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.     

Chlamydia trachomatis infections in heterosexuals attending sexually transmitted disease clinics in Slovenia

This study assessed the age and gender distribution of Chlamydia trachomatis infections among patients attending two clinics for sexually transmitted diseases (STDs) in Slovenia. Between January 1999 and December 2003, 1714 heterosexual male and 892 heterosexual female patients were tested for C. trachomatis. The prevalence of C. trachomatis infection was 19.5% (n = 334) for male patients and 10.7% (n = 96) for female patients, with the highest prevalence in the group aged 15-30 years. The prevalence decreased between 2000 and 2003 among female patients. The results support the implementation of routine screening for C. trachomatis genital infection among male and female patients aged < 30 years attending STD clinics in Slovenia.     

Unclassified serovars of Chlamydia trachomatis isolated from Japanese infants

Objective: To analyze antigenic and genetic variations of Chlamydia trachomatis among the serovars obtained from Japanese infants.
Methods: The polymerase chain reaction (PCR) was used to amplify a large part of the major outer-membrane protein gene, and restriction fragment length polymorphism (RFLP) was used to identify the serovars of C. trachomatis from nasopharyngeal and conjunctival swabs from Japanese infants and neonates.
Results: The typing of 10 nasopharyngeal isolates gave the following results: seven E, one H, and two unclassified serovars. The typing of seven conjunctival isolates gave the following results: five D, one F, and one unclassified serovar. Reactive patterns of these unclassified strains, determined by PCR-RFLP, to monoclonal antibodies were different from those of 15 reference serovars.
Conclusions: Characterization of unclassified variants will allow more detailed epidemiologic studies of perinatal C. trachomatis infections in Japan.     

Chlamydia trachomatis infection, Fallopian tube damage and a mannose-binding lectin codon 54 gene polymorphism

BACKGROUND: Mannose-binding lectin (MBL), a component of the innate immune system, provides a first-line defense against invading microorganisms. Polymorphisms in the MBL gene have been associated with increased risk of infection. Chlamydia trachomatis genital tract infections are a major cause of Fallopian tube occlusion. Our objective was to test whether an MBL codon 54 polymorphism might contribute to development of C. trachomatis-associated tubal damage. METHODS: In a case-control study, 97 women with occluded and 104 women with patent Fallopian tubes were tested for a history of chlamydial infection by serology and for their MBL codon 54 genotype by PCR and restriction fragment length polymorphism analysis. Clinical data were blinded to those performing all laboratory analyses. RESULTS: Women with tubal occlusion who also had a positive chlamydial serology had the highest rate of variant MBL B allele carriage (P<0.001). Among women who were chlamydial antibody negative, allele B carriage was also more frequent in those with blocked, as opposed to patent, Fallopian tubes (P<0.01). CONCLUSIONS: Wild-type allele A homozygosity is protective against, while carriage of the variant allele B is a risk factor for, Fallopian tube occlusion in women who are seropositive or seronegative for C. trachomatis.     

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.     

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     

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.     

拟诊为淋病患者中泌尿生殖道沙眼衣原体基因分型及序列分析

目的 了解深圳市拟诊为淋病患者中泌尿生殖道沙眼衣原体的合并感染情况及其基因型分布和序列变异特点.方法 采集401例拟诊为淋病患者的泌尿生殖道分泌物样本,应用Roche Amplicor全自动核酸检测系统对样本进行淋球菌和沙眼衣原体双检,提取DNA,应用巢式聚合酶链反应(nested-PGR)扩增沙眼衣原体主要外膜蛋白基因(omp1)中的VS1～VS2片段,并对其进行序列测定,所获得的序列利用Mega4.0软件与标准参考株进行比对,分析确定其基因型及序列变异情况.结果 401例拟诊为淋病患者中淋球菌的感染率为82.3%(330/401),沙眼衣原体的感染率为24.2%(97/401),淋球菌和沙眼衣原体的合并感染率为21.7%(87/401).97份沙眼衣原体阳性样本中获得73份沙眼衣原体基因片段序列,共检出8个基因型,分别为E型(27.4%)、G/Ga型(23.3%)、D/Da型(16.4%)、F型(13.7%)、J型(11.0%)、H型(5.5%)、B和K型(各1.4%).序列分析发现3例(4.1%)菌株发生错义突变,分别为D/Da型、E型、G/Ga型;F型、H型、J型和K型序列虽多见碱基突变,但均为同义突变.结论淋病患者合并感染沙眼衣原体的比例较高,且泌尿生殖道沙眼衣原体的基因型以E、G/Ga、D/Da和F型为主.序列分析可以为泌尿生殖道沙眼衣原体的分子流行病学研究提供依据.     

