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The role of serology, antibiotic susceptibility testing and serovar determination in genital chlamydial infections
Best Practice & Research Clinical Obstetrics and Gynaecology Vol. 16, No. 6, pp. 801±814, 2002
doi:10.1053/beog.2002.0321, available online at http://www.idealibrary.com on
4 The role of serology, antibiotic susceptibility testing and serovar determination in genital chlamydial infections Kenneth Persson
MD, PhD
Senior consultant Department of Clinical Microbiology, MalmoÈ University Hospital, SE 205 02 MalmoÈ, Sweden
Systemic and local antibodies regularly develop in genital infections caused by Chlamydia trachomatis. Such antibodies cannot be used as a sign of current infection as they often persist for years after the infection has resolved. Chlamydial antibodies have, however, been extremely useful for demonstrating associations between C. trachomatis and clinical conditions such as ectopic pregnancy and tubal factor infertility. In particular, antibodies to the chlamydial heat shock protein 60 predict the presence of tubal scarring. C. trachomatis has been divided into 15 (or 18) serovars, with many genotypes within each serovar. Dierences in pathogenicity between serovars have been reported but no general pattern has emerged. Genotyping is a powerful epidemiological tool but is not yet ready for routine clinical use. C. trachomatis infections can be successfully treated by tetracycline or macrolides. Some resistant strains have been reported, causing treatment failures, and the problem of emerging antibiotic resistance cannot be neglected. Key words: chlamydia; antibodies; serovars.
Chlamydia trachomatis (Ct) causes symptomatic and asymptomatic genital infections in men and women. Manifestations involve urethritis, cervicitis, endometritis, salpingitis with late sequelae such as ectopic pregnancy and infertility in women. In men, urethritis and sometimes epididymitis occur. Chlamydial infection in pregnant women may cause prematurity or small-for-date babies, and the infection can be transmitted to the infant, resulting in inclusion conjunctivitis and pneumonia. Intra-amniotic infection by Ct has been observed. Chlamydial conjunctivitis in adolescents and adults may also occur, indicating concurrent genital chlamydial infection. Detection of current Ct infection is based on demonstration of the organism. Tissue culture methods, direct ¯uorescent antibody tests or enzyme-linked immunosorbent assays have now been largely replaced by nucleic acid ampli®cation tests such as PCR (Roche), LCR (Abbott), SDA (Becton Dickinson) and TMA (Gen-Probe Inc.). c 2002 Elsevier Science Ltd. All rights reserved. 1521±6934/02/$ - see front matter *
802 K. Persson
SEROLOGY IN GENITAL CHLAMYDIAL INFECTIONS Systemic and local antibodies in genital chlamydial infection Both systemic and local antibodies in secretions can be detected in C. trachomatis (Ct) infection. Early studies showed that serum IgG antibodies could be detected in 40±100% of women with genital chlamydial infection1±4 and in 79% of men with chlamydial urethritis5, but 16±87% of chlamydia-negative women used as controls also had such antibodies ± as had 43% of chlamydia-negative males. Serum IgM antibodies were reported in 17±49% among Ct-positive women but also among 3±23% of Ctnegative controls. IgA antibodies in cervical secretions are commonly found in Ct-infected women. Such antibodies have been observed in 57±64% of Ct-infected women but were also detected in 12±47% of Ct-negative controls. All of these studies were based on cell cultures for the detection of Ct infection. This technique has a sensitivity of about 70% or even less compared to nucleic acid ampli®cation tests (NAAT). Recently, serological results from PCR-positive and PCRnegative women were reported.6 Serum IgG antibodies were demonstrated in 56±74% of PCR-positive women compared to 25±29% among negative controls. An inverse relationship between local IgA antibodies and the number of organisms (measured as inclusion-forming units, IFUs) has been demonstrated, suggesting a partial immunity conferred by these antibodies.7 Serum IgG antibodies do not provide any protection for future re-infection but have been shown to prevent ascending infections after legal abortion where curettage had been performed.8 Population-based sero-surveys have shown that antibodies to Ct start to appear between 15 and 20 years of age, peak between 40 and 50 years of age, and start to decline after middle age.9 This is in contrast to the age-speci®c antibody rates of C. pneumoniae where antibodies start to appear after 5 years of age and sharply increase up to 15 years of age after which age antibody prevalence continues to increase for the rest of life, but at a lower rate. Both IgG and IgA antibodies to Ct seem to persist over many years although titres decline10 at least in some situations. Tests for antibodies to C. trachomatis Antibody testing for Ct has traditionally been performed by the microimmuno¯uorescence test (MIF) developed by Wang et al during the 1960s.11 Antigen dots of egg- or cell-culture-grown organisms are placed on a glass slide and then incubated with serum and after washing with an anti-human globulin antibody with a ¯uorescent tag. Dilutions of the serum are then tested and an antibody titre thus determined. The test is laborious, and demands expertise, but has been very useful in delineating antigenic dierences between strains and also for clarifying the antibody response in genital chlamydial infection. In order to standardize the MIF test, and thus get better and comparable results in dierent laboratories, panels of sera have been distributed to laboratories in dierent parts of the world. Testing for antibodies to C. pneumoniae has been the prime objective and the overall agreement has improved in later panels. Similar pro®ciency panels for Ct would be helpful. Cross-reactive antibodies for the dierent species of Chlamydia used in MIF have been a concern. The most important of the common antigens is the lipopolysaccharide (LPS), which is part of the cell wall of all chlamydiae. This antigen has been puri®ed and used in a complement ®xation (CF) test for chlamydial antibodies. The CF test has been widely used to diagnose ornithosis or
Serology, antibiotic susceptibility and serovar determination 803
C. pneumoniae infections but has not been useful for genital chlamydial infections, except for lymphogranuloma venereum (LGV). Antibodies to the LPS can be detected by MIF as well. In some commercially available MIF tests the LPS has been removed from the antigens to reduce cross-reactivity. LPS antibodies appear in high titres mainly during the acute phase of ornithosis or C. pneumoniae infection but disappear later on. High titres of antibodies in the CF test are therefore considered diagnostically signi®cant, suggesting current infection of C. psittaci or C. pneumoniae. In the MIF tests the LPS cross-reactive antibodies might be mistaken for speci®c antibodies to any of the antigens. The removal of LPS is therefore an obvious advantage when speci®c antibodies to the dierent species of chlamydia are of interest. For the diagnosis of current infection of ornithosis or C. pneumoniae infections LPS antibodies may be diagnostically useful. Both the 60-kDa Omp-2 and the hsp60 antigens are conserved proteins with shared epitopes between chlamydial species but are not surface-exposed and should therefore not cause cross-reactivity in the MIF test. A commercial test based on recombinant chlamydial LPS is available. The test is more sensitive than the CF test and antibodies can be detected even after genital chlamydial infection. In our own experience LPS-IgG antibodies were detected (rElisa, Medac, Hamburg) in 62% of culture-positive women and in 81% of culture-positive men, while culture-negative women had such antibodies in 37% and men in 33%. IgM antibodies were detected by this test in culture-positive and -negative women in 22% and 3% respectively and in men in 19% and 3%. IgA antibodies in serum were demonstrated in 60% and 22% in positive and negative women and in 75% and 17% in men. These ®ndings are in general agreement with what has been reported by other groups using other methods as mentioned above. Chlamydiae form inclusions in cell culture. Such infected cell cultures have been established on microscopic slides and then used to detect chlamydial antibodies. Whole inclusion ¯uorescence test (WIF), often with an LGV-2 strain as antigen, has been described for antibody testing in genital Ct infection.12 The test performance usually diers from that of MIF.13 Recombinant MOMP from Ct has been used as antigen to detect antibodies to Ct with a speci®city of 91% and a sensitivity of 80%.14 Recombinant Omp-2 has also been tried as antigen for the detection of Ct antibodies.15 Sensitivity has been high but speci®city lower as Omp-2 is a conserved protein with shared epitopes between the dierent chlamydial species. Recently, commercial tests in the ELISA format based on speci®c peptides have become available. Several of these tests have shown good speci®cities and sensitivities. Although MIF is still considered the gold standard these new tests hold the promise to become useful alternatives to MIF. There are few comparisons between MIF and these ELISA tests. One such recent comparison showed sensitivities between 71 and 85% and speci®cities better than 96% for three dierent ELISA kits.6 IgG antibodies were detected in about 70% of PCRpositive women but also in 25±29% of PCR-negative women. IgA antibodies were found in serum in 13% of PCR-positive and in 6% of the PCR-negative cases. In another study antibodies to Ct were detected in 84% of culture-positive women and in 61% of positive men. Titre changes were observed in a minority of the cases.16 Ct infection is often asymptomatic and may have been present for weeks or months when ®nally detected and treated. Titre changes of IgG or IgM antibodies may therefore not be demonstrated. The antibody response in re-infections is poorly known. The antibody prevalence will vary with the population studied but the data
804 K. Persson
