E型沙眼衣原体重组主要外膜蛋白诱导出的小鼠免疫效应

目的 检验自行研制的E型沙眼衣原体重组主要外膜蛋白(rMOMP)诱导小鼠产生的特异性免疫应答效应.方法 3～4周清洁级BALB/C雌鼠36只,分为佐剂组、单独组和对照组,每组12只.佐剂组采用纯化的rMOMP 50 μg和等体积的弗氏佐剂、单独组单用纯化的rMOMP 50μg、对照组单用PBS200μL,于第0、2、4周分别双侧股四头肌进行免疫.ELISA法检测小鼠血清中沙眼衣原体特异性IgG抗体以及阴道冲洗液中沙眼衣原体特异性sIgA抗体,ELISA法检测细胞因子IFN-γ,MTT法检测小鼠脾淋巴细胞特异性增殖反应;同源攻击后阴道宫颈脱落细胞沙眼衣原体培养,观察小鼠的迟发型超敏反应以及小鼠的血清抗体中和试验.结果 佐剂组小鼠血清沙眼衣原体特异性IgG抗体A_(405)值为0.641±0.059,淋巴细胞增殖指数5.085±1.291,迟发型超敏反应右足足垫增厚0.324 ±0.054 mm.单独组小鼠血清特异性IgG抗体A_(405)值为0.424±0.015,淋巴细胞增殖指数3.123±0.840,迟发型超敏反应右足足垫增厚0.272±0.064mm.佐剂组各项指标的检测结果都高于单独组(P<0.05)和对照组(P<0.01).结论 沙眼衣原体rMOMP能刺激机体产生有效的特异性体液和细胞免疫.     

Genotyping of Portuguese Chlamydia trachomatis urogenital isolates.

OBJECTIVE: To determine the prevalence of the different Chlamydia trachomatis genotypes in Portuguese patients. METHODS: Urogenital isolates (n = 240) derived from attenders of various clinics in the Lisbon area were differentiated into genovars by genotyping with restriction fragment length polymorphism (RFLP) analysis of the PCR amplified omp1 gene. RESULTS: Genotype E was the most common for both men (47.9%) and women (43.8%). Genotypes D and F were the second most prevalent for men (11.3%) and genotype H was the second most prevalent for women (19.5%). Genotypes F, G, D, in women and H, G, I, in men, were found in a lower percentage of cases. Genotypes B, Ba, J, K, L1 and L2 were very rarely detected. CONCLUSIONS: With one exception, the overall distribution of Chlamydia trachomatis genotypes in our study is similar to what has been observed in other western countries. The only exception is the unusual prevalence of genotype H among women. The clinical manifestations associated with this and other genotypes were similar.     

沙眼衣原体E型MOMP基因原核表达载体的构建和纯化

目的:构建沙眼衣原体E型主要外膜蛋白(MOMP)基因的原核表达载体,并在大肠杆菌(BL-21)中融合表达,为沙眼衣原体疫苗的研究提供材料。方法:用PCR技术扩增E型沙眼衣原体MOMP基因片段,再将其定位插入到原核表达载体pGEX中,构建重组表达质粒。然后将重组表达质粒转化入大肠杆菌(BL-21)中,并用酶切分析、PCR扩增及部分序列测定等方法对重组质粒进行了鉴定。然后诱导表达,用SDS-PAGE及蛋白印迹进行鉴定,然后进行蛋白纯化。结果:PCR扩增出约1202bp DNA片段,序列测定证实与GenBank登陆的E型沙眼衣原体一致;表达产物的相对分子量为66KD,与预期分子量相符,蛋白印迹证实表达产物为特异性蛋白,并纯化获得大量蛋白。结论:成功的构建了原核表达载体pGEX/MOMP,在大肠杆菌中得到了表达,并得到了纯化后的蛋白。     

Typing of Chlamydia trachomatis strains from urine samples by amplification and sequencing the major outer membrane protein gene (omp1)

