Evaluation of molecular typing for epidemiological study of Chlamydia trachomatis genital infections.

Molecular typing and serotyping were compared for 150 Chlamydia trachomatis strains isolated from genital sources, belonging to 10 different serovars. Because of the general agreement of the two methods, molecular omp1 genotyping was applied to the epidemiological study of C. trachomatis isolates from genital infections in Bordeaux (France), during a 29-month period. The most prevalent omp1 genotypes were E (51.7%), F (17.3%), D (8.8%), and G (8.4%). Restriction enzyme analysis allowed identification of a serovar D variant (Dv), whereas serovar E strains were homogeneous.     

Urogenital Chlamydia trachomatis serovars in men and women with a symptomatic or asymptomatic infection: an association with clinical manifestations?

To determine whether certain Chlamydia trachomatis serovars are preferentially associated with a symptomatic or an asymptomatic course of infection, C. trachomatis serovar distributions were analyzed in symptomatically and asymptomatically infected persons. Furthermore, a possible association between C. trachomatis serovars and specific clinical symptoms was investigated. C. trachomatis-positive urine specimens from 219 asymptomatically infected men and women were obtained from population-based screening programs in Amsterdam. Two hundred twenty-one C. trachomatis-positive cervical and urethral swabs from symptomatically and asymptomatically infected men and women were obtained from several hospital-based departments. Serovars were determined using PCR-based genotyping, i.e., restriction fragment length polymorphism analysis of the nested-PCR-amplified omp1 gene. The most prevalent C. trachomatis serovars, D, E, and F, showed no association with either a symptomatic or asymptomatic course of infection. The most prominent differences found were (i) the association of serovar Ga with symptoms in men (P = 0.0027), specifically, dysuria (P < 0.0001), and (ii) detection of serovar Ia more often in asymptomatically infected people (men and women) (P = 0.035). Furthermore, in women, serovar K was associated with vaginal discharge (P = 0.002) and serovar variants were found only in women (P = 0.045).     

Use of PCR and reverse line blot hybridization assay for rapid simultaneous detection and serovar identification of Chlamydia trachomatis

The aim of this study was to develop and evaluate multiplex and nested PCR-reverse line blot (RLB) hybridization assays for detection and serovar identification of Chlamydia trachomatis. Two sets of primers targeting the VD2 region of the omp1 gene and one set targeting the cryptic plasmid were designed for use in multiplex (both targets) and nested PCR (omp1 only). For the RLB assay, labeled omp1 amplicons were hybridized to a membrane containing probes specific for 15 C. trachomatis serovars. The assays were used to test 429 clinical specimens, which had been previously tested for C. trachomatis using the COBAS AMPLICOR system. Specimens were tested without knowledge of the COBAS AMPLICOR result. Of 205 specimens that were positive by COBAS AMPLICOR, 201 (98%) were positive by multiplex PCR-RLB and 188 (92%) were also positive by omp1 nested PCR-RLB. In addition, three of 224 COBAS AMPLICOR-negative specimens were positive by omp1 nested PCR-RLB. One hundred sixty-six of 191 (87%) specimens in which C. trachomatis serovars were identified contained only one serovar and 25 (13%) contained two or three serovars. Serovars D, E, and F were found in 31 (16%), 83 (43%), and 51 (27%) specimens, respectively. Serovar E (41%) was the most commonly identified single serovar. Serovars J and K were found alone uncommonly (<2% each), but 18 of 25 (72%) specimens with multiple C. trachomatis serovars contained one or both (10 specimens) of these serovars. The nested (ompI) PCR-RLB is a specific and sensitive method for simultaneous detection and serovar identification of C. trachomatis, which can reliably identify mixed C. trachomatis serovars. It is suitable for use in epidemiological studies.     

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.     

Prevalence and serovar distribution of asymptomatic cervical Chlamydia trachomatis infections as determined by highly sensitive PCR.

