Chlamydia trachomatis is a leading cause of sexually transmitted infection (STI) in the United States, affecting more than one million women in 2008 alone.1 C trachomatis infection is often asymptomatic and, thus, undiagnosed and untreated. Untreated, C trachomatis infection may lead to ascending infection, causing complications such as ectopic pregnancy and tubal factor infertility.1,2 However, not all women infected with C trachomatis develop tubal damage. Women with normal fallopian tubes on laparoscopy or with normal fertility can have high titers of antibodies to C trachomatis.3 It is unclear what determines whether a woman exposed to C trachomatis will have development of tubal pathology. Host immune responses to C trachomatis infection are thought to contribute significantly to tubal damage.
Extensive efforts have been made to identify biomarkers that correlate with tubal factor infertility. Previous studies have revealed a strong association of anti-C trachomatis and anti-HSP60 antibodies with tubal factor infertility.3 – 9 Using a high-resolution whole genome protein array, we previously compared the profiles of anti-C trachomatis antibodies between women with tubal factor infertility and women with infertility attributable to nontubal causes and confirmed the association of HSP60 with tubal factor infertility and identified new antigens associated with tubal factor infertility.5 However, these and other studies focused on well-defined study groups for comparison. When more diverse patient populations were included, the anti-C trachomatis and HSP60 antibodies were no longer able to differentiate women with tubal factor infertility from those with normal fertility and acute C trachomatis infection.5 The goal of this study was to use a whole genome proteome array to define distinct panels of C trachomatis antigens for predicting tubal pathology and acute infection.
MATERIALS AND METHODS
All participants were recruited at the University of Texas Health Science Center at San Antonio with Institutional Review Board approval. All participants underwent a single blood draw. Serum samples were stored at −20°C until analyzed. The tubal factor infertility group (n=24) were recruited from a university-based infertility clinic. Inclusion criteria were women at least 21 years of age with a pelvic laparoscopy demonstrating hydrosalpinx, fimbrial phimosis, or peritubal adhesions. Exclusion criteria included previous tubal ligation, surgical finding of endometriosis, or a history of pelvic infection or inflammation other than pelvic inflammatory disease, such as appendicitis. The fertile control group (n=25) was recruited from a university-affiliated county hospital. Fertile control group participants were at least 21 years of age and had at least one live birth and normal pelvic findings at the time of tubal ligation, performed using either laparoscopy or mini-laparotomy. The STI group participants (n=24) were referred by the health department to a university-based clinic after diagnosis of C trachomatis based on positive C trachomatis nucleic acid detection.10,11 The STI group participants were of reproductive age (15–45 years of age). Phlebotomy was performed within 2 weeks of the diagnosis. Clinical characteristics of the three study groups are summarized in Table 1.
C trachomatis serovar D or Chlamydia pneumoniae AR39 organisms used in the current study were grown, purified, and titrated as described previously.3,5,12,13 Antichlamydial organism antibodies in human sera were titrated using an immunofluorescence assay.3,5 HeLa cells infected with C trachomatis or C pneumoniae organisms were used as antigens for titrating human serum samples. The human antibody binding was visualized with a goat anti-human immunoglobulin (Ig) G conjugated with Cy3 and the fluorescence labeling was observed and photographed with an Olympus AX70 fluorescence microscope equipped with multiple filter sets, as previously described.14,15 The highest dilution of a serum that still gave a positive reactivity was defined as the titer of the given serum sample.
A glutathione S-transferase fusion protein microplate enzyme-linked immunosorbent assay-based proteome array was used to detect human antibody recognition of chlamydial proteins as previously described.10,11 Individual chlamydial glutathione S-transferase fusion proteins bound to 96-well microplates precoated with glutathione were used to react with human serum samples preabsorbed with a bacterial lysate containing glutathione S-transferase alone. The human antibody reactivity was detected with a goat anti-human IgG, IgA, and IgM conjugated with horseradish peroxidase, and the optical density was measured at 405 nm using a microplate reader. To confirm the antibody binding specificity, all human sera were further absorbed with lysates made from either HeLa cells alone or C trachomatis serovar D-infected HeLa cells before reacting with the fusion protein-coated plates. The antibody binding that remained positive after HeLa-alone lysate absorption but significantly reduced by Chlamydia−HeLa lysate absorption was considered true positive.
