生殖支原体感染与女性黏液脓性宫颈炎相关性研究

目的建立生殖支原体Taqman MGB荧光聚合酶链反应(PCR)检测方法,检测宫颈炎患者生殖支原体感染情况,以期发现生殖支原体感染与女性黏液脓性宫颈炎之间的相关性。方法参照国内、外文献建立Taqman MGB荧光PCR检测技术;采集大连市皮肤病医院性病门诊有黏液脓性宫颈炎的女性患者生殖道分泌物标本74例、体检中心对照组标本63例,应用上述检测方法检测标本中的生殖支原体。结果 Taqman探针荧光PCR法敏感性和特异性均较好。黏液脓性宫颈炎患者生殖支原体阳性率为24.3%,对照组未检测出生殖支原体;除不孕以外其他几项调查内容包括尿道炎症状、非婚性接触史、复发性宫颈炎以及治疗史均与生殖支原体感染有关联(P0.001),其阳性率分别为17.6%、52.9%、42.5%、100%。结论 Taqman MGB荧光PCR法是目前较为适用的生殖支原体检测方法。生殖支原体感染可能是黏液脓性宫颈炎的致病因素;生殖支原体的检测在指导复发性宫颈炎的治疗中有较为重要的意义。     

首段尿和尿道拭子标本应用PCR法检测生殖支原体的比较

目的：比较性病门诊男性患者PCR尿道拭子和首段尿标本对生殖支原体（Mg）的检测。方法：采集非淋菌性尿道炎（NGU）患者及无尿道炎就诊者的泌尿生殖道拭子标本及首段尿液标本,分别运用2对引物的PCR法检测Mg。结果：NGU组和无尿道炎者的拭子标本、首段尿标本Mg检出率分别为8.68%、5.90%;3.92%、3.92%。NGU组两种标本Mg的检出率间有统计学差异,P〈0.05;无尿道炎就诊者的两种标本的Mg检出率间无统计学差异（P〉0.05）。结论：首段尿标本检测生殖支原体可作为泌尿生殖道拭子标本检测的补充,提高临床检出率,还可作为无症状者的筛查手段。     

广西梧州和贺州不同场所暗娼泌尿生殖道沙眼衣原体感染及其基因型别分布

【摘要】 目的 探讨广西梧州和贺州暗娼人群（FSW）沙眼衣原体的流行情况及基因型别的分布。方法2009年7月至2010年9月间,在广西梧州和贺州的娱乐场所共招募810例符合条件的FSW。根据其不同场所分为低档场所、中档场所和高档场所。用罗氏Amplicor 沙眼衣原体核酸检测试剂对所有收集的宫颈拭子标本进行检测;用Qiagen DNA提取试剂对沙眼衣原体阳性标本的进行DNA提取,经巢式PCR方法扩增ompA基因的VS2区,扩增后的产物利用测序仪进行双向测序后分析基因型别。卡方检验分析沙眼衣原体感染率及其基因型别在来源于不同场所和不同地区的FSW间的差异。结果 经核酸检测161例沙眼衣原体阳性,沙眼衣原体感染率20.0%（161/805）。经卡方分析显示：高档场所和中档场所的FSW沙眼衣原体感染率低于低档场所,分别为χ2 = 3.97,P < 0.05,χ2 = 5.95,P < 0.05。基因型E在低档场所FSW人群中分布较中档场所FSW人群中多,χ2 = 5.02,P < 0.05,而基因型K在低档场所FSW人群中分布较中档场所FSW人群中少,Fisher 精确法,t = 0.048,P = 0.048。结论 低档场所的FSW沙眼衣原体的感染率较高,主要流行的基因型为E,而以症状为基础的筛查策略会漏检许多感染沙眼衣原体基因型E的患者,因此需要加大在低档FSW人群中沙眼衣原体的筛查的力度。 【关键词】 衣原体,沙眼; 基因; 卖淫     

