Interpretation of Chlamydia trachomatis antibody response in chlamydial oculogenital infection.

OBJECTIVE: To study: (a) the chlamydial antibody response (to the D-K serovars) using the micro-immunofluorescence (micro-IF) test in the following groups: (I) chlamydial genital infection only, (II) chlamydial ocular infection only, (III) combined chlamydial ocular and genital infection (oculo-genital infection), (IV) chlamydial ocular infection with chlamydia-negative non-gonococcal urethritis, (V) adenovirus conjunctivitis (control group 1), (VI) male partners of group I-IV with no chlamydial oculogenital infection or non-gonococcal urethritis (control group 2) (b) the cross reactivity of antibodies in patients' sera between the three chlamydial species and within the serovars of C trachomatis in those with culture-positive chlamydial oculo-genital infection. SETTING: oculogenital (diagnostic) clinic at Moorfields Eye Hospital, London, UK. SUBJECTS: 209 consecutive patients attending the clinic with Chlamydia trachomatis oculogenital infection and 86 patients with adenovirus conjunctivitis (control group 1) and 55 male partners with no evidence of chlamydial oculogenital infection or non-gonococcal urethritis (control group 2). RESULTS: Of all the patients with proven chlamydial oculogenital infection, 10.5% (22/209) and 94% (197/209) had IgM and IgG antibodies respectively. The geometric mean IgG antibody titres (GMT) were 1:98, 1:123, 1:245 and 1:101 in groups I to IV respectively. The IgG GMT values seen in control groups 1 and 2 were 1:45 and 1:36 respectively. Only 2/86(2%) patients in group V (control group 1) had IgG chlamydial antibodies of 1:32 and 1:64, whilst only 1/55(1.8%) and 4/55(7.3%) of patients in group VI(control group 2) had chlamydial IgG antibody titres of > or = 1:256 and > or = 1:128 respectively. A four-fold rise or fall in IgG antibody titre occurred in 56%(107/192) of patient groups I-IV over 2-6 weeks. Low titre cross-reactive antibody responses against different chlamydial species and serovars were commonly seen; 71%(148/209) of all patients showed cross-reactivity with Chlamydia pneumoniae or psittaci species or both, whilst 92% (193/209) of patients showed some level of cross reactivity to other pooled serovars of C trachomatis (A-C and L 1-3). CONCLUSIONS: Serological diagnosis of chlamydial infection as evidenced by a positive IgM antibody response, high IgG titre (> or = 1:256) or > or = 4-fold rise or fall in IgG antibody titre was seen in 78%(163/209) of patients with culture-positive chlamydial oculogenital infection. Chlamydial IgG antibody titres of > or = 1:256 had a sensitivity of 42.6%, specificity of 98.2%, positive predictive value of 98.8% and a negative predictive value of 31% for chlamydial infection at any site, when considering groups I-IV and control group 2. In this study of 216 patients with conjunctivitis, a positive IgG antibody response (titre > or = 1:16) had a sensitivity of 98.5%, specificity of 97.7%, positive predictive value of 98.5% and a negative predictive value of 97.7%, for chlamydial conjunctivitis. Patients with dual chlamydial infection of conjunctiva and genital tract had a higher IgG GMT titre than those with ocular or genital infection alone: infection at a second site may produce an anamnestic response. Although the micro-IF test is a useful adjunct for the diagnosis of chlamydial infection, cross-reactivity between different chlamydial species and serovars is common. Chlamydial seroepidemiological studies should be interpreted with caution, as studies may attribute a serological response to a particular species or serovar in a setting where two or more are prevalent.

Evidence for naturally occurring recombination in the gene encoding the major outer membrane protein of lymphogranuloma venereum isolates of Chlamydia trachomatis.

The nucleotide sequence of the major outer membrane protein gene (omp1) was determined for three geographically distinct lymphogranuloma venereum isolates which were serologically untypeable. The three omp1 sequences were hybrids of serovars L1 and L2, containing a putative DNA recombination site in variable segment 2. Efforts to manipulate the chlamydial genome in vitro by recombination should be intensified.

The differentiation of Chlamydia species by antigen detection in sputum specimens from patients with community-acquired acute respiratory infections.