Power calculations were based on the hypothesis that the sensitivity and specificity are both more than 0.5. If the true value of the sensitivity and specificity are both 0.8, and if the significance level is 5%, then this study will attain a power of 80% with 24 individuals per group.
Data were analyzed using Microsoft Excel 2007 and R 2.14.1. Kruskal-Wallis was used to compare anti-C trachomatis and anti-C pneumoniae antibody titers between all three groups of women. Pair-wise contrasts were compared using Kruskal-Wallis test with Dunn-Šidák correction for multiple testing. Results from enzyme-linked immunosorbent assay were analyzed using both Kruskal-Wallis (for comparing quantitative optical density value data) and Fisher exact test (for comparing the number of sera positively reacted with a given antigen). Dunn-Šidák correction was used to account for multiple testing. Combination of these two methods allowed us to identify C trachomatis antigens that are both clinically and statistically significant. When Kruskal-Wallis was used, the optical density values (after subtracting background from the same plate) were used. When the Fisher exact test was used, positive reactivity frequency was used. A reaction was determined positive when the optical density value was 2 standard deviations above the mean calculated from the same 96-well plate.5
Women in the subject group were recruited randomly from three distinct socioeconomic and demographic groups from January 1, 2006 to June 1, 2011. The tubal factor infertility group participants (n=24) were recruited from a university-based infertility clinic. The fertile control group participants (n=25) were recruited from a university-affiliated county hospital. The STI group participants (n=24) were referred by the health department to a university-based clinic after diagnosis of C trachomatis. Clinical characteristics of the study subject group participants are shown in Table 1.
Serially diluted serum samples from women with tubal factor infertility, fertile control, and STI were reacted with C trachomatis-infected and C pneumoniae-infected HeLa cells to measure the titers of the corresponding antibodies. The STI group women displayed the lowest levels of anti-C pneumoniae antibodies (7,785±6,296) compared with tubal factor infertility (23,000±23,084; P=.04) and fertile control group participants (17,136±19,709; P=.53), whereas tubal factor infertility group and fertile control group participants displayed similar levels of the antibodies (P=.25). The levels of anti-C pneumoniae antibodies correlated well with age among the three groups of women. STI women were significantly younger (mean age 21.8±3.1 years) than both tubal factor infertility group participants (34.6±4.4; P<.001) and fertile control group participants (32.5±5.7; P<.001), respectively, whereas the tubal factor infertility and fertile control group women had similar ages (P=.20). Thus, anti-C pneumoniae antibody levels increase with age. However, as shown in the bottom portion of Table 1, there was no significant difference in anti-C trachomatis antibody titers between the groups (P=.06). Titers were highest in STI group participants (65,500±70,078) compared with those in either the tubal factor infertility group (35,483±39,950; P=.52) or the fertile control group (23,760±23,974; P=.17). There was no significant difference between tubal factor infertility and fertile control groups (P=.53). In contrast to the age-dependent increase in anti-C pneumoniae antibody titers, the anti-C trachomatis antibody titers decreased as age progressed. Contrary to previous studies using anti-C trachomatis antibodies to differentiate tubal factor infertility from infertility of nontubal causes,3,5 we found that the overall anti-C trachomatis antibodies were not able to differentiate tubal factor infertility patients from the fertile control group or STI group. We next used a whole genome protein array to analyze the human serum samples to identify antibodies able to differentiate these groups of patients.
