间接免疫荧光法检测沙眼衣原体包涵体抗原的研究

目的探讨基于沙眼衣原体全菌体兔血清抗体的间接免疫荧光法检测沙眼衣原体包涵体抗原的研究。方法将6×103IFU沙眼衣原体接种于Hep-2细胞,培养72 h,PCR检测验证;以兔抗沙眼衣原体全菌体血清抗体为一抗,用免疫荧光法检测沙眼衣原体包涵体抗原,同时采用传统的Giemsa染色、Lugol′s碘染对沙眼衣原体包涵体进行鉴定,并统计和比较其包涵体的检出率。结果经PCR鉴定为阳性的沙眼衣原体感染的Hep-2细胞,间接免疫荧光检测在细胞浆中出现绿色荧光,检出率为28.84%;经Giemsa染色和Lugol′s碘染验证胞浆中存在典型的包涵体结构,检出率分别为15.19%和17.36%,与免疫荧光法比较差异有统计学意义(P0.01)。结论基于兔抗沙眼衣原体全菌体血清的间接免疫荧光法可显著提高沙眼衣原体包涵体的检出率。     

五种不同沙眼衣原体实验室筛查试剂检测性能评价

目的通过对不同商品化沙眼衣原体(CT)检测试剂的检测结果,与Roche Cobas Amplicor检测结果的分析比较,评价5种沙眼衣原体检测试剂的性能。方法采用分层按比例的方法抽取32家医疗机构,收集各抽样单位2009年9月至11月期间,皮肤(性病)科、妇产科、泌尿科门诊就诊者的泌尿生殖道标本1 986人份。比较各个医疗机构采用的检测方法与Roche Cobas Amplicor检测方法所得检测结果,分析各不同检测试剂的特异度和灵敏度。结果共对1 986例样本进行了Roche Cobas Amplicor沙眼衣原体核酸检测,沙眼衣原体阳性为367例(18.48%)。3种免疫层析法(ICA)检测试剂中,ICA1(国产)、ICA2(国产)、ICA3(进口)灵敏度分别为34.48%、15.22%、52.53%;特异度分别为99.45%、98.04%、98.93%;阳性预测值(Positive predictive value,PPV)分别为93.75%、63.64%、92.86%;阴性预测值(Negative predictive value,NPV)分别为86.36%、83.68%、88.73%。2种聚合酶链反应(PCR)法检测试剂中,PCR1(国产)、PCR2(国产)灵敏度分别为70.09%、96.43%;特异度分别为99.44%、100.00%;阳性预测值分别为96.15%、100.00%;阴性预测值分别为94.34%、99.31%。用免疫层析试剂检测的四种样本间的敏感性和特异性有明显差异;PCR检测方法中,各类型标本的评价相近。结论不同公司生产的免疫层析检测试剂的性能存在显著差异,且总体水平均低于PCR法检测试剂。另外,不同类型标本对沙眼衣原体的检测也有影响,免疫层析法检测试剂较适用于女性阴道拭子和宫颈拭子标本的检测。     

衣原体感染

应用超高倍多媒体显微仪检测泌尿生殖道沙眼衣原体——倪少娟等（广西南宁广西壮族自治区妇幼保健院检验科530003）；《中国医疗器械杂志》，2005，29（4）：308．309[应用超高倍多媒体显微仪直接镜检并动态观察衣原体，阳性标本及随机抽取的20例阴性标本与VIDAS CHL法进行比较。镜检法在365例泌尿生殖道标本中检出沙眼衣原体32例；52例标本（镜检法阳性32例，阴性20例）两种检测方法结果差异无统计学意义，P〉0．05。超高倍多媒体显微仪直接镜检法可用于泌尿生殖道沙眼衣原体感染的诊断。]     

