1 Research Institute Growth and Development (GROW), Department of Obstetrics and Gynaecology, Maastricht, 2 Department of Medical Microbiology, Maastricht and 3 Department of Epidemiology, Maastricht, The Netherlands
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Abstract |
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Key words: Chlamydia antibody titre/Chlamydia trachomatis/Chlamydia pneumoniae/screening/tubal factor subfertility
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Introduction |
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Chlamydia antibody testing (CAT), e.g. by micro-immunofluorescence (MIF) tests, has been introduced into the fertility work-up as a simple and inexpensive screening test for tubal factor subfertility. The clinical significance of CAT is limited by patients with chlamydial antibodies but without tubal pathology at laparoscopy (defined as false positive CAT results), and by patients without chlamydial antibodies but tubal pathology at laparoscopy (defined as false negative CAT results). False negative CAT results may be explained by non-chlamydial causes of tubal pathology. False positive CAT results increase health care costs by increasing the number of laparoscopies. Therefore, if CAT is applied for selecting patients for laparoscopy, the number of false positive CAT results should be minimized.
In discussing false positive CAT results (i.e. patients with positive antibody titres, without tubal pathology at laparoscopy), the possible cross-reactivity in MIF tests between C. trachomatis and C. pneumoniae antibodies is a major issue. Initially, in the MIF test, antigens from elementary bodies of each of the serotypes of C. trachomatis were included in the test, and provided serotype specific antibody testing (Wang and Grayston, 1970). The preparation of multiple immunotype antigens made the test technically difficult to perform and impractical, and therefore modifications were introduced (Treharne et al., 1977
). By pooling antigens of epidemiologically related serotypes, or using one broadly reacting serotype (usually L2), the number of antigens was reduced. Since MIF tests have been modified, and serotype-specific antigens are no longer used, cross-reactivity with other Chlamydia species (C. pneumoniae in particular) has been suggested (Mannion et al., 1991
). The aim of this study was to evaluate the significance of C. pneumoniae antibodies in false positive CAT results in female subfertility patients. Recently, new test methods have been developed, which are considered species-specific for C. trachomatis and C. pneumoniae. These tests are commercially available enzyme-linked immunosorbent assays (ELISA), which have not yet been evaluated in a clinical setting for CAT in subfertility patients. These tests were used to establish the prevalence of antibodies to C. pneumoniae in subfertile women. Secondly, cross-reactivity in the MIF test with C. pneumoniae antibodies was evaluated as a possible cause of false positive CAT results.
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Materials and methods |
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After thawing the spare sera of the participating patients, an ELISA for C. trachomatis antibodies (Labsystems, Helsinki, Finland) and an ELISA for C. pneumoniae antibodies (Bioclone Elegance, Marrickville, Australia) were performed. These ELISAs are considered specific for C. trachomatis antibodies (Närvänen et al., 1997) and for C. pneumoniae antibodies respectively.
Serological methods
For the indirect micro-immunofluorescent IgG antibody test, 20 µl of the serum was diluted eight times in phosphate buffered saline (PBS) and incubated on the C. trachomatis-spot immunofluorescence substitute slides (egg grown C. trachomatis biovar L2; BioMerieux, Hertogenbosch, The Netherlands) for 30 min at 37°C in a moist chamber. The slides were washed three times for 5 min in PBS and incubated with fluorescein-conjugated rabbit antihuman IgG (Dako, ITK Diagnostics BV, Uithoorn, The Netherlands) diluted in PBS for 30 min at 37°C. After three washings in PBS and one in ultra-pure water processed through a Milli-Q purifying system (Millipore, Bedford, MA, USA), slides were embedded in Fluoprep mounting medium (BioMerieux). A positive reaction was characterized by specific fluorescence of the C. trachomatis elementary bodies. For quantitative determination serial dilutions in PBS were performed. All slides were evaluated independently by two readers. In case of disagreement, the judgement of a third reader was decisive. According to receiver operating characteristic curves constructed, an antibody titre of 32 appeared to be the optimum threshold value in predicting tubal pathology (Land et al., 1998). Therefore, a positive MIF result was defined as a titre
32.
