WO2013007985A1

WO2013007985A1 - Method

Info

Publication number: WO2013007985A1
Application number: PCT/GB2012/051572
Authority: WO
Inventors: Myra O. MCCLURE; Patrick J. Horner
Original assignee: University of Bristol; Imperial Innovations Ltd
Current assignee: University of Bristol; Ip2ipo Innovations Ltd
Priority date: 2011-07-08
Filing date: 2012-07-05
Publication date: 2013-01-17
Anticipated expiration: 2014-01-08
Also published as: GB201111673D0

Abstract

The present invention relates to a method for determining whether an individual has, or is at increased risk of, a chronic sequela as a result of Chlamydia trachomatis infection. The method comprises detecting Pgp3 or anti-Pgp 3 antibodies in a sample from the individual. The presence of Pgp3 or anti-Pgp 3 antibodies in the sample indicates that the individual has, or is at increased risk of, chronic sequelae as a result of Chlamydia trachomatis infection.

Description

Method

The present invention relates to a method for determining whether an individual is at risk of chronic sequelae as a result of Chlamydia trachomatis infection. Chlamydia trachomatis is one of the most common bacterial sexually transmitted infections in Europe. Rates in sexually active young people are commonly between 5 % and 10 %, with rates decreasing thereafter ^4;5. People with genital chlamydia may experience symptoms of genital tract inflammation including urethritis and cervicitis, but the majority remain asymptomatic. Chlamydia is a significant public health problem because untreated chlamydia may lead to pelvic inflammatory disease (PID). This may result in subfertility, ectopic pregnancy and chronic pelvic pain in women and there is evidence that it may impair fertility in men as well^{4 5}. Overall, 1 1 % of women with PID develop tubal factor infertility and 9% develop ectopic pregnancies⁶. This risk seems to be higher for those with PID caused by infection with C. trachomatis compared with PID caused by other factors, such as infection with Neisseria gonorrhoeae⁷. At least one study using serology has linked Chlamydia with infertility in men^{8 9}.

In addition, Chlamydia can cause a sexually acquired reactive arthritis (SARA) in some individuals^{10 11}. Recent evidence suggests that Chlamydia SARA is underdiagnosed with many patients being diagnosed with undifferentiated oligoarthritis^12"14. Undifferentiated oligoarthritis has a similar clinical presentation to Chlamydia reactive arthritis and often these individuals have not been tested for Chlamydia at presentation¹⁴. In patients with SARA, there is evidence that this is due to persistent infection in the joints which cannot be detected by testing for Chlamydia at the lower genital tract^{13 14}. It is possible that new therapeutic agents may become available for SARA secondary to Chlamydia in the future. Thus, being able to identify individuals at increased risk of this condition using a simple blood test will be of clinical value.

There is evidence that many individuals infected with C. trachomatis become micro- organism-negative without treatment, probably because of an effective immune response. Approximately 50% of Chlamydia NAAT-positive women are negative after 1 yr and 90% after 3 yrs ^28;29. However, the immune response may also result in tissue damage and it is uncertain which antigens, if any, may be involved in such a deleterious immune response. Currently, there are no biomarkers available for identifying individuals at increased risk of chronic sequelae following Chlamydia infection. Although Chlamydia antibody titre (CAT) has been associated with an increased risk of tubal disease in women with infertility, this was using either the Micro-immunofluorescent (MIF) technique or whole immunofluoresence test (WIF)^{9 15}. Sensitivity and specificity of these tests are poor for detecting infertile women with tubal disease ~75%¹⁶. Both tests use whole organisms which are polyantigenic. Detection of C. trachomatis antibodies in patients is complicated by cross-reaction to other chlamydial species, particularly C. pneumoniae which is a common respiratory pathogen ^7;18. C.

pneumoniae antibody prevalence increases from a few per cent in pre-school children to 20- 30% in teenagers and 40-60% in adults ⁹. Both C. pneumoniae and C trachomatis express the genus-specific lipopolysaccharide (LPS), as well as sharing many genes with which there is considerable homology i.e. more than 50% amino acid between proteins encoded from these genes²⁰. There is variation in the literature as to what dilution titre is considered indicative of infection or tubal damage^{16 21 "24}. The Microimmunofluorescence (MIF) assay used to be considered the 'gold standard' for serodiagnosis of C. trachomatis²⁵. However, the procedure is subjective, not designed for high-throughput and its specificity compromised by cross-reactivity with other chlamydial species ^25;26. With the WIF assay, infected cultures, usually of the LGV strain, established on microscope slides are fixed and used as the antigen in a similar fashion to MIF to detect Chlamydia antibodies. The same

disadvantages of the MIF test hold true for the WIF assay which detects antibodies to proteins expressed in whole chlamydial inclusions in infected cells ^25;26.

