纳米银抗菌凝胶联合氟康唑治疗外阴阴道假丝酵母菌疗效观察

目的：探讨纳米银抗菌凝胶联合氟康唑治疗外阴阴道假丝酵母菌病疗效。方法选择门诊确诊的外阴阴道假丝酵母菌患者58例随机分为治疗组和对照组,治疗组氟康唑单次口服150mg,纳米银抗菌凝胶连用6d。对照组纳米银抗菌凝胶连用6d,对比疗效。结果两组停药后7d总有效率为93.3%和82.1%,两组在下次月经干净后总有效率为93.3%和78.6%。结论纳米银抗菌凝胶联合氟康唑治疗外阴阴道假丝酵母菌具有较好的疗效。     

86例外阴阴道假丝酵母菌病的临床疗效分析

目的讨论氟康唑联合克霉唑治疗白假丝酵母菌性阴道炎的效果。方法随机抽取来我院就诊的白假丝酵母菌性阴道炎的女性患者86例,随机分为研究组与常规组,每组各43例。针对常规给予苏打水溶液外阴清洗后置入克霉唑栓治疗;针对研究组给予在常规组治疗的基础上口服氟康唑胶囊治疗;两组用药均1次/d,以7d为一个疗程,进行4个疗程的观察。结果研究组总有效率为95.35％明显高于常规组的74.42％。所有数据均符合统计学差异（P＜0.05）。结论氟康唑联合克霉唑对于白假丝酵母菌性阴道炎的治疗效果好,值得推广。     

雌激素影响外阴阴道假丝酵母菌病的发生发展

白假丝酵母菌是外阴阴道假丝酵母菌病的主要条件致病菌,高水平的雌激素与外阴阴道假丝酵母菌病的高发病率密切相关。雌激素对菌体和宿主均有影响。雌激素通过结合白假丝酵母菌的雌激素结合蛋白增加1型菌丝壁蛋白、分泌型天冬氨酸蛋白酶和磷脂酶的表达,促进形态转换和热休克蛋白90的表达,从而增强白假丝酵母菌对宿主组织的粘附、入侵和环境适应力。同时雌激素通过与宿主的雌激素受体结合影响阴道的pH,抑制阴道上皮细胞和吞噬细胞介导的先天性抗假丝酵母菌免疫以及T细胞和B细胞介导的获得性抗假丝酵母菌免疫。雌激素是建立外阴阴道假丝酵母菌病动物模型的关键因素。此文章总结了在外阴阴道假丝酵母菌病的发生发展中雌激素对假丝酵母菌和宿主产生的影响。     

白假丝酵母菌DNA提取方法的研究

目的建立一种提取白假丝酵母菌DNA的有效方法。方法采用蜗牛酶消化破壁形成原生质体，饱和酚／氯仿抽提法提取基因组DNA，用琼脂糖凝胶电泳及PCR反应等进行鉴定。结果提取物经琼脂糖凝胶电泳检测到DNA主带，基本无DNA碎带，提取的DNA浓度为（1．18±0．36）ug／ul，纯度好（OD260／OD280〉1．7），不用RNase酶处理，无需任何纯化即可用于PCR扩增。结论本研究中提取DNA的方法简便易行．提取物可适用于各种分子生物学研究。     

