多重PCR诊断甲真菌病的实验研究

目的初步建立多重PCR诊断甲真菌病的方法。方法选择3对引物(真菌通用引物、皮肤癣菌特异性引物和酵母特异性引物)建立三重PCR体系,采用红色毛癣菌、白念珠菌和短帚霉摸索反应条件和验证体系的适用性;并用红色毛癣菌来测定反应的灵敏度。结果筛选得到的真菌通用引物NS、皮肤癣菌特异性引物CHS1和酵母特异性引物ACT1有较好的通用性和特异性;在适宜的反应条件下,无论对单模板,还是多模板,该反应体系均能扩增出目的片段,未见明显非特异片段的干扰;该反应体系的检测灵敏度为102cfu/ml。结论多重PCR技术是一种快速、敏感和特异诊断甲真菌病的方法。     

临床疑诊甲真菌病1036例真菌学分析

目的　了解近 5年本院甲真菌病病原菌的种类和构成 ,观察流行病学特点。方法　对近 5年临床疑诊的10 3 6例甲真菌病患者的真菌学实验室检查情况进行系统分析、总结。结果　共培养出真菌 63 1株 ,其中酵母菌占49.60 % ,以克柔念珠菌、红酵母、近平滑念珠菌为主 ;皮肤癣菌占 2 1.71% ,主要菌种为须癣毛癣菌和红色毛癣菌 ;其他丝状真菌占 19.81% ,主要菌种为曲霉和青霉 ;污染真菌占 8.87%。结论　本院近 5年甲真菌病患者病原菌依次为酵母菌、皮肤癣菌、其他丝状真菌 ,排前 5位的真菌分别是须癣毛癣菌 (11.2 5 % ) ,克柔念珠菌 (10 .14 % ) ,红酵母 (9.98% ) ,红色毛癣菌 (9.5 1% ) ,曲霉 (8.87% )。     

皮肤癣菌的分子生物学研究进展

皮肤癣菌是浅部真菌病的致病菌 ,对其分子生物学研究主要集中于基因结构的分析、基因诊断、基因分类等方面。目前 ,已在红色毛癣菌等某些癣菌的线粒体DNA上发现了重要的呼吸链酶 ,如细胞色素氧化酶、NADH脱氢酶的结构基因 ,以及诸多氨基酸的tRNA基因簇 ;建立了用真菌特异性通用引物或癣菌特异性引物聚合酶链反应诊断皮肤癣菌病的方法 ;应用限制性酶切片段多态性研究、随机扩增多态DNA分析、聚合酶链反应等方法进行皮肤癣菌的基因分类 ,分析癣菌的种间差异 ,对一些重要的皮肤癣菌 (如红色毛癣菌、须癣毛癣菌 )还进行了种内的分型研究。     

杭州地区973例甲真菌病分析

目的了解杭州地区甲真菌病病原菌的种类和构成情况,获取流行病学资料.方法我们于2004年6月-11月对直接镜检阳性的973例甲真菌病患者进行了真菌分离培养及流行病学调查.结果共培养出722株病原菌,皮肤癣菌、酵母、非皮肤癣菌霉菌(NDM)所占比例分别为43.63%、38.37%、18.01%.皮肤癣菌中红色毛癣菌占89.52%(282/315),酵母中近平滑念珠菌居首,占21.66%,非皮肤癣菌霉菌中以曲霉和青霉为主.结论杭州地区的甲真菌病病原菌为皮肤癣菌、酵母菌和非皮肤癣菌霉菌,其中以红色毛癣菌和近平滑念珠菌为主.要注意非皮肤癣菌的污染或寄生问题.     

甲真菌病400例致病真菌分析

目的:确定上海地区甲真菌病的致病菌种。方法:对本院皮肤科门诊就诊的直接镜检阳性的400例甲真菌病患者的甲标本做真菌分离培养和分析。结果:分离出致病真菌233株,其中皮肤癣菌120株(红色毛癣菌104株,须癣毛癣菌10株,犬小孢子菌3株,絮状表皮癣菌3株),酵母菌68株,非皮肤癣菌11株(曲霉6株,青霉5株),其余为丝状真菌。结论:上海地区甲真菌病的致病真菌以皮肤癣菌为主,酵母菌中非白念珠菌占有一定比例。     

