广州地区产ESBL肺炎克雷伯菌SHV型质粒的分子流行病学调查

目的 运用变性高效液相色谱（DHPLC）技术调查广州地区产SHV型β-内酰胺酶肺炎克雷伯菌各种亚型的流行分布情况,试图建立一种方便快捷的分子诊断及其流行病学监测的新方法.方法 对73份产超广谱β-内酰胺酶（ESBL）肺炎克雷伯菌进行SHV质粒基因扩增,分别采用变性高效液相色谱（DHPLC）和测序法对扩增产物进行分析,以明确基因类型,并建立各个已知SHV基因亚型的特征性DHPLC图谱库.结果 73株产ESBL的肺炎克雷伯菌中68株确定为SHV基因型,其中62株为已知基因型,分别为SHV-1225株,SHV-1a 14株,SHV-17株,SHV-2a 8株,SHV-28 5株,SHV-2 2株,SHV-26和SHV-33各1株;6株为新的SHV基因型,其中5株获得命名;非SHV型菌株5株,分别为LEN-4型1株,OKP型4株.经过DHPLC分析,全部样本均表现为异常的洗脱峰,各种亚型的异常洗脱峰的形态迥异,其敏感性达100%（68/68）,特异性为93.2%（68/73）.SHV型质粒基因的突变集中在nt92、nt324～nt402及nt703～nt786这3个区段.结论 SHV-12是广州地区产SHV型β-内酰胺酶肺炎克雷伯菌主要的基因亚型,高比例基因变异的检出预示本地区即将或已经面临肺炎克雷伯菌新耐药机制的抵抗和流行,因此必须加强对产ESBL肺炎克雷伯菌的分子流行病学监测,及时调整抗菌策略;DHPLC可作为一种快速敏感的筛查方法用于产ESBL肺炎克雷伯菌的分子流行病学监测.     

ESBL阳性肺炎克雷伯菌所致肺炎临床特点和耐药性分析

目的 研究产超广谱β内酰胺酶(ESBL)的肺炎克雷伯菌的耐药特性及其所致肺炎的临床特点及临床危险因素,为预防ESBL阳性肺炎克雷伯菌所致肺炎的发生和临床治疗中抗生素的合理使用提供参考依据.方法 回顾性收集首都医科大学附属北京同仁医院93例(其中包括感染ESBL阳性肺炎克雷伯菌48例,非ESBL阳性肺炎克雷伯菌45例)住院患者的病历资料包括患者性别、年龄、治疗所用抗生素、住院时长、基础疾病以及侵入性操作6方面的信息,以及培养肺炎克雷伯菌的临床药敏结果.并对其危险因素进行单因素和多因素的分析.结果 ESBL阳性肺炎克雷伯菌菌株对阿米卡星的耐药率最低为41.6％,其次为复方新诺明和亚胺培南分别为56.3％和58.3％;对头孢菌素类药物(除头孢替坦外)耐药率为100％.临床治疗用药中单独用药最多见的是哌拉西林、美罗培南;联合用药最常见的是哌拉西林联合舒巴坦、美罗培南联合莫西沙星.感染ESBLs阳性肺炎克雷伯菌的肺炎患者,60岁以上占79.2％,平均住院天数为28.5 ±11.6天;死亡率为31％;合并高血压的有18例,合并糖尿病的有9例,合并脑梗的有3例,合并呼吸系统疾病的有10例;治疗过程中采用侵入性操作的有35例;感染ESBLs阴性肺炎克雷伯菌的肺炎患者,60岁以上33.4％,平均住住院天数为15.5 ±5.0天;死亡率6％;合并高血压的有18例,合并糖尿病的有9例,合并脑梗的有3例,合并呼吸系统疾病的有10例;治疗过程中采用侵入性操作的有16例.结论 住院时长(≥20天)、侵入性操作(包括气管插管、鼻导管吸氧、鼻饲等)、治疗过程中阿米卡星的使用,都是造成感染ESBL阳性肺炎克雷伯菌肺炎的临床感染危险因素(P＜0.05).在治疗感染ESBL阳性肺炎克雷伯菌的肺炎患者时应根据药敏结果调整临床用药.控制和预防感染ESBL阳性肺炎克雷伯菌的临床高危因素,可减少感染的可能性.     

