PCR-RFLP方法检测淋球菌及opa基因分型研究

目的研究聚合酶链反应-限制性长度多态性分析技术进行淋球菌opa基因分型及广西地区淋球菌opa基因分型情况。方法采用质粒PCR对80株淋球菌临床分离株进行检测,后用RFLP方法对其扩增产物进行opa基因分型。结果80株淋球菌经PCR测定后,有效进行分型的有70株淋球菌,这些菌株经opa基因分型后共产生63个独立opa基因型别,其中58个opa基因型各由单一株菌组成,另外5个opa基因型由2株或2株以上的菌株组成。结论聚合酶链反应-限制性长度多态性分析技术可对淋球菌进行检测和opa基因分型。opa基因分型方法是分子流行病学研究的重要工具。     

淋球菌opa基因分型方法的建立与评估

目的 探讨建立淋球菌opa基因分型方法,并对建立的实验室方法进行流行病学资料验证。方法 采用PCR-RFLP建立淋球菌opa基因分型方法,opa基因多态性分析使用特定的Gelcompar软件。选取淋病患者26例,其中有三对患者两两之间具有明确的性关系,而其他患者之间无明确性接触史。研究用收集到的已知流行病学资料验证opa基因分型方法的可靠性。结果 建立PCR-RFLP为基础的opa基因分型方法。26株淋球菌opa基因经过两种限制性内切酶（TaqⅠ与HpaⅡ）分别酶切后,发现两两之间存在明确性联系的3对菌株其opa基因被酶切后具有相同（100％）的长度多态性特性,而无明确性联系的菌株其opa基因被酶切后未发现具有类似相同的长度多态性。结论 实验室结果与患者所提供的性接触信息完全吻合。opa基因分型方法是一种较可靠的性网络调查工具。     

广东省珠海地区淋球菌多抗原序列分型研究

 目的:了解广东省珠海地区淋球菌基因型的分布,为淋病的防控提供实验室依据。方法：采用淋球菌多抗原序列分型法（NG-MAST）对2018-2019年珠海市淋病监测网络收集的172株淋球菌菌株进行基因分型,并构建系统进化树。结果：172株淋球菌中,porB基因型82种,tbpB基因型44种;porB和tbpB基因型别前三位分别为por2978（13株）、por1135（11株）、por3215(9株) 和tbp10（37株）、tbp21（26株）、tbp110（13株）。NG MAST型别79种,未知基因型占33.7%,优势基因型为ST9659（9株）、ST5308（6株）、ST8140（6株）,系统进化树显示菌株具有高度多态性。结论：本地区NG MAST型别具有多样性,新基因型菌株较多。     

广州淋球菌基因特征及其多抗原序列分型与环丙沙星耐药相关性研究

目的 探讨2014年广州市97株淋球菌环丙沙星耐药株的基因特征及其多抗原序列分型（NG-MAST）与淋球菌耐环丙沙星的相关性。 方法 用琼脂稀释法测定淋球菌对环丙沙星的最低抑菌浓度（MIC）,PCR分别扩增淋球菌的gyrA、parC基因和NG-MAST分型基因porB、tbpB基因并测序,获取耐药菌株的ST型别。 结果 97株淋球菌中95株（97.9%）对环丙沙星耐药。95株环丙沙星耐药菌株均在gyrA基因对应丝氨酸的第91和95位点上发生了突变,其中93株菌出现了parC基因突变。41株高水平耐药株（MIC ≥ 16 mg/L）中35株（85.4%）出现了parC基因87位点突变,54株低水平耐药株中32株（59.3%）出现此突变,差异有统计学意义（χ2 = 7.64,P < 0.05）。96株淋球菌分离株配对后,50株为网站已编号型别,共35个不同的ST型,其中10个ST型含有2 ~ 4个不同的分离株,ST型别中最常见ST5309。对淋球菌菌株系统进化树分析,淋球菌流行株可分为两群,第1群84株中MIC ≥ 16 mg/L的菌株39株占46.4%,第2群12株中只有1株MIC值为16 mg/L,差异有统计学意义（χ2 = 6.27,P = 0.012）。 结论 淋球菌对环丙沙星的高水平耐药主要与parC基因87位点突变相关。NG-MAST分型与环丙沙星耐药程度高低可能存在相关性。     