indicate that neither IgG nor IgA antibodies can be used to diagnose current genital infection by Ct or to exclude such an infection. Serology ± a powerful research tool in chlamydial disease Antibody studies have been seminal to demonstrate associations between clinical conditions such as tubal factor infertility17,18 or ectopic pregnancy19,20 and prior infection by Ct. Several studies have reported increased rates of Ct antibodies in these conditions compared to controls, suggesting that previous pelvic in¯ammatory disease (PID) caused by Ct is common in these cases. The involvement of Ct in spontaneous miscarriages has also been studied. Some studies have not detected any association between antibodies to Ct and early miscarriages21,22 but a few reports did observe such an association.23,24 It is possible that Ct antibodies can be an indirect marker for this condition in certain populations. Ct has been detected in endometrial biopsies. It is therefore possible that endometrial Ct infection can cause problems when fertilized eggs are implanted in the uterine cavity after in vitro fertilization (IVF). Again, results are con¯icting. A few studies indicated that Ct antibodies were associated with poor outcome in IVF25±27 although other studies did not detect such an eect.28±30 Hsp60 antibodies have also been studied in IVF. The rate of take-home babies does not seem to be aected by such antibodies in serum. Local antibodies in cervical secretion to hsp60 may, however, predict a poor outcome.31 These observations need further con®rmation. Antibody studies have also been used to explore the role of Ct infection for the development of cervical cancer. In such studies antibodies to human papilloma virus (HPV) of certain types are strongly associated with cancer development, but Ct antibodies were also an independent predictor of cervical cancer. In one study serovar G was more strongly associated with cancer than were other serovars.32 This serovar usually represents less than 5% of the strains circulating in the population. As antibodies to HPV are found in only half of the patients with cervical cancer ± even though HPV DNA can be detected in almost every case ± an independent role for Ct in cancer development has been questioned. In fact, when HPV DNA is introduced in the statistical analysis Ct may no longer be a signi®cant predictor of cancer. Recently it was reported that HPV-infected patients with antibodies to Ct had a doubled risk of cervical cancer compared to those without Ct antibodies.33 Ct might thus be a cofactor for the development of squamous carcinoma of the cervix. An alternative view would suggest that Ct is only a marker of an increased risk for sexually transmitted infections (STIs) in general and thus correlated with an increased risk of acquiring carcinogenic types of HPV. The potential role of Ct in cervical cancer is therefore still uncertain. Antibodies to the 60-kDa heat shock protein of Ct predict tubal scarring For some years the heat shock proteins (hsps) have been particularly incriminated in the development of tubal scarring after Ct infection. There are four groups of hsps based on molecular weight. They are proteins which are found in all living organisms. Thermal or chemical stress induces production of these proteins, which help protein folding or refolding in order to preserve the function of cellular components. Studies in guinea pigs demonstrated that a triton X-100 extract from Ct induced an in¯ammatory response in the conjunctiva of previously immunized animals but not in naõÈ ve controls.34 The extract was later shown to contain the hsp60 of Ct, and recombinant hsp60 was able to induce the same in¯ammatory response as the crude extract.35 Clinical studies
Serology, antibiotic susceptibility and serovar determination 805
disclosed that antibodies to the hsp60 of Ct were more common in women with ectopic pregnancy than in women with PID.36 Later studies have corroborated and extended these ®ndings, and antibodies to chsp60 independently predict tubal factor infertility and ectopic pregnancy even better than MIF antibodies.37±39 Hsp60 seems to have a unique role in the development of tubal scarring and would not just re¯ect the force of infection as MIF antibodies. Bacterial and human hsp60 are quite similar and share epitopes. An autoimmune component has therefore been suggested but has not been veri®ed in animal model systems of autoimmune disease.40 In the animal model systems the hsp60 response was found to be genus-related rather species-speci®c. Antibodies to Chlamydia pneumoniae in humans start to increase during adolescence, usually before exposure to C. trachomatis. A synergistic eect on the hsp60 response to C. trachomatis infection in those with prior exposure to C. pneumoniae could be suspected. Two studies did not ®nd any relation between hsp60 antibody response and C. pneumoniae antibody status.41,42 In one study such an association was observed in a subgroup of women with tubal infertility43 and in yet another study the presence of antibodies to C. pneumoniae was a prerequisite for tubal damage in C. trachomatis infection but the studied groups were small.44 As data are not in agreement the in¯uence of C. pneumoniae on the hsp60 response in C. trachomatis infection is still uncertain. Serology as a clinical tool for the diagnosis of Ct infection In order to be considered as a diagnostic test the speci®city must be high. The MIF test is considered the gold standard but is dicult to perform and the speci®city of the test has been questioned after C. pneumoniae was discovered. Antibodies to the chlamydial LPS could cause cross-reactivity but speci®c antibodies to Ct and C. pneumoniae can usually be distinguished by the test. When LPS has been removed from the antigens, as is the case in some commercial tests, cross-reactivity should be a minor problem. Modern commercial ELISA tests based on peptides from the MOMP of Ct have so far showed high speci®cities. Serum antibodies in current lower genital tract infection by Ct Although IgG antibodies can be detected in serum in 40±100% of infected women, demonstrated by cell culture or NAAT, 16±87% of Ct-negative women also have such antibodies. The situation is similar for serum IgA antibodies but at a lower level. The predictive values therefore become unacceptably low to use IgG or IgA antibodies in serum to diagnose current lower genital tract infection. The shorter half-life of IgA antibodies compared to IgG has suggested that IgA antibodies could re¯ect persistent infection. A similar notion has been proposed for C. pneumoniae. There is no solid ground as yet for the use of IgA antibodies as a marker of persistent or unresolved infection by either C. trachomatis or C. pneumoniae. IgM antibodies may have a better positive predictive value but the sensitivity is too low, which precludes their use for the diagnosis of genital chlamydial infection. IgA antibodies in genital secretions Local antibodies have been detected in cervical secretions of women with Ct infection in 57±64% of the cases but also in 12±47% of Ct-negative women. Again, the predictive values are too low to make such antibodies diagnostically useful.
806 K. Persson
Ct in upper genital tract infection There is a concern that Ct infection in the Fallopian tubes or in the endometrial wall will not be detected in samples from the cervix or urethra. Serology might assist in such situations. One study compared four dierent serological tests in patients with pelvic pain where endometrial biopsies had been performed.45 Fourteen biopsies of the 53 tested were PCR-positive for Ct while only four cervical cultures detected Ct (cervical samples for PCR were not available). The MIF±IgM test had a sensitivity of 79% and a speci®city of 94% while the WIF test had a speci®city and a sensitivity of 100 and 81% respectively. Hsp60 antibodies had a sensitivity of 43% and a speci®city of 100%. The role of serology in these special situations has still to be determined. Ct antibodies in infertility The titre of Ct antibodies seems to correlate with tubal damage, probably re¯ecting the force of infection in tubal infertility.46 Ct antibody testing has been suggested as part of the routine work-up in infertility cases although it seems to have a moderate ability to predict tubal factor infertility in individual cases. Hsp60 antibodies in serum or secretions Antibodies to hsp60 of Ct independently predict tubal scarring in women and an unfavourable course of urethritis in men.47 Hsp60 antibodies in local secretions have also been associated with a decreased rate of take-home babies after IVF. The use of hsp60 antibodies has been limited to research settings but the introduction of commercial tests will change this situation. Sexual assault Although serology is vital for the detection of infection with HIV, HBV and HCV after sexual assault, antibody tests for Ct have no accepted place in this situation. Cell culture for the detection of Ct infection has been recommended but NAATs may soon replace detection by culture. IgM antibodies in neonatal chlamydial infection Neonatal inclusion conjunctivitis has been known for almost a century when typical inclusions in scrapings from the conjunctiva of infants with neonatal non-gonorrhoeal conjunctivitis were described. Similar inclusions were also found in cells from the genital tract of the parents, indicating that this was an STI. During the 1970s it was shown that neonatal chlamydial infection could also lead to pneumonia, often occurring from 3 to 12 weeks after birth. Later on it was reported that IgM antibodies to Ct were regularly found in infant pneumonia.48 SEROVAR DETERMINATION Sero- and genotyping of C. trachomatis During the late 1960s and 1970s, when isolates of C. trachomatis were compared by antisera produced in mice it became clear that dierent serotypes existed.
Serology, antibiotic susceptibility and serovar determination 807
The serotypes were designated A±K, including two that are very closely related, the B and the Ba serotypes. In addition, three types of lymphogranuloma venereum were identi®ed. The distinction between serotypes was possible by polyclonal mouse sera where a new strain was tested with existing sera and an antiserum raised against the new candidate type and used against existing type strains. In this laborious way 15 dierent serovars were identi®ed, and these still form the basis of characterization of C. trachomatis. The serovars were also grouped in two major categories, the B complex, including B, Ba, D and E, and the C complex with serovars A, C, H, I, J and K; an intermediate group is formed by F and G. Later, monoclonal antibodies were developed and further types were suggested; these were named Da, Ia and L2a. When the MOMP gene had been cloned and sequenced by Stephens at al, new methods for typing were developed.49 Ampli®cation of the MOMP gene by the polymerase chain reaction (PCR) formed the basis for further characterization either by restriction enzyme digestion patterns, called restriction fragment length polymorphism (RFLP), or by complete sequencing of the gene product. Sometimes, RLFP is used for primary characterization with sequencing to resolve unusual patterns. For RFLP, primers and restriction enzymes were chosen so that the established serovars would still be the recognized entities. A good agreement between RFLP and serotyping by monoclonal antibodies was demonstrated by Rodriguez et al.50 In a similar way, sequencing of the MOMP gene has been used to characterize isolates of C. trachomatis.51±53 The typing of clinical isolates has disclosed that trachoma is associated with serovars A, B, Ba and C where households often share the same serovar, although dierent serovars can be in circulation in the same village. After population-based treatment trials the pattern may be more complex where dierent serovars can be reintroduced, leading to mixed infections even within households.