OBJECTIVES: To develop a novel protocol for the extraction, amplification, and sequencing of Chlamydia trachomatis MOMP gene (omp1) from urine, a non-invasive source, and apply it to an epidemiological study on the distribution of C trachomatis strains in a population of pregnant women in Thailand. METHODS: The C trachomatis DNA was extracted from culture stocks and urine using a slightly modified commercially available kit, the High Pure PCR Template Preparation Kit (Roche Molecular Biochemicals, IN, USA). The PCR and sequencing primers used for the amplification and sequencing of the omp1 were designed based on the nucleotide sequence of multiple C trachomatis strains found in GenBank. The protocol for the extraction, amplification, and sequencing was tested on laboratory culture stocks of reference strains of all C trachomatis serovars and on urine samples collected in a cross sectional study designed to assess the prevalence of C trachomatis infections in the cities of Bangkok and Chiang Rai, Thailand. RESULTS: The omp1 gene was successfully amplified and sequenced from 18 laboratory C trachomatis reference strains and from 45 C trachomatis positive urine clinical samples collected from asymptomatic pregnant women. Among clinical samples, we found nine different C trachomatis genotypes: F (11, 25%), D (10, 22.6%), H (5, 11.7%), K (5, 11.7%), E (4, 9.3%), Ia (3, 7%), B (3, 7%), Ja (2, 4.5%), and G (1, 2.3%). One specimen generated an omp1 DNA sequence pattern indicating the presence of a mixed infection with at least two different serovars. CONCLUSIONS: Urine is a convenient and reliable source for genotyping C trachomatis strains. A clear advantage of urine over traditional samples, such as cervical swabs, is that urine is a non-invasive source which makes collection easier and thus facilitates the enrolment of patients in clinical and epidemiological studies. In addition to typing, urine is increasingly used for diagnosis of C trachomatis infection by several commercially available nucleic acid amplification assays which represents a distinct advantage for collecting, transport, storage, and laboratory handling of samples.     

Evaluation of a new chemiluminometric immunoassay, Magic Lite Chlamydia, for detecting Chlamydia trachomatis antigen from urogenital specimens.

The chemiluminometric sandwich immunoassay, Magic Lite Chlamydia by Ciba Corning (Medfield, MA), using one Chlamydia trachomatis-specific monoclonal antibody conjugated with acridinium ester and one polyclonal immobilized to magnetic particles, was compared to cell culture in 291 urogenital specimens from 92 men and 102 women tested both in the cervix and urethra. The proportion of patients with positive culture results ranged from 11.8% among the women to 15% among the men, using microculture plates and peroxidase-antiperoxidase staining procedure with genus-specific monoclonal antibody (Orthodiagnostics, Raritan, NJ). The chemiluminometric method for detecting Chlamydia trachomatis antigen showed an overall sensitivity of 72.4%, specificity of 97.7%, positive predictive value of 77.7%, and negative predictive value of 96.9%. A higher sensitivity of Magic Lite was found in clinical specimens yielding more than 10 inclusions by well in cell culture. The newly developed chemiluminometric test, easy to perform, had the same limits in terms of sensitivity and positive predictive value than the other widely-used existing tests for detecting Chlamydia trachomatis antigen from urogenital specimens.     

Genotyping of Chlamydia trachomatis serovars derived from heterosexual partners and a detailed genomic analysis of serovar F.

OBJECTIVE--To investigate C trachomatis serovars in contact-traced heterosexual partners. METHODS--Urogenital Chlamydia trachomatis isolates (n = 112) derived from 35 heterosexual patients (index patients) and their 37 chlamydia positive partners (contact patients) were differentiated into serovars by genotyping with restriction fragment length polymorphism (RFLP) analysis of the PCR amplified omp1 gene. In order to investigate whether different strains within the frequently prevalent serovar F were transmitted, two pairs of serovar F (n = 4) were further analysed by genomic DNA fingerprinting with arbitrary primer PCRs (AP-PCRs). RESULTS--Identical C trachomatis serovars were found in 31 of the 35 pairs, serovars E, F, D, and G being most prevalent. In the remaining four pairs different serovars (either D, E, F or G) were found between the index and the contact patients. By AP-PCR analysis the strains of serovar F were found to be identical between the index and the contact patients, but were different between the two pairs in all AP-PCRs used. CONCLUSION--A majority of heterosexual partners, once traced positive for C trachomatis infections, are infected with identical serovars. Identical strains of serovar F found in partners as found by DNA fingerprinting confirms the sexual transmission of C trachomatis.     

