1 Research Institute Growth and Development (GROW) and Department of Obstetrics and Gynaecology, 2 Maastricht Infection Centre (MINC) and Department of Medical Microbiology and 3 Department of Clinical Epidemiology and Medical Technology Assessment (KEMTA), Academisch Ziekenhuis Maastricht, P.O.Box 5800, 6202 AZ Maastricht, The Netherlands
4 To whom correspondence should be addressed. Email: jedh{at}sgyn.azm.nl
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Abstract |
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Key words: Chlamydia trachomatis/chlamydia heat shock protein 60/C-reactive protein/immunoglobulin A/persistent infection
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Introduction |
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Previous studies, in which evidence of persistent C. trachomatis infections has been found in the upper genital tract of women with tubal damage, support this hypothesis. Gérard et al. (1998) have found viable C. trachomatis micro-organisms in seven out of ten tubes of patients with ectopic pregnancies. Furthermore, C. trachomatis has been detected in 5679% of the tubes of women with tubal factor subfertility, who underwent reconstructive surgery (Campbell et al., 1993
; Patton et al., 1994b
). Previously, we have demonstrated that genus-specific immunoglobulin (Ig) G antibodies to chlamydia lipopolysaccharide, which are supposed to be markers of persistent infections, are significantly more often detectable in sera of subfertile women with distal tubal pathology (62.7%) as compared to those without distal tubal pathology (33.9%) (Den Hartog et al., 2004
).
Since the association between C. trachomatis-specific IgG antibodies and tubal pathology has been noted (Punnonen et al., 1979), measuring C. trachomatis IgG antibodies in serum is used as a screening method for tubal pathology. Although species-specific C. trachomatis IgG antibodies are markers of previous infections, their presence does not reflect the course of the infection. Therefore, measuring IgG antibodies to C. trachomatis is not useful in discriminating between clearance or persistence of the infection.
We hypothesize that persistent C. trachomatis infections play an important role in the development of tubal pathology, and have studied known serological markers of persistent infections in subfertile women with and without tubal pathology. Elevated levels of IgA antibodies and C-reactive protein (CRP), in the absence of an acute infection, have been suggested to be markers of chronic inflammation and infection, and have been evaluated previously in studies on the relationship between chronic C. pneumoniae infections and respiratory and cardiovascular disease (Roivainen et al., 2000; Gattone et al., 2001
; Johnston et al., 2001
; Falck et al., 2002
; Wong et al., 2002
). IgG antibodies to chlamydia heat shock protein 60 (cHSP60) have also been associated with chronic inflammation (Morrison et al., 1989
), and have been studied previously in subfertile women with tubal pathology (Toye et al., 1993
; Arno et al., 1995
; Freidank et al., 1995
; Claman et al., 1997
).
Sera of subfertile women were tested for the presence of IgG and IgA antibodies to C. trachomatis, IgG antibodies to cHSP60 and CRP. All women underwent a laparoscopy with tubal testing. We correlated the serological test results with the presence of tubal pathology at laparoscopy, and evaluated the role of single tests and test combinations in predicting the risk of persistent C. trachomatis infections and tubal pathology.
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Materials and methods |
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Two independent investigators, who were unaware of the CAT results, scored 313 successive laparoscopy reports to assess the grade of tubal pathology. In cases of disagreement, consensus was reached by consultation. For the sake of the study, tubal pathology was defined as extensive peri-adnexal adhesions and/or distal occlusion of at least one tube (Land et al., 1998). Subfertile women without distal tubal pathology served as controls. The controls had unexplained subfertility, partners with mild male factor subfertility, or proximal occlusion of at least one tube.
Serological methods
IgG antibodies to C. trachomatis were detected using the species-specific Chlamydia pneumoniae IgG micro-immunofluorescence (MIF) test (AniLabsystems, Finland), as described previously (Den Hartog et al., 2004). This species-specific test, which is the currently used screening test for C. trachomatis IgG antibodies in our clinic, has been found to be a good predictor of tubal pathology (Land et al., 2003
). The threshold titre used for a positive test was 32.
IgA antibodies to C. trachomatis were detected using the Chlamydia trachomatis IgA enzyme immunoassay (EIA) (AniLabsystems, Finland). The test was used according to the manufacturer's instructions. The threshold index for a positive test was 1.4.
IgG antibodies to cHSP60 were detected using the cHSP60 IgG enzyme-linked immunosorbent assay (ELISA; Medac, Germany), which is available for research use only. The test was used according to the manufacturer's instructions. The threshold index for a positive test was 1.11.