As a result of these disadvantage, the authoritative website Chlamydiae.com concluded in 2003²⁷ that: "There is inadequate scientific justification for making serious clinical decisions about patient management on the basis of chlamydial serology. New generation

recombinant or peptide-based tests for antibody to C. trachomatis are likely to be more specific than tests based on whole chlamydial antigens. Where other diagnostic methods are lacking or it is not possible to obtain relevant samples, the new serology tests with their high negative predictive value may be of use for identifying patients in whom it is unlikely that C. trachomatis infection is playing a role." Thus, the perception in the art is that the lack of specificity of conventional serological tests for C. trachomatis infection, such as the MIF and WIF assays, means that they are simply not specific enough and that recombinant or peptide-based tests are preferred. According to the present invention, there is provided a method for determining whether an individual has, or is at increased risk of, a chronic sequela as a result of Chlamydia trachomatis infection, comprising:

detecting Pgp3 or anti-Pgp 3 antibodies in a sample from the individual,

wherein presence of Pgp3 or anti-Pgp 3 antibodies in the sample indicates that the individual has, or is at increased risk of, chronic sequela as a result of Chlamydia trachomatis infection.

The inventors have, surprisingly, found that Pgp3 is a marker for chronic sequelae as a result of Chlamydia trachomatis infection. They have also found that the greater the level of Pgp3 or anti-Pgp 3 antibodies detected, the greater the risk of chronic sequelae as a result of Chlamydia trachomatis infection. The present invention provides the first evidence supporting the use of Pgp3 as a marker for chronic inflammatory damage in humans.

Although previous work indicates that the antibody titre of polyclonal antigen assays such as the WIF and MIF are associated with risk of tubal disease, it is not known which antigens are involved. Moreover, these assays are considered to be unreliable because of the

reproducibility problems and potential cross-reactivity with C. pneumoniae ²⁷ , described above.

As discussed above, many individuals infected with C. trachomatis become micro-organism- negative without treatment. Detection of anti-Pgp3 antibodies in a sample provides an indication that the patient has or has had Chlamydia trachomatis infection. The inventors have found that, as the anti-Pgp 3 antibody titre increases, the risk of an individual having chronic sequelae as a result of Chlamydia trachomatis infection increases, regardless of whether there is an existing Chlamydia trachomatis infection. This means that the present invention can be used to diagnose chronic sequelae as a result of Chlamydia trachomatis infection, even if there is no existing infection. This is particularly important where the sequela is fallopian tubal damage in women which may result in ectopic pregnancy and/or tubal factor infertility or subfertility. Thus, the present invention can provide an indication of, for example, whether a woman may be at risk of fertility problems owing to a prior Chlamydia trachomatis infection. "Increased risk" means risk increased relative to a person who has not been infected with Chlamydia trachomatis.

In addition, the present invention allows Chlamydia trachomatis infection to be ruled in, or out, as being the cause of symptoms in an individual. As a result, treatment of the symptoms can be tailored to the causes identified. For example, a patient may present with symptoms of arthritis and the present invention can determine whether the symptoms are potentially caused by SARA or not, and an appropriate treatment can be prescribed. Thus, the present invention can be used to screen for individuals at increased risk of chronic sequelae as a result of Chlamydia trachomatis infection, and for the diagnostic management of individuals under investigation for chronic sequelae as a result of Chlamydia trachomatis infection, such as subfertility or seronegative undifferentiated arthritis.

Pgp3 is a C. trachomatis-spectf\c antigen, which is encoded on the 7.5kB C. trachomatis plasmid that is not present in C. pneumoniae. The Pgp3 protein, (PubMed Accession number: YP 001569038) is encoded by open reading frame 5 (orf 5) of the Chlamydia plasmid (PubMed Accession number: NC_010029). According to the present invention,

Pgp3 may be detected by an immunoassay, such as an ELISA as described by Wills et al². In such an assay, wells of a 96-well microtitration plate were coated with 20ng/well of Pgp3 protein and blocked. Anti-sera were assayed at a dilution of 1 :100 in blocking buffer and plates developed, and the absorbance read spectrophotometrically at 450nm. Samples were considered to be positive if the absorbance at 450nm was greater than or equal to 0.473.

