细胞培养、连接酶链反应和六种聚合酶链反应试剂盒检测沙眼衣原体的比较研究

目的 与细胞培养和连接酶链反应（LCR）比较考察6种国产聚合酶链反应（PCR）试剂盒在检测性传播疾病门诊患者标本沙眼衣原体的诊断价值。方法 在北京、上海、南京、天津5家临床医院性病门诊收集到673份尿道／宫颈拭子标本,分别进行沙眼衣原体培养和PCR检测,对结果不相符合的标本采用LCR复检,将各种PCR检测结果分别与培养、LCR以及综合结果进行比较分析。结果 合格病例616例,培养法检测阳性率6．3％,PCR检测阳性率分别为23．5％－28．7％。与培养结果比较,各种PCR检测的敏感性均在90％以上,其中PCR1、PCR2和PCR5均达到100％。LCR复核标本200份,与之相比,PCR检测的敏感性为83．9％－98．6％,特异性66．7％－94．7％,YI指数0．523－0．881。其中PCR2结果符合性最好,其它依次为PCR4、PCR1、PCR5、PCR3及PCR6。综合分析证明国产PCR检测沙眼衣原体的敏感性增在85％以上,特异性均在95％以上。YI指数由高到低分别为PCR2、PCR1、PCR3、PCR5、PCR4、PCR6。结论 国产PCR检测尿道／宫颈拭子沙眼衣原体具有较高的敏感性与特异性,可以用于临床检验,实验室质控与监督是本方法得以正确应用的关键。     

连接酶链反应检测性病患者沙眼衣原体感染

目的 评价连接酶链反应(LCR)诊断性病患者尿道/宫颈中沙眼衣原体(Ct)的意义。方法 STD门诊尿道(宫颈)炎患者276例,取尿道/宫颈拭子,以LCR分析法检测Ct。采用每4份标本相混合的方法分别以LCR分析法检测尿道/宫颈拭子标本中的Ct,其中56例患者同时进行尿道/宫颈拭子Ct细胞培养。差异性结果由PCR法扩增Ct的主要外膜蛋白基因来进行确认,确定LCR分析法检测Ct的敏感性、特异性。结果 LCR分析法检测尿道/宫颈拭子Ct的敏感性、特异性分别为96.7%和100%。采用每4份标本相混合的方法分别以LCR分析法检测尿道/宫颈拭子标本中的Ct,与单独用每份标本逐一进行LCR检测比较,结果完全一致,符合率为100%。结论 以尿道/宫颈拭子为标本,LCR分析法检测Ct的敏感性、特异性高。适用于诊断泌尿生殖道Ct感染。用标本相混合的方法LCR分析检测尿道/宫颈拭子标本中的Ct,适用于Ct感染的普查。     

男性泌尿道沙眼衣原体三种检测方法的比较

我们对104例拟似衣原体性尿道炎患者的尿道拭子，采用衣原体快速免疫测定法(EIA)、PCR法和LCR法进行沙眼衣原体检测。结果三种方法的敏感性和特异性，PCR分别为100％和95．6％；LCR为94．4％和100％；EIA为86．1％和100％。PCR的敏感性最高，特异性最低(95．6％)；LCR和EIA敏感性均低于PCR，但特异性均高于PCR。     

LCR技术检测男性首段尿中的淋病奈瑟菌和沙眼衣原体

目的：应用连接酶链反应（LCR）技术检测男性尿标本中的淋病奈瑟菌和沙眼衣原体,初步评价其敏感性和特异性。方法：采集受检者晨起或较长时间（2小时以上）不排尿后的首段尿（FVU）标本1131份,利用LCR技术对此尿液标本进行淋病奈瑟菌和沙眼衣原体检测,对Cut-off值在灰区以上的标本进行PCR检测。对LCR和PCR结果相异的标本,用另-LCR试剂进行复检,参照“扩大的金标准”来确定检测结果。结果：LCR技术检测淋病奈瑟菌的敏感性和特异性分别为100％和99．9％,沙眼衣原体分别为97．5％和98．4％。结论：应用LCR技术筛检男性尿液中淋病奈瑟菌和沙眼衣原体,是一种既敏感又特异的非侵入诊断方法,可避免取尿道标本给患者带来的痛苦。     