CRP was determined using the CRP ELISA (DiaMed Eurogen, Belgium). The test was used according to the manufacturer's instructions. In order to reliably detect low CRP concentrations, this high-sensitivity (hs) CRP test was used. CRP levels between 1.0 and 10.0 mg/l (slightly raised levels, but still within the normal range) are assumed to reflect a persistent infection, and were considered as positive. CRP levels <1.0 mg/l (low risk of persistent infection) or >10.0 mg/l (probably acute infection) were considered as negative (Pearson et al., 2003).
Statistical methods
Characteristics of women with and without distal tubal pathology were compared using the MannWhitney U-test. For comparison of the prevalence of IgG and IgA antibodies to C. trachomatis, IgG antibodies to cHSP60 and hs-CRP in women with and without distal tubal pathology, the 2-test was used. The prognostic value of single tests as well as test combinations for distal tubal pathology was determined by calculating sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), odds ratio (OR) and 95% confidence interval (CI). A forward stepwise logistic regression analysis was used to select the best combination of tests. The bootstrap technique was used to test the difference between OR (Efron and Tibshiram, 1993
). P<0.05 was considered statistically significant.
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Results |
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CRP levels between 1.0 and 10.0 mg/l (slightly raised levels, but still within the normal range) were assumed to reflect a persistent infection, and were considered as positive. CRP values >10.0 mg/l were considered as negative, since these values were assumed to reflect acute infections. Twelve women (3.8%) had CRP levels >10.0 mg/l (median 16.1, range 11.2 > 130.3). Of these 12 patients, two had severe endometriosis, which is a known cause of elevated CRP levels (Abrão et al., 1997). In the remaining ten patients, no clinical evidence of acute infections or other underlying inflammatory diseases could be found. Since seven out of these ten serum samples were obtained in autumn or winter, minor or subclinical infections (e.g. influenza-like infections) might have caused the elevated CRP levels.
First, we evaluated the prevalence of IgG and IgA antibodies to C. trachomatis, IgG antibodies to cHSP60 and a positive hs-CRP in subfertile women with and without distal tubal pathology. As shown in Table I, IgG and IgA antibodies to C. trachomatis, IgG antibodies to cHSP60 and a positive hs-CRP test were found significantly more often in women with distal tubal pathology as compared to women without distal tubal pathology.
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Discussion |
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IgA antibodies have been associated with chronic inflammation and infection. With respect to chlamydia, previous studies have demonstrated an association between C. pneumoniae IgA antibodies and its chronic sequelae, e.g. respiratory and cardiovascular morbidity. Falck et al. (2002) have found that the prevalence of symptoms of chronic respiratory tract disease increases parallel to the increase in C. pneumoniae IgA titre. In patients with coronary symptoms, a positive C. pneumoniae IgA titre significantly increases the risk of myocardial injury (OR 1.95) (Wong et al., 2002
). It has been suggested that serum IgA antibodies, as compared to IgG antibodies, may be more reliable markers of persistent C. pneumoniae infections (Saikku, 1999
).
cHSP60 is a chlamydia genus-specific protein, serving as a strong antigenic target for the immune system (Morrison et al., 1989; Kaufmann, 1990
). It has been suggested that antibodies to cHSP60 are markers of chronic inflammation (Kaufmann, 1990
). Studies have shown a strong association between anti-cHSP60 antibodies and tubal factor subfertility. Anti-cHSP60 antibodies are significantly more prevalent in subfertile women with tubal disease (4476%) as compared to those without tubal disease (819%) (Freidank et al., 1995
; Claman et al., 1997
). Among subfertile women with antibodies to C. trachomatis, anti-cHSP60 antibodies are significantly more prevalent in women with tubal pathology (7681%) as compared to those without tubal pathology (043%) (Toye et al., 1993
; Arno et al., 1995
).
CRP is an acute phase protein. Slightly raised CRP concentrations, but still within the normal range, are known indicators of chronic inflammation. Research on the pathophysiology of coronary heart disease has shown that the association between C. pneumoniae infections and the risk of cardiovascular events is stronger if CRP is slightly raised, but within the normal range (Gattone et al., 2001). As compared to patients without C. pneumoniae antibodies and a low CRP, the risk of coronary events increased when C. pneumoniae antibodies were present (OR 1.22; 95% CI 0.742.01), but increased even more when both C. pneumoniae antibodies and a slightly elevated CRP were present (OR 5.40; 95% CI 2.3512.43) (Roivainen et al., 2000
). Serum CRP levels were significantly higher in patients with C. pneumoniae-infected atherosclerotic plaques (8 mg/l) as compared to patients with non-infected atherosclerotic plaques (undetectable CRP) (Johnston et al., 2001
). The role of CRP in tubal factor subfertility has not yet been studied.
