老年患者白假丝酵母菌基因分型与药敏试验分析

目的了解老年慢性阻塞性肺炎（COPD）患者白假丝酵母菌基因分型及药敏结果差异。方法对320株白假丝酵母菌进行25SrDNA基因分型,用ATB Fungus3进行药敏实验。结果320株白假丝酵母菌分成了A、B、C3型。A型对5-氟胞嘧啶耐药率高于B型和C型;C型对两性霉素全都敏感,敏感率高于A型和B型;C型对氟康唑、伊曲康唑的耐药率高于A型和B型;B型对伏立康唑耐药率高于A型和C型。结论白假丝酵母菌PCR分型法有较强的特异性,且快速简便;白假丝酵母菌基因型别对不同药物的敏感率有影响。     

REP-PCR技术在光滑假丝酵母菌基因分型中的应用价值

目的评估重复序列PCR(REP-PCR)技术在光滑假丝酵母菌基因分型中的作用。方法收集深部真菌感染患者体内分离的光滑假丝酵母菌34株,以API 20C AUX酵母菌鉴定板条鉴定菌种及生化表型。采用REP-PCR对34株光滑假丝酵母菌进行基因分型:提取真菌基因组DNA,以Care-2重复元件设计引物,PCR扩增产物电泳后运用NTSYS软件进行聚类分析,聚类树状图相似系数(SI)≥97%且无明显条带差异定为同一基因型,SI≥97%且仅相差一条条带定为亚型。比较REP-PCR分型与多位点序列分型(MLST)的辨别力指数(DP),分析具有不同生化表型光滑假丝酵母菌的REP-PCR基因分型情况。结果 REP-PCR基因分型结果显示:34株光滑假丝酵母菌分为17个基因型,其中A型8株,B型6株,C、D型各3株,E型2株,F～Q型各1株,未发现亚型。REP-PCR和MLST基因分型的DP(95%CI)分别为0.911(0.770～0.980)和0.369(0.220～0.560),两者比较差异有统计学意义(χ2=21.68,P0.01)。34株光滑假丝酵母菌有两种生化表型,生化表型代码分别为2000040(32株)和6000040(2株),生化表型代码为60000402的2株经REP-PCR分型结果分别为D型和K型(SI=0.43)。结论 REP-PCR对光滑假丝酵母菌基因分型的辨别力较强且操作简便,适用于临床实验室对光滑假丝酵母菌的流行病学研究。     

白假丝酵母菌感染及分型方法的研究进展

白假丝酵母菌，是人体正常菌群之一，在口腔、皮肤、肠道、阴道、肛门等中均可分离到，同时是引起人体真菌感染的主要条件致病菌。随着医学科学的发展，各种新药物、新技术如广谱抗生素、激素、各种导管插管技术等的广泛使用，导致深部真菌病的发病率愈来愈高，以白假丝酵母菌感染为主。     

白假丝酵母菌25SrDNA基因分型与粘附力的相关性

目的 调查白假丝酵母菌25SrDNA基因型分布,并分析白假丝酵母菌各基因型与粘附力之间的相关性.方法 收集76株白假丝酵母菌,转沙保罗氏培养液纯培养24 h,裂解法提取基因组DNA,用特异性引物扩增白假丝酵母菌25SrDNA基因,根据PCR产物电泳图谱进行基因分型;分别测定各基因型粘附力.结果 PCR产物电泳结果显示76例白假丝酵母菌可分为A、B、C 3种基因型(其中A型48例,B型8例,C 型20例).测序结果显示,B型和C型产物比A型产物多出1个379 bp的序列(Ⅰ类内含子).根据有无内含子将菌株分为无内含子组(A型)和有内含子组(B型+C型),无内含子组粘附力明显强于有内含子组,两组比较差异有统计学意义(P<0.05).结论 白假丝酵母菌25SrDNA基因型与粘附力之间可能存在相关性,内含子的插入可能影响白假丝酵母菌的粘附力.     

