腹部大手术术后深部真菌感染的临床与真菌学分析

目的探讨腹部大手术术后深部真菌感染的危险因素及诊治措施。方法回顾性分析2006年1月～2009年6月期间我科腹部大手术术后合并深部真菌感染48例患者的临床和真菌学资料。结果 48例真菌感染患者共分离出56株菌株,其中白念珠菌占41.1%,是最主要的致病菌株。患者基础疾病和术后长期、多种广谱抗生素联合使用是深部真菌感染的重要因素。结论深部真菌感染是腹部大手术术后的重要并发症,白念珠菌仍然是主要病原菌。对术后患者深部真菌感染应采取积极预防、及时发现和有效治疗。     

Detection of Candida species by polymerase chain reaction (PCR) in blood samples of experimentally infected mice and patients with suspected candidemia

In this study, we have established and evaluated a genus-specific polymerase chain reaction (PCR) and species-specific nested PCRs for the detection of Candida species in blood samples of neutropenic mice and patients suspected of candidemia. DNA segments of the gene encoding cytochrome P450 L1A1 were targeted for amplification by using genus and species-specific primers. As compared to the genus-specific PCR, the species-specific nested PCRs improved the sensitivity by 10 times with the detection limit < 10 yeast cells. Of the 18 blood samples tested daily over a period of 8 days following Candida albicans infection in neutropenic mice, four samples were positive by genus-specific PCR and 11 were positive by species-specific nested PCR. The PCR results were correlated with culture findings obtained on blood samples. Two of the three blood culture-positive samples were positive by genus-specific PCR and all the three with species-specific nested PCR. Among 15 mice, which were negative by blood culture but had C. albicans isolated from visceral organs, 2 and 8 mice yielded positive results by genus-specific PCR and species-specific nested PCR, respectively. Consistent with the results of the animal study, species-specific nested PCR yielded much higher positivity as compared to culture (52.2% versus 21.2%) in patients suspected for candidemia. Moreover, 8 specimens which were negative for Candida by genus-specific PCR became positive by species-specific nested PCR. No correlation was apparent between PCR positivity and Candida antigen titers. The results suggest that nested PCR is a sensitive technique for the detection of Candida species from blood samples, and thus it may have application in the diagnosis of suspected cases of candidemia and candidiasis.     

濮阳市部分浅部真菌病中癣菌和念珠菌混合感染的分析

目的了解濮阳地区部分浅部真菌病中两种及两种以上真菌混合感染的情况。方法对临床确诊为浅部真菌病的患者456例,取标本行10%KOH直接镜检、真菌培养及菌种鉴定。登记确诊患者相关临床资料,分析其中混合感染的发病特点。结果确诊皮肤癣菌和念珠菌混合感染病例36例,分离率为7.89%,多见于股部(58.33%)和足部(27.78%);分离菌株72株,皮肤癣菌中以红色毛癣菌为主(75.00%),念珠菌中以非白念珠菌为主(72.22%)。结论濮阳地区部分浅部真菌病中混合感染主要为皮肤癣菌和念珠菌的混合感染,多见于股部和足部;致病菌以红色毛癣菌和非白念珠菌为主。     

白念珠菌临床分离调查及基因分型研究

目的 了解白念珠菌临床分离情况,并探讨其药敏结果与基因分型的相关性.方法 回顾性分析本院2011年3～11月间临床分离白念珠菌分布及耐药性;随机选取232株,采用PCR方法扩增白念珠菌25S rDNA基因内含子区进行基因分型研究;采用ATB真菌药敏试剂条进行药敏分析;统计分析药敏结果与基因分型的相关性.结果 期间共检出酵母样真菌973例,占病原菌阳性样本数比率为15.7％ (973/6196);其中分离白念珠菌562株,占58％ (562/973),主要分布科室为呼吸科(39.1％)、老年科(13.2％)、ICU(7.7％)、神经内科(7.5％)、免疫科(6.0％)以及其他科室(26.5％);标本类型以下呼吸道为主(81.7％),其次为尿路(9.4％)、血液(1.8％)等.对氟胞嘧啶、两性霉素B、氟康唑、伊曲康唑及伏立康唑的耐药率分别为0.9％、0％、1.4％、1.6％和1.1％.随机选取的232株白念珠菌经PCR方法可分为3型:A型125株,B型96株,C型11株.各型在5种药物的耐药性上并无差异.结论 临床分离酵母样真菌以白念珠菌为主,感染部位以下呼吸道为主;临床分离株对5种抗真菌药物敏感度较高,主要基因型为A和B型,不同基因分型间药敏结果并无统计学差异.     