吉林地区大肠埃希菌和肺炎克雷伯菌ESBLs的基因分型

目的对产超广谱β-内酰胺酶(ESBLs)的大肠埃希菌和肺炎克雷伯菌携带的ESBLs耐药基因进行分型,为临床合理应用抗生素提供理论依据。方法收集住院患者标本中分离出的51株大肠埃希菌和32株肺炎克雷伯菌,经PCR对上述菌株所携带的ESBLs耐药基因进行分型。结果产ESBLs大肠埃希菌中耐药质粒编码TEM型、SHV型和非TEM非SHV型超广谱β-内酰胺酶基因的百分率依次为80.4%,7.8%和11.8%;而在肺炎克雷伯菌中的百分率依次为78.1%,71.9%和25.0%。多数产ESBLs肺炎克雷伯菌同时产生一种以上的β-内酰胺酶。结论获得了吉林地区大肠埃希菌和肺炎克雷伯菌ESBLs的不同基因型,ESBLs基因型具有地区性差异。     

产超广谱β-内酰胺酶和质粒AmpC酶肺炎克雷伯菌的耐药性及基因型检测

通过PCR基因测序分析两种酶的基因类型，用接合转移试验了解肺炎克雷伯菌耐药基因转移方式，并用纸片扩散试验和微量稀释法测定细菌对17种抗生素的敏感性，现报道如下。     

北京发现同时产DHA-1质粒AmpC型及CTX-M型超广谱β-内酰胺酶的肺炎克雷伯菌

目的研究大肠埃希菌和肺炎克雷伯菌同时产超广谱β内酰胺酶(ESBL)及质粒型Ampc酶菌株的比率及其基因型。方法收集北京两家教学医院2001--2002年产ESBL且对头孢西叮耐药的59株大肠埃希菌和21株肺炎克雷伯菌，采用等电聚焦电泳测定β内酰胺酶的等电点；接合试验证实酶基因有无可转移性，并用碱裂解法提取质粒；采用多重聚合酶链式反应(multiplex polymerase chain reaction，MPCR)及序列分析确定质粒AmpC酶的基因型。脉冲场凝胶电泳(pulsed field gelel ectrophoresis，PFGE)确定耐药菌株的亲缘关系。结果北京两家医院ESBL中质粒型AmCZ酶的发生率，大肠埃希菌分别为0和2％，肺炎克雷伯菌则分别为9．7％和17．1％。1株大肠埃希菌及9株肺炎克雷伯菌产生DHA-1型质粒AmpC酶，同时也产CTX-3型(6株)或CTX—M-14(1株)或SHV-12(3株)型ESBL。10株中，3株肺炎克雷伯菌可将头孢西叮耐药性传给受体菌。这10株菌均至少携带1个约33—36kb的大质粒，未发现质粒的传播。PFGE发现这10株菌来自不同的克隆株。结论北京地区发现同时产DHA-1质粒AmpC型及CTX—M型ESBL的肺炎克雷伯菌，它们来自不同的克隆。     

产超广谱β-内酰胺酶肺炎克雷伯菌药物敏感性和基因型研究

【摘要】 目的 研究产超广谱β-内酰胺酶（ESBLs）肺炎克雷伯菌基因型和药敏特征。方法 对2003年12月～2004年12月哈尔滨地区临床分离的74株产ESBLs肺炎克雷伯菌，测定菌株对常用β-内酰胺类和非β-内酰胺类抗菌药物的敏感性，并采用SHV型和TEM型特异性引物检测ESBLs的基因型别。结果 产ESBLs细菌对头孢吡肟、氨曲南、阿米卡星和环丙沙星的敏感率依次71.6%、20.3%、54.1%、31.1%，对头孢哌酮/舒巴坦的敏感率为54.1%，69株（93.2%）对亚氨培南敏感。74株产ESBLs细菌中携带blaSHV和blaTEM基因的阳性例数分别是45（60.8%）和23（31.1%）。结论 本地区产ESBLs肺炎克雷伯菌对常用β-内酰胺类和非β-内酰胺类抗菌药物的敏感性降低，临床上需慎用酶抑制剂复合物及第四代头孢菌素，亚氨培南仍是治疗产ESBLs菌感染的首选药物。产ESBLs肺炎克雷伯菌blaSHV的流行频率高于blaTEM。     

