Chlamydia antibody testing in screening for tubal factor subfertility: the significance of IgG antibody decline over time*

Anna P. Gijsen1,3, Jolande A. Land1, Valère J. Goossens2, Marlea E.P. Slobbe2 and Cathrien A. Bruggeman2

1 Research Institute Growth and Development (GROW), Department of Obstetrics and Gynaecology, Maastricht and 2 Cardiovascular Research Institute Maastricht (CARIM), Department of Medical Microbiology, Maastricht, The Netherlands


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: Chlamydia (C.) trachomatis antibody testing in screening for tubal factor subfertility is limited by false negative results, i.e. negative Chlamydia antibody tests in patients with tubal pathology at laparoscopy. The present study was performed to determine whether decline in C. trachomatis IgG antibodies over time is responsible for those false negative results. METHODS: A total of 39 women with an initial titre of >=64 were re-studied after 4–7 years. A new serum sample was collected from each patient. The initial and the second serum sample were tested for C. trachomatis IgG antibodies using a micro-immunofluorescence assay (MIF). A species-specific enzyme-linked immunosorbent assay (ELISA) was used to validate the MIF test results. All patients filled out a questionnaire to determine risk factors for renewed C. trachomatis infection between the initial and second serum sample. RESULTS: Seven of the 39 patients (18.0%) showed a decline (>2 titre steps) in IgG antibodies by MIF over a period of 4–7 years, but IgG antibodies never became undetectable. In the 7/39 patients who showed a decline by MIF, signal/cut-off values by ELISA did not change. CONCLUSION: A decline in IgG antibody titre is not a significant cause of false negative Chlamydia antibody test results.

Key words: IgG antibody/Chlamydia trachomatis/diagnostic test/screening/tubal factor subfertility


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
In women, up to 80% of Chlamydia infections remain asymptomatic (Quinn et al., 1996Go). These untreated women are at risk of developing chronic sequelae such as periadnexal adhesions and tubal occlusion, and of presenting at fertility clinics ~10–15 years after the primary infection.

Chlamydia immunoglobulin (Ig) G antibodies are thought to persist for years and have therefore been used as markers of a previous C. trachomatis infection (Ngeow, 1996Go). Chlamydia antibody testing has been incorporated in fertility work-up on a large scale, and has been shown to be a non-invasive and cost-effective screening method for tubal factor subfertility (Mol et al., 1997Go).

The clinical significance of Chlamydia antibody testing, however, has its limitations, due to false positive and false negative test results (Land et al., 1998Go). Patients with false negative Chlamydia antibody test results (i.e. negative Chlamydia antibody tests in patients with tubal pathology at laparoscopy) may have non-Chlamydia-related causes of adhesions or tubal occlusion, or no antibodies may be found despite previous C. trachomatis infection. As a cause of false negative Chlamydia antibody tests, it has been postulated that IgG antibodies may decline over time after C. trachomatis infection, in view of the time span between primary infection in adolescence and fertility work-up in adulthood.

So far, little is known about the course of Chlamydia IgG antibodies over time. Four studies have been published on this subject. The post-therapeutic course of serum Chlamydia IgG antibody titres has been studied in 47 women with acute salpingitis (Henry-Suchet et al., 1994Go), who had positive IgG antibody titres and were treated with doxycycline. Twelve months after treatment, the IgG titre had decreased in 15 of the 47 (31.9%) women. However, only two of the 47 women (4.3%) reverted from IgG seropositive to seronegative in the 1 year interval studied. Another report on the effect of treatment with doxycycline on serum Chlamydia IgG antibodies was a study performed in 33 women, who had had an episode of acute salpingitis associated with high IgG titres (>64) (Piura et al., 1993Go). Mean follow-up time was 34.2 months (range 24–49). No significant change in IgG titres was demonstrated in 21 women (63.6%), a significant increase in titre (>=4-fold) was found in eight women (24.2%), and a significant decrease in four women (12.1%). The authors concluded that post-treatment titres remain high in most cases, even when treatment resulted in complete resolution of clinical signs and symptoms of the disease. A third published study dealt with the long-term persistence of serum Chlamydia IgG antibodies in patients with high initial IgG antibody titres (>=1:128) in serum after pelvic inflammatory disease (Puolakkainen et al., 1986Go). The follow-up time was between 3.1 and 6.3 years. Twenty-six out of 60 patients (43.3%) showed an insignificant change in Chlamydia IgG serum antibody levels, eight (13.3%) showed a significant increase and 26 (47.3%) showed a significant decrease. The fourth study published on titre course has been performed in asymptomatic patients during a community-oriented screening programme among 860 women of reproductive age (Chaim et al., 1992Go). Twenty-one patients (2.4%) were found to have Chlamydia IgG antibody titres >64. These 21 patients were treated with doxycycline, after which IgG titres were followed in 17 of the cases. Over a 5 year interval, IgG titres were unchanged in one case (5.9%), had increased 2-fold in one case (5.9%), decreased >=3-fold in 11 cases (64.7%) and decreased 2-fold in four cases (23.5%).

