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.     

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).     

Differences in growth characteristics and elementary body associated cytotoxicity between Chlamydia trachomatis oculogenital serovars D and H and Chlamydia muridarum

AIM: In vitro growth and elementary body (EB) associated cytotoxicity of two Chlamydia trachomatis strains belonging to serovars D and H and C muridarum were compared to identify difference(s) that correlate with virulence variations between these strains in the mouse model of human female genital tract infection, and phenotypic characteristics that could explain human epidemiological data on serovar prevalence and levels of shedding during serovar D and H infection. METHODS: Replication cycle kinetics, inclusion characteristics, and EB associated cytotoxicity were assessed in McCoy cell monolayers using culture, light microscopy, and lactate dehydrogenase release. RESULTS: Over 72 hours, more rapid production and release of inclusion forming units (ifu) allowed C muridarum to initiate two replication rounds, resulting in 4-8 times more ifu/input unit of infection than with serovars D and H. Although C muridarum EBs were significantly more cytotoxic to McCoy cell monolayers than serovar D at moderate and high multiplicity of infection ratios (MOI), serovar H EBs were significantly more cytotoxic than C muridarum, even at the lowest MOI tested. CONCLUSIONS: These phenotypic differences are consistent with the more invasive course and severe pathological outcome of infection in mice infected with C muridarum, providing an objective basis for questioning the appropriateness of C muridarum as a surrogate for the human biovar of C trachomatis in the murine model of female genital tract infection. The differences seen between the human strains could help explain human epidemiological data relating to differences in prevalence and level of shedding that occurs during infection with oculogenital serovars D and H.     

Prior genital tract infection with a murine or human biovar of Chlamydia trachomatis protects mice against heterotypic challenge infection

We sought to assess the degree of cross-protective immunity in a mouse model of chlamydial genital tract infection. Following resolution of genital infection with the mouse pneumonitis (MoPn) biovar of Chlamydia trachomatis, mice were challenged intravaginally with either MoPn or human serovar E or L2. The majority of animals previously infected with MoPn were solidly immune to challenge with either of the two human biovars. Surprisingly, approximately 50% of animals became reinfected when homologously challenged with MoPn, although the secondary infection yielded significantly lower numbers of the organism isolated over a shorter duration than in the primary infection. Primary infection with serovar E also protected against challenge with MoPn or serovar L2, although the degree of immune protection was lower than that resulting from primary infection with MoPn. Blast transformation and assessment of delayed-type hypersensitivity indicated that mice previously infected with either human or murine biovars produced broadly cross-reactive T cells that recognized epitopes of either murine or human biovars of C. trachomatis. Immunoblotting demonstrated that primary MoPn infection produced immunoglobulin G (IgG) antibody to antigens of MoPn as well as at least three distinct antigenic components of human serovar E, one of which was identical in molecular weight to the major outer membrane protein (MOMP). Primary infection with serovar E produced IgG antibody reactive against serovar E but not MoPn MOMP and against at least one ca. 60-kDa protein of both chlamydial strains. Our results indicate that primary genital infection of mice with murine C. trachomatis induces immunity against challenge with either of two human biovars.     

Differences in the association of Chlamydia trachomatis serovar E and serovar L2 with epithelial cells in vitro may reflect biological differences in vivo.

Chlamydia trachomatis serovar E is one of the most common bacterial sexually transmitted pathogens. Since it is an obligate intracellular bacterium, efficient colonization of genital mucosal epithelial cells is crucial to the infectious process. Serovar E elementary bodies (EB) metabolically radiolabeled with 35S-Cys-Met and harvested from microcarrier bead cultures, which significantly improves the infectious EB-to-particle ratio, provided a more accurate picture of the parameters of attachment of EB to human endometrial epithelial cells (HEC-1B) than did less infectious 14C-EB harvested from flask cultures. Binding of serovar E EB was (i) equivalent at 35 and 4 degrees C, (ii) decreased by preexposure of EB to heat or the topical microbicide C31G, (iii) comparable among common eukaryotic cell lines (HeLa, McCoy), and (iv) significantly increased to the apical surfaces of polarized cells versus nonpolarized cells. In parallel experiments with C. trachomatis serovar L2, serovar E attachment was not affected by heparin or heparan sulfate whereas these glucosaminoglycans dramatically reduced serovar L2 attachment. These data were confirmed by competitive inhibition of serovar E binding and infectivity by excess unlabeled live and UV-inactivated serovar E EB but not by excess serovar L2 EB. The noninvasive serovar E strains in the lumen of the genital tract enter and exit the apical domains of target columnar epithelial cells to spread canalicularly in an ascending fashion from the lower to the upper genital tract. In contrast, the invasive serovar L2 strains are primarily submucosal pathogens and likely use the glucosaminoglycans concentrated in the extracellular matrix to colonize the basolateral domains of mucosal epithelia to perpetuate the infectious process.     

