Genomics and Proteomics as Compared to Conventional Phenotypic Approaches for the Identification of the Agents of Invasive Fungal Infections

There is overwhelming evidence that prompt diagnosis coupled with timely instigation of appropriate antifungal therapy are critical determinants of clinical outcome in invasive fungal infections. However, since the clinical symptoms of infection are often nonspecific, the number and diversity of potential aetiological agents is vast, and many fungi exhibit species-specific differences in antifungal susceptibility, the accurate identification of the responsible pathogen is a cornerstone of the therapeutic decision pathway. Traditionally, identification was achieved by examination of the phenotypic characteristics of the fungus obtained in pure culture, ideally from a normally sterile site/sample. However, this standard culture-based approach lacks sensitivity and obtaining appropriate specimens for culture is often difficult. Moreover, numerous recent studies have demonstrated the existence of clinically relevant cryptic species within well-established morphospecies that can not be differentiated by phenotypic methods. Here we discuss recent advances in genomic and proteomic approaches for the rapid and accurate identification of the principal pathogenic fungi associated with invasive fungal infections.     

血液或无菌体液培养阳性标本中病原菌检测结果分析

目的：了解血培养（包括血液、无菌体液培养,下同）阳性标本中病原菌的分布及阳性报警时间,为临床及时明确病原菌和正确用药提供参考依据。方法：无菌条件下采集血培养标本注人相应的培养瓶,经仪器扫描后放入血培养仪进行检测,报警后及时进行菌种鉴定和药敏试验。结果：364例阳性报警标本中真阳性标本为176例,其中革兰阳性菌占61．7％,革兰阴性菌占35．0％,真菌占3．3％;188例为假阳性标本,其中革兰阳性菌占54％,革兰阴性菌占41．9％,真菌占4．0％;新生儿科的感染阳性率最高;不同种类病原菌的阳性报警时间多重叠;临床医生经验用药正确或根据药敏结果更换用药的百分比为78．2％。结论：本院引起血液、无菌体液感染的病原菌以革兰阳性细菌为主,病原菌种类较多,存在一定的污染;当新生儿有局部感染时要警惕脓毒血症;单独靠血培养仪报警时间来鉴定区分病原菌与污染菌不一定可靠,及时了解血培养结果及标准药敏结果可以辅助找出感染性疾病的病因,尽早正确合理的使用抗菌药物,从而优化抗菌治疗。     

非侵袭性真菌性鼻窦炎的实验室诊断及致病菌分析

目的探讨非侵袭性真菌性鼻窦炎的实验室诊断方法,分析其致病菌,为鼻窦炎合并真菌感染的临床诊断、治疗提供依据。方法对我院临床及鼻内镜下所诊断的10例真菌性鼻窦炎患者,鼻内镜手术时直接吸取病变的鼻窦黏膜及窦腔内容物,通过直接镜检、真菌培养、传统鉴定及分子生物学鉴定和组织病理学检查对其进行检查。结果 10例病例中,直接镜检阳性者8例;病理学检查可见真菌菌丝或者孢子者8例;接种培养及基因鉴定阳性者5例(感染菌株包括2例烟曲霉复合体、1例杂色曲霉、1例枝孢样枝孢霉、1例帚霉)。不同方法检测出的阳性病例并非完全重叠。结论真菌镜检、真菌培养、真菌分子生物学鉴定、组织病理学检查在诊断真菌感染时可以互补,有助于明确诊断及发现新菌株。     

