珠菌感染人体时,刺激机体产生固有免疫及特异性细胞免疫和体液免疫应答.其中特异性细胞免疫占主导地位.了解认识白色念珠菌感染的免疫应答对诊断、预防及治疗白色念珠菌感染具有重要意义.     

白色念珠菌感染对小鼠免疫功能的抑制作用

本实验观察了白色念球菌感染小鼠的细胞免疫及体液免疫的变化。实验结果显示白色念珠菌感染小鼠的PHA诱发的体内淋巴细胞转化率以及免疫玫瑰花形成细胞率均显著低于正常鼠(分别为P<0.01及P<0.05),感染小鼠的溶血素产生水平也显著降低(P<0.001)。本文还对白色念珠菌感染的免疫抑制机理进行了初步的探讨。     

白假丝酵母菌基因分型技术的研究

白假丝酵母菌是假丝酵母菌性外阴阴道病最常见的致病菌种。对致病菌快速准确地分型在研究其所致的感染性疾病的发病机制及诊治措施中发挥重要作用,并且有利于致病菌的流行病学调查。因此本文就用于白假丝酵母菌研究的基因分型技术作一综述。     

In vitro activity of ibrexafungerp and comparators against Candida albicans genotypes from vaginal samples and blood cultures

ObjectivesEmergence of azole resistance may contribute to recurrences of vulvovaginal candidiasis. Thus, new drugs are needed to improve the therapeutic options. We studied the in vitro activity of ibrexafungerp and comparators against Candida albicans isolates from vaginal samples and blood cultures. Furthermore, isolates were genotyped to study compartmentalization of genotypes and the relationship between genotype and antifungal susceptibility.MethodsCandida albicans unique patient isolates (n = 144) from patients with clinical suspicion of vulvovaginal candidiasis (n = 72 isolates) and from patients with candidaemia (n = 72) were studied. Antifungal susceptibility to amphotericin B, fluconazole, voriconazole, posaconazole, isavuconazole, clotrimazole, miconazole, micafungin, anidulafungin and ibrexafungerp was tested (EUCAST 7.3.2). Mutations in the erg11 gene were analysed and isolates genotyped.ResultsIbrexafungerp showed high activity (MICs from 0.03 mg/L to 0.25 mg/L) against the isolates, including those with reduced azole susceptibility, and regardless of their clinical source. Fluconazole resistance rate was 7% (n = 5/72) and 1.4% (n = 1/72) in vaginal and blood isolates, respectively. Some amino acid substitutions in the Erg11 protein were observed exclusively in phenotypically fluconazole non-wild type. Population structure analysis suggested two genotype populations, one mostly involving isolates from blood samples (66.3%) and the mostly from vaginal samples (69.8%). The latter group hosted all fluconazole non-wild-type isolates.DiscussionIbrexafungerp shows good in vitro activity against Candida albicans from vaginal samples including phenotypically fluconazole non-wild-type isolates. Furthermore, we found a certain population structure where some genotypes show reduced susceptibility to fluconazole.     

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.     

Species distribution and drug susceptibility of candida in clinical isolates from a tertiary care centre at Indore

Background: The incidence of fungal infections has increased significantly, contributing to morbidity and mortality. This is caused by an alarming increase in infections with multi-drug resistant bacteria leading to overuse of broad-spectrum antimicrobials, which lead to overgrowth of Candida, thus enhancing its opportunity to cause disease. Candida are major human fungal pathogens that cause both mucosal and deep tissue infections. Objective: The aim of our study was to identify the distribution of Candida species among clinical isolates and their sensitivity pattern for common antifungal drugs. Materials and Methods: Two hundred and thirty-seven different clinical isolates of Candida were collected from patients visiting to a tertiary care centre of Indore from 2010 to 2012. Identification of Candida species as well as antifungal sensitivity testing was performed with Vitek2 Compact (Biomerieux France) using vitek 2 cards for identification of yeast and yeast like organisms (ID-YST cards). Antifungal susceptibility testing was performed with Vitek2 “Fungal Susceptibility Card (AST YS01) kits respectively. Results: We found that the non-albicans Candida were more prevalent than Candida albicans in paediatric (<3 year) and older (>60 year) patients than other age group (4-18, 19-60 years) patients and also in intensive care unit (ICU) patients as compared to out patient department (OPD) patients. Resistance rates for amphotericin B, fluconazole, flucytosine, itraconazole, and voriconazole were 2.9%, 5.9%, 0.0%, 4.2% and 2.5%%, respectively. All the strains of C. krusei were found resistant to fluconazole with intermediate sensitivity to flucytosine. Conclusion: Species-level identification of Candida and their antifungal sensitivity testing should be performed to achieve better clinical results.     

