High prevalence and incidence of sexually transmitted diseases in urban adolescent females despite moderate risk behaviors.

To better understand the prevalence, incidence, and risk factors for sexually transmitted diseases (STDs) among female adolescents, a prospective 6-month cohort study was conducted at four teen clinics in a southeastern city. At enrollment, 260 (40%) of 650 sexually active females ages 14-19 years had an STD: chlamydia, 27%; herpes simplex virus type 2 (HSV-2), 14%; gonorrhea, 6%; trichomoniasis, 3%; and hepatitis B, 2%. At follow-up, 112 (23%) of 501 participants had an incident infection: chlamydia, 18%; HSV-2, 4%; gonorrhea, 4%; and trichomoniasis, 3%. At either enrollment or follow-up, 53% had >/=1 STD; of those with 1 lifetime partner, 30% had an STD. Having a new partner (odds ratio [OR], 2.2; 95% confidence interval [CI], 1. 1-4.2) or friends who sell cocaine (OR, 1.6; CI, 1.0-2.6) was independently associated with incident infection. STD incidence and prevalence were extremely high in this population, even in teenagers with only 1 lifetime partner. Individual risk behaviors appeared less important for STD risk than population factors.

Head-to-head evaluation of five chlamydia tests relative to a quality-assured culture standard.

Nucleic acid amplification tests offer superior sensitivity for the detection of Chlamydia trachomatis infection, but many laboratories still use nonamplification methods because of the lower cost and ease of use. In spite of their availability for more than a decade, few studies have directly compared the nonamplification tests. Such comparisons are still needed in addition to studies that directly compare individual nonamplification and amplification tests. The purpose of this study was to evaluate and compare the performance characteristics relative to culture of five different tests for the detection of C. trachomatis with and without confirmation of positive results. The tests were applied to endocervical specimens from 4,980 women attending family planning clinics in the northwestern United States. The five nonculture tests included Chlamydiazyme (Abbott), MicroTrak direct fluorescent antibody (DFA) (Syva), MicroTrak enzyme immunoassay (EIA) (Syva), Pace 2 (Gen-Probe), and Pathfinder EIA (Sanofi/Kallestad). All positive results obtained with a nonculture test (except MicroTrak DFA) were confirmed by testing the original specimens with a blocking antibody test (Chlamydiazyme), a cytospin DFA (MicroTrak EIA and Pathfinder EIA), and a probe competition assay (Pace 2). The prevalence of culture-proven chlamydia was 3.9%. The sensitivities of the nonculture tests were in a range from 62 to 75%, and significant differences between tests in terms of sensitivity were observed. The positive predictive value for each test was 0.85 or higher. The specificities of the nonculture tests without performance of confirmations were greater than 99%. Performing confirmatory tests eliminated nearly all of the false positives.

Transmission of Chlamydia trachomatis and Neisseria gonorrhoeae among men with urethritis and their female sex partners.

Transmission of Chlamydia trachomatis and Neisseria gonorrhoeae among infected men and their female sex partners was examined using a design enhancing the likelihood that spread was directed from men to women. Chlamydia culture-negative specimens were examined using DNA amplification tests. Infection rates in women exposed to male sex partners with Chlamydia only were 65% (20/31) and with gonorrhea only were 73% (33/45). Infection of women by either agent was not influenced by the number of sexual exposures to or coinfection in men. There was a 98% (40/41) concordance of N. gonorrhoeae isolates among partners by auxotype and serovar. Chlamydia isolates were serotyped using ELISA and immunofluorescence testing and confirmed by nested polymerase chain reaction: 50% (6/12) of men and 57% (8/14) of women yielded mixed serovars. Sixty-four percent of pairs (9/14) were infected with identical serovars and an additional 28% shared at least one serovar. Multiple serovars of C. trachomatis, but not of N. gonorrhoeae, were common in sex partners and exchanged frequently.

Correlation of infecting serovar and local inflammation in genital chlamydial infections.