297例浅部真菌病临床及病原菌菌种分析

目的 探讨皮肤浅部真菌病致病真菌菌种的构成.方法 对297例真菌涂片阳性和培养阳性的浅部真菌病患者,取标本进行分离培养及菌种鉴定,培养阳性标本在形态学上不能准确鉴定的,进行梅里埃API 20C AUX酵母菌鉴定试剂盒或核糖体DNA （rDNA） ITS区序列测定,确切鉴定菌种.使用SPSS 17.0统计软件对于结果进行统计分析.结果 共分离培养出致病菌13种,其中红色毛癣菌86株（29.0％）,须癣毛癣菌68株（22.9％）,念珠菌属59株（19.9％）,暗色真菌属13株（4.4％）,曲霉菌属13株（4.4％）,红酵母菌12株（4.0％）,青霉菌属9株（3.0％）,毛霉菌9株（3.0％）,犬小孢子菌5株（1.7％）,浅白隐球菌3株（1.0％）,毛孢子菌属2株（0.7％）,絮状表皮癣菌1株（0.3％）,混合感染17株（5.7％）.结论 本地区浅部真菌病以甲癣为主,主要致病真菌是红色毛癣菌,但其他种类真菌感染尤其是念珠菌属有明显上升趋势.     

A key to the genera of medically important fungi

A simplified method for the rapid identification of medically important fungi is presented in the form of a dichotomous key. By noting the presence or absence of diagnostic features, one can effectively use the key to separate medically important fungi from other fungi. An unfamiliar fungus of medical importance in tissue or culture can be identified to genus within minutes with a high degree of accuracy.Variable characteristics such as color, size, texture, and shape of colonies are not used in the key. Colony morphology varies with different culture media, age, incubation temperature, and other factors. Biochemical studies are required for the identification of yeast-like organisms.     

Characterization of an extracellular protease and its cDNA from the nematode-trapping fungus Monacrosporium microscaphoides

To better exploit the biocontrol potential of nematophagous fungi, it is important to fully understand the molecular background of the infection process. In this paper, several nematode-trapping fungi were surveyed for nematocidal activity. From the culture filtrate of Monacrosporium microscaphoides, a neutral serine protease (designated Mlx) was purified by chromatography. This protease could immobilize the nematode Penagrellus redivivus in vitro and degrade its purified cuticle, suggesting that Mlx could serve as a virulence factor during infection. Characterization of the purified protease revealed a molecular mass of approximately 39 kDa, an isoelectric point of 6.8, and optimum activity at pH 9 at 65 degrees C. Mlx has broad substrate specificity, and it hydrolyzes protein substrates, including casein, skimmed milk, collagen, and bovine serum albumin. The gene encoding Mlx was also cloned and the nucleotide sequence was determined. The deduced amino acid sequence contained the conserved catalytic triad of aspartic acid--histidine--serine and showed high similarity with two cuticle-degrading proteases (PII and Aoz1), which were purified from the nematode-trapping fungus Arthrobotrys oligospora. Research on infection mechanisms of nematode-trapping fungi has thus far only focused on A. oligospora. However, little is known about other nematode-trapping fungi. Our report is among the first to describe the purification and cloning of an infectious protease from a different nematode-trapping fungus.     

Challenges in Laboratory Detection of Fungal Pathogens in the Airways of Cystic Fibrosis Patients

Study of the clinical significance of fungal colonization/infection in the airways of cystic fibrosis (CF) patients, especially by filamentous fungi, is challenged by the absence of standardized methodology for the detection and identification of an ever-broadening range of fungal pathogens. Culture-based methods remain the cornerstone diagnostic approaches, but current methods used in many clinical laboratories are insensitive and unstandardized, rendering comparative studies unfeasible. Guidelines for standardized processing of respiratory specimens and for their culture are urgently needed and should include recommendations for specific processing procedures, inoculum density, culture media, incubation temperature and duration of culture. Molecular techniques to detect fungi directly from clinical specimens include panfungal PCR assays, multiplex or pathogen-directed assays, real-time PCR, isothermal methods and probe-based assays. In general, these are used to complement culture. Fungal identification by DNA sequencing methods is often required to identify cultured isolates, but matrix-assisted laser desorption/ionization time-of-flight mass spectrometry is increasingly used as an alternative to DNA sequencing. Genotyping of isolates is undertaken to investigate relatedness between isolates, to pinpoint the infection source and to study the population structure. Methods range from PCR fingerprinting and amplified fragment length polymorphism analysis, to short tandem repeat typing, multilocus sequencing typing (MLST) and whole genome sequencing (WGS). MLST is the current preferred method, whilst WGS offers best case resolution but currently is understudied.     

