巢式PCR扩增梅毒螺旋体polA及其临床应用的研究

目的　建立一种检测临床标本中微量梅毒螺旋体 (Tp)的敏感、特异的方法。方法　运用常规及巢式PCR扩增了Tp的DNA多聚酶I基因 (polA)的特异性片段 ,检测了两方法的特异性和敏感性 ;与血清学方法比较 ,评价了两方法在诊断梅毒中的意义。结果　常规及巢式 polAPCR只在扩增Tp时有特异性产物 ,灵敏度分别约为 40个 /10 0 μl及 1个 /10 0 μl。检测 13 1份拟诊为梅毒的血清标本 ,血清学方法与巢式 polAPCR结果无显著性差异 ;巢式和常规 polAPCR结果有显著性差异 ,巢式PCR敏感性高于常规PCR。结论　巢式 polAPCR具有高度敏感、特异、标本处理简便等优点 ,适用于检测血清等标本中微量Tp ,可作为血清学方法的补充用于梅毒诊断。     

低温对梅毒筛查试验RPR影响的研究

观察低温对梅毒血清学筛选试验RPR敏感性的影响。严格控制可能影响试验结果的其它因素 ,将同一病人的血清在普通实验室 (2℃～ 15℃ )和标准实验室 (2 5℃± 2℃ )内分别检测一次 ,阳性结果经梅毒血清学确证试验TPPA检测排除假阳性。结果 :共有 5 6 6例筛查者的标本完成本研究 ,标准实验室检测出 6 9例RPR真阳性 ,普通实验室检测出 6 2例RPR真阳性者 ,因此 ,普通实验室有 7例 (10 .1% )为RPR假阴性 ,其滴度均在 1:4或以下。结论 :低温 (2℃～15℃ )影响RPR的敏感性。     

性传播疾病

20053947梅毒螺旋体的巢式PCR检测与基因分型/郑和平(广东省皮肤性病防治中心),欧志英,胡玉山…∥中华皮肤科杂志.2005,38(9).546～548根据Pillay标准基因分型,共检测2002～2004年间广州地区62例疑似硬下疳病例,33例(53.2%)暗视野镜检发现梅毒螺旋体;54例PCR检测阳性,阳性率为87.1%。47例arp基因分型以14型为主(36例占76.6%),49例tpr基因分型以d型为主(39例占79.6%),47例双重基因分型发现7个基因型,依次为14d31例占66.0%,13d5例占10.6%,14b4例占8.5%,12b3例占6.4%,12d2例占4.3%,15d和14i各1例占2.2%。提示巢式PCR法检测梅毒螺旋体具有较高的敏感性,该地区梅毒螺旋体基因型以14d型为优势型。图3参8(俞晓林)20053948应用TP IgM WB法诊断早期先天梅毒/温贵华(深圳市宝安区慢性病防治院),彭石潜,张荣…∥中国艾滋病性病.2005,11(4).285～286采用梅毒螺旋体明胶颗粒凝集试验(TP IgM WB)方法对58例梅毒孕妇所产的60例(2例双胞胎)新生儿血清进行检测,并与...     

梅毒螺旋体的巢式PCR检测与基因分型

目的探讨广州地区梅毒螺旋体基因型分布,确定其分子流行病学特点。方法收集2002-2004年间连续就诊的疑似梅毒患者的生殖器溃疡标本,用暗视野显微镜和巢式PCR检测梅毒螺旋体,阳性者进行巢式PCR扩增酸性重复蛋白基因(arp)和苍白螺旋体重复基因族(tpr),凝胶电泳分析arp基因重复序列个数和tpr基因的MseⅠ酶切片段多态性。根据Pillay标准基因分型。结果共检测62例疑似硬下疳病例,33例(53．2％)暗视野镜检发现梅毒螺旋体；54例PCR检测阳性,阳性率为87.1％。47例arp基因分型以14型为主(36例占76.6％),49例tpr基因分型以d型为主(39例占79.6％),47例双重基因分型发现7个基因型,依次为14 d 31例占66.0％,13 d 5例占10.6％,14 b 4例占8.5％,12 b 3例占6.4％,12 d 2例占4.3％。15 d和14 i各1例占2.2％。结论巢式PCR法检测梅毒螺旋体具有较高的敏感性,广州地区梅毒螺旋体基因型以14 d型为优势型。梅毒的早期诊断和基因分型对于梅毒的防治有重要意义。     

