Characterization of Chlamydia trachomatis omp1 gene among sexually transmitted disease patients in south China

Chlamydia trachomatisinfections are the leadingcause of bacterial sexually transmitted diseases(STD) [1] .Fifteen prototypic serovarslabelled Ato Kand L1,L2,and L3were initiallyrecognisedby polyclonal antibodies ,and additional immuno-variants (Ba ,Da ,Ia ,L2a ,etc) ,whichin somepublications are referred to as distinct serovars ,have been identified by monoclonal antibodies .Most serovars can cause urogenital infections andare associated with a spectrumof clinical diseases[2] ,including ur…     

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。     

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）。     

Apoptosis of ejaculated human sperm is induced by co-incubation with Chlamydia trachomatis lipopolysaccharide

BACKGROUND: Previous work has shown that co-incubation of human sperm with Chlamydia trachomatis serovars E and LGV leads to premature sperm death and that this is due primarily to chlamydial lipopolysaccharide (LPS). Here, we investigated the possible involvement of apoptosis in this premature sperm death. METHODS: Highly motile preparations of sperm from normozoospermic patients were co-incubated for 6 h with extracted LPS from C. trachomatis serovars E and LGV. Three different methods were used to determine if LPS-treated sperm underwent apoptosis, including: (i) flow cytometry; (ii) measurement of ADP:ATP ratios; and (iii) measurement of mono- and oligonucleosomal DNA fragments. Caspase activity was also investigated by fluorimetry and by use of a pan-caspase inhibitor and caspase-3 inhibitor. RESULTS: All three methods used for detection indicated that C. trachomatis LPS induced some apoptosis in sperm after 6 h when compared with a staurosporine (apoptosis-positive) control. Moreover, a greater degree of apoptosis was seen with C. trachomatis serovar E than with serovar LGV. It was also shown that C. trachomatis LPS-induced apoptosis of sperm could be blocked with a pan-caspase inhibitor and a caspase-3 inhibitor. Moreover, by using a fluorogenic substrate, apoptosis was shown to be caspase-mediated. CONCLUSIONS: In general it is believed that apoptosis does not occur in C. trachomatis-infected host cells. However, using three different methods, our findings clearly indicate that co-incubation of sperm with C. trachomatis LPS results in cellular death which is in part due to apoptosis and is caspase-mediated. These findings provide an explanation as to how C. trachomatis can mediate premature death in human sperm.     

Co-incubation of human spermatozoa with Chlamydia trachomatis serovar E causes premature sperm death

The aim of this work was to investigate the effect of elementary bodies (EB) of Chlamydia trachomatis serovars E and LGV on sperm motility, viability and acrosomal status. Highly motile preparations of spermatozoa from normozoospermic patients were co-incubated for 6 h with 0.54x10(6) EB per ml. At 1, 3 and 6 h of incubation, sperm motility was determined by computer-assisted semen analysis (CASA) and the proportion of dead cells determined by the hypo-osmotic swelling (HOS) test. Acrosomal status was also examined using a standard monoclonal antibody assay. In the absence of EB, the percentage of motile spermatozoa remained >69% over the 6h incubation and the proportion of dead spermatozoa at <12%. However, during the incubation with EB of serovar E there was a significant decline in the percentage of motile spermatozoa (P < 0.05), and a corresponding increase in the proportion of dead spermatozoa (P < 0.05) at all time-points. However, following incubation with serovar LGV, only the percentage of dead spermatozoa after 6 h incubation was significantly different from the control (P < 0.05). The amount of acrosome-reacted spermatozoa remained unchanged (<16%) in all incubations at all time-points. Dose-response experiments indicated that increasing the concentration of EB to 2.5x10(6) per ml did not significantly alter the results. Furthermore, co-incubation of spermatozoa with dead EB (killed by heat treatment) abolished the chlamydia-mediated response, indicating that the effect is a result of the live organism and not soluble components or membrane elements. These data suggest that a detrimental effect on sperm function by some serovars may be an as yet unrecognized component of infertility problems.     

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.     

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.     

Value of papanicolaou smear in detection of Chlamydia trachomatis infection

This study was undertaken to assess the value of Papanicolaou smear for the diagnosis of Chlamydia trachomatis infection. The study was both retrospective (groups I and II) and prospective (group III). Group I consisted of 41 smears with cytomorphological changes proposed by Gupta, Kiviat, or Shiina. Group II was a control group, consisting of 30 cytologically normal smears. All these smears were subjected to specific immunofluorescent (IF) staining under identical conditions to confirm the diagnosis. In group III, 40 consecutive duplicate cervical smears were collected from patients attending the Sexually Transmitted Disease Clinic. One smear was routinely examined, and the specific IF staining was done on the other smear. The results in all the three groups were analysed. It was concluded that Papanicolaou smear is not useful in the detection of Chlamydia trachomatis infection.     

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.     