An amplified enzyme immunoassay (IDEIA III: Dako Diagnostics Ltd) for detecting genus-specific chlamydia antigen was evaluated prospectively on 286 respiratory specimens from 275 patients presenting with community-acquired pneumonia or persistent chest infection. Nineteen patients had evidence of recent chlamydial infection, having two or more positive sputum or serological markers. Sputa from two other patients were ELISA-positive in the absence of other positive criteria and were regarded as false-positive results. When compared with a direct immunofluorescence test for chlamydial elementary bodies (EBs) using a genus-specific monoclonal antibody, the ELISA gave a positive predictive value of 91% and a negative predictive value of 99%. Non-specific problems with a wide variety of other micro-organisms isolated from the sputa were not encountered. Attempts to differentiate between Chlamydia psittaci, Chlamydia pneumoniae and Chlamydia trachomatis using genus-specific lipopolysaccharide reactive--and species-specific major outer membrane protein--monoclonal antibodies were encouraging and results were substantiated, in most patients, by the species-specific serological assays of the whole-cell-inclusion immunofluorescence or micro-immunofluorescence assays. The study demonstrated that antigen detection techniques offer scope for routine laboratories to diagnose chlamydial respiratory infections rapidly and reliably and may enable differentiation to species level. Although immunofluorescence offers marginally greater sensitivity and specificity when compared with ELISA, the latter is less subjective and less demanding. Sixty-eight per cent of these infections would have remained undiagnosed despite the general availability of ELISA tests.

Serological diagnosis with the Chlamydia Spot-IF test.

Using a set of sera for which full chlamydial micro-immunofluorescence results suggested a clear diagnosis, we have evaluated the Chlamydia Spot-IF test (bioMerieux), which allows a comparison of titres to Chlamydia trachomatis and C. psittaci antigens. A modification of the test in which the antigen slides were pre-treated with a monoclonal antibody to chlamydial lipopolysaccharide, improved its ability to differentiate infections with C. trachomatis from those with C. psittaci or C. pneumoniae.

Lymphocyte proliferative responses to chlamydial antigens in human chlamydial eye infections.

In order to study the relationship between cell-mediated immune responses to Chlamydia trachomatis and the pathogenesis of human chlamydial eye disease, we have measured the peripheral blood lymphocyte proliferative responses to whole chlamydial elementary bodies in 40 subjects with oculogenital chlamydial infection of varying severity, 13 subjects with genital chlamydial infections and 12 healthy seronegative controls. The mean stimulation index was significantly higher in those with oculogenital infections than in controls. There was a strong correlation between the response to C. trachomatis serotypes B and L1. We studied the relationship between proliferative responses and four clinical parameters: follicular conjunctivitis, papillary hypertrophy, corneal pannus and epithelial punctate keratitis, but were unable to show a significant association with any of these. Nor was there any association between proliferative response and serum antibody titre to C. trachomatis (pooled serotypes D-K), duration of disease or quantitative isolation of chlamydia from the conjunctiva. The depletion of CD8+ cells had no consistent effect on proliferative responses to serotype L1 in 13 subjects.

Intra-articular chlamydial antigen and inflammatory arthritis.

Joint material from 133 patients with well-characterized inflammatory arthritis, including individuals likely to have suffered reactive arthritis, was studied. The majority of patients were also examined for the presence of genital tract infection with Chlamydia trachomatis. Fluorescein-conjugated monoclonal antibodies demonstrated the presence of C. trachomatis antigen in synovial fluid cell deposits or synovial sections from inflamed knee joints of seven patients with reactive arthritis. The significance of these findings is discussed, as is the low rate of detection of chlamydial antigen in either the genital tract or the joint from patients in this study. We emphasize the need for further work aimed at identifying the relevant immunogenic chlamydial antigens responsible for the initiation of reactive arthritis.

Nucleotide sequence and taxonomic value of the major outer membrane protein gene of Chlamydia pneumoniae IOL-207.

Chlamydia pneumoniae IOL-207 genomic DNA was hybridized with a 1.5 kb labelled DNA probe containing the 3' region of the coding sequence for the major outer membrane protein (MOMP) of C. trachomatis serovar L1. An 8.5 kb Bg/II fragment containing the complete MOMP gene was cloned into lambda EMBL3. Two hybridizing EcoRI fragments were sub-cloned into the lambda ZAP II cloning vector and the resulting plasmids were used as templates for sequencing both strands of the C. pneumoniae MOMP gene. Computer taxonomic studies using the nucleotide and inferred amino acid sequence of the MOMP of C. pneumoniae IOL-207 and all known chlamydial MOMP sequences supported the designation of C. pneumoniae as a new species, but electron microscope studies suggested that the presence of pear-shaped elementary bodies (EBs) may not be a reliable taxonomic criterion.