For the C. trachomatis IgG ELISA (Labsystems, Helsinki, Finland), sera were diluted 1:10 with Tris-buffer and tested in microtitre plates coated with synthetic peptides derived from the major outer membrane proteins of C. trachomatis (L2). The plates were incubated for 30 min at 37°C. The strips were washed five times in 300 µl washing solution (citrate buffered PBS) and dried. To each well 200 µl conjugate was added (sheep antihuman IgG/horseradish peroxidase). The plate was incubated again for 30 min at 37°C. The washing steps were repeated five times. Citrate-acetate buffered substrate in a volume of 200 µl was pipetted in each well, the plate was incubated at room temperature in the dark for 15 min. Finally, 50 µl stop reagent (2 mol/l H2SO4) was added. The absorbance of the plates was measured in a spectrophotometer at 405 nm. Threshold indexes were calculated according to the manufacturer's instructions. The threshold index for a positive test was 1.1.
For the C. pneumoniae IgG ELISA (Elegance, Marrickville, Australia), human sera were diluted 1:210 with washing solution and tested in strips coated with highly purified C. pneumoniae-specific outer membrane complexes. The plate was covered and incubated for 30 min at 37°C. The strips were washed three times with 300 µl washing solution. To each well, 100 µl C. pneumoniae IgG antibody reagent (labelled anti-human IgG polyclonal antibodies) was added and the plate was incubated again for 60 min. The plate was washed as described above. To each well 100 µl substrate solution (diethanolamine) was added. The plate was incubated at room temperature for 10 min. Finally, 25 µl Chlamydia stop solution was added and the fluid on the plate gently mixed. The optical density of the plates was measured in a spectrophotometer at 405 nm. The threshold index for a positive test, as recommended by the manufacturer, was 1.1.
Statistical methods
Data obtained at laparoscopy were correlated to serological test results, using the 2 test. P values <0.05 were considered significant. Differences between proportions were calculated by using proportion tests for two independent proportions. 95% confidence intervals for proportion differences and P values were calculated. P values <0.05 were considered significant.
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Results |
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C. pneumoniae antibodies were detected by ELISA in 75% of patients. In 40% of patients a positive MIF test for C. trachomatis antibodies was found. C. pneumoniae antibodies were found in 83/95 (87%) of patients who had a positive MIF test for C. trachomatis, and in 96/145 (66%) of patients with a negative MIF test (P < 0.0005). C. pneumoniae antibodies were found in 66/76 (86%) of patients who had a positive ELISA for C. trachomatis antibodies, and in 51/164 (69%) of patients who had a negative ELISA for C. trachomatis antibodies (P < 0.0005). Table I summarizes positive serological test results in patients with and without tubal pathology at laparoscopy respectively.
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Discussion |
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From Table I it can be concluded that the prevalence of C. trachomatis antibodies in the group of subfertile women studied here was comparable using MIF and ELISA, 40 and 32% respectively. Using ELISA the prevalence of C. pneumoniae IgG antibodies was 75%. In patients with tubal pathology at laparoscopy the prevalence was 76%. It was 74% in women without tubal factor subfertility (Table I
). Several reports on the prevalence of C. pneumoniae antibodies in Western European countries have been published. In 21% of over 4500 women attending a genitourinary clinic C. pneumoniae antibodies were found using MIF (Moss et al., 1993
). In adolescents the prevalence of C. pneumoniae antibodies using MIF was 69% in men, and 57% in women (Freidank and Brauer, 1992
). Another study confirmed the prevalence of C. pneumoniae antibodies to be higher in men (72%) than in women (56%), and showed the prevalence to increase with increasing age (Karvonen et al., 1994
). In female subfertility patients the prevalence of C. pneumoniae antibodies was significantly higher in patients with bilaterally occluded tubes at laparoscopy (75%), as compared to patients with normal tubes (56%) (Freidank et al., 1995
). The prevalence of C. pneumoniae antibodies found in the current study (75%) is comparable with the prevalence reported in the literature in asymptomatic women and subfertility patients.
In discussing false positive test results for CAT, cross-reactivity in MIF tests with C. pneumoniae is a major issue. Since MIF tests have been modified and serotype specific antigens are no longer used, cross-reactivity with Chlamydia species other than C. trachomatis has been demonstrated (Mannion et al., 1991).
Comparison of subfertility patients with positive and negative MIF test results respectively, revealed a significant difference in the prevalence of C. pneumoniae antibodies (87 and 66% respectively, P < 0.0005). A possible explanation for the difference found is cross-reactivity between C. pneumoniae and C. trachomatis antibodies in the MIF test. To explore the likelihood of cross-reactivity further, the prevalence of C. pneumoniae antibodies was analysed in two subgroups of patients (Tables II and III). In patients without tubal factor subfertility but a positive MIF test result (Table II
), C. pneumoniae antibodies were found more frequently than in patients without tubal factor subfertility and negative MIF test results. These data suggest that C. pneumoniae antibodies can be a cause of false positive CAT results. Analysis of the same groups using C. trachomatis ELISA instead of MIF (Table III
) showed the same results.