血清标本10546份非梅毒螺旋体抗原血清学试验假阳性结果分析

目的了解非梅毒螺旋体抗原血清学试验假阳性发生率及原因。方法对本院2007-2008年门诊及住院病人送检的血清标本进行快速血浆反应素(RPR)环状卡片试验和梅毒螺旋体颗粒凝集试验(TP-PA),对结果进行统计学分析。结果 2007-2008年合计检测了10546份血清标本,RPR假阳性率为0.21%(15/7002)。2007年(0.32%)与2008年(0.11%)的RPR假阳性率差异无统计学意义(P0.05)。15例RPR假阳性血清标本中10例为有高危性行为的常规检查者,2例为孕妇,皮肌炎、尖锐湿疣和龟头破溃者各1例。结论 RPR假阳性有一定的发生率。建议RPR阳性者以TPPA等梅毒螺旋体抗原血清学试验进行确认。     

快速乳胶试验检测梅毒螺旋体抗体

目的：评价快速乳胶试验检测梅毒螺旋体抗体的可行性。方法：采用快速乳胶试验和TPPA平行检测50例梅毒患者血清和30例对照组血清的梅毒螺旋体抗体(TP—Ab)。结果：50例梅毒患者血清中，快速乳胶试验阳性47例，阴性3例；30例对照组血清快速乳胶试验阳性1例，阴性29例；以TPPA结果为标准，快速乳胶试验的敏感性为94．0％(47／50)，特异性为96．7％(29／30)。结论：快速乳胶试验检测TP—Ab具有一定的敏感性和特异性，操作简单，快捷。     

应用巢式聚合酶链反应检测一期梅毒

目的:探讨巢式聚合酶链反应(PCR)在一期梅毒早期诊断中的应用价值。方法:应用巢式PCR检测一期梅毒溃疡组织液和血清中TP-DNA,与暗视野检查(DF)、TPPA结果进行比较。结果:31例临床诊断为一期梅毒患者,溃疡组织液巢式PCR、血清巢式PCR、暗视野检查、TPPA检测的阳性分别为28例(90.32%)、4例(12.90%)、17例(54.84%)和27例(87.10%)。组织液巢式PCR检测结果与暗视野检查结果比较,差异有统计学意义(P<0.01),与TPPA结果比较,差异无统计学意义(P>0.05)。结论:巢式PCR检测一期梅毒溃疡组织液中TP-DNA敏感、特异,优于DF,而检测血清阳性率低,血清不适合作为巢式PCR检测的样本。     

聚合酶链反应对一期梅毒患者的诊断

目的:建立聚合酶链反应(PCR)用于快速检测梅毒螺旋体。方法:根据梅毒螺旋体47ku膜蛋白基因序列,自行设计筛选一对特异寡核苷酸引物,建立梅毒螺旋体的PCR。同时对其进行实验室评价和临床标本的检测验证。结果:PCR对梅毒螺旋体能产生特异性扩增,片段大小为252bp,而对其他生殖道常见菌及人基因组DNA不能扩增出任何片段。检测65例临床标本,PCR检查阳性54例(83.1%),梅毒螺旋体明胶颗粒凝集试验(TPPA)检查阳性52例(80%),梅毒螺旋体暗视野显微镜检查阳性40例(61.5%)。PCR检测结果,与暗视野显微镜检查结果比较,差异有显著性(P<0.01);与梅毒血清学检查结果比较,差异无显著性(P>0.05)。结论:PCR可特异、快速地检测梅毒螺旋体,并具有较好的敏感性。     

