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Abstract
Objectives Population-based Chlamydia trachomatis seroepidemiological studies help to identify trends in chlamydia infection. However, an improved understanding of the antibody response to infection is required when using serology to estimate cumulative incidence. Thus, the objectives of this longitudinal, retrospective, biobank-based study were to assess the appearance and persistence of C. trachomatis major outer membrane protein (MOMP)-specific serum IgG antibodies after infection and to evaluate the role of antibodies in providing protective immunity against recurrent infection.
Methods Data of notified C. trachomatis infections in Finland were obtained from the National Infectious Diseases Register. Serum samples were acquired from the Finnish Maternity Cohort. 411 women with single chlamydia infection and 62 women with recurrent infections, and for whom suitable paired serum samples were available, were included in the study. Antibody appearance, persistence after infection and the impact of recurrent infections were evaluated. IgG antibodies specific for MOMP were measured from serum using an ELISA method.
Results Anti-C. trachomatis MOMP-specific IgG antibodies were detected in 65.5% (269/411) of women within 3 months of notification of infection. In the absence of recurrent infection, seroprevalence declined to 34.5% (142/411) 3–10 years after the initial infection. The serum antibody levels at baseline correlated positively with seroprevalence at follow-up. Reinfection boosted the humoral immune response by increasing seroprevalence and the serum antibody levels. Seroprevalence within 3 months after first notification of infection was 65.5% (19/29) in women who were later diagnosed with recurrent infection, comparable with women with single notification of infection (65.5%, 269/411).
Conclusions Approximately one-third of women with single notification of chlamydia infection remain seropositive 3–10 years after the initial infection. The concentration of antibodies remained stable during the follow-up. Recurrent infection boosted the humoral immune response, but reinfection occurred despite the presence of pre-existing antibodies.
- chlamydia trachomatis
- antibodies
- epidemiology (general)
- chlamydia infection
- serology
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Introduction
Chlamydia trachomatis is a small, obligate intracellular pathogen that induces an antigen-specific immune response in an infected host. C. trachomatis serology is a useful tool with which to quantify the prevalence and incidence of chlamydia infection.1 2 In Finland, as in many countries, chlamydia surveillance is based on the reporting of current infections using a nucleic acid amplification test (NAAT). Lack of information on the number of patients tested results in underestimation of the true incidence of chlamydia. Knowledge of population-level chlamydia prevalence and incidence is crucial for optimal screening and treatment services and an evaluation of the impact of chlamydia control interventions.1 3 C. trachomatis serology could potentially be used as a screening test in an infertility work-up to estimate the likelihood of tubal factor infertility (TFI) and minimise delays performing further investigations and treatment.4–6 Serology can also be used to estimate the population excess fraction of TFI and ectopic pregnancies. These estimates are useful when contemplating the need and cost-effectiveness of control interventions.1 7
The use of serology for diagnostic purposes and in seroepidemiological studies requires knowledge of the sensitivity and specificity of the test, as well as information about the persistence of serum antibodies after acute infection resolution. The majority of commercially available serological tests have limitations in their sensitivity, specificity or cross-reactivity with a common respiratory pathogen C. pneumoniae.2 The microimmunofluorescence test, previously considered the gold standard for a serological diagnosis of chlamydia,8 lacks specificity and reproducibility, is labour-intensive, and its interpretation is observer-dependent.9 Commercially available ELISA tests that use a synthetic peptide from the immunodominant region of the chlamydial major outer membrane protein (MOMP) are specific due to the presence of species-specific and serotype-specific epitopes in the MOMP.10 11 A novel ‘in-house’ IgG ELISA based on the C. trachomatis-specific antigen Pgp3 appears to be a sensitive serological test, with a growing interest as an epidemiological tool.1 12
There is wide interindividual variation in the type and intensity of the immune response evoked by C. trachomatis. The appearance of antibodies is influenced by differences in the genetic background of the host13 14: pathogen factors, such as serotype and infectious burden; tissue tropism; and the number of infection episodes.12 15 Studies in which different methods and antigenic epitopes were used have consistently demonstrated that a number of individuals do not develop specific IgG antibodies following the identification of chlamydia infection using NAAT.16 17 This might be partly due to transient infection,18 and it has been hypothesised that early diagnosis and treatment interrupts the formation of a specific antibody response.19 However, infection clearance is dependent on the activation of a cell-mediated immune response,20 which can be intensive also when the antibody response is undetectable.21
In Finland, the registration of diagnosed infections is comprehensive as laboratories are obliged to provide notification of positive C. trachomatis test results with a personal identity number to the National Infectious Diseases Register (NIDR). Screening for C. trachomatis in Finland is not systematic, but there are national guidelines for opportunistic screening, for example, during pregnancy if considered necessary. The testing and treatment of chlamydia are free of charge in public healthcare services. Registry database information on notified chlamydia infections and samples of pregnant women acquired from the Finnish Maternity Cohort (FMC) were combined in the present research.