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。     

假丝酵母菌药敏实验及影响药物敏感性的因素分析

目的了解深圳地区育龄妇女外阴阴道假丝酵母菌的耐药情况及药物敏感性,探讨影响其药物敏感性的因素,为外阴阴道假丝酵母菌病（vulvovaginal candidiasis,VVC）的治疗提供参考依据。方法从体检妇女人群中阴道分泌物培养念珠菌感染阳性病例中选择103例为研究对象,对样本采用氟康唑、两性霉素B、伊曲康唑、酮康唑、益康唑进行药物敏感性实验;并回顾性调查检测对象病史、用药情况等。结果研究对象平均年龄（36.16±6.72）岁,文化程度以初中、高中和大专为主,分别占14.3%、29.7%和52.0%。对5种药物都敏感的样本有55例,占53.5%,对1种以上药物不敏感的样本有48例,占46.5%。药物敏感性依次是：两性霉素B（97.0%）、酮康唑（94.2%）、伊曲康唑（92.2%）、氟康唑（80.6%）、益康唑（72.8%）。近期使用抗生素更加容易使菌株对伊曲康唑和益康唑产生不敏感。使用抗生素的对象对两性霉素B、伊曲康唑、益康唑的耐药率分别为7.1%、14.3%和35.7%;而没有使用抗生素的对象对两性霉素B、伊曲康唑、益康唑的耐药率分别为2.3%、6.7%和25.8%。反复感染者的样本对两性霉素B（100.0%）和酮康唑（100.0%）非常敏感,但是会增加对伊曲康唑（8.7%）和益康唑（30.4%）的不敏感性。结论深圳地区外阴阴道假丝酵母菌整体耐药性较少,但近期使用抗生素和反复感染者对药物的敏感性有影响。临床治疗宜首选两性霉素B和酮康唑,反复感染者建议联合用药治疗。     

药物流出泵抑制剂联合咪康唑清除白假丝酵母菌生物被膜的研究

目的 观察咪康唑分别与两种药物流出泵抑制剂联用清除耐药株(persister)的效果.方法 白假丝酵母菌参考株YEM30,在96孔板中形成生物被膜(biofilm),CDR1抑制剂Enniatin B、CDR1/CDR2抑制剂Milbemycins α25单独或联合与咪康唑作用后,采用菌落形成单位(CFU)计数的方法统计耐药株的数量.采用SAS8.0统计软件包对数据进行q检验.结果 咪康唑分别联合两种药物流出泵抑制剂清除生物被膜耐药株的效果明显优于咪康唑单独使用(P<0.001),其中咪康唑与Enniatin B联用效果更佳.结论 咪康唑与药物流出泵抑制剂联合应用具有清除生物被膜中表现型耐药株的作用,这为提高抗真菌治疗的效果提供了一条新途径和新思路.     

流式细胞术快速检测假丝酵母菌药物敏感性方法的建立及应用北大核心CSCD

目的：建立基于流式细胞分析技术快速检测假丝酵母菌临床菌株药物敏感性或耐药性的方法。方法以碘化丙锭（ PI）为荧光染料,采用白假丝酵母菌ATCC90029株确定流式药敏试验门设置及最佳实验条件。采用所建立的真菌流式药敏试验检测110株假丝酵母菌临床菌株对氟康唑和伏立康唑的敏感性或耐药性并经典的M27-A3常量稀释法进行比较。结果调整电压可将白假丝酵母菌ATCC90029株活菌与死菌在流式细胞仪SS/log（ FL3）门中分为边界清晰的两群细胞,不同比例死菌与活菌混合液与流式药敏试验检测值之间有高度相关性（r＝0．999）。流式药敏试验30 min内可出结果,其最适菌液浓度为1．0×106/ml、药物与真菌最适孵育时间为3 h、最佳染色方法及时间为脱氧胆酸钠预处理后PI染色5 min。流式药敏试验与M27-A3常量稀释法对110株假丝酵母菌临床菌株对氟康唑和伏立康唑敏感或耐药总符合率分别为98．2％和87．3％。结论较之经典的常量稀释法,流式药敏试验用于检测真菌对药物的敏感性或耐药性具有快速、准确、敏感等优点,具有临床应用前景。     