生殖支原体对大环内酯类抗生素耐药突变位点研究

【摘要】 目的 了解生殖支原体对大环内酯类抗生素耐药相关的23S rRNA基因突变情况。 方法 就诊于我院性病门诊的91例近期应用过大环内酯类药物治疗的持续性和复发性尿道炎患者,取尿道拭子和前段尿标本进行生殖支原体（Mg）、沙眼衣原体（Ct）、解脲脲原体（Uu）等病原体检测,筛选出单一生殖支原体阳性患者标本,应用套式PCR扩增与大环内酯类抗生素耐药性相关的23S rRNA基因V区片段,扩增产物进行DNA测序,测得序列与美国国立生物信息中心已登记的生殖支原体标准株G37的相应基因序列比对。 结果 91例的标本中,Mg阳性21例,Ct阳性18例（19.8%）,Uu阳性10例,Mg与UU同时阳性2例,Ct与Uu同时阳性3例,Mg、Ct和Uu检测均为阴性者37例。21例Mg阳性标本中,18例的标本成功扩增出23S rRNA基因V区片段,除1例未发现基因突变外,其余17例均发现2058和2059位点突变,其中A2059G突变10例,A2058G突变5例,A2058T突变2例。 结论 23S rRNA基因V区片段基因突变可能与南京及周边地区Mg对大环内酯类药物耐药性相关。     

多重PCR结合反向线点杂交方法检测七种性病病原体

目的 建立多重PCR结合反向线点杂交技术同时检测淋球菌、 沙眼衣原体、生殖支原体、人型支原体、微小脲原体、解脲脲原体和毛滴虫的方法。方法 设计7对生物素标记的上述病原体特异性引物同时扩增病原体靶基因。扩增产物与预先标记在尼龙膜上的特异性探针杂交、显影，判读结果。多重PCR结合反向线点杂交方法检测211个尿道、宫颈分泌物临床标本（男107例，女104例），并与传统单一引物PCR检测结果比较，评价反向线点杂交的敏感性和特异性。结果 多重PCR结合反向线点杂交技术能敏感和特异地检测所有这7种病原体标准株，能敏感地检测病原体100拷贝的靶基因。在211个临床标本的检测中，2.8% （6 /211）的标本使用多重PCR结合反向线点杂交技术检测为阳性，而单一引物PCR检测为阴性，再使用巢式PCR检测这6个标本，结果为阳性。结论 多重PCR结合反向线点杂交技术能快速、敏感和特异地检测多种性病病原体。     

巢式-荧光定量PCR技术快速检测生殖支原体

目的建立生殖支原体巢式-荧光定量PCR快速检测技术。方法针对生殖支原体MgPa基因的保守片段(1 414～1 561bp),利用Primer express 3.0设计引物及探针,巢式PCR扩增临床样本,纯化后克隆到载体,制备阳性标准品。优化PCR反应条件,研究其特异性、灵敏性和重现性。结果构建了含MgPa基因的重组质粒,以不同浓度的重组质粒制作标准曲线,在10~1～10~9拷贝数之间有较好的线性关系。该方法检测阳性率高达15.4%,能特异性检测生殖支原体,而人型支原体、解脲脲原体、沙眼衣原体等检测为阴性;灵敏度高,可检测到10个拷贝数;3组重复试验的变异系数均小于3%。结论本研究建立了生殖支原体的快速检测技术,具有检测阳性率高、特异性强、灵敏度高、重现性好等特点。     

反向斑点杂交技术快速鉴定泌尿生殖道致病性支原体的研究

目的 建立PCR反向斑点杂交方法(PCR-RDB)快速鉴定泌尿生殖道常见致病性支原体.方法 以4种支原体[微小脲原体(Up)、解脲脲原体(Uu)、生殖支原体(Mg)、人型支原体(Mh)]的16SrRNA基因为靶序列设计通用引物和探针,将4种特异的寡核苷酸探针固定于尼龙膜上.利用巢式PCR扩增Up、Uu、Mg和Mh,扩增产物变性后与各特异性探针进行杂交、显色并分析结果,并对检测体系的敏感性、特异性进行测试.同时检测分析60例临床拭子标本.结果 4种特异性探针只与相应的支原体DNA杂交,与其他病原体无交叉反应,其敏感性是1 CFU.60例临床拭子标本PCR-RDB方法共筛选出支原体阳性19例,其中3例为支原体混合感染的标本(Up+Uu 2例,Uu+Mg 1例).结论 该方法可快速、敏感、准确地鉴定引起泌尿生殖道感染的致病性支原体.     