采用改良(1,3)-β-D-葡聚糖检测法监测某部中心医院侵袭性毛孢子菌感染情况

目的分析不同标本、不同检测方法的灵敏性与特异性,评价某部中心医院特殊人群侵袭性毛孢子菌感染情况。方法 (1)筛选某部中心医院毛孢子菌感染高发部门可疑感染者,分段收集不同体液标本。(2)对比传统G试验与改良G试验检测(1,3)-β-D-葡聚糖(BG)含量的敏感性与假阳性率差异。(3)巢式聚合酶链反应(PCR)法检测标本中毛孢子菌特异性DNA片段。结果改良G试验的敏感性和准确度均较传统G试验提高(P0.05),假阳性和假阴性率均下降。入院1周时,采用改良G试验获得的血浆、支气管肺泡灌洗液和尿液的阳性率高于入院2周时。入院1周时改良G试验在血浆和支气管肺泡灌洗液中的敏感性高于巢式PCR,而巢式PCR的特异性优于前者,差异有统计学意义(P 0.05)。结论改良G试验与巢式PCR扩增法相结合,可以提高毛孢子菌感染的检出率、降低假阳性率,并缩短检出时间。     

免疫印迹法在老年人梅毒诊断中的应用

目的采用免疫印迹法(WB)对临床梅毒血清学检测TPPA阳性且RPR阴性的老年人进行复测,探讨该人群是否存在梅毒感染。方法采集2012年11月-2014年10月就诊于贵州省人民医院患者,对84例RPR(-)、TPPA(+)的老年人血清标本运用免疫印迹法(WB)(TP-IgG)进行复测,对实验结果进行统计学分析。结果 WB法(TP-IgG)复测有79例阳性,5例可疑阳性,阳性率94.05%,与TPPA法符合率94.05%。WB(TP-IgG)法四种特异性蛋白均有表达。在TPPA滴度不同的情况下,TP17和TP15表达较高,TP47表达较其他3种蛋白低。结论在对TPPA阳性且RPR阴性的老年人群梅毒检测结果分析中,TPPA法与WB法检测有较高符合率。对用WB(TP-IgG)复测结果为可疑阳性的标本,在临床上需长期随访,必要时建议用TP-IgM检测,为是否进行驱梅治疗提供依据。     

聚合酶链反应检测梅毒螺旋体的进展

梅毒是由苍白螺旋体感染引起的一种常见系统性性传播疾病,早诊早治梅毒有利于减少传播、减轻系统损害.直接暗视野检查苍白螺旋体或血清抗体检测是诊断梅毒的主要手段,但有局限性.聚合酶链反应通过扩增苍白螺旋体靶序列诊断梅毒,而polA、tpp47基因为聚合酶链反应法检测苍白螺旋体常用靶基因.反转录聚合酶链反应和巢式聚合酶链反应的敏感性高于其他聚合酶链反应法.聚合酶链反应法检测一期梅毒拭子中苍白螺旋体敏感性可达60％以上,而检测血液及脑脊液苍白螺旋体敏感性不超过60％(胎传梅毒可达83％),特异性93％以上.患者HIV感染与聚合酶链反应检测敏感性无关.     