In the present study, all evaluated serological markers of persistent infections were significantly more prevalent in women with tubal pathology as compared to women without tubal pathology. However, as single tests, the markers of persistent infections performed poorly as compared to the current screening test for tubal pathology (IgG to C. trachomatis). Odds ratios of IgA antibodies to C. trachomatis (6.1), IgG antibodies to cHSP60 (5.9) and hs-CRP (2.0) were significantly lower as compared to IgG antibodies to C. trachomatis (13.9).
The low OR of the C. trachomatis IgA and cHSP60 IgG tests might be explained by these antibodies being poorer markers of chronic inflammation than is currently presumed. The significantly lower OR of the cHSP60 IgG test, as compared to the C. trachomatis IgG test, might be explained by cross-reaction with the highly similar C. pneumoniae. Anti-C. pneumoniae antibodies are highly prevalent in subfertile women (detectable in 83.1% of women with distal tubal pathology and in 72.8% of women without distal tubal pathology), and are not associated with tubal disease (Den Hartog et al., 2004). The manufacturer of the cHSP60 IgG test mentions that cross-reaction with other chlamydia species may occur, due to the homology of >95% between cHSP60 of the different species. The low OR of the hs-CRP test as a single test might be explained by CRP being a general, and not a chlamydia-specific, marker of chronic inflammation.
A forward stepwise logistic regression analysis was performed in order to determine if the prognostic value of the best single test (i.e. C. trachomatis IgG) could be significantly improved by adding one or more tests. Only combining the C. trachomatis IgG test and the hs-CRP test resulted in a significantly higher OR (39.7) as compared to the C. trachomatis IgG test only (13.9). Measuring C. trachomatis IgG antibodies (markers of a previous infection), in combination with hs-CRP (a marker of the course of the infection), seems to identify a subset of subfertile women with the highest risk of persistent infections and distal tubal pathology. These results are comparable to previous studies, in which risk factors for cardiovascular disease were studied. In these studies, the association between C. pneumoniae and cardiovascular disease, which is commonly known, is stronger when elevated CRP levels, but within the normal range, are also detectable (Roivainen et al., 2000; Gattone et al., 2001
; Johnston et al., 2001
). Using the forward stepwise logistic regression model, no significant increase in test performance was noted when adding a third and fourth test to the combination C. trachomatis IgG/hs-CRP.
A limitation of this study is selection and referral bias. Only women who had undergone a laparoscopy with tubal testing, which is the reference standard in diagnosing tubal pathology, were included in the present study. This inclusion criterion will cause selection bias, as has been discussed previously (Den Hartog et al., 2004). This bias will be worsened by referral bias, since the C. trachomatis IgG test was used in the decision regarding who received a laparoscopy. However, it is hard to prevent selection and referral bias in a study like this, since a laparoscopy, which has costs and risks, is not a routine procedure in all subfertility patients.
The clinical purpose of serial testing is to find a combination of tests which can estimate the risk of tubal disease more accurately. The ultimate goal would be a test combination with a PPV and NPV of 100%. In these cases, invasive tubal testing may no longer be indicated. This goal has not yet been achieved in the present study. However, if our results could be confirmed in a larger study, the test combination C. trachomatis IgG/hs-CRP might be a better screening method for tubal pathology as compared to the current method (C. trachomatis IgG only). In daily practice, all C. trachomatis-positive samples could be retested with the hs-CRP test, in order to identify those women who are at highest risk of a persistent C. trachomatis infection and tubal disease.
In summary, we hypothesized that the risk of tubal pathology is increased in subfertile women with persistent C. trachomatis infections. We have studied serological markers of persistent infections in subfertile women. All evaluated serological markers of persistent C. trachomatis infections are significantly more common in subfertile women with tubal pathology as compared to women without tubal pathology. C. trachomatis IgG-positive subfertile women with raised CRP concentrations, but still within the normal range, are supposed to have persistent C. trachomatis infections and are at highest risk of tubal pathology.
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Acknowledgements |
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References |
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Submitted on October 26, 2004; accepted on December 2, 2004.
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