连接酶链反应检测宫颈沙眼衣原体感染

为评价连接酶链反应（LCR）诊断宫颈沙眼衣原体（CT）感染的意义，用质粒LCR和聚合酶链反应（PCR）检测170例STD门诊女性就诊者宫颈试子标本中的CT，对两项检测结果不一致的标本进行校正，对PCR阳性而LCR阴性者，将LCR标本稀释10倍重复LCR或用PCR反应的DNA模板作LCR检测。对PCR阴性而LCR阳性者，用另一对针对沙眼衣原体主要外膜蛋白（MOMP）基因的引物作PCR测试。将以上两项检测结果阳性的标本判断为真阳性，确定LCR和PCR的敏感性和特异性。发现170例患者中24例LCR检测阳性（14.2%)，26例PCR阳性（15.3%），对8例两项结果不一致的标本作了校正。LCR检测的敏感性和特异性分别为92%,99.3%；而PCR分别为92%，97.9%。结果提示LCR诊断女性宫颈CT感染具有较高的敏感性和特异性。     

连接酶链反应检测男性尿标本中沙眼衣原体

目的 评价连接酶链反应（LCR）检测男性尿标本中沙眼衣原体（CT）的敏感性和特异性。方法 取性病专科门诊162例男性就诊者尿道拭子作CT培养；同时取患者晨起或较长时间（2h以上）不排尿后的首次尿（FVU）标本，用质粒LCR检测CT。对培养和质粒LCR结果相异的标本，用另一针对CT主要外膜蛋白基因的LCR进行确证，参照“扩大的金标准”来确定检测结果。结果 质粒LCR检测的敏感性和特异性分别为100％和99．2％，而培养法分别为82．8％和100％。讨论 LCR是一种既敏感又特异的CT诊断方法。用尿标本作检测对象，可避免取尿道标本给患者带来的痛苦，可作为筛检男性CT感染的一种非损伤性方法。     

4179例性病门诊患者沙眼衣原体ELISA法检测结果分析

目的：了解ELISA方法在检测性病门诊患者标本沙眼衣原体的意义。方法：收集本中心1999年～2003年性病门诊患者共4179例尿道或宫颈拭子标本，进行衣原体抗原酶免疫(ELISA)测定法检测。结果：4179例患者，沙眼衣原体检出率11．0％，其中男性检出率9．2％，女性检出率14．1％。高于快速胶体金法的5．75％，高于细胞培养法的6．33％，低于荧光PCR法27．1％，结论：ELISA法检测泌尿生殖道沙眼衣原体具有操作简便、敏感性和特异性较高，在性病门诊中是值得推荐的一种可行有效的实验方法。     

三种方法检测性病患者泌尿生殖道沙眼衣原体感染的对比观察

目的：了解EUSA、酶联荧光测定技术(VIDAS)和荧光定量PCR三种方法检测泌尿生殖道沙眼衣原体感染结果。方法：分别用EUSA和VIDAS检测300例性病患者宫颈或尿道拭子沙眼衣原体抗原，同时以荧光定量PCR检测结果为对照。结果:ELISA与荧光定量PCR检测结果和VIDAS和荧光定量PCR检测结果比较，符合率均为98．33％。结论：荧光定量PCR、ELISA和VIDAS三种方法均可用于检测泌尿生殖道沙眼衣原体感染，其中ELISA不需要特殊仪器设备，更适合临床推广应用。     

Vidas CHL法在女性宫颈和尿液标本中检测沙眼衣原体的应用

目的：为研究能否用Vidas衣原体试验（CHL）检测女性尿液中沙眼衣原体。方法：采用Vidas CHL法检测126例有症状和220例无症状女性患者宫颈拭子及尿沉渣中沙眼衣原体。结果：与宫颈拭子沙眼衣原体细胞培养相比，Vidas CHL法检测有症状女性患者宫颈拭子、尿沉渣中沙眼衣原体敏感性分别为95．8％（P＞0．05）、87．5％（P＞0．05），特异性分别为98．0％（P＞0．05）、99．0％（P＞0．05）；检测无症状女性患者宫颈拭子、尿沉渣中沙眼衣原体敏感性分别为92．9％（P＞0．05）、50．0％（P＜0．05），特异性分别为97．9％（P＞0．05）、99．0％（P＞0．05）。结论：Vidas CHL法具有高度敏感性和特异性，可用来检测有症状女性患者尿沉渣中沙眼衣原体，但不能用来检测无症状女性患者尿沉渣中沙眼衣原体。     