颗粒酶在大鼠白假丝酵母菌肺炎中的作用探讨

目的:观察大鼠白假丝酵母菌肺炎颗粒酶的表达,探讨颗粒酶在大鼠白假丝酵母菌肺炎发展变化过程中可能起到的作用及其意义。方法:选用清洁级健康雄性SD大鼠,随机分为实验组与对照组,各组40只。实验组从气管注入白假丝酵母菌菌液制备白假丝酵母菌肺炎模型,分别于1、3、7、11天心室腔穿刺抽血,经离心后取血清,用双抗体夹心ABC-ELISA法测定血清颗粒酶、并同步检测血清IFN-γ、II-2水平。采用SPSS13.0统计软件进行统计学分析与统计学图表绘制。结果:白假丝酵母菌肺炎模型组第1、3、7、11天组均有炎症反应,Szapiel评分分别为1.70±0.48、3.60±0.70、2.90±0.57、1.30±0.52(P0.05),菌体负荷数分别为3.50±1.08、4.60±1.08、1.30±0.48、0.10±0.32(106CFU/ml),(P0.05)。白假丝酵母菌肺炎模型组第1、3、7天颗粒酶、IFN-γ、IL-2值较正常对照组增高,两组存在显性著差异(P0.05)。11天模型组与对照组比较无显著差异(P0.05)。血清颗粒酶表达与IFN-γ、IL-2水平变化呈正相关,相关系数(r)分别为0.845和0.808均P0.05。结论:颗粒酶在消除大鼠白假丝酵母菌肺炎病原体方面可能起着积极的作用并与IFN-γ、IL-2可能起协同作用。     

妊娠期外阴阴道假丝酵母菌病症状与妊娠结局分析

目的：研究妊娠期外阴阴道假丝酵母菌病(VVC)症状与妊娠结局的关系。方法选取我院收集的136例妊娠>35 w患者阴道分泌物假丝酵母菌涂片阳性诊断VVC,其中有症状者60例作为观察组,无症状者76例作为对照组。观察两组妊娠结局及不良妊娠结局情况。结果观察组胎膜早破、产褥感染和剖宫产率与对照组比较差异有统计学意义(<0.05);而胎儿窘迫和新生儿感染率比较差异无统计学意义(>0.05)。结论妊娠期有症状VVC可致胎膜早破、剖宫产率等不良妊娠结局增高,早期诊断和治疗十分必要。     

特比萘芬与氟康唑或伊曲康唑联合对氟康唑诱导耐药稳定白念珠菌的影响

目的探讨特比萘芬与氟康唑或伊曲康唑联合抗氟康唑诱导产生的耐药稳定白念珠菌的作用.方法采用多步诱导法,在YEPD培养基中,利用氟康唑诱导白念珠菌敏感株产生耐药稳定菌株.根据美国国家临床实验标准委员会（NCCLS）制定的标准,采用棋盘微量稀释法测定特比萘芬与氟康唑或伊曲康唑对耐药稳定菌株的联合药敏试验,并对诱导耐药稳定菌株ERG11基因的编码区序列进行DNA测序.结果临床敏感菌株和标准敏感菌株能被诱导形成耐药菌株,但大部分不稳定,诱导耐药稳定株ERG11基因的编码区DNA测序有突变点存在,特比萘芬与氟康唑或伊曲康唑联用对诱导耐药稳定株可产生协同作用.结论特比萘芬与氟康唑或伊曲康唑联合应用对基因突变产生的耐药株有协同作用,可阻止或延迟氟康唑诱导的耐药性白念珠菌菌株的产生.     

Doxorubicin selects for fluconazole-resistant petite mutants in Candida glabrata isolates