The prevalence rates and serovar distributions of Chlamydia trachomatis cervical infections were investigated in two different groups of women. Group I consisted of 393 asymptomatic young women (aged 17 to 30 years) who were invited to participate in a C. trachomatis screening program. Group II consisted of 734 randomly selected patients (aged 17 to 68 years) attending an inner-city gynecological outpatient clinic. C. trachomatis was detected in cervical scrapes by PCR specific for endogenous plasmid. These plasmid PCR-positive samples were subsequently subjected to genotyping by C. trachomatis-specific omp1 PCR-based restriction fragment length polymorphism analysis (J. Lan, J. M. M. Walboomers, R. Roosendaal, G. J. van Doornum, D. M. MacLaren, C. J. L. M. Meijer, and A. J. C. van den Brule, J. Clin. Microbiol. 31:1060-1065, 1993). The overall prevalence rates of C. trachomatis found in patients younger than 30 years were 9.2 and 11.8% in groups I and II, respectively. A clear age dependency was seen in group II, with the highest prevalence rate (20%) found in patients younger than 20 years, while the rate declined significantly after 30 years of age (5.9%). In women younger than 30 years, the genotyping results showed that serovars E, I, and D (in decreasing order) were frequent in group I, while serovars F, E, and G (in decreasing order) were predominantly found in group II. The study shows that C. trachomatis infections are highly prevalent in asymptomatic young women. The different serovar distributions found most likely reflect the different compositions of the study groups, but additional analysis of the case histories of individual patients suggests that certain serovars might be associated with symptomatic (i.e., serovar G) or asymptomatic (i.e., serovars D and I) infections.     

Evaluation of a novel PCR-based assay for detection and identification of Chlamydia trachomatis serovars in cervical specimens

The aims of this study were to compare a novel PCR-based Chlamydia trachomatis detection and genotyping (Ct-DT) assay with the FDA-approved, commercially available C. trachomatis detection Hybrid Capture 2 (HC2) assay and to investigate the C. trachomatis serovar distribution among young women in a rural Costa Rican study population. A total of 5,828 sexually active women participating in a community-based trial in Costa Rica were tested for C. trachomatis by HC2. A sample of 1,229 specimens consisting of 100% HC2 C. trachomatis-positive specimens (n = 827) and a random sample of 8% HC2 C. trachomatis-negative specimens (n = 402) were tested with the Ct-DT assay. Agreement between the two assays was determined by the unweighted kappa statistic. Discrepant specimens were tested with a second commercially available test (COBAS TaqMan). The Ct-DT-positive specimens were further analyzed with the Ct-DT genotyping step to investigate the distribution of 14 different C. trachomatis serovars (A, B/Ba, C, D/Da, E, F, G/Ga, H, I/Ia, J, K, L1, L2/L2a, and L3). After accounting for the sampling fraction selected for Ct-DT testing, crude agreement with the HC2 assay was 98% and the kappa was 0.92 (95% confidence interval [CI], 0.89 to 0.97). The 33 discordant samples that were further analyzed with the COBAS TaqMan test showed better agreement with the Ct-DT assay (31/33, P < 0.001). Among the 806 Ct-DT-positive samples, serovar E was the most common serovar (31%), followed by serovars F and D (both 21%) and serovar I (15%). In conclusion, the novel Ct-DT assay permits reliable detection and identification of C. trachomatis serovars.     

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.     

Specific-pathogen-free pigs as an animal model for studying Chlamydia trachomatis genital infection

The purpose of the present study was to evaluate pigs as a large-animal model for female genital infection with two Chlamydia trachomatis human serovar E strains. Sixteen-week-old specific-pathogen-free female pigs (gilts) were intravaginally infected with the trachoma type E reference strain Bour or the urogenital serovar E strain 468. Several conclusions can be drawn from our findings on the pathogenicity of a primary C. trachomatis genital infection in gilts. First of all, we demonstrated that the serovar E strains Bour and 468 could ascend in the genital tract of gilts. The serovar E strains could replicate in the superficial columnar cervical epithelium and in the superficial epithelial layer of the uterus, which are known to be the specific target sites for a C. trachomatis genital infection in women. Second, inflammation and pathology occurred at the replication sites. Third, the organisms could trigger a humoral immune response, as demonstrated by the presence of immunoglobulin M (IgM), IgG, and IgA in both serum and genital secretion samples. Our findings imply that the pig model might be useful for studying the pathology, pathogenesis, and immune response to a C. trachomatis infection of the genital system.     