应用VIDAS—酶联免疫荧光法（CHL）检测泌尿生殖道沙眼衣原体

目的:用VIDAS-酶联免疫荧光法(CHL)法检测男性尿道及女性宫颈管标本的沙眼衣原体。方法:用细胞培养、VIDAS—CHL法、“立明”衣原体快速免疫法(C—C法)平行检测539例有症状和277例无症状患者的拭子标本。结果:与扩大金标准比较,VIDAS—CHL法检测有症状患者拭子标本的敏感性为90.70％,特异性为96.65％;C—C法的敏感性为74.17％,特异性为98.45％;两者差异有显著性(x2=7.07,P<0.05);VIDAS—CHL法检测无症状患者拭子标本的敏感性为92.00％,特异性为98-81％;C—C法的敏感性为76.00％,特异性为98.41％;两者差异无显著性(x2=0.581,P>0.05);结论:VIDAS—CHL法具有高度敏感性和特异性,可用来检测男性尿道和女性宫颈管中沙眼衣原体。     

Detection of Chlamydia trachomatis by ligase chain reaction compared with polymerase chain reaction and cell culture in urogenital specimens.

OBJECTIVE--The aim of this study was to evaluate the newly developed ligase chain reaction (LCR) assay for the detection of Chlamydia trachomatis in urogenital specimens using cell culture and Amplicor PCR for comparison. SUBJECTS--Two hundred and eighty patients attending hospital or urban STD clinics (high-risk population, 62 men and 84 women) and obstetric/gynaecology clinics (low-risk population, 134 women) in Bordeaux, France. METHODS--Specimens from men were tested with LCR on urethral swabs and urine, with Amplicor or urine, with cell culture on urethral swabs. Specimens from women were tested with LCR, Amplicor and cell culture on endocervical swabs and with LCR on urine. When the three methods generated different results, the LCR and Amplicor tests were repeated on the remaining samples. Samples with discordant LCR and Amplicor results and a negative culture were further analysed by major outer membrane protein gene omp1-PCR. RESULTS--After analysis of discrepant results, the overall prevalence was 7.5% (21/280) calculated on the basis of an expanded "gold standard" defined as culture positive or LCR plus Amplicor positive or omp1-PCR positive for discrepant results between LCR and Amplicor tests. Of the 21, 20 were detected by LCR, 17 by Amplicor and culture. The specificity of LCR and Amplicor was 99.6%. CONCLUSION--The LCR Chlamydia trachomatis test is a highly sensitive nonculture technique and a good alternative test for the detection of chlamydial infections.     

Comparison of urine, first and second endourethral swabs for PCR based detection of genital Chlamydia trachomatis infection in male patients