基因分型在假丝酵母菌研究中的应用

近年来临床上假丝酵母菌属感染的发生率逐年上升,尤其是白假丝酵母菌.基因分型技术因便捷、分辨率高和重复性强被广泛应用,成为假丝酵母菌有效的流行病学研究工具.目前基因分型方法主要包括多位点酶电泳、DNA指纹图谱技术(如限制性片段长度多态性技术、随机引物扩增多态性DNA)、脉冲电场凝胶电泳以及单链构象多态性分析、微卫星标记技术和多位点序列分析等新兴技术.文章就目前主要基因分型方法及其在假丝酵母菌研究中的应用进行综述.     

基因分型在假丝酵母菌研究中的应用

近年来临床上假丝酵母菌属感染的发生率逐年上升,尤其是白假丝酵母菌.基因分型技术因便捷、分辨率高和重复性强被广泛应用,成为假丝酵母菌有效的流行病学研究工具.目前基因分型方法主要包括多位点酶电泳、DNA指纹图谱技术(如限制性片段长度多态性技术、随机引物扩增多态性DNA)、脉冲电场凝胶电泳以及单链构象多态性分析、微卫星标记技术和多位点序列分析等新兴技术.文章就目前主要基因分型方法及其在假丝酵母菌研究中的应用进行综述.     

白假丝酵母菌检测技术的发展及评价

<正>白假丝酵母菌(candida albicans)俗称白色念珠菌,在一定条件下可侵犯皮肤、黏膜、内脏和血液,引起继发性感染,表现为急性、亚急性或慢性炎症。近年来,医学诊断和治疗技术的迅猛发展,使得各种危重症患者的病死率有所下降,与此同时各种新医疗技术越来越多地被应用,以及免疫缺陷患者增多,人口老龄化日益加剧,条件致病性真菌引发的深部真菌性疾病发生率愈来愈高,严重威胁着患者的身体健康和生命安全,受到医学界的高度重视[1]。对人类致病的深部真     

白假丝酵母菌最优化RNA提取策略的探索

目的 探索白假丝酵母菌总RNA简单、快捷、高效的提取方法。方法 选取昆明医科大学第一附属医院妇产科门诊2011年7月-2013年3月有临床症状且阴道分泌物镜检阳性的外阴阴道假丝酵母菌病（VVC）患者的白假丝酵母菌40株,选用不同时间与温度组合下的4种溶壁酶（Lyticase）破壁方案（A方案：37 ℃、12 h,B方案：25 ℃、12 h,C方案：37 ℃、24 h,D方案：25 ℃、24 h）进行RNA提取,将所得RNA进行荧光定量PCR（qPCR）反应验证RNA的浓度及纯度,并进行管家基因18S rRNA的检测。结果 D方案提取获得的RNA浓度高于A、B、C方案（P＜0.05）|B方案提取获得的RNA浓度高于A、C方案（P＜0.05）|而A、C两方案提取获得的RNA浓度比较,差异无统计学意义（P＞0.05）。B、D方案获得的RNA纯度高于A、C方案（P＜0.05）|但A、C方案获得的RNA纯度比较,差异无统计学意义（P＞0.05）|B方案提取获得的RNA纯度高于D方案（P＜0.05）。A、B、C、D方案提取的RNA扩增产物Ct转化值比较,差异有统计学意义（P＜0.05）|其中B、D方案qPCR扩增产物Ct转化值高于A、C方案（P＜0.05）|但B、D方案及A、C方案之间的qPCR扩增产物Ct转化值比较,差异无统计学意义（P＞0.05）。管家基因18S rRNA的引物扩增出的产物片段大小为150 bp。结论 以Lyticase破壁提取白假丝酵母菌具有简单易行及RNA产量高、纯度好的优点,值得临床推广应用。     

RAPD技术及其在假丝酵母菌研究中的应用

随着医学科学的发展及人们对深部真菌感染的重视,越来越多的新技术用于临床真菌的检测和鉴定,随机扩增DNA多态性（RAPD）是在PCR基础上发展起来的一种新的基因分型方法.本文综述了假丝酵母菌RAPD分析技术应用的研究进展及前景.     