When antibodies from 24 tubal factor infertility, 25 fertile control, and 24 STI participants were reacted with 908 C trachomatis proteins (Fig. 1), the 73 patient sera recognized C trachomatis antigens distributed across the entire genome (Fig. 1A), with 541 antigens recognized by at least one serum and 367 antigens not detected by any serum (Fig. 1B). Many C trachomatis antigens were recognized in all three groups of women, although some antigens were preferentially recognized by individual or combinations of groups (Fig. 1C). As shown in Figure 1D, regardless of which groups the women were in, 50% or more of the 73 patient sera recognized a total of 13 antigens, including pCT03 (Pgp3, a plasmid-encoded hypothetical protein that is secreted into host cell cytosol),16,17 CT858 (CPAF, a chlamydial protease or proteasome-like activity factor known to be secreted into host cell cytosol),18 CT101 (hypothetical protein), CT841 (FtsH, ATP-dependent zinc protease), CT240 (recombination protein RecR), CT443 (outer membrane complex protein B),19 CT142 and CT143 (both hypothetical proteins), CT813 and CT529 (both inclusion membrane proteins),20,21 CT694 (a putative effector of the type III secretion pathway),22 CT022 (50S ribosomal protein L31 type B), and CT806 (insulinase family, protease III). These proteins were designated as immunodominant antigens in these women.
The reactivity of each of the 908 C trachomatis antigens with 73 human sera was compared between the three groups of women both quantitatively (intensity as measured in optical density values) and qualitatively (frequency of recognition). Antigens that displayed statistically significant differences in reactivity (either quantitatively or qualitatively) and were recognized by 10% or more of the sera from at least one group of women were selected. There were 97 antigens that met both these requirements (data not shown). We first focused on antigens that displayed statistically significant differences recognized in tubal factor infertility group women compared with fertile control group women (Table 2). Among the antigens preferentially recognized by tubal factor infertility sera, five failed to react with any sera from fertile control group women under the serum dilution of 1:1,000, suggesting that these antigens can distinguish tubal factor infertility group women from fertile control group women with 100% specificity. When the reactivity patterns of these five antigens in tubal factor infertility group women were revealed (Fig. 2), we found that HSP60 reacted with sera from 9 out of 24 tubal factor infertility group women, with a detection sensitivity of 38%. Antigens CT376 reacted with an additional three tubal factor infertility samples, whereas CT557 reacted with another two. Thus, the tri-antigen panel could detect tubal factor infertility samples with a sensitivity of 58% while still maintaining 100% specificity. The remaining two antigens (CT111 and CT579) failed to react with any additional unique tubal factor infertility sera (data not shown). It is worth noting that CT443, the outer membrane complex protein B, was recognized by tubal factor infertility group women with the highest frequency and intensity (both were significantly higher compared with fertile control group women). We next tested whether a combination of human serum dilution and CT443 fragmentation could help to further differentiate tubal factor infertility group women from fertile control group women. The CT443 full-length proteins were expressed in 12 different fragments, all of which were used to react with both tubal factor infertility group women and fertile control group women sera at different dilutions (Fig. 2 and data not shown). Only the fragments representing the C-terminal portion of CT443, including fragment 9, fragment 11, and fragment 12, were reactive with human sera. At each human serum dilution, the CT443 fragments produced significantly greater differences than the full-length CT443 did in antibody reactivity between tubal factor infertility group samples and fertile control group samples. For example, at 1:1,000 dilution of human sera, the full-length CT443 reacted with 88% of tubal factor infertility group samples and 60% of fertile control group samples (a 32% decrease in reactivity with fertile control group sample antibodies), whereas fragment 11 reacted with 58% of tubal factor infertility group samples and 24% fertile control group samples (59% decrease). When the human serum was used at 1:10,000 dilution, neither CT443 full-length nor fragment 11 reacted with fertile control sera, but both reacted with 38% and 17% of tubal factor infertility sera, respectively. Among the tubal factor infertility sera recognized by CT443, one was unique and not detected by any of the already identified three tubal factor–associated antigens. Thus, a combination of serum sample dilution with the four antigens (HSP60, CT376, CT557, and CT443) could differentiate tubal factor infertility from fertile control groups with a sensitivity of 63% (95% confidence interval [CI] 0.41–0.77) while maintaining 100% specificity (95% CI 0.91–1). Thus, these four antigens were designated as tubal factor infertility antigens. However, these four antigens also strongly reacted with STI serum samples even when the sera were highly diluted (Fig. 2E and F). We next reanalyzed proteome array data to identify C trachomatis antigens that could differentiate STI group women from tubal factor infertility group women. A total of 21 C trachomatis antigens strongly reacted with STI sera but reacted poorly with serum samples of either tubal factor infertility group women or fertile control group women (data not shown) and thus were designated as STI or acute infection-associated antigens. When human serum samples were diluted at 1:10,000, 13 of the 21 STI-associated antigens failed to react with any sera from either tubal factor infertility group women or fertile control group women but maintained positive reactivity with some STI serum samples (Fig. 3). Notably, antigen CT875 reacted with 14 out of the 24 STI serum samples. Antigen CT147 reacted with an additional unique STI sample. A combination of CT875 and CT147 can distinguish women with acute infection from those with tubal factor infertility and normal fertility with a detection sensitivity of 63% (95% CI 0.41–0.77) and a specificity of 100% (95% CI 0.95–1).