淋球菌对头孢曲松的敏感性及淋球菌多抗原测序分型研究

【摘要】 目的 探讨南京市淋球菌对头孢曲松的敏感性以及相应菌株的淋球菌多抗原测序分型（NG-MAST）基因型别。 方法 2007年和2012年在中国疾病预防控制中心性病控制中心临床防治基地分别收集了204株和81株淋球菌,经过分离纯化及鉴定后,用琼脂稀释法测定其对头孢曲松的最小抑菌浓度（MIC）;菌株培养后利用试剂盒提取DNA,并进行淋球菌多抗原测序分型（NG-MAST）。 结果 测试的285株淋球菌MIC ≥ 0.060 μg/ml的菌株比例为63.2%,MIC ≥ 0.125 μg/ml的比例为31.6%。2012年MIC ≥ 0.060 μg/ml和MIC ≥ 0.125 μg/ml的菌株比例分别为44.4%和11.1%,2007年MIC ≥ 0.060 μg/ml和MIC ≥ 0.125 μg/ml的菌株比例分别为70.6%和39.7%。NG-MAST分型研究显示,285株淋球菌共有166个型别,菌株多样性较高,其中73种为已知型别,93种为新型别。2007年测定的所有菌株中以ST568（n = 13）,ST270（n = 9）,ST421（n = 7）,ST2288（n = 5）,ST1731（n = 4）,ST1766（n = 4）,ST1866（n = 4）,ST1870（n = 4）等为主。2012年测定的所有菌株中以ST2318（n = 5）,ST1053（n = 4）,ST5990（n = 4）,ST8726（n = 4）为主。相同NG-MAST型别的菌株具有相同或相近的MIC值。 结论 2012年与2007年菌株的优势型别有较大变化,某些型别与头孢药敏值有较强对应关系。NG-MAST分型可能作为分子生物学标记用于淋球菌耐药监测。     

尿液标本淋球菌多抗原序列分型研究

【摘要】 目的 探讨江苏常州、扬州,广西梧州、贺州,海南三亚、琼海,广东江门、茂名淋球菌多抗原序列分型（NG-MAST）的型别分布。方法 88份淋球菌核酸检测阳性的男性性病门诊就诊者（MSP）的尿液标本,用Qiagen试剂盒提取DNA,并进行2次PCR扩增porB、tbpB基因片段,扩增成功的标本测序后登陆NG-MAST网站进行序列比对,获取菌株基因型别信息。 结果　对88份阳性标本提取DNA进行单次PCR,扩增porB、tbpB基因的效率分别为13.6%和14.8%,共有12份标本比对成功,得到菌株基因型别,分型效率为13.6%（12/88）;进行2次PCR扩增后,porB、tbpB基因的PCR扩增效率分别为71.6%和72.7%,分别增长了58.0%和57.9%,共有62份标本比对成功得到菌株基因型别,分型效率为70.5%（62/88）,分型效率提高了56.9%。62份标本共分出45个基因型,其中40个型为已知型别,5个为新型别。所有型别中,ST1866型丰度最高,有6份标本为此型别,ST1972、ST3356各有4份标本,都集中于江苏项目点。ST532有3份,集中于广东项目点。ST2221有2份,集中于广西项目点,其余各基因型均只有1份标本,散在分布于各省。5个新型别为：porB基因型为892、tbpB基因型98%相似于46型的标本,且丰度较高,仅发现于江苏项目点;porB基因型为903、tbpB基因型99%相似于958型;porB基因型为130、tbpB基因型96% 相似于504型;porB基因型为2790、tbpB基因型99%相似于32型;porB基因型为1053、tbpB基因型99%相似于856型。结论　尿液标本可以用于NG-MAST分型研究,2次PCR扩增可以提高尿液标本的分型效率,不同地区淋球菌NG-MAST型别表现为多样性。 【关键词】 奈瑟球菌,淋病; 基因型; 淋球菌多抗原序列分型     