Epidemiology of Chlamydia trachomatis using analysis of gene encoding of the major outer membrane protein]

One hundred and eight clinical strains and 24 reference strains of C. trachomatis were typed using differential restriction mapping of omp1, the gene which encodes the major outer membrane protein. The gene was obtained by polymerase chain reaction (PCR). This molecular typing method correlated well with serological typing. Eighty-four per cent of clinical strains were typed using the enzyme AluI alone. Heterogeneity was looked for among the most common serovars (E, F, and D; 62%, 17%, and 9%, respectively). Analysis of the PCR-amplified fourth variable domain of omp1 using denaturing gradient gel electrophoresis followed by direct sequencing of the variants disclosed substantial heterogeneity within the D serovar. Conversely, serovars E and F were homogeneous, with however a single variant strain of serovar E.     

Combination of PCR targeting the VD2 of omp1 and reverse line blot analysis for typing of urogenital Chlamydia trachomatis serovars in cervical scrape specimens

In this study we developed and evaluated a new PCR-based typing assay, directed to the VD2 region of the omp1 gene, for the detection and typing of urogenital Chlamydia trachomatis infections. A nested VD2 PCR-reverse line blot (RLB) assay was developed for the typing of nine different urogenital serovars of C. trachomatis. The assay developed was tested with reference strains of C. trachomatis serovars and cervical scrapes of 86 Colombian women previously found to be positive for C. trachomatis by using plasmid PCR. Two sets of primers directed to the VD2 region of the omp1 gene of C. trachomatis were designed, and fragments of 220 and 166 bp were generated in the primary and nested PCRs, respectively. In addition, an RLB assay was developed to identify nine different urogenital serovars of C. trachomatis (Ba, D, E, F, G, H, I, J, and K) and group controls, including group B (Ba, D, and E), group C (I, J, K, and H), and an intermediate group (F and G). Using this assay, we were able to type 81 of the 86 samples (94.2%). Of these samples, 91.3% were single C. trachomatis infections, and 8.7% were multiple infections. The most common serovars identified were serovars D (22.2%), F (18.5%), G (13.6%), and E (12.3%). Of the women with multiple C. trachomatis infections, >50% contained both serovars D and E. The nested VD2 PCR-RLB developed is a simple, fast, and specific method for the identification of individual urogenital C. trachomatis serovars previously detected by using plasmid PCR. Moreover, it is an appropriate method for studying multiple C. trachomatis infections and for use in large epidemiological studies.     

PCR assessment of Chlamydia trachomatis infection of semen specimens processed for artificial insemination

In order to ascertain the microbiological quality of stored semen specimens processed for artificial insemination by a donor (AID), we developed a PCR assay targeting the chlamydial plasmid to detect Chlamydia trachomatis in semen. The lower limit of detection of this assay corresponded to 2.5 to 5 elementary bodies per microl of semen. A total of 669 cryopreserved ejaculates from 97 asymptomatic donors were tested for C. trachomatis infection. Twelve ejaculates, originating from four donors, were found to be positive, indicating a 4% prevalence of C. trachomatis infection among the donor population studied. Cross-contamination between the cryopreserved specimens in the storage container was studied by typing using sequence analysis of PCR-amplified omp1 genes of the strains. Two donors were infected with serovar E, one was infected with serovar F, and one was infected with serovar K. For two donors, the duration of C. trachomatis positivity could be assessed. One donor donated C. trachomatis-positive semen for at least 4 successive months, and the other did so for at least 16 months. The occurrence of C. trachomatis infection in cryopreserved donor semen indicates that ejaculates from donors not tested for a C. trachomatis infection just prior to donation should be tested for infection by a direct test such as the PCR described here. Direct testing of semen specimens will detect not only donors with an active infection but also C. trachomatis-infected ejaculates already stored and will thus improve the microbiological quality of AID, since discrepancies in the presence of C. trachomatis in urine and semen specimens have been reported.     

