Clinical characteristics, serology and serovar studies on Chlamydia trachomatis infections Caroline Bax BW.indd 1 26-08-10 12:17 Clinical characteristics, serology and serovar studies on Chlamydia trachomatis infections. Thesis. Leiden University, the Netherlands. © 2010 C.J. Bax, Leiden, the Netherlands. Financial support for the printing of this thesis was provided by Wetenschapsfonds MC Haaglanden, Medac, Ferring BV. Cover Kees Boer Lay-out Optima Graﬁsche Communicatie BV Printed by Optima Graﬁsche Communicatie BV ISBN 978-90-8559-105-4 Caroline Bax BW.indd 2 26-08-10 12:17 Clinical characteristics, serology and serovar studies on Chlamydia trachomatis infections Proefschrift ter verkrijging van de graad van Doctor aan de Universiteit Leiden, op gezag van de Rector Magniﬁcus prof. mr. P.F. van der Heijden, volgens besluit van het College voor Promoties te verdedigen op woensdag 13 oktober 2010 klokke 13.45 uur door Caroline Johanna Bax geboren te Tilburg in 1970 Caroline Bax BW.indd 3 26-08-10 12:17 Promotiecommissie Promotor Prof. dr. J.B. Trimbos Co-promotores Dr. P.J. Dörr (Medisch Centrum Haaglanden) Dr. S.A. Morré (VU Medisch Centrum) Overige leden Dr. P.M. Oostvogel (Medisch Centrum Haaglanden) Prof. dr. J.T. van Dissel Prof. dr. F.M. Helmerhorst Prof. dr. H.H.H. Kanhai Caroline Bax BW.indd 4 26-08-10 12:17 20 years from now you will be more disappointed by the things you didn’t do than by the ones you did. So, throw off the bow lines. Sail away from the safe harbour. Catch the trade winds in your sails. Explore. Dream. Discover. Caroline Bax BW.indd 5 Mark Twain 26-08-10 12:17 Caroline Bax BW.indd 6 26-08-10 12:17 Contents Chapter 1 11 General Introduction Part I Clinical characteristics; prevalence and risk factors Chapter 2 33 Clinical characteristics of Chlamydia trachomatis infections in a general outpatient department of Obstetrics and Gynaecology in the Netherlands. Sex Transm Infect. 2002;78(6):E6. Chapter 3 41 Analyses of multiple site and concurrent Chlamydia trachomatis serovar infections, and serovar tissue tropism for urogenital versus rectal specimens in male and female patients. Submitted Sex Transm Infect. 2010. Part II Serology Chapter 4 57 Comparison of serological assays for detection of Chlamydia trachomatis antibodies in different groups of obstetrical and gynecological patients. Clin Diagn Lab Immunol. 2003;10(1):174–6. Chapter 5 65 Chlamydia trachomatis heat shock protein 60 (cHSP60) antibodies in women without and with tubal pathology using a new commercially available assay. Sex Transm Infect. 2004;80(5):415-6. Part III Serovar Chapter 6 73 The serovar distribution of urogenital Chlamydia trachomatis strains among sexual transmitted disease clinic patients and gynaecological patients in the region of The Hague, The Netherlands: an ethnic epidemiological approach. Submitted Sex Transm Infect. 2010. Chapter 7 87 Signiﬁcantly higher serological responses of Chlamydia trachomatis B group serovars vs. C and I serogroups. Drugs Today. 2009;45(Suppl. B):135-40. Caroline Bax BW.indd 7 26-08-10 12:17 Part IV General Discussion and Summary Chapter 8 99 General Discussion Chapter 9 131 Summary en Nederlandse Samenvatting Abbreviations 149 Authors and afﬁliations 153 Acknowledgements/Dankwoord 157 Curriculum vitae 163 Caroline Bax BW.indd 8 26-08-10 12:17 Caroline Bax BW.indd 9 26-08-10 12:17 Caroline Bax BW.indd 10 26-08-10 12:17 Chapter 1 General Introduction Caroline Bax BW.indd 11 26-08-10 12:17 Caroline Bax BW.indd 12 26-08-10 12:17 General Introduction 13 Contents Studies included in the thesis 13 Introduction to the thesis 14 Classiﬁcation 14 Characteristics 15 Pathogenesis 16 Prevalence and risk factors 17 Clinical course of infection 17 - Women - Neonates and infants - Men - Sequelae - Transmission and duration of infection - Treatment and resistance Detection methods 19 - Culture - Antigen detection - Nucleic acid detection methods - Sample sites - Serologic tests Serotyping-genotyping 22 Aims and outline of this thesis 23 Studies included in the thesis In search of epidemiological risk factors for Chlamydia trachomatis (CT) and prevention of tubal pathology we performed two prospective-cohort studies. The results are presented in this thesis. The ﬁrst study describes the epidemiological data of patients with CT infections using a well deﬁned cohort of patients visiting an outpatient department of Obstetrics and Gynaecology located in the central urban area of The Hague, and was reported in 2002-2004. To assess the value of different serological assays for the detection of CT antibodies a comparison is made in the same cohort. An evaluation of a new assay detecting heat shock protein 60 (HSP60) antibodies and its role in predicting tubal pathology has been made as well. In 2007 a second, sequential study was started at the same outpatient department of Obstetrics and Gynaecology and the nearby regional sexually transmitted disease clinic to ﬁnalise some of the aims of the study, which will be outlined at the end of this chapter. The time gap between the ﬁrst and second study has no consequences for the character of the study as a whole. Findings and conclusions of the ﬁrst part are still relevant and actual. The second part is a logical Caroline Bax BW.indd 13 26-08-10 12:17 consequence of the ﬁrst, within the same subject of research, although some epidemiological data are continuously changing over time. Introduction to the thesis CT infection is the most prevalent sexual transmitted disease in the Netherlands and worldwide. In the Netherlands approximately 60.000 people are infected each year of which 35.000 are women. In women approximately 70% of the infections are asymptomatic. However, infections caused by CT may lead to serious complications such as pelvic inﬂammatory disease (PID). Functional damage to the tubes may result in (tubal factor) subfertility and ectopic pregnancy. Infections in pregnant women may lead to neonatal conjunctivitis and pneumonia. The highest prevalence is found in young sexually active women, who are especially at risk for complications. It is unknown why some women have asymptomatic infections and why others develop symptomatic upper genital tract infections. It has been suggested that different serovars and/or host-factors may be involved in the clinical course of infection, the rate of upper genital tract progression, and clearance or persistence of infection. Treatment is simple and effective. Late complications can be prevented if treatment is given in time. Chapter 1 With the development of techniques to detect CT in urine samples screening programs have become 14 reality and large scale screening programs for asymptomatic infections have been implemented. In the population of obstetrical and gynaecological patients it is important to determine risk factors for infection. Uterine instrumentation such as intrauterine device (IUD) insertions and abortion curettage may cause upper genital tract infection if an asymptomatic cervical infection is present, by ascending infection or by reactivation of viable microorganisms in the upper genital tract. To optimise prevention strategies for complications we wanted to determine host risk factors for CT infections for obstetrical and gynaecological patients. The dynamics of CT serovars need further evaluation. Insight in changes of serovar distribution in different ethnic groups over time, in relation to clinical data, could contribute to understanding changes in the epidemiology of CT infections and have clinical implications. Classiﬁcation The genus Chlamydia consists of different species. Most well known are CT, Chlamydia psittaci, and Chlamydia pneumoniae (CP). Chlamydiae are associated with a broad spectrum of diseases, such as cardiovascular, pulmonary, and ocular disease, and urogenital tract infections1-3. CT infects mainly human and is divided into two biovars (table 1). The trachoma biovar consists of serovar A-C, causing trachoma, and the serovars D-K, which infect the urogenital tract. The LGV biovar, consisting of serovars L1-L3, causes lymphogranuloma venereum. Caroline Bax BW.indd 14 26-08-10 12:17 General Introduction 15 Table 1. Human Chlamydia trachomatis serovar classiﬁcation and site of inﬂammation Chlamydia trachomatis Biovar Serovar Host Trachoma A, B, Ba, C Human Site of inﬂammation Conjunctivae Urogenital tract (rare) D, Da, E, F, G, Ga, H, I, Ia, J, K Human Urogenital tract Conjunctivae Respiratory tract (rare) LGV L1, L2, L2a, L3 Human Inguinal nodes Urogenital tract Rectum Characteristics Chlamydiae are nonmotile, gram-negative, obligate intracellular bacteria with a unique developmental life cycle (ﬁgure 1)4. Figure 1. Developmental cycle of Chlamydia trachomatis. (©Thesis T. Huittinen 2003) The extracellular elementary body (EB) is the infectious, but metabolically inactive form, which is taken up by the host cell by inclusion. The EB transforms into the larger, non-infectious, metabolically active reticulate body (RB), which replicates by binary ﬁssion (a process which results in the reproduction of a living prokaryotic cell by DNA replication and division into two parts which each have the potential to grow to the size of the original cell). Within 40-48 hours the RBs differentiate back to EBs, which are Caroline Bax BW.indd 15 26-08-10 12:17 released from the inclusion vacuole by exocystosis or host cell lysis to infect other cells. The complete cycle from entry to release takes about 48-72 hours5,6. Chlamydiae have both RNA and DNA. The cell wall contains both proteins and lipids. The most prominent protein is the major outer membrane protein (MOMP). It has a function in maintaining the structural integrity of the cell wall and forms with Omp2 and Omp3 (cystine rich proteins located on the inner surface of the outer membrane) a disulﬁde cross-linked complex in the cell wall7. This transmembrane protein with surface antigenic components can be used to identify the different CT serovars. Lipopolysaccharide (LPS) is a genus-speciﬁc antigen which is located on the surface of the MOMP. Pathogenesis Although progress has been made in the past years, it is yet unknown why some women have asymptomatic infections and why others develop symptomatic upper genital tract infections. It is also unclear why in some women the upper genital tract infection causes serious damage to the fallopian tubes, while in other women the infection is cleared, apparently without clinical consequences. Differences in the clinical course of the infection could be due to interaction between bacterial (virulence factors among Chapter 1 different serovars, see serotyping), environmental (vaginal ﬂora composition, co-infections) and host 16 factors (genetic differences). The susceptibility for a CT infection is, among other factors, inﬂuenced by the vaginal ﬂora. The presence of co-infections, i.e. Neisseria gonorrhoeae (NG) or Candida albicans, may cause similar or dissimilar symptoms. An association with persistence of human papillomavirus (HPV) in the presence of a CT infection was found8. The host immune response plays an important role in the inﬂammation and pathological tissue scarring in chlamydial disease. It has been proposed that women with weak cell-mediated immune responses to CT and strong antibody responses to CT are those susceptible to reinfection, slow to resolve infection and have high levels of clinical inﬂammation (tissue scarring). Likewise, women with strong cell-mediated immune responses and comparatively low antibody responses are resistant to infection and less susceptible to disease9. The humoral immune response is represented by the presence of antibodies. Especially chlamydial 60 kDa heat shock protein (HSP60) is a predictor of upper genital tract infection10. Speciﬁc cytotoxic T cells are representations of the cellular immune response. These cells cause lysis of the CT infected cells through binding to CT antigens like MOMP, LPS and HSP6011,12. Therefore, it is proposed that chronic inﬂammation by repeated or persistent CT infection may lead to tissue scarring by release of cytokines or a continuous production of HSP60, which triggers the immune system leading to tissue damage13. Also the inﬂammation caused by the activation of cytotoxic T cells, to eliminate CT infected cells, by HSP60 leads to scarring. Caroline Bax BW.indd 16 26-08-10 12:17 General Introduction 17 Prevalence and risk factors The prevalence of CT infections depends on the population tested. Wilson et al. found prevalences ranging from 1.7-17% in asymptomatic women in Europe14. Among young women attending STD clinics rates are well above 10%15, and in population-based studies among women under 30 year of age the prevalence was between 2-6% in the Netherlands16, Denmark17 and the United Kingdom18. In the literature several risk factors for CT infections have been identiﬁed, such as young age, being of black race, recent new partner (< one year), number of sexual partners, condom use and complaints of postcoital bleeding in women19-21. In the Netherlands several similar risk factors have been identiﬁed, including Surinam or Netherlands Antilles ethnicity22. Higher prevalences are found in urban areas16. However, it is unclear whether the same risk factors apply for an obstetrical and gynaecological population. Clinical course of infection Women Nearly 70% of the CT infections in women are asymptomatic and will therefore not be treated with antibiotics23. These women may play an important role in the transmission of infections with the risk of longterm sequelae. Clinical manifestations of symptomatic infections include cervicitis, urethritis, endometritis and PID. Most common complaints related to CT infections are abnormal vaginal discharge, dysuria and postcoital bleeding. In case of PID, pelvic-, uterine-, and/or adnexal-pain are reported. Ascending CT infections are responsible for most of the morbidity due to tubal scarring, which may result in tubal factor subfertility and ectopic pregnancy24,25. During pregnancy CT infections may lead to complications such as spontaneous abortion, preterm labour, preterm premature rupture of membranes, low birth weight, chorioamnionitis and postpartum endometritis26,27. Although reports show conﬂicting results about the correlation as reviewed by Baud et al.28. Neonates and infants During delivery CT infections may be transmitted to the neonate causing conjunctivitis and pneumonia. The risk of maternal-fetal transmission is around 50%29-31. CT is the most common cause of neonatal conjunctivitis and one of the most common causes of pneumonia in early infancy. In the Netherlands more than 60% of the cases of neonatal conjunctivitis are causes by CT, and in 7% of the infants with respiratory complaints, CT was detected32,33. Symptoms of conjunctivitis develop within 2 weeks after delivery, and pneumonia at 4 to 17 weeks after birth. Infants with chlamydial pneumonia are at increased risk for later pulmonary dysfunction and possibly for chronic respiratory disease34. Besides maternally transmission during delivery, transmission of CT to neonates can also occur after birth. Caroline Bax BW.indd 17 26-08-10 12:17 Men Up to 50% of the urethral CT infections in men are asymptomatic23. CT infections in men may cause urethritis. Symptoms, such as dysuria and abnormal urethral discharge occur 1 to 3 weeks after exposure to CT. Untreated infections may lead to Reiter’s syndrome, a disorder more common in men than in women, consisting of arthritis, urethritis and conjunctivitis35. CT infections may also lead to epididymitis and prostatitis (1-4%)36. CT infections play an uncertain role in male subfertility37. Proctitis might occur, especially in men-who-have-sex-with-men (MSM) populations. Non-LGV-serotypes give less complaints (irritation, itching, (bloody) discharge, diarrhoea) than LGV strains. Sequelae In the Netherlands approximately 60.000 CT infections occur each year, of which 35.000 are in women. Upper genital tract infection might occur (PID 5.000-10.000 each year), resulting in secondary complications (tubal factor subfertility 1.000-2.000 and ectopic pregnancy 200-400 each year) (fact-sheet Chlamydia, stichting SOA bestrijding 1998; Screenen op Chlamydia, Rapport Gezondheidsraad 2004). In 50% of the PIDs, CT and/or Neisseria gonorrhoeae (NG) are found38,39. In the other 50% a variety of bacteria is found, including enteric organisms such as Mycoplasma hominis and Ureaplasma urealyticum40. The diagnosis of PID is sometimes difﬁcult. Around 50% of the PIDs are thought to be asymptomatic41,42. Chapter 1 Thirty percent of the clinical suspected PIDs cannot be conﬁrmed laparoscopically43,44. The role of CT 18 in the development of progressive upper genital tract pathology after repeated episodes of PID has been established45. There is a correlation between the severity of the PID and the incidence and the level of chlamydial antibody response41,42. The risk of PID following a CT infection is generally believed to be between 8-20%. A review of the literature showed that the PID rate differences depend on whether symptomatic infections were studied and the prior probability of having a sexually transmitted infection (STI)46. In asymptomatic women, diagnosed with a CT infection in a screening program, the PID rate was 0-4%, while in symptomatic women or women with a higher risk of having a STI, the PID rate was 12-30%. For tubal tissue damage to occur, prolonged exposure to Chlamydia is considered a major predisposing factor, either by chronic persistent infection or by frequent reinfection47,48. After a subclinical or clinical PID, the estimated risk of ectopic pregnancy and subfertility varies between 5-25%24,41,49,50 and 10-20%18,51, respectively. This is based on data from high-risk populations and symptomatic infections. Weström et al. showed that an increasing number of PID episodes leads to a higher prevalence of ectopic pregnancy and subfertility24. There is reason to believe that there is some overestimation of the risk estimates, due to incorrect diagnosis, misclassiﬁcation and mistakes in calculations of complication rates52. Morré et al. found that the infection resolved spontaneously in the ﬁrst year after diagnosis in 45% of the untreated women53. None of the women developed signs of a PID, but a subclinical PID with subsequent complications cannot be excluded. Therefore the exact prevalence of sequelae of CT infections is hard to establish. In a retrospective record-linkage cohort, which included Uppsala resident women aged 15-24 years between January 1985 and December 1989, results of laboratory tests for CT, hospital diagnoses, and socio-demographic data Caroline Bax BW.indd 18 26-08-10 12:17 General Introduction 19 were linked. The cumulative incidence of PID by age 35 was: 5.6% (95% CI 4.7-6.7%) in women who ever tested positive for Chlamydia, 4.0% (3.7-4.4%) in those with negative tests, and 2.9% (2.7-3.2%) in those who were never screened. The corresponding ﬁgures for ectopic pregnancy were: 2.7% (2.1-3.5%), 2.0% (1.8-2.3%), and 1.9% (1.7-2.1%); and for subfertility: 6.7% (5.7-7.9%), 4.7% (4.4-5.1%), and 3.1% (2.8-2.3%)82. This study suggests that the incidence of these complications of Chlamydia is substantially lower than believed54. Transmission and duration of infection The risk of transmission between partners is reported to be 45-75%, with lower frequencies in those patients having an asymptomatic infection55,56. Some patients clear the infection spontaneously; 25% within 1-3 months57, and 45% after 1-year follow-up53, while in others the infection persists for years; 46% after 1 year and 18% after 2 years58. However 94% of the women cleared the infection spontaneously at 4-years follow-up58. The presence of chlamydial antibodies does not mean protection against new infections. In contrast, it might even cause more damage because of a more severe immune response. Treatment and resistance The treatment of a CT infection is simple and effective. First choice is a single dose of 1 gram azithromycin. Alternatively doxycycline 100 mg twice a day, for 7 days is recommended. A meta-analysis showed that azithromycin and doxycycline are equally efﬁcacious in achieving microbial cure and have similar tolerability. The cure rate for azithromycin was 97%, and 98% for doxycycline59. In pregnant women erythromycin (1 gram twice a day) or amoxicillin (500 mg three times a day) are often prescribed as ﬁrst choice. However, azithromycin has been shown to be equally effective, safe and was associated with less side effects and better compliance60. In case of a symptomatic infection or PID, because of the polymicrobial nature, broad-spectrum regimens (oral or parenteral) are suggested, to provide adequate coverage against these microorganisms61. There is some evidence of the development of antibiotic resistance62,63. However, antimicrobial resistance of CT is unclear and needs further research64. Detection methods After the detection of Chlamydia inclusions by Giemsa staining in 1907, detection methods have improved with respect to sensitivity, speciﬁcity, time per assay and laboratory standardisation. From time-consuming and not very sensitive culture, to antigen detection methods such as enzyme immunoassay (EIA) and direct ﬂuorescent antibody assay (DFA), to more recently developed techniques such as nucleic acid ampliﬁcation tests (NAATs) and Rapid tests or Point Of Care (POC) tests. Test characteristics of the most widely used commercially available tests are summarised in table 2. Caroline Bax BW.indd 19 26-08-10 12:17 Table 2. Sensitivities and speciﬁcities of Chlamydia trachomatis detection assays (based on Bianchi et al.)65. (© J. Land, et al.)66. Test Sensitivity Speciﬁcity Detection limit (%) (%) (no. of organisms) NAAT 90-95 >99 1-10 DFA 80-85 >99 10-500 EIA 60-85 99 500-1000 DNA-probe 75-85 >99 500-1000 Cell culture 50-85 100 5-100 POC 25-55 >90 >10000 Culture Although cell culture is the deﬁnite proof of a Chlamydia infection, making the assay 100% speciﬁc, it has some major disadvantages. The technique is very laborious, expensive and relatively insensitive (50-85%) as compared to the more recently developed NAATs67,68. Furthermore, for culturing the clinical material has to be handled in a way (regarding time, transport and storage conditions) that does not affect the viability of the organism. Cell culture has been regarded as the gold standard for Chlamydia infections Chapter 1 but has lost this status due to the introduction of the NAATs. 