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Besides commercially available serological assays that detect antibodies to major outer membrane protein (MOMP)1 and lipopolysaccharide (LPS) “in-house” chlamydial heat shock protein 60 (cHSP60) assays are extensively used in assessing serological responses to urogenital Chlamydia trachomatis infection. Although comparison of the different “in-house” assays is difficult owing to a lack of standardisation, there is a consensus among the users of these assays that the anti-cHSP60 responses in women increase with the severity of C trachomatis associated disease, leading to the suggestion that the high amino acid sequence homology between chlamydial and human HSP60 results in autoimmune mediated fallopian tube damage. Owing to the significance of the possible association of the response to cHSP60 and progressive disease, a commercially produced assay that employs defined cHSP60 epitopes should allow for the comparison of results obtained in different laboratories, as well as forward the use of cHSP60 as a diagnostic tool if the assay proves to be relevant in predicting pathology or clinical outcome of a urogenital chlamydial infection.
This study evaluated a recently introduced commercially available cHSP60 serological assay and determined the anti-cHSP60 responses in three gynaecologically well defined groups of women.
Group 1 consisted of women without tubal pathology as assessed by either hysterosalpingography or laparoscopy (n = 21), group 2 consisted of pregnant women (unknown tubal status, proved fertility; n = 86), and group 3 consisted of women with confirmed (based on hysterosalpingography or laparoscopy) tubal pathology (n = 11). C trachomatis positivity was assessed previously using one of the following serological assays: microimmunofluorescence (MIF) (BioMérieux’s Hertogenbosch, Netherlands), BAG Chlamydia EIA (Biologische Analysensystem GmbH, Lich, Germany) and the CT-pELISA (Medac, Wedel, Germany). The study groups and techniques were described previously.2,3 The cHSP60 assay (Medac, Wedel, Germany) was performed according to the manufacturer’s instructions.
Results are shown in figure 1. C trachomatis IgG positivity was previously determined to be 19% for group 1, 40% for group 2, and 64% for group 3, showing the expected clear difference in IgG seroprevalence between women with and without procedure confirmed tubal pathology, while an intermediate prevalence observed in pregnant women. The same pattern but with lesser incidence was observed in the anti-cHSP60 responses being 4.8%, 16%, and 27%, for groups 1–3, respectively (χ2 test for trend: χ2 = 3.1, p = 0.079, group 1 v group 3: p = 0.096, OR 10.6). The incidences of anti-cHSP60 were increased in the CT IgG positive subgroups to 25%, 35%, and 43%, for groups 1–3, respectively (see lower panel in fig 1), while only 3.8% anti-cHSP60 titres were observed in the C trachomatis IgG negative subgroups, all in subgroup 2 (unknown tubal status, proved fertility). This indicates that the concordance between CT IgG and cHSP60 positivity is high, almost 90%; however, clearly a different subgroup of women is identified by the cHSP60 assay since only 40% of the C trachomatis IgG positive women has a cHSP60 response (measurement of agreement: kappa 0.371). Finally, the median cHSP60 titres increased from groups 1–3: 50, 100, and 200, respectively, suggesting an association between the level of cHSP60 response and tubal pathology.
As far as we know this is the first study evaluating the commercially available cHSP60 assay in women with different degrees of tubal pathology. Two abstracts were published in the ISSTDR meeting Vienna, Austria in 20024,5 on cHSP60 antibodies in women with pelvic inflammatory disease (85% in patients with C trachomatis positive swabs and patients with occluded tubes, 20% in blood donors) and in women with open or occluded fallopian tubes (31% and 70% respectively).
The standardisation provided through this new commercially available assay will potentially enhance the comparability of cHSP60 results between laboratories. The results presented here, although obtained in small but well defined groups, look suggestively promising. Indeed, power calculations (alpha = 0.5, beta = 0.1) show that doubling (1.7 times) the size of the (sub)groups would results in significant p values instead of clear trends. However, further studies are needed in larger groups with different degrees of pathology because of C trachomatis infections to further determine the diagnostic, prognostic, and clinical relevance of this new assay.
CJB, drafting of the manuscript, involved in the initial collection of the cohort, collection of the clinical data, and laboratory serology analyses for IgG C trachomatis, corresponding author; JS, C trachomatis heat shock protein 60 serology, data management, critically reading the manuscript; PMO, providing the setting for and supervision of all serology assays performed to determine C trachomatis IgG presence, critically reading the manuscript; JBT, supervision of the data collection, critically reading the manuscript; PJD, providing setting and logistics for cohort collection, supervision of the clinical data collection, critically reading the manuscript; ASP, providing the setting for JS to perform the C trachomatis work, critically reading the manuscript; SAM, principal investigator for this manuscript and the Chlamydia trachomatis research line, drafting of the manuscript, data analyses, and overall supervision.
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