We investigated the use of C. trachomatis MOMP-specific IgG antibody detection as an indicator of past infection, and examined seroprevalence, conversion from seropositive to a seronegative state, and changes in the antibody concentration levels of paired samples following NAAT-detected chlamydia infection. We also evaluated the effect of recurrent infection on the seroprevalence and antibody levels and the influence of pre-existing antibodies on the risk of recurrent infection.
Methods
Study population
The FMC of Northern Finland Biobank Borealis was established in 1983 as a nationwide effort by the National Institute for Health and Welfare, Finland. The FMC comprises two million serum samples that are collected during the first or early second trimester of pregnancy (5th–95th percentile: months 2–4 of pregnancy) from over 950 000 women. The FMC covers over 95% pregnancies in Finland, with archived prenatal serum specimens drawn for routine screening for congenital infections (HIV, hepatitis B and syphilis). After obtaining women’s informed consent, the remaining serum samples are stored at −25°C in a protected biorepository and are available for research.
Information on notified C. trachomatis infections from women who provided samples to the FMC during 2007–2014 was obtained from the NIDR of the National Institute for Health and Welfare. Positive test results with identification information, detection method and sampling date have been registered since 2004. When multiple notifications are received of the same individual, a recurrent infection is registered if the time interval between notifications is at least 3 months.
Inclusion criteria and selection of samples
The inclusion criteria for women and selection of samples to the study are depicted in figure 1. The total number of women who provided samples to the FMC during the study period was 309 819 (mean age when the samples were taken: 28.6 years; range: 13–54 years). Notification of at least one chlamydia infection was made for 8% (n=24 552) of the 309 819 women (mean age at the time of notification: 23.1 years; range: 11.6–53.4 years) between 2004 and 2014. Notification of 1, 2 and ≥3 chlamydia infections was made for 81% (n=19 816), 15% (n=3726) and 4% (n=1010) of the women, respectively.
A flow chart detailing the approach used to evaluate the appearance and persistence of Chlamydia trachomatis MOMP-specific IgG antibodies after infection and the role of antibodies in providing protective immunity against recurrent infection in the current study. FMC, Finnish Maternity Cohort; MOMP, major outer membrane protein.
To evaluate the appearance and persistence of MOMP after notification of a chlamydia infection, the following inclusion criteria were applied: the serum sample had to have been obtained 0–3 months after notification of the C. trachomatis infection, and a follow-up sample had to have been taken 3–10 years of the initial notification in women without any other C. trachomatis infections having been registered between the two samples. Accordingly, available samples were found for 411 women who were included in the study.
To study the effect of recurrent infections on seroprevalence and C. trachomatis-specific antibody levels, women notified as having at least two infections during the study period were selected. Reinfections were relatively rare in this study, and the inclusion criteria of two separate samples within a defined timeframe of two infections narrowed down the number of eligible women. Thus, the timeframe between notification of the infection and donation of the sample was extended to 12 months. Finally, 62 women who met these criteria were included in the study.