20 Antigen detection i) The two target antigens used in DFA and EIA are the Chlamydia species-speciﬁc MOMP and the genusspeciﬁc LPS (ﬁgure 2). Cross-reactivity is noticed on the basis of MOMP69 and LPS70, and non-speciﬁc binding occurs by reactions with host proteins. The sensitivity of DFA is 80-90% and the speciﬁcity 98-99% relative to culture71-73. The DFA test was widely used as a conﬁrmation test. Most (commercially available) EIA techniques are based on the immunochemical detection of LPS genus-speciﬁc antigen. To improve the speciﬁcity, some manufacturers have developed a blocking assay in order to verify positive EIA results74. Currently most DFA and EIA have been replaced by NAATs. ii) Rapid tests or POC tests have been developed as tests which do not require sophisticated equipment and can be completed in about 30 minutes. Most POC tests employ EIA technology and use antibodies against, amongst others, LPS. They give rise to false-positive results due to cross-reactivity with LPS from other microorganisms. In general the POC tests are signiﬁcantly less sensitive and speciﬁc than laboratory-performed EIA and DFA. POC tests have been suggested to be of value for high prevalence ocular CT detection, but for urogenital CT infections these tests have a much too low sensitivity75. Nucleic acid detection methods Besides identifying Chlamydia by culture or by detecting parts of its membrane, CT can be identiﬁed by detecting the nucleic acid (either DNA or RNA) (ﬁgure 2). i) In the non-ampliﬁcation systems for the detection of Chlamydia DNA and RNA, for which the PACE 2 is the best available test, sensitivity is 85-98% as compared to cell culture with a speciﬁcity of around 99%76-78. Caroline Bax BW.indd 20 26-08-10 12:17 General Introduction 21 MOMP RNA PLASMIDS Chromosome (DNA) LPS Figure 2. Schematic representation of a Chlamydia particle with different targets for diagnostic tests: major outer membrane protein (MOMP), lipopolysaccharide (LPS), Chlamydia plasmids (DNA and RNA), Chlamydia chromosome (DNA and RNA). (© J. Land, et al.) 66. The PACE 2 (Gen-Probe) is a commercially available DNA hybridization probe for the detection of CT (simultaneously with NG). The probe hybridizes to a species-speciﬁc sequence of chlamydial 16S rRNA. A DNA-rRNA hybrid is formed and absorbed onto a magnetic bead. The chemiluminescent response is detected quantitatively by a luminometer. Actively dividing Chlamydiae contain up to 104 copies of 16S rRNA. The DNA probe can detect approximately 103 chlamydial elementary bodies. ii) In the past 25 years NAATs have been developed. In these ampliﬁcation tests the original amount of nucleic acid (either DNA or RNA) present in the clinical sample is multiplied by polymerase chain reaction (PCR). NAATs such as PCR are very sensitive and highly speciﬁc. They make it possible to use noninvasive sampling techniques. The assays detect the extra-chromosomal CT plasmid DNA existing in ten copies per Chlamydia particle79. Besides the commercially available PCR assays numerous so called ‘in-house’ PCR assays are used. They vary greatly in performances and speciﬁcity, and are often used in discrepancy analysis in comparing commercially available assays. For the detection of Chlamydia ribosomal RNA in ampliﬁcation systems, amongst others, template mediated ampliﬁcation (TMA) is used (GenProbe). Since all nucleic acid ampliﬁcation technologies detect nucleic acid targets, they do not depend on either viability or an intact state of the target organism for a positive result. This might explain discrepancies between culture and DNA ampliﬁcation tests due to nonviability of the target organism. Sample sites For women it is advised to test ﬁrst-void urine (FVU) or a urethral specimen in combination with a cervical specimen80. Other studies found the vaginal introitus also a representative site to detect CT infections, with the advantage of being noninvasive81. Rectal swabs should only be obtained in women at high risk of being infected. In male patients, urethral site sampling or FVU can be used for the detection of CT infections82. In MSM rectal samples should be taken as well, to detect more patients, Caroline Bax BW.indd 21 26-08-10 12:17 The pharyngeal sampling site does not seem to contribute much to the detection of CT infected patients83. It is not known whether this site has clinical implications, if standard treatment is effective and if the infection can be transmitted via oral sex or kissing. Serologic tests Different serological assays have been developed for the detection of antibodies to CT. Little is known about antibody proﬁles during acute or chronic genital Chlamydia infections. Therefore, the presence of CT antibodies will not distinguish a previous from a current infection. After an infection antibodies persist for a long period of time84. The microimmunoﬂuorescence (MIF) test is the most sensitive serologic test for Chlamydia species and considered the ‘gold standard’ for the serological diagnosis of CT. It detects species- and serovar-speciﬁc responses. It is used for the detection of prior exposure to CT by the presence of IgG antibodies. However, cross-reactivity with CP in the existing assays should be taken into account85. The most important disadvantages are that the test is laborious and costly, and the reading of the assay is subjective. Several new commercially available species-speciﬁc (peptide-based) EIAs have been developed for the detection of CT antibodies. They seem to perform equally compared to MIF in predicting tubal pathology86. The most accurate tests for Chlamydia antibody testing (CAT) have a sensitivity of approximately Chapter 1 60% for tubal pathology while their speciﬁcity is 85-90%87. The advantage is that the EIAs provide objec- 22 tive reading and allow the handling of more samples at the same time. IgG antibodies to Chlamydia heat shock protein 60 (cHSP60) have been suggested as markers of chronic inﬂammation, and may therefore be good predictors of tubal pathology. These antibodies were found in over 70% of women with occluded tubes and in <20% of women with patent tubes88. Chlamydia IgG antibody testing in serum is applied in reproductive medicine in the fertility work-up on a large scale, but it has no place in early diagnosis of Chlamydia infections. Serotyping-genotyping Currently, 19 different CT serovars and serovariants have been identiﬁed by sero- and genotyping89,90. In conventional serotyping, after culture, polyclonal and monoclonal antibodies are used against the MOMP of CT91. Nowadays genotyping is performed molecular on the Omp1 gene (which encodes for the MOMP) by PCR-based restriction fragment length polymorphism (RFLP)92-95. The serovars can be divided into three serogroups: the B-group (serovars B, Ba, D, Da, E, L1, L2, and L2a), the intermediate group (serovars F, G, and Ga) and the C-group (serovars I, Ia, J, K, C, A, H, and L2b). The most prevalent CT strains worldwide are serovars D, E, and F. They account for approximately 70% of the typed urogenital serovars. Conjunctivitis is mainly caused by serovars A-C. In urogenital infections serovars D-K are predominantly isolated. Serovars L1-L3 can be found in the inguinal lymph nodes96-99. It is still unknown why particular CT serovars run either a symptomatic or asymptomatic clinical course or have a more persistent versus quickly clearing course of infection. Several studies have shown Caroline Bax BW.indd 22 26-08-10 12:17 General Introduction 23 relationships between speciﬁc serovars and clinical disease, but the results are still contradictory. It is not unlikely that variation at the bacterial genomic level instead of only the ompA gene could give more insight in the differences in virulence, tissue tropism, disease pathogenesis and epidemiology. Furthermore, it could lead to the identiﬁcation of strains within one serovar with different pathogenic features which could explain the differences in disease manifestations found in the literature for the same serovar. Besides the use of serovar typing for epidemiology and transmission studies, also from a clinical point of view typing can be relevant since the LGV serovars need a three week treatment of doxycycline instead of one time azithromycin. Aims and outline of this thesis In the Netherlands there are no general prevention policies for uterine instrumentation, such as curettage, IUD insertion, or hysterosalpingography (HSG), for CT infections. Uterine instrumentation may lead to upper genital tract infections in women with CT infections. There is little information about the prevalence and risk factors for CT infections in a general outpatient department (OPD) of Obstetrics and Gynaecology (O&G) in the Netherlands. Are prevalence and risk factors for CT infections in a general OPD of O&G in the Netherlands comparable to other populations or other (European) countries? Therefore, we performed a prospective, observational study in the inner city of The Hague (Chapter 2). There is also discussion about which sample site in women is most effective in detecting CT infection. Is multiple site testing really necessary? We determined the incidence of multiple site or concurrent CT serovar infections and the prevalence of speciﬁc serovars in urogenital vs. rectal specimens in male and female patients attending a sexual transmitted disease (STD) clinic or OPD of Obstetrics and Gynaecology (Chapter 3). For the detection of (a past) CT infections several serological tests are available. So far they miss clinical evaluation. The MIF is considered the gold standard, but has several limitations. Can these new assays replace MIF in its role to identify those patients who need a laparoscopy for tubal pathology? Therefore, we compared the characteristics of two EIAs (pELISA and Chlamydia-EIA) in three well deﬁned populations of women (Chapter 4). Chlamydia antibody testing (CAT) is nowadays used in the fertility work-up. IgG antibodies to Chlamydia HSP60 (cHSP60) have been suggested as markers of chronic inﬂammation, and may therefore be good predictors of tubal pathology. We evaluated a commercially available cHSP60 serologic assay and determine the anti-cHSP60 responses in three groups of women with different degrees of tubal pathology (Chapter 5). Caroline Bax BW.indd 23 26-08-10 12:17 Data about speciﬁc serovars and the clinical course of infection, the rate of upper genital tract progression, and the clearance-persistence rate are inconclusive. We need to get more epidemiological data about the CT serovars and serogroups. Therefore, we focussed on the different CT serogroups and serovars, both to get insight in the serovar distribution within different ethnical groups (Chapter 6), and to get insight in the relations between the serological responses and serovars/serogroups (Chapter 7). If we know who is at risk for CT infections and if we can identify those women with a high risk of developing upper genital tract infection and the forthcoming sequelae, we can possibly take precautionary measurements when intrauterine instrumentation is required and also prevent unnecessary antibiotic use. Hopefully in the future we will be able to describe what consequences a (previous) CT infection will have in an individual patient. Chapter 1 The aim of this thesis was to gain more insight into all of these aspects. 24 Caroline Bax BW.indd 24 26-08-10 12:17 General Introduction 25 References 1. Falck G, Gnarpe J, Hansson LO, et al. Comparison of individuals with and without speciﬁc IgA antibodies to Chlamydia pneumoniae. Respiratory morbidity and the metabolic syndrome. Chest. 2002;122:1587-93. 2. Smieja M, Mahony J, Petrich A, et al. Association of circulating Chlamydia pneumoniae DNA with cardiovascular disease: a systematic review. BMC Infect Dis. 2002;2:21. 3. Numazaki K, Asanuma H, Niida Y. Chlamydia trachomatis infection in early neonatal period. BMC Infect Dis. 2003;3:2. 4. AbdelRahman YM, Belland RJ. The chlamydial developmental cycle. FEMS Microbiology Reviews. 2005;29:949-59. 5. Hackstadt T, Fischer ER, Scidmore MA, et al. Origins and functions of the chlamydial inclusion. Trends Microbiol. 1997;5:288-93. 6. Fields KA, Hackstadt T. The chlamydial inclusion: escape from the endocytic pathway. Annu Rev Cell Dev Biol. 2002;18:221-45. 7. Sacks DL, MacDonald AB. Isolation of a type-speciﬁc antigen from Chlamydia trachomatis by sodium dodecyl sulphate polyacrylamide gel electrophoresis. J Immunol. 1979;122:136-9. 8. Silins I, Ryd W, Strand A, et al. Chlamydia trachomatis infection and persistence of human papillomavirus. Int J Cancer. 2005;116:110-5. 9. Brunham RC, Peeling RW. Chlamydia trachomatis antigens: role in immunity and pathogenesis. Infect Agents Dis. 1994;3:218-33. 10. den Hartog JE, Land JA, Stassen FR, et al. Serological markers of persistent C. trachomatis infections in women with tubal pathology subfertility. Hum Reprod. 2005;20:986-90. 11. Pfeifer JD, Wick MD, Roberts RL, et al. Phagocytic processing of bacterial antigens for class I MHC presentation to T cells. Nature. 1993;361:359-62. 12. Witkin SS, Jeremias J, Toch M, et al. Cell-mediated immune response to the recombinant 57 kDa heat shock protein of Chlamydia trachomatis in women with salpingitis. J Infect Dis. 1993;167:1379-83. 13. Beatty WL, Byrne GI, Morrison RP. Repeated and persistent infection with Chlamydia and development of chronic inﬂammation and disease. Trends Microbiol. 1994;2:94-8. 14. Wilson JS, Honey E, Templeton A, et al. A systematic review of the prevalence of Chlamydia trachomatis among European women. Hum Reprod. 2002;8:385-94. 15. van Bergen JE, Spaargaren J, Götz HM, et al. and the PILOT CT study-group. Population prevalence of Chlamydia trachomatis and Neisseria gonorrhoeae in the Netherlands. Should asymptomatic persons be tested during population-based Chlamydia screening also for gonorrhoea or only if chlamydial infection is found? BMC Infectious Diseases. 2006;6:42. 16. van Bergen J, Götz HM, Richardus JH, et al. and PILOT CT study-group. Prevalence of urogenital Chlamydia trachomatis increases signiﬁcantly with level of urbanisation and suggests targeted screening approaches: results from the ﬁrst national population based study in the Netherlands. Sex Transm Infect. 2005;81:17-23. 17. Andersen B, Olesen F, Møller JK, et al. Population-based strategies for outreach screening of urogenital Chlamydia trachomatis infections: a randomized, controlled trial. J Infect Dis. 2002;185:252-8. 18. Macleod J, Salisbury C, Low N, et al. Coverage and uptake of systematic postal screening for genital Chlamydia trachomatis and prevalence of infection in the United Kingdom general population: cross sectional study. BMJ. 2005;330:940. 19. Williams H, Tabrizi SN, Lee W, et al. Adolescence and other risk factors for Chlamydia trachomatis genitourinary infection in women in Melbourne, Australia. Sex Transm Infect. 2003;79;31-4. 20. Radcliffe KW, Ahmad S, Gilleran G, et al. Demographic and behavioural proﬁle of adults infected with Chlamydia: a case-control study. Sex Transm Infect. 2001;77:265-70. 21. Verhoeven V, Avonts D, Meheus A, et al. Chlamydia infection: an accurate model for opportunistic screening in general practice. Sex Transm Infect. 2003;79:313-7. Caroline Bax BW.indd 25 26-08-10 12:17 Chapter 1 26 22. Götz HM, van Bergen JE, Veldhuijzen IK, et al. A prediction rule for selective screening of Chlamydia trachomatis infection. Sex Transm Infect. 2005;81:24-30. 23. Zimmerman HL, Potterat JJ, Dukes RL, et al. Epidemiologic differences between Chlamydia and gonorrhea. Am J Public Health. 1990;80:1338-42. 24. Weström L, Joesoef R, Reynolds G, et al. Pelvic inﬂammatory disease and fertility: a cohort study of 1844 women with laparoscopically veriﬁed disease and 657 control women with normal laparoscopic results. Sex Transm Dis. 1992;19:185-91. 25. Lan J, van den Brule AJ, Hemrika DJ, et al. Chlamydia trachomatis and ectopic pregnancy: retrospective analysis of salpingectomy specimens, endometrial biopsies and cervical smears. J Clin Pathol. 1995;48:815-9. 26. Gencay M, Koskiniemi M, Ammala P, et al. Chlamydia trachomatis seropositivity is associated both with stillbirth and preterm delivery. APMIS. 2000;108:584-8. 27. Mårdh PA. Inﬂuence of infection with Chlamydia trachomatis on pregnancy outcome, infant health and life-long sequelae in infected offspring. Best Pract Res Obstet Gynaecol. 2002;16:847-64. 28. Baud D, Regan L, Greub G. Emerging role of Chlamydia and Chlamydia-like organisms in adverse pregnancy outcomes. Curr Opin Infect Dis. 2008;21:70-6. 29. Wu S, Shen L, Liu G. Study on vertical transmission of Chlamydia trachomatis using PCR and DNA sequencing. Chin Med J. 1999;112:396-9. 30. Gencay M, Koskiniemi M, Fellman V, et al. Chlamydia trachomatis infection in mothers with preterm delivery and in their newborn infants. APMIS. 2001;109:636-40. 31. Bell TA, Stamm WE, Kuo CC, et al. Risk of perinatal transmission of Chlamydia trachomatis by mode of delivery. J Infect. 1994;29:165-9. 32. Rours GI, Hammerschlag MR, van Doornum GJ, et al. Chlamydia trachomatis respiratory infection in Dutch infants. Arch Dis Child. 2009;94:705-7. 33. Rours IG, Hammerschlag MR, Ott A, et al. Chlamydia trachomatis as a cause of neonatal conjunctivitis in Dutch infants. Pediatrics. 2008;121:e321-6. 34. Weiss SG, Newcomb RW, Beem MO. Pulmonary assessment of children after chlamydial pneumonia of infancy. J Pediatr. 1986;108:659-64. 35. Rahman MU, Cheema MA, Schumacher HR. Molecular evidence for the presence of Chlamydia in the synovium of patients with Reiter’s syndrome. Arthritis Rheum. 1992;35:521-9. 