沙眼衣原体诊断技术研究进展

沙眼衣原体（Ct）是一种特殊的病原体,具有与革兰氏阴性细菌相似的细胞壁,含有DNA和RNA两种类型的核酸,严格寄生于宿主细胞内,沙眼衣原体是一种能够通过滤器,以二分裂方式繁殖的原核细胞型微生物.它具有两种形态：在细胞外具有高度传染性的为原体 在细胞内进行复制、无传染性的为始体.它可以引起非淋球菌尿道炎等许多泌尿生殖道相关疾病,近年来其感染率和危害性已超过淋病奈瑟菌而居性传播疾病之首,眼部衣原体侵入人体眼结膜和角膜引起沙眼和包涵体结膜炎,是世界范围致盲的首要病因.约80%的被感染女性无临床症状,感染反复迁移,造成病理改变,可导致复杂的并发症.因此,早期、简便、快速、特异地发现Ct,对临床的诊断,疾病的早期治疗和预防其流行等具有重要的意义.目前,对沙眼衣原体的诊断方法主要有培养法,免疫学法和分子生物学法.     

Cytopathologic detection of Chlamydia trachomatis in vaginopancervical (Fast) smears

The value of the Papanicolaou-stained vaginopancervical (Fast) smear in the detection of chlamydial infection has been disputed. We examined 116 satisfactory Fast smears from 203 women enrolled in the Johns Hopkins Fertility Control Clinic and compared tissue-culture results with cytopathologic detection using various published morphologic criteria. All Chlamydia culture-positive cases were reviewed, and certain cytologic features considered helpful in the detection of chlamydial infection in cervical smears obtained from this selected high-risk population were identified. The changes that had the highest correlation with tissue culture included fine vacuolation of metaplastic endocervical cells, giving their cytoplasm a rarefied "moth-eaten" appearance. Using these criteria, cytopathologic changes of chlamydial infection were observed in 24 of 28 cases of tissue-culture-positive cases and in 8 of 88 tissue-culture-negative cases. The sensitivity and specificity of the Fast-smear cytodiagnosis of Chlamydia infection utilizing these morphologic changes and compared with tissue culture were 86% and 91%, respectively. Other cytologic features, including inflammatory background and intracytoplasmic structures consistent with initial and intermediate chlamydial bodies within the metaplastic cells, were found to be useful although less specific and less sensitive. The implications of these diagnostic features, the conditions to be considered in their differential diagnosis, and the pitfalls of chlamydial cytodiagnosis and the chlamydia culture studies have been critically reviewed. Study design and the high unsatisfactory cervical smear rate are discussed.     

Use of the ligase chain reaction on urine of men and their female sexual partners for detection of genital Chlamydia trachomatis infection

Objective: The purpose of the present study was to evaluate an in vitro DNA amplification assay named the ligase chain reaction (LCR) for the detection of Chlamydia trachomatis cryptic plasmid DNA in urine from men and women, in comparison with urethral swab culture in men and cervical swab culture in women.
Methods: 591 patients (394 men with urethritis and 197 female sex partners) attending a center for sexually transmitted diseases in northern Italy between January 1994 and January 1995 were enrolled in this study. A cervical swab was collected from women and a urethral swab from men for standard tissue cell culture. From each patient 20 mL of the first stream of the urine (FVU), taken at least 2 h after the last urination, were collected for LCR analysis. Discrepant results were further analyzed by direct fluorescence and a LCR with alternative primers.
Results: In men the prevalence of C. trachomatis infection by urethral culture was 13.45% and, after resolution of discordant results, the LCR method performed on FVU showed a sensitivity, specificity, positive predictive value and negative predictive value of 89.4%, 100%, 100% and 98.2%, respectively; the sensitivity of tissue cell culture was 92.8%. In female sex partners, the prevalence of C. trachomatis infection by cervical culture was 3.04%; LCR detected eight true positive samples, two more than tissue cell culture, and no false-negative results.
Conclusion: LCR analysis of FVU is a rapid, non-invasive technique and represents a good alternative to tissue cell culture. Further study is needed to investigate possible LCR inhibitors present in urine samples.     