生殖系多种病原体感染与男性不育的关系

目的 探讨男性不育患者弓形体，解脲支原体，沙眼衣原体感染情况和抗精子抗体感染情况和抗精子抗体在男性不育中的作用。方法 应用多聚酶链反应（PCR）技术分别对112例男性不育病人和62例正常生育者的精液进行精浆弓形体DNWA，解脲支原体和沙眼衣原体DNA的检测，用金标免疫斑点试验检测精浆抗精子抗体（AsAb)。结果 男性不育组弓形体阳性20例（17．86％），解脲支原体阳性35例（31．25％），沙眼衣原体阳性18例（16．07％），抗精子抗体阳性33例（29．46％）。3种病原体感染率与抗精子抗体的阳性率均明显高于正常对照组（P＜0．01，且多种病原体感染患者抗精子抗体的阳性率明显高于单项感染者。结论 男性不育患者多种病原体感染后，导致抗精子抗体拉生是男性不育的重要免疫因素。     

泌尿生殖道标本中沙眼衣原体的直接检测及基因分型

应用聚合酶链反应（PCR）和限制性片段长度多态性（RFLP）分析，建立了沙眼衣原体（Ct）直接检测及基因分型的方法。与细胞培养法比较，评价其检测的敏感性。400份性病门诊患者的泌尿生殖道标本同时用培养和质粒PCR检测Ct，结果29份标本培养阳性，其中28份标本质粒PCR阳性，另有5份培养阴性的标本质粒PCR阳性。对培养和质粒PCR阳性的标本进一步做主要外膜蛋白基因（ompl）PCR或ompl巢式PCR，结果32份标本omplPCR或ompl巢式PCR阳性。将阳性标本的ompl产物用AluI和MspI等限制性内切酶酶切，产生的酶切带谱与标准株带谱比较，以鉴定阳性标本的基因型。结果E型为13份标本，F：6、G：4、D：3、J：2、K：2、H：1、非典型：1。研究表明：PCR－RFLP可直接检测及基因分型泌尿生殖道标本中的Ct，为一种敏感的血清分型替代方法。     

一种基于momp基因的衣原体分子生物学甄别技术的初步建立

目的利用PCR方法建立一种基于momp基因的衣原体分子甄别方法。方法根据衣原体momp基因的恒定区和可变区分别设计衣原体科特异性引物和种特异性引物，利用PCR方法对本实验室保存的衣原体进行扩增，以达到甄别的目的。利用限制性片段长度多态性（RFLP）分析科特异性PCR扩增产物，研究momp基因的多态性。结果利用建立的PCR方法对本实验室保存的九株衣原体进行了研究，结果表明八株衣原体都是鹦鹉热嗜衣原体，一株为沙眼衣原体。利用限制性片段长度多态性（RFLP）分析科特异性PCR扩增产物，分析了D34、B11001、CW2和CP12四株衣原体，结果可以产生两种不同的带型。结论利用建立的PCR方法可以达到检测甄别衣原体的目的，并可以通过RFLP分析衣原体的侵袭性。     

11254例生殖道沙眼衣原体感染荧光PCR检测结果分析

目的对宝安区人民医院门诊病人生殖道沙眼衣原体感染,经荧光聚合酶链式反应（PCR）检测结果进行分析,探讨其感染现状及临床检测意义。方法采用荧光PCR方法检测11254例患者的沙眼衣原体（CT）的脱氧核糖核酸（DNA）．结果11254例患者标本中共检出832例阳性,总阳性率为7．39％;其中男性检测3612例,阳性数426例,女性7640例,阳性数406例,阳性率分别为11．79％,5．31％;男性女性发病比率差异有统计学意义（x^2=150．167,P〈0．001）。沙眼衣原体阳性患者高发年龄为20～39岁,占84．86％;支原体的合并感染率为14．18％。结论临床医生应注意临床病人多重感染的情况,应对全社会进行积极的健康的性教育与宣传,普及检测手段。     