产超广谱β内酰胺酶肺炎克雷伯菌医院感染肺炎临床危险因素的病例对照研究

目的探讨产超广谱β内酰胺酶(ESBLs)肺炎克雷伯菌医院感染肺炎的临床危险因素。方法用SPSS11.5软件进行统计,对101例肺炎克雷伯菌医院感染肺炎的临床危险因素进行病例对照研究。结果单因素分析发现住院时间大于20天、入住重症监护病房、气管插管或切开、留置导管、机械通气、头孢噻肟的使用是产ESBLs肺炎克雷伯菌医院感染肺炎的主要危险因素。多因素非条件logistic回归分析表明,头孢噻肟的使用是产ESBLs肺炎克雷伯菌医院感染肺炎的独立危险因素。结论合理使用头孢噻肟、采用替换性抗生素治疗策略是防止产ESBLs肺炎克雷伯菌感染肺炎流行的重要措施。     

儿童感染肺炎克雷伯菌产AmpC酶和ESBLs的检测及耐药性分析

目的了解广州地区儿童感染肺炎克雷伯菌产质粒介导的AmpC酶和超广谱β-内酰胺酶（ESBLs）的情况及其耐药特征,为临床合理用药提供参考依据。方法采用标准纸片扩散法检测ESBLs,头孢西丁三维试验法检测AmpC酶,K—B纸片法测定肺炎克雷伯菌对抗菌药物的敏感性。结果共检出248株肺炎克雷伯菌,其中46株产AmpC酶,阳性率为18．5％;157株产ESBLs,阳性率为63．3％;同时产AmpC酶和ESBLs菌株阳性率为18．1％。产AmpC酶肺炎克雷伯菌对第三代头孢菌素高度耐药。耐药率达80％～100％;对头孢吡肟、含酶抑制剂复合药的耐药率也在56．5％～93．5％之间：但对环丙沙星、阿米卡星的耐药率则在30％以下,对亚胺培南全部敏感。产ESBLs菌株对头孢菌素、含酶抑制剂复合药的耐药率也较高,在50％-91．7％之间,但对阿米卡星、环丙沙星、亚胺培南仍高度敏感。ESBLs阴性肺炎克雷伯菌对所测抗生素的敏感率均在81．2％以上。产酶菌株耐药率明显比非产酶菌株高。结论广州地区儿童感染肺炎克雷伯菌产ESBLs和AmpC酶的状况已十分突出：产酶菌株对常用抗生素的耐药率较高;碳青霉烯类抗生素可作为治疗产AmpC酶和／或ESBLs肺炎克雷伯菌感染的经验用药。     

产生超广谱β-内酰胺酶的肺炎克雷伯菌的药物敏感性及其基因型研究

目的研究产生超广谱β-内酰胺酶（ESBLs）的肺炎克雷伯菌基因型及其药敏特征。方法对2003年12月～2004年12月哈尔滨地区临床分离的74株产ESBLs肺炎克雷伯菌,测定菌株对常用β-内酰胺类和非β-内酰胺类抗菌药物的敏感性,并采用SHV型和TEM型特异性引物检测ESBLs的基因型。结果产ESBLs细菌对头孢吡肟、氨曲南、阿米卡星和环丙沙星的敏感率依次为71．6％、20．3％、54．1％、31．1％,对头孢哌酮／舒巴坦的敏感率为54．1％,69株（93．2％）对亚胺培南敏感。74株产ESBLs细菌中携带blaSHV和6kTEM基因的阳性例数分别是45例（60．8％）和23例（31．1％）。结论哈尔滨地区产ESBLs肺炎克雷伯菌对常用β-内酰胺类和非β-内酰胺类抗菌药物的敏感性降低,临床上需慎用酶抑制剂复合物及第4代头孢菌素,亚胺培南仍是治疗产ESBLs菌感染的首选药物。产ESBLs肺炎克雷伯菌blaSHV的流行频率高于blaTEM.     