实-时PCR定量检测尿道炎和无症状男性首段尿中的生殖支原体

生殖支原体 (Mg)可能是引起非淋球菌性尿道炎 (NGU)的病原体之一。由于Mg很难培养 ,常规PCR难以对支原体进行定量检测 ,迄今为止 ,尚无有关Mg含量与支原体对泌尿生殖道致病性之间关系的研究。因此 ,作者建立了TaqMan实 -时PCR定量检测Mg的方法。材料和方法 :①采集 30 0例男性尿道炎患者和76例无症状男性首段尿标本 2 0mL ,用AMPLICORSTD - 1检测淋球菌和沙眼衣原体 ;用PCR及种系发生试验检测支原体和脲原体。所有Mg阳性尿标本用TaqMan实 -时PCR定量MgDNA ;②选用一对引物扩增Mg的 16SrRNA ,克隆、纯化PCR产物 ,制备MgD…     

实时荧光定量PCR检测白念珠菌的可靠性评价

目的:评价实时荧光定量PCR检测白念珠菌的可靠性.方法:自行设计白念珠菌种特异性引物,利用标准菌株构建重组质粒,进行实时荧光定量PCR检测,并建立标准曲线.结果:荧光定量PCR检测白念珠菌最低能检测到10个拷贝的基因,即相当于1～5 CFU/mL,同时与其他真菌、细菌及病毒等无交叉阳性反应.结论:实时荧光定量PCR法检测白念珠菌不仅具有较高的敏感性和特异性,可以对白念珠菌进行定量检测.     

生殖支原体与黏液脓性宫颈炎相关性的研究

目的 探讨生殖支原体与女性非衣原体非淋球菌感染的黏液脓性宫颈炎的相关性。方法 对象包括性病门诊就诊的女性非衣原体非淋球菌感染的黏液脓性宫颈炎患者226例及健康体检人群118例。采集宫颈拭子标本，运用PCR检测生殖支原体。一般情况、病史和性行为等采用问卷调查。结果生殖支原体在非衣原体非淋球菌感染的黏液脓性宫颈炎患者中的检出率为11.06%（25/226），健康体检人群中的检出率为0.85%（1/118），两组间差异有统计学意义（χ2 = 11.58，P < 0.001）。分析226例黏液脓性宫颈炎患者显示，有异位妊娠史、宫颈糜烂、附件压痛及宫颈内管分泌物镜检多型核白细胞≥10个/油镜视野的患者生殖支原体的感染率分别为27.78%，16.36%，18.28%，14.12%，而无异位妊娠史、宫颈糜烂、附件压痛及宫颈内管分泌物镜检多型核白细胞 < 10个/油镜视野的患者分别为9.62%，6.03%，6.02%，1.79%，生殖支原体的感染率差异在四组人群间均有统计学意义（P < 0.05），而多性伴数和宫颈口有黏液脓性分泌物在两组间差异均具有统计学意义（P < 0.01）。结论 性病门诊就诊的非衣原体非淋球菌感染黏液脓性宫颈炎患者中，生殖支原体感染率高于普通人群。     

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。     

广西壮族、汉族系统性硬化病与HLA-DQA1、DQB1等位基因相关性研究

目的 探讨广西壮族、汉族系统性硬化病(SSC)与HLA-DQA1、-DQB1等位基因的相关性.方法 用PCR-序列特异性引物(PCR-SSP)方法,对壮、汉族Sse患者各50例和壮、汉族健康人各100例的HLA-DQA1、-DQB1基因进行研究.结果 与正常人对照组相比,壮族SSc患者组中HLA-DQA1*0401、-DQB1*0501、-DQB1*0601基因频率显著升高(分别为RR:4.06,χ2=15.41,Pc<0.01;RR=4.47,χ2=10.65,Pc<0.01和RR=3.47,χ2=10.06,Pc<0.01),汉族SSc患者组中HLA-DQA1*0401、-DQA1*0601、-DQB1*0601基因频率显著升高(分别为RR=9.33,χ2=8.37,Pc<0.05;RR=8.071,χ2=20.13,Pc<0.01和RR=3.76,χ2=10.76,Pc<0.01).壮、汉族SSc患者组中HLA-DQA1*0201基因频率均显著降低(χ2=13.58,Pc<0.01和χ2=12.21,Pc<0.01).结论 HLA-DQA1*0401、-DQB1*0601可能是广西壮族、汉族SSc患者的易感基因,HLA-DQB1*0501可能是广西壮族SSc患者的易感基因,HLA-DQA1*0601可能是广西汉族SSc患者的易感基因.