通用PCR法在痰标本真菌检测中的临床应用价值分析

目的:探讨通用PCR法在痰标本真菌检测中的临床应用价值。方法:选择2015年9月至2017年9月本院收治的免疫力低下患者178例作为研究资料,各收集痰标本2份,其中一份用于PCR扩增,另一份经培养后若观察到真菌或可疑菌落,则提取后在此进行PCR扩增测序。比较3种方法对真菌的检出情况、真菌阳性率,与组织病理学结果进行比较,分析3种方法的诊断效能。结果:178份痰标本经平板培养可观察到107份真菌生长,其余71份未见真菌生长。挑取真菌及可疑菌落行PCR结果显示阳性、阴性分别115、63份。对痰标本直接PCR结果显示,阳性、阴性分别124、54份。PCR扩增产物测序结果显示大多是白色念珠菌感染,仅3份是曲霉菌感染。内对照扩增验证结果显示有1份阴性标本存在扩增抑制现象。3种方法的真菌阳性率:直接PCR法痰培养后PCR法痰培养,但组间无显著差异(P0.05)。3种方法诊断效能总体趋势:直接PCR法痰培养后PCR法痰培养,其中直接PCR法与痰培养后PCR法的特异度、准确度均显著高于痰培养法(P0.05),直接PCR法的敏感度显著高于痰培养法(P0.05)。结论:通用PCR法在痰标本真菌检测中的临床应用价值较高,直接PCR具有操作简单、快速等优势。     

家族传播的口腔白色念珠菌基因型多态性分析

目的检测家族传播的口腔白色念珠菌基因多态性。方法采集35个家庭（119个样本）的口腔牙菌斑,采用PCR ITS1-ITS2基因分型方法,检测、分析家族传播的口腔白色念珠菌基因多态性。结果 18个家庭（18/35,61%）,34个样本（34/119,28.6%）有白色念珠菌感染,11个家庭存在家族传播（11/18,61%）。在5个母子（父子）垂直传播的家庭成员中,白色念珠菌基因型均不一致。在3个呈水平传播的家庭成员中,两家基因型一致,1家不一致。在3个垂直-水平传播的家庭成员中,两家基因型一致,1家不一致。白色念珠菌家族传播基因型差异有显著统计学意义（χ2=26.571,P〈0.01）。白色念珠菌感染与年龄、性别、学历、吸烟、饮酒、义齿和龋病均无显著相关。结论白色念珠菌在口腔定植,受宿主自身遗传背景影响较大,在家族垂直传播中呈明显的基因多态性。呈水平传播的白色念珠菌菌种具有较高的传染性,基因型可保持不变。     

Nucleic acid sequence-based amplification (NASBA) detection of medically important Candida species.

