Genotyping of Chlamydia trachomatis from endocervical specimens in Brazil

BACKGROUND: There is no data concerning genotyping of Chlamydia trachomatis from Brazilian samples. GOAL: To characterize the genotype of C. trachomatis detected in women assisted at a STD public clinic and establish the prevalence of this infection in that population. STUDY DESIGN: Endocervical samples of a group of 100 women were tested for chlamydial infection with PCR directed to C. trachomatis cryptic plasmid. Genotyping of positive samples were done after omp1 amplification and sequencing. RESULTS: The overall prevalence of C. trachomatis infection was 19%, with the highest prevalence in women between 15 and 25 years old (68.4%). Four genotypes were found associated with endocervical infections: D, E, F, and K. Sequence analysis revealed a coinfection of genotypes D and E in 1 woman. CONCLUSIONS: To our knowledge this is the first study to characterize Brazilian C. trachomatis endocervical samples and Brazilian C. trachomatis genotype coinfection. Our results also emphasize the importance of routine diagnosis of C. trachomatis for the control of this STD.     

男性尿道沙眼衣原体血清型与临床症状的相关性研究

目的：探讨沙眼衣原体（CT）血清型与男性尿道CT感染患者临床表现的相关性,分析不同型沙眼衣原体的毒力。方法：采用巢式PCR扩增ompl基因的VS1-VS2序列,并进行酶切限制性片段长度多态性分析（RFLP）;涂片法检测阴道毛滴虫、念珠菌和炎性细胞,培养法分离鉴定淋球菌。结果：94例男性尿道感染患者CT株基因分型检出9种血清型,主要流行型别是F（28．7％）,E（26．6％）,J（19．1％）和D（10．6％）。无症状患者的CT感染率为28．3％,其中以F和E型为主（65．4％）。剔除2例伴有淋球菌感染患者后,分析各血清型（群）与临床症状的相关性,均无统计学差异（P〉0．05）。F和J型引起的炎症反应较重。结论：CT血清型与男性尿道CT感染患者临床症状无明显相关性。     

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.     

泌尿生殖道沙眼衣原体感染的临床表现特点与基因型分布

目的了解沙眼衣原体引起的泌尿生殖道感染临床表现与其分离株的基因型的关系。方法应用聚合酶链反应和限制性片段长度多态性分析,对来自妇产科和皮肤性病门诊的泌尿生殖道感染沙眼衣原体患者的92份标本和来自健康体检者沙眼衣原体检测为阳性的68例标本进行了基因分型,并分析其临床表现特点与基因分型的关系。结果共检出D、E、F、G、H、I、J、K等基因型;检出D、E混合感染3例和D、J混合感染1例。有症状和无症状患者中E型、D型、F型、G型检出均较多;H型只在有症状的患者中检出;J型多在无症状的患者中检出。在不同年龄段中,以31～40岁为最多,但有症状患者和无症状患者之间无统计学差异。结论泌尿生殖道沙眼衣原体感染的临床表现与不同基因型感染有关。     

Chlamydia trachomatis variant not detected by plasmid based nucleic acid amplification tests: molecular characterisation and failure of single dose azithromycin