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.     

Characterization of Chlamydia trachomatis omp1 Genotypes among Sexually Transmitted Disease Patients in Sweden

A method for detection and genotyping of genital Chlamydia trachomatis infections based on omp1 gene amplification and sequencing was developed. DNA was extracted from urogenital or urine samples using a Chelex-based method, and an approximately 1,100-bp-long fragment from the omp1 gene was directly amplified and sequenced. Genotyping was performed by BLAST similarity search, and phylogenetic tree analysis was used to illustrate the evolutionary relationships between clinical isolates and reference strains. The method was used to determine the genotypes of C. trachomatis in 237 positive urogenital and/or urine specimens collected at a Swedish sexually transmitted disease clinic during 1 year. The most common genotypes corresponded to serotypes E (47%) and F (17%). The omp1 gene was highly conserved for genotype E (106 of 112 samples without any mutation) and F (41 of 42 samples without any mutation) strains but appear slightly less conserved for genotypes G (n = 6) and H (n = 6), where the sequences displayed one to four nucleotide substitutions relative to the reference sequence. Genotyping of samples collected at the follow-up visit indicated that two patients had become reinfected, while three other patients suffered treatment failure or reinfection. One woman appeared to have a mixed infection with two different C. trachomatis strains. This omp1 genotyping method had a high reproducibility and could be used for epidemiological characterization of sexually transmitted Chlamydia infections.     

Diagnosis of Chlamydia trachomatis infections in a sexually transmitted disease clinic: evaluation of a urine sample tested by enzyme immunoassay and polymerase chain reaction in comparison with a cervical and/or a urethral swab tested by culture and polymerase chain reaction

Objective   To evaluate the value of a urine sample for diagnosing Chlamydia trachomatis infection in an STD clinic in a prospective study of samples collected from 410 consecutive STD patients (167 female and 243 male).
Methods   Urine samples were tested by enzyme immunoassay (EIA) and polymerase chain reaction (PCR) in comparison with cervical and/or urethral swabs tested by PCR and cell culture. A questionnaire was completed for a total of 320 patients concerning symptoms, and determining whether they were controls, contacts or were being tested subsequent to legal abortion.
Results   The overall prevalence of C. trachomatis infection was 11.5%. At least 40% of patients were asymptomati c . Of the C. trachomatis -positive patients, 85% were diagnosed by testing urine, compared to 91% by testing swabs. For urine tests, the sensitivities of PCR were 66.7% and 71.9% for female and male patients, respectively, and the sensitivities of EIA were 40.0% and 62.5%, or 46.7% and 71.9%, for female and male patients, respectively, by including a 30% gray zone below the cut-off value. For swabs, the sensitivities of PCR were 93.3% and 87.5% for female and male patients, respectively, and equal to the sensitivities of culture. In total, 3.3% of controls and 35% of contacts were found to be C. trachomatis positive.
Conclusion   The use of urine samples for the diagnosis of C. trachomatis infections was effective, but urine samples should be additional to conventional swab(s) instead of replacing. Partner notification and a confirmation of cure is recommended.     

Characterization of Apoptotic Activities During Chlamydia trachomatis Infection in Primary Cervical Epithelial Cells

Chlamydiae are obligate intracellular bacteria that infect human epithelial cells. It has been reported that Chlamydia trachomatis, induces apoptosis in epithelial cells, however, the molecular mechanisms responsible for host cell death especially in primary epithelial cells remained largely unknown as most of the studies are in cell line like HeLa. In this study we demonstrated that C. trachomatis induces apoptosis signaling pathway and apoptosis in primary cervical epithelial cells in a time and dose dependent manner. Live cervical epithelial cells were isolated from endocervical cells and induction was done with chlamydial EBs. Our results demonstrated that apoptosis in infected epithelial cells was associated with an increased activity of caspase 8; however, caspase 9 was activated to a lesser extent. Analysis of apoptosis pathway revealed that expression level of McL-1, Bcl-2, CASP8, and TRADD genes were found to be significantly upregulated (P?<?0.01), where as levels of Caspase 1, Caspase 10 and BRIC2 were found to be significantly downregulated (p?<?0.01). Our results showed that Chlamydia induces apoptosis and caspase activation in epithelial cells through caspase 8, with an increased expression of the McL-1, which confers a block at the mitochondrial level.     

Prevalence of Chlamydia trachomatis infection among patients attending infertility and sexually transmitted diseases clinic (STD) in Kano,North Western Nigeria

Background

Chlamydia trachomatis is the most common bacterial sexually transmitted disease in the world with severe complications. The aim of this study was to determine the prevalence and possible risk factors of C. trachomatis in Kano. There is dearth of information on this subject in this locality.