To study the protection provided by pre-existing MOMP-specific IgG antibodies against recurrent chlamydia infection, the baseline serological status (and antibody levels) of women who experienced repeated chlamydia infections was compared with that of women with single notification of chlamydia infection (n=411). To ensure that the groups were comparable with regard to the timeframe used to obtain the baseline samples, women with several notifications who had donated a second sample within 3 months of notification of the first infection (n=29) were included in this part of the study (figure 1).
Laboratory analysis
Serum IgG antibodies specific for C. trachomatis were analysed (serum dilution of 1:50) using a commercial ELISA ( Chlamydia trachomatis-IgG-ELISA plus, Medac, Wedel, Germany) based on synthetic peptide-containing immunogenic region of the MOMP, according to the manufacturer’s instructions. The MOMP-specific ELISA is specific (≥95%) with minimal cross-reactivity with C. pneumonia.22 The measured optical density (OD) values of the samples and the positive control were corrected based on the nominal and measured OD value of the calibrator, according to the manufacturer’s instructions. The results were expressed as a concentration of arbitrary unit values in millilitres (AU/mL). Samples with a unit value of ≥22 AU/mL were considered positive.
Statistical analysis
Statistical analysis was performed using IBM SPSS Statistics V.24 for Windows. The χ2 and McNemar’s tests were employed to compare the categorical variables, and the continuous variables were compared by non-parametric Mann-Whitney U or Wilcoxon signed-rank test.
Results
Sensitivity of the antibody test up to 3 months after diagnosis with NAAT
MOMP-specific IgG antibodies were observed in 269 (65.5%, 95% CI 60.9% to 70.1%) of the 411 samples obtained within 3 months of notification of C. trachomatis infection. Seroprevalence was highest (67.1%) in the samples obtained within 1 month of C. trachomatis infection notification (n=167/249, 95% CI 61.3% to 72.9%), declining slightly to 65.4% (n=34/52) after 1–2 months (95% CI 52.5% to 78.3%), to 63.4% (n=26/41) after 2–3 months (95% CI 48.7% to 78.1%) and to 60.9% (n=42/69) after 3–4 months (95% CI 49.4% to 72.4%). The decline in seroprevalence was not statistically significant (p=0.318), and at each time point the 95% CI covered the mean seroprevalence level of 65.5%. Differences in the concentration levels of the antibodies were not observed in relation to sampling time of <4 months from diagnosis.
C. trachomatis-specific IgG antibody findings 3–10 years after infection
Seroprevalence decreased from 65.5% at baseline (ie, the first samples taken within 3 months of notification of chlamydia infection) to 34.5% (n=142/411) in the follow-up samples obtained 3–10 years after notification of infection. Approximately half of the women who were seropositive at baseline had measurable levels of C. trachomatis antibodies in their serum samples 3–10 years after infection, while seroprevalence was stable over the years (table 1). Seroconversion was observed in 4.9% (n=7/142) of women who were negative at baseline.
Seroprevalence of Chlamydia trachomatis MOMP-specific IgG and follow-up time*
The median IgG MOMP antibody concentration was 38.5 AU/mL at baseline, and 14.4 AU/mL, 10.8 AU/mL and 13.4 AU/mL when assessed at 3–5 years, 5–7 years and 7–10 years, respectively, after notification of infection. The serum IgG MOMP-specific antibody concentration ratio between the follow-up and baseline samples was analysed in women who were seropositive at baseline (figure 2). The mean antibody concentration ratio was similar at different follow-up time intervals. The antibody concentration level 3–10 years after infection was 40%–45% that at baseline.
A depiction of the mean Chlamydia trachomatis MOMP-specific IgG concentration ratio (follow-up to baseline sample). Women who were seropositive at baseline (n=269) were included in the analysis and grouped according to follow-up duration. The circle represents the estimates, and the error bars indicate 95% CIs. MOMP, major outer membrane protein.