In conclusion, the four studies published on the course of Chlamydia IgG antibody titres describe 153 patients, of which 56 (36.6%) had a substantial titre decline during the study period. However, all papers concern the post-therapeutic course of C. trachomatis levels in patients with active genital infections, immediately after the institution of therapy. The objective of the present study was to investigate the course of Chlamydia IgG antibody levels in asymptomatic subfertility patients, in whom no active C. trachomatis infection is assumed to be present. A micro-immunofluorescence (MIF) test was used, as this test is still considered the best available standard, and a species-specific enzyme-linked immunosorbent assay (ELISA) was used to validate the MIF test results.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
In our hospital, Chlamydia antibody testing has been part of the fertility work-up since 1992. Blood is drawn at the patient's first visit and Chlamydia antibody testing is performed. Between 1992 and 2000, spare serum (sample 1) from 253 patients was stored for research purposes. Of those 253 patients, 67 had an antibody titre of >=64, which we considered positive (Land et al., 1998Go), and had a follow-up period of at least 4 years. These women were approached for a second blood sample (sample 2). Forty-five women could be traced, and 39 women agreed to participate in the study. After a mean follow-up time of 5 years and 7 months (range 4 years and 0 months to 7 years and 2 months), sample 2 was obtained from these 39 patients. In all 39 patients, sample 1 was re-determined simultaneously with the determination of sample 2 by MIF. In 33 patients, sufficient sample 1 and sample 2 serum was available for additional antibody testing by ELISA.

An indirect MIF antibody technique for C. trachomatis IgG antibodies was used. In short, 20 µl of serum was diluted eight times in phosphate-buffered saline (PBS) (pH 7.4) and incubated on the C. trachomatis-spot immunofluorescence substitute slides (egg grown C. trachomatis biovar L2; BioMerieux, 's 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 B.V., Uithoorn, The Netherlands) diluted in PBS for 30 min at 37°C. After three washings in PBS and one in ultrapure water, processed through a milli-Q purifying system (Millipore, Bedford, MA, USA), slides were embedded in Fluoprep mounting medium (Biomerieux) under a cover slip. A positive reaction was characterized by specific fluorescence of the C. trachomatis elementary bodies. For a quantitative determination of the antibody titre, 2-fold serum dilutions in PBS were made. The reciprocal value of the highest dilution giving a positive signal in the MIF was considered as the antibody titre. All slides were evaluated independently by two readers. In case of disagreement, the independent judgement of a third reader was decisive. Differences in titres were expressed in titre steps. A decline of two titre steps or more between sample 1 and sample 2 was considered significant.

For validation of the MIF results, a C. trachomatis IgG ELISA (Labsystems, Helsinki, Finland) was performed. 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 30 min at 37°C. The strips were washed five times in 300 µl washing solution (citrate buffered PBS) and the inverted strips were tapped a few times on a paper towel to remove excess fluid. To each well, 200 µl conjugate was added (sheep antihuman IgG/horse-radish peroxidase) and the plate 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 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 450 nm. Cut-off indexes and signal/cut-off (S/C) values were calculated and the results interpreted according to the manufacturer's instructions. The results were interpreted to be negative (N) when S/C was <1.0, equivocal (E) when S/C was >=1.0 and <1.4, positive (P) when S/C was >=1.4 and <2.5 and highly positive (HP) when S/C was >=2.5. Results were considered different when the S/C value of sample 1 was significantly different from the S/C value of sample 2.

At the time of sample 2, all patients filled out a questionnaire in order to determine risk factors for a renewed C. trachomatis infection after sample 1. Questions were asked about abdominal complaints compatible with pelvic inflammatory disease, fertility treatment in which cervical manipulation is involved, the use of contraceptive methods, and new sexual partners since sample 1. Furthermore, social habits such as smoking and alcohol use were asked for, since these factors have been associated with the risk of tubal factor subfertility (Bahamondes et al., 1994Go). For statistical analysis a Fisher's exact test was used. P < 0.05 was considered significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Initially, in all 39 women Chlamydia antibody testing was performed as part of their fertility work-up after they had been trying to achieve pregnancy for at least 12 months. At sample 1, patients had a mean age of 29 years (range 19–38) and at sample 2 they had a mean age of 36 years (range 26–45). IgG antibody titres by MIF in sample 1 and sample 2 are given in Table IGo. Also in Table IGo, S/C values of sample 1 and sample 2 are shown, to allow comparison of MIF and ELISA results. In 32/39 (82.0%) patients, no significant change (i.e. more than two titre steps) in Chlamydia IgG antibody titre by MIF was demonstrated between sample 1 and sample 2. In 7/39 patients (18.0%) a decline in IgG antibodies of at least two titre steps was found over a period of 4–7 years. However, in no patient IgG antibodies became undetectable.