The relationship between acute inflammation and serovar of Chlamydia trachomatis was evaluated in patients with genital chlamydial infection who attended a sexually transmitted diseases clinic. Polymorphonuclear leukocytes (PMNLS) were enumerated on Gram's-stained smears of endourethral contents in men; cervicitis was scored by visual observation of the endocervix in women. Isolates were serotyped with a monoclonal antibody-based radioimmunoassay. The distribution of serovars in 99 women did not differ in the presence or absence of cervicitis or concurrent gonorrhea. An overall difference (P = .037) was observed when serovar distributions in men with less than or equal to 3 PMNLs (n = 42), greater than or equal to 10 PMNLs (n = 41), and gonococcal urethritis (n = 42) were compared. Follow-up pairwise comparisons revealed that men with less than or equal to 3 PMNLs had fewer isolates of serovars F and G than did men with greater than or equal to 10 PMNLs (P less than .05). No strong overall association was observed between inflammation and serovar.

Humoral immune response to chlamydial genital infection of mice with the agent of mouse pneumonitis.

The objective of this study was to characterize the humoral immune response to chlamydial genital infection of mice with the mouse pneumonitis agent (MoPn). With an enzyme-linked immunoabsorbent assay, immunoglobulin G antibodies to MoPn were first detected in plasma by day 14. Peak plasma antibody concentrations were reached by day 49, and this response did not decline significantly throughout the 300-day monitoring period. Immunoglobulin A against MoPn could first be detected in pooled vaginal washes by day 21 after infection and had reached peak concentrations by day 28, but anti-MoPn immunoglobulin G was not consistently present in secretions. The antibody response in secretions had declined slightly by day 300. Immunoblot analysis revealed that the early phase of the plasma antibody response to MoPn as a result of genital infection was against lipopolysaccharide, the major outer membrane protein, and a 62-kilodalton (kDa) protein. In secretions, early-phase immunoglobulin A antibodies were directed to the major outer membrane protein and lipopolysaccharide. Late reactions to 15-, 22-, and 83-kDa proteins in plasma were noted. Late reactions to the 62-kDa protein in secretions were also noted. The cause of these late responses remains unexplained. When mice were challenged intravaginally with MoPn at 50-day intervals after the primary infection, it was found that mice inoculated on day 100 or after were susceptible to reinfection. Susceptibility could not be related to a decline in the antibody concentration in plasma or secretions or in the antibody response to specific components of MoPn as measured by immunoblot analysis.

Association of recurrent chlamydial infection with gonorrhea.

To determine whether concurrent gonorrhea reactivates latent chlamydial infection, we studied 74 recurrent chlamydial infections and the effect of concurrent gonorrhea at the recurrent episode on the chlamydial serovar identified. Serotyping of 74 recurrent pairs of chlamydial isolates from patients attending a sexually transmitted diseases clinic indicated that 47.1% (16 of 34) with gonorrhea at the time of recurrence harbored chlamydiae of the same serovar as at the initial infection, while only 22.5% (9 of 40) without gonorrhea had the same serovar (P = .03). The proportion of recurrences by the same serovar in the group without gonorrhea did not differ from the proportion predicted by a random exposure model (22.2% vs. 18.4%, P = .46), while the proportion in the gonorrhea group did (47.1% vs. 19.8%, P less than .0001). The possibility of reinfection did not appear more likely in the group with gonorrhea than in the group without. These observations support the hypothesis that concurrent gonorrhea can reactivate latent chlamydial infection.

Detection of surface-exposed epitopes on Chlamydia trachomatis by immune electron microscopy.