Epidemiology of dierent serovars The genital serovars are: D, E, F, G, H, I, J, K and some of the B/Ba strains. In addition, L1±3 are associated with lymphogranuloma venereum. Typing of clinical isolates has shown that D, E and F account for 60±80% of the strains, with E being the most common (Table 1). This pattern is very similar in dierent parts of the world and has been reported from the USA54, Sweden, Holland, Canada and New Zealand. One hundred isolates up to over 1700 strains were typed in these studies. Similar reports have also appeared from other countries in Europe although the number strains has been fewer. A dierent pattern has been reported from Greece where a high prevalence of Da strains was observed. In another study of rectal isolates from gay men, D-strains were the most common, accounting for 53% of the rectal isolates, while only 18% were D serovars of cervical strains.55 There was a signi®cant decline in the proportion of D serovars of rectal infections during the study period, suggesting an epidemiological clustering within a particular group. Sequencing of the MOMP gene has disclosed many dierent genotypes within the framework of the established serovars. Some of these genotypes seem to have been disseminated to dierent parts of the world while others have been detected only locally so far. The genotypes re¯ect a gradual evolutionary change in C. trachomatis but even recombination has been observed in chimaeras.56 Whether such evolutionary changes are in¯uenced by immunological pressure is uncertain. The protection after infection seems to be weak and of short duration. The serovar distribution found
808 K. Persson Table 1. Distribution of serovars of C. trachomatis in 2326 unselected patients at MalmoÈ University Hospital during 1989 and 1990. Infections were characterized as ®rst infections when no previous test had been positive, although the patient may still have had undetected infection. All re-infections had been veri®ed by previous positive tests. Women Serovar B D E F G H I J K Total
1st infection 7 142 476 307 44 43 15 67 86
(0.6%) (12) (40) (26) (4) (4) (1) (6) (7)
1187
Men
Re-infection 1 35 94 76 11 15 4 18 19
(0.4%) (13) (34) (28) (4) (6) (1) (7) (7)
273
1st infection 6 77 288 229 30 25 7 46 61
(0.8%) (10) (37) (30) (4) (3) (1) (6) (8)
769
All
Re-infection 2 14 38 21 7 1 1 7 6
(2%) (14) (39) (22) (7) (1) (1) (7) (6)
97
1st infection 13 219 764 536 74 68 22 113 147
(0.7%) (11) (39) (27) (4) (3) (1) (6) (8)
1956
Re-infection 3 (0.8%) 49 (13) 132 (36) 97 (26) 18 (5) 16 (4) 5 (1) 25 (7) 25 (7) 370
among patients with re-infections is the same as that found among those with their ®rst detected infection (Table 1), also suggesting low serovar-speci®c protection. Pathogenicity of dierent serovars It has been reported that serovar may also in¯uence infectivity as measured by the number of inclusion-forming units (IFUs) in clinical samples. It was found that the number of IFUs was independently related to age, sex, race and serovar where samples with B-complex strains had more IFUs than C-complex strains.57 Similar ®ndings were also reported by Frost et al.58 Women have higher numbers of IFUs than do men, and younger persons shed more organisms than do older ones. Recently, persistent infection in patients was reported to be associated with C-complex strains.59 The important question is whether there is a serovar-speci®c morbidity or whether some serovars are more likely to cause symptoms or speci®c manifestations than others. It was found by Batteiger et al that the frequency of cervicitis and easy bleeding in women with C. trachomatis infection was not related to the infecting serovar; however, it was found that males with a lower number of polymorphonuclear white cells in urethral scrapings were more likely to have the intermediate serovars F or G.60 In another study, the F strains seemed less prone to produce cervicitis than other serovars.61 In San Francisco, an F variant was found only in women with PID and was associated with severe symptoms.62 In yet another study we were not able to detect any serovars that were particularly associated with salpingitis.63 Recently it was reported that symptomatic males were more likely to have variant Da while women with vaginal discharge had an excess rate of serovar K.64 In aggregate, these studies do not disclose a general pattern. It is possible that certain genotypes may be more pathogenic than others ± such as the F-variants mentioned above. It is also possible that pathogenicity is not associated with the MOMP gene that has been studied so far and that other genes may be more important. One such potentially important gene activity was recently reported when a cytotoxin produced by C. trachomatis was described. The dichotomy between the trachoma and the genital strain serovars seems to re¯ect a biological dierence, however.
Serology, antibiotic susceptibility and serovar determination 809
ANTIBIOTIC RESISTANCE OF C. Trachomatis The drugs-of-choice for treatment of infections involving C. trachomatis have been tetracycline or doxycycline and the macrolides such as erythromycin and, recently, azithromycin. These antibiotics have minimal inhibitory concentrations (MIC) of 0.1 to 0.25 mg/ml, which is considered sucient for ecient treatment. The MIC value for rifampicin is also very low but this drug has been reserved for treatment of mycobacterial infections. Despite the lack of cell wall components similar to those which bind penicillin in other bacteria it has been found that this group of antibiotics can arrest the development to mature and infectious organisms of Ct in the cellular inclusions. Ampicillin has thus been used to treat genital chlamydial infections in pregnant women where microbiological eradication can reach 90% or more. The ¯uoroquinolones, such as o¯oxacin, are also clinically eective, with MIC values of 0.5 mg/ml. While treatment ecacy studies have usually reported microbiological eradication in 95% or more of the infected patients, long-term reappearance of infection has been demonstrated in 6±17% or even up to 29% in young persons.65±67 Recurrences of C. trachomatis may be due to treatment failure or re-infection. Serovar determination would suggest both mechanisms. One of the reasons for treatment failure could be development of antibiotic resistance. There are few reports of antibiotic resistance. The technique used to determine antibiotic sensitivity is laborious and cannot be used routinely. An underestimation of the magnitude of the problem is therefore possible. Antibiotic-resistant strains Some information is available. Mourad described two strains of C. trachomatis with MIC values of 1 mg/ml and thus having a relative resistance to erythromycin.68 Resistance to tetracyclines has also been reported.69 Recently, strains which had elevated MIC values for doxycycline, azithromycin and o¯oxacin at concentrations of 44 mg/ml were described from three patients with treatment failures.70 Some years earlier a series of strains isolated from patients with persistent infection after treatment were reported, although the infections were ®nally treated successfully.17 Strains from ®ve patients were resistant to tetracycline at 4 to 48 mg/ml. Resistance to erythromycin was also detected among these strains. It was noted that only a fraction of the inoculated organisms (less than 1%) showed resistance. After passage in a medium containing 8 mg/ml of tetracycline, 100% of the progeny showed resistance. Attempts to passage such fully resistant strains in antibiotic-free medium were unsuccessful in most cases but at least one strain could be serially propagated; this strain was then completely sensitive to tetracycline. These observations have been described as heterotypic resistance. In vitro induction of antibiotic resistance Resistance to quinolone has been produced in vitro by growing C. trachomatis in cell culture in the presence of the antibiotic studied.72 Two resistant strains of an LGV prototype strain were obtained with MIC values 1000-fold higher than for sensitive strains. A mutation was demonstrated in a gyrase gene (gyrA) speci®c to the resistant strain. From the data reported, C. trachomatis seems to be able to develop antibiotic resistance to drugs often used in the treatment of this infection. Although
810 K. Persson
the prevalence of resistant strains seems to be low at present the problem cannot be neglected. When infection persists despite repeated courses of antibiotic treatment resistance may have developed. When other possible explanations for the treatment failure have been ruled out susceptibility testing of the strain is indicated but few laboratories are prepared to perform such testing.
SUMMARY Serology has been very helpful for elucidating associations between Ct and clinical conditions such as tubal factor infertility and ectopic pregnancy. Although antibodies, both systemic and local, often develop after Ct infection, such antibodies are not useful for detecting current infection, although they might be considered in the work-up of female infertility. Typing of Ct strains, either by antibodies or by DNA methods, has been useful for studying the pathogenicity and epidemiology of these organisms. Antibiotic resistance is still uncommon but the situation could change.