Candida infections are a permanent threat to immunocompromised individuals such as cancer patients, and Candida glabrata has emerged as a major problem in recent years. Resistance may develop during lengthy antifungal therapies and is often mediated by upregulation of fungal drug efflux pumps. During chemotherapy the yeast cell is also exposed to cytotoxic agents that may affect its drug susceptibility. Four C. glabrata isolates, three susceptible and one resistant to fluconazole (FLU), were incubated with 20 μg/ml of doxorubicin (DOX) for 90 min. In a second experiment, the isolates were cultured with DOX for ten days. Samples were taken on subsequent days to determine the minimal inhibitory concentration (MIC) of FLU and to analyze expression of CgCDR1, CgCDR2, CgSNQ2 and CgPDR1. Samples were also used to assess the petite phenotype. Short-term DOX exposure did not induce efflux pump gene expression, but genes were consistently overexpressed in FLU-susceptible isolates during long-term exposure. An increase in MIC values on day 6 in two of the isolates coincided with the first occurrence of petite mutants in all susceptible isolates. The respiratory deficiency of selected petite mutants was confirmed by culturing mutants on agar containing glycerol as the sole carbon source. FLU MIC values for respiratory-deficient clones were ≥64 μg/ml, and efflux pump gene expression was greatly increased. The resistant isolate did not develop mitochondrial dysfunction. In summary, the cytotoxic agent DOX selects for FLU-resistant respiratory-deficient C. glabrata mutants, which may affect antifungal therapy.     

A computational study targeting the mutated L321F of ERG11 gene in C. albicans,associated with fluconazole resistance with bioactive compounds from Acacia nilotica

BackgroundEmergence of fluconazole resistance mediated by L321F mutation in the ERG11 gene poses a serious impediment in the candidal treatment process. This leads to the search of novel target proteins to develop newer drugs against fluconazole resistant C. albicans. The present investigation is thus aimed to explore the inhibitory potential of bioactive compounds from A. nilotica against the wild and mutated ERG11 gene of C. albicans.Materials and methodsHomology modelling of the wild and mutated ERG11 target gene was done by Modeller 9.2, with SDM I-mutant form of ERG11 together with SAVES server and Ramachandran plot validation. 2D and 3D structures of the bioactive compounds from A. nilotica were optimized by ACD chemsketch. Molinspirational assessments for the molecular properties of the ligands and their drug likeliness were estimated. In silico inhibitory potential of the selected ligands against wild and mutated ERG11 was done by AutoDock 2.0 and was visualized with Accelrys discovery studio tool.ResultsApigenin proved to be the best candidate to target mutant ERG11 with a binding energy of −8.33 Kcal/mol followed by catechin with six hydrogen bonds with more drug likeliness. Molinspiration assessments showed zero violations for all the bioactive compounds from A. nilotica and the TPSA scores of the ligands showed the values < 140 Å towards the best oral bio-availability.ConclusionThe findings of the study emphasize that kaempferol, apigenin and catechin from A. nilotica seem to possess a promising inhibitory effect against the wild and mutated ERG11 of C. albicans suggesting ERG11 as the best target to combat fluconazole resistant C. albicans with further in vivo validation targeting the same.     

Candida glabrata candidemia: An emerging threat in critically ill patients

Background:

Candidemia is an important nosocomial blood stream infection in critically ill patients. Although several studies have addressed candidemia, very few have reviewed the impact of Candida glabrata candidemia in Intensive Care Unit (ICU) patients.

Materials and Methods:

The medical records of ICU patients between 2006 and 2010 were reviewed retrospectively. The epidemiology, clinical features and mortality related risk factors among our adult ICU patients were seen.

Results:

Among 144 episodes of candidemia, C. glabrata (n = 26; 18.05%) was the third most common species isolated. The incidence of C. glabrata candidemia was 0.21/1000 ICU admissions. The most common risk factors were prior exposure to broad spectrum antibiotics (100%), central venous catheter (100%), mechanical ventilation (76.9%), diabetes mellitus (50%), age >65 years (46.15%). Urine (23%) was the most common source of C. glabrata candidemia. Overall in hospital 30 days mortality rate due to C. glabrata fungemia was 53.8%. Patients who were treated with fluconazole showed better outcome than patients treated with amphotericin B. Renal failure requiring hemodialysis was the significantly associated with mortality in our study.