Molecular epidemiology of genital Chlamydia trachomatis infection in high-risk women in Senegal, West Africa

The prevalence and heterogeneity of Chlamydia trachomatis infections in a cohort of female sex workers in Dakar (Senegal) were determined by using endocervical-swab-based PCR DNA amplification assays. The overall prevalence of cervical chlamydial infection was 28.5% (206 of 722), and most of these infections were asymptomatic. An increased number of sexual partners was significantly associated with infection (adjusted odds ratio [AOR] = 1.37; 95% confidence interval [CI] = 1.06 to 1.77), while the presence of a yeast infection was negatively associated with chlamydial infection (AOR = 0.28; 95% CI = 0.10 to 0.83). Six different C. trachomatis genotypes were identified based on phylogenetic analysis of the omp1 gene sequences. Interestingly, genotype E predominated (47.6%) and was not associated with visible signs of cervical inflammation compared to non-E genotypes (P < 0.05). Overall, the high rate of asymptomatic C. trachomatis infection by genotype E may suggest genotype-specific properties that confer a transmission advantage in this high risk population.     

Characterization of Chlamydia trachomatis omp1 genotypes detected in eye swab samples from remote Australian communities

Chlamydia trachomatis conjunctival samples collected over a 6-month period from individuals with clinical signs of trachoma and located in remote communities in the Australian Northern Territory were differentially characterized according to serovar and variants. The rationale was to gain an understanding of the epidemiology of an apparent increased prevalence of acute trachoma in areas thought to be less conducive to this disease. Characterization was performed through sequencing of a region of the omp1 gene spanning the four variable domains and encoding the major outer membrane protein. Nucleotide and deduced amino acid sequences were genotyped by using a BLAST similarity search and were examined by phylogenetic analyses to illustrate evolutionary relationships between the clinical and GenBank reference strains. The predominant genotype identified corresponded to that of serovar C (87.1%), followed by the genotype corresponding to serovar Ba (12.9%). All nucleotide and amino acid sequences exhibited minor levels of variation with respect to GenBank reference sequences. The omp1 nucleotide sequences of the clinical samples best aligned with those of the conjunctival C. trachomatis reference strains C/TW-3/OT and Ba/Apache-2. All clinical samples (of serovar C) exhibited four or five nucleotide changes compared with C/TW-3/OT, while all serovar Ba samples had one or two nucleotide differences from Ba/Apache-2. Phylogenetic analyses revealed close relationships between these Northern Territory chlamydial samples and the respective reference strains, although the high proportion of sequence variants suggests an evolutionarily distinct C. trachomatis population causing eye infections in Australia. Given that such genotypic information has gone unreported, these findings provide knowledge and a foundation for trachoma-associated C. trachomatis variants circulating in the Northern Territory.     

Chlamydia trachomatis serovar E isolates from patients with different clinical manifestations have similar courses of infection in a murine model: host factors as major determinants of C trachomatis mediated pathogenesis

BACKGROUND: Some investigators have proposed an association between certain Chlamydia trachomatis serovars and the clinical course of infection in humans. A recent study of over 1100 patients with culture confirmed and serotyped C trachomatis urogenital infection detected no such association. AIMS: To corroborate these results using a murine model of female genital tract infection. METHODS: Various parameters of infection were assessed in mice intravaginally infected with human genital isolates of C trachomatis serovar E from four cases with either a clear symptomatic or asymptomatic clinical course in both the patient and their partner. RESULTS: No differences were seen among the strains in the incidence or duration of infection, polymorphonuclear granulocyte responses, or upper genital tract progression. CONCLUSIONS: An investigation to determine the correlation between the clinical manifestations of different isolates of C trachomatis serovar E in humans and certain parameters of microbial pathogenesis in a mouse model failed to reveal an association between the measured parameters and the tendency of serovar E to produce symptomatic versus asymptomatic infections in humans. These findings suggest that differences in the clinical course of infection in humans seen with these strains may be more related to host factors than to genetic variation among strains.     