Practice points . antibodies develop in response to genital chlamydial infection in most cases . antibodies, systemic or local, are not useful for demonstrating current lower genital tract infection . the demonstration of IgM antibodies is a good diagnostic marker in neonatal chlamydial pneumonia . antibodies to hsp60 predict tubal damage in chlamydial infection . C. trachomatis is divided into 15 (or 18) dierent serovars, from A through L, and can be subdivided into many more genotypes within this framework . the most common serovars are E, D and F, which represent 60±80% of isolates from the genital tract, with few exceptions worldwide . some particularly pathogenic variants of C. trachomatis may occur, but a general pattern suggesting that some serovars are more pathogenic than others has not emerged . by DNA sequencing of the MOMP gene a very powerful epidemiological tool has become available, although it is not yet ready for clinical use . so far, few strains of C. trachomatis are resistant to commonly used antibiotics . quinolone resistance has been induced in vitro
Research agenda . the association between C. trachomatis and cervical cancer as an independent risk factor, or as a cofactor, needs to be explored further . surveillance for new and more pathogenic types of C. trachomatis . clinical adaptation of MOMP gene sequencing for epidemiological purposes . continuous surveillance for signs of development of antibiotic resistance in cases where treatment failure is observed
Serology, antibiotic susceptibility and serovar determination 811
Although a lot of information has been presented during the last few years on the association between Ct and cervical cancer, more corroborating data are needed. The heat shock proteins seem to be important in the development of scarring after Ct disease but the mechanisms are still elusive. REFERENCES 1. Osser S & Persson K. Immune response to genital chlamydial infection and in¯uence of Chlamydia pneumoniae (TWAR) antibodies. European Journal of Clinical Microbiology & Infectious Diseases 1989; 8: 532±535. 2. Richmond SJ, Milne JD, Hilton AL & Caul EO. Antibodies to Chlamydia trachomatis in cervicovaginal secretions: relation to serum antibodies and current chlamydial infection. Sexually Transmitted Diseases 1980; 7: 11±15. * 3. Schachter J, Cles L, Ray R & Hines PA. Failure of serology in diagnosing chlamydial infections of the female genital tract. Journal of Clinical Microbiology 1979; 10: 647±649. 4. Treharne JD, Darougar S, Simmons PD & Thin RN. Rapid diagnosis of chlamydial infection of the cervix. British Journal of Venereal Diseases 1978; 54: 403±408. 5. Terho P & Meurman O. Chlamydial serum IgG, IgA and local IgA antibodies in patients with genitaltract infections measured by solid-phase radioimmunoassay. Journal of Medical Microbiology 1981; 14: 77±87. * 6. Morre SA, Munk C, Persson K et al. Comparison of three commercially available peptide-based immunoglobulin G (IgG) and IgA assays to microimmuno¯uorescence assay for detection of Chlamydia trachomatis antibodies. Journal of Clinical Microbiology 2002; 40: 584±587. 7. Brunham RC, Kuo CC, Cles L & Holmes KK. Correlation of host immune response with quantitative recovery of Chlamydia trachomatis from the human endocervix. Infection and Immunity 1983; 39: 1491±1494. 8. Osser S & Persson K. Postabortal pelvic infection associated with Chlamydia trachomatis and the in¯uence of humoral immunity. American Journal of Obstetrics and Gynecology 1984; 150: 699±703. 9. Saikku P. Diagnosis of acute and chronic Chlamydia pneumoniae infections. In Or®la J, Byrne G, Chernesky M et al (eds) Chlamydial Infections. Proceedings of the Eighth International Symposium on Human Chlamydial Infections, pp 163±172. Bologna: Societa Editrice Esculapio, 1994. 10. Puolakkainen M, Vesterinen E, Purola E et al. Persistence of chlamydial antibodies after pelvic in¯ammatory disease. Journal of Clinical Microbiology 1986; 23: 924±928. *11. Wang S. The microimmuno¯uorescence test for Chlamydia pneumoniae infection: technique and interpretation. Journal of Infectious Diseases 2000; 181 (supplement 3): S421±S425. 12. Richmond SJ. Immuno¯uorescence testing for chlamydial antibodies. Lancet 1979; 2: 1016. 13. Peterson EM, Oda R, Tse P et al. Comparison of a single-antigen microimmuno¯uorescence assay and inclusion ¯uorescent-antibody assay for detecting chlamydial antibodies and correlation of the results with neutralizing ability. Journal of Clinical Microbiology 1989; 27: 350±352. 14. Mygind P, Christiansen G, Persson K & Birkelund S. Detection of Chlamydia trachomatis-speci®c antibodies in human sera by recombinant major outer-membrane protein polyantigens. Journal of Medical Microbiology 2000; 49: 457±465. 15. Mygind P, Christiansen G, Persson K & Birkelund S. Analysis of the humoral immune response to Chlamydia outer membrane protein 2. Clinical and Diagnostic Laboratory Immunology 1998; 5: 313±318. 16. NaÈrvaÈnen A, Poulakkainen M, Hao W et al. Detection of antibodies to Chlamydia trachomatis with peptide-based species-speci®c enzyme immunoassay. Infectious Diseases in Obstetrics and Gynecology 1997; 5: 349±354. 17. Gump D, Gibson M & Ashikaga T. Evidence of prior pelvic in¯ammatory disease and its relationship to Chlamydia trachomatis antibody and intrauterine contraceptive device use in infertile women. American Journal of Obstetrics and Gynecology 1983; 148: 153±159. 18. Moore D, Foy HM, Daling J et al. Increased frequency of serum antibodies to Chlamydia trachomatis in infertility due to distal tubal disease. Lancet 1982; ii: 574±577. 19. Sheeld PA, Moore DE, Voigt LF et al. The association between Chlamydia trachomatis serology and pelvic damage in women with tubal ectopic gestations. Fertility and Sterility 1993; 60: 970±975. 20. Svensson L, MaÊrdh P, Ahlgren M & NordenskjoÈld F. Ectopic pregnancy and antibodies to Chlamydia trachomatis. Fertility and Sterility 1985; 44: 313±317. 21. Coste J, Job-Spira N & Fernandez H. Risk factors for spontaneous abortion: a case-control study in France. Human Reproduction 1991; 6: 1332±1337.
812 K. Persson 22. Osser S & Persson K. Chlamydial antibodies in women who suer miscarriage. British Journal of Obstetrics and Gynaecology 1996; 103: 137±141. 23. Quinn PA, Petric M, Barkin M et al. Prevalence of antibody to Chlamydia trachomatis in spontaneous abortion and infertility. American Journal of Obstetrics and Gynecology 1987; 156: 291±296. 24. Witkin SS & Ledger WJ. Antibodies to Chlamydia trachomatis in sera of women with recurrent spontaneous abortions. American Journal of Obstetrics and Gynecology 1992; 167: 135±139. 25. Licciardi F, Grifo JA, Rosenwaks Z & Witkin SS. Relation between antibodies to Chlamydia trachomatis and spontaneous abortion following in vitro fertilization. Journal of Assisted Reproduction and Genetics 1992; 9: 207±210. 26. Lunenfeld E, Sarov B, Sarov I et al. Chlamydial IgG and IgA in serum and follicular ¯uid among patients undergoing in vitro fertilization. European Journal of Obstetrics, Gynecology, and Reproductive Biology 1990; 37: 163±173. 27. Rowland GF, Forsey T, Moss TR et al. Failure of in vitro fertilization and embryo replacement following infection with Chlamydia trachomatis. Journal of in vitro Fertilization and Embryo Transfer 1985; 2: 151±155. 28. Driscoll GL, Dodd J, Tyler JP et al. Positive chlamydial serology and its eect on factors in¯uencing outcome of IVF treatment. Australian & New Zealand Journal of Obstetrics & Gynaecology 1991; 31: 145±147. 29. Osser S, Persson K, Wramsby H & Liedholm P. Does previous Chlamydia trachomatis infection in¯uence the pregnancy rate of in vitro fertilization and embryo replacement? American Journal of Obstetrics and Gynecology 1990; 162: 40±44. 30. Torode HW, Wheeler PA, Saunders DM et al. The role of chlamydial antibodies in an in vitro fertilization program. Fertility and Sterility 1987; 48: 987±990. 31. Witkin SS, Sultan KM, Neal GS et al. Unsuspected Chlamydia trachomatis infection and in vitro fertilization outcome. American Journal of Obstetrics and Gynecology 1994; 171: 1208±1214. 32. Anttila T, Saikku P, Koskela P et al. Serotypes of Chlamydia trachomatis and risk for development of cervical squamous cell carcinoma. Journal of the American Medical Association 2001; 285: 47±51. 33. Smith JS, Munoz N, Herrero R et al. Evidence for Chlamydia trachomatis as a human papillomavirus cofactor in the etiology of invasive cervical cancer in Brazil and the Philippines. Journal of Infectious Diseases 2002; 185: 324±331. *34. Watkins N, Hadlow W, Moos A & Caldwell H. Ocular delayed hypersensitivity: A pathogenic mechanism of chlamydial conjunctivitis in guinea pigs. Proceedings of the National Academy of Sciences of the USA 1986; 83: 7480±7484. *35. Morrison RP, Belland RJ, Lyng K & Caldwell HD. Chlamydial disease pathogenesis. The 57-kD chlamydial hypersensitivity antigen is a stress response protein. Journal of Experimental Medicine 1989; 170: 1271±1283. *36. Wagar EA, Schachter J, Bavoil P & Stephens RS. Dierential human serologic response to two 60,000 molecular weight Chlamydia trachomatis antigens. Journal of Infectious Diseases 1990; 162: 922±927. 37. Arno JN, Yuan Y, Cleary RE & Morrison RP. Serologic responses of infertile women to the 60-kd chlamydial heat shock protein (hsp60). Fertility and Sterility 1995; 64: 730±735. 38. Dieterle S & Wollenhaupt J. Humoral immune response to the chlamydial heat shock proteins hsp60 and hsp70 in Chlamydia-associated chronic salpingitis with tubal occlusion. Human Reproduction 1996; 11: 1352±1356. 39. Toye B, Laferriere C, Claman P et al. Association between antibody to the chlamydial heat-shock protein and tubal infertility. Journal of Infectious Diseases 1993; 168: 1236±1240. 40. van Eden W, van der Zee R, Alberta G et al. Do heat shock proteins control the balance of T-cell regulation in in¯ammatory disease? Immunology Today 1998; 19: 303±307. 41. Peeling R, Toye B, Claman P et al. Seropositivity to Chlamydia pneumoniae and antibody response to the chlamydial heat shock protein. In Or®la J, Byrne G, Chernesky M et al (eds) Chlamydial Infections. Proceedings of the Eighth International Symposium on Human Chlamydial Infections, pp 502±505. Bologna: Societa Editrice Esculapio, 1994. 42. Persson K, Osser S, Birkelund S et al. Antibodies to Chlamydia trachomatis heat shock proteins in women with tubal factor infertility are associated with prior infection by C. trachomatis but not by C. pneumoniae. Human Reproduction 1999; 14: 1969±1973. 43. Freidank HM, Clad A, Herr AS et al. Immune response to Chlamydia trachomatis heat-shock protein in infertile female patients and in¯uence of Chlamydia pneumoniae antibodies. European Journal of Clinical Microbiology & Infectious Diseases 1995; 14: 1063±1069. 44. Gijsen AP, Land JA, Goossens VJ et al. Chlamydia pneumoniae and screening for tubal factor subfertility. Human Reproduction 2001; 16: 487±491. 45. Chernesky M, Luinstra K, Sellors J et al. Can serology diagnose upper genital tract Chlamydia trachomatis infections? Studies on women with pelvic pain, with or without chlamydial plasmid DNA in endometrial biopsy tissue. Sexually Transmitted Diseases 1998; 25: 14±19.