巢式-实时PCR检测梅毒初诊患者不同样本中梅毒螺旋体靶DNA

目的 探讨巢式-实时PCR(NR-PCR)检测初诊梅毒患者不同样本中梅毒螺旋体(Tp)靶DNA的可行性及应用前景.方法 以Tp polA为靶基因,用NR-PCR检测梅毒患者初诊时皮损拭子、血清、全血、脑脊液、末梢耳垂血等样本中的靶DNA,用统计软件SPSS.13分析结果.结果 NR-PCR检测Tp polA靶DNA的极限为2个Tp/ml,总体阳性率为71.7%,检测不同类样本的阳性率从高到低依次为:耳垂血92.0%>脑脊液90.2%>拭子74.3%>血清66.9%>全血64.2%.NR-PCR结果与血清学检查结果的一致性为76.0%(152/200).进一步分析显示:一期、二期梅毒拭子DNA阳性率(63.2%比87.5%)差异无统计学意义(χ2=2.62,P>0.05);血清样本中,二期梅毒DNA阳性率高于一期梅毒(χ2=3.6,P=0.06);全血样本中,二期梅毒DNA阳性率高于其他各类型梅毒;神经梅毒耳垂末梢血阳性率与脑脊液比较,P=0.06.隐性梅毒血清(RPR)滴度≥1:8组的Tp DNA阳性率高于RPR≤1:4组.结论 NR-PCR方法检测梅毒患者不同样本中Tp DNA是可行的,不同类型梅毒、不同类型样本以及其血清RPR滴度水平都影响Tp DNA阳性率.     

Performance of routine syphilis serology in the Ethiopian cohort on HIV/AIDS

OBJECTIVES: To assess the performance of routine syphilis screening during 5 year follow up of Ethiopian factory workers, participating in a cohort study on HIV/AIDS. METHODS: Syphilis serology test results of factory workers, who each donated at least six blood samples were evaluated. Screening in 1997-8 had been performed by the Treponema pallidum particle agglutination (TPPA) assay and in 1999-2001 by the rapid plasma reagin (RPR) test. TPPA had been followed by RPR or RPR by TPPA, in case of a positive screening result. Samples of study subjects showing inconsistent sequential TPPA and/or RPR results were retested independently by three laboratory technicians. RESULTS: A total of 540 cohort participants (8.3% HIV positive at enrollment) donated 4,376 blood samples (mean 8.3 per subject). From 93 of the 176 participants with at least one positive TPPA result during follow up, 152 samples were retested by RPR and/or TPPA. Based on the revised syphilis test results, the 540 cohort participants were classified as having no (70.5%), past (20.6%), prevalent (6.9%), or incident (2.0%) syphilis. The RPR screening test was difficult to interpret and yielded 8.2% biological false positive (BFP) RPR results, or 3.2% if weak positive results were excluded. There was no correlation between HIV infection and BFP RPR reactions. Sample mix-ups were detected in 1.2%. CONCLUSION: Evaluation of routine syphilis screening as performed in a long term cohort study on HIV/AIDS in Ethiopia showed difficulties encountered in syphilis screening programmes such as a high percentage of BFP RPR, inconsistencies in interpretation of the RPR test, and sample mix ups. The findings stress the need to develop a syphilis screening assay that is easy to perform and interpret and to implement quality assurance programmes.     

四种梅毒血清学检测方法的比较

目的评价梅毒甲苯胺红不加热血清反应素试验(TRUST)、快速血浆反应素环状卡片试验(RPR)、梅毒酶联免疫吸附试验(TP-ELISA)和梅毒螺旋体明胶凝集试验(TPPA)在梅毒检测中的应用价值。方法同时用RPR、TRUST、TP-ELISA和TPPA法检测确诊为梅毒患者的血清87份、健康查体人员血清100份及非梅毒患者的血清50份,并评价TRUST、RPR、TP-ELISA和TPPA法检测结果的敏感性和特异性等。ELISA法结合TRUST法双法筛查梅毒抗体,阳性标本再用TPPA法甄别生物学假阳性。结果TRUST、RPR和TPPA法对87份TP-ELISA法检测为阳性标本的敏感性分别为75.86%,74.71%,98.90%,特异性为98.67%,98.67%和100.00%。RPR法与TRUST法、TPPA法与ELISA法比较差异均无显著性意义(P均>0.05);TRUST法与TPPA法、RPR法与TPPA法比较差异均有显著性意义(P均<0.01)。100份健康查体人员血清,RPR、TRUST、TP-ELISA和TPPA法检测均为阴性,50份非梅毒患者血清进行RPR和TRUST法检测,2份阳性。结论TP-ELISA可替代TPPA使用;RPR法和TRUST法存在不同程度的假阳性,TP-ELISA结合TRUST双法筛查血液,用TPPA报告确证试验阳性。     