Nucleic Acid Sequence Based Amplification (iNASBA), an isothermal amplification technique for nucleic acids, was evaluated for the identification of medically important Candida species using primers selected from 18S rRNA sequences conserved in fungi. An RNA fragment of 257 nucleotides was amplified for Candida albicans. Nineteen different fungi were tested for rRNA amplification with the NASBA. All were positive when analyzed on agarose gel, whereas human RNA was negative. For the identification of Candida species, NASBA amplification products were analyzed in an enzyme bead-based detection format, using species-specific biotinylated probes and a generic Candida HRPO probe or a membrane-based system using biotinylated probes and avidin-HPRO. Discrimination of the major human pathogenic Candida spp. was based on a panel of biotinylated probes for C. krusei, C. tropicalis, C. albicans, C. glabrata, and C. lusitaniae. Using rRNA dilutions obtained from pure cultures of C. albicans, the combination of NASBA and the enzymatic bead-based detection yielded a sensitivity equivalent to 0.01 CFU. In a model system using 1 ml of artificially contaminated blood as few as 1-10 CFU of C. albicans could be detected. Testing of 68 clinical blood samples from patients suspected of candidemia showed that eight samples were positive for C. albicans and one for C. glabrata. Testing of 13 clinical plasma samples from patients suspected of fungemia identified the presence of C. albicans in two specimens. The whole procedure of sample preparation, amplification and identification by hybridization can be performed in 1 day. This speed and the observed sensitivity of the assay make the NASBA a good alternative to PCR for the detection of candidemia.     

Presence of fungi in stool of children

A total of 258 children were tested for the presence of fungi in stool. One group consisted of 148 children with non-specific gastrointestinal tract disorders while the other was a group of 110 asthmatics. A quantitative method of enzymatic and mechanical homogenisation was used. The findings were divided into three ranges as follows: < 10(3), 10(3)-10(5), > 10(5) fungal cells in one gram of stool. The number of > 10(5) fungal cells in one gram of stool was considered as pathogenic and requiring treatment. Such a number of fungi in stool was found in 48.1% of children in the first group and in 35.9% in the second one. However, the percentage of fungal presence was higher in the group of asthmatics (83.6% vs. 70.3%). Candida albicans considerably outnumbered the remaining fungal species in the isolates. It was found out that other than C. albicans Candida species were more resistant to the antifungals.     

Detection of Candida dubliniensis in patients with candidiasis in Caracas, Venezuela

Candida species are responsible for 80% of all nosocomial fungal infections. In 1995 a new yeast species was described, Candida dubliniensis which shares with Candida albicans characteristics. We have studied 109 yeast isolates identified as C. albicans to investigate the presence of C. dubliniensis by microbiological studies and PCR using DUBR/DUBF primers. Positive results using microbiological tools were between 90 and 98%. Two morphological and physiological of the 80 DNA examined samples (2.5%) showed a PCR product of 288 bp which allow the identification of C. dubliniensis. This is the first report in Venezuela of identification of this species using a PCR approach.     

Identification of Candida albicans by polymerase chain reaction amplification of CaYST1 gene intron fragment

A single pair of primers, deduced from the intron nucleotide sequence of the Candida albicans CaYST1 gene, was used in PCR analysis performed with both genomic DNA and whole cells of clinical isolates of Candida species and other microorganisms. All the clinical C. albicans isolates generated the expected 310 bp amplicon; other Candida species as well as laboratory strains belonging to other fungal genera failed to amplify any DNA fragment, except for Candida pseudotropicalis (amplicon of 1200 bp), Kluyveromices marxianus (amplicon of 1250 bp) and Cryptococcus neoformans (several amplicons longer than 1200 bp). Unusual C. albicans isolates from Africa also yielded the expected 310 bp amplicon. These results indicate that genes containing intron sequences may be useful to design species-specific primers for identification of fungal strains by PCR. The sensitivity of the method was evaluated for C. albicans genomic DNA by using both various DNA concentrations (224 ng to 2.7 pg) and different cell amounts (10(7); to 5 cells). The results obtained may be useful in earlier detection of candidiasis.     

Diagnosis of candidosis by amplification of small subunit of 18S rRNA gene

A PCR assay for the diagnosis of infection produced by Candida sp. was developed. The primers, designated 520 and 522, were selected from highly-conserved areas of the small subunit (ssu) 18S rRNA gene of Candida spp. To check the value of the results a Candida albicans oligonucleotide probe, digoxigenin-labeled, and a general Candida probe were used in hybridization experiments with the amplified products. We were able to detect a Candida- specific fragment of 1800 bp from different clinical samples. The procedure described could provide an interesting complement to present diagnostic methods of detecting Candida sp in clinical samples.     