Serology, antibiotic susceptibility and serovar determination 813 46. Punnonen R, Tehro P, Nikkanen V & Meurman O. Chlamydial serology in infertile women by immuno¯uorescence. Fertility and Sterility 1979; 31: 656±659. 47. Horner PJ, Cain D, McClure M et al. Association of antibodies to Chlamydia trachomatis heat-shock protein 60 kD with chronic nongonococcal urethritis. Clinical Infectious Diseases 1997; 24: 653±660. 48. Schachter J, Grossman M & Azimi PH. Serology of Chlamydia trachomatis in infants. Journal of Infectious Diseases 1982; 146: 530±535. 49. Stephens RS, Mullenbach G, Sanchez-Pescador R & Agabian N. Sequence analysis of the major outer membrane protein gene from Chlamydia trachomatis serovar L2. Journal of Bacteriology 1986; 168: 1277±1282. 50. Rodriguez P, de Barbeyrac B, Persson K et al. Evaluation of molecular typing for epidemiological study of Chlamydia trachomatis genital infections. Journal of Clinical Microbiology 1993; 31: 2238±2240. 51. Dean D & Millman K. Molecular and mutation trends analyses of omp1 alleles for serovar E of Chlamydia trachomatis. Implications for the immunopathogenesis of disease. Journal of Clinical Investigation 1997; 99: 475±483. 52. Jurstrand M, Falk L, Fredlund H et al. Characterization of Chlamydia trachomatis omp1 genotypes among sexually transmitted disease patients in Sweden. Journal of Clinical Microbiology 2001; 39: 3915±3919. 53. Lampe MF, Suchland RJ & Stamm WE. Nucleotide sequence of the variable domains within the major outer membrane protein gene from serovariants of Chlamydia trachomatis. Infection and Immunity 1993; 61: 213±219. 54. Kuo CC, Wang SP, Holmes KK & Grayston JT. Immunotypes of Chlamydia trachomatis isolates in Seattle, Washington. Infection and Immunity 1983; 41: 865±868. 55. Barnes RC, Rompalo AM & Stamm WE. Comparison of Chlamydia trachomatis serovars causing rectal and cervical infections. Journal of Infectious Diseases 1987; 156: 953±958. 56. Hayes LJ, Yearsley P, Treharne JD et al. Evidence for naturally occurring recombination in the gene encoding the major outer membrane protein of lymphogranuloma venereum isolates of Chlamydia trachomatis. Infection and Immunity 1994; 62: 5659±5663. 57. Eckert LO, Suchland RJ, Hawes SE & Stamm WE. Quantitative Chlamydia trachomatis cultures: correlation of chlamydial inclusion-forming units with serovar, age, sex, and race. Journal of Infectious Diseases 2000; 182: 540±544. 58. Frost EH, Deslandes S & Bourgaux-Ramoisy D. Chlamydia trachomatis serovars in 435 urogenital specimens typed by restriction endonuclease analysis of ampli®ed DNA. Journal of Infectious Diseases 1993; 168: 497±501. 59. Dean D, Suchland RJ & Stamm WE. Evidence for long-term cervical persistence of Chlamydia trachomatis by omp1 genotyping. Journal of Infectious Diseases 2000; 182: 909±916. 60. Batteiger BE, Lennington W, Newhall WJ et al. Correlation of infecting serovar and local in¯ammation in genital chlamydial infections. Journal of Infectious Diseases 1989; 160: 332±336. 61. Workowski KA, Stevens CE, Suchland RJ et al. Clinical manifestations of genital infection due to Chlamydia trachomatis in women: dierences related to serovar. Clinical Infectious Diseases 1994; 19: 756±760. *62. Dean D, Oudens E, Bolan G et al. Major outer membrane protein variants of Chlamydia trachomatis are associated with severe upper genital tract infections and histopathology in San Francisco. Journal of Infectious Diseases 1995; 172: 1013±1022. 63. Persson K & Osser S. Lack of evidence of a relationship between genital symptoms, cervicitis and salpingitis and dierent serovars of Chlamydia trachomatis. European Journal of Clinical Microbiology & Infectious Diseases 1993; 12: 195±199. 64. Morre SA, Rozendaal L, van Valkengoed IG et al. Urogenital Chlamydia trachomatis serovars in men and women with a symptomatic or asymptomatic infection: an association with clinical manifestations? Journal of Clinical Microbiology 2000; 38: 2292±2296. 65. Kjaer HO, Dimcevski G, Ho G et al. Recurrence of urogenital Chlamydia trachomatis infection evaluated by mailed samples obtained at home: 24 weeks' prospective follow up study. Sexually Transmitted Infections 2000; 76: 169±172. *66. Whittington WL, Kent C, Kissinger P et al. Determinants of persistent and recurrent Chlamydia trachomatis infection in young women: results of a multicenter cohort study. Sexually Transmitted Diseases 2001; 28: 117±123. 67. Xu F, Schillinger JA, Markowitz LE et al. Repeat Chlamydia trachomatis infection in women: analysis through a surveillance case registry in Washington State, 1993±1998. American Journal of Epidemiology 2000; 152: 1164±1170. 68. Mourad A, Sweet RL, Sugg N & Schachter J. Relative resistance to erythromycin in Chlamydia trachomatis. Antimicrobial Agents and Chemotherapy 1980; 18: 696±698. 69. Lefevre JC, Lepargneur JP, Guion D & Bei S. Tetracycline-resistant Chlamydia trachomatis in Toulouse, France. Pathologie-biologie 1997; 45: 376±378.
814 K. Persson *70. Somani J, Bhullar VB, Workowski KA et al. Multiple drug-resistant Chlamydia trachomatis associated with clinical treatment failure. Journal of Infectious Diseases 2000; 181: 1421±1427. *71. Jones RB, Van der Pol B, Martin DH & Shepard MK. Partial characterization of Chlamydia trachomatis isolates resistant to multiple antibiotics. Journal of Infectious Diseases 1990; 162: 1309±1315. 72. Dessus-Babus S, Bebear CM, Charron A et al. Sequencing of gyrase and topoisomerase IV quinoloneresistance-determining regions of Chlamydia trachomatis and characterization of quinolone-resistant mutants obtained in vitro. Antimicrobial Agents and Chemotherapy 1998; 42: 2474±2481.
doi:10.1053/beog.2002.0321, available online at http://www.idealibrary.com on
4 The role of serology, antibiotic susceptibility testing and serovar determination in genital chlamydial infections Kenneth Persson
MD, PhD
Senior consultant Department of Clinical Microbiology, MalmoÈ University Hospital, SE 205 02 MalmoÈ, Sweden
Systemic and local antibodies regularly develop in genital infections caused by Chlamydia trachomatis. Such antibodies cannot be used as a sign of current infection as they often persist for years after the infection has resolved. Chlamydial antibodies have, however, been extremely useful for demonstrating associations between C. trachomatis and clinical conditions such as ectopic pregnancy and tubal factor infertility. In particular, antibodies to the chlamydial heat shock protein 60 predict the presence of tubal scarring. C. trachomatis has been divided into 15 (or 18) serovars, with many genotypes within each serovar. Dierences in pathogenicity between serovars have been reported but no general pattern has emerged. Genotyping is a powerful epidemiological tool but is not yet ready for routine clinical use. C. trachomatis infections can be successfully treated by tetracycline or macrolides. Some resistant strains have been reported, causing treatment failures, and the problem of emerging antibiotic resistance cannot be neglected. Key words: chlamydia; antibodies; serovars.