分室培养装置在丛枝菌根真菌研究中的应用及其发展

重点围绕玻璃珠分室培养系统、H形分室培养系统、根排斥室培养系统、供体自养植物的双分室体外培养系统、丛枝菌根(AM)真菌与普通植物根器官的双重培养系统、AM真菌与Ri T-DNA转型根的双重单胞无菌培养系统、AM真菌与Ri T-DNA转型根双重培养的改良分室单胞培养系统等7个不同的分室培养装置, 对AM真菌的培养类型及其应用进行了系统的评述。其中, 采用玻璃珠分室培养装置易于将AM真菌与培养基质分开, 能获得大量纯净的AM真菌繁殖体, 用于研究AM真菌对矿质元素和微量元素的吸收, 具有不可替代的作用。H形分室培养系统和根排斥室(RECs)培养系统均能够获得连续的、可切断的共生菌根网络(CMNs), 可用于研究植物-植物、植物-昆虫之间化感作用产生的信息交流。供体自养植物的双分室培养系统有益于研究AM真菌对宿主植物在单作和混作条件下生长效应的影响。AM真菌与植物根器官的双重培养系统为研究AM真菌的侵染过程及生理、生化特性提供了极大的方便, 同时为纯培养研究提供了重要的理论依据。AM真菌与Ri T-DNA转型根的双重单胞无菌培养体系可以获得AM真菌纯净菌体, 是研究AM真菌遗传、生理、生化等特性的理想方法。以AM真菌与Ri T-DNA转型根的双重单胞无菌培养系统为基础, 可以在菌丝生长室置换培养基、在根室中补充适量碳源, 并多次收获AM真菌繁殖体。转型根改良双重培养系统是提高AM真菌孢子接种剂产量的有效方法。综上所述, AM真菌的分室培养系统已经取得显著进展, 为开展个体、种群、群落等不同层次的菌根生态学研究提供了依据。     

联合接种法在假丝酵母菌培养中的应用

目的寻找一种在最短时间内能为假丝酵母菌的鉴定提供最多信息的简易培养方法。方法在同一个平板内用分区划线法、多点接种法及小培养对标本进行培养,分离假丝酵母菌。结果一个平板内同时使用三种方法接种培养。可从分区划线法、多点接种法观察到菌落的全貌(如颜色、质地、形态)、种类、数量及生长特性等。可用显微镜从小培养中看到假丝酵母菌的孢子和菌丝的结构。结论由于假丝酵母菌种类繁多导致假丝酵母菌临床感染表现不同,临床诊断和治疗在很大程度上依赖于实验室的检查结果,因此寻找快速有效的鉴定方法至关重要。一个平板内同时用三种方法接种标本进行培养,可大大提高假丝酵母菌的检出率及鉴定效率,并能判定分离出的假丝酵母菌是目的菌株还是污染菌。     

临床标本中酵母样真菌的分离鉴定和药敏结果分析

目的调查目前临床标本中酵母样真菌感染现状及耐药性情况。方法常规分离临床标本中的酵母样真菌,用科玛嘉显色培养基及法国生物-梅里埃API20CAUX鉴定系统鉴定菌种,并用K—B法进行药敏试验。结果研究期间共分离到193例有临床意义的酵母样真菌,检出率较高的依次为白假丝酵母菌（62．7％）,热带假丝酵母菌（20．2％）,光滑球拟酵母菌（9．3％）。在6种抗真菌药中,对伊曲康唑、氟康唑的耐药率增高。结论近年来由酵母样真菌感染引起的疾病及其对常用抗真菌药物的耐药率有明显增加,应加强真菌的检测和耐药性监测工作,以指导临床合理使用药物。     