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.     

Distribution study of Chlamydia trachomatis serovars among high-risk women in China performed using PCR-restriction fragment length polymorphism genotyping

This was one of the first epidemiological studies in China focused on genital Chlamydia trachomatis serotype distribution in high-risk female populations using omp1 gene-based restriction fragment length polymorphism analysis. One thousand seven hundred seventy cervical swab samples from women attending sexually transmitted disease clinics and female sex workers in six cities in China (Shenzhen and Guangzhou in southern China, Nanjing and Shanghai in eastern China, and Nanning and Chengdu in southwestern China) were subjected to serovar genotyping. The proportion of omp1 genes successfully amplified in 240 C. trachomatis plasmid-positive samples was 94.2% (226/240). Serotypes E (n = 63; 27.9%), F (n = 53; 23.5%), G (n = 28; 12.4%), and D (n = 25; 11.1%) were most prevalent. Though there was no significant difference in the geographic distribution of C. trachomatis, serotype E was predominant in the South (32.1%) and East (27.1%), while serotype F was predominant in the Southwest (28.3%). Serotype F infection was associated with young age and single status. Serovar G was associated with lower abdominal pain; 47.5% of asymptomatic patients were infected with serovar E. These results provide information on distribution of genital C. trachomatis serotypes among high-risk women in China and indicate that high-risk women, including those who are asymptomatic, can be infected with multiple serovars of C. trachomatis, revealing exposure to multiple sources of infection. Although the scope for generalizations is limited by our small sample size, our results showing clinical correlations with genotypes are informative.     

Comparison of multiple genital tract infections with Chlamydia trachomatis in different strains of female mice.

We have previously shown that female outbred CF-1 mice are susceptible to prolonged genital tract infection with the oculogenital serovars (D-K) of Chlamydia trachomatis, and that partial homotypic and heterotypic protection against reinfection is induced. To understand the possible role of inherent T-helper 1 (Th1)/Th2 polarity bias on both the course of infection and the level of acquired immunity induced by infection, 2 immunologically different and well-characterized inbred strains of mice, BALB/c and C57BL/6, were studied in this model. Groups of mice were inoculated intravaginally with C. trachomatis serovar D (Ct D) and monitored by culture to determine the duration of initial infection. Two months later, mice were reinfected, and monitored along with age- and condition-matched control groups. Plasma and vaginal secretions were collected for serologic analysis and specific delayed-type hypersensitivity was assessed by footpad swelling. Initial infection in C57BL/6 mice was comparable in duration to outbred CF-1 mice (median duration 42 versus 43.5 days), while BALB/c mice had a shorter median duration of initial infection (12 days). All strains had significantly shorter durations of infection following reinfection. BALB/c mice shed 4-10 times more inclusion-forming units (IFU) than both C57BL/6 and CF-1 mice on sample days during the first week of infection and all strains shed less IFU during reinfection. C57BL/6 and BALB/c mice had significantly lower anti-Ct D immunoglobulin G titers in both plasma and vaginal secretions than CF-1 mice following resolution of infection; the frequency of immunoglobulin A seropositive vaginal secretions was less in both inbred strains, being significantly less in the case of C57BL/6 mice. Qualitative analysis of the antigen specificity and isotype composition revealed differences among the mouse strains. All 3 strains had detectable levels of specific footpad swelling on day 14 of infection, whereas only BALB/c mice showed a significant response at 70 days post-infection. Significant differences between 2 strains of mice that differ in Th1/Th2 polarity bias were observed in: 1) the duration of infection; 2) the level of bacterial shedding during infection; and 3) the quantitative and qualitative cellular and humoral responses made in response to female genital tract infection with a human oculogenital isolate of C. trachomatis. In addition, a similar and significant level of partial acquired immunity to reinfection was observed in both strains, suggesting that inherent Th1/Th2 polarity bias present upon initial infection does not prevent the development of a protective immune response within the genital tract during infection with an oculogenital isolate of C. trachomatis.     