OBJECTIVE: To characterise a Chlamydia trachomatis variant strain from a patient with non-gonococcal urethritis (NGU) whose first void urine (FVU) displayed discrepant Ctrachomatis test results and describe the clinical response to treatment. METHODS: The FVU specimen was assayed with an immune based Chlamydia Rapid Test (CRT) and various nucleic acid amplification tests (NAATs) to establish C trachomatis infection. Sequencing of the major outer membrane protein gene (omp1 also known as ompA) was undertaken to identify the serovar of the variant strain. Polymerase chain reaction (PCR) analysis was also conducted to determine whether the strain harboured deletions in the cryptic plasmid or was plasmid free. RESULTS: The FVU specimen was strongly reactive in CRT but negative with the plasmid based Amplicor PCR (Roche) and ProbeTec ET (Becton-Dickinson) assays. However, NAATs for 16S RNA (Aptima Combo 2, GenProbe), omp1 (RealArt CT PCR, Artus and in-house NAATs) or the outer membrane complex B protein gene (omcB) established C trachomatis infection. Sequencing of omp1 showed that the variant belonged to serovar I. PCR analysis indicated that the variant was plasmid free. The patient did not respond to single dose azithromycin treatment but subsequently responded to a course of doxycycline. CONCLUSIONS: A pathogenic plasmid free C trachomatis variant was identified. Clinicians should be alerted to the possibility of undetected C trachomatis infection caused by such variants and the potential of azithromycin failure in patients with recurrent chlamydial NGU. The occurrence of this variant is rare and should not form the basis for judgment of the performance or usefulness of plasmid based NAATs for C trachomatis detection.     

New Chlamydia trachomatis L2 strains identified in a recent outbreak of lymphogranuloma venereum in Vienna, Austria

BACKGROUND: Since 2003, an ongoing outbreak of lymphogranuloma venereum (LGV), caused by Chlamydia trachomatis biovar L2b, has been reported among men who have sex with men. METHODS: Twenty-four samples positive for C. trachomatis were analyzed for specific biovars and genovariants by genotyping of the variable segment (VS) 4, VS2 and VS1 regions of the outer membrane protein (omp) A. In addition we assessed the patients' sociodemographic background and clinical signs and symptoms. RESULTS: Twenty-four men who have sex with men presented with either anorectal or inguinal symptoms and tested positive for C. trachomatis DNA. Of these, the L2 genotype accounted for 15 patients, with a high coinfection rate with HIV (73.3%) and other sexually transmitted infections (53.4%). Analysis of the VS1, VS2, and VS4 regions of the ompA gene revealed the variant L2b in 8 patients. In 4 patients, 3 new L2 sequences were identified with nucleotide changes in the VS1, VS2, and VS4 region, respectively, defining new strains designated L2c, d, e. CONCLUSIONS: This outbreak of LGV represents the further spread of C. trachomatis L2 infection. Sequence analysis of ompA regions shows heterogeneity of L2 variants, suggesting more than 1 source of the LGV infections diagnosed in Vienna.     

反向斑点杂交技术检测泌尿生殖道沙眼衣原体及基因分型

目的 建立反向斑点杂交技术,对泌尿生殖道沙眼衣原体感染进行检测和基因分型。方法 用Oligo软件设计寡核苷酸探针,采用巢式聚合酶链反应(PCR)扩增omp1基因的VS1-VS2序列,反向斑点杂交技术对沙眼衣原体进行检测和基因分型。结果 巢式PCR扩增omp1基因的VS1-VS2序列的敏感性与质粒PCR符合率为98.2%(56/57)。优选出11条型特异性(A、B+Ba、C、D、E、F、G、H、I、J和K)和3条群特异性寡核苷酸探针:B群(B、Ba、D和E型),C群(A、C、H、I、J和K型)和中间群(F和G型)。特异性经过基因库中的BLAST程序比较和试验优化,结果显示各探针均与标准株呈特异性杂交。56例VS1-VS2 PER阳性的临床标本杂交法检测均阳性,共检出59株沙眼衣原体,包括8个基因型,其中以E、F、D和H型为主,占77.9%,分别为25.4%,22.0%,16.9%和13.6%。发现3例混合感染占5.4%,分别为D/E、D/F和F/K。结论 反向斑点杂交技术简便且快速,可直接对临床标本沙眼衣原体进行检测和基因分型。     

Screening for Chlamydia trachomatis in an anonymous and confidential HIV counseling and testing site: feasibility and prevalence rates