Abstract：

Objective To explore the potential associations of HLA-DQA1 and DQB1 alleles with systemic scleroderma (SSc) in Zhuang and Han nationalities in Guangxi Zhuang Autonomous Region. Methods Genomic DNA was extracted from the peripheral blood of SSc patients of Zhuang (n=50) and Han (n=50) nationality,normal controls of Zhuang (n=100) and Han (n=100) nationality in Guangxi Zhuang Autonomous Region.PCR with sequence-specific primers (PCR-SSP) was used to detect HLA-DQA1 and -DQB1 alleles in these subjects. Results There was a significant increase in the frequency of HLA-DQA1*0401, -DQBl*0501 and -DQB1*0601 alleles in the patients of Zhuang nationalty(RR=4.056,χ2=15.407,PC=0.001;RR=4.472,χ2=10.653,Pc=0.004;RR=3.473,χ2=10.06,Pc=0.008)compared with normal controls of Zhuang nationality,and in the frequency of HLA-DQA1*0401,DQA1*0601 and DQB1*0601 alhles in patients of Han nationality (RR=9.333,χ2=8.371,Pc=0.036;RR=8.071,χ2=20.130,Pc=0.000;RR=3.764,χ2=10.755,Pc=0.004)compared with normal control of Han nationality.However,the frequency of HLA-DQA1*0201 allele was statistically lower in the patients of Zhuang and Han nationality than in the controls of corresponding nafionality (χ2=13.583,Pc=0.002;χ2=12.209,Pc=0.004).Conclusions HLA-DQA1*0401 and-DQB1*0601may be susceptible genes for SSc in Zhuang and Han nationalities,HLA-DQB1*0501 for Sse in Zhuang nationality,and HLA-DQAl*060l for SSc in Han nationality in Guangxi Zhuang Autonomous Region.     

Comparison of first void urine and urogenital swab specimens for detection of Mycoplasma genitalium and Chlamydia trachomatis by polymerase chain reaction in patients attending a sexually transmitted disease clinic

OBJECTIVE: The objective of this study was to compare urogenital swab specimens and first void urine (FVU) specimens from male and female patients at a sexually transmitted disease clinic for the detection of Mycoplasma genitalium and Chlamydia trachomatis infections using in-house, inhibitor-controlled polymerase chain reaction (PCR). STUDY DESIGN: Urethral swabs and FVU were collected from 1856 men and 753 women who also had a cervical swab collected. A positive diagnosis of infection was made if any 1 of the specimens tested positive and were confirmed in a second PCR assay targeting independent genes. RESULTS: M. genitalium DNA and C. trachomatis DNA were detected in 126 (6.8%) and 246 (13.3%) of the male sample sets and in 51 (6.8%) and 73 (9.7%) of the female specimen sets, respectively. Using our in-house PCR and sample preparation methods, FVU was found to be the most sensitive diagnostic specimen for both pathogens, but for optimal sensitivity, it should be supplemented with a cervical specimen in women. In a small subset of female FVUs, storage at -20 degrees C led to false-negative M. genitalium PCR results in 27% of specimens found positive when a sample preparation was performed before freezing. The age-specific prevalence of M. genitalium in men was almost constant between 18 and 45 years of age in contrast to C. trachomatis infections, which were more common in younger men. CONCLUSION: Urine appeared to be a better diagnostic specimen than the urethral swab for M. genitalium and C. trachomatis detection by PCR in this cohort of sexually transmitted disease clinic attendees but should be supplemented with a cervical specimen in women.     

Associations between Mycoplasma genitalium,Chlamydia trachomatis,and pelvic inflammatory disease

OBJECTIVE: To evaluate the association between Mycoplasma genitalium, Chlamydia trachomatis, and pelvic inflammatory disease (PID) METHODS: A case-control methodology was used. Swab eluates were processed using the QIAamp DNA mini kit. Polymerase chain reaction (PCR) for M. genitalium was carried out using a real time in-house 16S based assay. An endocervical swab was taken and tested for the presence of C. trachomatis (ligase chain reaction, Abbott Laboratories), and a high vaginal swab was taken and tested for the presence of Neisseria gonorrhoeae and bacterial vaginosis. RESULTS: Of the PID cases 13% (6/45) had evidence of M. genitalium infection compared to none of the controls (0/37); 27% (12/45) of the cases had C. trachomatis infection compared to none of the controls; and 16% (7/45) of cases only had serological evidence of C. trachomatis infection compared to 5% (2/37) of controls. Cases were more likely to present with M. genitalium and/or C trachomatis than controls (p<0.001). CONCLUSIONS: This study indicates that there may be an association between M. genitalium and PID, and that this relation is largely independent of C. trachomatis. Future studies need to investigate the pathological basis of the relation between M. genitalium and PID using samples from women with PID diagnosed using laparoscopy and endometrial biopsy. Little is known about the epidemiology of M. genitalium: large scale epidemiological investigations are needed to determine the prevalence, incidence, and factors associated with this emerging infection.     