阴道白假丝酵母菌基因型与体外药物敏感性研究

目的 研究不同基因型阴道假丝酵母菌对克霉唑、咪康唑和制霉菌素的体外药物敏感性.方法 以特异引物PCR法(INT-PCR)对151株致病白假丝酵母菌进行基因分型,参考美国临床和实验室标准协会(CLSI)抗真菌药敏实验标准(M27-A方案)以微量稀释法检测不同基因型菌株的药物敏感性.结果 根据扩增条带中是否存在450 bp和840 bp片段可将白假丝酵母菌分为A、B、C 3种基因型;克霉唑和咪康唑对A基因型菌株的最小抑菌浓度(MIC)值明显高于C基因型菌株,差异有统计学意义(P＜0.05);制霉菌素对不同基因型菌株的MIC值间差异无统计学意义(P＞0.05).结论 不同基因型白假丝酵母菌株对抗真菌药物的敏感性有明显差异,这种差异可能是临床上假丝酵母菌性外阴阴道炎治疗效果存在个体差异的原因之一.     

Multilocus sequence typing indicates diverse origins of invasive Candida tropicalis isolates in China

Background According to data from the China Hospital Invasive Fungal Surveillance Net (CHIF-NET) 2010, Candida tropicalis (C. tropicalis) is the third most common pathogen causing invasive candidiasis. Moreover, the majority of fluconazole-resistant C. tropicalis isolates were from a single hospital. Therefore, a molecular epidemiological survey is necessary to investigate the genetic relatedness of C. tropicalis isolates in China.

Methods In this study, 48 C. tropicalis isolates causing invasive fungal infections from four tertiary hospitals in China were studied. All the isolates were identified by sequencing the internal transcribed spacer region. Antifungal susceptibility to triazoles, amphotericin B, and caspofungin was determined by the Clinical and Laboratory Standards Institute standard broth microdilution method. Multilocus sequence typing (MLST) was performed, and phylogenetic analysis was further performed by the eBURST and maximum parsimony (MP) methods to characterize the genetic relatedness of isolates.

Results MLST discriminated 40 diploid sequence types (DSTs) among 48 isolates, including 36 novel DSTs, and the XYR1 gene showed the highest discriminatory power. The DSTs obtained from this study were compared with those of previously reported C. tropicalis isolates, and there was poor type alignment with regional strains. Nine groups and 11 singletons were identified by eBURST, whereas two groups and 10 subgroups were clustered by MP analysis. Generally, there were no obvious correlations between clonal clusters generated and the specimen source or hospital origin. Seven fluconazole-resistant isolates were confirmed and assigned to three distinguishable branches.

Conclusions The results suggested diverse origins of invasive C. tropicalis isolates in China. Although most invasive C. tropicalis strains in the mainland of China were clustered with previously characterized Asian isolates, major C. tropicalis clusters identified in this study were genetically distinct from those of other geographic regions.

     

Optimization of Bartonella henselae multilocus sequence typing scheme using single-nucleotide polymorphism analysis of SOLiD sequence data

Background  Multi-locus sequence typing (MLST) is widely used to explore the population structure of numerous bacterial pathogens. However, for genotypically-restricted pathogens, the sensitivity of MLST is limited by a paucity of variation within selected loci. For Bartonella henselae (B. henselae), although the MLST scheme currently used has been proven useful in defining the overall population structure of the species, its reliability for the accurate delineation of closely-related sequence types, between which allelic variation is usually limited to, at most, one or two nucleotide polymorphisms. Exploitation of high-throughput sequencing data allows a more informed selection of MLST loci and thus, potentially, a means of enhancing the sensitivity of the schemes they comprise.

Methods  We carried out SOLiD resequencing on 12 representative B. henselae isolates and explored these data using single nucleotide polymorphism (SNP) analysis. We determined the number and distribution of SNPs in the genes targeted by the established MLST scheme and modified the position of loci within these genes to capture as much genetic variation as possible.

Results  Using genome-wide SNP data, we found the distribution of SNPs within each open reading frame (ORF) of MLST loci, which were not represented by the established B. henselae MLST scheme. We then modified the position of loci in the MLST scheme to better reflect the polymorphism in the ORF as a whole. The use of amended loci in this scheme allowed previously indistinguishable ST1 strains to be differentiated. However, the diversity of B. henselae was still rare in China.

Conclusions  Our study demonstrates the use of SNP analysis to facilitate the selection of MLST loci to augment the currently-described scheme for B. henselae. And the diversity among B. henselae strains in China is markedly less than that observed in B. henselae populations elsewhere in the world.     