中国部分地区淋球菌耐药监测点产青霉素酶淋球菌及其blaTEM-135突变体的流行调查

目的 了解中国不同地区淋球菌耐药监测点产青霉素酶淋球菌（PPNG）的比例及blaTEM-135突变体在PPNG中的分布,比较PPNG及blaTEM-135突变体淋球菌多抗原测序分型（NG-MAST）的型别分布,了解不同地区PPNG blaTEM-135突变体的差异与联系。 方法 2012年在江苏、上海、浙江、天津、广东、广西6个淋球菌耐药监测点共收集572株淋球菌,经过分离纯化及鉴定后,采用头孢噻吩纸片法测定PPNG;菌株培养后利用试剂盒提取DNA,通过错配扩增突变分析PCR（MAMA PCR）鉴定blaTEM-135突变体,采用NG-MAST进行分型研究。 结果 572株淋球菌中PPNG总阳性率为38.1%（218/572）,PPNG中相应blaTEM-135突变体的总比例为52.3%（114/218）。监测点中PPNG阳性率从高至低分别为：浙江（45/87,51.7%）、上海（36/79,45.6%）、广东（78/205,38.0%）、广西（12/32,37.5%）、江苏（24/77,31.2%）、天津（23/92,25%）;PPNG中相应blaTEM-135突变体的阳性率从高至低分别为：浙江（31/45,68.9%）、江苏（14/24,58.3%）、广东（39/78,50.0%）、上海（17/36,47.2%）、天津（9/23,39.1%）、广西（4/12,33.3%）。NG-MAST分型研究显示,blaTEM-135突变体中流行菌株型别有ST2318、ST1768、ST1866、ST1053、ST8726等,其中ST1768、ST1053和ST8726与blaTEM-135突变体有较强的对应关系。天津PPNG菌株及blaTEM-135突变体ST分布与其他各监测点有显著差异,江浙沪地区菌株ST有一定联系。 结论 中国淋球菌耐药监测点PPNG及相应blaTEM-135突变体阳性率处于较高水平,不同地区间PPNG及相应blaTEM-135突变体阳性菌株ST型别分布差异有统计学意义。     

质粒介导产β －内酰胺酶淋球菌的检测及其TEM-1基因质粒分型

【摘要】 目的 调查质粒介导的产? －内酰胺酶淋球菌 (PPNG)在淋球菌临床分离株中流行情况及TEM-1基因的分子流行病学情况。方法 收集2000-2008年从性病门诊患者中分离的 934株淋球菌,纸片酸度法测定PPNG,多重PCR方法鉴定?－内酰胺酶质粒并行TEM-1基因分型。 结果 2000-2008年共检测934株淋球菌,检出230株PPNG(24.6%),流行率逐年增多,由2000年的17.1%上升到2008年的42.1%;PPNG的TEM-1基因质粒分型以亚洲型质粒流行为主,只在2008年检测出1株含非洲型质粒,未见多伦多型质粒。结论 PPNG流行率逐年增高,产? －内酰胺酶的TEM-1基因流行以亚洲型质粒为主,偶见非洲型质粒。     

广西性病门诊患者泌尿生殖道沙眼衣原体株高分辨多位点测序分型研究

目的 了解广西性病门诊患者中泌尿生殖道沙眼衣原体型别分布,同时对随访患者沙眼衣原体的感染情况进行分析。方法 在广西壮族自治区皮肤病防治所收集泌尿生殖道沙眼衣原体感染患者,采集男性尿道和女性宫颈拭子标本,并对初筛阳性患者在治疗完成后进行随访和标本采集,同时采集患者基本信息和临床信息。应用QIAxtractor全自动核酸纯化仪提取DNA,巢式PCR扩增沙眼衣原体主要外膜蛋白基因（ompA）和MLST（高分辨多位点测序分型）所需的CT046 （hctB）、CT058、CT144、CT172和 CT682 （pbpB）5个基因。对PCR产物进行序列测定,经序列比对和MLST型别分析获得菌株的ompA基因分型和型别,并用BioNumerics7软件对广西和意大利沙眼衣原体株绘制最小生成树。结果 在44份来自初诊患者和6份来自随访患者的沙眼衣原体阳性样本中,42份成功进行沙眼衣原体ompA和MLST分型。共发现7种ompA基因型和15种hr?MLST分型的ST型别,其中有3种ST型别为首次报道。广西地区沙眼衣原体基因型别较意大利地区具有特征性。6例随访患者经分型方法鉴定3例为再次的新发感染,3例因未能成功进行基因分型而未能确诊。结论 广西性病门诊患者感染的沙眼衣原体具有独特的型别,在随访中出现泌尿生殖道沙眼衣原体再感染病例。     

广州地区沙眼衣原体omp1基因分型及突变研究

目的 探讨不同omp1基因型沙眼衣原体（Ct）在广州地区非淋菌性尿道（宫颈）炎患者中的分布情况。方法 从性病和妇科门诊及外展现场采集男性尿道和女性宫颈拭子,提取Ct基因组DNA为模板,巢式PCR扩增Ct omp1基因VD1 ~ VD3并测序分型,分析Ct型别决定簇VD1 ~ VD2区域的氨基酸突变情况。结果 共检测1208份拭子,检出Ct 132株,对其中130株进行测序分型,共发现10个omp1基因型,分别为E型38株（29.23%）、D型25株（19.23%）、J型24株（18.46%）、F型21株（16.15%）、G型7株（5.38%）、H型5株（3.85%）、K型5株（3.85%）、B型、Ja型各2株（各1.54%）、I型1株（0.77%）;可见E、D、J、F 4个型别为主要感染型别,共占83%。序列分析发现,D、B、K 3个型别在VD1 ~ VD2区域的氨基酸存在突变。另有2例为混合感染未能确定型别。结论 广州地区Ct omp1基因型别感染以E、D、J、 F 4种型别为主,共占83%,Ct B型也发现于男性尿道和女性宫颈。     