实时荧光定量PCR检测沙眼衣原体的研究

目的采用TaqMan探针建立检测沙眼衣原体的实时荧光定量PCR（real-time PCR）方法。方法根据沙眼衣原体外膜蛋白A的基因（ompA）序列设计引物和探针,以克隆的ompA部分基因片段作DNA模板,建立实时荧光定量检测方法。结果建立的荧光定量PCR检测方法的最低检出限为5 copies/反应,检测线性范围100～107线性关系良好（r2=0.997）,比巢式PCR敏感100倍;且与鹦鹉热衣原体、淋球菌、解脲脲原体、大肠杆菌等病原菌DNA以及人基因组DNA均无交叉反应,表明该方法具有良好的特异性。以巢式PCR作参比,建立的荧光定量PCR法检测沙眼衣原体的阳性符合率为100.00%,阴性符合率为95.09%,总符合率为96.81%。结论建立的检测沙眼衣原体实时荧光定量PCR具有特异性强和敏感性高的特点,可快速检测样本中微量沙眼衣原体DNA,适用于对沙眼衣原体进行大规模筛选。     

Chlamydia trachomatis antigen detection in pregnancy and its verification by antibody blocking assay

Purpose: To detect the prevalence of genital infection caused by Chlamydia trachomatis in pregnant women and also to confirm the positive results using blocking antibody assay. Methods: Endocervical specimens were collected from 200 symptomatic and asymptomatic pregnant women attending the ANC OPD at M P Shah Medical College, Jamnagar. The samples were tested for presence of Chlamydia trachomatis antigen using the monoclonal antibody. Blocking antibody assay was used to further verify the positive results. Results: Out of 200 pregnant women, 38 (19%) were found positive for Chlamydia trachomatis antigen. Out of the 68 symptomatic patients, C. trachomatis antigen was detected in 26.4%. After verification of the positive samples 13.6% of the asymptomatic pregnant women were found to be harbouring the infection in their genital tract. Two (5.2%) out of the 38 positive samples, on verification with the blocking antibody assay, were found to be false positive by IDEIA,^TM thus the specificity of the IDEIA^TM being 94.8%. In patients with previous history of abortions, 27.7% were tested positive for C. trachomatis infection. Conclusions: Significant number of pregnant women shad C. trachomatis antigen in their endocervical canal, which can be easily diagnosed by this simple enzyme immuno assay having a specificity of 94.8%. Verification of positive results by antibody blocking assay can further improve the specificity of this non-culture test. Asymptomatic patients should also be screened for the infection. History of previous abortions places the patient at a higher risk for C. trachomatis infection thus such patients should be definitely tested for chlamydia infection.     

Usefulness of transvaginal hydrolaparoscopy in investigating infertile women with Chlamydia trachomatis infection

BACKGROUND: A new technique called transvaginal hydrolaparoscopy (THL) was recently developed for the exploration of the tubo-ovarian structures in infertile patients without obvious pelvic pathology. This study was performed to investigate the usefulness of THL to evaluate Chlamydia trachomatis tubal infertility. METHODS: Forty-one women with primary and secondary infertility participated in this study. Fourteen had past C. trachomatis infection. In 38 (92.7%) of the 41, access to the pouch of Douglas was obtained. In total, 71 (93.4%) out of 76 adnexa were clearly visualized. Thirty-seven patients were analysed and compared their tubal passages and peritubal adhesions using both hysterosalpingography (HSG) and THL. Twenty-four tubes from 14 patients with past C. trachomatis infection and 44 tubes from 23 patients without a history of C. trachomatis infection were compared. RESULTS: For the diagnosis of the tubal passage, there were no significant differences in the discrepancy rates between HSG and THL, in patients with and without past C. trachomatis infection. In 14 (58.3%) of the 24 tubes from patients with past C. trachomatis infection and in eight (18.2%) of the 44 tubes from patients without infection, peritubal adhesion was diagnosed only by THL. There was a significant difference in the discrepancy rates of the diagnosis of peritubal adhesion between HSG and THL in the two groups (P = 0.0007 ). CONCLUSIONS: These results suggest that C. trachomatis infection is highly associated with peritubal adhesion which is difficult to diagnose by HSG. Therefore, in C. trachomatis antibody-positive patients, exclusion of tubal pathology by THL or standard laparoscopy should be carried out to consider appropriate treatments. Although THL is not a substitute for laparoscopy, it can be proposed as a first line procedure in the early stages of the infertility investigation.     