36. Peipert JF. Clinical practice. Genital chlamydial infections. N Engl J Med. 2003;349:2424-30. 37. Cunningham KA, Beagley KW. Male genital tract chlamydial infection: implications for pathology and infertility. Biol Reprod. 2008;79:180-9. 38. Wasserheit JN, Bell TA, Kiviat NB, et al. Microbiol causes of proven pelvic inﬂammatory disease and efﬁcacy of clindamycin and tobramycin. Ann Intern Med. 1986;104:187-93. 39. Bowie WR, Jones H. Acute pelvic inﬂammatory disease in outpatients: association with Chlamydia trachomatis and Neisseria Gonorrhoeae. Ann Intern Med. 1986;95:686-8. 40. Weström L. Introductory address: treatment of pelvic inﬂammatory disease in view of etiology and risk factors. Sex Transm Dis. 1984;11:437-40. 41. Cates W, Wasserheit JN. Genital chlamydial infections: epidemiology and reproductive sequelae. Am J Obstet Gynecol. 1991;164:1771-81. 42. Wolner-Hanssen P. Silent pelvic inﬂammatory disease: is it overstated? Obstet Gynecol. 1995;86:312-5. 43. Morcos R, Frost N, Hnat M, et al. Laparoscopic versus clinical diagnosis of acute pelvic inﬂammatory disease. J Reprod Med. 1993;38:53-6. 44. Bevan CD, Johal BJ, Muntaz G, et al. Clinical, laparoscopic and microbiological ﬁndings in acute salpingitis: a report on a United Kingdom cohort. Br J Obstet Gynaecol. 1995;102:407-14. 45. Simms I, Stephenson JM. Pelvic inﬂammatory disease epidemiology: what do we know and what do we need to know? Sex Transm Infect. 2000;76:80-7. 46. Boeke AJ, van Bergen JE, Morré SA, et al. De kans op pelvic inﬂammatory disease bij urogenitale infectie met Chlamydia trachomatis; literatuuronderzoek. Ned Tijdschr Geneeskd. 2005; 149:878-84. Caroline Bax BW.indd 26 26-08-10 12:17 General Introduction 27 47. Brunham RC, Peeling RW. Chlamydia trachomatis antigens: role in immunity and pathogenesis. Infect Agents Dis 1994;3:218-33. 48. Mårdh PA. Tubal factor infertility, with special regard to chlamydial salpingitis. Curr Opin Infect Dis. 2004;17:49-52. 49. Haddix AC, Hillis SD, Kassler WJ. The cost effectiveness of azithromycin for Chlamydia trachomatis infections in women. Sex Transm Dis. 1995;22:274-80. 50. Weström L, Bengtsson LP, Mårdh PA. Incidence, trends, and risk factors of ectopic pregnancy in a population of women. Br Med J (Clin Res Ed). 1981;282:15-8. 51. McCormack WM. Pelvic inﬂammatory disease. N Engl J Med. 1994;330:115-9. 52. van Valkengoed IG, Morré SA, van den Brule AJ, et al. Overestimation of complication rates in evaluations of Chlamydia trachomatis screening programs-implications for cost-effectiveness analyses. Int J Epidemiol. 2004;33:416-25. 53. Morré SA, van den Brule AJ, Rozendaal L, et al. The natural course of asymptomatic Chlamydia trachomatis infections: 45% clearance and no development of clinical PID after one-tear follow-up. Int J STD AIDS. 2002;13:Suppl 2:12-8. 54. Low N. Natural history: have we overestimated the incidence of Chlamydia complications? ISSTDR. Amsterdam 2005;TW-306. 55. Manavi K, McMillan A, Young H. Genital infections in male partners of women with chlamydial infections. Int J STD AIDS. 2006;17:34-6. 56. Markos AR. The concordance of Chlamydia trachomatis genital infection between sexual partners, in the era of nucleic acid testing. Sex Health. 2005;2:23-4. 57. Parks KS, Dixon PB, Richey CM, et al. Spontaneous clearance of Chlamydia trachomatis infection in untreated patients. Sex Transm Dis. 1997;24:229-35. 58. Molano M, Meijer CJ, Weiderpass E, et al. The natural course of Chlamydia trachomatis infection in asymptomatic Colombian women: a 5-year follow-up study. J Infect Dis. 2005;191:907-16. 59. Lau CY, Qureshi AK. Azithromycin versus doxycycline for genital chlamydial infections: a meta-analysis of randomized clinical trials. Sex Transm Dis. 2002;29:497-502. 60. Pitsouni E, Lavazzo C, Athanasiou S, et al. Single-dose azithromycin versus erythromycin or amoxicillin for Chlamydia trachomatis infection during pregnancy: a meta-analysis of randomised controlled trials. Int J Antimicrob Agents. 2007;30:213-21. 61. Sweet RL. Treatment strategies for pelvic inﬂammatory disease. Expert Opin Pharmacother. 2009;10:82337. 62. Somani J, Bhullar VB, Workowski KA, et al. Multiple drug-resistant Chlamydia trachomatis associated with clinical treatment failure. J Infect Dis. 2000;181:1421-7. 63. Misyurina OY, Chipitsyna EV, Finashutina YP, et al. Mutations in a 23S rRNA gene of Chlamydia trachomatis associated with resistance to macrolides. Antimicrob Agents. 2004;48:1347-9. 64. Wang SA, Papp JR, Stamm WE, et al. Evaluation of antimicrobial resistance and treatment failures for Chlamydia trachomatis: a meeting report. J Infect Dis. 2005;191:917-23. 65. Bianchi A, De Barbeyrac B, Bebear C, et al. Multi-laboratory comparison of 28 commercially available Chlamydia trachomatis tests. In: Chlamydial Infections. Proceedings of the 9th International Symposium on Human Chlamydia Infection, Napa, California, USA, pp 587-594, 1998. 66. Land JA, van Bergen JEAM, Morré SA, et al. Epidemiology of Chlamydia trachomatis infection in women and the cost-effectiveness of screening. Hum Reprod Update. 2010;16:189-204. 67. Goessens WHF, Mouton JW, van der Meijden WI, et al. Comparison of three commercially available ampliﬁcation assays, AMP CT, LCx and COBAS AMPLICOR, for detection of Chlamydia trachomatis in ﬁrst-void urine. J Clin Microbiol. 1997;35:2628-33. 68. Puolakkainen M, Hiltunen-Back E, Reunala T, et al. Comparison of performances of two commercially available tests, a PCR assay and a ligase chain reaction test, in detection of urogenital Chlamydia trachomatis infection. J Clin Microbiol. 1998;36:1489-93. Caroline Bax BW.indd 27 26-08-10 12:17 Chapter 1 28 69. Fox AS, Saxon EM, Doveikis S, et al. Chlamydia trachomatis and parainﬂuenza 2 virus: a shared antigenic determinant? J Clin Microbiol. 1989;27:1407-8. 70. Nurminen M, Leinonen M, Saikku P, et al. The genus-speciﬁc antigen of Chlamydia: resemblance to the lipopolysaccharide of enteric bacteria. Science. 1983;220:1279-81. 71. Chernesky MA, Mahoney JB, Castriciano S, et al. Detection of Chlamydia trachomatis antigens by enzyme immunoassay and immunoﬂuorescence in genital specimens from symptomatic and asymptomatic men and women. J Infect Dis. 1986;154:141-8. 72. Quinn TC, Gupta PK, Burkman RT, et al. Detection of Chlamydia trachomatis cervical infection: a comparison of Papanicolaou and immunoﬂuorescent staining with cell culture. Am J Obstet Gynecol. 1987;157:394-9. 73. Smith JW, Rogers RE, Katz BP, et al. Diagnosis of chlamydial infection in women attending antenatal and gynaecologic clinics. J Clin Microbiol. 1987;25:868-72. 74. Newhall WJ, Johnson RE, DeLisle S, et al. Head-to-head evaluation of ﬁve Chlamydia tests relative to a quality-assured culture standard. J Clin Microbiol 1999;37:681-5. 75. Michel CE, Solomon AW, Magbanua JP, et al. Field evaluation of a rapid point-of-care assay for targeting antibiotic treatment for trachoma control: a comparative study. Lancet. 2006;367:1585-90. 76. Yang JQ, Tata PV, Park-Turkel HS, et al. The application of AmpliProbe in diagnostics. Biotechniques. 1991;11:392-7. 77. Clavel C, Masure M, Putaud I, et al. Hydrid capture II, a new sensitive test for human papillomavirus detecion. Comparison with hybrid capture I and PCR results in cervical lesions. J Clin Pathol. 1998;51:73740. 78. Iwen PC, Blair TM, Woods GL. Comparison of the Gen-Probe PACE 2 system, direct ﬂuorescentantibody, and cell culture for detecting Chlamydia trachomatis in cervical specimens. Am J Clin Pathol. 1991;95:578-82. 79. Black CM, Marrazzo J, Johnson RE, et al. Head-to-head multicenter comparison of DNA probe and nucleic acid ampliﬁcation tests for Chlamydia trachomatis infection in women performed with an improved reference standard. J Clin Microbiol. 2002;40:3757-63. 80. Brokenshire MK, Say PJ, van Vonno AH, et al. Evaluation of the microparticle enzyme immunoassay Abbott IMx Select Chlamydia and the importance of urethral site sampling to detect Chlamydia trachomatis in women. Genitourin Med. 1997;73:498-502. 81. Wiesenfeld HC, Heine RP, Rideout A, et al. The vaginal introitus, a novel site for Chlamydia trachomatis testing in women. Am J Obstet Gynecol. 1996;174:1542-6. 82. Chernesky MA, Martin DH, Hook EW, et al. Ability of new APTIMA CT and APTIMA GC assays to detect Chlamydia trachomatis and Neisseria gonorrhoeae in male urine and urethral swabs. J Clin Microbiol. 2005;43:127-31. 83. Carré H, Edman AC, Boman J, et al. Chlamydia trachomatis in the throat: is testing necessary? Acta Derm Venereol. 2008;88:187-8. 84. Gijsen AP, Land JA, Goossens VJ, et al. Chlamydia antibody testing in screening for tubal factor subfertility: the signiﬁcance of IgG antibody decline over time. Hum Reprod. 2002;17:699-703. 85. Gijsen AP, Land JA, Goossens VJ, et al. Chlamydia pneumoniae and screening for tubal factor subfertility. Hum Reprod. 2001;16:487-91. 86. Gijsen AP, Goossens VJ, Land JA, et al. The predictive value of chlamydia antibody testing (CAT) in screening for tubal factor subfertility: micro-immunoﬂuorescence (MIF) versus ELISA, p.101. In P. Saikku (ed.), Proceedings Fourth Meeting of the European Society for Chlamydia Research, Helsinki, Finland, 2000. 87. Land JA, Gijsen AP, Kessels AG, et al. Performance of ﬁve serological Chlamydia antibody tests in subfertile women. Hum Reprod. 2003;18:2621-7. 88. Freidank HM, Clad A, Herr AS, et al. Immune response to Chlamydia trachomatis heat-shock protein in infertile female patients and inﬂuence of Chlamydia pneumoniae antibodies. Eur J Clin Microbiol Infect Dis. 1995;14:1063-9. Caroline Bax BW.indd 28 26-08-10 12:17 General Introduction 29 89. Morré SA, Ossewaarde JM, Lan J, et al. Serotyping and genotyping of genital Chlamydia trachomatis isolates reveal variants of serovars Ba, G, and J as conﬁrmed by omp1 nucleotide sequence analysis. J Clin Microbiol. 1998;36:345-51. 90. Dean D and Miller K. Molecular and mutation trend analysis of omp1 alleles for serovar E of Chlamydia trachomatis. Implications for the immunopathogenesis of disease. J Clin Invest. 1997;99:475-83. 91. Ossewaarde JM, Rieffe M, de Vries A, et al. Comparison of two panels of monoclonal antibodies for determination of Chlamydia trachomatis serovars. J Clin Microbiol. 1994;32:2968-74. 92. Frost EH, Deslandes S, Veilleux S, et al. Typing of Chlamydia trachomatis by detection of restriction fragment length polymorphism in the gene encoding the major outer membrane protein. J Infect Dis. 1991;163:1103-7. 93. Meijer A, Morré SA, van den Brule AJ, et al. Genomic relatedness of Chlamydia isolates determined by ampliﬁed fragment length polymorphism analysis. J Bacteriol. 1999;181:4469-75. 94. Molano M, Meijer CJ, Morré SA, et al. Combination of PCR targeting the VD2 of opm1 and reverse line blot analysis for typing of urogenital Chlamydia trachomatis serovars in cervical scrapes specimens. J Clin Microbiol. 2004;42:2935-9. 95. Morré SA, Moes R, van Valkengoed I, et al. Genotyping of Chlamydia trachomatis in urine specimen will facilitate large epidemiological studies. J Clin Micobiol. 1998;36:3077-8. 96. van der Laar MJ, van Duynhoven YT, Fennema JS, et al. Differences in clinical manifestations of genital chlamydial infections related to serovars. Genitourin Med. 1996;72:261-5. 97. Morré SA, Rozendaal L, van Valkengoed IGM, et al. Urogenital Chlamydia trachomatis serovars in men and women with symptomatic or asymptomatic infection: an association with clinical manifestation? J Clin Microbiol. 2000;38:2292-6. 98. 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 Fransisco. J Infect Dis. 1995;172:1013-22. 99. Yang CL, Zhang Y-X, Watkins NG, et al. DNA sequence polymorphism of the Chlamydia trachomatis omp1 gene. J Infect Dis. 1993;168:1225-30. Caroline Bax BW.indd 29 26-08-10 12:17 Caroline Bax BW.indd 30 26-08-10 12:17 Part I Clinical characteristics; prevalence and risk factors Caroline Bax BW.indd 31 26-08-10 12:17 Caroline Bax BW.indd 32 26-08-10 12:17 part I | Chapter 2 Clinical characteristics of Chlamydia trachomatis infections in a general outpatient department of Obstetrics and Gynaecology in the Netherlands C.J. Bax P.M. Oostvogel J.A.E.M. Mutsaers R. Brand M. Craandijk J.B. Trimbos P.J. Dörr Sex Transm Infect. 2002;78(6):E6. Caroline Bax BW.indd 33 26-08-10 12:17 Abstract Objectives: Evaluation of prevalence and risk factors of Chlamydia trachomatis infections (CTI) in an outpatient obstetrical and gynaecological population. Methods: A prospective, observational study was performed at an inner city hospital in The Hague, the Netherlands. Thirteen hundred and sixty-eight women attending the outpatient department of Obstetrics and Gynaecology participated in the study. For detection of CTI we used: ampliﬁcation of CT rRNA in urine samples (Gen Probe/AMPLIFIED-CT), and DNA-probe for detection of CT rRNA from a urethral, endocervical and anal swab (Gen Probe/PACE 2). Results: The overall prevalence of CTI in our general obstetrical and gynaecological population was 4.5%. The prevalence in women under 30 years of age was 8.1%. We found age and postcoital bleeding to be signiﬁcant risk factors. We did not ﬁnd signiﬁcant differences between women from different ethnic origin or between women using different kinds of contraceptives. Twelve (19.4%) CTI-patients were part I | Chapter 2 found positive by urine test only, and 15 (24.2%) only by DNA-probe. 34 Conclusions: Age is the most important risk factor in our population (overall prevalence 4.5%, prevalence in women under 20 years of age 15.8%). Analyses of urine and of endocervical specimens are complementary for the determination of the prevalence of CT infections in women. Cost-effectiveness analysis is needed to determine to what extent age-based screening and/or antibiotic prophylaxis before uterine instrumentation is indicated. Caroline Bax BW.indd 34 26-08-10 12:17 Clinical characteristics of Chlamydia trachomatis infections 35 Introduction Chlamydia trachomatis infections (CTI) are the most common sexually transmitted infections1. CTI in women may cause pelvic inﬂammatory disease (PID) and subsequently result in tubal factor subfertility and ectopic pregnancy2. Finally CTI in pregnancy may cause neonatal conjunctivitis and pneumonia3. Approximately 70% of the CTI in women are asymptomatic. The prevalence in asymptomatic obstetrical and gynaecological patients in Europe ranges from 0.8 to 4.8%4,5. Uterine instrumentation such as curettage and a hysterosalpingogram in women with a CTI may cause PID as well6. In The Netherlands there are no general policies for uterine instrumentation. We would like to establish general policies to prevent intra-abdominal CTI. Therefore, information about the clinical aspects of CTI, patient characteristics and data on the prevalence of CTI in an outpatient department of Obstetrics and Gynaecology are needed. Methods Patients From June 1996 to September 1997, new patients attending the outpatient department of Obstetrics and Gynaecology of the Westeinde Hospital were requested to participate in the study. Approval was obtained by the ethics committee. The Westeinde Hospital is an inner city hospital, with a high percentage of patients from different ethnic origin. After informed consent a questionnaire was completed and a gynaecologic examination was performed. Detection methods For detection of CTI two methods were used: (i) A probe hybridisation assay from a urethral, an endocervical and an anorectal swab (PACE 2 assay) [Gen-Probe]. Swabs were analysed within 24 hours according to Gen-Probe’s packet insert instructions. (ii) Ampliﬁcation of CT-rRNA by Transcription-Mediated Ampliﬁcation (TMA) in urine samples with the Gen-Probe AMP CT assay. Urine specimens were collected before swab specimens were gathered and stored at +4 ºC. The urine was analysed on a weekly basis according to Gen-Probe procedures. Deﬁnition of CTI: when either swab- or urine analysis was positive, a patient was considered positive for CTI at the time of sampling. Statistical analysis For descriptive purposes we used cross tabulations. To estimate the probability of chlamydial infections as a function of the various possible risk factors we used a logistic regression model. Risk factors, as found in the literature and assessed by the questionnaire and gynaecological examination were entered into the model after which a backwards elimination was performed. Based on the ﬁnal model we Caroline Bax BW.indd 35 26-08-10 12:17 computed the effects of the remaining risk factors as an estimated odds ratio and its associated 95% conﬁdence interval. Results During the study period 1684 patients were enrolled. A total of 276 patients refused participation for different reasons (16.4%), data on 40 patients (2.4%) could not be analysed (incomplete data). With regard to age non-responder analysis did not reveal signiﬁcant differences with responders. Data on 1368 patients were examined (table 1A). Sixty-two patients (4.5%) were found to be positive by DNA-probe and/or urine analysis. Of the 62 patients with a CTI the DNA-probe was positive in 48 cases. Fifteen (24.2%) patients with a positive DNA-probe turned out to be negative in urine analysis. Of the CTI-patients 45 were found positive in urine analysis. Twelve (19.4%) CTI-patients were detected by positive urine test only. Table 2 shows the number of CT infected women, the number of women tested and the according prevalence for various risk factors. The CT prevalence was inversely associated with age. An increase of part I | Chapter 2 age of one year reduces the probability of infection by 10% (OR 0.90, 95 % CI 0.87-0.94, p<0.001). There was also a signiﬁcant association with postcoital bleeding. In terms of relative risk the probability of a CTI in patients with postcoital bleeding is 2.6 times as high compared to patients without this complaint (OR 2.6, 95% CI 1.06-6.6, p<0.04). Table 1. Results of Gen Probe PACE 2 assay and the AMP CT assay in the detection of Chlamydia trachomatis (n). 