Conclusion:

Candida glabrata was the 3^rd most common Candida causing candidemia in our ICUs with a incidence of 0.21/1000 ICU admissions. The outcome of ICU acquired C. glabrata candidemia was poor with 30 days mortality rate of 53.8%. Renal failure requiring hemodialysis was the only risk factor associated with mortality. Further studies are required to identify the other risk factors associated with mortality in C. glabrata candidemia.     

Candida glabrata fungaemia in intensive care units.

Candidaemia is increasingly important in intensive care units (ICUs). Compared with Candida albicans fungaemia, the impact of C. glabrata fungaemia on ICU patients is not well-known. The aim of this study was to investigate the clinical features, the antifungal susceptibility and the treatment outcomes of C. glabrata fungaemia in ICU patients. The medical records of ICU patients with candidaemia between 2000 and 2005 were reviewed retrospectively, and antifungal susceptibility testing was performed for isolates of C. glabrata. Among 147 episodes of candidaemia occurring in adult ICUs, C. glabrata was the second most common species and accounted for 45 (30%) episodes of candidaemia. The incidence of C. glabrata fungaemia was 1.3/1000 ICU admissions. Fluconazole resistance was found in 11% of C. glabrata isolates. The 30-day all-cause mortality rate was 58%. Therapeutic regimens containing amphotericin B were associated with better outcome. Despite higher fluconazole resistance, C. glabrata candidaemia was not associated with greater mortality than non-glabrata candidaemia in the ICU setting.     

Structural analysis of a Candida glabrata centromere and its functional homology to the Saccharomyces cerevisiae centromere

A 451-bp fragment exhibiting centromere activity had been previously isolated from Candida glabrata genomic DNA. It contains three elements, CgCDEI, CgCDEII and CgCDEIII, highly homologous to those of Saccharomyces cerevisiae. In this study, the requirement of each element for centromere function was analyzed in detail. Deletion analysis identified a small fragment of 153 bp, which included all three elements, to be sufficient for centromere activity. Linker substitution analysis of CgCDEI and CgCDEIII revealed that both elements are required for centromere function. Some of the substitution mutations in CgCDEIII caused a complete loss of centromere activity. These results suggested a functional similarity of centromeres between C. glabrata and S. cerevisiae. However, the C. glabrata centromere did not function in S. cerevisiae cells, suggesting species specificity of the C. glabrata centromere. To examine whether species specificity of the centromeres between these two yeasts does exist, chimeric centromeres between the two species were constructed. Exchange of CgCDEII or CgCDEIII with CDEII or CDEIII of S. cerevisiae, respectively, increased C. glabrata centromere activity in S. cerevisiae, indicating participation of the two elements in determining the species specificity of centromere function. Received: 2 July / 4 October 1996     

Acquired fluconazole resistance and genetic clustering in Diutina (Candida) catenulata from clinical samples

ObjectivesDiutina (Candida) catenulata is an ascomycetous yeast isolated from environmental sources and animals, occasionally infecting humans. The aim of this study is to shed light on the in vitro antifungal susceptibility and genetic diversity of this opportunistic yeast.MethodsForty-five D. catenulata strains isolated from various sources (including human and environmental sources) and originating from nine countries were included. Species identification was performed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and confirmed via internal transcribed spacer ribosomal DNA barcoding. In vitro antifungal susceptibility was determined for seven systemic antifungals via the gradient strip method after 48 hours of incubation at 35°C using Etest® (Biomérieux) or Liofilchem® strips. Isolates exhibiting fluconazole minimal inhibitory concentrations (MICs) of ≥8 μg/mL were investigated for mutations in the ERG11 gene. A novel microsatellite genotyping scheme consisting of four markers was developed to assess genetic diversity.ResultsMIC ranges for amphotericin B, caspofungin, micafungin, isavuconazole, and posaconazole were 0.19–1 μg/mL, 0.094–0.5 μg/mL, 0.012–0.064 μg/mL, 0.003–0.047 μg/mL, and 0.006–0.032 μg/mL, respectively. By comparison, a broad range of MICs was noted for fluconazole (0.75 to >256 μg/mL) and voriconazole (0.012–0.38 mg/L), the higher values being observed among clinical strains. The Y132F amino acid substitution, associated with azole resistance in various Candida species (C. albicans, C. tropicalis, C. parapsilosis, and C. orthopsilosis), was the main substitution identified. Although microsatellite typing showed extensive genetic diversity, most strains with high fluconazole MICs clustered together, suggesting human-to-human transmission or a common source of contamination.DiscussionThe high rate of acquired fluconazole resistance among clinical isolates of D. catenulata is of concern. In this study, we highlight a link between the genetic diversity of D. catenulata and its antifungal resistance patterns, suggesting possible clonal transmission of resistant isolates.     