The cut-off value of ≥0.473 (absorbance at 450nm) was selected for the pgp3 ELISA in order to give a specificity of≥96% with 740 C. trachomatis -negative paediatric sera ². Receiver operating characteristic (ROC) analysis was also undertaken using these 740 negative sera and those from 356 C. trachomatis -positive patients ². Wills et al. compared the Pgp3 ELISA against three commercial immunogenic major outer membrane protein (MOMP) peptide ELISA assays ². Sensitivities and specificities were determined using sera from 356 high risk patients, attending departments of genitourinary medicine (GUM), in whom C.

trachomatis had been detected at least one month prior to the sample being tested and from 722 chlamydia-negative children aged 2-13 years. This ELISA was significantly more sensitive than the best performing MOMP peptide ELISA (57.9% vs 49.2% P=0.003). This is consistent with the findings of Wang et al. who observed that Pgp3 was the protein against which anti-chlamydial antibodies were most likely to be produced in people infected with C. trachomatis ³. The Pgp3 assay is also as sensitive as MIF assay. In the study by Wills et al. ², the sensitivity and specificity of the LPS-depleted MIF assay which was used in the study of tubal factor infertility by Land et al ⁴⁷ was investigated. The sensitivity was similar in women (see Tables 2 and 3 in Wills et al. ²). The study also demonstrated that the MIF assay with LPS depletion is specific for C. trachomatis (see Table 4 in Wills et al. ²).

Chronic sequelae as a result of Chlamydia trachomatis infection include chronic inflammatory damage secondary to Chlamydia trachomatis infection. Such damage may include pelvic inflammatory disease, fallopian tubal damage in women, such as adhesions which may result in ectopic pregnancy and/or tubal factor infertility or subfertility. Adhesions persisting at follow-up are a marker for persistent tubal damage which did not resolve following effective treatment of the infection. These adhesions obstruct the passage of the sperm up the fallopian tube to fertilise the ovum and the passage of the fertilised egg down the fallopian tube prior to implantation in the uterus. Complete obstruction results in tubal factor infertility and partial obstruction may result in subfertility and/or ectopic pregnancy. In addition, sequelae include infertility in men and sero-negative undifferentiated arthritis due to chlamydia in men and women. Diagnostic assays for Pgp3 polypeptides typically involve incubating a sample in the presence of a detectably labelled antibody capable of identifying a Pgp3 polypeptide and detecting the antibody by any suitable immunoassay, including without limitation, competitive and non-competitive assay systems using techniques, such as Western blots,

radioimmunoassays, ELISAs (enzyme linked immunosorbent assays), "sandwich" immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays and protein A immunoassays.

Equally, anti-Pgp3 antibodies can be detected in similar assays, using recombinant Pgp3 antigen to capture such antibodies and then detect antibodies binding to recombinant Pgp3 in a manner known to those skilled in the art. Such assays may be carried out in the conventional way using a laboratory analyzer or with point-of-care or home testing device, such as the lateral flow immunoassay, as described in EP291 194.

In one embodiment, anti-Pgp3 antibodies, or antibodies used to detect anti-Pgp3 antibodies, can be detectably labelled by linking the antibodies to an enzyme and used in an enzyme immunoassay (EIA). This enzyme, in turn, when later exposed to an appropriate substrate, will react with the substrate in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorometric or by visual means. Enzymes which can be used to label detectably the antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha- glycerophosphate dehydrogenase, those phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta- galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. The detection can be accomplished by colorimetric methods which employ a chromogenic substrate for the enzyme. Detection may also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards.

Detection may be accomplished using any of a variety of other immunoassays. For example, by radioactivity labelling the antibodies, it is possible to detect Pgp3 through the use of a radioimmunoassay (RIA) - see Laboratory Techniques and Biochemistry in Molecular Biology by Work, T. S. et al., North Holland Publishing Company, NY (1978) with particular reference to the chapter entitled "An Introduction to Radioimmune Assay and Related Techniques" by Chard, T. The radioactive isotope can be detected by such means as the use of a gamma counter or a scintillation counter or by autoradiography. It is also possible to label an antibody with a fluorescent compound. When the fluorescently labelled antibody is exposed to light of the proper wavelength, its presence can then be detected due to fluorescence. Among the most commonly used fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o- phthaldehyde and fluorescamine. The antibody can also be detectably labelled using fluorescence emitting metals such as ⁵²EU, or others of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as