Opa typing of Neisseria gonorrhoeae strains isolated from patients attending sexually transmitted disease clinics in China

BACKGROUND: Gonorrhoea has been one of the most common sexually transmitted diseases (STDs) in China. A clear understanding of its transmission dynamics is important in formulating prevention and control measures. GOAL: To investigate the distribution of opa types in patients attending at STD clinics in China and to evaluate the concordance between epidemiologic data and opa-typing results. STUDY DESIGN: Opa typing was conducted for 330 Neisseria gonorrhoeae strains isolated from the patients at 4 STDs clinics in China, and the epidemiologic data were collected as well. RESULTS: A total of 309 opa types were detected from the 330 isolates. Two hundred ninety-two opa types were unique, and 17 opa types were found in more than 1 patient. Opa typing confirmed all 9 sexual links that were revealed by epidemiologic information and further identified 9 opa clusters and 8 similar pairs. CONCLUSIONS: Opa typing is a discriminatory tool that can be used in epidemiologic studies on gonococcal infections. This technique is more powerful than epidemiologic data to identify sexual links and improve our understanding of the transmission dynamics of gonorrhoea.     

中山地区淋球菌头孢菌素敏感性及NG-MAST研究

目的: 研究中山辖区淋球菌对头孢曲松与头孢克肟的敏感性,及淋球菌多抗原测序分型(NG-MAST)的基因型别.方法: 收集2017-2018年中山市辖区医疗机构淋球菌菌株,经分离、鉴定和纯化后,采用琼脂稀释法检测其对头孢曲松与头孢克肟的最小抑菌浓度(MIC);应用NG-MAST进行淋球菌porB和tbpB外膜蛋白基因分型...     

Molecular epidemiology of recently emergent ciprofloxacin-resistant Neisseria gonorrhoeae in South Africa

OBJECTIVES: Syndromic management guidelines for male urethritis syndrome and female discharge syndrome (nonpregnant) in South Africa advocate the use of ciprofloxacin for potential infection with Neisseria gonorrhoeae. In 2003, reports of clinical failure of gonorrhea following ciprofloxacin treatment prompted a clinic-based surveillance to detect the presence of resistant isolates. STUDY: Urethral samples for the isolation of N gonorrhoeae were obtained from consecutive male patients with urethral discharge attending the largest sexually transmitted disease clinic in KwaZuluNatal. Molecular typing of isolates was performed by means of N gonorrhoeae multiantigen sequence typing (NG-MAST). RESULTS: Of 139 isolates, 31 (22%) were resistant to ciprofloxacin (minimum inhibitory concentration >or=1 mg/l). NG-MAST revealed novel, as well as previously described, sequence types (ST). The largest cluster of our isolates belonged to ST 217. This ST has been reported among ciprofloxacin-resistant isolates from Europe. CONCLUSION:: The results underscore the urgency of review of the current treatment guidelines for discharge disease in KwaZuluNatal.     

Global transmission of prolyliminopeptidase-negative Neisseria gonorrhoeae strains: implications for changes in diagnostic strategies

BACKGROUND: Species confirmation of Neisseria gonorrhoeae is commonly performed with biochemical kits, rely on the activity of the enzyme prolyliminopeptidase (PIP). This enzyme has previously been considered to be almost universally present in N gonorrhoeae. However, increasing numbers of N gonorrhoeae isolates lacking PIP activity have been identified. OBJECTIVES: To investigate the possibility of a widespread transmission of one or several N gonorrhoeae PIP-negative strains among several countries worldwide. METHODS: PIP-negative N gonorrhoeae isolates cultured from 2001 to 2004 in Australia, New Zealand and Scotland were comprehensively characterised and compared with previous data from England and Denmark. All isolates were characterised by antibiotic susceptibility testing, serovar determination, pulsed-field gel electrophoresis (PFGE), opa-typing, sequencing of the entire porB gene and N gonorrhoeae multiantigen sequence typing (NG-MAST). RESULTS: Most (83%) of the viable Australian isolates, and all the New Zealand and Scottish isolates were assigned serovar IB-4, with similar antibiograms, nearly identical porB1b gene sequences, identical (ST210) or highly related (ST292, ST1259) NG-MAST STs, and indistinguishable or related PFGE fingerprints as well as opa-types. The isolates showed characteristics indistinguishable or highly related to the previously described English and Danish outbreak strain. CONCLUSIONS: A comprehensive characterisation indicates a widespread dissemination, mainly among men who have sex with men (MSM), of indistinguishable and highly related genotypes that have evolved from a single N gonorrhoeae PIP-negative serovar IB-4 strain among several countries worldwide. An increased awareness of PIP-negative N gonorrhoeae strains is crucial and changes in the diagnostic strategies may need to be considered.     