生殖系多种病原体感染与男性不育的关系

目的　探讨男性不育患者弓形体、解脲支原体、沙眼衣原体感染情况和抗精子抗体在男性不育中的作用。　方法　应用多聚酶链反应 (PCR)技术分别对 112例男性不育病人和 6 2例正常生育者的精液进行精浆弓形体 DNA、解脲支原体和沙眼衣原体 DNA的检测 ,用金标免疫斑点试验检测精浆抗精子抗体 (As Ab)。　结果　男性不育组弓形体阳性 2 0例 (17.86 % ) ,解脲支原体阳性 35例 (31.2 5 % ) ,沙眼衣原体阳性 18例 (16 .0 7% ) ,抗精子抗体阳性 33例 (2 9.4 6 % )。3种病原体感染率与抗精子抗体的阳性率均明显高于正常对照组 (P<0 .0 1) ,且多种病原体感染患者抗精子抗体的阳性率明显高于单项感染者。　结论　男性不育患者多种病原体感染后 ,导致抗精子抗体的产生是男性不育的重要免疫因素。     

精液沙眼衣原体感染与男性不育的关系

目的研究精液沙眼衣原体（CT）感染与男性不育的关系。方法选择精液CT阳性的男性不育者57例作为CT阳性组,另选择正常对照组15例。检测精液常规,免疫层析法检测CT感染情况,末端脱氧核苷酸转移酶（TdT）介导的TUNEL法及瑞—姬染色检测细胞凋亡,常规细胞形态学和透射电镜检测凋亡精子形态。结果CT阳性组精子凋亡率（33.03±14.98）%,正常对照组为（9.68±2.78）%,CT阳性组精子凋亡率增高（P〈0.01）。CT阳性组精子凋亡率与不育时间呈正相关,与精液量、精液活力、精子密度及正常形态率呈负相关（P均〈0.01）。结论精液CT感染诱导精子凋亡率增加。不育时间越长、精子数量越少、活力越差、正常形态率越低,精子凋亡率越高。     

性传播疾病

女性生殖道性传播疾病病原体混合感染调查——申建维等（武钢第二职工医院湖北武汉430085）；《中华医院感染学杂志》2007，17（5）：533-534[目的：探讨女性生殖道性传播疾病病原体混合感染的检测现状及临床意义。方法：分别采用革兰染色镜检、金标法、培养法、酶标法进行淋菌、沙眼衣原体、解脲脲支原体、人支原体、     

The clinical significance of specific IgA antibodies in local secretions in cases with chlamydial urogenital infections--comparison with serum antibodies and analysis of antigen specificity by immunoblotting assay]

IgA antibody titers to C. trachomatis in local secretions were measured by immunoperoxidase assay (Savyon kit) in male and female cases with various urogenital infections, and the clinical significance of IgA antibody in the local secretion was discussed. In addition, the antigen specificity of the IgA for C. trachomatis in the local secretions was analyzed by immunoblotting assay. 1) In female cases with cervicitis and male cases with urethritis, the positive rate of IgA antibody in their secretions was higher in cases with C. trachomatis antigen than in those without it. In addition, the IgA antibody titers in their secretions tended to be higher than in serum, suggesting that the result reflected a local immune response at the site of infection. 2) In cases with chronic prostatitis, a condition in which detection of antigen at the site of infection was difficult, the positive rate of IgA antibody in prostatic secretion was 23.6%. We confirmed that most of the IgA antibodies in prostatic secretions were of the secretory type. 3) IgA antibodies in secretions reacted to the major outer membrane protein (MOMP) and 60-Kd polypeptides of the outer membrane of C. trachomatis by immunoblotting assay, proving that they were the secretory IgA antibodies specific for C. trachomatis. These results described above confirmed that measurement of IgA antibody titers in local secretions by immunoperoxidase assay and immunoblotting assay was useful for the diagnosis of chlamydial urogenital infections such as chronic prostatitis, which the antigen detection was usually difficult. Examination of IgA antibody in local secretions was considered to be useful for making a correct diagnosis even in cases who were suspected to have C. trachomatis infection but showed negative antigen.     