用线虫模型研究白念珠菌生物膜中滞留菌对抗真菌治疗效果的影响

目的 念珠菌生物膜能够产生一定比例的可以耐受高浓度药物冲击的滞留菌(persisters).以秀丽隐杆线虫作为模式生物感染产生不同比例滞留菌的白念珠菌临床株并用两性霉素B处理,观察比较各组存活率,阐明滞留菌对抗真菌治疗效果的影响.方法 将同步化的成熟线虫与不同的白念珠菌临床株共培养2 h后,洗脱转移至96孔板中,加入梯度稀释的两性霉素B,并设不加药孔对照.培养5 d后计算各孔线虫存活率.结果 相同药物浓度下,感染产生高比例滞留菌的临床株组较同组不加药的线虫存活率的提高量明显低于感染低比例滞留菌组的提高量,差异有统计学意义(P＜0.01).结论 秀丽隐杆线虫是研究滞留菌和抗真菌药物药效学的可用模型;滞留菌的耐药性可能是抗真菌治疗失败和复发性感染的重要原因.

Abstract：

Objective To illuminate the effect of persisters on antifungal therapy by infecting Caenorhabditis elegans as a live-animal model with Candida albicans isolates of different persister levels, treating them with amphotericin B and comparing the survival rate. Methods Glp-4 (bn2ts); sek-1 (km4) worms were synchronized and grown to sterile to L4-stage, put on different Candida albicans strains lawns separately for 2 h. Dispensed 15-20 worms per well of the 96-well plate, and added serial dilutions of amphotericin B for each strain group. Wells filled without any amphotericin B were used as negative controls intra-group. Incubated the plate at 25C for 5 days, counted the survival rate of each well. Results Compared with negative controls, survival rate of drug wells in each group increased. In the same drug concentration, the increase for high-persister group was significantly lower than that for low-persister group (P＜0.01). Conclusion Caenorhabditis elegans provides a model for the study of persisters and antifungal pharmacodynamics.The drug tolerance of persisters may be a critical component responsible for antifungal drug failure and relapsing infections.     

Assessment of genetic relatedness of vaginal isolates of Candida albicans from different geographical origins

PCR fingerprinting with single non-specific primers was used to type vaginal isolates of C. albicans from Portugal, Angola, Madagascar, and two regions of Germany (Berlin and Munich). In addition to analysing isolates that exhibited the normal biotype of C. albicans, the study included atypical strains that failed to assimilate glucosamine and N-acetylglucosamine, which were isolated from women in Angola and Madagascar. A total of 212 strains of C. albicans were studied, representing 87 different multi-locus genotypes. The genotypes of strains from each geographical population were highly similar but not identical. There was one exception: a strain from Portugal grouped with the typical strains from Angola. The typical and especially the atypical populations from Africa displayed less genotype variation than the populations from Europe. The Portuguese samples exhibited the greatest genotypic heterogeneity. Distance analysis (UPGMA) revealed a statistically weak correlation between genotype and geographical origin of the C. albicans isolates.     