BACKGROUND: Chlamydia trachomatis is the most common bacterial sexually transmitted disease (STD) in the United States. The development of nucleic acid amplification tests for C trachomatis in urine specimens allows for screening outside traditional clinic settings. Persons visiting an HIV counseling and testing site may be at increased risk for STDs, including C trachomatis. GOAL: To measure the acceptance of C trachomatis urine screening and the prevalence of C trachomatis infection among clients at an HIV counseling and testing site. STUDY DESIGN: Site HIV counselors offered urine C trachomatis screening to clients, administered a questionnaire, and collected urine samples. RESULTS: Of 808 counseling and testing site clients approached for C trachomatis screening, 572 (71%) accepted. The most common reasons for declining screening were absence of symptoms (33%) and recent STD testing (32%). Men were more likely to accept urine screening than women (risk ratio, 1.31; 95% CI, 1.06-1.62), as were clients who practiced oral sex, had a history of STD, or who had never been screened for STD. Of 560 urine specimens processed, only 8 (1.43%; 95% CI, 0.66-2.91%) were infected with C trachomatis. CONCLUSIONS: Sites offering HIV testing and counseling are a feasible alternative to clinical settings for C trachomatis screening. Prevalence may be too low for screening to be cost effective unless higher-risk subpopulations can be identified.     

First evaluation of six nucleic acid amplification tests widely used in the diagnosis of Chlamydia trachomatis in Russia

Background  In Russia, nationally developed nucleic acid amplification tests (NAATs), which have never been validated to international commercially available NAATs, are mainly used in the diagnosis of Chlamydia trachomatis infection.
Objective  To evaluate the performance characteristics of six NAATs widely used to diagnose C. trachomatis infection in Russia.
Materials and methods   In total, 446 consecutive symptomatic patients (319 females and 127 males) were included. Five polymerase chain reaction (PCR) assays and one real-time nucleic acid sequence-based amplification (NASBA) assay were evaluated on cervical and vaginal samples from females and on urethral and first voided urine samples from males. As reference methods, the Cobas Amplicor PCR, as the main 'gold standard' method, and LightMix 480HT PCR were used.
Results  The overall prevalence of C. trachomatis infection was 12.6%. The Russian NAATs and the reference methods displayed a high level of concordance (97.9% to 99.2%). In comparison with the reference methods, the sensitivities, specificities, positive predictive values and negative predictive values of the Russian tests in different specimens ranged from 86.1% to 100%, 99.1% to 100%, 92.3% to 100% and 98.2% to 100%, respectively.
Conclusions  According to the reference methods, C. trachomatis NAATs developed and used in Russia have relatively good performance characteristics for both invasive and non-invasive samples. However, larger studies that include symptomatic and asymptomatic patients as well as genital and extra-genital samples, and in comparison with other internationally well-recognized, validated, and ideally Food and Drug Administration–approved C. trachomatis NAATs performed strictly according to the manufacturer's instructions, need to be conducted.

Conflicts of interest

None declared     

Screening of Chlamydia trachomatis urogenital infections among the male and female population of the Republic of Macedonia

BACKGROUND: Noninvasive urine screening for Chlamydia trachomatis infections offers a valuable public health tool, that could be of vast importance in Chlamydia control programs. OBJECTIVE: The goal was to determine the prevalence of C. trachomatis infections among a sexually active population, to define the epidemiological factors associated with it, and to develop potential selective screening strategies among asymptomatic individuals in the Republic of Macedonia, using a highly sensitive and specific DNA amplification method for C. trachomatis. STUDY DESIGN: A total of 1435 urine samples, divided into two main groups: asymptomatic individuals (n = 1210) and symptomatic patients (n = 225), were tested. Samples from the asymptomatic group were collected during routine screening programs, while the symptomatic group consisted of patients with symptoms of urogenital tract infection, attending sexually transmitted diseases (STD) clinics. The presence of C. trachomatis was determined using commercial AMPLICOR C. trachomatis Assay (Roche Diagnostic Systems, Inc., Branchburg, NJ, USA). RESULTS: The prevalence of C. trachomatis infections among different groups was: recruits 0%, soldiers 0.4%, policemen 3.5%, clerks 4.6%, pregnant women 4%, and students 4.4%. The average prevalence for both groups (asymptomatic and symptomatic) was 2.3%[95% confidence interval (CI): 1.5-3.1%]. The average prevalence for the asymptomatic group was 1.6% (95% CI: 0.8-2.4%), while the average prevalence for the symptomatic group was 6.2% (95% CI: 3.1-9.3%) which were significantly different (P = 0.00003). CONCLUSION: Testing first void urine specimens by AMPLICOR C. trachomatis assay is a highly sensitive and specific method for diagnosing C. trachomatis infections in men and women. This method provides health care workers and public health officials with a new molecular amplification assay that uses noninvasive urine specimens for population-based screening purposes. The prevalence of C. trachomatis was relatively low among asymptomatic individuals. However, selective screening strategies are highly recommended for testing the student population in the Republic of Macedonia.     

