多重PCR快速诊断甲真菌病

目的探讨多重PCR技术从临床标本中检测甲真菌病病原真菌的方法。方法采用蛋白酶K消化法和煮沸法处理临床标本，从中提取致病菌DNA，然后用真菌通用引物NS，皮肤癣菌特异性引物CHS1和酵母特异性引物ACT1同时进行PCR扩增检测，并与直接镜检和培养法作对比。结果共收集104例临床标本，PCR检测、直接镜检和培养的敏感度分别为93．3％、100％和64．4％，特异度分别为100％、86．4％和100％，阳性预测值分别为100％、84．9％和100％，阴性预测值分别为95．2％、100％和78．7％。PCR方法在24小时内即可完成从标本处理至结果判读的全过程。结论多重PCR检测具有较好的特异性和准确性，可初步将甲真菌病的三大类病原菌区分开，且比培养缩短了近2周的时间，为临床快速诊断和及时、合理地治疗甲真菌病提供参考。     

真菌病

20060113聚合酶链反应快速诊断浅部真菌病/李筱芳(中国医科院、协和医大皮研所),陈辉,崔凡…∥中华皮肤科杂志.-2005,38(12).-722~724将临床标本用蛋白酶K消化法和煮沸法处理,从中提取致病菌DNA,然后用引物ITSI进行PCR扩增检测,并与直接镜检和培养法作对比。结果显示PCR检测具有较好的特异性和准确性,可在24h内完成从标本处理至结果判读的全过程,比培养缩短近2周的时间,认为该方法可为临床快速诊断和及时治疗浅部真菌病提供参考。图1表2参7(李文彬)20060114多点接种真菌培养法在甲真菌病病原学诊断中的应用研究/黄怀球(中山大学附属第…     

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。     

浅部真菌感染临床诊断与真菌镜检及培养结果的差异性比较

目的:了解仅根据临床表现诊断浅部真菌病的准确率及临床诊断、真菌镜检和培养结果之间的符合情况.方法:随机选择2007年5-10月至本科门诊就诊,诊断为浅部真菌感染的患者,取皮损鳞屑做真菌镜检和培养,计算其临床诊断、真菌镜检和培养结果之间的符合率.结果:确诊患者中临床诊断和真菌镜检结果的符合率为75.00%,临床诊断和真菌培养结果的符合率为49.67%,真菌镜检和真菌培养结果的符合率为56.33%.另外,皮肤癣菌和念珠菌混合感染率为12.00%.结论:仅根据临床表现诊断浅部真菌病的准确率较低,真菌培养是浅部真菌感染诊断的金标准;皮肤癣菌和念珠菌的混合感染值得重视.     

甲真菌病组织病理学检查的研究

甲真菌病是甲病变最常见的原因,皮肤病中发病率为2%~13%,占浅部真菌感染的30%.长期以来甲真菌病的公认诊断标准是临床表现加至少一项真菌检查,即甲真菌直接镜检和(或)真菌培养阳性.从以往研究资料中发现甲真菌培养法的敏感性和特异性均较低,而目前尚缺乏可靠资料对直接镜检法的敏感性做出准确评价^[1].传统的组织病理学方法敏感性很高,但由于耗时较长,故不便于临床应用.因此,寻找出一种更快速、可靠的方法确诊甲真菌病是十分必要的.     

浅部真菌病2600例病原菌分析

目的了解昆明地区浅部致病真菌的分布情况。方法对本科2010年1月-2011年6月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 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,红色毛癣菌1088株(69.52%),马拉色菌216株(13.80%),须癣毛癣菌118株(7.54%)。镜检和(或)培养阳性的2600例浅部真菌病患者中,足癣803例(30.88%),甲真菌病424例(16.31%),股癣386例(14.85%),体癣364例(14.00%),花斑癣259例(9.96%),手癣194例(7.46%),马拉色菌毛囊炎83例(3.19%)和头癣46例(1.77%),同时患有手癣和足癣41例(1.58%)。结论镜检结合培养法的阳性率显著高于单一镜检或培养法,昆明地区浅部真菌的病种以足癣、甲真菌病、股癣较多见,浅部致病真菌以红色毛癣菌和马拉色菌为主。     