Usefulness of real-time PCR in detecting Chlamydia trachomatis and Neisseria gonorrhoeae in endocervical swabs and first-voided urine specimens

We evaluated performance of Abbott RealTime CT/NG assay (real-time PCR, Abbott Japan) for detect Chlamydia trachomatis and Neisseria gonorrhoeae by real-time PCR in 88 female patients with cervicitis symptoms seen at gynecological clinics and 100 male patients with urethritis symptoms seen at urological or dermatology clinics in Kitakyushu, Japan. Endocervical swab and first-voided urine (FVU) specimens were then collected from women and FVU specimens from men. Detection rates of C. trachomatis and N. gonorrhoeae by real-time PCR in the 3 types of specimens were compared to those by ProbeTec ET assay (ProbeTec, BD Diagnostic System). The overall positive concordance between real-time PCR and ProbTec were 97.1% (66/68) for C. trachomatis and 100% (33/33) for N. gonorrhoeae, C. trachomatis detection yielded 3 discordant results in endocervical specimens and 1 discordant result in male FVU by real-time PCR and ProbTec. Three of 4 reexamined using Aptime Combo 2 Assay (Fuji Rebio Inc.) were positive for C. trachomatis. Endocervical swab and FVU specimen results for C. trachomatis were discordant in 3 cases in real-time PCR and 4 in ProbeTec. Subjects with 2 or more positive endocervical awab results in female or male FVU specimens were assumed to be "true positive" for C. trachomatis. The sensitivities of real-time PCR for detecting C. trachomatis was 94.4% in endocervical swabs, 77.8% in female FVU and 97.4% in the male FVU. The sensitivities for real-time PCR for detecting N. gonorrhoeae was 100% in all 3 specimentypes. Abbott RealTime CT/NG assay was useful for detecting C. trachomatis using endocervical swabs or male FVU specimens and for detecting N. gonorrhoeae using endocervical swabs and all FVU specimens.     

Detection of anti-Chlamydia trachomatis antibody by means of enzyme immunoassay using synthetic peptide antigen]

Newly developed diagnostic kits for the detection of Anti-Chlamydia trachomatis, Peptide-Chlamvdia (LOY: Meiji Milk Products Co., Ltd., Tokyo; for IgG and IgA), were evaluated using the microimmunofluorescence assay (MIF) as the gold standard. These results were also compared to results of testing by Sero-IPALISA and immunoblot (I-B). Detection by LOY in based on enzyme immunoassay with synthetic peptides as the antigen. Thirty serum samples from pediatric patients and 130 serum samples from gynecology patients were used. All 26 pediatric samples that were positive for Chlamydia pneumoniae IgG antibody tested negative with LOY, indicating that the presence of the antibody against C. pneumoniae did not affect the assay by LOY. For 90 gynecological samples, the total, the positive and the negative agreement rates for IgG were quite high; i.e. 87.8%, 90.0% and 70.0% (LOY vs MIF), 85.6%, 85.0% and 90.0% (Sero-IPALISA vs MIF), and 92.0%, 94.9% and 70.0% (I-B vs MIF), respectively. On the other hand, many cases of MIF (-) and LOY (+) discrepancy were seen in IgA detection. In order to better understand the basis for such disagreement. 34 serum samples were collected from patients whose cervical samples were negative for the Chlamydia group antigen based on the assay with IDEIA-Chlamydia. They were then assayed by MIF and LOY. The total, the positive and the negative agreement rates for IgG were 91.2%, 100% and 90.9%, while the total and the negative agreement rates for IgA were 88.2% and 88.2% (there were no IgA positive cases). Furthermore, 6 serum samples (1 case of MIF (+) LOY (+) and 5 cases of MIF (-) LOY (+)) were provided to determine whether LOY detects C. trachomatis specific IgA antibody. Increasing amounts of C. trachomatis serovar L2 were added to the serum samples resulting in a progressive decrease in their reactivity in the LOY assay. These results lead us to speculate that LOY can reveal even low levels of C. trachomatis specific IgA antibody. In conclusion, LOY can be used as an useful kit for detecting C. trachomatis antibody.     