Association between Chlamydia trachomatis and abnormal uterine bleeding

PROBLEM: The purpose was to identify distinct inflammatory markers in endometrial tissues of women with abnormal uterine bleeding (AUB) and Chlamydia trachomatis infection. METHOD OF STUDY: Archived endometrial specimens from 92 randomly selected premenopausal women with AUB were examined for C. trachomatis using the species-specific monoclonal antibody against major outer membrane protein (MOMP) and for histopathology associated with inflammation. Statistical analyses included single and multiple logistic regression. Diagnostic accuracy was summarized using receiver operating characteristic (ROC) curves. RESULTS: Chlamydia trachomatis was detected in 44 (48%) of 92 AUB specimens. There were statistically significant correlations of positive MOMP with higher counts of plasma cells (P < 0.01), macrophages (P < 0.0001), and lymphocytic foci (P = 0.01). The ROC curve for macrophages was the strongest predictor (area under the curve = 0.82) for C. trachomatis. CONCLUSION: The prevalence of C. trachomatis in women with AUB is under-estimated. Macrophages appear to be a strong marker for the presence of C. trachomatis in the endometrium.     

沙眼衣原体套式（Nested）PCR检测研究

本文报告沙眼衣原体（ＣＴ）的套式（Ｎｅｓｔｅｄ）ＰＣＲ检测方法，本方法ＣＴ隐匿性质粒为靶基因，外套引物采用国外学者所报告灵敏度和特异性较高的序列，内套引物自行设计，经方法学考核表明本法灵敏度和特异性极高。１４６例临检标本套式ＣＴ，ＰＣＲ阳性检出率为３６．３％而市售ＰＣＲ试剂盒（其引物序列与本文外套引物相同）阳性检出率仅为４．１％，前者明显高于后者（Ｐ〈０．０１）。     

A distinct cellular profile is seen in the human endocervix during Chlamydia trachomatis infection

Problem The endocervix is a major target of Chlamydia trachomatis infection, but little is known about the immune repertoire in this tissue, or its response to these common bacteria. Method of study Using a cytobrush, we isolated cells from the endocervix of 20 women during C. trachomatis infection, and post‐antibiotic treatment. Endocervical swabs and blood were taken in parallel. Endocervical cells were enumerated, and endocervical and blood T cells immunophenotyped. Chlamydia trachomatis was genotyped by sequence analysis of the OmpA gene, and quantified by culture. Results Chlamydia trachomatis genotypes were D, E, F and Ia, and infectious burden varied considerably. Endocervical T cell and neutrophil numbers were highly elevated during infection, with both CD4 and CD8 T‐cell subsets accumulating. Regardless of the presence or absence of infection, the endocervical cell infiltrate was dominated by effector memory T cells, and the numbers of CCR5 and CD103 expressing T cells was significantly higher than in the blood. Human leukocyte antigen (HLA‐DR) expression by endocervical T cells was significantly increased during infection. Conclusion The human endocervix exhibits a distinct cellular response to C. trachomatis infection that can be longitudinally evaluated by cytobrush sampling. Infecting organisms can be sampled and analyzed in parallel.     

炎症性细胞因子在沙眼衣原体肺感染中的表达及其与机体防御的关系

目的 探讨炎症性细胞因子TNF-a，IL-1β和IL-6在沙眼衣原体肺感染中的产生及与机体防御的关系。方法 用沙眼衣原体小鼠肺炎株(MoPn)通过鼻腔感染小鼠，用过氧化物酶连接的鼠抗衣原体脂多糖单抗染色HeLa229细胞检测衣原体在肺组织的生长；通过测定中性粒细胞髓过氧化物酶(MPO)检测中性粒细胞在小鼠肺组织中的聚集；用RT-PCR检测小鼠肺组织炎症性细胞因子mRNA表达。结果 MoPn感染后第2天，肺组织有衣原体生长，于感染后第7天达高峰，第21天清除感染的衣原体。感染后第3天，前炎症细胞因子TNF-α，IL-B和IL-6在小鼠肺组织中的表达明显增高，IL-1β mRNA表达于感染第7天后降低，而TNF-a和IL-6 mRNA的表达至感染后第14天仍维持较高水平。高水平的TNF-α，IL-lβ和IL-6表达出现于中性粒细胞浸润的高峰期。结论 衣原体肺感染诱导前炎症细胞因子TNF-α，IL-1β和IL-6的高表达，可能具有中性粒细胞趋化活性，并参与衣原体感染的免疫防御。     