diethylenetriamine pentaacetic acid (EDTA). The antibody also can be detectably labelled by coupling it silver or gold particles or to a chemiluminescent compound. The presence of the chemiluminescent-tagged antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of particularly useful chemiluminescent labelling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester. Likewise, a bioluminescent compound may be used to label the antibody of the present invention. Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Important bio luminescent compounds for purposes of labelling are luciferin, luciferase and aequorin. Antibody detection can be enhanced by modifying the antigen, for example by linking it to silver or gold particles. An antibody molecule may also be adapted for use in a immunometric assay, also known as a "two-site" or "sandwich" assay. In a typical immunometric assay, a quantity of unlabelled antibody (or fragment of antibody) is bound to a solid support or carrier and a quantity of detectably labelled soluble antibody is added to permit detection and/or quantitation of the ternary complex formed between solid-phase antibody, antigen, and labelled antibody. Typical immunometric assays include "forward" assays in which the antibody bound to the solid phase is first contacted with the sample being tested to extract the antigen from the sample by formation of a binary solid phase antibody-antigen complex. After a suitable incubation period, the solid support or carrier is washed to remove the residue of the fluid sample, including unreacted antigen, if any, and then contacted with the solution containing an unknown quantity of labelled antibody (which functions as a "reporter molecule"). After a second incubation period to permit the labelled antibody to complex with the antigen bound to the solid support or carrier through the unlabeled antibody, the solid support or carrier is washed a second time to remove the unreacted labelled antibody. In another type of "sandwich" assay, the so-called "simultaneous" and "reverse" assays are used. A simultaneous assay involves a single incubation step as the antibody bound to the solid support or carrier and labelled antibody are both added to the sample being tested at the same time. After the incubation is completed, the solid support or carrier is washed to remove the residue of fluid sample and uncomplexed labelled antibody. The presence of labelled antibody associated with the solid support or carrier is then determined as it would be in a conventional "forward" sandwich assay. In the "reverse" assay, stepwise addition first of a solution of labelled antibody to the fluid sample followed by the addition of unlabeled antibody bound to a solid support or carrier after a suitable incubation period is utilized. After a second incubation, the solid phase is washed in conventional fashion to free it of the residue of the sample being tested and the solution of unreacted labelled antibody. The determination of labelled antibody associated with a solid support or carrier is then determined as in the "simultaneous" and "forward" assays.

As mentioned, an assay may be used to identify the presence of an antibody that is immunologically reactive with Pgp3. Briefly, a biological sample may be obtained from a subject. The sample may include whole blood, serum, plasma, urine, saliva, interstitial fluid and genital tract secretions, such as cervical mucus, vaginal fluid and semen. The sample may then be incubated with a solid support containing bound recombinant Pgp3. Finally, the antibody-antigen complex may be detected by conventional means, including the means described above. A suitable immunoassay for detecting anti-Pgp3 antibodies is described in Wills et al ².

The term "solid support" is intended to include any support or carrier capable of binding Pgp3 antigen. Well-known supports or carriers, include glass, polystyrene, polypropylene, polyethylene, dextran, nylon amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite. The nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention. The support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to an antigen. Thus, the support or carrier configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod.

Alternatively, the surface may be flat such as a sheet, test strip, etc. Preferred supports or carriers include polystyrene beads. Those skilled in the art will know many other suitable carriers for binding antigen, or will be able to ascertain the same by use of routine experimentation. Suitable methods and reagents for detecting an antibody-antigen complex in assays of the present invention are commercially available or known in the relevant art.

The method of the present invention may further comprise detection of one or more further markers of chronic sequelae as a result of Chlamydia trachomatis infection. Such markers include chlamydia heat shock proteins, such as hsp60, and the immunogenic major outer membrane protein (MOMP)

Previously, immune response to the chlamydia hsp60 has been linked to the development of chronic sequelae in animal models ^{25;26;3t 37}. Heat shock proteins are molecular chaperones and are among the most highly-conserved proteins in phylogeny with respect to structure and function ³⁸. Chlamydia hsp60 has been demonstrated to elicit a delayed type

hypersensitivity reaction. It can also directly activate endothelial cells, smooth muscle cells and macrophages to produce proinflammatory cytokines and as it has -50% homology with human hsp60 which could lead to break down in self tolerance (see below) ^38~40. A variety of Chlamydia hsp60 serology ELISAs, using either whole recombinant or purified antigen or recombinant peptides, has been developed in order to study the association with disease in humans including at least one which is commercially-available assay ^{3 ;33;41 "44}. n remains unclear how important chlamydial hsp60 is in disease pathogenesis in humans ^{3840 45 46} . The immunogenic major outer membrane protein (MOMP) makes up 60% of the total outer membrane protein of Chlamydia and is the basis of several commercially-produced ELISAs which use C. trachomatis specific MOMP peptides. Land et al. showed an association with tubal disease but this association was not as strong as that observed with an LPS depleted MIF assay⁴⁷. No details of MOMP peptide antibody ELISA absorbance were provided.