The cell surfaces of two Chlamydia trachomatis serovars were explored by immune electron microscopy with monoclonal antibodies that recognize a number of chlamydial outer-membrane components. Species, subspecies and serovar-reactive epitopes on the major outer-membrane protein (MOMP) of a lymphogranuloma venereum biovar strain, L2/434/Bu, and a trachoma biovar strain, F/UW-6/Cx, were exposed on the surfaces of both elementary bodies (EBs) and reticulate bodies (RBs). Three epitopes on MOMP were inaccessible on EBs and RBs of both strains. These included a genus-reactive, species-reactive, and a subspecies-reactive epitope. In contrast, genus-specific epitopes on lipopolysaccharide (LPS) were not detected on the EB surface, but were clearly expressed on RBs of both L2/434/Bu and F/UW-6/Cx chlamydiae. Antibodies specific for the 60 kDa and 12 kDa 'cysteine-rich' outer-membrane proteins did not react with surface epitopes on either EBs or RBs. These data provide evidence that MOMP is a major surface antigen of both morphological forms, whereas some portions of the LPS molecule are exposed on the RB surface but become inaccessible to antibody after conversion to the infectious EB form.

Antigenic structure of surface-exposed regions of the major outer-membrane protein of Chlamydia trachomatis.

The major outer-membrane protein (MOMP) of Chlamydia trachomatis displays a number of surface-exposed epitopes. Some of these are shared by all or some of the 15 known serovars, whereas others are serovar-specific. With use of epitope-mapping analysis of limited proteolytic digests of purified MOMPs with monoclonal antibodies, the present study assessed whether the same or different surface-exposed regions of the protein express these different epitopes. Results with both chymotrypsin and the staphylococcal V8 protease for MOMP of serovar F indicated that many of the shared surface epitopes are clustered in one region whereas the serovar-reactive epitope is located in a separate region. Analysis of MOMPs from all the serovars with a species-specific monoclonal antibody indicated that each MOMP possesses a structurally analogous region that expresses this epitope. Such a region might play a role in pathogenic mechanisms that are shared by all serovars.

Biosynthesis and disulfide cross-linking of outer membrane components during the growth cycle of Chlamydia trachomatis.

The synthesis and accumulation of Chlamydia trachomatis outer membrane proteins within infected HeLa 229 host cells were monitored by assessing the uptake of [35S]cysteine into chlamydial proteins during the 48-h growth cycle of a lymphogranuloma venereum strain, L2/434/Bu. Synthesis of the major outer membrane protein, a protein that accounts for about 60% of the outer membrane protein mass of elementary bodies (EB), was first detected between 12 and 18 h after infection. The uptake of [35S]cysteine into the 60,000-molecular-weight doublet (60K doublet) and 12.5K cysteine-rich proteins was not observed until 30 h after infection, when the intracellularly dividing reticulate bodies were beginning to transform into infectious EBs. By using a more sensitive immunoblotting method in conjunction with monoclonal antibodies specific for the 60K doublet proteins, synthesis of these proteins was detected even earlier, by 18 h after infection. These data suggest that the time and extent of synthesis of these outer membrane proteins are regulated by processes that coincide in time with the transformation of reticulate bodies into EBs. Additional studies were performed to determine the extent of disulfide cross-linking of outer membrane proteins during the growth cycle. Both the major outer membrane protein and the 12.5K protein became progressively cross-linked to about 60% during the last 24 h of the growth cycle, whereas the 60K doublet proteins were extensively cross-linked during most of the cycle. These data may indicate an intracellular cross-linking mechanism, possibly enzymatic, that exists in addition to an auto-oxidation mechanism that occurs upon host cell lysis and exposure to the extracellular environment.

Effect of blind passage and multiple sampling on recovery of Chlamydia trachomatis from urogenital specimens.