Development and performance of a microwell-plate-based polymerase chain reaction assay for Mycoplasma genitalium

BACKGROUND: Mycoplasma genitalium is associated with, and could be the cause of, idiopathic cases of urethritis, endometritis, and cervicitis. Further epidemiologic studies on this organism are needed, but currently used polymerase chain reaction (PCR) assays are labor-intensive and culture is insensitive. GOAL: The goal was to develop and evaluate a microwell-plate-based PCR assay for M. genitalium. STUDY DESIGN: We adapted an M. genitalium PCR assay targeting the MgPa gene to a 96-microwell plate format with colorimetric detection of PCR products and incorporation of an internal inhibition control to determine the limit of detection of this assay (termed MgPa-IMW) for M. genitalium DNA and evaluate its performance on cervical and male urine specimens. RESULTS: The MgPa-IMW PCR assay detected 1 and 17 genome copies of M. genitalium (with 27% and 95% confidence) and was able to detect specimens inhibited for amplification. This assay was 100% concordant (50 positive and 50 negative) with the Southern-blot-based PCR assay with cervical specimens. Similarly, this test was 89% concordant with the Southern-blot-based assay for 64 male urine specimens (25 positive, 32 negative, 7 discordant), 97% concordant after correcting for specimens no longer positive by the Southern blot-based assay after freezer storage. CONCLUSION: The MgPa-IMW assay is sensitive and specific for the detection of M. genitalium in patient specimens and should facilitate large-scale screening for this organism.     

Mycoplasma genitalium: an organism commonly associated with cervicitis among west African sex workers

OBJECTIVES: To identify the contribution of Mycoplasma genitalium to the aetiology of cervicitis in sub-Saharan Africa and its relative importance in the overall burden of sexually transmitted infections among female sex workers (FSW). METHODS: The study population consisted of FSW recruited in Ghana and Benin during the initial visit of a randomised controlled trial. A questionnaire was administered, a pelvic examination carried out, and cervical samples obtained for detection of M genitalium, Neisseria gonorrhoeae, Chlamydia trachomatis, and Trichomonas vaginalis. Clinical signs potentially indicating cervicitis were cervical discharge, pus on the cervical swab, bleeding after sampling, and inflammatory cervix. RESULTS: Among 826 FSW, 26.3% were infected with M genitalium. N gonorrhoeae was strongly and independently associated with each of the four signs of cervicitis (adjusted odds ratios (AOR): 4.1 to 6.0). The AOR for C trachomatis were intermediate (1.3-4.1) and the AOR for M genitalium were lower (between 1.6 and 1.8) but statistically significant (p< or =0.05) for each sign. CONCLUSIONS: M genitalium is weakly associated with signs of cervicitis in west African FSW but is highly prevalent.     

自体血清皮肤试验阳性慢性荨麻疹与HLA-DRB1基因的相关性研究

目的 探讨广西地区自体血清皮肤试验阳性慢性荨麻疹与HLA-DRB1等位基因遗传易感性的关系。 方法 对144例广西地区慢性荨麻疹患者进行自体血清皮肤试验,按试验结果分为阳性组62例,阴性组82例。采用聚合酶链反应-序列特异性引物方法,对患者组和199例正常人对照组进行HLA-DRB1等位基因的分型,并分析DRB1基因在3个组中的分布。使用SPSS13.0统计软件分析。结果DRB1*01、*1401、*16等位基因频率在阳性组、阴性组和正常人对照组间比较,差异均有统计学意义（χ2 = 10.92,Pc = 0.03;χ2 = 35.34,Pc < 0.01;χ2 = 12.69,Pc = 0.03）。进一步在各组间进行两两比较,仅DRB1*1401等位基因频率在自体血清皮肤试验阳性组与对照组间（RR = 17.09,Pc < 0.01）及自体血清皮肤试验阳性组与阴性组间（RR = 7.20,Pc < 0.01）,差异均有统计学意义。结论 DRB1*1401等位基因可能是广西地区自体血清皮肤试验阳性慢性荨麻疹的易感基因或与其连锁。