2006-2013年海军总医院血培养真菌感染分析

目的：了解海军总医院检验科血培养标本中真菌感染状况及药敏结果,为临床诊断及合理用药提供参考。方法对本院2006年1月~2013年12月血培养真菌阳性标本进行回顾性分析。结果从临床送检的血培养标本检出真菌114株,菌株主要为白色念珠菌（37.7%）,热带念珠菌（28.9%）,光滑念珠菌（21.0%）;连续8年真菌感染血培养阳性呈逐年增长趋势,2013年检出最高（22株）;体外抗真菌药敏试验的敏感率从高到低依次为：两性霉素B、5-氟胞嘧啶、伏立康唑、伊曲康唑、氟康唑,其中两性霉素B抗菌活性最好,100%敏感。30株真菌感染血培养标本检测了血清（1,3）-β-D葡聚糖,阳性率为53.3%（16/30）。结论引起真菌血症的病原体主要是念珠菌,联合血清学检测有助于辅助诊断真菌血症,真菌药敏试验结果给临床治疗提供了有参考价值的信息。     

2158例深部真菌感染拟诊病例临床特点与病原菌检查

目的总结分析送检拟诊深部真菌感染病例临床特征和实验室结果,为临床更好的诊治深部真菌感染提供借鉴。方法回顾性分析我院(2012~2016)年真菌室深部真菌送检病例临床资料和真菌检查结果。结果 2 158例送检标本中,镜检查出菌丝和/或孢子的阳性标本共336例,镜检阳性率为15.57%,培养阳性标本共496例,培养阳性率为22.89%;鉴定出白假丝酵母菌406株(82.19%),光滑假丝酵母菌48株(9.72%),其他真菌的比例不足10%。深部真菌感染的部位主要为口腔,占26.91%;光滑假丝酵母菌和新型隐球菌感染更易发生在(31～40)岁年龄组;热带假丝酵母菌感染则易发生于(41～50)年龄段和71岁以上年龄段。不同年龄段真菌培养结果有统计学差异。结论深部真菌感染的主要病原菌为白假丝酵母菌,但光滑假丝酵母菌等非白假丝酵母菌的检出率逐年增高,各菌种在不同年龄段分布存在差异。     

真菌病

20121860浅部真菌病2600例病原菌分析/张道军(昆明医学院一附院皮肤科),黄云丽,张卫卫…∥中国皮肤性病学杂志.-2012,26(6).-501～503对拟诊为浅部真菌病患者的临床标本再次进行镜检和分离培养及菌种鉴定,并对结果进行统计学分析。结果:在7944份临床送验标本中,直接涂片镜检阳性率29.39%,培养阳性率19.70%,而镜检和(或)培养的阳性率为32.73%,显著高于单一的镜检或培养。上述3种方法的真菌检出率差异均有统计学意义(P均<0.005)。分离的1565株浅部致病真菌中,红色毛癣     

住院患者深部真菌感染的病原菌分布与药敏分析

目的 了解住院患者深部真菌感染的现状,病原菌的种类以及病原菌对常用抗真菌药物的敏感性。方法 采集我院2012年1月—2014年4月临床疑似感染的住院患者的31 305份血液及体液标本进行培养,对分离出的菌株,采用直接镜检、科玛嘉显色培养基和VITEK-32自动分析仪等进行菌种鉴定;纸片扩散法ATBFUNGUS3真菌药敏板条测试药敏,并收集临床资料进行分析。结果 共分离出真菌290株,分离率为0.92%;标本中真菌检出构成比以血液最高(41.03%),其次为尿液(21.38%);真菌检出最高的科室为普通外科(55.82%);真菌构成比依次为白念珠菌31.03%,热带念珠菌17.93%,光滑念珠菌17.59%,近平滑念珠菌13.10%。药敏试验中白念珠菌和近平滑念珠菌对唑类药物的敏感性相对较高。4种常见真菌对两性霉素B和氟胞嘧啶均表现出较高的敏感性。结论 血液标本真菌检出构成比最高;普通外科真菌感染患者的分布最多;分离菌株中念珠菌属多见,尤以白念珠菌感染率最高;两性霉素B和氟胞嘧啶的耐药率较低,唑类药物的耐药率较高,各菌种间耐药性存在差异。     