Antifungal susceptibility profile of Candida clinical isolates from 22 hospitals of São Paulo State,Brazil

This study aimed to evaluate the frequency of cryptic Candida species from candidemia cases in 22 public hospitals in São Paulo State, Brazil, and their antifungal susceptibility profiles. During 2017 and 2018, 144 isolates were molecularly identified as 14 species; C. parapsilosis (32.6%), C. albicans (27.7%), C. tropicalis (14.6%), C. glabrata (9.7%), C. krusei (2.8%), C. orthopsilosis (2.8%), C. haemulonii var. vulnera (2.1%), C. haemulonii (1.4%), C. metapsilosis (1.4%), C. dubliniensis (1.4%), C. guilliermondii (1.4%), C. duobushaemulonii (0.7%), C. kefyr (0.7%), and C. pelliculosa (0.7%). Poor susceptibility to fluconazole was identified in 6.4% of C. parapsilosis isolates (0.12 to >64 µg/mL), 50% of C. guilliermondii (64 µg/mL), 66.6% of C. haemulonii var. vulnera (16-32 µg/mL), and C. duobushaemulonii strain (MIC 64 µg/mL). Our results corroborated the emergence of C. glabrata in Brazilian cases of candidemia as previously reported. Importantly, we observed a large proportion of non-wild type C. glabrata isolates to voriconazole (28.6%; <0.015 to 4 µg/mL) all of which were also resistant to fluconazole (28.6%). Of note, C. haemulonii, a multidrug resistant species, has emerged in the Southeast region of Brazil. Our findings suggested a possible epidemiologic change in the region with an increase in fluconazole-resistant species causing candidemia. We stress the relevance of routine accurate identification to properly manage therapy and monitor epidemiologic trends.     

Reverse transcriptase polymerase chain reaction (RT-PCR) detection of HLP gene expression in Candida glabrata and its possible role in in vitro haemolysin production

Although haemolysins are known to be putative virulence factors contributing to pathogenicity in Candida species, the haemolytic activity of Candida glabrata and its genetic expression is ill understood at present. Thus, we studied a total of 34 Candida glabrata isolates for their in vitro haemolytic activity using a previously described plate assay system. The mRNA expression of HLP, a putative haemolysin gene, of these isolates was also evaluated using a semi-quantitative, non-competitive RT-PCR assay. All 34 C. glabrata isolates exhibited both partial (alpha) and complete (beta) haemolytic activity to varying degrees. In parallel with the haemolytic activity, all isolates were also positive for HLP mRNA expression. The expression levels of HLP mRNA (as relative units) ranged from 1.01 to 1.82, with a mean value of 1.32. On regression analysis of latter values and the haemolytic activity (in terms of the dimension of the haemolytic zone in the plate assay) of the C. glabrata isolates a highly significant positive correlation was noted (r=0.759, p<0.0001). Taken together, our data illustrate not only the phenotypic characteristics of haemolysin(s) and HLP expression of a battery of C. glabrata clinical isolates, but also, for the first time, evidence for a role of HLP in haemolysis.