浅部真菌病1948份临床标本的真菌学分析

目的 通过对浅部真菌病患者临床送检标本的病原真菌菌种进行系统分析,了解感染及病原真菌的分布情况。方法 采用直接镜检、培养及真菌鉴定等方法对临床送验标本进行检验和鉴定,大部分标本鉴定到种。结果 1948份临床送验标本中,直接涂片镜检阳性率53．41％,培养阳性率40．28％,而镜检＋培养的阳性率为66．98％。对上述3种方法的真菌检出率进行比较,均存在显著差异（χ^2检验P均〈0．005）。在培养的1944份标本中,共分离出18个属,36种真菌,其中,红色毛癣菌24．52％、须癣毛癣菌16．48％、白念珠菌12．64％。结论 ①镜检结合培养的阳性率显著高于单一的镜检或培养的阳性率。②在患者即时的真菌镜检阴性时,应选择培养方法进一步检测,不轻易排除浅部真菌病感染可能。③皮肤癣菌居患者浅部真菌病致病菌首位,而白念珠菌及酵母类菌也是重要病原菌。     

A Rare Case Report of Subcutaneous Phaeohyphomycotic Cyst Caused by Exophiala oligosperma in an Immunocompetent Host with Literature Review

We report a rare case of phaeohyphomycotic cyst in an immunocompetent patient caused by Exophiala oligosperma. This fungus is earlier known to cause infections in the immunocompromised. Identification of black fungi at species level is more challenging by conventional methods, and hence final identification of the fungi was based on sequencing of rDNA. The patient was managed with surgical excision. To the best of our knowledge, this is the first case report of E. oligosperma human infection from India.     

院内150例真菌感染及其耐药性分析

目的研究院内真菌感染的特点及耐药状况。方法对临床标本分离的真菌,用TAB Expression鉴定仪鉴定,E—test抗真菌药敏试纸条进行药敏试验。结果150株真菌共有14个种,以白色念珠菌为主．占45．3％。各种真菌对6种抗真菌药物均有不同程度的耐药。结论院内真菌感染呈上升趋势,并且产生一定的耐药株。     

Epidemiology of Emerging Fungi and Fungi-like Organisms

Over the past several decades, rare and unusual fungi have been reported increasingly as agents of infection in humans and animals. This increased frequency of unusual fungal infections may be related in part to increasing numbers of patients with immunosuppression, as well as increased opportunities for environmental exposure to the organisms. In addition, the advent of better diagnostic modalities has aided in the identification of unusual organisms. This review discusses the epidemiology of emerging and unusual fungi and other pathogens causing fungal-like infection.     

Applications of MALDI-TOF-MS in clinical microbiology laboratory

For twenty years, mass spectrometry (MS) has emerged as a particularly powerful tool for analysis and characterization of proteins in research. It is only recently that this technology, especially MALDI-TOF-MS (Matrix Assisted Laser Desorption Ionization Time-Of-Flight) has entered the field of routine microbiology. This method has proven to be reliable and safe for the identification of bacteria, yeasts, filamentous fungi and dermatophytes. MALDI-TOF-MS is a rapid, precise and cost-effective method for identification, compared to conventional phenotypic techniques or molecular biology. Its ability to analyse whole microorganisms with few sample preparation has greatly reduced the time to identification (1-2 min). Furthermore, this technology can be used to identify bacteria directly from clinical samples as blood culture bottles or urines. Future applications will be developed in order to provide direct information concerning virulence or resistance protein markers.     

Eukaryote-Made Thermostable DNA Polymerase Enables Rapid PCR-Based Detection of Mycoplasma,Ureaplasma and Other Bacteria in the Amniotic Fluid of Preterm Labor Cases

Background

Intra-amniotic infection has long been recognized as the leading cause of preterm delivery. Microbial culture is the gold standard for the detection of intra-amniotic infection, but several days are required, and many bacterial species in the amniotic fluid are difficult to cultivate.

Methods

We developed a novel nested-PCR-based assay for detecting Mycoplasma, Ureaplasma, other bacteria and fungi in amniotic fluid samples within three hours of sample collection. To detect prokaryotes, eukaryote-made thermostable DNA polymerase, which is free from bacterial DNA contamination, is used in combination with bacterial universal primers. In contrast, to detect eukaryotes, conventional bacterially-made thermostable DNA polymerase is used in combination with fungal universal primers. To assess the validity of the PCR assay, we compared the PCR and conventional culture results using 300 amniotic fluid samples.