Isolation of Chlamydia trachomatis from the male urethra.

Chlamydia trachomatis was isolated from 26% of urethral swabs taken from 509 men with urethritis. The highest yield of 68% was obtained from a selected group of men with nonspecific urethritis (NSU) who had a frank urethral discharge. This is a higher than in previous reports, and is significantly higher than the isolation of C. trachomatis from men with less severe urethritis. The higher yield was similar to C. trachomatis isolation rates reported among patients with severe trachoma in hyperendemic areas. Men with a previous history of NSU had low isolation rates. Overall, 30% of 385 men with NSU had positive chlamydial culture results, 7% of 59 men with gonococcal urethritis alone were Chlamydia-positive, 15% of 59 men with gonorrhoea followed by NSU (post-gonococcal urethritis) were Chlamydia-positive, and only 3% of 61 men without urethritis harboured Chlamydia. Swabs taken from the cervical os of 28 of 108 female contacts of men with NSU had a positive result for C. trachomatis. Significantly more pairs of sexual partners had the same chlamydial culture result than had different results. The chlamydial isolation rate was higher among men admitting a casual sexual contact than in men claiming only regular partnerships. The findings provide further evidence for the sexual transmission of C. trachomatis and for its aetiological role in NSU.     

Unusual prevalence of the rare serovar Da of Chlamydia trachomatis in Greece detected by monoclonal antibodies.

Twenty-seven monoclonal antibodies (mAb), eight with subclass-specificities and nineteen reacting with one or two C. trachomatis serovars, were developed and used to immunotype twenty-six clinical isolates from Greek patients with chlamydial infections. Twelve samples, first classified as serovar D, were identified as the recently established serovar Da on the basis of their negative reaction with mAb JG9, an mAb previously shown to distinguish between D and Da, and a new mAb 114D9 with similar specificity. The 12 Da strains represent the highest prevalence of the rare serovar Da that has been reported worldwide. Further characterization of mAbs JG9 and 114D9 revealed a unique cross-reactivity of the D-specific JG9 with the mouse biovar MoPn. Epitope mapping studies on overlapping synthetic peptides of the fourth variable domain of the MOMP of serovar D and Da localized the epitopes of JG9 and 114D9 and a D/Da specific mAb 113D5 within this domain. The results were consistent with the specificity of these mAbs observed with whole microorganisms and indicated their usefulness as epidemiologic markers.     

Comparison of the Amplicor Chlamydia trachomatis test and cell culture for the detection of urogenital chlamydial infections.

OBJECTIVE--To compare the polymerase chain reaction (PCR) Amplicor Chlamydia trachomatis test with the cell culture method, in diagnosing urogenital chlamydial infections. SUBJECTS--439 patients (327 women and 112 men) attending one STD clinic and Family Planning and Gynaecological Clinics in Lisbon, Portugal, between November 1993 and March 1994. METHODS--In women, two endocervical swab samples were collected: one for PCR Amplicor and one for standard culture technique. Men were asked to submit 20 ml of urine (first pass urine) for PCR Amplicor and one urethral specimen was taken for culture. The order of collection of the specimens was rotated every 50 patients. Discrepant results were further analysed by a second PCR with primers directed against the C trachomatis major outer membrane protein (MOMP) and by direct fluorescent antibody (DFA). RESULTS--After analysis of discrepancies, the adjusted sensitivity and specificity of PCR on endocervical specimens were 92.9% and 100% and the positive and negative predictive values were 100% and 99.7% respectively; on the urine samples these values were 100%, 99.1%, 100% and 99.1%, respectively. CONCLUSION--These results indicate that the PCR Amplicor test is a rapid sensitive and specific assay for the detection of C trachomatis in urogenital infections and provides a non-invasive technique for screening chlamydia infection in men.     