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     

浅部真菌病668例病原菌种类分析

目的分析我地区浅部真菌病致病菌及菌种构成情况。方法通过对近2年我院浅部真菌病患者临床送检标本进行直接镜检、培养及真菌鉴定,鉴定到种。结果 1 600份临床送检标本,668份直接涂片镜检阳性,阳性率41.75%,其中甲真菌病214例、体癣176例、足癣166例、手癣78例;将阳性标本进行真菌培养,共培养分离207株致病菌,培养阳性率30.98%,其中红色毛癣菌居首位,须癣毛癣菌第2位,犬小孢子菌居第3位。结论河南地区浅部真菌病主要为甲真菌病、体癣、足癣,致病菌种以红色毛癣菌为主。     

甲真菌病甲板真菌镜检方法的探讨

目的探索一种操作简便、结果可靠的甲真菌病检验方法。方法对341例拟诊甲真菌病的患者分别采用KOH直接镜检法和浸软法进行病损指、趾甲的真菌镜检。取病损指、趾甲末端标本少许放入标本小碗中,加入10%的KOH后加盖浸泡至病甲标本完全浸透软化呈蛋白胨状,取标本放在载玻片上并将标本压平、压薄呈云雾状或毛玻璃状,在普通光学显微镜下检查真菌成分。结果KOH直接镜检法和浸软法的真菌检出率分别为34.90%和100.00%。结论浸软法真菌镜检是一种适合临床的、检出率较高的甲真菌病诊断方法。     

大学生中皮肤浅部真菌病调查报告

1995届大学生共1731人,男1146人,女585人,逐个体检,临床诊断浅部真菌病者,于病损处取材直接镜检,阳性者做真菌培养。结果发现浅部真菌病113例,男92例,女21例。镜检阳性86例,阳性率76.1%。见表1。表1　113例浅部真菌病分类、镜检情况　例病　种发病数男女镜检阳性手癣　　　　3301足癣　　　　53322132股癣　　　　3939035股癣合并足癣1010010体癣　　　　2202花斑癣　　　6606总计　　　　113922186　　取80例镜检阳性标本做真菌培养(花斑癣未做)55例有真菌生长。结果见表2。表2　80例镜检阳性标本真菌培养结果　例病原菌手癣足癣股癣股癣合…     

Wood灯在皮肤浅表真菌感染诊断中应用价值评估

目的 评估Wood灯在皮肤常见浅表真菌感染诊断中的应用价值.方法 对129例根据临床病史及体征初步诊断为皮肤浅表真菌感染患者进行Wood灯和真菌实验室检查.结果 花斑糠疹、马拉色菌毛囊炎患者Wood灯检查的阳性率分别为84%、85.7%,同时阳性病例的真菌培养或镜检的阳性率达92.9%、87.5%,两者有高度的一致性.而临床诊断手足癣和体股癣者其荧光阳性率只有8.3%.真菌总检出率却高达85.4%(41/48),两者不具有一致性.结论 Wood灯在花斑糠疹、马拉色菌毛囊炎的检查上有较高的特异性和敏感性,临床诊断上有应用价值,而对手足癣和体股癣诊断则无意义.

Abstract：

Objective To estimate the performance of Wood's lamp examination in the diagnosis of superficial cutaneous fungal infections. Methods Totally, 129 patients, who were diagnosed with superficial cutaneous fungal infections according to clinical medical history and signs, were enrolled in this study. Wood's lamp examination of lesions was carried out. Cutaneous samples were obtained from the patients and subjected to microscopic examination and fungal culture. Results Wood's lamp examination was positive in 84% and 85.7% of patients with tinea versicolor and malassezia folliculitis, respectively; among these patients positive for Wood's lamp examination, 92.9% were positive for fungal culture, and 87.5% for microscopic examination. In patients clinically diagnosed with tinea manus and pedis, tinea corporis or tinea cruris, 8.3% were positive for Wood's lamp examination, while 85.4% were positive for fungal examination. There was a high consistency between Wood's lamp examination and fungal examination in patients with tinea versicolor and malassezia folliculitis, but not in those with tinea manus and pedis, tinea corporis or tinea cruris. Conclusions Wood's lamp examination shows a high specificity and sensitivity and is useful in the diagnosis of tinea versicolor and malassezia folliculitis, but seems unapplicable for the diagnosis of tinea manus and pedis, tinea corporis or tinea cruris.     