白色念珠菌诱导小鼠胸腺细胞凋亡

目的　研究白色念珠菌（ 白念菌） 在体内对小鼠胸腺细胞凋亡的诱导作用。方法　小鼠经尾静脉注射白念菌后,以流式细胞仪（ＦＣＭ） 分析、ＤＮＡ 琼脂糖凝胶电泳分析及细胞形态学改变为指标检测细胞凋亡。静脉注射ＮＯＳ 抑制剂观察对白念菌诱导胸腺细胞凋亡的影响。结果　白念菌能诱导小鼠胸腺细胞产生特征性的细胞凋亡形态学改变;流式细胞仪分析显示特征性的凋亡峰;琼脂糖凝胶电泳显示胸腺细胞出现典型的ＤＮＡ“梯状带”;用荧光染色（ＡＯ＋ ＥＢ） 以及ＦＣＭ 检测凋亡细胞百分率,发现白念菌注射后２４ 小时,胸腺细胞凋亡百分率随白念菌剂量增加而增高;用４ ×１０６ 白念菌经尾静脉注射后,胸腺细胞凋亡百分率于６ 小时开始增高,２４ 小时达高峰,以后迅速降低;小鼠胸腺萎缩,胸腺重量于１２ 小时明显降低,且于７２ 小时达到最低水平;ＮＯＳ 抑制剂氨基胍仅能部分抑制白念菌诱导的小鼠胸腺细胞凋亡;热灭活的白念菌不能诱导胸腺细胞凋亡。结论　白念菌菌血症能诱导小鼠胸腺细胞凋亡,而且呈时间和剂量依赖性;白念菌诱导小鼠胸腺细胞凋亡有赖于真菌的代谢;白念菌诱导小鼠胸腺细胞凋亡的过程可能与ＮＯ 部分相关。     

Multiple colony antifungal susceptibility testing detects polyresistance in clinical Candida cultures: a European Confederation of Medical Mycology excellence centers study

ObjectivesMany factors influence the outcome of in vitro antifungal susceptibility testing (AFST), including endpoint definition, inoculum sizes, time and temperature of incubation, and growth medium used. This European Confederation of Medical Mycology (ECMM) Excellence center driven study investigated multiple colony testing (MCT) of five separate colonies to investigate the prevalence of polyresistance (PR), defined as heterogeneous MICs from a same-species Candida culture irrespective of the underlying resistance mechanism.MethodsCandida spp. MCT for fluconazole and anidulafungin was performed by Etest prospectively comprising 405 clinical samples. MCT results were compared to the real-life routine MIC data and PR was assessed. Candida colonies displaying strong PR were selected for genotyping using multilocus sequence typing and random amplified polymorphic DNA assays for C. lusitaniae.ResultsCandida PR was observed in 33 of 405 samples (8.1%), with higher rates for non-albicans species (26/186, 14%) than for C. albicans (7/219, 3.2%), and for fluconazole than for anidulafungin. MCT detected acquired resistance more often than routine AFST (18/405, 4.5%) and 9 of the 161 investigated blood cultures showed PR (5.6%). Multilocus sequence typing and random amplified polymorphic DNA did not reveal a uniform genetic correlate in strains studied.ConclusionsThis study shows that Candida single MIC-values obtained in routine diagnostics may be incidental, as they fail to detect PR and resistant subpopulations reliably. The reasons for PR seem to be manifold and should be regarded as a phenotypical expression of genomic variability irrespective of the underlying resistance mechanism, which may help to interpret ambiguous and non-reproducible AFST results.     

Antifungal pharmacokinetics and pharmacodynamics: bridging from the bench to bedside

This review considers a way in which experimental data can be used to identify safe and effective antifungal regimens for humans. The process begins with experimental models of invasive fungal infections that enable definition of optimal dosages and schedules of antifungal drug administration to be defined. These preclinical models also enable the identification of drug exposure targets that are associated with therapeutic outcomes of interest. Human pharmacokinetic variability results in a considerable range of drug exposures following the use of fixed antifungal drug regimens. This variability can be quantified using population pharmacokinetic modeling techniques. Monte Carlo simulation can then be used to simulate pharmacokinetic variability and thereby estimate the proportion of patients with a therapeutic outcome of interest. Effective and safe regimens can thus be studied appropriately in clinical settings. This approach can, and should, be used to optimize antifungal therapy for a large number of clinical scenarios.