OBJECTIVES: To compare endourethral swabs and urine as diagnostic specimens for the detection of genital Chlamydia trachomatis infection using the polymerase chain reaction (PCR), in male patients attending a genitourinary clinic and to assess whether the first endourethral swab used solely for diagnosing gonococcal infection could be used for C trachomatis detection as well. METHODS: Two endourethral swabs were taken from 80 male patients, in whom the likelihood of genital C trachomatis infection was high. The first swab was used for microscopy and culture for Neisseria gonorrhoeae, before being used for C trachomatis detection. First voided urine specimens were collected from 61 of these patients. All three specimens were processed for C trachomatis DNA detection using the Roche Cobas Amplicor PCR. A diagnosis of genital C trachomatis infection was made if any one of the specimens tested reproducibly positive. Samples from 13 patients showing discrepant PCR results between swabs and/or urine were retested by ligase chain reaction (LCR). RESULTS: Chlamydia trachomatis DNA was detected in 35 (43.8%) of the 80 patients. In 17 of the 35 patients (48.6%), all the genital specimens were positive. However, in 18 (51.4%) patients, one or more of the genital specimens had negative PCR results. Among the 18 patients with discrepant results, urine was found to be a more sensitive diagnostic specimen than the second urethral swab picking up 13 out of 16 positives (81.3%) as opposed to five out of 18 (27.8%). There was no significant difference between the two swabs. Retesting by LCR, of the samples from 13 of the 18 patients with discrepant PCR results confirmed them all as true positives, although as with PCR, not all specimens in the set were concordantly positive. LCR detected all the 13 positives in urine, while there was no difference in the detection rate between the first and the second urethral swabs. CONCLUSIONS: Urine appeared to be a better diagnostic specimen than the conventional second endourethral swab for C trachomatis detection by PCR in this cohort of male patients. There was no difference between the first swab, intended primarily for N gonorrhoeae testing and the second swab intended for C trachomatis detection. This raises questions over the need for the conventional second swab for detecting C trachomatis.     

Detection of Chlamydia trachomatis in genital swabs: comparison of commercial and in house amplification methods with culture

AIMS: To evaluate the sensitivity of the Roche Cobas, Roche Amplicor plate kit, ligase chain reaction (LCR), and an in house polymerase chain reaction (PCR) by titration of purified elementary bodies (EB) and also to test 245 urethral and endocervical specimens for Chlamydia trachomatis by the four assays as well as conventional culture. STUDY DESIGN: EB titrations were run in duplicate in each commercial assay and six times in the in house PCR. Clinical samples were aliquoted and tested by each assay and were considered positive if C trachomatis was detected by two or more separate tests or if the sample was either culture or immunofluorescence positive. Major outer membrane protein (MOMP) specific primers were used as a confirmatory assay for the in house PCR. RESULTS: The in house PCR, Roche Cobas Amplicor, LCR, and Amplicor plate kit gave detection limits of approximately 1, 1-2, 2, and 2-4 EBs respectively. By the criteria described above for definition of a C trachomatis positive result in clinical samples we identified 23 true positives among the 245 clinical specimens. The in house PCR detected all 23 giving a sensitivity of 100% and a specificity of 98%. The Roche Cobas Amplicor, Roche Amplicor plate kit, and LCR detected 21, 19, and 19 of these respectively giving sensitivities of 87.5%, 82%, and 82% respectively and specificities of 99.5%, 99%, and 100% respectively. The culture gave a sensitivity of 78% and specificity of 100%. CONCLUSION: All four amplification assays had a greater sensitivity than the culture used routinely in this laboratory. The in house plasmid PCR had the greatest sensitivity and when combined with confirmation by immunofluorescence detected the greatest number of positives. This increased sensitivity is likely to have been achieved by the use of a DNA purification step and of nested primers in the amplification stage and their combined use in routine diagnostic assays for chlamydia might increase the frequency of C trachomatis detections. However, this assay is much less user friendly than the two semiautomated commercial assays investigated in this study.


     

Detection of Chlamydia trachomatis antigen with an enzyme immunoassay

Urogenital swabs (571) were investigated with a solid-phase enzyme immunoassay for the detection of Chlamydia trachomatis antigen (Chlamydiazyme, Abbott). The results were compared with the conventional cell culture method (McCoy cell culture). Urogenital C. trachomatis infections were diagnosed with the cell culture in 14 of 122 male STD patients (12%), in 12 of 79 female STD patients (15%), in 23 of 89 prostitutes (26%), and in 3 of 115 asymptomatic males (3%). In comparison with cell culture, the sensitivity of Chlamydiazyme for urethral specimens from male STD patients was 86%. In female STD patients, for urethral specimens a sensitivity of 83% was found and for cervical specimens a sensitivity of 80%. The corresponding values for specimens from prostitutes were 60% and 100%, respectively. The specificity of Chlamydiazyme for urethral specimens of male STD patients reached 95%. With respect to urethral and cervical specimens of female STD patients, the specificity was 88% and 82%, respectively, and in prostitutes 92% each. The low specificity in female patients cannot be ascribed only to Chlamydiazyme since, after subcultivation and detection of inclusions by the use of fluorescein-labeled monoclonal antibodies, some of the false-positive Chlamydiazyme results turned out culture positive. This means that the specificity of Chlamydiazyme is actually higher. Because it can be performed rapidly and simply and reaches detection rates approaching those of the cell culture method, the enzyme immunoassay is an improvement in the diagnosis of C. trachomatis infections.     