巢式PCR诊断甲真菌病中红色毛癣菌和须癣毛癣菌

目的：评价巢式PCR诊断甲真菌病中红色毛癣菌和须癣毛癣菌的敏感性和特异性。方法：液氮冷冻甲标本,微量法提取DNA,应用特异性引物,巢式PCR方法扩增DNA。结果：36例直接镜检和培养均为阳性的甲标本,培养显示24例为红色毛癣菌,12例为须癣毛癣菌。巢式PCR中FirstPCR34例阳性,34例标本均产生了650bp目的片段;NestPCR32例阳性,32例中有22例标本产生了137bp的片段,为红色毛癣菌;10例标本产生了102bp的片段,为须癣毛癣菌。PCR敏感性为88．9％,特异性为100％。结论：巢式PCR是一种快速、特异和敏感的诊断甲真菌病中红色毛癣菌和须癣毛癣菌的方法。     

皮肤癣菌的分子生物学研究进展

皮肤癣菌是浅部真菌病的致病菌，对其分子生物学研究主要集中于基因结构的分析、基因诊断、基因分类等方面。目前，已在红色毛癣菌等某些癣菌的线粒体ＤＮＡ上发现了重要的呼吸链酶，如细胞色素氧化酶、ＮＡＤＨ脱氢酶的结构基因，以及诸多氨基酸的ｔＲＮＡ基因簇；建立了用真菌特异性通用引物或癣菌特异性引物聚合酶链反应诊断皮肤癣菌病的方法；应用限制性酶切片段多态性研究、随机扩增多态ＤＮＡ分析、聚合酶链反应等方法进行皮肤     

宁波地区甲真菌病及其相关因素的调查分析

于2002年12月1日-2004年12月1日,对819例甲真菌病病例进行真菌分离培养及流行病学调查。结果：从819例患者1032个靶甲真菌培养分离出真菌615株,皮肤癣菌、酵母菌和霉菌三者分别占51.9%、41.6%和6.5%,且有部分患者存在混合感染情况。皮肤癣菌中红色毛癣菌居首位,占40.2%,酵母中白念珠菌居首,占14.1%,非皮肤癣菌霉菌中以曲霉为主。甲真菌病致病菌中酵母及霉菌日益增多,故治疗应个体化,宜选用广谱抗真菌药物治疗。     

深圳地区甲真菌病病原菌流行病学的多中心研究

目的 了解深圳地区最近4年甲真菌病病原菌的种类和构成,发现流行病学特点。方法 以多中心研究方式选取深圳地区6家不同区域的主要医院,选取临床表现典型、真菌镜检阳性的1162例甲真菌病患者进行真菌分离培养。结果共培养出致病真菌553株。皮肤癣菌占68.53％,以红色毛癣菌为主占52.26％,其次是须癣毛癣菌占11.21％;酵母菌占25.68％,其中以白念珠菌最多占9.22％;霉菌占5.79％,主要是曲霉属和青霉属;混合感染占4.30％。结论深圳地区最近4年甲真菌病病原菌为皮肤癣菌、酵母菌和霉菌。红色毛癣菌和白念珠菌所占比例最高。     

内蒙古医科大学附属医院47年浅部真菌病及病原菌分析

目的探讨内蒙古医科大学附属医院近50年浅部真菌病分布以及致病菌种构成的动态变化。方法对该院皮肤性病科47年真菌培养阳性的15 648例患者临床资料、真菌培养鉴定结果进行分析。结果浅部真菌病位列前3的是足癣、手癣和甲真菌病;甲真菌病病原菌以红色毛癣菌为主,白念珠菌和其他酵母菌随着年代的增加逐渐增多;体癣与头癣病原菌以犬小孢子菌为主,并随着年代逐渐增多;所有培养阳性菌株中,列前3位的是红色毛癣菌、须癣毛癣菌复合体和犬小孢子菌。结论从1970—2016年浅部真菌病的病原菌变化不大,皮肤癣菌仍占主要优势,以红色毛癣菌、须癣毛癣菌复合体为主。     