The invention also provides the use of PgP3 as a marker for chronic sequelae as a result of Chlamydia trachomatis infection. In addition, the present invention provides a kit comprising means for detecting two or more markers of chronic sequelae as a result of Chlamydia trachomatis infection, wherein one of the two or more markers is Pgp3. Further markers may be chlamydia hsp60 and MOMP.

Preferred features of each aspect are as for each other aspect mutatis mutandis. Example

The present invention will be illustrated further with reference to the following non-limiting example.

The association of the presence of antibodies against Pgp3, MOMP and hsp60 with adhesions was studied in 35 women with pelvic inflammatory disease confirmed on laparoscopy, who had a subsequent follow-up with a laparoscopic examination. Adhesions, persisting at follow-up, are a marker for persistent tubal damage, which did not resolve following effective treatment of the infection. These adhesions obstruct the passage of the sperm up the fallopian tube to fertilise the ovum and the passage of the fertilised egg down the fallopian tube prior to implantation in the uterus. Complete obstruction results in tubal factor infertility and partial obstruction may result in subfertility and/or ectopic pregnancy. Thirty five women presenting with pelvic pain and diagnosed with pelvic inflammatory disease were laparoscoped prior to treatment and following completion of treatment. It was found that the presence of adhesions at follow-up was weakly associated with antibody to Pgp 3; p=0.087. Thus, Chlamydia exposure was weakly associated with presence of tubal disease. This is most likely because some of the women would have been exposed previously to Chlamydia which had not resulted in tubal disease, thus weakening the association. The strongest association, however, was with increasing antibody titre as measured by absorbance (see Table). A similar picture was also observed with antibody to the major outer membrane protein (MOMP) using the SeroCT MOMP peptide ELISA and chlamydia heat shock protein 60 (hsp60) but the associations were weaker. Adhesions Adhesions

(Absorbance)

Yes No P (RR 95% CI) Yes No P

Pgp3 antibody 1 1 8 0.087 (2.3; 0.9- 1.5 0.67 0.006

+ve (58%) 5.9)

Pgp3 antibody 4 (25%) 12

-ve

SeroCT antibody 8 (32%) 5 0.16 (1 .9; 0.9-4.1 ) 1 .22 0.48 0.018

+ve

SeroCT antibody 7 (37%) 15

-ve

Hsp60 antibody 8(50%) 8 0.51 (1 .4; 0.6-2.9) 0.55 0.16 0.095

+ve

Hsp60 antibody 7 (37%) 12

-ve

Table 1 shows association of Chlamydia antibody using three ELISAs either as positive negative or using absorbance with persistence of adhesions following pelvic inflammatory disease.

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Claims

1 . A method for determining whether an individual has, or is at increased risk of, a chronic sequela as a result of Chlamydia trachomatis infection, comprising:

detecting Pgp3 or anti-Pgp 3 antibodies in a sample from the individual,

wherein presence of Pgp3 or anti-Pgp 3 antibodies in the sample indicates that the individual has, or is at increased risk of, chronic sequelae as a result of Chlamydia trachomatis infection.

2. A method as claimed in claim 1 , wherein the chronic sequela as a result of Chlamydia trachomatis infection is chronic inflammatory damage secondary to Chlamydia trachomatis infection, infertility or subfertility in men or women, ectopic pregnancy or sero-negative undifferentiated arthritis.

3. A method as claimed in claim 2, wherein Pgp3 or anti-Pgp 3 antibodies are detected by means of an immunoassay.

4. A method as claimed in claim 2, wherein the immunoassay is an ELISA.

5. A method as claimed in any preceding claim, further comprising detecting in the sample one or more further markers that indicate the risk of a chronic sequela as a result of Chlamydia trachomatis infection.

6. A method as claimed in claim 5, wherein said markers are selected from chlamydia heat shock protein60 (hsp60), and the immunogenic major outer membrane protein (MOMP).