PCR—RFLP用于甲真菌病病原菌诊断和鉴别

目的：用聚合酶链反应－限制性片段长度多态性（PCR－RFLP）方法提高甲真菌病诊断的敏感性和特异性,缩短病原诊断时间。方法：用18S-rRNA真菌特异性通用引物,对真菌菌株、甲标本基因组进行PCR扩增,产物行HaeⅢ酶切分析,并与KOH直接镜检、真菌培养结果进行比较。结果：真菌菌株用PCR－RFLP方法检测,能区分皮肤癣菌、酵母菌和霉菌。15例甲真菌现标本PCR扩增均为阳性,KOH直接镜检查,真菌培养阳性率分别为10／15、9／15。培养阳性的9例标本,PCR－RFLP分析结果均与培养符合。结论：PCR方法诊断甲真菌感染的敏感性明显高于直接镜检和培养。PCR－RFLP能够鉴别病原菌的种属。     

真菌四重PCR体系的建立及其临床应用

目的建立多重PCR体系并探讨其用于深部真菌感染诊断的可行性。方法用真菌通用引物内转录间区(ITS)联合曲霉属、镰刀菌属、毛孢子菌属、白念珠菌、新生隐球菌特异性引物建立多重PCR体系,用单重和四重PCR检测模拟体液和30例可疑深部真菌感染患者临床标本中的真菌。结果ITS可与5对属或种特异性引物任意组合(二重PCR),四重PCR组合为ITS+毛孢子菌属+白念珠菌+新生隐球菌。30例临床标本中,真菌培养、PCR检测的阳性率分别为26.67%和43.33%,两种方法的检出率差异无统计学意义(P0.05)。结论单重和四重PCR敏感性高、稳定性好、操作简单、耗时短,可为深部真菌感染的早期诊断提供病原学依据。     

荧光染色法和KOH湿片法、真菌培养法在皮肤浅部真菌感染诊断中的应用比较

目的： 评估荧光染色法在皮肤浅部真菌感染诊断中的价值。方法： 收集我院皮肤门诊疑似皮肤浅部真菌感染标本，同时采用荧光染色法、KOH湿片法和真菌培养法进行检测，比较检测结果。结果： 在236例标本中荧光染色法、KOH湿片法、真菌培养法总阳性率分别为90.6%、67.4%、60.2%，在179例皮屑标本中阳性率分别为92.1%，69.3%，64.2%，52例甲真菌病标本中阳性率分别为84.6%，57.7%，44.2%，荧光染色法与其他两种方法相比，差异均有统计学意义（均P<0.01）。5例毛发标本中阳性率分别为100%，100%，80%,三种方法阳性率无统计学差异（均P>0.05）。结论： 荧光染色法简单有效，可以降低假阴性率。     

PCR检测深部致病真菌感染的研究

目的探讨PCR在可疑深部真菌感染临床标本检测中的应用。方法对60份临床标本分别进行传统真菌培养以及真菌通用引物PCR和三重PCR检测。结果真菌培养阳性共19份，其中白念珠菌10份、烟曲霉2份、新生隐球菌2份、其他菌株5份（包括光滑念珠菌2份、热带念珠菌2份、克柔念珠菌1份）；真菌通用PCR检测共有21份阳性，经三重PCR检测共有15份阳性，分别为白念珠菌10份、烟曲霉3份、新生隐球菌2份。PCR方法与传统培养方法相比，检出率差异无统计学意义。结论PCR检测可疑深部真菌感染临床标本快速简便，稳定性好，敏感性和特异性均较理想，适合临床实验室常规使用。     