Serological and molecular capsular typing, antibiotic susceptibility and multilocus sequence typing of Streptococcus pneumoniae isolates from invasive and non-invasive infections

Background Streptococcus pneumoniae (S. pneumoniae) is a major causative agent of severe infections, including sepsis, pneumonia, meningitis, and otitis media, and has become a major public health concern. We report the pneumococcal serotype and sequence type (ST) distribution, and antimicrobial resistance of 39 S. pneumoniae strains from seven hospitals in China.

Methods Blood/cerebrospinal fluid (CSF) and sputum isolates from patients were analyzed to determine S. pneumoniae serotypes by polymerase chain reaction (PCR) and the Neufeld Quellung reaction, the multilocus sequence types (MLST) by PCR and sequencing, and susceptibility to antimicrobial agents by the VITEK Gram Positive Susceptibility Card.

Results A total of 39 isolates were collected including 21 blood/CSF and 18 sputum isolates. Conventional serotyping by the Quellung reaction required 749 reactions. In contrast, PCR based typing needed only 106 PCR reactions. The most frequent serotypes from the blood/CSF isolates were 14 (38.1%), 19A (14.3%), 23F (9.5%), and 18C (9.5%). In the sputum isolates the most frequent serotypes were 19F (33.3%), 23F (16.7%), 19A (11.1%), and 3 (11.1%). The incidence of penicillin resistance in the blood/CSF and sputum isolates was 66.7% and 55.6%, respectively. Statistical analysis showed that patients ≤5 years old had a higher resistance to penicillin when they compared with the patients ≥65 years old (P=0.011). Serotypes 14, 19A and 19F were significantly associated with penicillin resistance (P <0.001). ST320, ST271, and ST876 isolates showed high resistant rates to several antibiotics including penicillin (P=0.006). All of the isolates of serotype 19A were resistant to both penicillin and erythromycin, and they were all multi-drug resistant (MDR) isolates.

Conclusions The specificity and sensitivity of multiplex-PCR are good, and this method represents a substantial savings of time and money, and can be widely used in the laboratory and clinical practice. Data from this research showed an extremely high prevalence of penicillin resistance and an increasing prevalence of multi-drug resistant (MDR) rate in S. pneumoniae. A distinctive emergence of serotype 19A was observed which was also associated with the increasing prevalence of antimicrobial resistance. Therefore, nationwide surveillance of pneumococcal resistance and serotypes is strongly warranted.

     

Analysis of genital Candida albicans infection by rapid microsatellite markers genotyping

Background Candida albicans （C. albicans） infection, often occurring in genital candidiasis, has increased dramatically recently. Developing an efficient C. albicans typing method may contribute to understanding its epidemiological characteristics and guiding efficient treatment. We used rapid microsatellite genotyping assay for interstrain differentiation of C. albicans isolates and explored some characteristics of its spread. Methods DNA was extracted from C. albicans isolates from gentalia, recta and mouths of 39 female cases and 27 male cases of genital candidiasis. Three fluorescent primers for the microsatellite markers in conserved genes （CDC3, EF3 and HIS3） of Co albicans were used to amplify the isolates DNA by PCR. Fluorescent signals were read with an automatic sequencer and analyzed with GeneScan software. Results Analysis of the three microsatellites markers showed 18 gene allelic associations in genital C. albicans infected patients： 10 allelic associations in female and 11 allelic associations in male, of which 3 allelic associations shared by both genders covered 71% of infections. The most dominant allele association of pathogenic strains for both genders was 116：124, 122：131,160：200 that covered about 50% of infection. Gentalia and recta shared the same strains in 80% of female patients, but in only 3.8% of male patients. There were 2.7% female patients, but no males, with same strain in both gentalia and mouths. Five of seven genital Co albicans infected couples had the same allelic associations of which 4 were the dominant pathogenic C. albicans susceptible for both genders. Conclusions The predominant allelic association of the pathogenic strain in genital C. albicans infection is 116：124, 122：131, 160：200. Vaginal pathogenic strains are probably maintained from the rectal reservoir. Pathogenic strains of male patients are probably from frequent sexual intercourse. The aggressiveness of some strains varies with gender.     