Detection of chlamydial infections depends on the sensitivity of the techniques used. Variables include the number of body sites sampled, the number of samples obtained, and the number of passages in tissue culture. To assess these factors, microdilution plate cultures with a single blind passage were performed on specimens from 10,291 men and women attending a sexually transmitted disease clinic. Overall, 21% of the men and 30% of the women were culture positive. However, 18% of endocervical, 28% of female urethral, and 29% of male urethral cultures that were positive became so only after a single passage. Of culture-positive women, 23% were positive at the urethra only. Pooled urethral and endocervical specimens were positive more often than an endocervical specimen alone but less often than separately cultured endocervical and urethral specimens. A total of 221 specimens from 92 men and 66 women were subjected to five serial blind passages. Of 83 positive specimens, 29 (35%) were positive only after two or more passages. A total of 37 (46%) women were culture-positive, but only 12 (33%) of those who were positive and had an endocervical culture would have been detected by a single endocervical culture that was not passaged. The sensitivity of chlamydial culture is substantially less than 100% but can be improved by culturing samples from both the urethra and endocervix in women and by serial passage in tissue culture.

Antigenic analysis of the major outer membrane protein of Chlamydia trachomatis with murine monoclonal antibodies.

We prepared monoclonal antibodies against prototype strains of the 15 serovars of Chlamydia trachomatis and identified a subset of reagents that reacted with the major outer membrane protein(s) (MOMPs) of one or more serovars. We then determined the specificities of these anti-MOMP monoclonal antibodies by radioimmunoassay and immunoblot assays against the 15 serovars of C. trachomatis and a C. psittaci strain. We identified 14 different anti-MOMP antibody specificities, including serovar-, several orders of subspecies-, and species-specific determinants. In addition, one antibody reacted with all C. trachomatis serovars and a C. psittaci strain, indicating the presence of a genus-specific epitope on MOMP. Many of the cross-reactions of the subspecies-specific antibodies were similar to those previously reported by use of the microimmunofluorescence technique. We also observed a number of cross-reactions that were unexpected but consistent with data derived by the microimmunofluorescence test. All antibodies, except the genus-specific antibodies, reacted with whole elementary bodies in a radioimmunoassay, suggesting surface exposure of the epitopes. These data confirm and extend previous observations that MOMPs among C. trachomatis serovars are antigenically complex and diverse. In addition, these data indicate that the cross-reaction patterns of some monoclonal antibodies directed against MOMP are similar to those detected by the microimmunofluorescence test and are consistent with the hypothesis that such determinants are contained within MOMPs.

Serovar determination of Chlamydia trachomatis isolates by using type-specific monoclonal antibodies.

A panel of 15 monoclonal antibodies was prepared that could distinguish among the 15 serovars of Chlamydia trachomatis. Twelve of these antibodies were specific for a single serovar (A, B, C, D, E, F, G, H, I, K, L1, and L2) and three were specific for two serovars (B/Ba, C/J, and C/L3). Ten of the serovar-specific and two of the bispecific antibodies were shown by immunoblotting to recognize epitopes on the major outer membrane protein. These data provide evidence that such epitopes are closely correlated with and may be partly responsible for the antigenic variations detected by microimmunofluorescence that distinguish the currently recognized serovars. When used in a radioimmunoassay, these antibodies correctly identified the serovar of 17 strains that had been serotyped by the microimmunofluorescence test. In addition, we found that the chlamydial antigen derived from 1.0 cm2 of an infected HeLa cell monolayer was sufficient to allow serotyping with these antibodies. Thus, these monoclonal antibodies may provide a rapid and reliable alternative to mouse immunization and microimmunofluorescence for serotyping of clinical isolates.

Differences in outer membrane proteins of the lymphogranuloma venereum and trachoma biovars of Chlamydia trachomatis.