慢性前列腺炎患者假丝酵母菌感染及耐药性分析

目的 了解慢性前列腺炎与假丝酵母菌感染的相关性及假丝酵母菌的耐药性。方法 采用常规沙堡平板分离360例慢性前列腺炎患者的前列腺液标本中的假丝酵母菌,疑似菌落用ATB Expression鉴定仪进行鉴定。采用ATB-Fungus真菌药敏板,对假丝酵母菌株进行药敏试验。结果 11．7％前列腺液标本（42／360）似丝酵母菌阳性,其中自假丝酵母菌23例（54．7％）,近平滑假丝酵母菌13例（30．9％）,其它6例（14．3％）。假丝酵母菌菌株对两性霉素B和制霉菌素敏感率均为100％,其次为酮康唑,敏感率为97．0％;对5-氟胞嘧啶耐药性最强,其耐药率为56．5％。结论 白假丝酵母菌和近平滑假丝酵母菌是慢性前列腺炎假丝酵母菌感染的优势菌种,假丝酵母菌株最敏感的药物是两性霉素B和制霉菌素。     

儿童重症监护病房医院感染暴发白色念珠菌血流感染4例分析

摘要 目的：探讨儿童重症监护病房白色念珠菌血流感染暴发的临床表现、危险因素、控制措施等,为预防和控制院内白色念珠菌血流感染暴发提供科学依据。方法：以2018年7月我院儿童重症监护病房发生的4例白色念珠菌血流感染暴发患儿为研究对象,分析患儿临床情况、临床特征、危险因素、暴发原因以及采取的预防控制措施。结果：4例医院感染暴发白色念珠菌血流感染患儿均存在基础疾病、有机械通气史、存在中心静脉或动脉置管、静脉或动脉置管前后均使用碘伏消毒、曾使用广谱抗生素、输血制品,白色念珠菌血流感染后最突出的临床表现均是发热。药敏方面,医院感染暴发的4例白色念珠菌感染患儿对唑类及5-氟胞嘧啶均耐药,但对两性霉素B均敏感。经拔除血管置管、减少或者避免广谱抗菌药的应用,根据药敏使用卡泊芬净及两性霉素B抗真菌等积极治疗,1例患儿放弃治疗后死亡,3例患儿顺利出院。通过Fisher确切概率法分析可知,留置中心静脉或动脉置管是儿童重症监护病房发生医院感染暴发白色念珠菌血流感染的危险因素（P<0.05）。结论：留置中心静脉或动脉置管是儿童重症监护病房发生医院感染暴发白色念珠菌血流感染的危险因素,医院感染暴发白色念珠菌血流感染患儿最突出的临床表现是发热,唑类及5-氟胞嘧啶耐药的患儿使用卡泊芬净及两性霉素B可能获得较好的治疗效果。     

Etiologic agents of fungemia in hospitalized patients]

The aim of performed examinations was the analysis of fungi as etiological agents of blood infections in patients hospitalized in surgical wards, internal medicine wards and intensive care units of the Medical Academy Central Clinical Hospital in Warsaw. Blood samples from patients hospitalized in 1997 were examined. Peripheral blood samples were incubated in BacT/Alert system (Organon Teknika, USA). Positive blood samples were inoculated on Sabouraud medium with chloramphenicol (bioMerieux, France or Oxoid, England). The time of cultivation was from 48 hours to 7 days at 30 degrees C. Fungal strains were identified by standard mycological procedures with the use of chromogenic medium BBL CHROMagar Candida (Becton Dickinson, USA) and biochemical test ID 32 C (bioMerieux, France). Susceptibility of strains to antifungal agents was determined by ATB FUNGUS method (bioMerieux, France). The total number of positive blood cultures in 1997 was 1380. Forty-two fungal strains were isolated from blood samples (3%). Strains belonged to the following species: C. albicans (17 isolates), C. parapsilosis (15), C. glabrata (3), melibiosica (2), C. pelliculosa (2), C. guilliermondii (1), C. tropicalis (1) and T. beigelii (1). Among fungi cultured from patients hospitalized in operative wards dominated C. parapsilosis (11) and C. albicans (10) strains, whereas from patients hospitalized in conservative wards most often C. albicans (6) strains were isolated. Candida strains were mostly susceptible to antifungal agents tested. It was interesting to culture Trichosporon beigelii (T. cutaneum) strain as an etiological agent of fungemia. This strain was multidrug-resistant.     