性病门诊男性尿道炎患者沙眼衣原体基因分型

目的 了解性病门诊就诊的尿道炎男性患者中,沙眼衣原体血清型分布情况。 方法 采集2013年1 - 12月中国医学科学院皮肤病医院性病门诊有尿道炎症状的男性患者的尿液,荧光定量PCR检测沙眼衣原体,对沙眼衣原体阳性患者的尿液提取DNA,用巢式 PCR法扩增沙眼衣原体主要外膜蛋白基因ompA的VS1-VS2片段,然后对此片段测序,测序结果用DNAStar5.0软件与每种血清型的参考菌株做比对,分析其血清型。 结果 对2013年432例男性尿道炎患者进行了沙眼衣原体筛查,阳性标本143例,阳性率33.1%。143例沙眼衣原体阳性标本,127例扩增出ompA的VS1-VS2片段,16例未扩增出。127例阳性标本经测序分析获得9种血清型。血清型分布情况如下：E型29（22.83%）、F型28株（22.05%）、D型19（14.96%）、G型16株（12.60%）、J型16株（12.60%）、K型8株（6.30%）、H株5株（3.94%）、I型3株（2.36%）、B型3株（2.36%）,E、F、D、J、G型占85.02%。与标准菌种比对,发现127例菌株中有14株存在碱基突变,为同义突变。 结论 性病门诊男性尿道炎沙眼衣原体血清型主要是E型、F型、D型和G型,与20年前相比,E型菌株比例有所下降,J型菌株比例增高。     

Clinical features of Chlamydia trachomatis rectal infection by serovar among homosexually active men.

BACKGROUND AND OBJECTIVES: Because C. trachomatis serovars correlate with the clinical manifestations of cervical infection, we undertook this study to determine whether clinical, behavioral, and laboratory findings correlate with C. trachomatis serovars isolated from rectal infections. GOAL OF THIS STUDY: To correlate C. trachomatis serovar with signs and symptoms of rectal infection. STUDY DESIGN: A cross-sectional study of 454 men with rectal C. trachomatis infection attending an urban sexually transmitted disease (STD) clinic was undertaken. Isolates were thawed, passaged to high titer, and typed using a panel of monoclonal antibodies. Compared to men infected with B complex isolates (164), men with C complex isolates (55) were less likely to report symptoms (OR: 0.4; 95% CI: 0.1-0.8), or to have erythema, bleeding, or mucopus (OR: 0.3; 95% CI: 0.1-0.8). Among men with inclusion counts of more than 100, those infected with FG group versus B complex isolates were more likely to present with mucopus (OR: 10.5; 95% CI: 1.2-95.5), more than 15 polymorphonuclear leukocytes (OR: 19.2; 95% CI: 1.7-219.8), and proctitis (OR: 4.2; 95% CI: 1.1-16.7). CONCLUSION: Signs and symptoms of rectal infection correlate with the serovar of C. trachomatis isolates.     