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Table I. Chlamydia IgG titres by micro-immunofluorescence (MIF) with differences in titre steps, and signal/cut-off (S/C) values by enzyme-linked immunosorbent assay (ELISA) with interpretation of S/C values
 
The ELISA test showed no change in 26/33 patients (78.8%), a decline was found in 1/33 patient (3.0%) and an increase in 6/33 patients (18.2%). However, in all 7/39 patients in whom a significant decline in IgG antibody titre was found by MIF, no differences were found by ELISA. In comparing Chlamydia antibody test results by MIF and ELISA, a remarkable discrepancy was found. In contrast to MIF, which showed IgG antibodies in all patients for both samples 1 and 2, the ELISA showed negative values in 7/33 (21.2%) patients in both samples.

For analysis of the questionnaires, patients were divided into two groups, to determine risk factors for renewed C. trachomatis infection after sample 1. Group A consisted of patients in whom no titre decline (more than two titre steps) by MIF was found, and group B of patients with titre decline measured by MIF. Groups A and B were compared for the prevalence of risk factors for renewed infection. The results of the questionnaires are shown in Table IIGo. The mean time interval between sample 1 and sample 2 did not differ for the two groups: 5 years and 8 months in group A and 5 years and 10 months in group B. Comparison of the group of patients without titre decline (group A) with those with titre decline (group B) revealed no significant differences in prevalences of the factors associated with renewed infection. As many patients in group A (34.4%) as in group B (28.6%) reported an episode of abdominal complaints after sample 1. In group A, 2/32 patients (6.3%) had had pelvic or abdominal surgery as compared to 1/7 patients (14.3%) in group B. No patient had received antibiotic treatment for abdominal complaints in the period between samples 1 and 2. In group A, 14/32 patients (43.8%) had undergone fertility investigations and/or treatment that involved cervical manipulation, as compared with 4/7 patients (57.1%) in group B. No significant difference in use of contraceptives was found between the groups, and especially no intrauterine contraceptive device use was reported between sample 1 and sample 2. Twenty-eight out of 32 patients (87.5%) in group A and 5/7 patients (71.4%) in group B reported that they had had the same sexual partner between sample 1 and sample 2. Furthermore, 18/32 patients (56.3%) in group A reported cigarette smoking versus 4/7 patients (57.1%) in group B. Alcohol consumption was reported by 15/32 patients (46.9%) in group A and 3/7 patients (42.9%) in group B. At statistical analysis, the above-mentioned factors showed no significant differences between groups A and B.


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Table II. Anamnestic prevalence of variables presuming Chlamydia reactivation or re-infection between samples 1 and 2
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Chlamydia antibody testing has been introduced as a simple and inexpensive screening test for tubal factor subfertility. However, little attention has been paid to its limitations. Although it is assumed that Chlamydia infections give rise to persistent IgG antibody formation, the exact course of titres over time is still poorly understood. The four studies published to date on the course of Chlamydia IgG antibody titres together describe a significant decline of titres of 36.6% in 153 patients tested, but describe seroconversion from positive to negative as a rare occurrence. However, these studies have all concerned patients recently treated for acute pelvic inflammatory disease only. To our knowledge, the present study is the first to investigate the course of Chlamydia IgG antibody levels in asymptomatic subfertility patients.