Reports in the literature are rare and controversial concerning cross-reactivity between C. pneumoniae and C. trachomatis antibodies in MIF tests. In female patients with tubal factor subfertility and pregnant controls, the presence of C. trachomatis and C. pneumoniae antibodies has been studied (Persson et al., 1999). C. trachomatis antibodies were detected more often in patients (88%) than in controls (48%). In contrast, no difference was found for C. pneumoniae antibodies between patients (52%) and controls (58%). The authors concluded that cross-reactivity did not occur, as C. trachomatis antibodies were equally common in patients with and without C. pneumoniae antibodies (89 and 87% respectively). A study (Moss et al., 1993
) in over 4500 serum samples of women attending a genitourinary clinic, using a modified MIF test to detect type-specific antibodies to Chlamydia species, has been performed. The IgG responses to Chlamydia species were detected in three distinct forms: species-specific (where IgG was directed against one species only), cross-reactive (where the IgG concentration against one species was 2-fold or higher than the IgG concentrations against other species), or group-specific (where IgG concentrations were similar against all species tested). In 67% of women Chlamydia IgG antibodies were found. Species-specific or cross-reactive IgG against C. trachomatis, C. pneumoniae and C. psittaci was present in 38, 21 and 0.1% of the samples respectively. In 8% of samples, the IgG concentration was similar against all three species (group-specific). The authors conclude that antibodies to C. pneumoniae may account for up to half of all Chlamydia positive test results using MIF. From the latter study (Moss et al., 1993
) and the current study it can be concluded that, due to cross-reactivity, C. pneumoniae antibodies are probably major contributors to the low specificity of those MIF tests in which pooled antigens are used. Species-specific antibody tests for C. trachomatis are prerequisites for improving the predictive value of CAT.
In the current study, tubal pathology was more frequent in patients in whom both antibodies to C. trachomatis and to C. pneumoniae were found although the numbers of patients are small within the subgroups (Tables IV and V). The precise mechanism by which Chlamydia causes tissue damage remains to be elucidated, but immune-mediated inflammatory responses seem to be involved in the process (Brunham and Peeling, 1994
). It has been suggested that primary chlamydial infections do not necessarily cause tubal tissue damage, but that tissue damage will develop only after reinfection or after reactivation of a primary infection (Patton and Kuo, 1989
). Reinfection or reactivation of primary infections is considered to induce a booster immune response and subsequently tubal pathology. It has been postulated that this immune response may not be specific for C. trachomatis, and may be induced by C. pneumoniae antibodies as well (Freidank et al., 1995
). In their study Freidank et al. (1995) found the presence of both antibodies to C. trachomatis and C. pneumoniae in the MIF test to be associated with a significantly higher rate of tubal occlusion. The findings prescribed here are in agreement with the findings of Freidank et al. (1995), i.e. more tubal pathology was found in patients with both C. trachomatis and C. pneumoniae antibodies. This supports the contention that C. trachomatis infection and (highly prevalent) C. pneumoniae infections might have a synergistic effect on the development of tubal pathology. However, the exact role of C. pneumoniae in this process needs further exploration.
In conclusion, in 240 subfertility patients a prevalence of C. pneumoniae antibodies of 75%, using a specific ELISA for C. pneumoniae was found. Comparison of laparoscopic and serological data suggests cross-reactivity between antibodies to C. trachomatis and antibodies to C. pneumoniae in the MIF test, as a cause for low specificity of CAT. The simultaneous presence of antibodies to C. trachomatis and to C. pneumoniae was associated with a significantly increased rate of tubal factor subfertility. C. pneumoniae antibodies, which are widespread, may be involved in chlamydial upper genital tract infections and/or subsequent tubal damage.
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Acknowledgments |
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Notes |
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4 To whom correspondence should be addressed at: Academisch Ziekenhuis Maastricht, P.O. Box 5800, 6202 AZ Maastricht, The Netherlands. E-mail: agij{at}sgyn.azm.nl
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References |
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Submitted on June 19, 2000; accepted on November 17, 2000.