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 ompi PCR-based RFLP analysis to differentiate C. trachomatis serovars A to K, Ba, Da, and Li 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 ompl 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 ompi PCR; of these five, two were positive by a nested ompi PCR. Genotyping by RFLP analysis of the 35 ompi PCR-positive samples showed that serovars D, E, and F are the most prevalent types found in cervical scrapes, while serovariant Dwas 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.

Several primers flanking the four variable domains of the ompl gene were selected and tested for sensitivity in several nested PCRs with serial dilutions of serovar G. The optimal sensitivity obtained was 0.1 to 0.01 inclusion-forming units, similar to that obtained in the C. trachomatis plasmid PCR. With this approach, any C. trachomatis PCR-positive sample can be typed.

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 ompi 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 0-58 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 postinflammatory complication of an ascending C trachomatis infection resulting in a scarred fallopian tube. (J7 Clin Pathol 1995;48:815-819) Keywords: Chlamydia trachomatis, polymerase chain reaction, ectopic pregnancy.The incidence of ectopic pregnancy has increased in many industrialised countries during the last two decades.' About 98% of ectopic pregnancies are tubal, often as a result of tubal damage after one or more episodes of pelvic inflammatory disease.6 The increasing incidence of ectopic pregnancy has led to many investigations of its microbial aetiology. Chlamydia trachomatis, one of the most common bacterial pathogens of sexually transmitted diseases, has been reported to be associated with ectopic pregnancy,7-9 as shown by the significantly increased incidence (twoto sevenfold) of antichlamydial IgG antibodies in sera from patients as compared with controls.7"'5 Moreover, studies of the microbial aetiology of pelvic inflammatory disease have shown that C trachomatis was present in the fallopian tubes at the time of salpingitis.16'9 So far the mechanism by which C trachomatis induces tubal damage in ectopic pregnancy remains unclear. It is speculated that persistent C trachomatis infection may cause tubal damage directly, or the occlusion may otherwise be the indirect result of post inflammatory damage. However, isolation of C trachomatis by cell culture from the resected salpinx of ectopic pregnancies has not been successful to date.'32022 Since the existence of a non-cultivable form of C trachomatis has been suggested,2"25 the role of this microorganism a...

Urogenital isolates (n = 93) of Chlamydia trachomatis were differentiated into serovars and variants by serotyping with monoclonal antibodies and genotyping by restriction fragment length polymorphism (RFLP) analysis of the PCR-amplifiedomp1 gene, respectively. The types of 87 of the 93 isolates (94%) were identical, as determined by both methods. Among these 87 isolates, 3 isolates were identified as the recently described new serovariant Ga/IOL-238 by omp1 nucleotide sequence analysis of the variable domains. Of the remaining six isolates, three isolates serotyped as both L2 and Ba but were identified as Ba/A-7 by genotyping by RFLP analysis of omp1. The omp1 nucleotide sequences of variable domains VD1, VD2, and VD4 of these urogenital Ba strains were identical to the sequences of the variable domains of Ba/J160, an ocular Ba type. The three remaining isolates were serotyped as J, but the patterns obtained by RFLP analysis of omp1, which were identical for the three isolates, differed from that of prototype serovar J/UW36. omp1 nucleotide sequence analysis revealed that these strains are genovariants of serovar J/UW36. Nucleotide sequence differences between serovar J/UW36 and this J genovariant, designated Jv, were found in both variable and constant domains. In conclusion, this study shows that the PCR-based genotyping of clinical C. trachomatis isolates by RFLP analysis ofomp1 has a higher discriminatory power and is more convenient than serotyping. Variants of C. trachomatisserovars Ba, G, and J were identified and characterized.

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 (). 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.Chlamydia trachomatis is one of the most frequent causes of sexually transmitted diseases. Currently, 15 serotypes have been identified, i.e., serotypes A to K and L1 to L3. These are extended by serovariants and genovariants, such as Ba, Da, D Ϫ , Ia, and L2a. Serovars D to K are urogenital pathogens and are also responsible for neonatal conjunctivitis and pneumonia. C. trachomatis infections may lead to pelvic inflammatory disease (PID), resulting in tubal infertility, ectopic pregnancy, and chronic pain (3,17,24). A substantial number of chlamydial infections have an asymptomatic course, and the published prevalence varies between 1.6 and 19%, depending on the population studied and the techniques used (3,13,20,21,24). It is assumed that asymptomatic C. trachomatis infection can also spread to the upper genital tract, causing PID and longterm sequelae such as tubal factor infertility and increased risk of ectopic pregnancy. Therefore, the detection of asymptomatic C. trachomatis infections is important. To date, data about serovar distributions in asymptomatic C. trachomatis infection are lacking.Recently, the PCR method has been used to detect C. trachomatis infections. PCR has proved to be more sensitive and specific than the conventional microbiological assays (4,9,15,16,18,22,23,25). This highly sensitive PCR is therefore a valuable tool for studying the prevalence of asymptomatic C.