四种分枝杆菌快速检测方法的研究

目的 建立敏感、特异的快速检测4种分枝杆菌的方法。方法 用特异性引物对结核分枝杆菌、鸟分枝杆菌、胞内分枝杆菌和堪萨斯分枝杆菌菌悬液DNA进行PCR扩增,验证其敏感性和特异性。将上述4种分枝杆菌的特异性引物同时放入PCR扩增反应体系中,以此反应体系分别对该4种分枝杆菌菌悬液DNA、及其两两组合或特定的三重组合进行扩增,同时验证其敏感性。结果 4种分枝杆菌的特异性引物分别放入各自的PCR扩增反应体系中,可分别特异性地扩增出该4种分枝杆菌对应的DNA片段,敏感性达1×10¹～1×10²个菌细胞/mL。4种分枝杆菌的特异性引物同时放入同一PCR扩增反应体系中可分别特异性地扩增出对应的4种分枝杆菌单菌及其两两组合或三种分枝杆菌组合的DNA片段,敏感性达1×10²～1×10³个菌细胞/mL;4种分枝杆菌的特异性引物对其他分枝杆菌进行扩增,结果均为阴性。结论 多重PCR方法能敏感、特异地快速检测4种分枝杆菌。     

Detection and differentiation of causative fungi of onychomycosis using PCR amplification and restriction enzyme analysis

Background Onychomycosis, a fungal nail infection, has become one of the most important dermatophytoses. Unfortunately, a predictably successful diagnostic approach to onychomycosis does not yet exist. Objective The purpose of this study was to develop a deoxyribonucleic acid (DNA)-based diagnostic method to improve the sensitivity and specificity of the detection and differentiation of the pathogenic fungi of onychomycosis. Methods We attempted to detect fungi in the nail using polymerase chain reaction (PCR) primer systems that were designed in conserved sequences of the small ribosomal subunit 18S-rRNA genes shared by most fungi, and differentiated between species by restriction enzyme analysis of the amplified product. Results Fragments of the gene coding for 18S-rRNA were amplified successfully from medically important fungi species, but not from normal nails. Restriction fragment length polymorphism patterns using HaeIII endonuclease were sufficiently different to allow the recognition of individual species. Conclusions The PCR–restriction enzyme analysis method appears to be a more sensitive detection and identification technique for onychomycosis than conventional methods, and has considerable diagnostic value.     

Molecular detection of dermatophytes and nondermatophytes in onychomycosis by nested polymerase chain reaction based on 28S ribosomal RNA gene sequences

Background  Onychomycosis is often caused by dermatophytes, but the role of nondermatophytes is underestimated due to the difficulty of identifying them by conventional direct microscopy and culture.
Objectives  This study aims to detect nondermatophytes, as well as dermatophytes, in the nail samples of patients with onychomycosis using a polymerase chain reaction (PCR)-based culture-independent method.
Materials and methods  The nested PCR assay targeting the sequence of the 28S ribosomal RNA gene was used to amplify fungal DNAs from 50 microscopy-positive nail specimens. Newly designed primer sets for dermatophyte universal, Trichophyton rubrum , T. mentagrophytes , Aspergillus spp., Scopulariopsis brevicaulis , Fusarium solani , F. oxysporum , F. verticillioides , Candida albicans and C. tropicalis were used after confirmation of their specificity.
Results  Forty-seven cases (94%) were positive for fungal DNA, among which dermatophytes were detected in 39 cases (83·0%): T. rubrum in 35 cases (74·5%) and T. mentagrophytes in eight cases (17·0%). Surprisingly, nondermatophytes were detected in 18 cases (38·3%), both dermatophytes and nondermatophytes in 10 cases (21·3%) and nondermatophytes alone in eight cases (17·0%). Aspergillus spp. alone was observed in five cases (10·6%).
Conclusions  This study indicates that most of the affected nail plates of patients with onychomycosis were positive for specific fungal DNAs, and suggests that nondermatophytes detected at high rates may be involved in the pathogenesis of onychomycosis.     

Genetic identification and detection of human pathogenic Rhizopus species, a major mucormycosis agent, by multiplex PCR based on internal transcribed spacer region of rRNA gene