孕妇解脲支原体、沙眼衣原体感染的诊断与药物干预

目的探讨妊娠期孕妇解脲支原体（UU）、沙眼衣原体（CT）感染的发病率及其对感染者的治疗效果。方法对108例妊娠妇女采用核酸杂交的方法检测宫颈分泌物,对标本UU、CT-DNA进行DNA扩增、核酸杂交检测。结果 108例妊娠早中期孕妇宫颈分泌物UU、CT的阳性率分别是33.33%,7.41%,口服阿奇霉素治疗后检测UU、CT阳性率分别是17.59%,1.85%。两组间有显著性差异（P=0.001或P=0.002）。结论解脲支原体、沙眼衣原体是妊娠期妇女感染的常见病原体,临床治疗效果显著值得推广应用。     

Urethritis associated with Chlamydia trachomatis: comparison of leukocyte esterase dipstick test of first-voided urine and methylene blue-stained urethral smear as predictors of chlamydial infection

The use of nucleic acid amplification tests for the diagnosis of C. trachomatis has made it possible to send urine samples instead of urethral swab specimens to the laboratory. The sensitivity is very high, but not 100%, and we continue to perform a test for urethritis at our STD clinic. The aim of this study was to compare the performance of two alternative tests in the diagnosis of urethritis as predictors of C. trachomatis infection: the leukocyte esterase (LE) dipstick test of first-voided urine and polymorphonuclear leukocyte counts in a methylene blue-stained (MBS) urethral smear. Urine samples from 480 male patients attending an STD clinic were analysed using the LE test and LCR assay for C. trachomatis; urethral samples were analysed with MBS urethral smear and LCR. The majority (75.8%) of the 480 patients examined were asymptomatic. Chlamydial infection was detected in 50 patients. The sensitivity, specificity and positive predictive value of the LE test for predicting C. trachomatis infection were 46.0, 91.6 and 39.0%, respectively, among all patients examined and 25.9, 95.8 and 33.3%, respectively, among the asymptomatic patients. The corresponding values for the MBS urethral smear were 76.0, 82.1 and 33.0% among all patients and 63.0, 89.6 and 32.7% among the asymptomatic patients. At our STD clinic we chose to perform the examination of MBS urethral smears in the diagnosis of urethritis because of its higher sensitivity relative to the LE test for predicting C. trachomatis.     

Recovery of cytomegalovirus and Chlamydia trachomatis from vaginal tampons

Herpes simplex virus (HSV), cytomegalovirus (CMV), and Chlamydia trachomatis are important agents in venereal and neonatal disease. Vaginal tampon culture for HSV has previously been demonstrated to be a simple and effective technique for quantitative culture of cervical secretions. We have evaluated the tampon culture as a means of performing quantitative cultures for CMV and C trachomatis. Cell-free and cell-associated CMV were quantitatively recovered from vaginal tampons when extraction was performed within one hour of tampon inoculation. However, when tampons were stored, there was a rapid loss of infectivity over time at all storage temperatures except -70 degrees C. C trachomatis was quantitatively recovered from tampons stored at less than or equal to 4 degrees C for four days. When stored at -70 degrees C, C trachomatis was stable on tampons for more than one week. Because HSV, CMV, and C trachomatis are stable in a single transport medium, a tampon stored at 4 degrees C briefly or at -70 degrees C for one week could be utilized for the detection of all three agents.     

Comparison of strand displacement and ligase chain amplification for detection of Chlamydia trachomatis infection in urogenital specimens

Two amplification tests for the diagnosis of Chlamydia trachomatis infection, namely the ligase chain reaction (LCx) and the strand displacement assay (ProbeTec), were compared using samples from 1183 patients at sexually transmitted disease clinics. The overall prevalence of positive results was 8.0%, with agreement between the two assays of 98.8%. For endocervical, urethral and male urine samples, agreement was 99.3%, 99.4% and 97.7%, respectively. For ten discrepant samples, alternative amplification assays suggested that the LCx and ProbeTec assays gave erroneous results in six and four cases, respectively. Inhibition of amplification was observed with three (0.25%) urine specimens.     