36 AMPLIFIED CT (urine) PACE 2 DNA probe Negative Positive Not determined Total Negative 1223 12 73 1308 Positive 15 31 2 48 Not determined 10 2 - 12 Total 1248 45 75 1368 (A) Overall results (B) Speciﬁcation of positive sampling sites of the DNA probe Endocervical 9 12 1 22 Urethral 0 6 0 6 Anorectal 3 0 0 3 Endocervical/urethral 2 7 1 10 Endocervical/anorectal 1 0 0 1 Endocervical/urethral/anorectal 0 1 0 1 Urethral/anorectal 0 1 0 1 Unknown 0 4 0 4 Caroline Bax BW.indd 36 26-08-10 12:17 Clinical characteristics of Chlamydia trachomatis infections 37 Table 2. Prevalence in relation to the characteristics of the women enrolled in the trial CT positive (n) Tested (n) Prevalence (%) Age (years) <20 12 76 15.8 20-29 34 490 6.9 30-39 11 461 2.4 40-49 5 233 2.1 50-59 0 72 0 60-69 0 20 0 70-79 0 12 0 >80 0 1 0 Unknown 0 3 0 Yes 6 59 10.2 No 52 1241 4.2 Unknown 4 68 5.9 European 20 568 3.5 Mediterranean 10 275 3.6 African 4 51 7.8 Creole 5 67 7.5 Hindu 12 236 5.1 Asian 3 48 6.3 Hispanic 3 27 11.1 Other/Unknown 5 96 5.2 32 733 4.4 Postcoital bleeding <0.04 Ethnic origin NS Contraceptive method Non p <0.001 NS Condom 4 97 4.1 OAC 22 324 6.8 Other 3 162 1.9 Unknown 1 52 1.9 Discussion The overall prevalence of CTI in our outpatient department of Obstetrics and Gynaecology was 4.5%, according with other European studies4,5. Similar risk factors as identiﬁed in other populations were independently associated with CTI4,7. As found in other studies we found signiﬁcantly higher prevalences in younger women. If women only under 30 years of age had been tested we would have found approximately 75% of the CTI patients. If women only under 40 years of age had been tested we would have missed only 5 patients (8.1%). In the United States population screening is advised for women 15-24 years of age, other studies advise age-based screening for women under 30 years of age5,8. To prevent complications it might be sensible to screen women in the fertile age (<40). Caroline Bax BW.indd 37 26-08-10 12:17 Our study conﬁrms the association between CTI and postcoital bleeding. However, since all patients with the complaint of postcoital bleeding and a CTI were under 30 years of age, this did not help to reveal more CTI patients than age-based screening alone. We could not conﬁrm the relationship between CTI and ethnic origin as described in other studies, in which a signiﬁcantly higher prevalence was found in women from Suriname and the Netherlands Antilles9. But we do see a trend in a similar direction. An even distribution of CTI was found among patients using condoms or no contraceptive methods. Other studies have described lack of barrier contraceptive as a risk factor10. A slightly higher prevalence was found in women using oral contraceptives. Nearly half of the CTI patients (44%) were detected by only one of the two diagnostic tests. Since no discrepant analysis was performed some false positive or negative results cannot be excluded. With respect to the sampling site of the DNA-probe we found that most CTI patients were found positive on the endocervical sampling site (34 patients). Eighteen patients were found positive at the urethral sampling site. It is remarkable that 6 patients were found positive at the anorectal sampling site (table 1B). For women it is advised to test urine or a urethral specimen in combination with an endocervical specimen11. Other studies found the vaginal introitus also a representative site to detect CTI, with the part I | Chapter 2 advantage of being noninvasive12. Anorectal swabs should only be obtained in women at high risk of 38 being infected. We would have missed three CTI patients if we had used only the endocervical swab and the urine analysis. These three patients were found positive by anorectal swab only. If we had used only the endocervical and urethral swab we would have missed 15 CTI patients (12 patients only positive by urine analysis and three patients only positive by anorectal swab with negative urine analysis). Although population screening on CTI with urine analysis seems to be an easy method, we conclude that analysis of urine as well as endocervical specimens is essential for determination of prevalence of CTI in women. Although DNA ampliﬁcation methods used on urine specimen are reported to be so sensitive that they may reﬂect positivity at the endocervical sampling site, our study shows discrepancies. Therefore, both analyses are complementary. Cost-effectiveness analysis will show whether age-based screening and/or antibiotic prophylaxis before uterine instrumentation will be indicated. Acknowledgements We thank prof. dr. J.B. Vandenbroucke and prof. dr. O.P. Bleker for reviewing this paper and Claire Peters for her help in processing the data. Caroline Bax BW.indd 38 26-08-10 12:17 Clinical characteristics of Chlamydia trachomatis infections 39 References 1. Gerbase AC, Rowly JT, Heymann DHL, et al. Global prevalence and incidence estimates of selected curable STDs. Sex Transm Inf. 1998;74(Suppl I):12-6. 2. Cates W, Wasserheit JN. Genital chlamydial infections: Epidemiology and reproductive sequelae. Am J Obstet Gynecol. 1991;164:1771-81. 3. Schachter J, Grossman M, Sweet RL, et al. Prospective study of perinatal transmission of Chlamydia trachomatis. JAMA. 1986;255:3374-7. 4. Warszawski J, Meyer L, Weber P. Criteria for selective screening of cervical Chlamydia trachomatis infections in women attending private gynecologic practices. Eur J Obs Gyn Reprod Biol. 1999;86:5-10. 5. Macmillan S, McKenzie H, Flett G, et al. Which women should de tested for Chlamydia trachomatis? Br J Obstet Gynaecol. 2000;107:1088-93. 6. Penney GC, Thomson M, Norman J, et al. A randomized comparison of strategies for reducing infective complications of induced abortion. Br J Obstet Gynaecol. 1998;105:599-604. 7. Stergachis A, Scholes D, Heidrich FE, et al. Selective screening for Chlamydia trachomatis infections in a primary care population of women. Am J Epidemiol. 1993;138:143-53. 8. Centers for Disease Control and Prevention. Recommendations for the prevention and management of Chlamydia trachomatis infections, 1993. MMWR Morb Mortal Wkly Rep. 1993;42(RR-12):1-39. 9. van den Hoek JAR, Mulder-Folkerts DKF, Courtinho RA, et al. Opportunistische screening op genitale infecties met Chlamydia trachomatis onder de seksueel actieve bevolking van Amsterdam. I. Meer dan 90% deelname en bijna 5% prevalentie. Ned Tijdschr Geneeskd.1999;143:668-72. 10. Handsﬁeld HH, Jasman LL, Roberts PL, et al. Criteria for selective screening for Chlamydia trachomatis infections in women attending family planning clinics. JAMA. 1986;225:1730-4. 11. Brokenshire MK, Say PJ, van Vonno AH, et al. Evaluation of the microparticle enzyme immunoassay Abbott IMx Select Chlamydia and the importance of urethral site sampling to detect Chlamydia trachomatis in women. Genitourin Med. 1997;73:498-502. 12. Wiesenfeld HC, Heine RP, Rideout A, et al. The vaginal introitus: A novel site for Chlamydia trachomatis testing in women. Am J Obstet Gynecol. 1996;174:1542-6. Caroline Bax BW.indd 39 26-08-10 12:17 Caroline Bax BW.indd 40 26-08-10 12:17 part I | Chapter 3 Analyses of multiple site and concurrent Chlamydia trachomatis serovar infections, and serovar tissue tropism for urogenital versus rectal specimens in male and female patients C.J. Bax K.D. Quint R.P.H. Peters S. Ouburg P.M. Oostvogel J.A.E.M. Mutsaers P.J. Dörr S. Schmidt C. Jansen A.P. van Leeuwen W.G.V. Quint J.B. Trimbos C.J.L.M. Meijer S.A. Morré Submitted Sex Transm Infect. 2010 Caroline Bax BW.indd 41 26-08-10 12:17 Abstract Objectives: The aims of the current study were to determine the incidence of concurrent infections on a serovar level, to determine the incidence of multiple anatomical infected sites on CT detection and serovar level, and analysis of site speciﬁc serovar distribution, to identify tissue tropism in urogenital vs. rectal specimens. Methods: Chlamydia trachomatis (CT) infected patients in two populations were analysed in this study: 75 patients visiting the outpatient department of Obstetrics and Gynaecology of the MC Haaglanden and 358 patients visiting the outpatient STD clinic, The Hague, the Netherlands. The PACE 2 assay (GenProbe) was used for detection of CT from urethral, cervical, vaginal, oropharyngeal, and anorectal swabs. CT genotyping was determined on all CT positive samples, using the CT-DT genotyping assay. Results: Samples of 433 patients (256 female and 177 male) with conﬁrmed CT infection were analysed. In 11 patients (2.6%) concurrent serovars in one anatomical sample site were present. In 62 (34.1%) female part I | Chapter 3 and four (9.3%) male patients multiple sample site infections were found. A considerable percentage of 42 women tested on the cervical/vaginal and rectal site were found positive on both sites (36.1%, 22 out of 61). In men, D/Da and G/Ga serovars were more prevalent in rectal than urogenital specimens (p= 0.0081 and p=0.0033, respectively) while serovar E was more prevalent in urogenital specimens (p=0.0012). Conclusions: The prevalence of multiple serovar infections is relative low. Signiﬁcant differences in serovar distribution are found in rectal specimens of men with serovar G/Ga as the most prominent, suggesting tissue tropism. Caroline Bax BW.indd 42 26-08-10 12:17 Multiple site and concurrent serovar infections 43 Introduction Chlamydia trachomatis (CT) is the most prevalent bacterial sexually transmitted disease (STD) worldwide. Many infections are asymptomatic and, if undiagnosed and untreated, they provide a reservoir for the disease and long term complications1. The most common sample types are cervical, urethral, and vaginal swabs, and ﬁrst-void urine. Depending on sexual behaviour rectal and pharyngeal swabs can also be taken. Nineteen CT serovars have been identiﬁed causing different types of infections; A-C cause ocular infections, D-K anogenital infections, and the serovars L1-L3 cause the disease lymphogranuloma venereum (LGV)2-4. Based on similarities on the major outer membrane protein (MOMP), the serovars can be divided into three serogroups, namely the B-group (serovars B, Ba, D, Da, E, L1, L2, and L2a), the intermediate group (serovars F, G, and Ga), and the C-group (serovars I, Ia, J, K, C, A, H, and L3). The most prevalent CT strains worldwide are serovars D, E, and F, accounting for approximately 70% of the typed urogenital serovars4-8. Serovar identiﬁcation is clinically important since, for example, the LGV serovars needs different treatment than the other ano-urogenital serovars D-K9-11. Most of the previous studies on CT serovar distribution focused on one anatomical site, usually the urogenital tract. However, when there is a preference of speciﬁc serovars for speciﬁc sample sites, i.e. urogenital vs. rectal, serovar distributions might differ. Studies have reported 2-15% multiple serovar infections in one anatomical site and widespread percentages of concurrent anatomical site infection5-7,12-14. Lan et al. found 5/37 women with a single identical serovar infection in multiple sample sites and 2/37 women with different serovars in multiple sample sites. No concurrent infections were found in men15. It has been suggested that the prevalence of infection varies by anatomical site, and that serovar G/Ga infects more common the rectum, while others are more common in the cervix/vagina16-18. Since there is limited information on this subject, the current study has three aims: to determine the number of concurrent infections on a serovar level, to determine the percentage of multiple anatomical infected sites on CT detection and serovar level, and analysis of site speciﬁc serovar distribution, to identify tissue tropism in urogenital vs. rectal specimens. Methods Specimen collection From January - October 2008, 433 CT infected patients in two populations were analysed in this study: patients visiting the outpatient department of Obstetrics and Gynaecology (OPD O&G) of the MC Haaglanden and patients visiting the outpatient STD clinic in the centre of The Hague, the Netherlands. The population was described in more detail in a serovar distribution study19. Caroline Bax BW.indd 43 26-08-10 12:17 CT detection and genotyping For the detection of CT a probe hybridisation assay was used on urethral, cervical, vaginal, oropharyngeal, and anorectal swabs (PACE 2 assay, Gen-Probe). For urine analysis ampliﬁcation of CT-rRNA by transcription-mediated ampliﬁcation (TMA) was used in urine samples with the Gen-Probe AMP CT assay. CT ampliﬁcation and genotyping were performed on all samples positive for CT, using the CT-DT detection and genotyping assay (Labo Biomedical Products BV, the Netherlands). All analyses were performed according to the manufacturer’s instructions and described previously20. Statistical analysis Serogroup and serovar distributions were compared, using χ2 and Fisher’s Exact statistics. A p-value < 0.05 was considered as statistically signiﬁcant. part I | Chapter 3 Results 44 During the study period samples of 433 patients (256 female and 177 male) were collected sequentially and were used for CT serovar and typing. Three male patients were excluded because of gender and sample site mismatch. Concurrent serovar infections per sampling site In 11 patients (2.6%: 5.3 % in OPD O&G, and 2.0% (2.2% in F and 1.7% in M) in STD) concurrent serovar infections in one sample site were found (table 1). Eight of these eleven patients were infected with the serovars E, F, or serovar G/Ga. Nine patients had serovars from different serogroups. Three patients had different serovars from the same serogroup. In four patients it was only possible to identify the serogroup, but not the serovar. Multiple sample site infections on a CT detection and serovar level The DNA probe (PACE2) results of tested sample sites are shown in table 2. In our OPD O&G population all patients (n=71) were tested at both the cervical and urethral sampling sites. Twenty-seven patients were positive on the cervical sampling site only, 38 were positive on both sites, and six patients were positive on the urethral sampling site only. In the female STD clinic population (n=177), 168 patients were positive on the cervical or vaginal sampling sites. Of the remaining nine patients, ﬁve were positive on the rectal site, three on the pharyngeal site, and one in urine analysis. In 19.8% (n=35) of the patients only one sample was taken (27 vagina only, seven cervix only, and one urine only). A considerable percentage of women tested on the cervical/vaginal and rectal site were found positive on both sites (36.1%, 22 out of 61). In the male STD population (n=170), 146 were positive on the urethral sampling site or in urine analysis. Of the remaining 24 patients, 20 are positive on the rectal site, two on the pharyngeal site, and two Caroline Bax BW.indd 44 26-08-10 12:17 Multiple site and concurrent serovar infections 45 Table 1. The distribution of concurrent CT serovars per sampling site. Sample site Sex Cervix (n=87) Vagina (n=142) Urethra (n=86) M# Rectum (n=47) Urine (n=105) I-group (F) I-group (G/Ga) M# B-group (E) C-group (K) M# B-group (E) I-group (F) F# I-group (F) C-group (?) F# B-group (E) C-group (J) F# C-group (H) C-group (K) F# B-group (E) C-group (J) F* I-group (G/Ga) C-group (?) F* C-group (H) C-group (K) F* B-group (E) C-group (?) F* B-group (D/Da) C-group (?) M=male, F=female; Serovars presented as Serogroup with Serovar; ?=unknown/untypeable serovar; * patients from the OPD O&G; # patients from the STD clinic. on both rectal and pharyngeal site. In 74.7% (n=127) of the patients only one sample was taken (98 urine only, 28 urethra only, and one pharynx only). Serovars could not be determined in all DNA probe positive patients. Thirteen CT positive rectal samples were not available for serovar determination. In the remaining samples used for serovar determination multiple sample site infections were observed in 62 (34.1%) female (38 from the OPD O&G) and four (9.3%) male patients. In the female patient group 43 patients were positive on the cervix/vagina and urethra, ten on the cervix/vagina and rectum, eight on the cervix/vagina and pharynx, and one patient on the vagina, rectum, and pharynx. In the male patient group three patients were positive on the rectum and pharynx, and one patient on the urethra and pharynx. In all but one patient (male, rectum and pharynx) the same serovars were observed. Single serovar and anatomical sites Overall, the serovars D, E and F were the most prevalent in the cervical (12.6%, 42.5%, and 25.3%, respectively) and urethral (11.6%, 41.9%, and 22.1%, respectively) sample sites. In all other sites serovar G/Ga was the third most prevalent serovar (vagina: E 34.5%, F 23.9%, and G/Ga 16.2%; urine: E 41.9%, Caroline Bax BW.indd 45 26-08-10 12:17 part I | Chapter 3 Table 2. Multiple site infections (DNA probe (PACE2) positive) in male and female patients tested at multiple sample sites. 46 Female* All sites Sample sites Tested (n) positive (n) % Tested (n) Male‡ Positive (n) % Cervix/Vagina + Urethra 75 43 57,3 - - - Cervix/Vagina + Rectum 29 19 65,5 - - - Cervix/Vagina + Pharynx 139 7 5,0 - - - Cervix/Vagina + Rectum + Pharynx 31 2 6,5 - - - Cervix/Vagina + Urethra + Rectum 1 1 100 - - - Urethra/Urine + Rectum - - - 39 5 12,8 Urethra/Urine + Pharynx - - - 41 1 2,4 Rectum + Pharynx - - - 38 2 5,3 Urethra/Urine + Rectum + Pharynx - - - 39 1 2,6 All sites * All, but one female patient (tested urine only) were tested on the cervical/vaginal sample site. ‡ All, but one male patient (tested at pharyngeal site only) were tested at the urethral site/urine. This table shows combinations of sample sites. Some patients were tested at more sites than the two mentioned, but the third site was then always negative. i.e. female cx/vag + rc n= 32 tested, but 31 were also pharynx tested and 1 urethra. F 21.9%, and G/Ga 12.4%; oropharynx: D/Da 29.4%, E 41.2%, and G/Ga 11.8%; rectum: D/Da 27.7%, E 21.3%, and G/Ga 34.0%). In all sample sites (except rectum) serovar E was the most prevalent. When the serovar distribution between rectal and urogenital specimens was compared no differences for women were observed (Figure 1). In women, 16 serovars D/Da were identiﬁed among 167 urogenital (cervix and vagina) specimens (9.6%), while in rectal specimens 3 (33.3%) serovars D/Da were observed. Although in rectal specimens the percentage of serovars D/Da was three times the percentage in urogenital specimens, this difference was only borderline signiﬁcant, possibly due to the low sample size of rectal infections (p=0.0592). In men, signiﬁcant differences were found for the serovars D/Da, E, and G/Ga between 25 rectal and 140 urogenital (urethra and urine) specimens. In 28% (n=7) of the rectal specimens serovar D/Da was identiﬁed versus 7.9% (n=11) in urogenital specimens (p=0.0081; OR 4.6, 95% CI 1.6-13.3). For serovar E we found 40.7% (n=57) in urogenital specimens and 8% (n=2) in rectal specimens (p=0.0012; OR 7.8, 95% CI 1.8-35). And in the male rectal specimens 10 contained serovar G/Ga (40%), while 19 urogenital specimens (13.6%) contained serovar G/Ga identical to the percentage found in women (p=0.0033; OR 4.2, 95% CI 1.7-11). Caroline Bax BW.indd 46 26-08-10 12:17 Multiple site and concurrent serovar infections 47 6HURYDU ' ( * 3 XURJHQLWDO UHFWDO 6HURYDU ' 3 ( 3 * 3 XURJHQLWDO UHFWDO Figure 1. Serovar D/Da, E and G/Ga distribution in females and males in urogenital vs. rectal specimens. All 25 men of which rectal samples were taken were men-who-have-sex-with-men (MSM). Fourteen out of the 140 males were MSM in the group from which urogenital specimens were obtained. In these 14 men we found 6 serovars D/Da, 3 serovars G/Ga, 4 serovars J, and 1 serovar F. Discussion Concurrent serovar infections per sampling site A prevalence of concurrent serovar infections (2.6%) in the same (low) range as detected in other studies was found5-7,12-14. Barnes et al. describe seven (2%) multiple serovars; three (1.4%) of 213 cervical swabs of women visiting a STD clinic, three (10%) of cervical swabs of jailed women (mostly prostitutes), and one (0.9%) of 109 rectal swabs of MSM attending a STD clinic21. In the current study none of the women Caroline Bax BW.indd 47 26-08-10 12:17 with multiple serovars were engaged in prostitution. One of the three men with multiple serovars was bisexual, the remaining two were heterosexual. In some patients it was only possible to identify the serogroup, but not the serovar. This is probably due to a new genovariant of the serovar which does not respond to the speciﬁc serovar probe, but does respond to the more conservative serogroup probe. Also, an infection with multiple serovars within the same serogroup can be caused by a new genovariant. For example, a new genovariant of serovar K was revealed by sequencing potential multiple infections of serovar H&K (both serogroup C) in women visiting a women’s health clinic in Uganda20. This leads to the recommendation to sequence multiple infections belonging to the same serogroup, to exclude new variants (not performed in the current study). Multiple sample site infections In 72 (33.8%) of the female patients in which multiple samples were taken, DNA probe (PACE 2) results were positive on more than one site. In nine (20.9%) of the male patients in which multiple samples were taken, DNA probe (PACE 2) results were positive on more than one site. All male patients were MSM. Kent et al. describe multiple site infections in 48 (10.5%) patients in a population of MSM who have been part I | Chapter 3 tested at three sites (rectum, urethra, and pharynx). In MSM who had only been tested at the urethral and 48 pharyngeal site only two (1.6%) were found positive at two sites, possibly caused by a lower CT prevalence (7.1% vs. 13.3%)16. In 66 patients serovars at multiple sample sites were positive. Dean et al. found 21% (n=7) of women to be positive at the cervical and endometrial sampling site6. Since in all but one patient the same serovar was found, serovars analysis at one sample site seems to justiﬁable. In female patients, cervix or vagina sampling revealed 94.9% of the CT infected