衣原体噬菌体phiCPG1衣壳蛋白Vp1对豚鼠结膜炎衣原体及E型沙眼衣原体的抑制作用

目的 探讨豚鼠结膜炎衣原体（GPIC）噬菌体衣壳蛋白Vp1对GPIC及E型沙眼衣原体的抑制作用,为沙眼衣原体感染的治疗提供新的思路。 方法 用Vp1-pET30a（+）重组质粒菌表达Vp1蛋白,Western印迹法鉴定蛋白,透析袋纯化蛋白,BCA法测定蛋白浓度,将GPIC、E型沙眼衣原体分别与Vp1蛋白、Tris甘氨酸溶液、S蛋白及培养液室温孵育3 h,衣原体培养过程中分别 加入相同浓度的上述液体,72 h或48 h后,免疫荧光计数包涵体数。 结果 GPIC在Vp1蛋白组、Tris甘氨酸溶液组、S蛋白组及培养液组培养72 h,包涵体计数分别为5.0 ± 1.5、24 ± 1.2、25 ± 1.7及25 ± 1.5,各组包涵体数比较,差异有统计学意义（F = 476.632,P < 0.05）。Vp1蛋白组GPIC包涵体数显著低于Tris甘氨酸溶液组、S蛋白组及培养液组（P < 0.05）,而后3组之间差异无统计学意义（P > 0.05）。与阴性对照组（培养液组）相比,Vp1蛋白对GPIC的抑制率为（80.2 ± 3.99）%。此外,Vp1蛋白对E型沙眼衣原体的抑制率为（77.2 ± 1.79）%,t检验示Vp1对GPIC的作用与对E型沙眼衣原体的作用差异无统计学意义（t = 2.057,P > 0.05）。 结论 Vp1蛋白可明显抑制GPIC的感染,同时对E型沙眼衣原体有相似的抑制作用。     

Evaluation of a radioactive rRNA:cDNA-hybridisation assay for the direct detection of Chlamydia trachomatis in urogenital specimens.

A radioactive cDNA probe complementary to chlamydial ribosomal RNA was used to detect C trachomatis in urogenital specimens. Of 37 specimens positive with cell culture 31 were confirmed by the rRNA:cDNA hybridisation test, the sensitivity being 83.8%. The specificity of the hybridisation test was 94.4%, as 186 of 197 specimens that were negative by cell culture were also negative when assessed by the hybridisation method. Given a prevalence of 15.8% the predictive values for positive and negative results were 73.8% and 96.9%, respectively. In additional experiments the possible role of microorganisms added to the specimen collection medium was investigated. However, no indication for crosshybridisation was found; at high concentrations microorganisms interfered with the test procedure.     

Prevalence of urogenital Chlamydia trachomatis increases significantly with level of urbanisation and suggests targeted screening approaches: results from the first national population based study in the Netherlands

OBJECTIVES: Chlamydia trachomatis (Chlamydia) is the most prevalent sexually transmitted bacterial infection and can cause considerable reproductive morbidity in women. Chlamydia screening programmes have been considered but policy recommendations are hampered by the lack of population based data. This paper describes the prevalence of Chlamydia in 15-29 year old women and men in rural and urban areas, as determined through systematic population based screening organised by the Municipal Public Health Services (MHS), and discusses the implications of this screening strategy for routine implementation. METHODS: Stratified national probability survey according to "area address density" (AAD). 21 000 randomly selected women and men in four regions, aged 15-29 years received a home sampling kit. Urine samples were returned by mail and tested by polymerase chain reaction (PCR). Treatment was via the general practitioner, STI clinic, or MHS clinic. RESULTS: 41% (8383) responded by sending in urine and questionnaire. 11% (2227) returned a refusal card. Non-responders included both higher and lower risk categories. Chlamydia prevalence was significantly lower in rural areas (0.6%, 95% CI 0.1 to 1.1) compared with very highly urbanised areas (3.2%, 95% CI 2.4 to 4.0). Overall prevalence was 2.0% (95% CI 1.7 to 2.3): 2.5% (95% CI 2.0 to 3.0%) in women and 1.5% (95% CI 1.1 to 1.8) in men. Of all cases 91% were treated. Infection was associated with degree of urbanisation, ethnicity, number of sex partners, and symptoms. CONCLUSION: This large, population based study found very low prevalence in rural populations, suggesting that nationwide systematic screening is not indicated in the Netherlands and that targeted approaches are a better option. Further analysis of risk profiles will contribute to determine how selective screening can be done.     