Azithromycin resistance in Treponema pallidum in Reunion Island: A cross-sectional study

ObjectiveSince the beginning of the 21st century, Reunion Island has experienced a syphilis epidemic. Infected patients are mostly heterosexual, with a high proportion of women, suggesting that congenital syphilis is present on the island. To determine whether azithromycin can be used for mass treatment of syphilis on Reunion Island, we assessed the prevalence of macrolide resistance in Treponema pallidum (TP).MethodsThis monocentric cross-sectional study was conducted at the Reunion Island University Hospital. Samples were collected from lesions suggestive of primary or secondary syphilis. Samples positive for TP by multiplex polymerase chain reaction (PCR) were sent to the French National Reference Centre (NRC) for further analysis. Nested PCR-tpp47 was performed on these samples for detection of TP-DNA; 23s rRNA was amplified by PCR in confirmed positive samples. The Restriction Fragment Length Polymorphism (RFLP) technique was performed on samples with amplified 23s rRNA for detection of the A2058G mutation.ResultsA total of 129 samples were collected from 119 patients. Of these, 18 tested positive for TP using multiplex PCR and were sent to the NRC. Fifteen (83.3%) of the 18 samples were confirmed positive by nested PCR-tpp47, and 23s rRNA was amplified in only 7 (38.9%) samples. Azithromycin resistance was detected in all TP strains with amplified 23s rRNA.ConclusionAmplification of 23s rRNA was successful in only 7 TP strains, all of which displayed resistance to macrolides. Keeping in mind the small sample size of our study, this suggests that azithromycin should not be used for mass treatment of syphilis in Reunion Island.     

Antiphospholipid antibodies: new data in the pathogenesis of collagenosis

Antiphospholipid antibodies (APAB) were first detected as a result of the disturbances they cause in routine biological tests. These effects include lupus anticoagulant (LA) in APTT test and false serological reactions for syphilis in VDRL test. With more sensitive techniques using purified phospholipid antibodies (ELISA, RIA) antibodies directed specifically against cardiolipidin or other phospholipids can be assayed. Beside being responsible for positivity of the VDRL test in the context of syphilis, APAB (false serological reactions for syphilis, LA, anticardiolipin antibodies) have also been detected in systemic lupus erythematosus (SLE) and lupus-like syndromes, after intake of certain drugs, and, more rarely, in a number of diseases (table I). The specificity of APAB detected in syphilis (antiphosphatidylcholine) is different from that of APAB detected in SLE (antiphosphatidylserine and anticardiolipin). A given APAB can react with several phospholipids, but such crossreactions do not occur haphazardly with all phospholipids. Crossreactions are observed between APAB and polynucleotides on the one hand, and between antinuclear antibodies and phospholipids on the other. This pattern has led to speculate that APAB are directly involved in the pathogenesis of SLE; however since these crossreactions are not extensive, such speculation is controversial. The presence of APAB in the form of LA, false serological reactions for syphilis or anticardiolipin antibodies is associated with a number of problems including recurrent thrombosis, repeated abortions, thrombocytopenia and positive Coombs' tests or, more rarely, pulmonary hypertension, migraine, epilepsy, chorea, and transverse myelitis.(ABSTRACT TRUNCATED AT 250 WORDS)     