院内深部真菌感染92例临床分析

目的探讨院内深部真菌感染的临床表现、感染相关因素及诊断治疗措施.方法对1993年1月至2002年12月间经微生物检查证实的92例院内深部真菌感染的病例临床资料进行回顾性分析.结果在92例患者中,肺部疾病26例,糖尿病19例,血液病及肿瘤19例,外科手术后13例,消化系统疾病6例,慢性肾脏病6例,风湿性疾病3例.主要感染真菌为白色念珠菌和酵母菌.感染相关因素:长时间应用广谱抗生素病人65例,应用糖皮质激素22例,应用免疫抑制剂18例,应用气管切开或气管内插管以及中心静脉留置导管17例,进行血液净化治疗的9例.治疗痊愈59例(64.1%),好转10例(10.9%),死亡23例(25%).结论白色念珠菌是院内深部真菌感染的主要致病菌株,有逐年增加趋势,病死率高.与长时间应用广谱抗生素及免疫抑制剂等有相关性.     

Hydrophobic and adhesive capacity of Candida strains isolated from patients with fungal infection

The aim of this study was to evaluate hydrophobicity of fungal strains of Candida sp. isolated from clinical materials of patients with fungal infections. Two hundred and eighty one strains of C. albicans and 29 strains belonging to the other species of Candida were tested in salt aggregation test (SAT). Strong hydrophobicity (autoaggregation in the test) was found in 29.4% of tested strains. The majority of them was isolated from vagina. In 20 randomly selected strains hydrophobicity was measured not only by salt aggregation test but also by hydrophobic interaction chromatography (MIC). The ability to attach to buccal epithelial cells in vitro was compared between 50 strongly hydrophobic strains and 30 with low hydrophobicity. The former strains attached significantly stronger (p less than 0.001).     

白念珠菌基因分型的相关研究

白色念珠菌定植于大多数人群的口腔中,在一定条件下可成为优势菌种而导致感染。基因分型是近年来白念分子生物学研究中的一个热点,随着医学科学技术的发展,由于深部真菌感染比例不断增加,分子生物学方法已经越来越广泛的应用于临床真菌病的研究中,从而为控制白念感染及为早期诊断、治疗提供基础。本文综述了限制性片段长度多态性、随机扩增多态性DNA分析、ITS区域序列分析等分子生物学技术在白念珠菌基因分型方面的相关研究,比较了它们的优缺点,并且讨论了将基因分型研究应用于临床诊断、治疗及开发新型抗真菌药物的发展趋势和广阔前景。认为目前更倾向于多种分型方法联合应用,并借助计算机软件进行分析,但是仍需进一步探索。     

Development of an in vitro model for the multi-parametric quantification of the cellular interactions between Candida yeasts and phagocytes