肺炎克雷伯菌随机扩增多态性DNA法基因分型及意义

应用随机扩增多态性DNA(RAPD)技术对临床分离的199株肺炎克雷伯菌(K．pneumoniae)进行基因分型，旨在为该菌医院感染的分子流行病学分析提供科学依据。     

变性高效液相色谱技术快速检测下呼吸道致病性细菌的实验研究

目的 运用变性高效液相色谱(DHPLC)技术检测5种下呼吸道致病性细菌,建立一种快速检测下呼吸道致病性细菌的分子生物学方法.方法 以下呼吸道致病性细菌16S rRNA基因的保守区设计通用引物,并在引物前加上40-bpGC,特异性扩增该基因的保守区和可变区,运用DH-PLC技术对PCR产物进行分析.选取50株临床分离菌株验证该方法的有效性.结果 通用引物可特异性扩增细菌16S rRNA,PCR产物经DHPLC分析后每种细菌均能得到特征性的洗脱峰.DHPLC检测临床分离菌株结果显示,与常规培养方法的符合率为100%.结论 DHPLC技术具有准确、简便、快捷和高通量等特点,在临床检测下呼吸道致病性细菌中具有潜在的应用价值.     

Detection of AGXT gene mutations by denaturing high-performance liquid chromatography for diagnosis of hyperoxyluria type 1

Primary hyperoxaluria type 1 is an autosomal recessive disorder of glyoxylate metabolism, caused by a deficiency of alanine:glyoxylate aminotransferase, which is encoded by a single copy gene (AGXT). The aim of this research was to standardize denaturing high-performance liquid chromatography, a new, sensitive, relatively inexpensive, and automated technique, for the detection of AGXT mutation. Denaturing high-performance liquid chromatography was used to analyze in blind the AGXT gene in 20 unrelated Italian patients with primary hyperoxaluria type 1 previously studied by other standard methods (single-strand conformation polymorphism analysis and direct sequencing) and 50 controls. Denaturing high-performance liquid chromatography allowed us to identify 13 mutations and the polymorphism at position 154 in exon I of the AGXT gene. Hence the method is more sensitive and less time consuming than single-strand conformation polymorphism analysis for the detection of AGXT mutations, thus representing a useful and reliable tool for detecting the mutations responsible for primary hyperoxaluria type 1. The new technology could also be helpful in the search for healthy carriers of AGXT mutations amongst family members and their partners, and for screening of AGXT polymorphisms in patients with nephrolithiasis and healthy populations. Received: 18 January 2001 / Accepted: 7 May 2001     

Physical separation of HLA-A alleles by denaturing high-performance liquid chromatography

Genomic typing of polymorphic loci may be hampered by ambiguous typing results. Moreover, robust methods for simultaneous sequencing of two alleles present in a given sample may be difficult to establish. We used denaturing high-performance liquid chromatography (DHPLC) for physical separation of HLA-A alleles before sequence-based genomic typing (SBT). Physical separation was achieved by resolution of heteroduplexes between the sample alleles and a modified reference probe by DHPLC followed by selective reamplification of the sample alleles present in heteroduplexes. Complementary strands of the reference probe and sample alleles for heteroduplex induction were obtained by lambda-exonuclease digestion. HLA-A genotyping of 101 individuals using DHPLC-SBT yielded better typing resolution compared with serological typing and genotyping by the sequence-specific primer-polymerase chain reaction (SSP-PCR) method. Physical separation of alleles using a modified reference probe allows for development of fully automated methods for genomic typing of highly polymorphic loci such as HLA.     

应用变性高效液相色谱技术快速诊断儿童型脊髓性肌萎缩症

目的 介绍变性高效液相色谱(denaturing high-performance liquid chromatography，DHPLC)技术在儿童型脊髓性肌萎缩症(spinal muscular atrophy，SMA)基因诊断中的应用。方法 PCR扩增25名正常人、1份标准样品及25例SMA患者运动神经元生存基因(survival，motor rleuron，SMN)第7外显子及其侧翼区域，PCR产物变性、复性后直接上样于DHPLC系统。通过改变A、B缓冲液的比例来分离各种DNA成份。结果 各种不同DNA成份以色谱峰的形式表现出来。23名正常标本呈现3个峰，依次为SMN1／SMN2异源双链峰、SMN2同源双链峰、SMN1同源双链峰。2名正常标本及1份标准品只有SMN1峰，表明缺失了SMN2。22例SMA患者只有SMN2峰，表明缺失了SMN1。另3例SMA患者呈现3个峰，表明无SMN1或SMN2缺失。结论 DHPLC诊断SMA具有敏感、准确、快速、简便等优点。     