淋病奈瑟菌基因分型的研究进展

淋病奈瑟菌是引起淋病的病原体.淋病奈瑟菌基因分型方法在淋病奈瑟菌疫苗的研究、制定流行病学控制措施和淋病的治疗上均起到重要的作用.常见基因分型方法如:opa分型、脉冲电场凝胶电泳、por分型、随机扩增多态性DNA分型、多位点序列分型等.由于不同基因分型方法有着其各自不同的优点和不足,依据不同的需要,可选择合适有效的基因分型方法以达到实验的要求.     

Analysis of the antibiotic sensitivity of Neisseria gonorrhoeae in Guangzhou, Peoples Republic of China

BACKGROUND: Sexually transmitted diseases began to re-emerge in China in the mid 1980s. During the last one and a half decades, Neisseria gonorrhoeae infection has become one of the three most common sexually transmitted diseases in China. At present, resistant strains of N. gonorrhoeae are increasing each year. This study was undertaken to better understand the sensitivity of five antibiotics to N. gonorrhoeae isolates in Guangzhou, China. GOAL: To determine the frequency and diversity of antibiotic resistance, particularly to penicillin and tetracycline, on gonococcal strains in Guangzhou. STUDY DESIGN: Strains of N. gonorrhoeae isolates from 203 patients with uncomplicated urethral gonococcal infections from Guangzhou, China were reviewed from September 1997 to August 1998. All strains were characterized with five different antimicrobials for sensitivity, including penicillin, tetracycline, spectinomycin, ciprofloxacin, and ceftriaxone. RESULTS: Penicillin resistance was present in 121 of 203 isolated strains (59.6%). The plasmid-mediated strains and chromosome-mediated strains among the penicillin-resistant strains that were resistance to penicillin were 5.8% and 94.2%, respectively. Plasmid-mediated strains resistant to penicillin and tetracycline were each 3.4%. Most isolated strains were resistant to ciprofloxacin, accounting for 60.6%. All strains were sensitive to spectinomycin and ceftriaxone. CONCLUSION: N. gonorrhoeae isolates exhibited a high rate of resistance to penicillin and ciprofloxacin. Spectinomycin highly effective for penicillin-producing N. gonorrhoeae, tetracycline-resistant N. gonorrhoeae and the highly resistant strains of ciprofloxacin.     

The epidemiology of ciprofloxacin resistant isolates of Neisseria gonorrhoeae in Scotland 2002: a comparison of phenotypic and genotypic analysis

OBJECTIVES: To characterise all isolates with reduced susceptibility or resistance to ciprofloxacin received by the Scottish Neisseria gonorrhoeae Reference Laboratory (SNGRL) in 2002 using N gonorrhoeae multi-antigen sequence typing (NG-MAST); to compare NG-MAST with conventional typing and to describe the epidemiology of ciprofloxacin resistant gonorrhoea in Scotland in 2002. METHODS: Isolates were characterised on receipt by auxotyping and serotyping (A/S typing), and antibiotic susceptibility testing, and retrospectively by NG-MAST. Epidemiological data were requested for all isolates in the study. RESULTS: The 106 isolates were separated into more sequence types (ST) than A/S classes (44 versus 17). All isolates within a sequence type had the same serotype, were homogeneous with respect to ciprofloxacin resistance category, but were sometimes heterogeneous with respect to auxotype or plasmid borne resistance to penicillin. Combined NG-MAST and epidemiological data revealed sustained transmission of several gonococcal strains predominantly within Greater Glasgow and Lothian. Clusters of isolates were associated with transmission within the United Kingdom, whereas isolates with unique STs were associated with foreign travel (p < 0.0001). CONCLUSIONS: NG-MAST is more discriminatory than A/S typing. Ciprofloxacin resistant gonococcal isolates in Scotland are heterogeneous, with endemic spread of some strains occurring predominantly in Greater Glasgow and Lothian.