We developed a new in vitro model for a multi-parameter characterization of the time course interaction of Candida fungal cells with J774 murine macrophages and human neutrophils, based on the use of combined microscopy, fluorometry, flow cytometry and viability assays. Using fluorochromes specific to phagocytes and yeasts, we could accurately quantify various parameters simultaneously in a single infection experiment: at the individual cell level, we measured the association of phagocytes to fungal cells and phagocyte survival, and monitored in parallel the overall phagocytosis process by measuring the part of ingested fungal cells among the total fungal biomass that changed over time. Candida albicans, C. glabrata, and C. lusitaniae were used as a proof of concept: they exhibited species-specific differences in their association rate with phagocytes. The fungal biomass uptaken by the phagocytes differed significantly according to the Candida species. The measure of the survival of fungal and immune cells during the interaction showed that C. albicans was the more aggressive yeast in vitro, destroying the vast majority of the phagocytes within five hours. All three species of Candida were able to survive and to escape macrophage phagocytosis either by the intraphagocytic yeast-to-hyphae transition (C. albicans) and the fungal cell multiplication until phagocytes burst (C. glabrata, C. lusitaniae), or by the avoidance of phagocytosis (C. lusitaniae). We demonstrated that our model was sensitive enough to quantify small variations of the parameters of the interaction. The method has been conceived to be amenable to the high-throughput screening of mutants in order to unravel the molecular mechanisms involved in the interaction between yeasts and host phagocytes.     

Use of molecular methods in identification of Candida species and evaluation of fluconazole resistance

The aim of this study was to evaluate the use of one of the molecular typing methods such as PCR (polymerase chain reaction) following by RFLP (restriction fragment length polymorphism) analysis in the identification of Candida species and then to differentiate the identified azole susceptible and resistant Candida albicans strains by using AP-PCR (arbitrarily primed-polymerase chain reaction). The identification of Candida species by PCR and RFLP analysis was based on the size and primary structural variation of rDNA intergenic spacer regions (ITS). Forty-four clinical Candida isolates comprising 5 species were included to the study. The amplification products were digested individually with 3 different restriction enzymes: HaeIII, DdeI, and BfaI. All the isolates tested yielded the expected band patterns by PCR and RFLP analysis. The results obtained from this study demonstrate that Candida species can be differentiated as C. albicans and non-C. albicans strains only by using HaeIII restriction enzyme and BfaI maintains the differentiation of these non-C. albicans species. After identification Candida species with RFLP analysis, C. albicans strains were included to the AP-PCR test. By using AP-PCR, fluconazole susceptible and resistant strains were differentiated. Nine fluconazole susceptible and 24 fluconazole resistant C. albicans were included to the study. Fluconazole resistant strains had more bands when evaluating with the agarose gel electrophoresis but there were no specific discriminatory band patterns to warrant the differentiation of the resistance. The identification of Candida species with the amplification of intergenic spacer region and RFLP analysis is a practical, short, and a reliable method when comparing to the conventional time-consuming Candida species identification methods. The fluconazole susceptibility testing with AP-PCR seems to be a promising method but further studies must be performed for more specific results.     

深部真菌感染临床特点分析

目的了解深部真菌感染患者的性别、年龄、感染部位、住院科室、菌种分布及真菌耐药情况,为临床防治真菌感染提供研究依据。方法收集荆州市中心医院2009年1月至2009年12月微生物实验室分离的真菌446株,采用科马嘉显色琼脂及API220C Aux鉴定系统鉴定,并使用ATMTMFUNGUS3真菌药敏卡进行体外药敏试验。结果临床真菌感染男性占72%,以老年患者为主,大于60岁者占54.9%;感染的真菌主要分布于呼吸内科和ICU,分别占35.5%、24.9%;主要感染部位为呼吸道,占91.3%;主要菌种为白假丝酵母菌、热带念株菌、近平滑假丝酵母菌、光滑念株菌和克柔念株菌,分别占64.2%、13.2%、9.6%、7.6%和5.4%;合并细菌感染的感染真菌100株,占22.2%,细菌中以革兰阴杆性菌为主,占96%;药敏试验结果显示真菌对各抗真菌药具有较好的敏感性。结论临床真菌感染已日益突出,以呼吸科及ICU患者老年男性为主,儿童真菌感染亦不容忽视,感染菌种以白假丝酵母菌和热带念株菌为主,临床应加强对这些真菌感染的预防和监测,防止真菌感染。