patients. Sexual behaviour questionnaires help to reveal the other sample sites (rectum and oropharynx). In male patients, urethral site sampling or urine revealed 85.9% of the CT infected patients. In MSM rectal samples are taken as well, to detect more patients, since the proctum can be a reservoir of CT infections. The pharyngeal sampling site does not seem to contribute much to the detection of CT infected patients. Serovar and anatomical sites Worldwide, serovars D, E, and F are most prevalent, but there are some demographic changes4,5. Serovar E was the most prevalent, in our study, as well as in others4,5. In our study, we found serovar G/ Ga to be the third most prevalent serovar after D and E, in most sampling sites. CT has been identiﬁed as a co-factor of cervical neoplasia22. A previous study had demonstrated an association between CT serovar G antibodies and squamous cell carcinoma23. In adenocarinoma of the cervix no co-infection of CT and Human Papillomavirus was found24. The prevalence of serovar G/Ga was the lowest (5.7%) at the cervical sampling site in our study. Similar to our results, Lan et al. found serovar G as the third most prevalent serovar in young women visiting an OPD of Obstetrics and Gynaecology20,25. In some Asian countries higher prevalences of serovar G are observed (7-15%)26. These prevalences are observed mostly in STD clinic populations, but also in obstetrical and gynaecological patients (14.9%). Caroline Bax BW.indd 48 26-08-10 12:17 Multiple site and concurrent serovar infections 49 In men, serovars D/Da and G/Ga were signiﬁcantly more prevalent in rectal than in urogenital swabs (28% vs. 7.9% and 40% vs. 13.6%). In women a similar tendency was observed for these serovars, although not signiﬁcant (33.3% vs. 9.6% and 22.2% vs. 13.8%). In men, serovar E was more prevalent in the urogenital swabs than in the rectal swabs (40.7% vs. 8%), while in women it was approximately the same (35.3% vs. 44.4%), and in normal range as compared to other Dutch studies. The serovars B/Ba, H, I/Ia, and K were not determined in the rectal swabs in both men and women. In women serovar J and F were not found in rectal swabs. All 25 rectal samples obtained from male patients in our study were from MSM. Serovar G/Ga was signiﬁcantly more prevalent in this group, followed by serovar D/Da. In 14 MSM urogenital samples were taken. Serovar D/Da was most prevalent (42.9%) followed by serovar J (28.6%) and serovar G/Ga (21.4%). In female rectal specimens serovar E was most frequently detected. Similar results were described by Barnes et al.18. The rectal swabs were obtained from MSM. They also tested the rectal swabs of 32 women and found two serovars B, one I/Ia, and one K. In our study those serovars were not identiﬁed in men or women. It is suggested that these serovars are less viable in the rectum. The permeability to toxic substances could be inﬂuenced by the porin activity of the major outer membrane protein (MOMP), therefore serotype might reﬂect organism permeability18,27. Two explanations for the prevalence differences between rectal and urogenital specimens, not mutually exclusive are present: 1) serovars G/Ga and D/Da have a higher afﬁnity to epithelial cells of the rectum compared to urogenital epithelial cells, potentially partially mediated by the environment, suggesting tissue tropism, still based on unknown virulence factors, and 2) the high incidence of serovars G/Ga and D/Da in rectal specimens of MSM can ﬁnd its origin in differences in sexual behaviour and group dynamics compared to heterosexuals. However, since in the heterosexual women included in our study the same trend was found, serovar distribution linked to core groups is less likely as an explanation. Other studies ﬁnd similar results; most rectal Chlamydia infections were caused by serovar G/Ga (47.9%) in MSM, while in the same population the prevalence of urogenital serovar G/Ga for men and women was much lower (16% vs. 11% resp.)17,28. In San Francisco, rectal specimens of MSM were tested in two populations16. The prevalence of CT infections was 8.8% and 5.7% in patients visiting a STD clinic and a Gay men’s health centre, respectively. Unfortunately, no serovar analysis was performed. Barnes et al. describe signiﬁcant higher prevalences of serovar G/Ga in cervical isolates of heterosexual women and rectal isolates of MSM18. The prevalence of serovar G/Ga (13%) in the rectal isolates is however signiﬁcantly lower than in our study (40%) (p=0.0026). Recently, Jeffrey et al. demonstrated that polymorphisms in open reading frame sequences have a correlation with different tissue tropisms of serovars. Genome sequence analysis is an effective approach to discover variable loci in Chlamydiae that are associated with clinical presentation29. In conclusion: the prevalence of multiple serovar infections at different sites of the same individual is relatively low. Therefore serovar analysis could be performed on one positive sample site. Signiﬁcant differences in serovar prevalences are found between rectal and urogenital specimens in men. The serovar Caroline Bax BW.indd 49 26-08-10 12:17 distribution in rectal specimens of MSM showed signiﬁcant differences, with serovar G/Ga as the most prominent. Acknowledgements The aims of this work are in part in line with the European EpiGenChlamydia Consortium which is supported by the European Commission within the Sixth Framework Program through contract no. part I | Chapter 3 LSHG-CT-2007-037637. See www.EpiGenChlamydia.eu for more details about this Consortium. 50 Caroline Bax BW.indd 50 26-08-10 12:17 Multiple site and concurrent serovar infections 51 References 1. Morré SA, Spaargaren J, Ossewaarde JM, et al. Description of the ICTI consortium: An integrated approach to the study of Chlamydia trachomatis infection. Drugs Today (Barc). 2006;42(Suppl. A):107-14. 2. Morré SA, Ossewaarde JM, Lan J, et al. Serotyping and genotyping of genital Chlamydia trachomatis isolates reveal variants of serovars Ba, G, and J as conﬁrmed by omp1 nucleotide sequence analysis. J Clin Microbiol. 1998;36:345-51. 3. Dean D, Miller K. Molecular and mutation trend analysis of omp1 alleles for serovar E of Chlamydia trachomatis. Implications for the immunopathogenesis of disease. J Clin Invest. 1997;99:475–83. 4. van der Laar MJ, van Duynhoven YT, Fennema JS, et al. Differences in clinical manifestations of genital chlamydial infections related to serovars. Genitourin Med. 1996;72:261-5. 5. Morré SA, Rozendaal L, van Valkengoed IGM, et al. Urogenital Chlamydia trachomatis serovars in men and women with symptomatic or asymptomatic infection: an association with clinical manifestation? J Clin Microbiol. 2000;38:2292-6. 6. 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. J Infect Dis. 1995;172:1013-22. 7. Yang CL, Zhang Y-X, Watkins NG, et al. DNA sequence polymorphism of the Chlamydia trachomatis omp1 gene. J Infect Dis. 1993;168:1225-30. 8. Quint K, Porras C, Mahboobeh S, et al. Evaluation of a novel PCR-based assay for detection and identiﬁcation of Chlamydia trachomatis serovars in cervical specimens. J Clin Microbiol. 2007;45:3986-91. 9. De Vries HJC, Smelov V, Middelburg JG, et al. Delayed microbial cure of Lymphogranuloma venereum proctitis with doxycycline treatment. Clin Infect Dis. 2009;48:e53-6. 10. Spaargaren J, Schachter J, Moncada J, et al. Slow epidemic of lymphogranuloma venereum L2b strain. Emerg Infect Dis. 2005;11:1787-8. 11. Morré SA, Spaargaren J, Fennema JS, et al. Real-time PCR for the diagnosis of lymphogranuloma venereum. Emerg Infect Dis. 2005;11:1311-2. 12. Batteiger BE, Lennington W, Newhall WJ, et al. Correlation of infecting serovar and local inﬂammation in genital chlamydial infections. J Infect Dis. 1989;160:332-6. 13. Brunham RC, Kimani J, Bwayo J, et al. The epidemiology of Chlamydia trachomatis within a sexually transmitted disease core group. J Infect Dis. 1996;173:950-6. 14. Brunham RC, Yang C, Maclean I, et al. Chlamydia trachomatis from individuals in a sexually transmitted disease core group exhibit frequent sequence variation in the major outer membrane protein (omp1) gene. J Clin Invest. 1994;94:458-63. 15. Lan J, Meijer CJ, van den Hoek AR, et al. Genotyping of Chlamydia trachomatis serovars derived from heterosexual partners and a detailed genomic analysis of serovar F. Genitourin Med. 1995;71:299-303. 16. Kent CK, Chaw JK, Wong W, et al. Prevalence of rectal, urethral, and pharyngeal Chlamydia and Gonorrhea detected in 2 clinical settings among men who have sex with men: San Francisco, California, 2003. Clin Infect Dis. 2005;41:67-74. 17. Geisler WM, Whittington WLH, Suchland RJ, et al. Epidemiology of anorectal chlamydial and gonococcal infections among men having sex with men in Seattle: utilizing serovar and auxotype strain typing. Sex Transm Dis. 2001;29:189-95. 18. Barnes RC, Rompalo AM, Stamm WE. Comparison of Chlamydia trachomatis serovars causing rectal and cervical infections. J Infect Dis. 1987;156:953-8. 19. Bax CJ, Quint KD, Peters RP, et al. The serovar distribution of urogenital Chlamydia trachomatis strains among sexual transmitted disease clinic patients and gynaecological patients in the region of The Hague, The Netherlands: an ethnic epidemiological approach. Submitted Sex Transm Infect. 20. Quint KD, van Doorn LJ, Kleter B, et al. A highly sensitive, multiplex broad-spectrum PCR-DNA-enzyme immunoassay and reverse hybridization assay for rapid detection and identiﬁcation of Chlamydia trachomatis serovars. J Mol Diagn. 2007;9:631-8. Caroline Bax BW.indd 51 26-08-10 12:17 part I | Chapter 3 21. Barnes RC, Suchland RJ, Wang S-P, et al. Detection of multiple serovars of Chlamydia trachomatis in genital infections. J Infect Dis. 1985;152:985-9. 22. Lehtinen M, Lyytikäinen E, Koskela P, et al. Chlamydia trachomatis and risk for cervical neoplasia – a metaanalysis of longitudinal studies. ISHCI, Hof by Salzburg, 2010. 23. Anttila T, Saikku P, Koskela P, et al. Serotypes of Chlamydia trachomatis and risk for development of cervical squamous cell carcinoma. JAMA. 2001;285:47–51. 24. Quint KD, de Koning MNC, Geraets DT, et al. Comprehensive analysis of Human Papillomavirus and Chlamydia trachomatis in in-situ and invasive cervical adenocarcinoma. Gynecol Oncol. 2009;114:390-4. 25. Lan J, Melchers I, Meijer CJLM, et al. Prevalence and serovar distribution of asymptomatical cervical Chlamydia trachomatis infections as determined by highly sensitive PCR. J Clin Microbiol. 1995;33:3194-7. 26. Gao X, Chen X-S, Yin Y-P, et al. Distribution of Chlamydia trachomatis serovars among High-risk women in China performed using PCR-restriction fragment length polymorphism genotyping. J Clin Microbiol. 2007;45:1185-9. 27. Kuo C-C, Wang S-P, Holmes KK, et al. Immunotypes of Chlamydia trachomatis isolates in Seattle, Washington. Infect Immun. 1983;41:865-8. 28. Eckert LO, Suchland RJ, Hawes SE, et al. Quantitative Chlamydia trachomatis cultures: correlation of chlamydial inclusion-forming units with serovar, gender and race. J Infect Dis. 2000;182:540-4. 29. Jeffrey BM, Suchland RJ, Quinn KL, et al. Genome sequencing of recent clinical Chlamydia trachomatis strains identiﬁes loci associated with tissue tropism and regions of apparent recombination. Infect Immun. 2010;78:2544-53. 52 Caroline Bax BW.indd 52 26-08-10 12:17 Caroline Bax BW.indd 53 26-08-10 12:17 Caroline Bax BW.indd 54 26-08-10 12:17 Part II Serology Caroline Bax BW.indd 55 26-08-10 12:17 Caroline Bax BW.indd 56 26-08-10 12:17 part II | Chapter 4 Comparison of serological assays for detection of Chlamydia trachomatis antibodies in different groups of obstetrical and gynaecological patients C.J. Bax J.A.E.M. Mutsaers C.L. Jansen J.B. Trimbos P.J. Dörr P.M. Oostvogel Clin Diagn Lab Immunol. 2003;10(1):174–6. Caroline Bax BW.indd 57 26-08-10 12:17 Abstract New serological enzyme immunoassays (EIAs) were compared with microimmunoﬂuorescence (MIF) as ‘gold standard’ to detect Chlamydia trachomatis (CT) antibodies in different groups of obstetrical, gynaecological and subfertile patients. There were no signiﬁcant differences in seroprevalence rates, except for the group of CT-positive patients (p<0.01). Test characteristics were calculated for Chlamydia-EIA (Biologische Analysen-system GmbH, Lich, Germany) and pELISA (Medac, Wedel, Germany). pELISA part II | Chapter 4 seems to be a good alternative to MIF. It has high speciﬁcity and is easier to perform. 58 Caroline Bax BW.indd 58 26-08-10 12:17 Comparison of serological assays 59 Introduction Recently new commercially available species-speciﬁc (peptide-based) enzyme immunoassays (EIAs) have been developed for the detection of Chlamydia trachomatis (CT)-antibodies. So far they have missed clinical evaluation. Serological assays have been used to detect antichlamydial antibodies in the fertility workup10, predicting tubal pathology5,7. Their value for fertility evaluation remains the subject of debate. There is wide variation between various tests in the correlation of antichlamydial antibodies with current CT infections or tubal pathology. The species-speciﬁc microimmunoﬂuorescence assay (MIF) is considered to be the ‘gold standard’ for the serological diagnosis of CT infections12. Cross-reactivity with Chlamydia pneumoniae in the existing assays should be taken into account3. MIF is laborious and reading subjective, and therefore it is not suitable for a daily routine. EIAs provide objective reading and allow the handling of more samples at the same time. We compared two new serological assays with MIF to determine the performance of these assays in the routine serodiagnosis of CT infections. Methods For our serological studies, we divided sera from obstetrical and gynaecological patients into four different groups: subfertility patients (n=76), pregnant women (n=150), a control group which includes a randomly selected group of women who visited our outpatient department with various complaints, unrelated to subfertility or pregnancy (n=220), and women found positive for CT in a direct antigen assay (n=40). Some women in the last group were also represented in the subfertility or pregnant group (n=2 and n=5, respectively). For serological diagnosis, we used two EIAs. The CT-pELISA (Medac, Wedel, Germany) was used to perform species-speciﬁc serology by using a synthetic peptide from the immunodominant region of the major outer membrane protein. This highly speciﬁc antigen makes it possible to discriminate between CT-speciﬁc antibodies and the whole anti-chlamydia antibody response. The BAG-Chlamydia-EIA (Biologische Analysensystem GmbH, Lich, Germany) uses the ultrasonicated whole cell CT antigen (strain LGV Type 17). If CT antibodies are present in the specimen, they will react with the antigen. Both microtiter assays use peroxidase-conjugated antihuman immunoglobulin G (IgG) and IgA antibodies to bind to CT IgG and IgA antibodies. After incubation with tetramethylbenzidine substrate, the reaction is stopped by the addition of sulfuric acid. The absorption is read photometrically at 450 nm. The intensity of the colour is proportional to the concentration (or titer) of the speciﬁc antibody in the sample. Cut-off values were calculated according to the manufacturer’s instructions. Results in the grey-zone were considered negative in the calculations. Caroline Bax BW.indd 59 26-08-10 12:17 An indirect MIF antibody technique was used as a gold standard to detect CT IgG-antibodies (egg-grown CT biovar L2; BioMérieux, ‘s Hertogenbosch, the Netherlands). Sera were diluted to a titer of 1:64 in phosphate buffered saline (PBS). After incubation and washing in PBS, a conjugate (Fluoline-G; Evans blue diluted in PBS) was added to the samples. After 30 minutes incubation at 37ºC and washing in PBS, the slide was covered with a coverslip with a mounting medium. A ﬂuorescence microscope was used for the reading of the slides. A positive reaction is a ‘starry sky’ appearance: ﬂuorescent green spots on a slightly red background. Two experienced persons evaluated all samples. When discrepancies occurred a third person evaluated the sample. For comparison of the EIAs to the MIF assay and to detect tubal pathology, two-by-two tables were used to calculate sensitivity, speciﬁcity, positive predictive value (PPV) and negative predictive value (NPV). The chi-square test was used to test the signiﬁcance of the difference in frequency distribution. A p-value of < 0.05 was considered signiﬁcant. part II | Chapter 4 Results 60 The seroprevalence rates in the subfertility, pregnant and control group are described in table 1. No signiﬁcant differences in overall prevalence rates of CT IgG antibodies were found in all three assays. The prevalence of CT IgA antibodies is very low. Signiﬁcantly higher prevalences of CT IgG antibodies were found in the group of CT-positive patients (p<0.01) (table 1). Also, a signiﬁcant (p<0.05) increase of the prevalence of CT IgA antibodies was found. The Chlamydia-EIA has a good correlation with the prevalence of CT IgG antibodies. This assay detected the highest percentage of CT-positive patients (82.5%). The test characteristics of the two EIAs are described in table 2. Table 1. Prevalence of C. trachomatis antibodies (IgG and IgA) using different detection assays in different gynaecological patient groups. Prevalencea Group (n) Chlamydia-EIA pELISA MIF (IgG)b Subfertility (76) IgG IgA 31.6 (24) 2.6 (2) 21.1 (16) 1.3 (1) 31.6 (24) Pregnant (150) IgG IgA 24.7 (37) 2 (3) 17.3 (26) 3.3 (5) 23.3 (35) Control (220) IgG IgA 31.4 (69) 3.6 (8) 19.1 (42) 5 (11) 31.4 (69) CT positive (40) IgG IgA 82.5 (33) 12.5 (5) 65 (26) 17.5 (7) 62.5 (25) a Values are percentages. Each parenthetical value is n. b Sera diluted to a titer of 1:64. Tubal patency is essential for fertility. Patency is tested either by visualizing the tubes by X- ray with contrast ﬂuid (hysterosalpingography) or by direct observation by laparoscopy during which the tubes are pertubated. In 32 subfertility patients tubal patency was tested. Twenty-one patients had patent tubes. In Caroline Bax BW.indd 60 26-08-10 12:17 Comparison of serological assays 61 14 of these patients, no CT IgG antibodies were found (67%). In 11 patients, tubal pathology was found. However, in three patients, none of the assays showed presence of CT IgG antibodies (27%). There was a signiﬁcant difference in serology in the MIF assay between the patients with tubal pathology versus those without (p<0.02). Table 3 shows the test characteristics of the three assays as predictors of tubal pathology. Table 2. Test characteristics of the Chlamydia-EIA and pELISA in relation to MIF in different groupsa Subfertility Pregnant Control Parameterb Chlamydia-EIA pELISA Chlamydia-EIA pELISA Chlamydia-EIA pELISA Sensitivity 66.7 58.3 77.1 62.9 76.8 47.8 Speciﬁcity 84.6 96.2 91.3 96.5 89.4 94 PPV 66.7 87.5 73 84.6 76.8 78.6 NPV 84.6 83.3 92.9 89.5 89.4 79.8 a MIF (IgG) was used as the gold standard. b For all parameters, values are percentages. Table 3. Test characteristics of the Chlamydia-EIA, pELISA and MIF in relation to tubal pathology (IgG). Result Parametera Chlamydia-EIA pELISA MIF Sensitivity 54.6 36.4 63.6 Speciﬁcity 71.4 85.7 81 PPV 50 57.1 63.6 NPV 75 72 81 a For all parameters, values are percentages. Discussion There is little literature available describing performance of new assays. pELISA has been evaluated in infertility patients especially to describe its role in predicting tubal factor infertility4,8,9. Blood samples from healthy female blood donors or pregnant women were commonly used as controls. In our subfertility group, the prevalence rates of CT IgG and IgA antibodies according to the pELISA and MIF assay were lower than described in other studies using the pELISA1,3,6,8,9. However, in other studies, the titer at which the MIF assay is considered positive is often not mentioned. A lower titer will give more falsepositive results. Another reason for the lower prevalence rate in our subfertility group might be that this group includes only a small number of patients with tubal