应用变性高效液相色谱技术检测汉族人Ⅰ型多囊肾致病基因突变

目的通过采用变性高效液相色谱（denaturing high-pedormanee liquid chromatogtraphy，DHPLC）技术检测汉族人常染色体显性多囊肾病（autosomal dominant polycystic kidney disease，ADPKD）Ⅰ型致病基因PKD1的突变，建立更为快速、敏感的突变筛查系统。方法以来源于19个ADPKD家系的67名成员血样标本的基因组DNA为模板，通过长链PCR和巢式PCR联合扩增的方法扩增PKD1全编码区，然后采用DHPLC方法进行初步筛查，将存在异常色谱图的扩增产物经核苷酸测序，确定突变的具体位点和类型，并与以往采用单链构象多态性（single strand conformation polymorphism，SSCP）方法检测出的突变结果相比较。结果共检测出14个致病突变，包括10个错义突变、1个插入突变、1个缺失突变、2个无义突变，其中12个突变位点与之前SSCP的检测结果相同，另新发现nt32819G→A和nt37137T→C两个突变位点，突变检出率为73．7％。结论DHPLC方法可以作为更为有效筛查汉族人ADPKD PKD1突变位点的检测途径。     

应用变性高效液相色谱技术研究肿瘤转移抑制基因KAI1/CD82多态与结直肠癌发病及转移的相关性

目的 研究肿瘤转移抑制基因KAII/CD82第8内含子IVS(8)6C11A/6T11G多态与结直肠癌发病及转移的相关性.方法 提取135例健康人、115例结直肠癌患者的外周血DNA,收集其相应的临床及病理资料,应用变性高效液相色谱技术,采用病例对照分组研究统计分析KAI1基因第8内含子剪接区域多态与结直肠癌发病及转移的相关性.结果 KAI1/CD82基因IVS(8)6T11G突变基因型检出率在正常人群为30.9%(84/135),结直肠癌患者中为31.3%(72/155),两者的差异无统计学意义(P=0.963);在结直肠癌低龄(＜50岁)和高龄(≥50岁)患者之间以及性别之间差异均无统计学意义(均P＞0.05);在常见的病理类型管状/管状乳头状腺癌、黏液腺癌中差异亦无统计学意义(P=0.633);在病理分级中有一定的差异性,但无统计学意义(P=0.267),而在有无淋巴结转移之间,两者差异有统计学意义(P.0.032)[6C11A有转移62.5%(80/128),无转移76.5%(78/102);6T11G有转移37.5%(48/128),无转移23.5%(24/102)].结论 肿瘤转移抑制基因KAI1/CD82第8内含子剪接区域多态与结直肠癌的发病、病理类型无关,但可能影响肿瘤的淋巴结转移,提示KAI1/CD82基因IVS(8)6C11A/6T11G多态筛查可能成为结直肠癌患者预后风险评估的指标.     

变性高效液相色谱检测 PKD2基因突变

目的　利用变性高效液相色谱分析技术 ( denaturing high- performance liquidchromatography,DHPL C) ,检测 2型常染色体显性遗传性多囊肾病致病基因 ( polycystic kidney diseasegene 2 ,PKD2 )突变。方法　收集临床确诊的汉族常染色体显性遗传性多囊肾病 ( autosomal dominantpolycystic kidney disease,ADPKD) 94个家系 ,提取外周血白细胞 DNA,用聚合酶链反应 ( polymerasechain reaction,PCR)扩增目的基因的全编码区 ,DHPL C对 PCR产物进行突变筛选 ,出现异常峰型的DNA片段进行核苷酸序列测定 ,明确突变位点和类型。结果　以 5 0名健康志愿者为正常对照 ,从 94例患者家系中成功检测出 8种突变 ,包括 2种无义突变、3种移码突变、3种错义突变。无义突变分别位于第 5和13外显子 ( 12 4 9C→ T,2 4 0 7C→ T) ,编码氨基酸分别在 4 17和 80 3位形成终止密码子。移码突变分别位于第2、12和 13外显子 ( 6 36 - 6 37ins T,2 348- 2 35 1del AGAA,2 4 0 1del A)。错义突变分别位于第 1、4和 5外显子 ( 5 6 8G→ A,96 4 C→T,116 8G→A) ,其编码氨基酸发生改变 ( 190 Ala→ Thr,32 2 Arg→Trp,390 Gly→ Ser)。结论　所检测出的 8种突变 ,为 ADPKD患者的基因诊断、产前诊断和囊肿前诊断积累了资料     