Evaluation of the new nucleic acid amplification system for direct detection of Chlamydia trachomatis and Neisseria gonorrhoeae in women

The performance of a real-time DNA amplification assay, BD ProbeTec ET System (BDPT, BD Diagnostic Systems), to detect Chlamydia trachomatis and Neisseria gonorrhoeae on endocervical and oropharyngeal samples was evaluated. After obtaining informed consent, 364 endocervical, 363 urine and 247 oropharyngeal specimens were collected from 307 cases. The overall agreement rate of the BDPT and Amplicor (AMP, Roche) assays for the detection of C. trachomatis and N. gonorrhoeae in endocervical samples was 99.2% (361/364) for C. trachomatis and 99.5% (362/364) for N. gonorrhoeae. Assay of oropharyngeal swabs by the BDPT yielded 21 C. trachomatis positives, and 19 of them were C. trachomatis negative by the DNA probe assay (Gen-Probe PACE). The AMP assay showed that 16/19 (84.2%) of the BDPT +/DNA probe - samples were positive. The BDPT also yielded 21 N. gonorrhoeae positives, 15 of which were negative with the DNA probe. Additional testing showed that all 15 BDPT +/DNA probe - samples were positive by the established nested PCR method. Our data suggest that the performance of the BDPT is comparable to that of AMP for detection of C. trachomatis and N. gonorrhoeae in endocervical swab samples and that it may be a useful method for detecting of C. trachomatis and N. gonorrhoeae in oropharyngeal samples clinically.     

Detection of Chlamydia trachomatis from urethral swab in male urethritis by polymerase chain reaction]

A polymerase chain reaction (PCR) with Chlamydia trachomatis-specific primers was applied for detection of C. trachomatis from urethral swab in male urethritis. The results were compared with those of culture method for detection of C. trachomatis. Of 18 clinical specimens tested in this study, inclusion bodies of C. trachomatis were detected in 11 specimens by the culture method. For PCR, sample DNA was prepared from transport medium in which urethral smear was suspended and two oligonucleotides based on sequences within the major outer membrane protein gene from C. trachomatis serovar L2 were used as extension primers. In 12 of the 18 specimens, 242bp DNA fragment was amplified by PCR and demonstrated to be the DNA fragment of C. trachomatis by Southern blot hybridization. No DNA of 242bp was amplified by PCR from five specimens in which any inclusion bodies of C. trachomatis were observed or from a specimen in which one inclusion body per cover slip was detected by culture method. C. trachomatis DNA of 242bp was amplified from all specimens in which 14 and more inclusion bodies per cover slip were detected by culture method. In two specimens concluded s negative by culture method, amplified C. trachomatis DNA were detected by PCR. Thus, the PCR would be a more simple and sensitive method for detection of C. trachomatis, compared with the culture method.     

Use of a peptide based enzyme immunoassay in diagnosis of Chlamydia trachomatis triggered reactive arthritis.

OBJECTIVE: To assess the presence of circulating IgA and IgG antibodies to Chlamydia trachomatis in sera of patients with reactive arthritis (ReA) and other arthritides. METHODS: A peptide based enzyme immunoassay (EIA) was used to study 132 patients divided into 5 groups: C. trachomatis triggered ReA, uroarthritis, enteroarthritis, oligoarthritis, and rheumatoid arthritis (RA). Followup sera were available from 19 patients. RESULTS: An increased prevalence of C. trachomatis antibodies was observed in patients with ReA triggered by C. trachomatis; 18/23 (78%) had IgA and 19/23 (83%) had IgG antibodies. In patient groups with uroarthritis (n = 12), enteroarthritis (n = 56), oligoarthritis (n = 16), and RA (n = 25), C. trachomatis IgA/IgG antibodies were detected in 58%/75%, 27%/21%, 25%/31%, and 20%/32% of patients, respectively. Both the IgA and IgG antibodies were positive in 74%, 50%, 16%, 25%, and 12% of the patients with C. trachomatis triggered ReA, uroarthritis, enteroarthritis, oligoarthritis, and RA, respectively. Based on positivity of both isotypes the sensitivity of the assay was 74% and specificity 84%. In the followup sera, an association between circulating C. trachomatis-specific antibody concentrations and clinical disease outcome of the arthritis was seen in patients with culture-positive C. trachomatis triggered ReA. CONCLUSION: C. trachomatis species-specific peptide EIA correlates well with conventional diagnosis of primary C. trachomatis infection in patients with ReA. This assay may be a valuable contribution to the diagnosis of C. trachomatis triggered ReA.     