Highly discriminative genotyping of Chlamydia trachomatis using omp1 and a set of variable number tandem repeats

This article reports the development of a method for genotyping Chlamydia trachomatis , using PCR and sequencing of omp1 , supplemented with three new variable number tandem repeat (VNTR) loci of C. trachomatis . Typeability, reproducibility and discriminatory power were assessed using four groups of samples: two groups (I and II) of C. trachomatis -positive patients and their positive partner(s), one group (III) of patients with recurrent or persistent C. trachomatis infections, and one group (IV) comprising samples containing a newly discovered mutant strain with a 377-bp deletion in the cryptic plasmid, the new variant C. trachomatis (nvCT). The VNTR loci (designated CT1335, CT1299, and CT1291) were all single nucleotide repeats chosen for maximal mutability and variation. In the study material, nine variants of CT1335, eight variants of CT1299 and five variants of CT1291 were found. The discriminatory power ( D ) of omp1 in the present material was D _omp1  = 0.69. D s for VNTRs CT1335, CT1299 and CT1291 were 0.53, 0.74 and 0.74, respectively. The resolution power of the omp1 -VNTR assay was 0.94. Stability over time of the VNTRs was investigated and found to be adequate for epidemiological studies. Using this genotyping assay, it was confirmed that the nvCT strain was indeed a clone. These results indicate that, with this novel method, strains of C. trachomatis can be individually identified, and epidemiological associations established.     

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.     

Identification of Serovar-Specific Single-Nucleotide Polymorphisms of <Emphasis Type="Italic">C. Trachomatis omp1</Emphasis> Gene

Complete sequences of omp1 gene encoding chlamydial main outer membrane protein were analyzed in 76 clinical strains of C. trachomatis. Thirty-four serovar-specific single-nucleo-tide polymorphisms were identified, 20 of them meet two criteria: unique position of the nucleotide and unique nucleotide substitution. Evaluation of serovar-specific single-nucleotide polymorphisms of omp1 gene can appreciably simplify and accelerate genetic diagnosis of C. trachomatis serovars. __________ Translated from Byulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 140, No. 8, pp. 192–197, August, 2005     

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.     

新生儿疾病沙眼衣原体和解脲脲原体感染的PCR检测研究

为探讨沙眼衣原体（CT）和解脲脲原体（UU）与新生儿疾病的关系，我们采用套式聚合酶链式反应（PCR）技术，从100例住院新生儿的结膜拭子中共检出阳性者58例，阳性率58％（其中CT36倒、UU22例）：剖宫产7例（CT4例，UU3例）；经产道分娩51例（CT32例、UU19例）。结果表明：新生儿是CT、UU易感者．可引起新生儿肺炎、宫内感染、早产、低体重、结膜炎、肠炎。采用先进的套式PCR技术检测具有灵敏、特异、简便的优点。     

PCR检测沙眼衣原体与习惯性流产和女性不孕相关性研究

本文应用聚合酶链反应技术（ＰＣＲ），对１２０例不孕和１００例查体妇女的宫颈内膜标本进行沙眼衣原体检测，以了解女性沙眼衣原体感染与自发流产的关系，并与一般细胞培养法进行了比较。结果：不孕妇女中沙眼衣原体感染率为１８．３％，显著高于对照组（８％Ｐ＜０．０５），并且自发流产３次、４次的妇女沙眼衣原体阳性率明显高于对照组（Ｐ＜０．０１）．敏感性也较一般培养法高（Ｐ＜０．０１）。本研究提示：沙眼衣原体感染与自发性习惯性流产关系十分密切，而且ＰＣＲ法在检测沙眼衣原体感染方面较一般培养法更敏感、快速，是早期诊断生殖道沙眼衣原体感染的一项有价值的方法。     

Chlamydia trachomatis: time for screening?

Genital Chlamydia trachomatis infection is the leading cause of bacterial sexually transmitted disease in industrialised countries, particularly among young people. The consequences of chlamydial infection may involve urethritis, cervicitis, pelvic inflammatory disease, ectopic pregnancy, tubal factor infertility, epididymitis and prostatitis. In addition, chlamydial infection increases the risk of acquisition of human immunodeficiency virus and has been associated with cervical cancer. Although screening programmes exist in a number of countries, the continuously increasing prevalence of chlamydial infections demonstrates the necessity for health authorities to establish effective screening policies, and the importance of defining a comprehensive European screening policy is emerging.     

衣原体噬菌体phiCPG1衣壳蛋白Vp1对沙眼衣原体的作用研究

目的 将原核表达纯化的衣原体噬菌体phiCPG1衣壳蛋白Vp1作用于沙眼衣原体(Chlamydia trachomatis,Ct),以期发现该Vp1蛋白对Ct生长的影响.方法 原核表达并纯化噬菌体phiCPG1的衣壳蛋白Vp1,将纯化后的蛋白复性后与Ct的标准株或临床野生株(终浓度为53μg/ml)室温孵育3h后接种至单层致密McCoy细胞中,培养过程中均加入了了 Vp1蛋白,48 h后碘染色包涵体计数和透射电镜观察Vp1蛋白对Ct生长的影响;MTT法检测Vp1蛋白对McCoy细胞系的细胞毒性作用;液体培养基稀释法检测Vp1蛋白对大肠杆菌BL21和DH5α的抗菌作用.结果 衣壳蛋白Vp1对Q标准株E和D型的抑制率分别为78％和72％,对Ct临床野生株的抑制率为40％～70％,透射电镜结果显示Vp1蛋白能够抑制Ct的正常发育,使包涵体内出现异常增大的网状体.而该Vp1蛋白对非衣原体的其他生物体如大肠杆菌BL21、DH5α和真核细胞McCoy的生长没有影响.结论 噬菌体衣壳蛋白Vp1对Ct的生长具有明显的抑制作用,为临床上Ct感染的治疗提供了新的思路.