重庆地区2135例浅部真菌病患者病原学分析

目的：了解重庆地区浅部真菌病病原菌菌种分布特点。方法：对2005年1月-2006年12月收集的2135例浅部真菌病临床标本进行直接镜检、培养及菌种鉴定。结果：镜检阳性率为66．5％，培养阳性率为55．7％。菌种分布主要为：红色毛癣菌872株（68．2％），犬小孢子菌50株（3．9％），须癣毛癣菌48株（3．8％），念珠菌及酵母样菌173株（13．5％），曲霉26株（2．0％），青霉11株（0．9％）。结论：①重庆地区浅部真菌感染中，皮肤癣菌仍占主导地位。男性皮肤癣菌的镜检及培养阳性率均高于女性，冬季癣菌的检出率低于春、夏、秋三季。②红色毛癣菌为优势致病菌，但其在15岁以下年龄组患者中的感染率明显低于其他年龄组。     

医院内深部真菌感染113例分析

1990年 1月至 1999年 8月，我们检查了经我科在院内各病房会诊后疑深部真菌感染患者的标本 369份，确诊为深部真菌感染 113例。现将检查结果分析如下。 一、资料和方法 1．病例来源及标本： 113例均来自我院各病房经我科会诊后疑深部真菌感染患者。标本为痰、咽拭子、舌苔、口腔白膜、血液、胸腹水、腹膜透析液、耳鼻眼部溃疡或增生物、尿、粪等。标本经连续 3次直接镜检找到菌丝或孢子， 3次培养亦为同一真菌，结合临床确定为深部真菌感染。 2．方法：采用沙堡液基、 37℃温箱孵育。标本一式 2管，培养 2周， 4周不生长为阴性。菌种鉴定…     

Comparison of fungal fluorescent staining and ITS rDNA PCR‐based sequencing with conventional methods for the diagnosis of onychomycosis

Background

The current gold standard for diagnosing onychomycosis is direct microscopic examination and culturing. Fungal culture is a time‐consuming procedure, while direct microscopy of potassium hydroxide (KOH) mounts suffers from low sensitivity. More rapid and sensitive methods for the diagnosis of onychomycosis are in high demand.

Objective

To establish an effective method for the diagnosis of onychomycosis by assessing the efficacies of fungal fluorescent staining and internal transcribed spacer (ITS) ribosomal DNA (rDNA) polymerase chain reaction (PCR)‐based sequencing.

Methods

A total of 204 clinical specimens from patients with suspected onychomycosis were analysed. The gold standard for a true positive sample was positive by KOH, culturing or both methods. All specimens were also tested by fungal fluorescent staining and ITS rDNA PCR‐based sequencing. We compared the detection, sensitivity and specificity for these two methods with conventional methods.

Results

In total, 126 (62%) and 102 (50%) were detected by fluorescent staining and PCR‐based sequencing, respectively. According to the conventional diagnostic standard, the sensitivity of fluorescent staining and PCR‐based sequencing was 97% and 78%, respectively, and specificities of 89% and 90%, respectively. Use of fluorescence enhanced the sensitivity of direct examination by 12% compared with KOH. PCR‐based sequencing increased the sensitivity by 6% compared with culturing.

Conclusions

Fluorescence microscopy has a higher sensitivity for the detection of fungi in nail specimens compared with KOH and can be used as a rapid screening tool. PCR‐based sequencing was faster and more sensitive compared with culture and when used in conjunction with fluorescence microscopy resulted in higher efficiency.     