The overall goal of the current study was to identify C trachomatis antigens that can be used to differentiate women with tubal factor infertility, normal fertility, and acute infection. The three groups in this study are representative of the female population of reproductive age with different clinical diagnoses and diverse racial and socioeconomic backgrounds. The anti-C pneumoniae antibody levels increased with increasing age in our study population, consistent with observations made previously in other populations.23,24 Furthermore, anti-C trachomatis antibody levels are the highest in the youngest group (STI), in agreement with the consensus that younger patients are more susceptible to C trachomatis infection.2 However, the overall anti-C trachomatis antibody titers failed to differentiate tubal factor infertility group from fertile control group or STI group. Thus, we used a whole genome proteome array to search for C trachomatis antigens that might allow us to differentiate these groups. We have found that a four-antigen panel consisting of HSP60, CT376, CT557, and CT443 in combination with antiserum sample dilution can distinguish tubal factor infertility group from fertile control group with a detection sensitivity of 63% and specificity of 100%. However, this same set of antigens strongly reacted with sera from the STI group. We next identified 21 STI-associated antigens, two of which, CT875 and CT147, distinguished STI group women from other C trachomatis-exposed women with a detection sensitivity of 63% and specificity of 100%. Thus, it is possible to use sequential screening of different panels of C trachomatis antigens to differentiate women with tubal factor infertility, normal fertility, and acute infection. We also have presented evidence that antigen fragmentation and serum dilution can be used to further improve detection sensitivity and specificity.
As illustrated in Figure 4, these findings can be applied clinically not only in infertility clinics but also in general women's health care settings for identifying women with tubal factor infertility, acute infection, and previous exposure to C trachomatis, respectively. In an infertility clinic, the antibody detection-based testing may complement or replace the more expensive invasive approaches such as hysterosalpingogram and laparoscopy for diagnosing tubal factor infertility. Panels of C trachomatis antigens could be developed into a widely available commercial test kit at a price of approximately $10 each. The antibody testing kit also could be used as a screening tool in low-risk patient populations. Individuals with antibodies to tubal factor infertility antigens (HSP60, CT376, CT557, and CT443) but not STI antigens (CT875 and CT147) could be referred for hysterosalpingogram or laparoscopy for further confirmation of tubal factor infertility or for consideration of in vitro fertilization. Individuals with antibodies reactive with both tubal factor infertility and STI antigens could be referred for antibiotics treatment after confirmation with a DNA detection-based diagnosis of vaginal swab samples. The antibody detection-based kit also may provide prognosis for individuals with previous exposure to C trachomatis infection.
Identification of biomarkers associated with tubal pathology not only may provide a safer means for diagnosis or prognosis of C trachomatis infection but also may promote our understanding of the mechanisms of C trachomatis pathogenesis and development of C trachomatis vaccines. It is widely believed that HSP60 or host immune responses to HSP60 may play important roles in tubal pathology.25 – 29 Whether and how the tubal factor infertility-associated CT376, CT557, and CT443 also may contribute to tubal pathology are worthy of further investigation.
Finally, we should be cautious in interpretation and application of the current findings. The antigen panels already identified still need to be confirmed in larger and more diverse populations. Additionally, more careful titration of additional antigens may further improve detection sensitivity while maintaining high specificity.
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