白念珠菌CDR1基因上游调控序列与氟康唑耐药的关系初探

目的探讨白念珠菌耐药基因CDR1基因上游调控序列对氟康唑耐药的影响。方法分别抽提氟康唑耐药／敏感配对菌株DNA,通过特异性合成CDR1基因上游调控序列引物,分析该序列在氟康唑耐药／敏感配对菌株中是否存在基因突变。结果白念珠菌氟康唑耐药／敏感配对菌株中的CDR1基因上游调控序列未出现任何碱基突变和缺失。结论白念珠菌氟康唑耐药与CDR1基因上游调控序列是否突变无关。     

用PCR技术直接检测白色念珠菌DNA的实验研究

目的建立和评价直接用PCR从血标本中检测白色念珠菌核酸的方法。方法用红、白细胞裂解液、破壁酶和真菌DNA提取盒直接处理血标本,得到的微量靶DNA用白色念珠菌种特异性引物进行扩增。结果在白色念珠菌制备的人血标本中检测出靶DNA,其敏感性达10个孢子/ml以下,从处理标本到报告结果仅需6h。结论用PCR法可特异、敏感地从血标本中直接检测白色念珠菌核酸,有助于临床快速诊断深部白色念珠菌感染。     

PCR-SSP法HLA-A、B基因分型与血清学分型的比较

目的比较聚合酶链反应-序列特异性引物(PCR-SSP)进行HLA-Ⅰ类A、B抗原位点分型的准确性,并探讨血清学分型错误发生的原因。方法用PCR-SSP以及单克隆抗体血清学分型技术对HLA-A、B分型并比较。结果34例样本PCR-SSP基因分型无假阳性和假阴性出现。PCR-SSP法与血清学比较,血清学检出错误或漏检率分别为HLA-A位点23.5%,B位点26.5%。血清学发生错误或易混淆的抗原有:A2和A68、A32和A33,B5、B60和61。结论PCR-SSP法进行HLA-A、B抗原等位基因分型具有分辨率高、特异性强、重复性好、实验过程简捷快速、分型结果较血清学更加准确可靠的优点。     

重复片段引物PCR用于鲍曼不动杆菌分型研究

目的 运用重复片段引物PCR的分析方法对鲍曼不动杆菌进行分子分型研究。方法 选用肠杆菌科基因间的重复序列（ERIC）为引物进行PCR扩增，对22株鲍曼不动杆菌进行分型研究，并与生物学分型及抗生素耐药结果进行比较。结果 经重复片段引物PCR的方法，分离出6种不同基因型的鲍曼不动杆菌，而生物学分型只有2种，且与抗生素谱较为相关。该方法简便易行，且重复性好，适合于医院感染流行病学研究。     

白色念珠菌二相性与口腔致病性关系的实验研究

目的　探讨白色念珠菌二相性与口腔致病性的关系。方法　分别将孢子相、菌丝相白色念珠菌与人口腔颊粘膜细胞 (BEC)进行粘附试验 ,比较二相性白色念珠菌对BEC的粘附率 ;用兔抗人sIgA血清中和人唾液中sIgA ,12 5Ⅰ sIgA放射免疫分析法检测唾液中sIgA的浓度 ,比较含sIgA及不含sIgA的唾液对二相性白色念珠菌粘附BEC的影响。 结果　菌丝相白色念珠菌对BEC的粘附率显著高于孢子相 (P <0 .0 0 1) ;含sIgA的唾液抑制孢子相及菌丝相白色念珠菌粘附BEC的能力强于不含sIgA的唾液 (P <0 .0 0 5 ) ,且唾液中sIgA抑制孢子相白色念珠菌粘附BEC的能力强于抑制菌丝相白色念珠菌粘附BEC的能力 (P <0 .0 5 )。结论　菌丝相白色念珠菌的口腔致病性强于孢子相     

建立序列特异性引物PCR检测儿茶酚胺氧位转移酶与单胺氧化酶基因多态性的方法

目的建立用PCR-SSP检测儿茶酚胺氧位转移酶(COMT)与单胺氧化酶(MAOA)的基因多态性的方法。方法根据COMT与MAOA的基因序列,采用针对变异位点的点突变方式设计出顺序特异性引物(SSP)进行PCR扩增,并借助常规电泳确定基因型。结果用PCR-SSP来研究COMT与MAOA基因多态性的方法特异性强,重复性好,而且简便、经济、快速。结论PCR-SSP可对具有单核苷酸突变的基因分型,应用前景良好。