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.     

Cytoskeletal requirements in Chlamydia trachomatis infection of host cells.

Infection of genital epithelial cells by the closely related sexually transmitted pathogens Chlamydia trachomatis serovars E and L2 results in different clinical disease manifestations. Following entry into target host cells, individual vesicles containing chlamydiae fuse with one another to form one large inclusion. At the cellular level, the only obvious difference between these serovars is the time until inclusion maturation, which is 48 h for the invasive serovar L2 and 72 h for serovar E. To begin to define the intracellular events of these pathogens, the effect of cytoskeletal disruption on early endosome fusion and inclusion development in epithelial (HEC-1B) and fibroblast (McCoy) cells was analyzed by fluorescence microscopy. Disruption of microfilaments with cytochalasin D markedly reduced serovar E, but not serovar L2, infection of both cell lines. Conversely, microfilament as well as microtubule disruption, with colchicine or nocodazole, had no effect on serovar E inclusion development but resulted in the formation of multiple serovar L2 inclusions per cell during early and mid-development. Later in serovar L2 inclusion development (> 36 h postinfection), vesicles containing chlamydiae fused to form one large inclusion in the absence of an intact cytoskeleton. These results imply that (i) C. trachomatis serovar E may utilize a different pathway for uptake and development from serovar L2; (ii) these differences are consistent in both epithelial cells and fibroblasts; and (iii) the cytoskeleton plays a unique role in the infection of host cells by these two genital pathogens.     

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.     

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.     

A model of genital Chlamydia trachomatis infection using human xenografts in severe combined immunodeficiency mice.

We developed a new model of human genital Chlamydia trachomatis infection in order to characterize the pathogen-host relationship in a clinically relevant system using a human strain of C. trachomatis instead of the commonly employed mouse biovar (MoPn). Human endometrial tissue was xenografted into the skin of mice homozygous for the mutation severe combined immunodeficiency and inoculated with C. trachomatis serovar K. C. trachomatis efficiently infected the endometrium as shown by cell culture and immunofluorescence microscopy and persisted for more than 6 weeks. Chlamydial inclusions detected by direct immunofluorescence and electron microscopy appeared to be smaller than those produced by in vitro cell culture-grown chlamydiae. A pattern of localized mild infection prevailed, and infiltrative uncontrolled spread of chlamydiae was observed in only 1 of 10 infected grafts. This might correspond to the well-known tendency of the agent to cause asymptomatic infections. This model allows the study of a human genital infection resembling the clinical situation and offers the possibility to better characterize the host-parasite relationship with respect to pathogenicity and therapy.     

Chlamydia trachomatis infection: host immune responses and potential vaccines

Chlamydia trachomatis causes genital tract infections that affect men, women, and children on a global scale. This review focuses on innate and adaptive immune responses in the female reproductive tract (FRT) to genital tract infections with C. trachomatis. It covers C. trachomatis infections and highlights our current knowledge of genital tract infections, serovar distribution, infectious load, and clinical manifestations of these infections in women. The unique features of the immune system of the FRT will be discussed and will include a review of our current knowledge of innate and adaptive immunity to chlamydial infections at this mucosal site. The use of animal models to study the pathogenesis of, and immunity to, Chlamydia infection of the female genital tract will also be discussed and a review of recent immunization and challenge experiments in the murine model of chlamydial FRT infection will be presented.     

Isolates of Chlamydia trachomatis that occupy nonfusogenic inclusions lack IncA, a protein localized to the inclusion membrane