54 549例住院患者梅毒血清学检测结果分析

目的：了解我院2017年住院患者梅毒血清学检测情况。方法：采用酶联免疫吸附法（ELISA-TP）筛查, 筛查阳性者用甲苯胺红不加热试验（TRUST）试验和梅毒螺旋体明胶颗粒凝集试验(TPPA)检测。结果：共收集患者54 549例，ELISA-TP阳性者1625例(检出率为3.00%)，其中1145例再用TRUST和TPPA方法检测，双阳性172例，检出率为0.32%。结论：我院住院患者梅毒感染率高，应对入院患者进行梅毒常规筛查。     

Clinical evaluation of four recombinant Treponema pallidum antigen-based rapid diagnostic tests for syphilis

Objective  To evaluate the sensitivity, specificity and feasibility of four rapid tests in the diagnosis of syphilis in sexually transmitted disease diagnosis and treatment centre of Peking Union Medical College Hospital.
Methods  Tests were performed on consecutive clinic attendees, using whole blood in the clinic, and whole blood and serum in the laboratory. The sensitivity and specificity of each test was evaluated, using a standard treponemal test ( Treponema pallidum haemagglutination assay) as the gold standard.
Results  The results are as follows. Abbott Determine Syphilis TP test: for serum specimens, the sensitivity was 100% and specificity was 98.9%; for whole blood specimens, the sensitivity was 81.9% and specificity was 99.4%. SD Bioline Syphilis 3.0 test: for serum specimens, the sensitivity was 95.5% and specificity was 97.9%; for whole blood specimens, the sensitivity was 87.6% and specificity was 99.4%. VisiTect-Syphilis test: for serum specimens, the sensitivity was 94.0% and specificity was 98.1%; for whole blood specimens, the sensitivity was 73.5% and specificity was 99.7%. Syphicheck-WB test: for serum specimens, the sensitivity was 67.4% and specificity was 98.8%; for whole blood specimens, the sensitivity was 64.0% and specificity was 99.7%.
Conclusion  We therefore strongly believe that rapid serological tests for syphilis are an acceptable alternative to conventional laboratory tests. Since they do not require equipment or electricity, they could increase coverage of syphilis screening, and enable treatment to be given at the first clinic visit.

Conflicts of interest

None declared.     

The superiority of polymerase chain reaction over an amplified enzyme immunoassay for the detection of genital chlamydial infections

BACKGROUND/OBJECTIVES: The polymer conjugate enhanced enzyme immunoassay (IDEIA) and Cobas Amplicor polymerase chain reaction Chlamydia trachomatis (CT) (Amplicor PCR) are two commonly used assays for the diagnosis of CT infection. The performance of these assays was compared for the diagnosis of genital CT infection among 1000 consecutive patients attending a genitourinary medicine (GUM) clinic. Confirmation of positive results and the clinical significance of the absence of cryptic plasmid in chlamydia on the diagnosis of infection by Amplicor PCR were also investigated. METHODS: IDEIA, Amplicor PCR, and two nested in-house PCR assays targeting cryptic plasmid and omp1 gene were performed on all samples. DNA from Amplicor PCR negative samples was pooled for in-house PCR assays. Each pool contained DNA from seven Amplicor PCR negative samples. RESULTS: Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and efficiency of IDEIA in the diagnosis of genital CT infection were 80%, 97%, 80%, 97%, and 95%, respectively. Sensitivity, specificity, PPV, NPV and efficiency of Amplicor PCR were 99%, 98%, 89%, 100%, and 98%, respectively. 16 (11%) of 144 Amplicor PCR positive results were identified as false positive by in-house PCR assays. No isolate of plasmid free CT was detected among the study population. CONCLUSIONS: IDEIA should not be used for the diagnosis of CT infection because of its poor sensitivity. Although the analytic specificity of Amplicor PCR was 98%, because of the adverse medical, social, and psychological impact of false positive results for patients, confirmation of Amplicor PCR positive results by a different assay with comparable sensitivity is essential. Amplification assays targeting cryptic plasmid are appropriate for the diagnosis of genital CT infections.