Genotyping of Chlamydia trachomatis would improve contact tracing

BACKGROUND: The reported number of Chlamydia trachomatis genital infections has increased 15% annually since 1997 in Sweden. Inaccurate partner notification might be one reason. GOAL: The goals were to determine if genotyping of C trachomatis would improve partner notification and to study the duration of infection. STUDY DESIGN: Sexual networks were constructed. C trachomatis isolates from 231 individuals attending the Orebro STD clinic during 1 year were typed by sequencing of the omp1 gene. RESULTS: All individuals were traced and diagnoses were established in 30 of 161 networks. More than one genotype was seen in seven networks. The mean duration of C trachomatis infection in each network was calculated to be 23 weeks. CONCLUSION: Genotyping could be a useful tool in partner notification when there are discrepant or uncommon genotypes. Limited clinic catchment areas create information difficulties that obstruct accurate contact tracing.     

Serovar distribution of urogenital Chlamydia trachomatis strains in The Netherlands.

The distribution of serovars in 208 Chlamydia trachomatis strains of urogenital origin isolated from 185 patients attending a sexually transmitted disease clinic in Rotterdam, The Netherlands, was assessed. Typing by monoclonal antisera using a dot enzyme linked immunosorbent assay (ELISA) showed that the most common serovars were E (found in 45 strains), F (39), D (34), and K (28). Other serovars detected were H (21), G, I, I', J (two to 12), and B (one strain). Mixed infection with two serovars was detected in two patients. These results indicate that most genital infections with C trachomatis result from a small number of serovars, and that those serovars are similar in The Netherlands and Seattle, USA.     

Variation outside variable segments of the major outer membrane protein distinguishes trachoma from urogenital isolates of the same serovar of Chlamydia trachomatis.

OBJECTIVES--Whereas serovars A, B, Ba and C of Chlamydia trachomatis are usually associated with trachoma, two of these serovars (Ba and C) are occasionally observed in urogenital infections. Variation in the gene encoding the major outer membrane protein (MOMP) was explored to distinguish urogenital from trachoma specimens of the same serovar. METHODS--A large portion of the MOMP gene was amplified by nested PCR directly from clinical samples from trachoma or urogenital infection and the serovar of the infecting C trachomatis was determined by restriction fragment length polymorphism (RFLP). Amplified DNA from trachoma serovars B, Ba and C and from urogenital serovars Ba, C, D and E was sequenced by the dideoxy chain termination method. RESULTS--While almost identical in variable segment (VS)I, three urogenital Ba samples differed from all trachoma B and Ba samples at eight nucleotides including two sites which changed amino acids in the constant region upstream of VSI. An identical sequence in this region was observed for the reference urogenital D serovar. Variation in this same region upstream of VSI also distinguished 40% of serovar D samples from prototype D including three that were sequenced. Two urogenital C differed from trachoma C samples at four sites that changed the MOMP amino acid sequence including two changes in the constant region between VSII and III and single changes in VSII and III. On the basis of these sequence determinations, RFLP was predicted which allowed extension of these observations to 20 other urogenital Ba, 12 trachoma B or Ba, seven variant D, 12 D, four urogenital C and three trachoma C samples without further sequencing. CONCLUSION--Urogenital Ba and C samples have VSI or II and III sequences identical or very similar to trachoma strains of the same serovar, but resemble more closely other serovars in the constant regions. Urogenital serovar D samples can also be divided into two genotypes on the basis of sequence differences in the constant region preceding VSI.     

Susceptibility of different serovars of Chlamydia trachomatis to inactivation by normal human serum.

The ability of a panel of normal human serum samples to inactivate 12 strains of Chlamydia trachomatis, each of a different serovar, was investigated. A wide range of antichlamydial activity was observed, with survival rates of C trachomatis varying from less than 1% in some experiments to 100% in others. The strain specificity of the anti-chlamydial activity exhibited by individual serum samples was not, however, related to the antigenic cross reactivity between serovars demonstrable by microimmunofluorescence testing, which suggested that type specific antigens were not predominantly involved in the inactivation process.     