factor infertility (TFI) (n=11), where in other groups, larger numbers of TFI patients were found. Therefore, comparison with non-TFI patients might be more applicable. The prevalence rates of CT IgG and IgA antibodies found in our group of pregnant women are in the same range as found in other studies for the pELISA and slightly higher than those for the MIF assay1,8,9. Our control group differs from other control groups, since it includes women visiting the Obstetrics and Gynaecology Outpatient Department for various complaints. When we compare our group with Caroline Bax BW.indd 61 26-08-10 12:17 blood donors and other asymptomatic patients, we ﬁnd for the pELISA IgG and MIF a higher prevalence rate and for the pELISA IgA prevalence in the same range1,6,9. Regarding our group of CT-positive patients, the pELISA shows approximately the same prevalence rates, while using the MIF assay, we found a lower prevalence1,6. We have no explanation for the lower prevalence and sensitivity found using the pELISA in all patient groups. We did not ﬁnd any study describing the performance of the Chlamydia-EIA. By using the MIF assay as the gold standard, the test characteristics of the Chlamydia-EIA and pELISA for the determination of serological evidence of a recent or past CT infection therefore depended on the patient group tested (table 2). Both tests have reasonably high speciﬁcity and NPV and would therefore match the criteria of a screenings test. We have to consider that cross-reactivity with other Chlamydia species also occurs for the MIF assay 3,11. When serology is used to detect tubal pathology, high speciﬁcity is important. However, when we used tubal pathology as the gold standard, the speciﬁcity and NPV are somewhat lower (table 3). We have to consider that these results are based on a small number of patients (n=32). In the patients with part II | Chapter 4 patent tubes, 33% had CT IgG antibodies, and in patients with tubal pathology, 27% had no CT IgG 62 antibodies. Therefore, none of the three assays we used appeared to be perfectly able to predict tubal pathology. Gijsen et al. described no signiﬁcant differences between two peptide-based EIAs and the MIF in predicting tubal pathology4. Our test results were comparable, with the exception of the pELISA, which showed a lower sensitivity and a higher speciﬁcity. And what about the IgA antibodies? In patients with a current CT infection low prevalence rates of IgA antibodies are found (table 1). It seems that the IgA antibodies can persist for years, even after effective therapy2. Therefore, IgA antibodies do not indicate a current CT infection. The role of IgA antibodies in the serodiagnosis of CT with the currently available assays remains negligible. pELISA seems to be a good alternative for MIF for the detection of CT antibodies. pELISA is more speciesspeciﬁc than the Chlamydia-EIA. It is less laborious and less expensive than MIF. A screenings test needs high speciﬁcity and high NPV. When the two assays are compared with MIF as the gold standard, pELISA has the highest speciﬁcity, and in the subfertility group, it has a NPV comparable to that of ChlamydiaEIA. Caroline Bax BW.indd 62 26-08-10 12:17 Comparison of serological assays 63 References 1. Clad A, Freidank H, Plünnecke J, et al. Chlamydia trachomatis species-speciﬁc serology: ImmunoComb Chlamydia Bivalent versus Microimmunoﬂuorescence (MIF). Infect. 1994;22:165-73. 2. Clad A, Freidank HM, Kunze M, et al. Detection of seroconversion and persistence of Chlamydia trachomatis antibodies in ﬁve different serological tests. Eur J Clin Microbiol Infect Dis. 2000;19:932-7. 3. Gijsen AP, Land JA, Goossens VJ, et al. Chlamydia pneumoniae and screening for tubal factor subfertility. Hum Reprod. 2001;16:487-91. 4. Gijsen AP, Goossens VJ, Land JA, et al. The predictive value of chlamydia antibody testing (CAT) in screening for tubal factor subfertility: micro-immunoﬂuorescence (MIF) versus ELISA, p.101. In P. Saikku (ed.), Proceedings Fourth Meeting of the European Society for Chlamydia Research, Helsinki, Finland, 2000. 5. Johnson NP, Taylor K, Nadgir AA, et al. Can diagnostic laparoscopy be avoided in routine investigation for infertility? Br J Obstet Gynaecol. 2000;107:174-8. 6. Maass M, Franke D, Birkelund S, et al. Evaluation of pELISA medac for the detection of Chlamydia trachomatis-speciﬁc IgG and IgA, p.112. In P. Saikku (ed.), Proceedings Fourth Meeting of the European Society for Chlamydia Research, Helsinki, Finland, 2000. 7. Mol BWJ, Dijkman B, Wertheim P, et al. The accuracy of serum chlamydial antibodies in the diagnosis of tubal pathology: a meta-analysis. Fertil Steril. 1997;67:1031-7. 8. Persson K, Osser S. A new peptide-based ELISA for antibodies to Chlamydia trachomatis assessed in patients with tubal factor infertility, p.132. In P. Saikku (ed.), Proceedings Fourth Meeting of the European Society for Chlamydia Research, Helsinki, Finland, 2000. 9. Petersen EE, Bätz O, Böttcher M. Chlamydia trachomatis serology: prevalence of antibodies in women attending IVF centers, p.133. In P. Saikku (ed.), Proceedings Fourth Meeting of the European Society for Chlamydia Research, Helsinki, Finland, 2000. 10. Thomas K, Coughlin L, Mannion PT, et al. The value of Chlamydia trachomatis antibody testing as part of routine infertility investigations. Hum Reprod. 2000;15:1079-82. 11. Wagenvoort JHT, Koumans D, van de Cruijs M. How useful is the Chlamydia micro-immunoﬂuorescence (MIF) test for the gynaecologist? Eu J Obstet Gynaecol Reprod Biol. 1999;84:13-5. 12. Wang S-P, Grayston JT, Kuo C-C, et al. Serodiagnosis of Chlamydia trachomatis infection with the microimmunoﬂuorescence test, p.237-248. In D. Hobson and K.K. Holmes (ed.), Nongonococcal urethritis and related infections, American Society for Microbiology, Washington, D.C., 1977. Caroline Bax BW.indd 63 26-08-10 12:17 Caroline Bax BW.indd 64 26-08-10 12:17 part II | Chapter 5 Chlamydia trachomatis heat shock protein 60 (cHSP60) antibodies in women without and with tubal pathology using a new commercially available assay C.J. Bax P.J. Dörr J.B. Trimbos J. Spaargaren P.M. Oostvogel A.S Peña S.A Morré Sex Transm Infect. 2004;80(5):415-6. Caroline Bax BW.indd 65 26-08-10 12:17 Caroline Bax BW.indd 66 26-08-10 12:17 cHSP antibodies and tubal patholoy 67 Introduction Besides commercially available serological assays that detect antibodies to major outer membrane protein (MOMP)1 and lipopolysaccharide (LPS) ‘in-house’ chlamydial heat shock protein 60 (cHSP60) assays are extensively used in assessing serological responses to urogenital Chlamydia trachomatis (CT) infection. Although comparison of the different ‘in-house’ assays is difﬁcult owing to a lack of standardisation, there is a consensus among the users of these assays that the anti-cHSP60 responses in women increase with the severity of CT associated disease, leading to the suggestion that the high amino acid sequence homology between chlamydial and human HSP60 results in autoimmune mediated fallopian tube damage. Owing to the signiﬁcance of the possible association of the response to cHSP60 and progressive disease, a commercially produced assay that employs deﬁned cHSP60 epitopes should allow for the comparison of results obtained in different laboratories, as well as forward the use of cHSP60 as a diagnostic tool, if the assay proves to be relevant in predicting pathology or clinical outcome of a urogenital chlamydial infection. This study evaluated a recently introduced commercially available cHSP60 serologic assay and determined the anti-cHSP60 responses in three gynaecologically well deﬁned groups of women. Methods Group 1 consisted of women without tubal pathology as assessed by either hysterosalpingography (HSG) or laparoscopy (n=21), group 2 consisted of pregnant women (unknown tubal status, proven fertility; n=86), and group 3 consisted of women with conﬁrmed (based on HSG or laparoscopy) tubal pathology (n=11). CT positivity was assessed previously using one of the following serological assays: microimmunoﬂuorescence (MIF) (BioMérieux, ‘s Hertogenbosch, the Netherlands), BAG Chlamydia EIA (Biologische Analysensystem GmbH, Lich, Germany) and the CT-pELISA (Medac, Wedel, Germany). The study groups and techniques described previously2,3. The cHSP60 assay (Medac, Wedel, Germany) was performed according to the manufacturer’s instructions. Results Results are shown in ﬁgure 1. CT IgG positivity was previously determined to be 19% for group 1, 40% for group 2, and 64% for group 3, showing the expected clear difference in IgG seroprevalence between women with and without procedure conﬁrmed tubal pathology, while an intermediate prevalence was observed in pregnant women. The same pattern but with lesser incidence was observed in the anticHSP60 responses being 4.8%, 16% and 27%, for groups 1-3, respectively (χ2 test for trend: χ2=3.1, p=0.079, group 1 vs. group 3: p=0.096, OR 10.6). The incidences of anti-cHSP60 were increased in the CT IgG positive subgroups to 25%, 35% and 43%, for groups 1-3, respectively (see lower panel in ﬁgure Caroline Bax BW.indd 67 26-08-10 12:17 Figure 1. Chlamydia trach different degrees of tubal p omatis IgG and cHSP60 antibody responses in Dutch Caucasian women with athology 1), while only 3.8% anti-cHSP60 titers were observed in the CT IgG negative subgroups, all in subgroup part II | Chapter 5 2 (unknown tubal status, proven fertility). This indicates that the concordance between CT IgG and cHSP60 positivity is high, almost 90%, however, clearly a different subgroup of women is identiﬁed by the cHSP60 assay since only 40% of the CT IgG positive women has a cHSP60 response (measurement of agreement: kappa 0.371). Finally, the median cHSP60 titers increased from group 1-3: 50, 100 and 200, respectively, suggesting an association between the level of cHSP60 response and tubal pathology. 68 Discussion As far as we know this is the ﬁrst study evaluating the commercially available cHSP60 assay in women with different degrees of tubal pathology. Two abstracts were published in the ISSTDR meeting Vienna, Austria in 20024,5 on cHSP60 antibodies in women with pelvic inﬂammatory disease (85% in patients with CT positive swabs and patients with occluded tubes, 20% in blood donors) and in women with open or occluded fallopian tubes (31% and 70% respectively). The standardisation provided through this new commercially available assay will potentially enhance the comparability of cHSP60 results between laboratories. The results presented here, although obtained in small but well deﬁned groups, look suggestively promising. Indeed, power calculations (alpha =0.5, beta=0.1) show that doubling (1.7 times) the size of the (sub)groups would results in signiﬁcant p-values instead of clear trends. However, further studies are needed in larger groups with different degrees of pathology because of CT infections, to further determine the diagnostic, prognostic and clinical relevance of this new assay. Caroline Bax BW.indd 68 26-08-10 12:17 cHSP antibodies and tubal patholoy 69 References 1. Morré SA, Munk C, Persson K, et al. Comparison of three commercially available peptide-based immunoglobulin G (IgG) and IgA assays to microimmunoﬂuorescence assay for detection of Chlamydia trachomatis antibodies. J Clin Microbiol. 2002;40:584-7. 2. Bax CJ, Mutsaers JA, Jansen CL, et al. Comparison of serological assays for detection of Chlamydia trachomatis antibodies in different groups of obstetrical and gynecological patients. Clin Diagn Lab Immunol. 2003;10:174-6. 3. Bax CJ, Oostvogel PM, Mutsaers JA, et al. Clinical characteristics of Chlamydia trachomatis infections in a general outpatient department of obstetrics and gynaecology in the Netherlands. Sex Transm Infect. 2002;78:E6. 4. Petersen EE, Clad A, Pichlmeier U, et al. The extended Chlamydia trachomatis diagnosis in patients with pelvic inﬂammatory disease - a better approach for the diagnosis of upper genital tract infections. Int J STD AIDS. 2002;13 (Supplement 1);29. 5. Clad A, Petersen EE, Dettlaff S. Antibodies to Chlamydia trachomatis heat shock protein 60 (CHSP60) and Chlamydia trachomatis major outer membrane protein (MOMP) in women with different tubal status. Int J STD AIDS. 2002;13 (Supplement 1);28. Caroline Bax BW.indd 69 26-08-10 12:17 Caroline Bax BW.indd 70 26-08-10 12:17 Part III Serovar Caroline Bax BW.indd 71 26-08-10 12:17 Caroline Bax BW.indd 72 26-08-10 12:17 part III | Chapter 6 The serovar distribution of urogenital Chlamydia trachomatis strains among sexual transmitted disease clinic patients and gynaecological patients in the region of The Hague, the Netherlands: an ethnic epidemiological approach C.J. Bax K.D. Quint R.P.H. Peters S. Ouburg P.M. Oostvogel J.A.E.M. Mutsaers P.J. Dörr S. Schmidt C. Jansen A.P. van Leeuwen W.G.V. Quint J.B. Trimbos C.J.L.M. Meijer S.A. Morré Submitted Sex Transm Infect. 2010 Caroline Bax BW.indd 73 26-08-10 12:17 Abstract Objectives: The Chlamydia trachomatis (CT) serovar distribution is stable in time, however geographical differences in the distribution are observed. These differences might be due to differences in the ethnical composition of the population. The ﬁrst objective of this study is to determine the CT serovar distribution among different ethnic populations in the region The Hague in two cohorts and to compare the results to previous obtained Dutch serovars distribution studies. The current study will elucidate the potential association between ethnicity and presence of different serovars. Secondly, the association between serogroup distribution and various demographical, clinical, and behavioural parameters (i.e. population, symptoms and age) was investigated. Methods: A total of 418 CT infected patients in two populations were analysed in this study: patients visiting the outpatient department (OPD) of Obstetrics and Gynaecology (O&G) of the MC Haaglanden and patients visiting the outpatient STD clinic in the centre of The Hague, the Netherlands. part III | Chapter 6 Results: Serovar E, F, and G/Ga were the most prevalent serovars, accounting for 73.2% of the serovars. 74 No strong relation was found between ethnicity and serovars/serogroups. For serovar H and I/Ia differences were observed with earlier Dutch studies. Finally, no relation between the demographical, clinical, and behavioural variables and serovars were found. Conclusions: This is the ﬁrst Dutch serovar study taking ethnicity into account and the second largest study on CT serovar distributions in the Netherlands. No major differences were observed between the different ethnic groups studied. We did ﬁnd some differences in serovar distribution compared to previously published Dutch serovar studies. Finally, our results suggest that the infecting serovar does not have a major impact on the clinical presentation of infections. Caroline Bax BW.indd 74 26-08-10 12:17 Serovar distribution; an ethnic epidemiological approach 75 Introduction Chlamydia trachomatis (CT) infections are one of the most common bacterial sexually transmitted diseases (STD) worldwide. In many cases the infections are asymptomatic in both men and women, and therefore undiagnosed and untreated. Nevertheless, asymptomatic infections still contribute to the distribution of a CT infection to others, in which it can give symptoms. Also, untreated CT infections may lead to late disease like pelvic inﬂammatory disease (PID), a major cause of ectopic pregnancy and tubal factor subfertility1. Currently, 19 different CT serovars and serovariants have been identiﬁed by sero- and genotyping2,3. In conventional serotyping, after culture, polyclonal and monoclonal antibodies are used against the major outer membrane protein (MOMP) of CT4. The serovars can be divided into three serogroups: the B-group (serovars B, Ba, D, Da, E, L1, L2, and L2a), the intermediate group (serovars F, G, and Ga) and the C-group (serovars I, Ia, J, K, C, A, H, and L3). Based on tissue tropism, the majority of serovars A-C cause a conjunctivitis, while serovars D-K are predominantly isolated form the anogenital tract. The serovars L1-L3 are more invasive serovars and can cause a Lymphogranuloma venereum (LGV)5-8. Untill recently, genotyping was performed molecular on the Omp1 gene (which encodes for the MOMP) by polymerase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP)9-12. Nowadays, several reverse line blots have been developed mostly based on ampliﬁcation within the Omp1 gene13,14. An increasing number of isolates are typed worldwide and provide a wealth of information on the epidemiology of CT infections. Several studies have described inconclusive data about speciﬁc serovars and the clinical course of infection, the rate of upper genital tract progression, and the clearance-persistence rate. Information about the differences in serovar distribution could give information about the epidemiology of CT infections and might have clinical implications5,7,15-18. Certain serovars might be associated with upper genital tract infection. And, for example, LGV strains need a different and longer therapy (up to three weeks) than other serovars. A recent study about serovar distribution in the Netherlands showed no signiﬁcant serovar distribution shift over time, but geographical differences were observed. This could be the result of different ethnic composition19,20. The current study will contribute to our understanding if differences in the serovar distributions found are in part ethnicity-based. The objective of this study is to determine the CT serovar distribution among different ethnic populations in the region The Hague in two cohorts and to compare the results to previous obtained Dutch serovars distribution studies. The association between serovar distribution and various demographical, clinical, and behavioural parameters will be investigated as well. Caroline Bax BW.indd 75 26-08-10 12:17 Methods Specimen collection CT infected patients in two well deﬁned populations were analysed in this study: patients visiting the outpatient department (OPD) of Obstetrics and Gynaecology (O&G) of the MC Haaglanden and patients visiting the outpatient STD clinic in the centre of The Hague, the Netherlands. i) OPD Obstetrics and Gynaecology, MC Haaglanden, The Hague; MC Haaglanden is an inner city hospital with patients from various ethnic origins. Patients visited the OPD O&G for various reasons, for example pregnancy, discharge, menstrual disorders, subfertility, contraception etc. If required, cervical and urethral swabs were taken. Samples from CT infected patients were used in this study in the period January - October 2008. ii) STD clinic, The Hague; During the same study period samples from CT infected patients (men and women) visiting the STD clinic were analysed in the study. Patients could be visiting because of complaints, because they were warned by an infected partner, or for check-up. In women cervical or vaginal swabs were taken, and in part III | Chapter 6 some women urethral swabs or ﬁrst-void urine (FVU). In men, urethral swabs or FVU was collected. In 76 men-who-have-sex-with-men (MSM) anorectal and oropharyngeal swabs were taken as well. In women these swabs were taken when oral or anal sex was reported. In both clinics information was collected concerning age, gender (STD clinic only, OPD all female), ethnicity, symptoms (symptomatic, asymptomatic, or upper genital tract infection), and co-infections (such as gonorrhoea, hepatitis B, HIV, Syphilis, Mycoplasma, Gardnerella, and Candida). All patient and sample data were anonymised by each center and analysed according to local ethical regulations. CT detection For the detection of CT we used a probe hybridisation assay on urethral, cervical, vaginal, pharyngeal, and rectal swabs (PACE 2 assay, Gen-Probe). Swabs were analysed within 24 hours according to GenProbe’s packet insert instructions. For urine analysis we used ampliﬁcation of CT-rRNA by transcriptionmediated ampliﬁcation (TMA) in urine samples with the Gen-Probe AMP CT assay. Urine specimens were collected