Influence of the C. trachomatis MOMP-specific antibody levels on the persistence of detectable antibodies in the follow-up samples
Women who were seropositive within 3 months of notification of infection (n=269) were divided into quintiles based on MOMP-specific IgG antibody concentration unit values in the sample (AU/mL). Women with a high concentration of antibodies (≥158 AU/mL) at baseline were significantly more likely to be seropositive 3–10 years after diagnosis (OR 94.1; 95% CI 25.6 to 345.9) than those in the lowest quintile. Seroprevalence at follow-up was 9.4% (n=5/53) in the first quintile of baseline antibody concentration (22.0–49.1 AU/mL), 31.5% (n=17/54) in the second quintile (49.2–56.4 AU/mL), 48.1% (n=26/54) in the third quintile (56.7–89.5 AU/mL), 70.4% (n=38/54) in the fourth quintile (89.6–152.5 AU/mL) and 90.7% (n=49/54) in the highest quintile (158.6–424.9 AU/mL).
Effect of recurrent infection on C. trachomatis MOMP-specific antibody levels in the follow-up samples
Recurrent C. trachomatis infection enhanced the antibody response significantly. The median antibody level was 32.5 AU/mL after the first notification of infection and 55.3 AU/mL with recurrent infection (p=0.025). Seroprevalence increased significantly from 56.5% (n=35/62) to 72.6% (n=45/62) (p=0.031), and seroconversion was seen in 51.9% (n=14/27) of those who were seronegative after the first notification of infection. Four of the 35 women (11.4%) who were seropositive after the first infection were seronegative during sampling after recurrent infection. The duration between sampling times was longer for these four subjects following notification of recurrent infection compared with that for primary infection.
Pre-existing antibodies and recurrent infections
Within 3 months of notification of the first infection, the seroprevalence of repeatedly infected women was 65.5% (n=19/29), comparable with the baseline seroprevalence for women with only one notifiable infection (65.5%, n=269/411). The median antibody concentration was 44.3 AU/mL for the samples of repeatedly infected women. This was higher than the baseline antibody concentration (38.5 AU/mL) recorded for those who did not experience repeated infection, but this difference was without statistical significance (p=0.252) (table 2).
Seroprevalence and Chlamydia trachomatis MOMP-specific antibody concentration levels in the study subgroups at different time points
Discussion
Using paired samples of the same individuals, it was found that approximately two-thirds of individuals infected with C. trachomatis had detectable antibody levels within 3 months of infection, and half of them were seronegative at follow-up (ie, seroprevalence of 34.5% when measured 3–10 years after infection). The detectability of antibodies at follow-up had a strong positive association with the antibody level. Compared with baseline, the antibody concentration decreased sharply but remained stable over the follow-up time of 3–10 years. Recurrent chlamydia infection also occurred despite pre-existing C. trachomatis MOMP-specific antibodies. Reinfection was shown to enhance antibody response.
In the current study, samples were acquired from pregnant women. Most infection notifications date to early pregnancy since baseline samples of women with single infection and both samples of repeatedly infected women were selected as close as possible to the notification of infection. This could affect the results because immune tolerance in pregnancy may reduce normal immunological response to infection. However, the seroprevalence findings were similar to those of previous studies for MOMP-specific antibodies.17
A MOMP-specific ELISA (Medac) was used in the current study, which may have lower sensitivity than that of Pgp3 antigen-based ELISA.23 However, the use of a MOMP-specific ELISA was feasible owing to its commercial accessibility, specificity, and the nature of MOMP being the most prominent protein in the C. trachomatis outer membrane and a target for neutralising antibodies during infection.24 The current study findings are in accordance with those of the cross-sectional study performed by Horner et al,17 who reported a reduction in seroprevalence within 6 months after using several methods and a comparable reduction using MOMP-specific ELISA (Medac). By using serum samples from the same women at follow-up, a reduction in seroprevalence at the individual level could be confirmed. On diagnosis of the infection, the seroprevalence of MOMP antibodies was comparable with that reported of Pgp3 IgG after a single episode of infection.12 It seems that antibody detection is dependent on the antigen used and even the method. Pgp3-specific antibodies that were measured using a double-antigen sandwich ELISA were detectable more frequently compared with indirect Pgp3 or MOMP ELISA tests.12 23
Seroconversions were detected in 4.9% of women tested negative at baseline, indicating that recurrences occurred between baseline and follow-up despite the infection not being notified. Thus, it is plausible that stable serum antibody levels in some women could be explained by undetected recurrent infection or exposure to C. trachomatis.