The lymphogranuloma venereum (LGV) and trachoma biovars of Chlamydia trachomatis exhibit differences in biological properties both in vivo and in vitro. To identify analogous biochemical differences, we studied the molecular charges of chlamydial outer membrane proteins (OMPs) by means of isoelectric focusing and nonequilibrium pH gradient electrophoresis. Analysis of proteins of whole elementary bodies biosynthetically labeled with L-[35S]cysteine revealed that most chlamydial proteins were neutral or acidic. The major OMPs (MOMPs) of all strains tested were acidic and had apparent isoelectric points (pIs) that varied within narrow limits (approximately 5.3 to 5.5) despite differences in molecular mass of up to 3,000 daltons (Da). However, a low-molecular-mass cysteine-rich OMP analogous to that previously described for Chlamydia psittaci varied consistently in molecular mass (12,500 versus 12,000 Da) and pI (5.4 versus 6.9) between LGV strains and trachoma strains, respectively. OMPs with a molecular mass of 60,000 Da in the trachoma biovar strains had pIs in the 7.3 to 7.7 range. However, analogous OMPs in the LGV strains existed as a doublet with a molecular mass of about 60,000 Da. Both members of the doublet were basic (pIs greater than 8.5). Both proteins of this basic doublet in LGV strains and the neutral analog in trachoma strains bound a species-specific monoclonal antibody in an immunoblot assay. These data indicate substantial differences in biochemical characteristics of analogous OMPs in the LGV and trachoma biovars. Such differences are the first structural differences described between LGV and trachoma strains which support their distinction into separate biovars and may be related to some of their biological differences.

Disulfide-linked oligomers of the major outer membrane protein of chlamydiae.

The major outer membrane protein of chlamydial elementary bodies was identified in dimer, trimer, and other multimeric forms. These natural multimers were stabilized by disulfide-mediated cross-linking. Such cross-linking of outer membrane proteins may play an important role in the formation and evolution of chlamydial cell wall structure.

Comparison of reticulate and elementary body antigens in detection of antibodies against Chlamydia trachomatis by an enzyme-linked immunosorbent assay.

Reticulate bodies from a type C and elementary bodies from a type L2 strain of Chlamydia trachomatis were isolated and used as antigens in an enzyme-linked immunosorbent assay (ELISA). Results obtained for human sera with these two antigens used in the ELISA were compared with each other and with results obtained for the same sera by the micro-immunofluorescence test. Negative control populations included cloistered nuns and children with respiratory infections. Populations at risk for chlamydial infection consisted of 42 men with nongonococcal urethritis attending a sexually transmitted diseases clinic and 42 college women who had contact with men with nongonococcal urethritis. ELISAs done with the two antigens were equivalent to each other and to the micro-immunofluorescence test in the ability to predict the presence or absence of infection. None of the tests had high predictive values for the men with urethritis. However, the negative predictive value of both the micro-immunofluorescence test and the elementary body ELISA was 0.92 for the college women. Such serological tests may be of value in screening selected populations for subclinical infections with C. trachomatis.

Analysis of the human serological response to proteins of Chlamydia trachomatis.

Sera from individuals with culture-proven genital infection with Chlamydia trachomatis were analyzed for the presence of antibodies to chlamydial proteins by an immunoelectrophoretic transfer method. Protein antigens from representative strains of the 15 known serotypes were resolved by gel electrophoresis and transferred to a nitrocellulose solid support before being probed with serum. Sera from infected patients reacted with many different proteins. Most of these sera reacted with a 60,000- and a 62,000-molecular-weight protein which were present in each of the C. trachomatis serotypes and clinical isolates analyzed. In contrast, reactions with the major outer membrane protein were frequently observed but were usually weak. Sera from control groups of children, cloistered nuns, and college women, who were presumed not to have had prior chlamydial infections, did not usually have antibodies against the 60,000- or 62,000-molecular-weight protein, but did react with the major outer membrane protein and a 29,000-molecular-weight protein. These observations may have implications for the development of serodiagnostic tests as well as the identification of candidate antigens for vaccine development.

The use of Tween 20 as a blocking agent in the immunological detection of proteins transferred to nitrocellulose membranes.