Chlamydia trachomatis (Ct) causes symptomatic and asymptomatic genital infections in men and women. Manifestations involve urethritis, cervicitis, endometritis, salpingitis with late sequelae such as ectopic pregnancy and infertility in women. In men, urethritis and sometimes epididymitis occur. Chlamydial infection in pregnant women may cause prematurity or small-for-date babies, and the infection can be transmitted to the infant, resulting in inclusion conjunctivitis and pneumonia. Intra-amniotic infection by Ct has been observed. Chlamydial conjunctivitis in adolescents and adults may also occur, indicating concurrent genital chlamydial infection. Detection of current Ct infection is based on demonstration of the organism. Tissue culture methods, direct ¯uorescent antibody tests or enzyme-linked immunosorbent assays have now been largely replaced by nucleic acid ampli®cation tests such as PCR (Roche), LCR (Abbott), SDA (Becton Dickinson) and TMA (Gen-Probe Inc.). c 2002 Elsevier Science Ltd. All rights reserved. 1521±6934/02/$ - see front matter *
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SEROLOGY IN GENITAL CHLAMYDIAL INFECTIONS Systemic and local antibodies in genital chlamydial infection Both systemic and local antibodies in secretions can be detected in C. trachomatis (Ct) infection. Early studies showed that serum IgG antibodies could be detected in 40±100% of women with genital chlamydial infection1±4 and in 79% of men with chlamydial urethritis5, but 16±87% of chlamydia-negative women used as controls also had such antibodies ± as had 43% of chlamydia-negative males. Serum IgM antibodies were reported in 17±49% among Ct-positive women but also among 3±23% of Ctnegative controls. IgA antibodies in cervical secretions are commonly found in Ct-infected women. Such antibodies have been observed in 57±64% of Ct-infected women but were also detected in 12±47% of Ct-negative controls. All of these studies were based on cell cultures for the detection of Ct infection. This technique has a sensitivity of about 70% or even less compared to nucleic acid ampli®cation tests (NAAT). Recently, serological results from PCR-positive and PCRnegative women were reported.6 Serum IgG antibodies were demonstrated in 56±74% of PCR-positive women compared to 25±29% among negative controls. An inverse relationship between local IgA antibodies and the number of organisms (measured as inclusion-forming units, IFUs) has been demonstrated, suggesting a partial immunity conferred by these antibodies.7 Serum IgG antibodies do not provide any protection for future re-infection but have been shown to prevent ascending infections after legal abortion where curettage had been performed.8 Population-based sero-surveys have shown that antibodies to Ct start to appear between 15 and 20 years of age, peak between 40 and 50 years of age, and start to decline after middle age.9 This is in contrast to the age-speci®c antibody rates of C. pneumoniae where antibodies start to appear after 5 years of age and sharply increase up to 15 years of age after which age antibody prevalence continues to increase for the rest of life, but at a lower rate. Both IgG and IgA antibodies to Ct seem to persist over many years although titres decline10 at least in some situations. Tests for antibodies to C. trachomatis Antibody testing for Ct has traditionally been performed by the microimmuno¯uorescence test (MIF) developed by Wang et al during the 1960s.11 Antigen dots of egg- or cell-culture-grown organisms are placed on a glass slide and then incubated with serum and after washing with an anti-human globulin antibody with a ¯uorescent tag. Dilutions of the serum are then tested and an antibody titre thus determined. The test is laborious, and demands expertise, but has been very useful in delineating antigenic dierences between strains and also for clarifying the antibody response in genital chlamydial infection. In order to standardize the MIF test, and thus get better and comparable results in dierent laboratories, panels of sera have been distributed to laboratories in dierent parts of the world. Testing for antibodies to C. pneumoniae has been the prime objective and the overall agreement has improved in later panels. Similar pro®ciency panels for Ct would be helpful. Cross-reactive antibodies for the dierent species of Chlamydia used in MIF have been a concern. The most important of the common antigens is the lipopolysaccharide (LPS), which is part of the cell wall of all chlamydiae. This antigen has been puri®ed and used in a complement ®xation (CF) test for chlamydial antibodies. The CF test has been widely used to diagnose ornithosis or
Serology, antibiotic susceptibility and serovar determination 803
C. pneumoniae infections but has not been useful for genital chlamydial infections, except for lymphogranuloma venereum (LGV). Antibodies to the LPS can be detected by MIF as well. In some commercially available MIF tests the LPS has been removed from the antigens to reduce cross-reactivity. LPS antibodies appear in high titres mainly during the acute phase of ornithosis or C. pneumoniae infection but disappear later on. High titres of antibodies in the CF test are therefore considered diagnostically signi®cant, suggesting current infection of C. psittaci or C. pneumoniae. In the MIF tests the LPS cross-reactive antibodies might be mistaken for speci®c antibodies to any of the antigens. The removal of LPS is therefore an obvious advantage when speci®c antibodies to the dierent species of chlamydia are of interest. For the diagnosis of current infection of ornithosis or C. pneumoniae infections LPS antibodies may be diagnostically useful. Both the 60-kDa Omp-2 and the hsp60 antigens are conserved proteins with shared epitopes between chlamydial species but are not surface-exposed and should therefore not cause cross-reactivity in the MIF test. A commercial test based on recombinant chlamydial LPS is available. The test is more sensitive than the CF test and antibodies can be detected even after genital chlamydial infection. In our own experience LPS-IgG antibodies were detected (rElisa, Medac, Hamburg) in 62% of culture-positive women and in 81% of culture-positive men, while culture-negative women had such antibodies in 37% and men in 33%. IgM antibodies were detected by this test in culture-positive and -negative women in 22% and 3% respectively and in men in 19% and 3%. IgA antibodies in serum were demonstrated in 60% and 22% in positive and negative women and in 75% and 17% in men. These ®ndings are in general agreement with what has been reported by other groups using other methods as mentioned above. Chlamydiae form inclusions in cell culture. Such infected cell cultures have been established on microscopic slides and then used to detect chlamydial antibodies. Whole inclusion ¯uorescence test (WIF), often with an LGV-2 strain as antigen, has been described for antibody testing in genital Ct infection.12 The test performance usually diers from that of MIF.13 Recombinant MOMP from Ct has been used as antigen to detect antibodies to Ct with a speci®city of 91% and a sensitivity of 80%.14 Recombinant Omp-2 has also been tried as antigen for the detection of Ct antibodies.15 Sensitivity has been high but speci®city lower as Omp-2 is a conserved protein with shared epitopes between the dierent chlamydial species. Recently, commercial tests in the ELISA format based on speci®c peptides have become available. Several of these tests have shown good speci®cities and sensitivities. Although MIF is still considered the gold standard these new tests hold the promise to become useful alternatives to MIF. There are few comparisons between MIF and these ELISA tests. One such recent comparison showed sensitivities between 71 and 85% and speci®cities better than 96% for three dierent ELISA kits.6 IgG antibodies were detected in about 70% of PCRpositive women but also in 25±29% of PCR-negative women. IgA antibodies were found in serum in 13% of PCR-positive and in 6% of the PCR-negative cases. In another study antibodies to Ct were detected in 84% of culture-positive women and in 61% of positive men. Titre changes were observed in a minority of the cases.16 Ct infection is often asymptomatic and may have been present for weeks or months when ®nally detected and treated. Titre changes of IgG or IgM antibodies may therefore not be demonstrated. The antibody response in re-infections is poorly known. The antibody prevalence will vary with the population studied but the data
804 K. Persson
indicate that neither IgG nor IgA antibodies can be used to diagnose current genital infection by Ct or to exclude such an infection. Serology ± a powerful research tool in chlamydial disease Antibody studies have been seminal to demonstrate associations between clinical conditions such as tubal factor infertility17,18 or ectopic pregnancy19,20 and prior infection by Ct. Several studies have reported increased rates of Ct antibodies in these conditions compared to controls, suggesting that previous pelvic in¯ammatory disease (PID) caused by Ct is common in these cases. The involvement of Ct in spontaneous miscarriages has also been studied. Some studies have not detected any association between antibodies to Ct and early miscarriages21,22 but a few reports did observe such an association.23,24 It is possible that Ct antibodies can be an indirect marker for this condition in certain populations. Ct has been detected in endometrial biopsies. It is therefore possible that endometrial Ct infection can cause problems when fertilized eggs are implanted in the uterine cavity after in vitro fertilization (IVF). Again, results are con¯icting. A few studies indicated that Ct antibodies were associated with poor outcome in IVF25±27 although other studies did not detect such an eect.28±30 Hsp60 antibodies have also been studied in IVF. The rate of take-home babies does not seem to be aected by such antibodies in serum. Local antibodies in cervical secretion to hsp60 may, however, predict a poor outcome.31 These observations need further con®rmation. Antibody studies have also been used to explore the role of Ct infection for the development of cervical cancer. In such studies antibodies to human papilloma virus (HPV) of certain types are strongly associated with cancer development, but Ct antibodies were also an independent predictor of cervical cancer. In one study serovar G was more strongly associated with cancer than were other serovars.32 This serovar usually represents less than 5% of the strains circulating in the population. As antibodies to HPV are found in only half of the patients with cervical cancer ± even though HPV DNA can be detected in almost every case ± an independent role for Ct in cancer development has been questioned. In fact, when HPV DNA is introduced in the statistical analysis Ct may no longer be a signi®cant predictor of cancer. Recently it was reported that HPV-infected patients with antibodies to Ct had a doubled risk of cervical cancer compared to those without Ct antibodies.33 Ct might thus be a cofactor for the development of squamous carcinoma of the cervix. An alternative view would suggest that Ct is only a marker of an increased risk for sexually transmitted infections (STIs) in general and thus correlated with an increased risk of acquiring carcinogenic types of HPV. The potential role of Ct in cervical cancer is therefore still uncertain. Antibodies to the 60-kDa heat shock protein of Ct predict tubal scarring For some years the heat shock proteins (hsps) have been particularly incriminated in the development of tubal scarring after Ct infection. There are four groups of hsps based on molecular weight. They are proteins which are found in all living organisms. Thermal or chemical stress induces production of these proteins, which help protein folding or refolding in order to preserve the function of cellular components. Studies in guinea pigs demonstrated that a triton X-100 extract from Ct induced an in¯ammatory response in the conjunctiva of previously immunized animals but not in naõÈ ve controls.34 The extract was later shown to contain the hsp60 of Ct, and recombinant hsp60 was able to induce the same in¯ammatory response as the crude extract.35 Clinical studies
Serology, antibiotic susceptibility and serovar determination 805
disclosed that antibodies to the hsp60 of Ct were more common in women with ectopic pregnancy than in women with PID.36 Later studies have corroborated and extended these ®ndings, and antibodies to chsp60 independently predict tubal factor infertility and ectopic pregnancy even better than MIF antibodies.37±39 Hsp60 seems to have a unique role in the development of tubal scarring and would not just re¯ect the force of infection as MIF antibodies. Bacterial and human hsp60 are quite similar and share epitopes. An autoimmune component has therefore been suggested but has not been veri®ed in animal model systems of autoimmune disease.40 In the animal model systems the hsp60 response was found to be genus-related rather species-speci®c. Antibodies to Chlamydia pneumoniae in humans start to increase during adolescence, usually before exposure to C. trachomatis. A synergistic eect on the hsp60 response to C. trachomatis infection in those with prior exposure to C. pneumoniae could be suspected. Two studies did not ®nd any relation between hsp60 antibody response and C. pneumoniae antibody status.41,42 In one study such an association was observed in a subgroup of women with tubal infertility43 and in yet another study the presence of antibodies to C. pneumoniae was a prerequisite for tubal damage in C. trachomatis infection but the studied groups were small.44 As data are not in agreement the in¯uence of C. pneumoniae on the hsp60 response in C. trachomatis infection is still uncertain. Serology as a clinical tool for the diagnosis of Ct infection In order to be considered as a diagnostic test the speci®city must be high. The MIF test is considered the gold standard but is dicult to perform and the speci®city of the test has been questioned after C. pneumoniae was discovered. Antibodies to the chlamydial LPS could cause cross-reactivity but speci®c antibodies to Ct and C. pneumoniae can usually be distinguished by the test. When LPS has been removed from the antigens, as is the case in some commercial tests, cross-reactivity should be a minor problem. Modern commercial ELISA tests based on peptides from the MOMP of Ct have so far showed high speci®cities. Serum antibodies in current lower genital tract infection by Ct Although IgG antibodies can be detected in serum in 40±100% of infected women, demonstrated by cell culture or NAAT, 16±87% of Ct-negative women also have such antibodies. The situation is similar for serum IgA antibodies but at a lower level. The predictive values therefore become unacceptably low to use IgG or IgA antibodies in serum to diagnose current lower genital tract infection. The shorter half-life of IgA antibodies compared to IgG has suggested that IgA antibodies could re¯ect persistent infection. A similar notion has been proposed for C. pneumoniae. There is no solid ground as yet for the use of IgA antibodies as a marker of persistent or unresolved infection by either C. trachomatis or C. pneumoniae. IgM antibodies may have a better positive predictive value but the sensitivity is too low, which precludes their use for the diagnosis of genital chlamydial infection. IgA antibodies in genital secretions Local antibodies have been detected in cervical secretions of women with Ct infection in 57±64% of the cases but also in 12±47% of Ct-negative women. Again, the predictive values are too low to make such antibodies diagnostically useful.