Declining C. trachomatis seroprevalence is a natural consequence of a reduction in serum antibody concentration. The antibody concentration ratio in individuals who remained seropositive after baseline was approximately 40% that at baseline, and it remained stable at the follow-up intervals from 3 to 10 years. This result is in line with the findings of a previous study that showed conversion from seropositive to a seronegative state in some women while others stayed seropositive with constant antibody levels for years after chlamydia.25 The average lifespan of IgG antibodies in the serum is relatively short, and stable levels of specific antibodies over a long period are enabled by long-lived plasma cells that persist in the absence of a detectable antigen for decades after the infection episode.26
Seroprevalence in infected individuals 3–10 years after notification of infection (34.5%) was higher than that reported in pregnant female population in general (a little over 10%)3 27 and lower than the seroprevalence for women with TFI observed in our previous studies (40.9% and 43.2%).4 5 In the current study, information on spontaneous or treatment-related pregnancies was not available. However, it can be assumed that many of the selected women did not have fertility issues owing to the selection criteria. In addition, women who were not pregnant after C. trachomatis infection were not included in the current study. It is reasonable that seroprevalence was higher for patients with TFI than for the current study population, which represents a random sample of women diagnosed with chlamydia during pregnancy, because C. trachomatis-induced reproductive tract pathology is immune-mediated. This also indicates that the sensitivity of antichlamydial antibodies as a marker of previous infection is generally higher for women with TFI than those who have cleared the infection without long-term consequences.28
The second study objective was to evaluate the impact of recurrent chlamydia infection influences on MOMP-specific antibody response. Recurrent infections increased the seroprevalence and significantly escalated antibody levels. When seroprevalence and antibody concentration levels after the first notification of infection were compared in recurrently infected individuals and those with only one infection notification, the seroprevalence and intensity of the humoral response were equal. This finding suggests that pre-existing IgG antibodies that are specific to MOMP—which is a primary target for neutralising antibodies during C. trachomatis infection24—are unable to completely prevent reinfection. This is in harmony with the general concept that antibodies provide some degree of protection but not complete immunity.29 Immunity also requires efficient activation of the cell-mediated response.20 Further research is warranted to obtain a better understanding of the protective immune response through cell-mediated immune response analysis.
Interpretation of the serological test results is complex, as several factors, including the amount of elapsed time since the infection, the number of infection episodes, test methods used and target antigens, influence the detection of antibodies. The development of serological assays has enabled C. trachomatis serology to be used as a research tool to estimate the true prevalence of chlamydia at the population level. Serology is needed to evaluate the impact of chlamydia control interventions. By combining registry data with biobank samples and serological testing, it is possible to identify populations at risk and improve chlamydia control efforts.
Key messages
Two-thirds of Chlamydia trachomatis-infected women develop major outer membrane protein (MOMP)-specific IgG antibodies after infection, and one-third of women remain seropositive years after the initial infection.
Recurrent infections increase the antibody concentration level and seroprevalence.
Pre-existing serum MOMP-specific antibodies are unable to prevent recurrent infection.
Acknowledgments
We thank Sara Kuusiniemi for excellent technical assistance.
References
Footnotes
Handling editor Nigel Field
Contributors H-MS devised the project. H-MS and HÖ designed the study. HÖ performed case selection and data analysis. HÖ, H-MS, PJ-K, TR and AT were involved in the interpretation of findings. HÖ wrote the first draft of the manuscript, and all authors commented and contributed to subsequent revisions and approved the final version.
Funding This work was supported by a research grant from the Helsinki University Hospital (grant number TYH2016255).
Competing interests None declared.
Patient consent for publication Not required.
Ethics approval The study was approved by the National Institute for Health and Welfare (reg no THL/503/6.02.00/2016) and by the ethics committee of the Hospital District of Helsinki and Uusimaa (reg no 18/13/03/03/2016).
Provenance and peer review Not commissioned; externally peer reviewed.
Data availability statement No data are available.