Evaluation of PCR for the diagnosis of dermatophytes in nail specimens from patients with suspected onychomycosis

Background. Conventional methods for detecting fungi in nail specimens are either nonspecific (microscopy) or insensitive (culture). Recently, PCR has been used to improve sensitivity in detecting the causative fungi in nail specimens from patients with suspected onychomycosis. Aim. To compare the detection rates of PCR with those of microscopy (with potassium hydroxide; KOH) and culture for dermatophytes in nail specimens from patients with suspected onychomycosis. Methods. In total, 120 patients with clinically suspected onychomycosis were recruited, and using a topoisomerase II‐based PCR, we compared the detection rate of dermatophytes for the three methods. Results. KOH microscopy, culture and PCR respectively yielded positive rates of 35 (29.2%), 12 (10%) and 48 (40%), and negative rates of 85 (70.8%), 108 (90%) and 72 (60%). Two culture‐positive specimens were not detected by PCR, but PCR picked up 38 specimens missed by culture. Of the 35 specimens that were microscopy‐positive, 12 grew dermatophytes and 23 nondermatophytes. Conclusions. This study demonstrates that PCR has a higher positive and lower negative rate for detection of dermatophytes compared with KOH microscopy or culture. We suggest that PCR should be used as a complementary method for confirmation of clinically suspected dermatophytic onychomycosis.     

聚合酶链反应检测角化型手癣的病原菌DNA

目的为了研究ＰＣＲ技术应用检测浅部真菌病鳞屑中病原真菌的可能性。方法我们应用二次ＰＣＲ技术对２３例角化型手癣进行了检测。结果真菌直接镜检及培养阳性率之和为６９．６％,与二次ＰＣＲ阳性率相同。其中４例真菌直接镜检及培养均阴性者二次ＰＣＲ阳性,４例直接镜检阳性者二次ＰＣＲ阴性。结论二次ＰＣＲ可作为角化型手癣的辅助诊断指标之一。     

New reasons for histopathological nail‐clipping examination in the diagnosis of onychomycosis

Background Onychomycosis is a common problem. Today, the gold standard in the diagnosis of onychomycosis is the direct microscopy and fungal culture. But direct microscopy is regarded as having a low sensitivity and fungal culture takes long time. In addition, in cases of controlling the antifungal treatment course, the present gold standard is often not a reliable tool because of the inhibition of the fungal growth in the culture. Objective The purpose of this study was to compare the present gold standard with histological examination with periodic acid‐Schiff staining (PAS) of nail clippings in the diagnosis of onychomycosis in a large cohort. Materials and methods We prospectively evaluated 1146 nail samples from 851 patients with clinical signs of onychomycosis using direct microscopy and fungal culture in comparison with PAS‐stain. Results A total of 631 nail samples revealed a positive result in at least one test. The most sensitive single test for the diagnosis of onychomycosis was PAS with 82%, followed by culture (53%) and direct microscopy (48%). In 64 cases, in which a prediagnostic antimycotic treatment has been initiated, PAS showed to have by far the highest sensitivity (88%) in comparison with culture (33%) or direct microscopy (50%). Conclusions Periodic acid‐Schiff staining is the single method with the highest sensitivity in terms of detection of fungal elements (hyphae) in nail specimens. Especially in cases with prior antifungal treatment, histological analysis of PAS‐stained nail clippings should be considered as an appropriate diagnostic tool.     

Evaluation of a polymerase chain reaction-restriction fragment length polymorphism assay for dermatophyte and nondermatophyte identification in onychomycosis

Background  Dermatophytes are the main cause of onychomycoses, but various nondermatophyte filamentous fungi are often isolated from abnormal nails. The correct identification of the aetiological agent of nail infections is necessary in order to recommend appropriate treatment.
Objective  To evaluate a rapid polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay based on 28S rDNA for fungal identification in nails on a large number of samples in comparison with cultures.
Methods  Infectious fungi were analysed using PCR-RFLP in 410 nail samples in which fungal elements were observed in situ by direct mycological examination (positive samples). The results were compared with those previously obtained by culture of fungi on Sabouraud agar from the same nail samples.
Results  PCR-RFLP identification of fungi in nails allowed validation of the results obtained in culture when Trichophyton spp . grew from infected samples. In addition, nondermatophyte filamentous fungi could be identified with certainty as the infectious agents in onychomycosis, and discriminated from dermatophytes as well as from transient contaminants. The specificity of the culture results relative to PCR-RFLP appeared to be 81%, 71%, 52% and 63% when Fusarium spp., Scopulariopsis brevicaulis , Aspergillus spp. and Candida spp., respectively, grew on Sabouraud agar. It was also possible to identify the infectious agent when direct nail mycological examination showed fungal elements, but negative results were obtained from fungal culture.
Conclusions  Improved sensitivity for the detection of fungi in nails was obtained using the PCR-RFLP assay. Rapid and reliable molecular identification of the infectious fungus can be used routinely and presents several important advantages compared with culture in expediting the choice of appropriate antifungal therapy.     

