A polymerase chain reaction (PCR) for the detection of Chlamydia trachomatis was developed and evaluated. Two primer-probe sets were designed; one detected a specific sequence of the plasmid, and the other detected the gene encoding the major outer membrane protein. Both sets reacted species specifically and amplified sequences from all human serovars. A simple protocol was used for sample pretreatment. The PCR was optimized by addition of tetramethylammonium chloride and bovine serum albumin. The results of the PCR with the plasmid primer-probe set were compared with those of culture and the Chlamydiazyme and Gen-Probe PACE 2 tests for urogenital specimens from 220 patients. The rates of prevalence of infection with C. trachomatis were 22.7, 16.4, 15.0, and 14.5%, respectively. The sensitivities of the Chlamydiazyme and Gen-Probe PACE 2 assays compared with culture were 66.7 and 61.1%, respectively, and their sensitivities compared with PCR were 60.0 and 60.0%, respectively. The sensitivity of culture compared with PCR was 70.0%. Forty-eight of the 50 specimens positive by PCR with the plasmid primer-probe set could be confirmed by PCR with the major outer membrane protein primer-probe set or culture. It is concluded that the PCR is the most sensitive technique for laboratory detection of C. trachomatis.

The efficacy of single-dose azithromycin therapy in the treatment of cervical Chlamydia trachomatis infections was compared to that of a standard seven-day course of treatment with doxycycline. Cervical samples from 60 patients reacted positively in an enzyme immunoassay for detection of Chlamydia trachomatis. In 31 patients Chlamydia trachomatis was isolated from the sample taken before treatment. Fourteen of the 31 patients were treated with doxycycline and 17 with azithromycin. All cultures of samples taken one and four weeks after the start of therapy were negative. All 31 isolates showed a similar pattern of MICs for the seven antibiotics tested, including azithromycin and doxycycline. No differences were observed between isolates of different serovars. In samples from four patients chlamydial DNA could be detected by PCR one week after the start of the therapy and in two patients also after four weeks. No difference in microbiological parameters could be observed between the two treatment groups. It is concluded that single-dose azithromycin is as effective as a seven-day course of doxycycline in the therapy of cervical Chlamydia trachomatis infections.

A panel of monoclonal antibodies was developed for serovar typing of clinical isolates of Chlamydia trachomatis. The panel could distinguish all 15 established serovars from one another, although the hybridomas of the panel were developed by fusions of myeloma cells and spleen cells from mice immunized with antigen derived from the urogenital serovars D to L3. The typing assay was based on a dot enzyme immunoassay, and the monoclonal antibodies that were included in the panel reacted strongly in this assay. A collection of 289 clinical isolates from The Netherlands was typed. The observed serovar frequency distribution was 51 isolates of serovar D (17.6%), 103 isolates of serovar E (35.6%), 62 isolates of serovar F (21.5%), 28 isolates of serovar G (9.9%), 14 isolates of serovar H (4.8%), 2 isolates of serovar I' (0.7%), 20 isolates of serovar J (6.9%), and 9 isolates of serovar K (3.1%). These results were confirmed by typing these isolates with a panel of monoclonal antibodies purchased from the Washington Research Foundation, Seattle. No strain variation was observed within serovar D with both panels. However, restriction fragment length polymorphism analysis of the gene encoding the major outer membrane protein showed that 32 isolates were similar to the prototype D and 17 were similar to the variant D-. The two others showed a new restriction pattern. Our panel of monoclonal antibodies contained one monoclonal antibody that divided the serovar G isolates into two groups. This differentiation was confirmed by restriction fragment length polymorphism analysis, confining this difference to a known sequence variation in variable domain IV. These data support the subdivision of serovar G into serovars G (prototype strain UW-57) and Ga (prototype strain IOL-238).

A microtiter plate-based method to detect amplified DNA was developed. The method uses on biotin-labeled primer in the polymerase chain reaction (PCR) mixture. The labeled amplicon is bound to streptavidin-coated microtiter plates, denatured and hybridized to a digoxigenin-labeled probe. The specificity of the hybridization reaction was optimized by varying the temperature of the subsequent washing step and adding urea to the washing buffer. The digoxigenin label was detected using an enzyme immunoassay (EIA). This PCR-EIA was compared with a standard PCR assay that uses gel electrophoresis, blotting and hybridization to detect the amplicon, with isolation in cell culture, and with an antigen detection EIA (Chlamydiazyme) in the diagnosis of Chlamydia trachomatis infection in 309 female patients attending a sexually transmitted diseases outpatient clinic. The prevalence of Chlamydia trachomatis infection as determined by isolation in cell culture, EIA, PCR-EIA and standard PCR assay was 9.1%, 8.7%, 12.3%, and 12.9%, respectively. Compared with results of a reference set of confirmed-positive cases (defined by a positive result in two or more independent assay after analysis of discrepancies), the sensitivity and specificity was 71.1% and 99.6% for cell culture, 65.8% and 99.3% for the EIA, 92.1% and 98.9% for the PCR-EIA, and 97.4% and 98.9% for the standard PCR assay. It is concluded that the PCR-EIA described is a fast, sensitive and specific method for detecting Chlamydia trachomatis in clinical specimens.

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Determination of Chlamydia trachomatis specific antibodies in serum and tears, isolation of C. trachomatis in cell culture and C. trachomatis antigen detection by direct immunofluorescence test were evaluated for the laboratory diagnosis of Chlamydia trachomatis infection in 30 patients with chronic conjunctivitis. C. trachomatis specific secretory immunoglobulin A (s-IgA) was detected in the tears of all 8 patients with a positive result in culture and the direct immunofluorescence test. In 4 additional patients, the s-IgA assay of tears was also positive. A presumptive clinical diagnosis of Chlamydia conjunctivitis was correlated with the presence of s-IgA in tears. S-IgA was not detected in the serum of any of the patients, indicating that s-IgA is locally produced and not transudated from the serum. For the diagnosis of chronic Chlamydia conjunctivitis, we recommend the determination of s-IgA in tears in addition to a test for Chlamydia detection.