Murine cytotoxic T lymphocytes induced following Chlamydia trachomatis intraperitoneal or genital tract infection respond to cells infected with multiple serovars.

The obligate intracellular bacterium Chlamydia trachomatis is associated with human diseases ranging from blinding trachoma to sexually acquired genital infections and the systemic disease lymphogranuloma venereum (LGV). We have previously reported the isolation and culture of protective murine cytotoxic T lymphocytes (CTL) following intraperitoneal infection with C. trachomatis serovar L2, a serotype associated with human LGV. In this report, we now demonstrate that CTL can also be primed following introduction of C. trachomatis serovar L2 into the uterus or ovarian bursa of mice. We also describe Chlamydia-specific CTL lines isolated following murine infection with a typical human urogenital isolate of C. trachomatis (serovar D) and show that such CTL can be primed by intraperitoneal, intrauterine, or intrabursal infection. Last, we demonstrate that these murine CTL lines respond to multiple serovars, recognizing and lysing cells infected with C. trachomatis serovars B, C, D, F, J, K, L2, and L3, representative of organisms causing blinding trachoma, genital infection, and LGV.     

Chlamydia trachomatis and ectopic pregnancy: retrospective analysis of salpingectomy specimens, endometrial biopsies, and cervical smears.

AIMS--To examine the role of Chlamydia trachomatis in ectopic pregnancy by detection of DNA in archival salpingectomy specimens, and in their preceding cervical specimens and endometrial biopsies, by using the polymerase chain reaction (PCR). METHODS--Archival paraffin embedded salpingectomy tissues (n = 48) from 37 women with ectopic pregnancy were examined for the presence of C trachomatis plasmid and omp1 DNA by PCR. In addition, preceding cervical specimens (n = 58) stored either as cervical cell suspensions or as archival cervical smears, and preceding endometrial biopsies (n = 18), taken 0-5.8 years before the ectopic pregnancy, were examined by PCR for the presence of C trachomatis. RESULTS--C trachomatis DNA was detected in only one of the 48 salpingectomy specimens from 37 women. However, in six of the 37 women, C trachomatis DNA was detected in the genital specimens (cervix and/or endometrial) taken before salpingectomy. C trachomatis infections were mostly found in endometrial or cervical specimens taken more than three years before ectopic pregnancy. No chlamydial DNA was found in endometrial or cervical specimens taken at the same time of the ectopic pregnancy. CONCLUSIONS--Although no C trachomatis DNA was found in salpingectomy specimens, several women with ectopic pregnancy had C trachomatis infections in endometrial and cervical specimens in the past. This suggests that at least in these cases the ectopic pregnancy is a late post-inflammatory complication of an ascending C trachomatis infection resulting in a scarred fallopian tube.     

Monoclonal antibodies in serovar determination of 53 Chlamydia trachomatis isolates from Amiens, France

The serovar distribution of 53 Chlamydia trachomatis strains obtained from 53 clinical isolates in Amiens (France) was studied by a micro-immunofluorescence test with a panel of 15 monoclonal antibodies. The isolates were of ocular (babies) or urogenital origin (adults). This typing showed that E was the most common serovar (62.3%) followed by F (9.4%), Ba, D, J (5.6%), H (3.8%) and G, K (1.9%). Two mixed infections were detected (one EG and one FG). Consequently, the serovar distribution of C. trachomatis in Amiens (France), was characterized by a predominance of serovar E higher than in other European countries.     