Results

Based on the detection level (positive and negative), 93.3% (280/300) of Mycoplasma, 94.3% (283/300) of Ureaplasma, 89.3% (268/300) of other bacteria and 99.7% (299/300) of fungi matched the culture results. Meanwhile, concerning the detection of bacteria other than Mycoplasma and Ureaplasma, 228 samples were negative according to the PCR method, 98.2% (224/228) of which were also negative based on the culture method. Employing the devised primer sets, mixed amniotic fluid infections of Mycoplasma, Ureaplasma and/or other bacteria could be clearly distinguished. In addition, we also attempted to compare the relative abundance in 28 amniotic fluid samples with mixed infection, and judged dominance by comparing the Ct values of quantitative real-time PCR.

Conclusions

We developed a novel PCR assay for the rapid detection of Mycoplasma, Ureaplasma, other bacteria and fungi in amniotic fluid samples. This assay can also be applied to accurately diagnose the absence of bacteria in samples. We believe that this assay will positively contribute to the treatment of intra-amniotic infection and the prevention of preterm delivery.     

病原真菌生态学研究现状及方法

通过形态学、生理生化学及分子生物学等方法鉴定由自然界分离的真菌菌种,构建系统发生树,了解菌种间亲缘关系,评估种间和种内的进化规律,了解真菌与其所处环境的相互关系,是真菌生态学的主要研究目的之一。本文就真菌与自然界和动物宿主的相互关系,环境中真菌的分离和鉴定方法及系统发生学研究及现状进行综述,以期了解致病真菌的生存环境、种类发生、感染途径等,达到尽早控制感染源、探讨致病性及防治疾病传播的目的。     

651株深部真菌感染菌种分布及耐药性分析

目的了解真菌耐药性变化趋势,探讨预防措施。方法对疑为深部真菌感染患者采样,用沙保罗培养基进行真菌分离,用ATB鉴定卡作菌种鉴定,用ATBFUNGUS板进行药敏试验。结果真菌感染中以白色假丝酵母菌感染为主,占57.6%。真菌对两性霉素-B、5-氟胞嘧啶保持较高的体外抗真菌活性,敏感率分别为94.99%和98.99%。结论深部真菌感染主要由白色假丝酵母菌、热带假丝酵母菌引起,对两性霉素-B、5-氟胞嘧啶保持较高的体外活性,两者仍是治疗真菌的有效药物。     

MALDI‐TOF‐MS‐based species identification and typing approaches in medical mycology

MALDI‐TOF MS‐based species identification has found its place in many clinical routine diagnostic laboratories over the past years. Several well‐established commercial systems exist and these allow precise analyses not only among bacteria, but also among clinically important yeasts. This methodology shows higher precision than biochemical and microscopic methods at significantly reduced turnaround times. Furthermore, the differentiation of different filamentous fungi including most dermatophytes and zygomycetes has been established. The direct identification of yeasts from blood culture bottles will be possible in a routine fashion with new standardized procedures. In addition to species identification, the MALDI‐TOF MS technology offers several further possibilities, like assays to detect or predict resistance phenotypes in fungi as well as subtyping approaches to detect clinically relevant subgroups. The differences between the commercial systems are discussed with respect to fungi and an overview of their performances provided. Factors influencing outcome of MALDI‐TOF‐based species identification are discussed.     

Effect of Enterobacter aerogenes on the Rhizosphere Microflora of Apple Trees*

Fifty–seven fungi and 59 bacteria were isolated from soils collected from two apple orchards located in Kelowna and Summerland, British Columbia, Canada. Sixteen fungi and 35 bacteria, respectively, were significantly inhibited by the biological control agent Enterobacter aerogeness in a dual culture test. These results indicate that the antibiotic(s) produced by this organism has a wide spectrum of activity. This ability of E. aerogenes may protect apple trees from infection by other, soil microorganisms.