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。     

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.     

红色毛癣菌raubitschekii变种致Majocchi肉芽肿一例

目的 报道1例Majocchi肉芽肿,对其病原菌红色毛癣菌变种raubitschekii的形态学、生理学及分子学特征进行研究,并通过基因分型的方法分析其深在感染与浅表皮肤感染的关系.方法 进行临床和病理检查,真菌镜检和培养,尿素酶试验,内转录间隔区(ITS区)序列分析.对来源于该患者病变趾甲及组织的菌株及7株红色毛癣菌的rDNA非转录间隔区(NTS)串联重复亚单位1(TRS-1区)进行PCR.结果 48岁女性患者,背部、臀部、大腿出现红色丘疹、结节2个月.9个月前曾行肝移植术,甲癣病史3年,术后加重.经病理和真菌学检查,确诊为Majocchi肉芽肿.足趾甲和组织真菌培养菌落和显微镜下形态、尿素酶试验阳性,结合ITS区序列分析结果,证实致病菌为红色毛癣菌变种raubitschekii.TRS-1区扩增后显示,病变趾甲和组织来源的菌株基因型完全一致,与其余临床分离株有差异.结论 NTS区的TRS-1区显示基因多态性,病变趾甲和组织基因型完全一致,提示两者来源相同.     

红色毛癣菌变种raubitschekii变种致Majocchi肉芽肿一例

目的　报道1例Majocchi肉芽肿,对其病原菌红色毛癣菌变种raubitschekii的形态学、生理学及分子学特征进行研究,并通过基因分型的方法分析其深在感染与浅表皮肤感染的关系。方法 进行临床和病理检查,真菌镜检和培养,尿素酶试验,内转录间隔区（ITS区）序列分析。对来源于该患者病变趾甲及组织的菌株及7株红色毛癣菌的rDNA非转录间隔区（NTS）串联重复亚单位1（TRS-1区）进行PCR。 结果 48岁女性患者,背部、臀部、大腿出现红色丘疹、结节2个月。9个月前曾行肝移植术,甲癣病史3年,术后加重。经病理和真菌学检查,确诊为Majocchi肉芽肿。足趾甲和组织真菌培养菌落和显微镜下形态、尿素酶试验阳性,结合ITS区序列分析结果,证实致病菌为红色毛癣菌变种raubitschekii。TRS-1区扩增后显示,病变趾甲和组织来源的菌株基因型完全一致,与其余临床分离株有差异。结论 NTS区的TRS-1区显示基因多态性,病变趾甲和组织基因型完全一致,提示两者来源相同。     

共聚焦激光扫描显微镜在皮肤浅部真菌感染诊断中应用价值的评估

目的 探讨皮肤共聚焦激光扫描显微镜（CLSM）在常见浅部真菌感染诊断中的应用价值。 方法 根据临床病史及体征初步诊断为浅部真菌感染患者59例,选定3处典型皮损做CLSM检查,记录各项指标的扫描结果,再行真菌镜检。结果 CLSM检测的25例手足癣患者中,14例（56%）角质层内可见菌丝,真菌镜检均阳性;11例角质层内未发现菌丝,真菌镜检8例阳性;新皮损（＜ 3周） 共8例,CLSM阳性7例,真菌镜检均阳性;旧皮损（＞ 3周）共17例,CLSM阳性7例（41%）,真菌镜检阳性14例（82%）。在检测的24例股癣患者中,19例（79.17%）角质层内可见菌丝,真菌镜检均阳性;5例角质层内未发现菌丝,真菌镜检4例阳性;新皮损（＜ 3周） 共17例,CLSM阳性16例（94.12%）,真菌镜检均阳性;旧皮损（＞ 3周）共7例,CLSM阳性3例,真菌镜检阳性6例。在检测的10例花斑糠疹患者中,CLSM均未发现菌丝,而真菌镜检阳性8例。外用联苯苄唑乳膏2周后的10例手足癣、股癣患者,CLSM检查均未见菌丝,角质层完整,真菌镜检阴性。结论 CLSM在手足癣、股癣的新发皮损的检查上与真菌镜检具有较高的一致性,在临床诊断上有一定的应用价值。     

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.     