The antigenic specificity of the humoral immune response to primary and repeated ocular infections of the guinea pig with the GPIC agent (Chlamydia psittaci).

The antigenic specificity of the humoral immune response in guinea pigs to primary and repeated ocular infections with the guinea pig inclusion conjunctivitis (GPIC) chlamydial agent was analysed using microbiological, serological and Western blotting techniques. The results indicate that although there was a response to many minor polypeptide antigens, there was a marked lack of reactivity to the major outer membrane protein (MOMP), particularly following reinfection of guinea pigs. It is suggested that, lack of a good antibody response to the MOMP, may be one of the reasons why guinea pigs are susceptible to repeated ocular infections with this chlamydial agent.

A study of human respiratory tract chlamydial infections in Cambridgeshire 1986-88.

Human respiratory tract chlamydial infections have been studied in Cambridgeshire for many years, but until recently we have been unable to distinguish between infection with Chlamydia psittaci or Chlamydia pneumoniae (TWAR). In this study, we have employed the micro-immunofluorescence (micro-IF) test for this purpose and to look for the relative incidence of C. psittaci and C. pneumoniae infections in Cambridgeshire. Among 50 patients with community-acquired respiratory tract symptoms whose serum samples had Chlamydia complement fixation test titres greater than or equal to 64, 25 had evidence of recent C. psittaci or C. pneumoniae infection. Nineteen (76%) of the 25 patients had evidence of recent C. psittaci infection and of these 16 (84%) had recently had contact with birds. Six patients (24%) had evidence of recent C. pneumoniae infection, and of these, only two (33%) had recently had contact with birds. While C. psittaci was grown from several of the birds associated with human C. psittaci infection, it was not cultured from any of the birds in contact with the two human C. pneumoniae cases.

In vitro studies of Chlamydia trachomatis susceptibility and resistance to rifampin and rifabutin.

Although rapid emergence of antibiotic-resistant mutants of Chlamydia trachomatis occurs when the organism is grown in subinhibitory concentrations of rifampin, no such mutants could be demonstrated when the organism was propagated under the same conditions in subinhibitory levels of the related drug rifabutin.

Colposcopy, biopsy, and cytology results in women with chlamydial cervicitis.

Colposcopy and biopsy were carried out at diagnosis and at follow up of 222 women. Of 322 cervical biopsy specimens taken, 174 were from women with chlamydial cervicitis (patients) and 48 from control women (both at diagnosis) and 100 from 76 patients and seven controls at follow up. Of the 174 patients with chlamydial cervicitis, 158 (91%) had erythema compared with 9/48 (19%) controls, and 140 (81%) had "follicles" and lumps compared with three (6%) controls. The cervical polymorphonuclear leucocyte (PMNL) count in a high power (x 1000) field (HPF) was 85 in patients compared with 47/HPF in controls. Cervical ectopia was found in 154 (89%) patients compared with 32 (67%) controls. After treating the 174 patients, we found erythema in nine (5%) and "follicles" in 16 (9%); both conditions were disappearing. Lymphocytic germinal follicles were found on histology in only 5/165 patients compared with none in controls or in patients at follow up examination (after treatment for chlamydial cervicitis). Inclusions were found in 6/165 patients compared with none in controls or patients at follow up. Chlamydiae were found on electron microscopy in slides from seven out of 159 patients compared with none from controls or 81 subjects at follow up. Estimating numbers of inflammatory cells and measuring vascularity showed diffuse increases in lymphocytes and plasma cells and increases in vascularity in both endocervical and exocervical tissue of patients with chlamydial cervicitis. These increases were lessened by treatment.

The microbial epidemiology of trachoma.

Detailed longitudinal studies of the microbiology of endemic trachoma in field situations have provided useful information concerning the epidemiology of this blinding disease. This knowledge should be of particular value for the further development of diagnostic techniques, and for the design of future trachoma control programs.

Diagnosis of ophthalmia neonatorum.