溶甲涂片镜检结合组化染色对甲真菌病病原学诊断的意义

目的：探讨溶甲涂片镜检结合组化染色对甲真菌病病原学诊断的意义。方法：采用KOH直接镜检,真菌培养,溶甲涂片直接镜检,溶甲涂片PAS染色和凝集素组化染色。结果：129例KOH直接镜检,真菌培养和溶甲涂片镜检的阳性率分别为73．3％,56．6％,95．3％,32例溶甲涂片真菌7个属与12种凝集素的结合形式具有属间区别。结论：溶甲涂片镜检结合组化染色阳性率高并有助于鉴别真菌菌属。     

聚合酶链反应检测深部致病真菌的实验研究

目的 建立能用于临床实践的检测常见致病真菌的聚合酶链反应(PCR)方法.方法 设计了以热启动PCR为基础的实验方法,首先用真菌通用引物对标本进行单重PCR,若阳性,再用白念珠菌、烟曲霉和新生隐球菌的种特异性引物进行三重PCR来检测这3种常见致病真菌.结果 对9属55种78株常见深部真菌均扩增出260bp的DNA片段,而对细菌和人DNA均未扩增出目的 片段,具有高度特异性和敏感性,该方法操作简便且成本低.结论 以热启动PCR为基础的单重PCR和三重PCR方法可能成为临床上深部真菌感染理想的快速诊断工具.     

Evaluation of a newly-developed immunochromatography strip test for diagnosing dermatophytosis

Background Traditionally, dermatophytosis, a common disease affecting millions of people world‐wide, has been diagnosed by direct microscopy and fungal culture. The immunochromatography (ICG) strip test was recently developed. Methods We compared the performance of the ICG strip test for the detection of dermatophytes in samples from human skin and nails with direct microscopy. The 160 samples, consisting of 88 skin and 72 nail specimens, were subjected to direct microscopy study using a 20% KOH solution and to examination with the ICG strip test. Of 160 samples, 18 were examined by fungal culture using Sabouraud dextrose agar medium. Results We found that the overall sensitivity and specificity of the ICG test were 83.5% and 66.7%; they were 82.1% and 76.2% for the 88 skin and 85.4% and 58.3% for the 72 nail specimens, respectively. Conclusions Our findings indicate that the efficacy of the ICG test is comparable to direct microscopy for the detection of dermatophytes. Performance of the assay was easy, and results were available quickly. We suggest that it is an effective tool for dermatophytosis screening.     

液体培养法、PCR法和SAT法在解脲支原体检测中的应用比较

目的：比较液体培养法、聚合酶链式反应（PCR）和实时荧光核酸恒温扩增检测技术（SAT）在解脲支原体(Uu)检测中的诊断价值。方法：应用液体培养法、PCR法和SAT法对77例疑似男性泌尿生殖系统感染患者尿道和23例女性宫颈的分泌物进行解脲支原体的检测。结果：100例患者中阳性29例（29.00%）,其中男15例,感染率19.48%（15/77）,女14例,感染率60.87%（14/23）。SAT法的敏感性为100%高于液体培养法89.66%和PCR法86.21%,差异无统计学意义(P>0.05),SAT法的特异性为84.50%低于液体培养法的98.59%和PCR法的98.59%,差异有统计学意义(P<0.05)。结论：以上三种方法均可用来检测Uu,SAT敏感性强,特异性低,适用于检测病原微生物含量不高的标本。