Genotyping of Chlamydia trachomatis strains from cultured isolates and nucleic acid amplification test-positive specimens

Urogenital strains of Chlamydia trachomatis are divided into several serogroups (D-K). Since these serovars are represented with differing prevalence in the population a serotyping of strains is necessary, when characterising the epidemiological situation. The aim of this study was the genotyping of C. trachomatis strains, the comparison of the results with those of serotyping, and the genotyping of positive specimens using commercial nucleic acid amplification tests (NAAT). The Chlamydia trachomatis major outer membrane protein gene (omp1) from 55 isolated strains and 36 NAAT-positive specimens was amplified by polymerase chain reaction (PCR). The restriction fragment length polymorphism (RFLP) patterns of these amplicons were compared with those of reference strains. The genotypes E and F were found to be most prevalent. The results are discussed considering other studies, genovariants and epidemiology.     

Multicenter comparative evaluation of two rapid immunoassay methods for the detection of Chlamydia trachomatis antigen in endocervical specimens

Objective: To evaluate two rapid immunoassay methods, QuickVue-Chlamydia (Quidel Corp., San Diego California) and Kodak SureCell (Kodak Corp., Rochester, NY) for the detection of Chlamydia trachomatis antigen in endocervical swabs from high- and low-risk females.
Methods: Seven hundred and twenty-four females attending three clinics were enrolled in the study. The results were compared to McCoy's or BGMK cell culture and discrepancies resolved with polymerase chain reaction and direct fluorescent antibody tests performed on left-over culture specimens.
Results: The sensitivity, specificity, predictive value of a positive and predictive value of a negative of the QuickVue Chlamydia assay were 92.0%, 99.1%, 92.0% and 99.1%, respectively. The sensitivity, specificity, predictive value of a positive and predictive value of a negative of the SureCell assay were 90.0%, 99.8%, 98.6% and 98.8%, respectively.
Conclusions: The performances of the two immunoassay methods were similar, and slight differences in sensitivity and specificity were not statistically significant. Both immunoassay methods performed well in high- and low-risk patient groups, both for symptomatic and for asymptomatic patients.     

聚合酶链反应检测丝虫病患者泌尿道沙眼衣原体及解脲支原体的研究

本文采用聚合酶链反应方法,检测了５０ 例丝虫病患者尿中沙眼衣原体（ＣＴ） 及解脲支原体（ＵＵ）ＤＮＡ,同时选择３０ 例正常人作为对照。结果显示,丝虫病患者尿中ＣＴＤＮＡ、ＵＵ ＤＮＡ 和两者混合感染检出率分别为３０ ％ 、２５ ％ 和２０ ％ ,高于正常对３６３％ （Ｐ＜０－０１）,ＣＴ及ＵＵ 感染与病人的病情、病程有一定的关联。提示乳糜尿的发生、发展与泌尿道ＣＴ 和ＵＵ感染有关。     

Genital Chlamydia trachomatis infections

Chlamydia trachomatis infections affect young, sexually active persons. Risk factors include multiple partners and failure to use condoms. The incidence of infection has increased in the past 10 years. Untreated C .  trachomatis infections are responsible for a large proportion of salpingitis, ectopic pregnancy, infertility and, to a lesser extent, epididymitis. Screening is a possible intervention to control the infection, which is often asymptomatic. The emergence of lymphogranuloma venereum proctitis in men who have sex with men, in Europe, and of a variant with a deletion in the cryptic plasmid, in Sweden, are new features of C .  trachomatis infections in the last years. A diagnosis is best made by using nucleic acid amplification tests, because they perform well and do not require invasive procedures for specimen collection. Single-dose therapy has been a significant development for treatment of an uncomplicated infection of the patient and his or her sexual partner.     

Screening for Chlamydia trachomatis in subfertile women

Chlamydia trachomatis is the most common sexually transmitted disease in the UK and Europe. The majority of female infections are asymptomatic and recognized sequelae include pelvic inflammatory disease, infertility, and ectopic pregnancy. Women with chlamydial infection who undergo uterine instrumentation are recognized to be at risk of ascending infection. Most patients attending for infertility investigations and treatment will undergo some form of uterine instrumentation. Published data regarding the prevalence of chlamydial infection in the subfertile are few and conflicting. In this study, more than 400 consecutive women presenting for infertility investigation and treatment at a single regional fertility centre were screened for Chlamydia: Half were screened using enzyme immunoassay (EIA) and half by ligase chain reaction (LCR). Prevalence by diagnostic test was 0% with EIA and 1.9% with LCR. Overall, the low prevalence was at least partly explained by older age. Until more evidence comes from studies testing consecutive subfertile patients both with EIA and a DNA amplification method such as LCR, centres using EIA should consider using prophylactic antibiotics prior to uterine instrumentation.