The impact on accuracy and cost of ligase chain reaction testing by pooling urine specimens for the diagnosis of Chlamydia trachomatis infections.

BACKGROUND AND OBJECTIVES: Nucleic acid amplification testing is the most accurate approach to diagnosing Chlamydia trachomatis infections. Our objective was to compare the accuracy and cost savings of pooling urines as opposed to individual testing. STUDY DESIGN: Strategies of pooling urine specimens into groups of four (4x pool) or eight (8x pool) followed by testing the positive pools individually were compared to individual specimen testing to determine if significant cost savingS could be realized without compromising the sensitivity and specificity of the LCx C. trachomatis Assay (Abbott Laboratories, Abbott Park, Chicago, IL) performed in a busy private medical laboratory. RESULTS: A total of 1,220 patient urine samples, 1,187 male (97%) and 33 female (3%), were tested using the normal LCx specimen to cutoff ratio (S/CO) of 1.0 and a decreased S/CO value of 0.2. Individual testing identified 98.2% (109/111) of positive urines. The 4x pooling maneuver identified 92.8% (103/111) of positive patients with the regular cutoff and 96.4% (107/111) when the cutoff was decreased. These values were 95.9% (47/49) and 97.9% (48/49), respectively, when eight urines were pooled. Both pooling and individual testing strategies identified all the negative samples accurately. Cost savings of pooling were calculated to be 44.5% for pools of four and 37.5% for pools of eight, applying the lowered cutoff. CONCLUSIONS: Pooling urine specimens for testing with the C. trachomatis LCx system is a simple, accurate, and cost-saving approach that can significantly reduce the cost of amplified nucleic acid testing with minimal sacrifice of testing accuracy.     

Differences in clinical manifestations of genital chlamydial infections related to serovars.

OBJECTIVES: To study the association of serovars of Chlamydia trachomatis with clinical manifestations of genital tract infection and socio-demographic characteristics. METHODS: In 1986-88 the C trachomatis isolates from 159 heterosexual men and 116 women attending a sexually transmitted disease (STD) clinic were collected and typed accordingly. A medical history was recorded, a physical examination took place and samples were taken for laboratory diagnostics. RESULTS: Serovars E, F and D were the most common for both men (75%) and women (67%). Men infected with serovars of the C-complex had more often a history of STD (p = 0.06). The opposite was demonstrated in women (p = 0.07). In addition, women younger than 18 years at first intercourse were more often infected with C-complex serovars (p = 0.05). For men, the serovars F/G less often produced symptoms of urethral discharge (p = 0.01) than the serovars of the B-complex and C-complex and were less often associated with the presence of 10 or more leukocytes in a Gram-stained smear (p = 0.04). CONCLUSIONS: In this study, infections with serovars F and G caused less obvious symptoms and signs of inflammation in men; in women no differences were found in the clinical manifestation of infections with different serovars.     

The molecular epidemiology of genital Chlamydia trachomatis in the greater Reykjavik area,Iceland

BACKGROUND: The diversity in MOMP (major outer membrane protein) of Chlamydia trachomatis is thought to be necessary for the bacteria to survive in its environment. The rate of change in the omp1 gene (coding for MOMP) is not known. Iceland offers a good opportunity to study the epidemiology of chlamydial infections because the population is small (280,000) and geographically well defined. GOAL: The goal was to determine the number and distribution of genotypes in a population attending the STD clinic in Reykjavík and to assess changes in omp1 sequences over a period of 2 years. STUDY DESIGN: Three-hundred thirty isolates of C trachomatis collected periodically from January 1999 to January 2001 were omp1 genotyped with nested PCR and sequencing. RESULTS: The serotypes found, in descending order of prevalence, were E, D, J, F, K, G, H, and I. Eighteen distinctive genotypes were found. During the study period no significant changes in frequency of genotypes were noted, and introduction of new or changed genotypes was not observed. CONCLUSION: The results indicate a relatively stable situation of genotypes and suggest an ecological advantage of serotype E.