The determination of the immunoreactivity of protein antigens in complex mixtures has been greatly facilitated by combining their separation via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with electrophoretic transfer to nitrocellulose membrane (NCM), and probing of bound proteins with specific antisera. Methods using various buffers and blocking agents have been published, but no studies have been published which compare these methods with each other or with others of potential merit. We have performed such a comparative study using protein antigens from Chlamydia trachomatis and Neisseria gonorrhoeae. In addition, we describe a method that blocks unoccupied protein binding sites on NCM with the nonionic detergent Tween 20, rather than proteins. This system proved to be equivalent or superior to other methods evaluated in the detection of immunoreactive proteins, and permitted staining of the NCM for protein after immunological probing. Such staining allowed precise identification of immunoreactive proteins. In addition, individual stained proteins could be excised and assessed for bound antibody in an indirect radioimmunoassay.

Resolution of basic gonococcal outer membrane proteins by nonequilibrium pH gradient electrophoresis.

Outer membrane proteins from opaque and transparent colonial variants of strain F62 of Neisseria gonorrhoeae were analyzed by two-dimensional electrophoresis with isoelectric focusing in the first dimension and sodium dodecyl sulphate-polyacrylamide gel electrophoresis in the second. Most of the higher-molecular-weight proteins focused sharply in the acidic region of the gel. In contrast, the principal outer membrane protein, a 31,000-molecular-weight protein, and the opacity-associated proteins remained near the origin (at the basic end of the gel) without focusing. However, when the samples were loaded on the acidic end of an isoelectric focusing gel and subjected to nonequilibrium pH gradient electrophoresis, these proteins behaved as basic proteins. In addition, three distinct opacity-associated heat-modifiable proteins could be identified. No other differences in the protein composition of outer membranes from opaque and transparent variants were apparent. Amino acid analysis of the principal outer membrane protein indicated that its net positive charge may result from partial amidation of its acidic residues. The unexpected observation that the major surface proteins of the gonococcus are basic may have implications for intragonococcal adhesion and for gonococcal interactions with mammalian cells.

Cross-linking analysis of the outer membrane proteins of Neisseria gonorrhoeae.

The organization of outer membrane proteins of Neisseria gonorrhoeae was investigated by using two-dimensional dodecyl sulfate-polyacrylamide gel electrophoresis and cross-linking agents. A naturally occurring protein aggregate, which may be composed of two proteins of 50,000 molecular weight, was detected in all strains. Treatment of whole cells with cross-linking agents yielded several additional complexes, suggesting that other proteins are arranged in the outer membrane as near neighbors. The principal outer membrane protein (molecular weight, 34,000) cross-linked (i) to itself to form a complex whch appeared to be trimeric, (ii) to the 28,000-molecular-weight outer membrane protein to form a bimolecular comlex, and (iii) to the 28,000-molecular-weight outer membrane protein in a 3:1 ratio. The formation of these complexes was independent of (i) colony type, (ii) colony opacity, (iii) pH during growth, and (iv) presence of markers for drug resistance or hypersensitivity.

High-molecular-weight antigenic protein complex in the outer membrane of Neisseria gonorrhoeae.

The outer membrane of Neisseria gonorrhoeae contains approximately 15 proteins, with 2 or 3 accounting for over 75% of the total protein mass. Samples of outer membrane from strain 2686 T4 analyzed by electrophoresis in 2% polyacrylamide gels revealed a band with an apparent molecular weight of 800,000. The band was protein material, as indicated by trypsin and pronase sensitivity and by L-[3H]proline incorporation. Peptidoglycan, nucleic acids, and carbohydrate were not detected in the band. Dye binding, L-[3H]proline incorporation, and labeling of solubilized outer-membrane proteins with 125I-labeled Bolton-Hunter reagent indicated that the band made up 10 to 13% of the total protein mass of isolated outer membranes. The material in the band was purified by gel filtration and, after reduction and alkylation, quantitatively recovered as subunits with an apparent molecular weight of 76,000. The protein in complex form was exposed at the cell surface, as evidenced by labeling whole cells with 125I by using a lactoperoxidase-catalyzed reaction and with CNBr-activated dextran. Rabbit serum raised against whole 2686 T4 gonococci contained antibody which reacted with the protein complex. The protein complex was detected in all gonococcal strains tested, but its presence could not be demonstrated in several other gram-negative species.