Study of serological diagnosis in ocular chlamydial infection with IPAZYME

We employed a indirect immunoperoxidase assay (IPAZYME in the evaluation of IgG and IgA antibody for Chlamydia trachomatis in serum samples from 218 patients such as cicatricial trachoma 55 cases, culture-positive adult inclusion conjunctivitis 48 cases and culture-negative conjunctivitis 47 cases, aged people, 68 cases as controls respectively. Frequency of positive IgG antibody showed a significant difference between adult inclusion conjunctivitis or cicatricial trachoma and controls. IgA antibody was positive in 25/48 (52%) in adult inclusion conjunctivitis and in 7/55 (12%) in cicatricial trachoma cases. Serum IgA antibody against Chlamydia trachomatis is of value to be an index of active ocular chlamydial inflammation. The correlation between severity of conjunctival cicatrix or corneal punnus and titers of IgG antibody was also significant.     

性病门诊人群泌尿生殖道沙眼衣原体混合感染及分型研究

目的 了解性病门诊人群泌尿生殖道沙眼衣原体混合感染及基因型分布特点。方法 对2048例性病门诊就诊者分别取尿道或宫颈拭子，实时聚合酶链反应（PCR）检测沙眼衣原体（CT）感染，阳性标本用套式PCR扩增ompl VD2区片断，反向线点杂交（RLB）检测特异性型别。结果 CT总体感染率为15．9％（325／2048）。对287份阳性标本成功分型，型别分布为：F21．6％，E20．9％，J19．9％，D12．9％，G8．4％，K4．2％和H1．4％，31份（10．8％）为混合感染。结论 在性病门诊人群中CT感染率较高，主要流行型别为F、E和J型，且具有较高的混合感染率。     

Simultaneous detection of Neisseria gonorrhoeae and Chlamydia trachomatis by PCR in genitourinary specimens from men and women attending an STD clinic

Neisseria gonorrhoeae and Chlamydia trachomatis are the two most common bacterial sexually transmitted infections that manifest primarily as urethritis in males and endocervicitis in females, though the infection may be asymptomatic especially in women. Since complications may occur in untreated symptomatic and asymptomatic infected individuals, early diagnosis and treatment of infected individuals is required to prevent severe sequelae and spread of these diseases. Recently molecular amplification assays like Polymerase Chain Reaction (PCR) and Ligase Chain Reaction (LCR) have been found to be highly sensitive and specific methods for detection of N. gonorrhoeae and C. trachonmatis not only in urethral and cervical specimens but also in urine. The objective of this study was to screen male and female Sexually Transmitted Disease (STD) clinic attenders, with and without symptoms suggestive of urethritis and cervicitis for presence of N. gonorrhoeae and C. trachomatis using a multiplex PCR based assay, to compare its performance with culture for N. gonorrhoeae and Direct Fluorescent Antibody (DFA) staining for C. trachomatis and also to compare the efficacy of PCR test performed on urine and genital swab specimens collected from this high risk group. Genital specimens and urine was collected from STD clinic attenders. N. gonorrhoeae and C. trachomatis was detected in genital specimens by culture and DFA respectively. Multiplex PCR was used to detect N. gonorrhoeae and C. trachomatis infection in both genital and urine specimens. Among men with urethritis, N. gonorrhoeae was detected in 70% by culture and 77% by PCR, while C. trachomatis as detected in 7.5% by DFA and 17.5% by PCR. Among females with endocervicitis, N. gonorrhoeae was detected in 7.7% by culture and 30.7% by PCR, while C. trachomatis was detected in 7.7% by DFA and in 15.4% by PCR. None of the asymptomatic males were positive for N. gonorrhoeae and C. trachomatis by conventional methods, while 43.9% were positive for N. gonorrhoeae and 7.5% for C. trachomatis by PCR. Fifty per cent of asymptomatic women were positive for C. trachomatis by PCR alone. We encountered PCR positive but culture/DFA negative results and also PCR negative but culture/DFA positive results. In view of this a single PCR test cannot be used for diagnosis and treatment of N. gonorrhoeae and C. trachomatis infection unless confirmed by a second test.