Interactions of human serum albumin with a modified poly(vinyl alcohol) gel packing for high-performance liquid chromatography

—The interactions of human serum albumin (HSA) with a poly(vinyl alcohol) gel packing (Asahipak GS-520) for high-performance liquid chromatography of proteins were investigated. Under certain conditions, the elution of HSA from the GS-520 column was retarded and its chromatogram was split into two peaks, indicating weak adsorption of HSA onto the gels and also the existence of two subfractions, i.e. human mercapto-albumin (HMA) and human non-mercapto-albumin (HNA). The chromatograms were confirmed to be greatly influenced by the salt composition, the pH, and the temperature of the isocratic mobile phase. It is characteristic for the adsorption of HSA onto the gels to be suppressed at a pH near its isoelectric point. The HSA-gel interaction parameters calculated using an adsorption chromatography theory demonstrate that the adsorption of HSA is caused by enthalpy-driven interactions, which are depressed by lowering the pH, in addition to hydrophobic interactions. Under the recommended chromatographic conditions for high resolution of HMA/HNA, it was found that the HSA samples possessed some subfractions besides HMA and HNA fractions.     

Detection of alpha-galactosidase a mutations causing Fabry disease by denaturing high performance liquid chromatography

Mutations in the alpha-galactosidase A (alpha-Gal A, GLA) gene cause Fabry disease, an X-linked recessive lysosomal storage disease. The majority of mutations are private, and confirmation of carrier status in females requires the definitive identification of a DNA mutation. In addition, knowledge of a family's mutation enables rapid and precise preimplantation and prenatal genetic testing. Here we report the development and use of DHPLC to rapidly and cost-effectively screen for alpha-Gal A mutations. Optimal DHPLC partial denaturing conditions for mutation detection were established for each PCR amplicon corresponding to the seven alpha-Gal A exons and their adjacent intronic/flanking sequences. At least five known mutations in each exon (45 in total) were screened by DHPLC to validate the method. Mutation detection was then performed in 14 affected males diagnosed by enzyme assay and 39 at-risk females, and the amplicons with abnormal DHPLC profiles were sequenced. In all affected males, and in 32 of the 39 at-risk females, four and 16 previously reported and 10 and 15 new mutations were identified, respectively. Sequencing all seven alpha-Gal A gene amplicons in the seven at-risk females who had normal DHPLC profiles excluded them as mutation carriers. Only one mutation (p.P362L) was not initially identified by its DHPLC profile, but in retrospect the profile was abnormal, emphasizing the need for experience in inspecting the profiles. In addition, this technique detected two new intronic polymorphisms, c.640-16A>G and c.1000-22C>T, with frequencies of 0.14 and 0.25 in both normal individuals and Fabry patients, respectively. This DHPLC method should improve the rapidity and cost-effectiveness of alpha-Gal A mutation identification in affected males and carrier females for Fabry disease.     

变性高效液相色谱法分析非综合征型耳聋人群SLC26A4基因突变

目的 研究非综合征型耳聋(nonsyndromic hearing loss,NSHL)患者SLC26A4基因的突变情况,为临床上NSHL患者基因诊断提供指导.方法 PCR分别扩增SLC26A4基因的21个外显子及其侧翼序列,所得目的 片段用变性高效液相色谱(denaturing high-performance liquid chromatorgraphy,DHPLC)进行突变筛查,有异常峰形的样本进行DNA测序.结果 在所选30例无血缘关系且GJB2基因检测未发现突变的NSHL患者中,共检测出10种SLC26A4基因变异,其中包括7种已知突变,2种未见报道的新突变(F572L和D87Y),及一种已知多态(Ivs11+47T＞C),其中Ivs7-2A＞G是最常见的突变,约占总突变的40%.结论 SLC26A4基因为仅次于GJB2的导致NSHL的相关基因,在(GJB2基因检测未发现突变的NSHL人群中SLC26A4基因的检出率达到23.3%,其中Ivs7-2A＞G是其最常见的突变.