Evaluation of serotyping using monoclonal antibodies and PCR-RFLP for Chlamydia trachomatis serotype identification

We compared genotyping by restriction fragment length polymorphism (RFLP) analysis of the amplified omp1 gene with serotyping by dot enzyme-linked immunosorbent assay (dot-ELISA) to determine the suitability of RFLP analysis for epidemiologic study. Fifteen prototypes of Chlamydia trachomatis and 30 clinical isolates were used in this study. To serotype with dot-ELISA, chlamydia antigen was spotted onto a series of replicate nitrocellulose membrane patches and reacted with 11 mAbs that distinguish the 15 known serovars of C. trachomatis. For RFLP analysis, the amplified chlamydia omp1 gene was digested with AluI to differentiate serovars A to K and L1 to L3. Serovars of C, H, I, J, and L3 were further typed by RFLP analysis after digestion with HinfI, and a combination of EcoRI and DdeI. PCR-based RFLP could identify serotype of 28 among 30 clinical isolates tested. The remaining two untypical isolates were probably due to double infections or mechanical transferring error. Serotyping of C. trachomatis isolates shows that serovars E, D, F, and H are the most prevalent types found in urogenital samples in Korea. In this study, we show that RFLP analysis of amplified omp1 gene may be useful in genotyping C. trachomatis isolates.     

Chlamydia trachomatis serovar differentiation by direct sequence analysis of the variable segment 4 region of the major outer membrane protein gene.

The polymerase chain reaction method was used to amplify DNA from the fourth variable segment of the gene encoding the major outer membrane protein of Chlamydia trachomatis. Direct sequencing of the amplified DNA from prototype strains confirmed previously identified nucleotide sequence differences that were specific for each serovar. This analysis revealed differences in the DNA sequences of prototype strains C/UW-1 and G/IOL-238 from those of prototype strains C/TW-3 and G/UW-57, sequenced previously. This method was also used to determine the serovar types of C. trachomatis in 125 urogenital specimens from infected patients. The most common serovars were E (38%), F (17%), and G and D (14% each). Serovar D was found significantly more often in specimens from men than in specimens from women (P = 0.004). Conversely, serovar G was found significantly more often in specimens from women than in specimens from men (P = 0.026). Only two serovar G isolates gave sequences identical to that of the prototype strain G/IOL-238, suggesting that this strain may be a serovar variant. Three isolates (D+, G-, and J') gave sequences which have not been reported previously. One isolate had the same sequence as the D- serovar variant. Sequence analysis of amplified DNA reveals subtle differences between C. trachomatis strains and provides a very sensitive method for molecular epidemiological analysis.     

Direct detection and genotyping of Chlamydia trachomatis in cervical scrapes by using polymerase chain reaction and restriction fragment length polymorphism analysis.

Detection and genotyping of Chlamydia trachomatis were optimized by using a polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis performed directly with crude cells of cervical scrapes. Different PCR pretreatment methods were evaluated on samples which were positive for C. trachomatis by cell culture. In comparison with DNA extraction and different proteolytic digestion methods, a simple pretreatment of 10 min of boiling appeared to be optimal for PCR amplification. Crude samples (n = 209) were first screened for C. trachomatis by both cell culture and plasmid PCR. Subsequently, positive samples found by plasmid PCR were subjected to a direct omp1 PCR-based RFLP analysis to differentiate C. trachomatis serovars A to K, Ba, Da, and L1 to L3 and serovariant D-. All cervical scrapes that were found positive for C. trachomatis by cell culture (n = 30) were also positive by plasmid PCR and omp1 PCR and could be easily genotyped. In addition, of the culture-negative group, eight samples were found positive by plasmid PCR. Five of these eight samples were also positive by omp1 PCR; of these five, two were positive by a nested omp1 PCR. Genotyping by RFLP analysis of the 35 omp1 PCR-positive samples showed that serovars D, E, and F are the most prevalent types found in cervical scrapes, while serovariant D- was also detected. This study shows that direct PCR and PCR-based RFLP analysis are feasible for detection and genotyping of C. trachomatis in cervical scrapes and are more sensitive than culture-based serotyping.