In our study, by using MIF a decline of IgG antibody titres of at least two titre steps was found in 7/39 patients (18.0%) over a period of 4–7 years. However, in no patient did IgG antibodies become undetectable. The persistently high Chlamydia IgG antibody titres in 82.0% of patients might theoretically be explained by re-infections by Chlamydia. Firstly, Chlamydia re-infection might occur by endogenous reactivation of the micro-organism that has become latent after a previous infection (Forsey et al., 1990Go). Subfertility patients are considered to be at risk for endogenous reactivation by cervical manipulation during fertility investigations (e.g. hysterosalpingography) or treatment (e.g. intrauterine insemination, IVF). Secondly, exogenous re-infection after a solved primary infection (Stamm et al., 1986Go) may occur. A questionnaire was composed to estimate the risks of C. trachomatis re-infection. We are aware of the fact that a questionnaire is a weak instrument, but there was no better method available to determine the risk of re-infection between the two time points studied. The questionnaire showed that there were as many patients who had undergone cervical manipulation in the group with titre decline as in the group without titre decline. Furthermore, the prevalence of active cervical C. trachomatis infections in subfertility patients has been reported to be <2% (Eggert-Kruse et al., 1997Go; Macmillan and Templeton, 1999Go). This also suggests that the incidence for endogenous reactivation in subfertility patients could be small. Our estimation of the risk of exogenous re-infection had to rely on patients' answers in the questionnaire, as patients were not screened for active C. trachomatis infections. Questions concerning risk factors associated with infection or re-infection were not answered differently by patients with no titre decline (group A) as compared to patients with titre decline (group B). Nevertheless, although the questionnaire indicated exogenous re-infection between the time points studied to be unlikely, re-infection cannot be excluded completely. Summarizing the results of the questionnaire, it can be concluded that in our study C. trachomatis re-infection by either endogenous reactivation or exogenous re-infection appears to be no major cause of persistence of Chlamydia IgG antibody titres in the subfertile women studied.

MIF is the most widely used test method for Chlamydia antibody testing in subfertility patients for screening for tubal pathology due to earlier C. trachomatis infections. Chlamydia antibody testing by MIF has been shown to have clinical value in predicting tubal pathology (Land et al., 1998Go). During C. pneumoniae infection however, antibodies are also formed against the common antigenic epitopes present in all members of the genus Chlamydia. As the MIF test is not species-specific, cross-reaction between C. trachomatis and C. pneumoniae might occur (Mannion et al., 1991Go; Moss et al., 1993Go; Gijsen et al., 2001Go). Furthermore, interpretation of the MIF results is subjective, and inter-observer differences are inevitable. Recently, ELISA tests have become available which are more species-specific, and the results can be objectively analysed by computer. In the present study, an ELISA was used to validate the results on titre decline found by MIF. Overall, in 78.8% of cases, the ELISA test showed no change in the interpretation of S/C values between the two samples, and in particular no decline in S/C value was found in the 7/39 patients that showed a significant titre decline by MIF. Remarkably, however, in 7/33 patients (21.2%) who had Chlamydia IgG antibodies by MIF, ELISA showed negative S/C values. The discrepancy found between MIF and ELISA test results might be due to cross-reaction in the MIF test with other Chlamydia species than C. trachomatis. On the other hand, since in ELISA tests synthetic peptides derived from different domains of the major outer membrane are used as antigens, these peptides will affect the specificity of the test. Therefore, patients without antibodies against the particular peptide used in the assay will have negative ELISA test results. Although the ELISA test has been shown to be highly sensitive in detecting Chlamydia IgG antibodies in patients with proven acute C. trachomatis infections (Närvänen et al., 1997Go), it has not yet been evaluated in screening for tubal factor subfertility in patients with previous subclinical infections.

In conclusion, this is the first paper to report on the course of C. trachomatis IgG antibodies over time in asymptomatic and untreated female subfertility patients. In 39 patients with initial C. trachomatis IgG antibody titres >=64, 7 (18.0%) showed a decline of at least two titre steps over a 4–7 year interval. However, in spite of this decline, all patients continued to test positive for IgG antibodies. Responses to a questionnaire showed that reactivation or re-infection does not seem to be responsible for the persistence of Chlamydia IgG antibodies, since patients without titre decline did not differ from those with titre decline as regards the risk factors associated with renewed C. trachomatis infection. We therefore conclude from this study that in subfertility patients, decline in IgG antibody titres over time is not a significant cause of false negative Chlamydia antibody test results (i.e. patients testing negative for Chlamydia antibodies, but with tubal factor subfertility found at laparoscopy).


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The authors acknowledge Rik de Jongh, medical student, for his help in collecting the data.


    Notes
 
3 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 Back

* Part of the work was presented at the 15th Annual Meeting of the European Society of Human Reproduction and Embryology, Tours, France, June 27-30, 1999. Back

Submitted on December 30, 1999; resubmitted on September 3, 2001


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Eggert-Kruse, W., Rohr, G., Demirakca, T., Rusu, R., Näher, H., Petzoldt, D. and Runnebaum, B. (1997) Chlamydial serology in 1303 asymptomatic subfertile couples. Hum. Reprod., 12, 1464–1475.[Abstract]

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Gijsen, A.P., Land, J.A., Goossens, V.J., Leffers, P., Bruggeman, C.A. and Evers, J.L.H. (2001) Chlamydia pneumoniae and screening for tubal factor subfertility. Hum. Reprod., 16, 487–491.[Abstract/Free Full Text]

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Submitted on December 30, 1999; resubmitted on September 3, 2001; accepted on October 29, 2001.