before swab specimens were gathered and stored at +4 ºC. The urine was analysed on a weekly basis according to Gen-Probe procedures. Ampliﬁcation, detection, and genotyping using the CT DT assay The CT-DT ampliﬁcation and genotyping assay was performed on all previous determined CT positive samples according to the manufacturer’s instructions (Labo Biomedical Products BV, The Netherlands). The CT-DT genotyping assay is a reverse hybridization probe line blot (RHA) with a probe for the detection of the cryptic plasmid, and probes to detect the 3 different CT serogroups (B, C, and Intermediate) and the 14 major serovars (A, B/Ba, C, D/Da, E, F, G/Ga, H, I/Ia, J, K, L1, L2/L2a, and L3)14. Caroline Bax BW.indd 76 26-08-10 12:17 Serovar distribution; an ethnic epidemiological approach 77 Studies used for comparison Spaargaren et al. describe the serovar distribution in a STD population in Amsterdam19. They also compare serovar distributions in the Netherlands, found in nine studies between 1986-2002. Populations included were a STD population, asymptomatic screenings population and mixed symptomatic and asymptomatic patients in Amsterdam, and a STD population in Rotterdam. The type of cohort did not inﬂuence the analyses. However, serovar distribution differences were found between Amsterdam and Rotterdam (C-group, especially serovar K, Intermediate group, especially serovar F). Morré et al. describe CT serovar distributions in symptomatically and asymptomatically infected patients in Amsterdam, identiﬁed in a general practitioners screenings program and a hospital outpatient-based CT infected population6. Statistical analysis Serogroup and serovar distributions were compared between men and women, and between Dutch cohorts, using χ2 statistics. A p < 0.05 was considered statistically signiﬁcant. Bonferroni correction was used for multiple comparisons and Mann-Whitney U test, if applicable, using Graph Pad InStat 3. Results During the study period samples were collected sequentially. Samples of 433 patients could be used for CT serovar detection and typing. Patients were excluded because of concurrent serovar infection in one sample site (n=11), different serovars in different sample sites (n=1), and mismatch sex and sample site (n=3). A total of 418 patients were used for further analysis. Ethnicity We compared serogroup distributions in different ethnic groups (Dutch Caucasian (DC)), Surinam, Netherlands Antilles (NA) and Turkish/Moroccan). Results are shown in table 1. No signiﬁcant differences were found between the ethnic groups, although one comparison was borderline signiﬁcant; the B-group and the intermediate group vs. the C-group for DC vs. NA patients (p=0.09; OR 2.1 (95% CI 0.95-4.8). In CT infected patients, no differences were observed in the age distribution between patients visiting the O&G department and women visiting STD outpatient clinic. In the STD clinic population signiﬁcant differences were found between male and female patients for DC and NA patients (p<0.0001 and p=0.0416, respectively). Male patients were older. In the STD clinic patients it was found that Surinam patients were signiﬁcantly younger than DC patients (p=0.0324), and this was mainly caused by the male Surinam STD clinic patients (p=0.0158). Within the O&G patients no signiﬁcant differences between ethnic groups were determined. Caroline Bax BW.indd 77 26-08-10 12:17 Table 1. Serogroup distribution and median age in different ethnic groups. Serogroups Ethnicity B n(%) Intermediate n(%) C n(%) Dutch Caucasian 125 (52,3) 80 (33,5) 34 (14,2) Total n 239 Netherlands Antilles 14 (36,8) 14 (36,8) 10 (26,3) 38 Surinam 11 (42,3) 12 (46,2) 3 (11,5) 26 Turkish/Moroccan 12 (46,2) 11 (42,3) 3 (11,5) 26 Ethnicity O&G STD F STD M Median age (range) STD total Dutch Caucasian 22 (17-47) 24 (16-72) 22 (16-61) 27 (18-72) Netherlands Antilles 22.5 (15-35) 23.5 (18-36) 22 (19-27) 27 (18-36) Surinam 27.5 (18-44) 21.5 (15-42) 21.5 (15-37) 21.5(19-42) Turkish/Moroccan 25 (18-44) 25.5 (19-39) 23 (19-39) 26 (24-36) Table 2. Serovar distribution in Dutch patients divided in serovars and serogroups and by gender. Previously published results in Dutch cohorts (19,6) are given for comparison. part III | Chapter 6 This study Serovar Women n (%) Men n (%) Spaargaren et al. 2004 Total Morré et al. 2000 Women n (%) Men n (%) Total B group 78 B 2(0.8) - 2(0.5) 4(1.0) - - - D/Da 32(12.9) 19(11.2) 51(12.2) 50(12.3) 41(12.9) 12(10.8) 53(12.4) E 95(38.3) 60(3 ) 155(37.1) 136(33.4) 130(41.0) 47(42.3) 177(41.4) Subtotal 129(52.0) 79(46.5) 208(49.8) 190(46.7) 171(53.9) 59(53.2) 230(53.7) 93(21.7) I group F 58 (23.4) 32(18.8) 90(21.5) 95(23.3) 72(22.7) 21(18.9) G/Ga 31(12.5) 30(17.7) 61(14.6) 38(9.3) 24(7.6) 12(10.8) 36(8.4) Subtotal 89(35.9) 62(36.5) 151(36.1) 133(32.7) 96(30.3) 33(29.7) 129(30.1) C group H 3(2.1) - 3(0.7) 34(8.4) 9(3.4) 7(6.3) 16(3.7) I/Ia 6(2.4) 3(18) 9(2.2) 30(7.4) 11(3.5) 6(5.4) 17(4.0) 19(4.4) J 16(6.5) 18(10.6) 34(8.1) 12(2.9) 17(5.4) 2(1.8) K 5(2.0) 8(4.7) 13(3.1) 8(2.0) 13(4.1) 4(3.6) 17(4.0) Subtotal 30(12.1) 29(17.1) 59(14.1) 84(20.6) 50(15.8) 19(17.1) 69(16.1) Total 248 170 418 407 317 111 428 Caroline Bax BW.indd 78 26-08-10 12:17 Serovar distribution; an ethnic epidemiological approach 79 Serovar distribution compared to other Dutch studies The most prevalent serovars were E, F, and G/Ga, they accounted for 73.2% of the serovars. There was no signiﬁcant difference in serovar distribution between male or female patients, nor between patients from the STD clinic or OPD Obstetrics and Gynaecology. The distribution of serovars is shown in table 2. We compared our serovar distribution to serovar distributions of other Dutch cohorts. [6,19] Serovar H and I/Ia showed signiﬁcant differences with Spaargaren et al. (H: p<0.0001 and I/Ia: p=0.0004). After Bonferroni correction this was still signiﬁcant. Serovar distribution and various variables Various demographic, clinical, and behavioural parameters were analysed for a possible association with the infecting serogroup. Serogroup distributions within these variables are shown in table 3. For nearly all variables the serogroup distribution showed that serogroup B was most prevalent. No signiﬁcant serogroup differences were found between symptomatic vs. asymptomatic patients. Remarkably, all 7 females in the STD clinic population engaged in prostitution had serovar E. This results in a signiﬁcant difference between these women and the STD women not engaged in prostitution for serovar E (p=0.0007). Their ethnicity was as follows: two were DC, one was Turkish, three were other European, and one was unknown. No other signiﬁcant differences were found. Table 3. Demographical, clinical and behavioural parameters in relation to serogroup in two populations (OPD O&G and STD clinic). Population Serogroup Variables OPD O&G B n(%) Int n(%) STD Female C n(%) n B n(%) Int n(%) STD Male C n(%) n B n(%) Int n(%) C n(%) n - - - - Sex Female 37(52.1) 24(33.8) 10(14.1 71 92(51.8) 65(36.7) 20(11.3) 177 Male - - - - - - - - 79(46.5) 62(36.5) 29(17.1) 170 -Heterosexual - - - - - - - - 59(48.0) 45(36.6) 19(15.4) 123 -Bisexual - - - - - - - - 3(33.3) 1(11.1) 9 -Homosexual - - - - - - - - 14(40.0) 12(34.3) 9(25.7) 35 51 29(47.5) 20(32.8) 12(19.7) 61 5(55.6) Symptoms Symptomatic 24(46.2) 19(36.5) 9(17.3) Asymptomatic 13(68.4) 5(26.3) Abdom. pain† 11(55.0) Co-infections* 5(25.0) 1(5.3) 52 27(52.9) 19(37.3) 4(20.0) 20 12(66.7) 6(33.3) 29(47.5) 25(41.0) 7(11.5) 5(9.8) 19 57(50.9) 41(36.6) 14(12.5) 61 9(37.5) 12(50.0) 112 46(44.7) 40(38.8) 17(16.5) 103 - 18 - - 3(12.5) 24 15(50.0) 10(33.3) - - 5(16.7) 30 History STD - - - - 19(54.3) 12(34.3) 4(11.4) 35 15(40.5) 10(27.0) 12(32.4) 37 History CT - - - - 16(51.6) 11(35.5) 4(12.9) 31 9(34.6) 26 No partners‡ - - - - 2 [1-8] 1 [0-7] 2 [1-5] Prostitution - - - - 7(100) - - 8(30.8) 9(34.6) 3 [1-25] 3 [0-80] 4 [1-10] 7 - - - - *We combined all co-infections (n) OPD O&G: gonorrhoea (3), syphilis(1), HIV(1), hepatitis B(1), Candida(10), Mycoplasma(13), Gardnerella(32); Female STD: gonorrhoea(7), syphilis(-), HIV(1), hepatitis B(2), Candida/bacterial vaginosis(14); Male STD: gonorrhoea(10), syphilis(2), HIV(13), hepatitis B(5). ‡ Median [range]. Caroline Bax BW.indd 79 26-08-10 12:17 The percentage of patients in various age-groups in relation to serogroup is shown in ﬁgure 1. No signiﬁcant differences were found. No age differences were found within the serogroups in the two populations (data not shown). The male STD patients were slightly older than the female STD patients (median 26-27 years of age vs. median 22-23 years of age in all serogroups). Serovar E was the most prevalent serovar in all age-groups. In the age-group >40 serovar G and E were evenly present (26.3%). 60 50 40 B I 30 C part III | Chapter 6 20 10 0 <20 20-29 30-39 >40 Figure 1. Serogroup distribution in different age-groups. X-axis: age-groups in years; Y-axis: frequency of serogroups (%). B=B-group, I=Intermediate group and C=C-group. 80 Discussion This is the ﬁrst study in the Netherlands taking ethnicity into account inside CT serovar distributions. It has been suggested that differences in serovar distribution might be the result of different ethnic compositions of the population. This suggestion was not conﬁrmed in the current cohort-based study, since no major differences in serovar distributions were observed. Questionnaire based ethnicity data were collected, however, mixed ethnic background cannot be excluded. The median age in most ethnic groups were comparable, although the Surinam male STD clinic patients were slightly younger than the DC male STD clinic patients. Male DC and NA STD clinic patients were signiﬁcantly older than female DC and NA STD clinic patients. The prevalence and age distribution in the general patient population visiting the O&G department and the STD outpatient clinic were determined. On average patients visiting the O&G department were older than the women visiting the STD outpatient clinic. We determined the prevalence in our OPD O&G population, and it was comparable to a previous study21, with an expected higher prevalence in the STD population (3.1% vs. 10.7%). We used the STD clinic annual report for their prevalences. The highest Caroline Bax BW.indd 80 26-08-10 12:17 Serovar distribution; an ethnic epidemiological approach 81 prevalences were found in patients from Netherlands Antilles origin, in both populations, as well as in male and female patients. Over time, no signiﬁcant changes in serovar distribution were observed in the past. Nevertheless, stable and clear differences in CT serovars distributions were found previously in different geographical Dutch regions19. Worldwide serovars D, E, and F are the most prevalent serovars, encounting for approximately 70% of the urogenital CT infections5,6. In the current study serovar G/Ga was the third most prevalent serovar after E and F, possibly due to a patient bias, since MSM are also included. The predominance of the B-group serovars in most studies conducted in different geographic locations and at different times suggests that these serovars have a biological advantage over the other serovars. The overall distribution of serovars closely resembles that found in other studies. When we compared our serovar distribution to the study of Spaargaren et al. we found only signiﬁcant differences in the incidence of the most infrequent serovars H and I/Ia. Geisler et al. found an association between serovar Ia and (high-risk) black population in a STD clinic population, also described by others20,22,23. The overall serovar distribution in the predominantly black population was similar to that reported elsewhere. These racial differences in serovar distribution might be due to behavioural, geographical, or biological factors. It has been suggested that one of the reasons could be that there is less mixing with partners from a different race (relatively closed populations)20. Also intrinsic biological differences among persons of different race or among chlamydial serovars could inﬂuence acquisition, transmission, and duration of infection (host susceptibility or immune response)20,24. In only one other Dutch study serogroup distribution was described between Dutch Caucasians (DC) and Surinam patients in a STD clinic population18. They found that, for both men and women, serovars F and G/Ga were less common among (high-risk) Surinam patients. Surinam men were more often infected with serovar I and E, Surinam women more often with serovar J. We could not conﬁrm these ﬁndings. In our study the serovars for Surinam men were 40% in the B-group, 40% in the intermediate group, and 20% in the C-group. For Surinam women the serovars were 43.8% in the B-group, 50% in the intermediate group, and were 6.3% in the C-group. In the Netherlands higher prevalences of CT infections are found in Surinam and NA patients25. One might compare these high risk groups with high risk black patients. However, in our NA patients the prevalence of CT serovars is lowest in the C-group (prevalence serovar I/Ia in NA men 7.7% and in NA women 8%). A previous study on ethnicity in The Hague suggested a different spread in ethnicity, i.e. less DC and more Turkish/Moroccan patients21. The previous study showed that 32.3% of the CT positive patients was of DC ethnicity, while the current study revealed that 57.2% of the patients had a DC ethnicity. However, when we looked at the OPD O&G patients separately, we found 32.3% DC patients. Apparently there are more DC patients visiting the STD clinic than patients from other ethnic background. Our ethnic groups Caroline Bax BW.indd 81 26-08-10 12:17 were smaller than expected, based on the ethnical distribution in the study performed previously, and therefore slightly underpowered21. Since one comparisons between DC and Netherlands Antilles patients was borderline signiﬁcant, larger studies might reveal clearer differences. It is important to take population, and demographical, clinical, and behavioural parameters into account for comparison with other studies. We looked at two well deﬁned populations and will describe them separately, with special attention to serogroup differences and symptoms. In the OPD O&G patients we found no signiﬁcant difference in the serogroup distribution between the symptomatic and asymptomatic patients. Persson et al. found in an OPD O&G population in Sweden a serogroup distribution similar to our study16. They found that symptoms were not associated with any serovar. Lan et al. found an association between serovar G and symptomatic infection in OPD O&G patients in the Netherlands26. We found in this population the highest prevalence of the intermediate group serovars in the symptomatic patients group, but only 3 out of 19 were serovar G/Ga. part III | Chapter 6 In the female STD clinic population no signiﬁcant serogroup differences were found. An earlier Dutch 82 study showed a different serogroup distribution compared to our study; B-group similar (54.1% vs. 51.8%), intermediate group lower prevalence (14.8% vs. 36.7%), and C-group higher prevalence (29.6% vs. 11.3%)18. There might be an increase of serovar G over time, as found by Suchland et al. in Seattle; (over a 9-year period signiﬁcant increase of serovar G)23. We found a prevalence of serovar G/Ga of 15.3% vs. 6.7% found by van Duynhoven et al. in 199818. A similar tendency was found by Geisler et al.27. Others found that serovars F and G (intermediate group) were associated with fewer signs of cervical infection. No differences in serovar distribution were found between patients with PID and those with lower genital tract infections17. Lower abdominal pain (referring to possible PID) in women was more often associated with serovars F (30%) and G (33%) (Intermediate group overall 32%, B-group 6%, and C-group 13%; OR 5.1). A similar observation was made for serovar K. Also a tendency was found towards fewer clinical signs of cervical infection with serovar F and G (not signiﬁcant)7,18. Reason could be that serovar F produces less signs of cervical infection and is therefore more often unrecognised and may lead to upper genital tract infection17. However, van der Laar et al. found no association between infecting serovar and clinical signs of cervical or urethral infection in women5. Two other Dutch studies found an association between asymptomatic infections and serovar Ia in women6,26. We could not conﬁrm these ﬁndings. In the male STD clinic populations we found no signiﬁcant differences. As was found in the female STD clinic population the prevalence of the intermediate serogroup seems to increase over time18,27. In men studies on speciﬁc serovar and clinical manifestations are contradictory5,6,18,28. Age is an important risk factor for CT infections. It is known that the prevalence of CT infections declines with increasing age, in an OPD O&G population, as well as in a STD clinic population26. Although behavioural factors, such as age at ﬁrst sexual intercourse, frequency of partner change, and failure to Caroline Bax BW.indd 82 26-08-10 12:17 Serovar distribution; an ethnic epidemiological approach 83 use barrier contraception, clearly are important in contributing to the increased prevalence of chlamydial infections in younger women. The number of inclusion forming units (IFUs) in culture has also been shown to be higher in younger women20. Higher inclusion counts in younger patients may imply greater transmissibility of infection and may be a factor contributing to the very high age-speciﬁc prevalence of CT infection in adolescents. These infections may represent initial encounters with CT in immunologically naïve individuals, while priori immunity in older individuals may reduce the IFU counts in recurrent infection. In addition, if IFU counts decline with increasing duration of infection, older individuals would be expected to have lower counts. Eckert et al. found that women have signiﬁcantly higher IFU counts than men, black patients had signiﬁcantly lower IFU counts than whites, and C-group serovars had signiﬁcantly lower IFU counts than B-group serovars24. Suchland et al. found serovar B, Ia, and mixed serovars to be associated with younger age in a Seattle Public Health clinic. Serovar D, F, H, and K were associated with older age, serovar G tended to be associated with the oldest patients. C-group serovars might be more common in older patients because immunity against the more prevalent B-group serovars developed earlier in life23. This is consistent with the ﬁnding of lower IFUs in C-group serovars and in older patients24. In our study serovar E was the most prevalent serovar in all age-groups. In the age-group >40, serovar G was evenly present. We could not conﬁrm the ﬁndings that C-group serovars were more prevalent in older women (<20=16,7%, 20-29=13,8%, 30-39=12,3%, >40=15,8%). We did ﬁnd a higher prevalence of serovar J in women >40 years of age. The serovar distribution in different age-groups showed no signiﬁcant differences. A similar distribution was found by others23. Lan et al. found serovar E to be the most prevalent one (33,3%) in asymptomatic women <30 years of age, and serovar F to be the most prevalent one (30%) in OPD O&G patients <30 years of age26. In general, the differences in the clinical course of infection can be explained by the interaction between the host (host factors) and the pathogen (virulence factors). This is an interaction which will be inﬂuenced by environmental factors such as co-infections and the serological responses induced by infection. Further studies on the differences in clinical course should include analyses of host genetic factors. Host variation might play an important role in the development of clinical disease. In conclusion, this is the ﬁrst Dutch study taking ethnicity into account and the second largest, study on serovar distribution in the Netherlands. No major differences were found between different ethnic groups. It did show some clear differences in serovar distribution for serovar H and I/Ia, compared to previously published Dutch serovar studies. The clinical course of infection does not seem to be inﬂuenced much by the infecting serovar. Caroline Bax BW.indd 83 26-08-10 12:17 part III | Chapter 6 References 84 1. Cates W, Wasserheit JN. Genital chlamydial infections: Epidemiology and reproductive sequelae. Am J Obstet Gynecol. 