806 K. Persson
Ct in upper genital tract infection There is a concern that Ct infection in the Fallopian tubes or in the endometrial wall will not be detected in samples from the cervix or urethra. Serology might assist in such situations. One study compared four dierent serological tests in patients with pelvic pain where endometrial biopsies had been performed.45 Fourteen biopsies of the 53 tested were PCR-positive for Ct while only four cervical cultures detected Ct (cervical samples for PCR were not available). The MIF±IgM test had a sensitivity of 79% and a speci®city of 94% while the WIF test had a speci®city and a sensitivity of 100 and 81% respectively. Hsp60 antibodies had a sensitivity of 43% and a speci®city of 100%. The role of serology in these special situations has still to be determined. Ct antibodies in infertility The titre of Ct antibodies seems to correlate with tubal damage, probably re¯ecting the force of infection in tubal infertility.46 Ct antibody testing has been suggested as part of the routine work-up in infertility cases although it seems to have a moderate ability to predict tubal factor infertility in individual cases. Hsp60 antibodies in serum or secretions Antibodies to hsp60 of Ct independently predict tubal scarring in women and an unfavourable course of urethritis in men.47 Hsp60 antibodies in local secretions have also been associated with a decreased rate of take-home babies after IVF. The use of hsp60 antibodies has been limited to research settings but the introduction of commercial tests will change this situation. Sexual assault Although serology is vital for the detection of infection with HIV, HBV and HCV after sexual assault, antibody tests for Ct have no accepted place in this situation. Cell culture for the detection of Ct infection has been recommended but NAATs may soon replace detection by culture. IgM antibodies in neonatal chlamydial infection Neonatal inclusion conjunctivitis has been known for almost a century when typical inclusions in scrapings from the conjunctiva of infants with neonatal non-gonorrhoeal conjunctivitis were described. Similar inclusions were also found in cells from the genital tract of the parents, indicating that this was an STI. During the 1970s it was shown that neonatal chlamydial infection could also lead to pneumonia, often occurring from 3 to 12 weeks after birth. Later on it was reported that IgM antibodies to Ct were regularly found in infant pneumonia.48 SEROVAR DETERMINATION Sero- and genotyping of C. trachomatis During the late 1960s and 1970s, when isolates of C. trachomatis were compared by antisera produced in mice it became clear that dierent serotypes existed.
Serology, antibiotic susceptibility and serovar determination 807
The serotypes were designated A±K, including two that are very closely related, the B and the Ba serotypes. In addition, three types of lymphogranuloma venereum were identi®ed. The distinction between serotypes was possible by polyclonal mouse sera where a new strain was tested with existing sera and an antiserum raised against the new candidate type and used against existing type strains. In this laborious way 15 dierent serovars were identi®ed, and these still form the basis of characterization of C. trachomatis. The serovars were also grouped in two major categories, the B complex, including B, Ba, D and E, and the C complex with serovars A, C, H, I, J and K; an intermediate group is formed by F and G. Later, monoclonal antibodies were developed and further types were suggested; these were named Da, Ia and L2a. When the MOMP gene had been cloned and sequenced by Stephens at al, new methods for typing were developed.49 Ampli®cation of the MOMP gene by the polymerase chain reaction (PCR) formed the basis for further characterization either by restriction enzyme digestion patterns, called restriction fragment length polymorphism (RFLP), or by complete sequencing of the gene product. Sometimes, RLFP is used for primary characterization with sequencing to resolve unusual patterns. For RFLP, primers and restriction enzymes were chosen so that the established serovars would still be the recognized entities. A good agreement between RFLP and serotyping by monoclonal antibodies was demonstrated by Rodriguez et al.50 In a similar way, sequencing of the MOMP gene has been used to characterize isolates of C. trachomatis.51±53 The typing of clinical isolates has disclosed that trachoma is associated with serovars A, B, Ba and C where households often share the same serovar, although dierent serovars can be in circulation in the same village. After population-based treatment trials the pattern may be more complex where dierent serovars can be reintroduced, leading to mixed infections even within households.
Epidemiology of dierent serovars The genital serovars are: D, E, F, G, H, I, J, K and some of the B/Ba strains. In addition, L1±3 are associated with lymphogranuloma venereum. Typing of clinical isolates has shown that D, E and F account for 60±80% of the strains, with E being the most common (Table 1). This pattern is very similar in dierent parts of the world and has been reported from the USA54, Sweden, Holland, Canada and New Zealand. One hundred isolates up to over 1700 strains were typed in these studies. Similar reports have also appeared from other countries in Europe although the number strains has been fewer. A dierent pattern has been reported from Greece where a high prevalence of Da strains was observed. In another study of rectal isolates from gay men, D-strains were the most common, accounting for 53% of the rectal isolates, while only 18% were D serovars of cervical strains.55 There was a signi®cant decline in the proportion of D serovars of rectal infections during the study period, suggesting an epidemiological clustering within a particular group. Sequencing of the MOMP gene has disclosed many dierent genotypes within the framework of the established serovars. Some of these genotypes seem to have been disseminated to dierent parts of the world while others have been detected only locally so far. The genotypes re¯ect a gradual evolutionary change in C. trachomatis but even recombination has been observed in chimaeras.56 Whether such evolutionary changes are in¯uenced by immunological pressure is uncertain. The protection after infection seems to be weak and of short duration. The serovar distribution found
808 K. Persson Table 1. Distribution of serovars of C. trachomatis in 2326 unselected patients at MalmoÈ University Hospital during 1989 and 1990. Infections were characterized as ®rst infections when no previous test had been positive, although the patient may still have had undetected infection. All re-infections had been veri®ed by previous positive tests. Women Serovar B D E F G H I J K Total
1st infection 7 142 476 307 44 43 15 67 86
(0.6%) (12) (40) (26) (4) (4) (1) (6) (7)
1187
Men
Re-infection 1 35 94 76 11 15 4 18 19
(0.4%) (13) (34) (28) (4) (6) (1) (7) (7)
273
1st infection 6 77 288 229 30 25 7 46 61
(0.8%) (10) (37) (30) (4) (3) (1) (6) (8)
769
All
Re-infection 2 14 38 21 7 1 1 7 6
(2%) (14) (39) (22) (7) (1) (1) (7) (6)
97
1st infection 13 219 764 536 74 68 22 113 147
(0.7%) (11) (39) (27) (4) (3) (1) (6) (8)
1956
Re-infection 3 (0.8%) 49 (13) 132 (36) 97 (26) 18 (5) 16 (4) 5 (1) 25 (7) 25 (7) 370
among patients with re-infections is the same as that found among those with their ®rst detected infection (Table 1), also suggesting low serovar-speci®c protection. Pathogenicity of dierent serovars It has been reported that serovar may also in¯uence infectivity as measured by the number of inclusion-forming units (IFUs) in clinical samples. It was found that the number of IFUs was independently related to age, sex, race and serovar where samples with B-complex strains had more IFUs than C-complex strains.57 Similar ®ndings were also reported by Frost et al.58 Women have higher numbers of IFUs than do men, and younger persons shed more organisms than do older ones. Recently, persistent infection in patients was reported to be associated with C-complex strains.59 The important question is whether there is a serovar-speci®c morbidity or whether some serovars are more likely to cause symptoms or speci®c manifestations than others. It was found by Batteiger et al that the frequency of cervicitis and easy bleeding in women with C. trachomatis infection was not related to the infecting serovar; however, it was found that males with a lower number of polymorphonuclear white cells in urethral scrapings were more likely to have the intermediate serovars F or G.60 In another study, the F strains seemed less prone to produce cervicitis than other serovars.61 In San Francisco, an F variant was found only in women with PID and was associated with severe symptoms.62 In yet another study we were not able to detect any serovars that were particularly associated with salpingitis.63 Recently it was reported that symptomatic males were more likely to have variant Da while women with vaginal discharge had an excess rate of serovar K.64 In aggregate, these studies do not disclose a general pattern. It is possible that certain genotypes may be more pathogenic than others ± such as the F-variants mentioned above. It is also possible that pathogenicity is not associated with the MOMP gene that has been studied so far and that other genes may be more important. One such potentially important gene activity was recently reported when a cytotoxin produced by C. trachomatis was described. The dichotomy between the trachoma and the genital strain serovars seems to re¯ect a biological dierence, however.