The value of urine samples from men with non-gonococcal urethritis for the detection of Chlamydia trachomatis.

Chlamydia trachomatis was sought at first and subsequent clinic visits in urethral swabs and urines from 112 heterosexual men with acute non-gonococcal urethritis (NGU). In comparison with a urethral swab tested by Micro Trak (MT), a urine deposit tested in the same way was 90% as sensitive. Examining a urine deposit by the enzyme immunoassay IDEIA was a little less sensitive (89%) than examining a similar deposit by MT, and was less sensitive (82%) than examining a urethral swab by MT. The results of testing urines were little influenced by collecting them either before or after swabbing the urethra, and there was evidence that examining all of a urine sample by IDEIA would have increased sensitivity. Overall, 55 (49%) of the men were diagnosed as C trachomatis-positive based on the results of testing both a urethral swab and a urine sample. Furthermore, a small numbers of chlamydiae were detected by examining urine by MT and, to a lesser extent, by IDEIA, so that there is no reason why this non-invasive approach should not be successful in men other than those with acute NGU.     

Chlamydial infection in homosexual men. Frequency of isolation of Chlamydia trachomatis from the urethra, ano-rectum, and pharynx.

Urethral, rectal, and pharyngeal material from 150 men who had had exclusively homosexual contact and who consecutively attended a sexually transmitted diseases clinic was cultured for Chlamydia trachomatis. The organism was isolated from at least one site in 15 (105) patients. The isolation rates from the urethra, rectum, and pharynx were 6 . 7%, 4%, and 1 . 3% respectively.     

Effects of broadening the gold standard on the performance of a chemiluminometric immunoassay to detect Chlamydia trachomatis antigens in centrifuged first void urine and urethral swab samples from men.

Traditionally, evaluations of nonculture assays for Chlamydia trachomatis are based on a comparison with urethral culture in men and cervical culture in women as the standard for positivity of infection, but it is known that culture may be less than 100% sensitive. A chemiluminometric immunoassay, Magic Lite (Ciba Corning, Medfield, MA) that detects C. trachomatis antigens was performed on centrifuged first void urine samples and urethral swabs collected from men attending a sexually transmitted disease (STD) clinic. Immunoassay performance was compared to urethral culture and also to a broader gold standard: an infected patient with positive culture results or a confirmed positive Chlamydiazyme enzyme immunoassay (Abbott, Chicago) result. Two studies were performed on a retrospective group of stored first void urine samples from 200 men and a prospective group of urethral swabs and first void urine samples from 199 men. Expanding the gold standard showed that a urethral swab assayed by culture had a sensitivity between 70.3% and 87.5%, with the following effects on immunoassay performance in the prospective study: the sensitivity of urethral swabbing was reduced from 96.2% to 78.4% (specificity increased from 96.0% to 98.1%) and first void urine sensitivity increased from 92.3% to 94.6% (specificity went from 87.9% to 93.8%). In the retrospective study, sensitivity of first void urine testing went from 91.4% to 92.5%, with a corresponding increase in specificity from 93.9% to 96.9%. This maneuver had relatively little impact on the negative predictive values, but dramatically increased the positive predictive values, for both samples.(ABSTRACT TRUNCATED AT 250 WORDS)