Method

Urine samples, Endocervical swabs and Urethral swab were collected from consecutive patients attending the Infertility and STD clinics in Aminu Kano Teaching Hospital (AKTH) between June and December 2012, after administering a questionnaire by the attending physician and also obtaining an informed consent.Samples were analyzed using Diaspot Chlamydia kit, a rapid immunoassay test for the detection of genital chlamydial antigen in urinogenital samples.

Results

A total of 125 consecutive samples were collected, comprising 69 females and 56 males aged between 14 – 55 years. Twelve samples tested positive for C. trachomatis antigen giving a prevalence rate of 9.6%. The age group prevalence were as follows 25 – 29 yrs (17.1%), 20 – 24 (16.7%), 15 – 19 (12.5%), 30 – 34 (11.1%) and > 49 years (9.0%). Married patients were associated with higher infection rate than single (8.3%), and divorced patients (33.3%). A higher percentage of the patients (95.2%) were not aware of the existence of C. trachomatis infection and its complications. Previous STD exposure was associated with increased risk of Chlamydia infection.

Conclusion

C. trachomatis infection if unchecked will continue to pose a threat to reproductive life with its established complications. Since asymptomatic cases are common in the population regular screening should be encouraged for every adult especially before commencement of marital life.     

密码修饰可增强沙眼衣原体MOMP DNA质粒免疫小鼠的免疫应答

目的 探讨密码修饰对沙眼衣原体主要外膜蛋白（MOMP）基因表达和DNA质粒免疫的影响。方法 根据人类密码子使用偏好对鼠肺炎沙眼衣原体（Chlamydia trochomatis mouse pneumonitis）MOMP基因进行优化修饰，设计合成人源化MOMP基因（HuMOMP）。构建以pcDNA3为载体的含HuMOMP及含野生型MOMP基因（WtMOMP）的真核表达质粒。瞬时转染COS-1细胞，Western blot方法比较HuMOMP基因及WtMOMP基因在哺乳动物细胞中的蛋白表达水平。DNA质粒肌内免疫BALB／c小鼠，检测小鼠血清特异性抗体、DTH和淋巴细胞增殖反应，比较优化密码MOMP基因和野生型MOMP基因DNA质粒免疫的免疫效果。结果 人工合成HuMOMP基因，成功构建重组真核表达质粒pcDNA3-HuMOMP及pcDNA3-WtMOMP。转染细胞Western blot结果显示，HuMOMP基因的蛋白表达水平明显高于WtMOMP基因。ELISA结果表明，HuMOMP DNA质粒免疫组小鼠血清特异性IgG抗体有所升高；细胞免疫检测结果显示，HuMOMP DNA质粒免疫组小鼠足垫肿胀程度增强、淋巴细胞增殖实验刺激指数（SI）增高，两者与WtMOMP DNA质粒免疫组相比P〈0．05，差异均有统计学意义。结论 人源化密码修饰能够增强沙眼衣原体MOMP基因在哺乳动物细胞中的蛋白表达及DNA质粒免疫小鼠的免疫反应，这对于新型衣原体DNA疫苗的研究具有重要意义。     

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.     

100例不育症患者解脲支原体和沙眼衣原体定量分析

目的为了分析不育症和非淋茵性尿道炎患者感染解脲支原体和沙眼衣原体定量检测结果的差异.方法荧光定量多聚酶链式反应(FQ-PCR)技术连续对100例(有97例作了UU检测,84例作了CT检测)本院生殖中心不育症患者及同时时662例(有470例作了UU检测,540例作了CT检测)普通性病和妇科门诊非淋患者CTDNA和UUDNA进行了定量检测.结果不育组UU阳性率=57/97(58.76%),UU拷贝对数值6.016±1.044;CT阳性率=7/84(8.33%),CT拷贝对数值4.968±2.190.普通非淋组UU阳性率=209/470(46.60%),UU拷贝对数值6.131±1.385;CT阳性率=62/540(11.48%),CT拷贝对数值5.894±1.943.结论UU是引起非淋和不育症的主要病原体.UU相对于CT对人类生育的危害更为严重.不育组和普通非淋组UU的定量结果没有统计学差异,而普通非淋组的CT的拷贝数则高于不育组,P＜0.05.     

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。     

Expression and Purification of the Major Outer Membrane Protein of Chlamydia Trachomatis in Prokaryotic Cell

Chlamydiatrachomatis(C.trachomatis)isanobligate intracellularbacterialpathogen.Ocularinfectionwith C.trachomatisserovarsA,B,BaandCleadstotracho ma,aleadingcauseofpreventableblindnessinmanyde velopingcountries[1].Urogenital tractinfectionwithC. trachomat…