The chlamydiae are obligate intracellular pathogens that occupy a nonacidified vacuole, termed an inclusion, throughout their developmenal cycle. When an epithelial cell is infected with multiple Chlamydia trachomatis elementary bodies, they are internalized by endocytosis into individual phagosomal vacuoles that eventually fuse to form a single inclusion. In the course of large-scale serotyping studies in which fluorescent antibody staining of infected cells was used, a minority of strains that had an alternate inclusion morphology were identified. These variants formed multiple nonfusogenic inclusions in infected cells, with the number of independent inclusions per cell varying directly with the multiplicity of infection. Overall the nonfusogenic phenotype was found in 1.5% (176 of 11,440) of independent isolates. Nonfusing variants were seen in C. trachomatis serovars B, D, D-, E, F, G, H, Ia, J, and K. The nonfusing phenotype persisted through repeated serial passage, and the phenotype was consistent in four mammalian host cell lines. Fluorescence microscopy and immunoblotting with antisera directed at proteins in the C. trachomatis inclusion membrane revealed that one such protein, IncA, was not detected in the inclusion membrane in each tested nonfusogenic strain. The distributions of other chlamydial proteins, including one additional Inc protein, were similar in wild-type and variant strains. The incA coding and upstream regions were amplified and sequenced from the prototype serovar D and two nonfusing serovar D((s)) strains. Three nucleotide changes were discovered in the D((s)) incA gene, leading to two amino acid changes within the predicted D((s)) IncA sequence. These studies demonstrate a subgroup of variant C. trachomatis isolates that form nonfusing inclusions; the variant phenotype is associated with the absence of detectable IncA and with an altered incA sequence that modifies the characteristic hydrophobic domain of the IncA protein.     

A systematic review of the prevalence of Chlamydia trachomatis among European women

The study aim was to establish by systematic review the prevalence of asymptomatic Chlamydia trachomatis infection of the lower female genital tract in Europe and also to assess the extent and effect of screening. The search process was wide ranging, using the electronic databases Medline, Embase and Aidsline and the Internet using the search engines Netscape and Euro-ferret. Studies published in any language during 1980-2000 were included if they unambiguously reported prevalence of C. trachomatis infection in asymptomatic women, and were assessed qualitatively. From >300 papers which quantified C. trachomatis urogenital infection, only 14 studies met the inclusion criteria: four from the UK, two from Sweden, two from The Netherlands, and one each from Bulgaria, France, Finland, Hungary, Italy and Spain. In only one study had screening taken place. The prevalence of C. trachomatis in unscreened asymptomatic women in Europe ranges from 1.7 to 17% depending upon the setting, context and country. The mode was -6% for women seeking contraception, and 4% for women having cervical smears. In conclusion, this review confirms high prevalence rates of C. trachomatis infection among asymptomatic women in many European settings.     

Experimental Chlamydia trachomatis infection causes apoptosis in human sperm

BACKGROUND: Chlamydia trachomatis is responsible for a widespread sexually transmitted infection. In men, it is associated with a wide clinical spectrum causing infertility. Furthermore, C. trachomatis serovar E infection decreases motility and increases the number of non-viable sperm. No other effects of C. trachomatis have been reported on sperm despite the crucial role of DNA integrity for sperm function. The aim of this study was to investigate the effects of C. trachomatis on sperm apoptosis. METHODS: Sperm from eight normozoospermic men were incubated with increasing concentrations of C. trachomatis serovar E elementary bodies (EB) for 6 and 24 h. Sperm were then collected to evaluate phosphatidylserine (PS) membrane translocation and DNA fragmentation by Annexin V-propidium iodide staining, TUNEL assay and flow cytometry. RESULTS: After 6 h of incubation, C. trachomatis had no effect on the percentage of sperm showing PS externalization. However, a significant effect on this parameter was observed after 24 h. C. trachomatis also significantly increased the number of sperm with DNA fragmentation both after 6 and 24 h of incubation. CONCLUSIONS: C. trachomatis causes sperm PS externalization and DNA fragmentation. These effects may explain the negative direct impact of C. trachomatis infection on sperm fertilizing ability.     

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.     

Plasma cell endometritis is associated with Chlamydia trachomatis infection.

The presence of plasma cells in endometrial tissue has been linked to Chlamydia trachomatis infection. The aim of our work was to determine the strength of the association between C trachomatis infection and plasma cell endometritis. C trachomatis infection was detected by polymerase chain reaction (PCR) or immunohistochemistry in 5 (24%) of 21 endometrial tissue samples with plasma cell endometritis and in 1 (4%) of 28 tissue samples with no evidence of plasma cell endometritis (P < .02). Patients with plasma cell endometritis were also more likely to have symptoms and signs consistent with upper genital tract chlamydial infection; thus there is an association between endometrial C trachomatis infection and the presence of plasma cells in the endometrium. The histopathologic finding of plasma cell endometritis should encourage further examination of the tissue sample for simultaneous chlamydial infection. Plasmid-based polymerase chain reaction and immunohistochemical staining of paraffin-embedded samples are useful methods for detecting C trachomatis in endometrial tissue.     

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.