Ninety consecutively seen babies with eye discharge in the first three weeks of life were examined. Four babies had "sticky eyes" with no evidence of ophthalmia and had uniformly negative cultures and test results for antichlamydial antibody; these babies were excluded. Of the 86 babies with ophthalmia neonatorum, Neisseria gonorrhoeae was isolated from eight, Chlamydia trachomatis from 44, other bacteria alone from 20, and 14 had negative cultures. Three babies with negative cultures had longstanding conjunctivitis and had been treated with chloramphenicol eye ointment; all had antichlamydial IgM antibodies, indicating that the conjunctivitis was chlamydial. Hence the total number of babies whose conjunctivitis was chlamydial was 47. The result of the Gram stained conjunctival smear correlated well with that of culture and final assessment by the microimmunofluorescence test, enabling an immediate presumptive diagnosis to be made of gonococcal, chlamydial, or bacterial conjunctivitis. Prompt and effective treatment of babies was started. Explanation to the mother and contact tracing were carried out when the confirmatory cultures and antibody tests were completed. The Gram stained conjunctival smear is a highly sensitive, specific, and predictive test for the aetiological agent of ophthalmia neonatorum. By virtue of its simplicity and rapidity the test may be useful in developing countries.

Antigenic specificity of human antibody to chlamydia in trachoma and lymphogranuloma venereum.

An understanding of the molecular basis of the humoral immune response to chlamydial infections in man requires the identification of target antigens to which antibodies are directed. The antigenic specificity of antibody from patients with lymphogranuloma venereum (LGV) or trachoma was therefore assessed by Western blotting. Surface polypeptides were first identified using purified chlamydial outer membrane complex as antigen. Antibodies in sera from patients with LGV but not from control negative sera reacted with a wide range of chlamydial surface polypeptides with molecular masses of 19, 29, 41, 58, 63 and 65 kDa. The major component of the antibody response detected by both immunoblotting and immunoprecipitation assay was directed against the major outer membrane protein (MOMP). Antibody to MOMP was species-specific on Western blotting, whereas antibody to several other polypeptides recognized common immunodeterminants on polypeptides of C. psittaci Cal-10 of equivalent molecular mass. Immunologically C. psittaci Cal-10 was more closely related to LGV strains of C. trachomatis than a guinea pig inclusion conjunctivitis strain of C. psittaci. Trachoma sera collected from a village in southern Iran showed predominantly type-specific antibody on micro-immunofluorescence to serotype A or B trachoma agents. These sera showed a weak immune response to MOMP, a pronounced response to a polypeptide of 36 kDa and much less widespread reactivity with other chlamydial polypeptides. The lack of an immune response to SDS-stable immunodeterminants on MOMP might contribute to the susceptibility of trachoma patients to repeated cycles of ocular infection with chlamydiae.

Sequential antibody changes following ulcerative herpetic keratitis.

Blood and tear levels of immunoglobulins against herpes simplex virus (HSV) were examined in 28 patients with dendritic keratitis over a period of 28 days. By means of an indirect micro-immunofluorescent technique blood and tear HSV IgG were detected, but neither circulating HSV IgM nor local HSV IgA were found. Over a four-week interval non-diagnostic fluctuations of HSV IgG occurred in most patients, though seven (25%) developed a rising blood IgG titre. Tear IgG appeared to be an exudate from blood. HSV was isolated from 68% of corneal swabbings and 11% of conjunctival swabbings. This study provides guidelines for laboratory testing in recurrent herpetic keratitis.

Prevalence in human beings of antibodies to Chlamydia IOL-207, an atypical strain of chlamydia.

Samples of serum from 18353 persons from the U.K. and abroad, were tested for type-specific antibodies to Chlamydia spp. Antibodies to an atypical strain, Chlamydia IOL-207, were detected in each population tested. Overall, the prevalence of these antibodies (19.9%) was similar to that of antibodies to C. trachomatis (21.6%). In the U.K., antibodies to C. IOL-207 were rare in children under the age of 5 years but became more common with increasing age. In adults the prevalence of these antibodies increased annually between 1979 and 1984. Antibodies to C. IOL-207 were not associated with sexually transmitted disease and only rarely with ocular disease. Their association with a common complaint such as mild respiratory illness is postulated. The presence of these antibodies within a population may lead to the over-diagnosis of C. trachomatis infections or psittacosis if only genus-specific serological tests are used.

The community epidemiology of trachoma.

Effective eradication of avoidable blindness due to trachoma requires an understanding of the epidemiologic determinants that promote the disease. These determinants include both host factors, such as age, sex, and race, and environmental determinants, including climate, water availability, and general socioeconomic conditions. All of these factors may drastically modify the incidence, prevalence, and severity of trachoma within a community. Longitudinal seroepidemiologic studies on the transmission of the infection have helped to elucidate the more important risk factors in trachomatous communities, and this information must be carefully considered in the assessment of proposed programs for trachoma control.