Urine and the laboratory diagnosis of Chlamydia trachomatis in males.

OBJECTIVE--To determine whether the use of urine samples from male patients can replace urethral swabs for the rapid detection of Chlamydia trachomatis by the Pharmacia EIA. SETTING--The STD clinic, Adelaide, South Australia. PATIENTS--There were two separate groups of male patients. Group A (398) patients provided urethral specimens for the EIA and culture tests. The patients in Group B (356) provided an urethral swab and a urine sample for the EIA test. METHODS--The urine samples and urethral swabs were tested for the presence of C trachomatis by the Pharmacia Chlamydia EIA. In addition, the urethral swabs from Group A patients were cultured for the organism by standard cell cultures. The infected cell cultures were identified by an immunofluorescence test using a FITC-monoclonal antibody to C trachomatis (Kallestad). RESULTS--When the EIA was validated against culture, it showed a sensitivity of 100% and a specificity of 95% with the urethral swabs from Group A patients. The urine specimens were positive in 24% of those patients who yielded a positive EIA result in the urethral swabs. CONCLUSIONS--Although the EIA test on urethral swabs showed high sensitivity and specificity when validated against culture, our results showed that the use of urine samples cannot replace urethral swabs for the laboratory diagnosis of this sexually transmitted disease.     

连接酶链反应检测女性尿标本中的奈瑟淋球菌

目的 评价连接酶链反应（ＬＣＲ）技术诊断女性淋病的价值。方法 对１７０例ＳＴＤ门诊女性就诊者作宫颈拭予淋球菌培养和尿标本ＬＣＲ检测,对两项检测结果不一致的标本作聚合酶链反应（ＰＣＲ）检测,参照“扩大的金标准”确定ＬＣＲ技术和培养的敏感性和特异性。结果 １７０例患者中１６例ＬＣＲ检测阳性（９．４％）,１４例培养阳性（８．２％）,对８例两项结果不一致的标本作了ＰＣＲ检测。ＬＣＲ检测的敏感性、特异性、阳性预期值和一预期值分别为９４．１％、１００％、１００％和９９．４％;而培养法分别为８２．４％、１００％、１００％和９８．１％。结论 尿标本ＬＣＲ检测对诊断女性淋病具有较高的敏感性和特异性,适宜作大规模的ＳＴＤ普查,使筛查女性无症状感染者的机会大大增加。     

Evaluation of the Amplicor Chlamydia trachomatis test versus culture in genital samples in various prevalence populations.

OBJECTIVE--To evaluate a newly developed polymerase chain reaction (PCR) assay, Amplicor C trachomatis for the detection of C trachomatis in genital samples using cell culture for comparison. SUBJECTS--501 patients (431 women and 70 men) attending an STD clinic in Hôpital Pellegrin (high-risk population) and gynaecological clinics (low-risk population) in Bordeaux, France. METHODS--The genital samples (cervical and urethral) were tested for the presence of C trachomatis using the Amplicor test and using standard cell culture identified by the immunofluorescence test using a monoclonal antibody to C trachomatis. Discrepancies between the results of culture and Amplicor were further analysed by major outer membrane protein gene (omp1)-PCR of the specimens taken in transport media and by direct fluorescent antibody (DFA) staining of elementary bodies in culture transport tubes. RESULTS--After analysis of discrepancies, the revised sensitivity and specificity of PCR were 95.3% and 100% and the positive and negative predictive values were 100% and 99.5%, respectively. CONCLUSION--The present results indicate that the Amplicor assay is rapid, specific and more sensitive than the culture method. This test provides an excellent non-culture method for the detection of C trachomatis in various prevalence populations.