A rationale for systemic treatment in onychomycosis with negative results on fungal examination

Background. Fungal infection of the nail affects millions of people worldwide, and has an estimated prevalence of about 10% of the general population. Laboratory confirmation of fungal infection is currently accepted as a requirement before initiation of antifungal treatment in clinical practice. Aim. To examine the rationale for systemic treatment in cases of clinical onychomycosis with negative results on fungal examination (potassium hydroxide test and fungal culture). Methods. In total, 147 patients with suspected clinical toenail onychomycosis but with negative results on fungal examination underwent up to three consecutive fungal examinations of the affected nails. Patients who were negative after these examinations underwent a fourth set of investigations, including PCR. Results. Of the 147 cases initially thought to be negative, 138 (94%) were rated as positive after up to four consecutive sets of laboratory mycological investigations including PCR. Trichophyton rubrum was by far the commonest dermatophyte cultured from all samples. Conclusions. In the majority of cases of initially negative examinations, consecutive laboratory fungal tests will eventually produce positive results. These findings suggest that systemic antifungal treatment should be started in patients with suspected fungal infections, even if they have negative laboratory fungal examinations.     

江西省农村地区浅部真菌病现场调查及致病菌的研究

目的 了解江西省农村地区居民浅部真菌病构成情况及致病菌的种类。方法 2006年9月至2007年6月对江西省的11个地市、54个县区的66个乡（镇）、村居民以义诊的形式进行浅部真菌病现场调查，同时对513例具有典型临床表现且真菌镜检阳性的皮损进行真菌培养。结果 共调查浅部真菌病患者1284例，其中务农者947例，占73.75%。浅部真菌病包括足癣736例（57.32%）、手癣202例（15.73%）、甲真菌病72例（5.61%）、体癣47例（3.66%）、股癣29例（2.26%）、花斑癣23例（1.79%）、头癣1例、皮肤念珠菌病1例、手足癣135例（10.51%）、手癣合并指甲真菌病12例、足癣合并趾甲真菌病10例、手足癣合并指趾甲真菌病8例、体股癣3例、手癣合并体癣2例，以及手足癣合并体癣、足癣合并花斑癣、足癣合并体癣各1例。分离出致病真菌480株，其中红色毛癣菌301株、白念珠菌88株、须毛癣菌41株、其他菌50株。结论 浅部真菌病的致病菌以红色毛癣菌占优势，白念珠菌第二，各地市之间菌种分布构成差异无统计学意义。     

疑似深部真菌感染患者血中(1→3)β-D-葡聚糖的检测

目的 探讨疑似深部真菌感染患者血中(1→3)-β-D-葡聚糖的浓度与感染的关系。方法 采用日本生化学工业株式会社的(1→3)-β-D-葡聚糖测定G-testTE试剂盒检测了13例患者血浆中的(1→3)-β-D-葡聚糖的含量,以UV-2450紫外可见光分光光度仪检测波长545nm的吸光度值,根据特定检测浓度换算公式得出标本的(1→3)-β-D-葡聚糖含量。结果 13例被检测患者中9例经培养证实为深部真菌感染者血浆的β-D-葡聚糖含量皆较高,最高者可达352.94pg/mL(为真菌混合感染患者),平均可达203.47pg/mL;4例真菌培养阴性患者血浆β-D-葡聚糖水平,3例超过54.40pg/mL,1例为16.16pg/mL。所有标本采用UV-2450紫外可见光分光光度仪在波长545nm检测,皆可出现较明显的吸收峰。采用GtestTE试剂盒检测13例疑似深部真菌感染患者血浆的β-D-葡聚糖含量,分析诊断深部真菌感染的有效性,结果其敏感度为92.31%,特异度100%,阳性预测值100%,阴性预测值为98.36%。结论 GtestTE方法简便,反应快速,可用于真菌感染的早期诊断。