Serology, antibiotic susceptibility and serovar determination 809
ANTIBIOTIC RESISTANCE OF C. Trachomatis The drugs-of-choice for treatment of infections involving C. trachomatis have been tetracycline or doxycycline and the macrolides such as erythromycin and, recently, azithromycin. These antibiotics have minimal inhibitory concentrations (MIC) of 0.1 to 0.25 mg/ml, which is considered sucient for ecient treatment. The MIC value for rifampicin is also very low but this drug has been reserved for treatment of mycobacterial infections. Despite the lack of cell wall components similar to those which bind penicillin in other bacteria it has been found that this group of antibiotics can arrest the development to mature and infectious organisms of Ct in the cellular inclusions. Ampicillin has thus been used to treat genital chlamydial infections in pregnant women where microbiological eradication can reach 90% or more. The ¯uoroquinolones, such as o¯oxacin, are also clinically eective, with MIC values of 0.5 mg/ml. While treatment ecacy studies have usually reported microbiological eradication in 95% or more of the infected patients, long-term reappearance of infection has been demonstrated in 6±17% or even up to 29% in young persons.65±67 Recurrences of C. trachomatis may be due to treatment failure or re-infection. Serovar determination would suggest both mechanisms. One of the reasons for treatment failure could be development of antibiotic resistance. There are few reports of antibiotic resistance. The technique used to determine antibiotic sensitivity is laborious and cannot be used routinely. An underestimation of the magnitude of the problem is therefore possible. Antibiotic-resistant strains Some information is available. Mourad described two strains of C. trachomatis with MIC values of 1 mg/ml and thus having a relative resistance to erythromycin.68 Resistance to tetracyclines has also been reported.69 Recently, strains which had elevated MIC values for doxycycline, azithromycin and o¯oxacin at concentrations of 44 mg/ml were described from three patients with treatment failures.70 Some years earlier a series of strains isolated from patients with persistent infection after treatment were reported, although the infections were ®nally treated successfully.17 Strains from ®ve patients were resistant to tetracycline at 4 to 48 mg/ml. Resistance to erythromycin was also detected among these strains. It was noted that only a fraction of the inoculated organisms (less than 1%) showed resistance. After passage in a medium containing 8 mg/ml of tetracycline, 100% of the progeny showed resistance. Attempts to passage such fully resistant strains in antibiotic-free medium were unsuccessful in most cases but at least one strain could be serially propagated; this strain was then completely sensitive to tetracycline. These observations have been described as heterotypic resistance. In vitro induction of antibiotic resistance Resistance to quinolone has been produced in vitro by growing C. trachomatis in cell culture in the presence of the antibiotic studied.72 Two resistant strains of an LGV prototype strain were obtained with MIC values 1000-fold higher than for sensitive strains. A mutation was demonstrated in a gyrase gene (gyrA) speci®c to the resistant strain. From the data reported, C. trachomatis seems to be able to develop antibiotic resistance to drugs often used in the treatment of this infection. Although
810 K. Persson
the prevalence of resistant strains seems to be low at present the problem cannot be neglected. When infection persists despite repeated courses of antibiotic treatment resistance may have developed. When other possible explanations for the treatment failure have been ruled out susceptibility testing of the strain is indicated but few laboratories are prepared to perform such testing.
SUMMARY Serology has been very helpful for elucidating associations between Ct and clinical conditions such as tubal factor infertility and ectopic pregnancy. Although antibodies, both systemic and local, often develop after Ct infection, such antibodies are not useful for detecting current infection, although they might be considered in the work-up of female infertility. Typing of Ct strains, either by antibodies or by DNA methods, has been useful for studying the pathogenicity and epidemiology of these organisms. Antibiotic resistance is still uncommon but the situation could change.
Practice points . antibodies develop in response to genital chlamydial infection in most cases . antibodies, systemic or local, are not useful for demonstrating current lower genital tract infection . the demonstration of IgM antibodies is a good diagnostic marker in neonatal chlamydial pneumonia . antibodies to hsp60 predict tubal damage in chlamydial infection . C. trachomatis is divided into 15 (or 18) dierent serovars, from A through L, and can be subdivided into many more genotypes within this framework . the most common serovars are E, D and F, which represent 60±80% of isolates from the genital tract, with few exceptions worldwide . some particularly pathogenic variants of C. trachomatis may occur, but a general pattern suggesting that some serovars are more pathogenic than others has not emerged . by DNA sequencing of the MOMP gene a very powerful epidemiological tool has become available, although it is not yet ready for clinical use . so far, few strains of C. trachomatis are resistant to commonly used antibiotics . quinolone resistance has been induced in vitro
Research agenda . the association between C. trachomatis and cervical cancer as an independent risk factor, or as a cofactor, needs to be explored further . surveillance for new and more pathogenic types of C. trachomatis . clinical adaptation of MOMP gene sequencing for epidemiological purposes . continuous surveillance for signs of development of antibiotic resistance in cases where treatment failure is observed
Serology, antibiotic susceptibility and serovar determination 811
Although a lot of information has been presented during the last few years on the association between Ct and cervical cancer, more corroborating data are needed. The heat shock proteins seem to be important in the development of scarring after Ct disease but the mechanisms are still elusive. REFERENCES 1. Osser S & Persson K. Immune response to genital chlamydial infection and in¯uence of Chlamydia pneumoniae (TWAR) antibodies. European Journal of Clinical Microbiology & Infectious Diseases 1989; 8: 532±535. 2. Richmond SJ, Milne JD, Hilton AL & Caul EO. Antibodies to Chlamydia trachomatis in cervicovaginal secretions: relation to serum antibodies and current chlamydial infection. Sexually Transmitted Diseases 1980; 7: 11±15. * 3. Schachter J, Cles L, Ray R & Hines PA. Failure of serology in diagnosing chlamydial infections of the female genital tract. Journal of Clinical Microbiology 1979; 10: 647±649. 4. Treharne JD, Darougar S, Simmons PD & Thin RN. Rapid diagnosis of chlamydial infection of the cervix. British Journal of Venereal Diseases 1978; 54: 403±408. 5. Terho P & Meurman O. Chlamydial serum IgG, IgA and local IgA antibodies in patients with genitaltract infections measured by solid-phase radioimmunoassay. Journal of Medical Microbiology 1981; 14: 77±87. * 6. Morre SA, Munk C, Persson K et al. Comparison of three commercially available peptide-based immunoglobulin G (IgG) and IgA assays to microimmuno¯uorescence assay for detection of Chlamydia trachomatis antibodies. Journal of Clinical Microbiology 2002; 40: 584±587. 7. Brunham RC, Kuo CC, Cles L & Holmes KK. Correlation of host immune response with quantitative recovery of Chlamydia trachomatis from the human endocervix. Infection and Immunity 1983; 39: 1491±1494. 8. Osser S & Persson K. Postabortal pelvic infection associated with Chlamydia trachomatis and the in¯uence of humoral immunity. American Journal of Obstetrics and Gynecology 1984; 150: 699±703. 9. Saikku P. Diagnosis of acute and chronic Chlamydia pneumoniae infections. In Or®la J, Byrne G, Chernesky M et al (eds) Chlamydial Infections. Proceedings of the Eighth International Symposium on Human Chlamydial Infections, pp 163±172. Bologna: Societa Editrice Esculapio, 1994. 10. Puolakkainen M, Vesterinen E, Purola E et al. Persistence of chlamydial antibodies after pelvic in¯ammatory disease. Journal of Clinical Microbiology 1986; 23: 924±928. *11. Wang S. The microimmuno¯uorescence test for Chlamydia pneumoniae infection: technique and interpretation. Journal of Infectious Diseases 2000; 181 (supplement 3): S421±S425. 12. Richmond SJ. Immuno¯uorescence testing for chlamydial antibodies. Lancet 1979; 2: 1016. 13. Peterson EM, Oda R, Tse P et al. Comparison of a single-antigen microimmuno¯uorescence assay and inclusion ¯uorescent-antibody assay for detecting chlamydial antibodies and correlation of the results with neutralizing ability. Journal of Clinical Microbiology 1989; 27: 350±352. 14. Mygind P, Christiansen G, Persson K & Birkelund S. Detection of Chlamydia trachomatis-speci®c antibodies in human sera by recombinant major outer-membrane protein polyantigens. Journal of Medical Microbiology 2000; 49: 457±465. 15. Mygind P, Christiansen G, Persson K & Birkelund S. Analysis of the humoral immune response to Chlamydia outer membrane protein 2. Clinical and Diagnostic Laboratory Immunology 1998; 5: 313±318. 16. NaÈrvaÈnen A, Poulakkainen M, Hao W et al. Detection of antibodies to Chlamydia trachomatis with peptide-based species-speci®c enzyme immunoassay. Infectious Diseases in Obstetrics and Gynecology 1997; 5: 349±354. 17. Gump D, Gibson M & Ashikaga T. Evidence of prior pelvic in¯ammatory disease and its relationship to Chlamydia trachomatis antibody and intrauterine contraceptive device use in infertile women. American Journal of Obstetrics and Gynecology 1983; 148: 153±159. 18. Moore D, Foy HM, Daling J et al. Increased frequency of serum antibodies to Chlamydia trachomatis in infertility due to distal tubal disease. Lancet 1982; ii: 574±577. 19. Sheeld PA, Moore DE, Voigt LF et al. The association between Chlamydia trachomatis serology and pelvic damage in women with tubal ectopic gestations. Fertility and Sterility 1993; 60: 970±975. 20. Svensson L, MaÊrdh P, Ahlgren M & NordenskjoÈld F. Ectopic pregnancy and antibodies to Chlamydia trachomatis. Fertility and Sterility 1985; 44: 313±317. 21. Coste J, Job-Spira N & Fernandez H. Risk factors for spontaneous abortion: a case-control study in France. Human Reproduction 1991; 6: 1332±1337.
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