BACKGROUND: Mucormycosis is an invasive opportunistic infection caused by fungi belonging to the order Mucorales. Due to the lack of laboratory tests, the diagnosis of mucormycosis is notoriously difficult. Added with its rapid progression as well as the debilitated state of the patients who contract the disease, mortality is extremely high. OBJECTIVE: The goal of this study was to genetically identify human pathogenic Rhizopus species, a major mucormycosis agent, by the internal transcribed spacer (ITS) region of rRNA gene. METHODS: Primers were designed to identify five Rhizopus species known to cause human disease by multiplex PCR. PCR was done not only with test strains and clinical isolates, but also with clinical samples from cutaneous mucormycosis patients. Sporangiospore morphology was observed by scanning electron microscopy to confirm the correlation of phenotypic and genotypic features. RESULTS: Multiplex PCR identified five Rhizopus species including Rhizopus oryzae, where R. azygosporus could only be distinguished from R. microsporus by certain polymorphisms that were present in its sequence. When this multiplex PCR was applied to clinical samples from three mucormycosis patients (paraffin sections from all and sera from one patient), Rhizopus DNA corresponding to the isolated pathogens were specifically detected. CONCLUSION: While fungal DNA detection from clinical samples is a rigorously studied area, this is the first report to genetically identify and detect Rhizopus species from human mucormycosis specimens. This may expand the possibility of this multiplex PCR system not only to identify isolated fungi, but also as a screening method for visceral mucormycosis.     

Direct detection and differentiation of causative fungi of onychomycosis by multiplex polymerase chain reaction-based assay

A rapid and reliable triplex PCR procedure was developed to detect pathogenic fungi directly from specimens of onychomycosis. One hundred and four patients were included in this study. Of them, forty-five (43.3%) were finally diagnosed with onychomycosis according to the diagnostic criteria. The sensitivity of PCR, microscopy and culture were 93.3%, 100% and 64.4%, respectively; the specificities were 100%, 86.4% and 100%, respectively; the positive predictive values were 100%, 84.9% and 100%, respectively; the negative predictive values were 95.2%, 100% and 78.7%, respectively. This molecular diagnostic process could distinguish the 3 groups of pathogens in onychomycosis (dermatophyte, yeast and mold) and could be completed within 8?h. This multiplex PCR assay could used in laboratories with no mycological specialization for rapid etiologic diagnosis and treatment selection, especially in suspected fungus cases if they can not be detected by conventional methods or if a rapid diagnosis of onychomycosis is needed.     

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.     

多点平皿培养法和常规试管培养法分离206例甲真菌病病原菌比较

目的比较多点平皿培养法和常规试管培养法在甲真菌病病原菌分离中的差异。方法同时采用多点平皿培养法和常规试管培养法对206例甲真菌病患者的253份靶甲标本进行病原菌分离及鉴定。结果多点平皿培养法和常规试管培养法甲真菌病病原菌的阳性率分别为72.73%和52.96%(P0.05),污染率分别为8.70%和4.74%(P0.01)。多点平皿培养法和常规试管培养法致病菌分离鉴定结果显示,皮肤癣菌分别为92.00%和93.15%、酵母菌分别为6.50%和6.85%、霉菌分别为1.50%和0%。多点平皿培养法和常规试管培养法分别检出混合感染16例和12例。结论多点平皿培养法分离甲真菌病病原菌优于常规试管培养法,该方法可提高甲真菌病病原菌的检出率。     

PCR法和传统方法对体液标本中致病真菌的检测比较

目的评价PCR方法诊断深部真菌感染的敏感性和特异性。方法应用真菌通用引物ITS4和ITS86对83例临床体液标本中的致病真菌进行PCR法快速检定,并与传统方法检验的结果进行比较。结果直接PCR扩增的阳性率与传统方法差异无统计学意义。结论采用ITS通用引物PCR法可准确、特异、快速、敏感地检测深部致病真菌。     

A nail drilling method suitable for the diagnosis of onychomycosis

We describe a nail drilling method suitable for the diagnosis of onychomycosis. Thirty-three patients with onychomycosis in which the big toenail had a white band or spike were enrolled in this study. We drilled a hole about 3-mm-wide in the most proximal part of the white band or spike using a ball-shaped metal file and then, through the hole, sampled the underlying nail material softened by fungi after removing the superficial hard nail plate. Fungi in 32 (97.0%) of the nail samples were detected by direct KOH examination. When incubated on Sabouraud's dextrose agar slant with chloramphenicol and cycloheximide, fungal cultures were obtained from 27 (81.8%) of the 33 nail samples. Fourteen of the fungal isolates were identified as T. rubrum, 11 as T. mentagrophytes, and 2 as Acremonium sp. The nail drilling method is suitable for diagnosing onychomycosis with a white band or spike, because it gives a high isolation rate and leaves the patients' nail relatively more intact compared with other methods.