1991;164:1771-81. 2. Morré SA, Ossewaarde JM, Lan J, et al. Serotyping and genotyping of genital Chlamydia trachomatis isolates reveal variants of serovars Ba, G, and J as conﬁrmed by omp1 nucleotide sequence analysis. J Clin Microbiol. 1998;36:345-51. 3. Dean D and Miller K. Molecular and mutation trend analysis of omp1 alleles for serovar E of Chlamydia trachomatis. Implications for the immunopathogenesis of disease. J Clin Invest. 1997;99:475-83. 4. Ossewaarde JM, Rieffe M, de Vries A, et al. Comparison of two panels of monoclonal antibodies for determination of Chlamydia trachomatis serovars. J Clin Microbiol. 1994;32:2968-74. 5. van der Laar MJ, van Duynhoven YT, Fennema JS, et al. Differences in clinical manifestations of genital chlamydial infections related to serovars. Genitourin Med. 1996;72:261-5. 6. Morré SA, Rozendaal L, van Valkengoed IGM, et al. Urogenital Chlamydia trachomatis serovars in men and women with symptomatic or asymptomatic infection: an association with clinical manifestation? J Clin Microbiol. 2000;38:2292-6. 7. 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. J Infect Dis. 1995;172:1013-22. 8. Yang CL, Zhang Y-X, Watkins NG, et al. DNA sequence polymorphism of the Chlamydia trachomatis omp1 gene. J Infect Dis. 1993;168:1225-30. 9. Frost EH, Deslandes S, Veilleux S, et al. Typing of Chlamydia trachomatis by detection of restriction fragment length polymorphism in the gene encoding the major outer membrane protein. J Infect Dis. 1991;163:1103-7. 10. Meijer A, Morré SA, van den Brule AJ, et al. Genomic relatedness of Chlamydia isolates determined by ampliﬁed fragment length polymorphism analysis. J Bacteriol. 1999;181:4469-75. 11. Molano M, Meijer CJ, Morré SA, et al. Combination of PCR targeting the VD2 of opm1 and reverse line blot anlysis for typing of urogenital Chlamydia trachomatis serovars in cervical scrapes specimens. J Clin Microbiol. 2004;42:2935-9. 12. Morré SA, Moes R, van Valkengoed I, et al. Genotyping of Chlamydia trachomatis in urine specimen will facilitate large epidemiological studies. J Clin Micobiol. 1998;36:3077-8. 13. Zheng HP, Jiang LF, Fang DY, et al. Application of an oligonucleotide array assay for rapid detecting and genotyping of Chlamydia trachomatis from urogenital specimens. Diagn Microbiol Infect Dis. 2007;57:16. 14. Quint KD, van Doorn LJ, Kleter B, et al. A highly sensitive, multiplex broad-spectrum PCR-DNA-enzyme immunoassay and reverse hybridization assay for rapid detection and identiﬁcation of Chlamydia trachomatis serovars. J Mol Diagn. 2007;9:631-8. 15. Barnes RC, Rompalo AM and Stamm WE. Comparison of Chlamydia trachomatis serovars causing rectal and cervical infections. J Infect Dis. 1987;156:953-8. 16. Persson K and Osser S. Lack of evidence of a relationship between genital symptoms, cervicitis and salpingitis and different serovars of Chlamydia trachomatis. Eur J Microbiol Infect Dis. 1993;12:195-9. 17. Workowski KA, Stevens CE, Suchland RJ, et al. Clinical manifestations of genital infection due to Chlamydia trachomatis in women: differences related to serovars. Clin Infect Dis. 1994;19:756-60. 18. van Duynhoven YTHP, Ossewaarde JM, Derksen-Nawrocki RP, et al. Chlamydia trachomatis genotypes: correlation with clinical manifestations of infections and patients’ characteristics. Clin Infect Dis. 1998;26:314-22. 19. Spaargaren J, Verhaest I, Mooij S, et al. Analysis of Chlamydia trachomatis serovar distribution changes in the Netherlands (1986-2002). Sex Transm Inf. 2004;80:151-2. 20. Workowski KA, Suchland RJ, Pettinger MB, et al. Association of genital infection with speciﬁc Chlamydia trachomatis serovars and race. J Infect Dis. 1992;166:1445-9. Caroline Bax BW.indd 84 26-08-10 12:17 Serovar distribution; an ethnic epidemiological approach 85 21. Bax CJ, Oostvogel PM, Mutsaers JAEM, et al. Clinical characteristics of Chlamydia trachomatis infections in a general outpatient department of obstetrics and gynaecology in the Netherlands. Sex Transm Inf. 2002;78(6):E6. 22. Geisler WM, Suchland RJ and Stamm WE. Association of Chlamydia trachomatis serovar Ia infection with black race in a sexually transmitted disease clinic patient population in Birmingham, Alabama. Sex Transm Dis. 2006;33:621-4. 23. Suchland RJ, Eckert LO, Hawes SE, et al. Longitudinal assessment of infecting serovars of Chlamydia trachomatis in Seattle public health clinics: 1988-1996. Sex Transm Dis. 2003;30:357-61 24. Eckert LO, Suchland RJ, Hawes SE, et al. Quantitative Chlamydia trachomatis cultures: correlation of chlamydial inclusion-forming units with serovar, gender and race. J Infect Dis. 2000;182:540-4. 25. van der Hoek JAR, Mulder-Folkerts DKF, Courtinho RA, et al. Opportunistisch screening op genitale infecties met Chlamydia trachomatis onder de seksueel actieve bevolking van Amsterdam. Meer dan 90% deelname en bijna 5% prevalentie. Ned Tijdschr Geneeskd. 1999;143:668-72. 26. Lan J, Melchers I, Meijer CJLM, et al. Prevalence and serovar distribution of asymptomatical cervical Chlamydia trachomatis infections as determined by highly sensitive PCR. J Clin Microbiol. 1995;33:3194-7. 27. Geisler WM, Suchland RJ, Whittington WLH, et al. The relationship of serovar to clinical manifestations of urogenital Chlamydia trachomatis infection. Sex Transm Dis. 2003;30:160-5. 28. Batteiger BE, Lennington W, Newhall WJ, et al. Correlation of infecting serovar and local inﬂammation in genital chlamydial infections. J Infect Dis. 1989;160:332-6. Caroline Bax BW.indd 85 26-08-10 12:17 Caroline Bax BW.indd 86 26-08-10 12:17 part III | Chapter 7 Signiﬁcantly higher serological responses of Chlamydia trachomatis B group serovars vs. C and I serogroups S.P. Verweij C.J. Bax K.D. Quint W.G.V. Quint A.P. van Leeuwen R.P.H. Peters P.M. Oostvogel J.A. Mutsaers P.J. Dörr J. Pleijster S. Ouburg S.A. Morré Drugs Today 2009;45(Suppl. B):135-40. Caroline Bax BW.indd 87 26-08-10 12:17 Abstract Chlamydia trachomatis (CT) serovars are divided in three serogroups, namely serogroup B, serogroup I (intermediate) and serogroup C, and subsequently into 19 different serovars. Worldwide, serogroup B is the most prevalent, followed by serogroup I. Clear differences have been observed in the duration of infection and growth kinetics between serovars from different serogroups in murine and cell culture models. Reasons for these observed differences are bacterial and host related, and are not well understood. The aim of this study was to determine the differences in immunoglobulin (Ig) G responses between the three serogroups in a group of patients infected with different serovars. Serovars were assessed from 235 CT positive patients and quantitative IgG responses were determined. Analyses of variance were used to compare the IgG responses between the three serogroups. Of the serovars, 46% were B group (with serovar E the most prevalent: 35.3%), 39.6% were I group and 14.3% were C group. A highly signiﬁcant difference in serologic response was shown when comparing the mean IgG concentrations (AU/ml) of patients having serovars in the most prevalent serogroup compared to the other serogroups: B= 135, C= 46 and I= 60 (B vs. C and B vs. I, p<0.001) part III | Chapter 7 In conclusion, the most prevalent serovars generate the highest serologic responses. 88 Caroline Bax BW.indd 88 26-08-10 12:17 Differences in serological responses of serogroups 89 Introduction Chlamydia trachomatis (CT) is one of the most common bacterial sexually transmitted diseases (STDs) worldwide. In most cases, infected patients undergo an asymptomatic and uneventful course of infection, and are thus likely to remain untreated. Untreated CT can give rise to late complications, including pelvic inﬂammatory disease (PID), ectopic pregnancy, and tubal pathology resulting in tubal factor subfertility1. Research devoted to the bacterium has provided insight in the structural components of CT. Strains of CT are classiﬁed into serovars based on nucleotide sequence differences in the omp1 gene, encoding the major outer membrane protein (MOMP)2. To date, 19 serovars of CT are known, generally causing conjunctival and urogenital infections3. The different serovars are divided in serogroups, based on phylogenetic mapping (table 1). Table 1: Serovar distribution into serogroups. Serogroup Serovar B B, Ba, D, Da, E, L1, L2, L2a C A, C, H, I, Ia, J, K, L3 I (Intermediate) F, G, Ga Conventional serotyping involves CT culture and both monocloncal and polycloncal antibodies against the MOMP protein. Currently, polymerase chain reaction (PCR)-based techniques allow rapid identiﬁcation of different serovars4-6. Geographical distributions of serovars/serotypes are very similar worldwide, except in small core groups (e.g. men-having-sex-with-men (MSM), and small communities)7,8. Relations between speciﬁc serovars and the clinical course of infection have been observed9-11, although conﬂicting data have been reported12-15. Ito et al. demonstrated that serovars D and E (belonging to serogroup B) cause the longest duration of infection in a murine model. Furthermore, a comparison of the invasiveness of strains D and H demonstrated a much higher frequency of uterine horn infection with serovar D16. In addition, they studied the in vitro growth, and elementary body (EB)-associated cytotoxicity of CT serovars D and H, in order to identify the above mentioned differences. These differences correlate with virulence variations between these strains in the mouse model of human female genital tract infection, and with phenotypic characteristics that could explain human epidemiological data on serovar prevalence and levels of shedding during serovar D and H infection. They showed that serovar H EBs were signiﬁcantly more cytotoxic compared with serovar D EBs, which have a longer duration of infection in the murine model and are much more prevalent in humans17. The data suggest a relation between the different serovars and the serologic responses in the murine model. This relation has not yet been studied in humans, but the murine and in vitro data suggest different serologic responses could be observed. Different commercial serologic assays to detect IgG against CT are currently available18. Medac Diagnostika has recently developed a new CT IgG ELISA kit (Chlamydia trachomatis-IgG-ELISA plus) which Caroline Bax BW.indd 89 26-08-10 12:17 allows quantitative measurement of IgG levels in serum, enabling to study the relation between host serologic responses and speciﬁc serovars. The aim of this study was to elucidate serologic IgG responses in patients infected with different serogroups. Methods Patient populations The study included 235 CT-infected patients, who visited either the outpatient department (OPD) of Obstetrics and Gynaecology at the MC Haaglanden Clinic, or the STD outpatient clinic in The Hague, the Netherlands from January to October 2008. In the Department of Obstetrics and Gynaecology clinical samples were obtained from patients visiting the OPD for various reasons including pregnancy, discharge, menstrual disorders, subfertility, and contraception. At the STD outpatient clinic reasons for visiting were STD-related complaints, partner notiﬁcation or STD check-up at the client’s request. part III | Chapter 7 Information was collected about age, gender (STD clinic only, OPD all female), ethnicity, and symptoms 90 (i.e. asymptomatic, symptoms, or upper genital tract infection). Serum samples were collected from all patients. CT detection and genotyping For the detection of CT we used a probe hybridisation assay from urethral and endocervical swabs (PACE 2 assay, Gen-Probe). Swabs were analysed within 24 hours according to Gen-Probe’s packet insert instructions. Ampliﬁcation, detection and genotyping using the CT DT assay CT detection and genotyping were determined on all samples positive for CT, using the CT-DT detection and genotyping assay8. The CT-DT ampliﬁcation, detection and genotyping steps were performed according to the manufacturer’s instructions (Labo Biomedical Products BV, the Netherlands). Brieﬂy, ﬁrst the CT-DT ampliﬁcation step was performed on extracted DNA to amplify all serovars available in GeneBank. Secondly, the CT detection step was performed to conﬁrm the results detected with the PACE 2 assay. The CT detection step detects all serovars, subserovars, and genovariants in GeneBank, but cannot differentiate between serovars. Borderline samples were considered positive. Finally, all PCR products that were positive with the CT detection step were further analysed with the CT-DT genotyping assay. The CT-DT genotyping assay is a reverse hybridization probe line blot (RHA) with a probe for the detection of the cryptic plasmid, and probes to detect the three different CT serogroups (B, C, and Intermediate) and the different serovars (A, B/Ba, C, D/Da, E, F, G/Ga, H, I/Ia, J, K, L1, L2/L2a, and L3). Caroline Bax BW.indd 90 26-08-10 12:17 Differences in serological responses of serogroups 91 Serology Determination of IgG levels in serum of all patients was done with ELISA, as described below. The ELISA procedure using the Chlamydia trachomatis-IgG-ELISA plus kit (Medac Diagnostika), was performed according to the manufacturer’s protocols. The assay employed was a quantitative IgG serology kit. Calculations to determine concentrations of IgG (Arbitrary Units (AU) /ml) in the serum were performed according to the protocols provided. Concentrations below 22 AU/ml were considered negative, concentrations between 22 AU/ml and 28 AU/ml were considered equivocal, and concentrations above 28 AU/ml were considered positive, as per the manufacturer’s instructions. Statistical analyses Analysis of variance (ANOVA) statistics were used to compare the IgG serum levels of the three CT serogroups. Equivocal values were excluded from these calculations. Analyses were performed with GraphPad Instat 3; p values < 0.05 were considered signiﬁcant: p values between 0.05 and 0.1 were considered a statistical trend. Results Serovars The serovar distribution in the cohort is shown in table 2. Serovar E (35.3%; serogroup B) was the most prevalent, followed by serovar F (25.1%) and serovar G/Ga (14.5%; serogroup I) (Table 2). It was found that 108 of the samples (46.0%) contained serovars belonging to serogroup B, 93 samples (39.6%) belonged to serogroup I, and 34 samples belonged to serogroup C (14.5%). The serovars A, C (both ocular serovars), and L1 – L3 were not observed in the studied cohort. Table 2: Serovar distribution and mean concentrations of IgG per serogroup. no. of patients % Total patients per serogroup % mean [IgG]* B/Ba 2 0.9 108 46.0 134.9 D/Da 23 9.8 E 83 35.3 H 3 1.3 34 14.5 45.9 I/Ia 4 1.7 J 21 8.9 K 6 2.6 F 59 25.1 93 39.6 60.2 G/Ga 34 14.5 235 100% 235 100% Serogroup Serovar B C I *Mean IgG concentrations in Arbitrary Units per ml (AU/ml), as per manufacturer’s instructions. Caroline Bax BW.indd 91 26-08-10 12:17 Serological IgG responses Serum CT IgG concentrations were measured for all patients using the Medac Chlamydia trachomatis-IgGELISA plus assay and mean CT IgG levels (AU/ml) were calculated per serogroup, as shown in table 2. Serogroup B, containing the most prevalent serovar E, had the highest mean IgG concentration. Highly signiﬁcant differences were observed comparing serogroup B to the other serogroups (B vs. C: p < 0.001; B vs. I: p < 0.001), while no statistically signiﬁcant differences were observed between serogroups C and I (C vs. I: p > 0.05). Discussion This is the ﬁrst study to compare serologic IgG responses in urogenital CT infections based on serogroup. As expected, serovar E is the most prevalent serovar in our study, followed by serovars F, and G/Ga. The mean CT IgG response was signiﬁcantly the highest in serogroup B (including the most prevalent serovar E), compared with the other serogroups. No differences were observed in the mean CT IgG concentra- part III | Chapter 7 tions between serogroups C and I. 92 This study clearly shows that serovar E (in the B serogroup) results in the highest serologic responses. This result is partly in line with a previous study showing that variable segments of the serovar E MOMP induce high serologic responses19. A similar increase in serologic responses to a speciﬁc serovar (D) has recently been described in an Indian population20. Furthermore, Morré et al. have shown that serovar E is more frequent in persistent infections compared with resolving infections21. In the study by van der Snoek et al. it was shown that rectally L2-infected men had signiﬁcantly higher serologic titers compared with men not infected rectally with lymphogranuloma venereum (LGV)22. The authors concluded that these signiﬁcantly increased titers, which can slowly diminish over time, probably represent the severe, more invasive, and more often chronic inﬂammation of the rectum caused by LGV serovars, compared with other serovars. Murine studies have shown that serovar D results in a longer and more invasive infection compared to serovar H, but resulted in less cytotoxicity17. Combining the results from this study with the published literature clearly shows that speciﬁc serovars may elicit stronger serologic responses compared with other serovars, and that this might be due to a more aggressive, or invasive course of infection. Speciﬁcally, serovars in the B serogroup (i.e. serovars D, E, and L2) seem to result in more severe infections. These results and those on the toxicity of CT serovars can be combined with data on development of complications and symptoms to gain more insight into the immunological responses underlying Chlamydia pathogenesis17,23. In conclusion, the present study demonstrates that serovars of serogroup B induce higher serologic responses than serovars of serogroups C and I. The results of this study will be included in a much larger EU Framework study to conﬁrm these results, to further enhance the power of the study, and enable Caroline Bax BW.indd 92 26-08-10 12:17 Differences in serological responses of serogroups 93 multivariate analyses to obtain further insight in the immunopathogenesis of CT infections. This will enable risk proﬁling of at-risk patients to prevent development of long-term complications. Acknowledgements This work was supported by the European Commission within the Sixth Framework Program through the EpiGenChlamydia project (contract no. LSHG-CT-2007-037637). See www.EpiGenChlamydia.eu for more details Caroline Bax BW.indd 93 26-08-10 12:17 part III | Chapter 7 References 94 1. Morré SA, Spaargaren J, Ossewaarde JM, et al. Description of the ICTI consortium: An integrated approach to the study of Chlamydia trachomatis infection. Drugs Today (Barc) 2006;42(Suppl. A):107-14. 2. Brunele BW, Sensabaugh GF. The ompA gene in Chlamydia trachomatis differs in phylogeny and rate of evolution from other regions of the genome. Infect Immun. 2006;74:578-85. 3. van der Laar MJ, van Duynhoven YT, Fennema JS, et al. Differences in clinical manifestations of genital chlamydial infections related to serovars. Genitourin Med. 1996;72:261-5. 4. Meijer A, Morré SA, van den Brule AJ, et al. Genomic relatedness of Chlamydia isolates determined by ampliﬁed fragment length polymorphism analysis. J Bacteriol. 1999;181:4469-75. 5. Molano M, Meijer CJ, Morré SA, et al. Combination of PCR targeting the VD2 of opm1 and reverse line blot anlysis for typing of urogenital Chlamydia trachomatis serovars in cervical scrapes specimens. J Clin Microbiol. 2004;42:2935-9. 6. Morré SA, Moes R, van Valkengoed I, et al. Genotyping of Chlamydia trachomatis in urine specimen will facilitate large epidemiological studies. J Clin Micobiol. 1998;36:3077-8. 7. White JA. Manifestations and management of lymphogranuloma venereum. Curr Opin Infect Dis. 2009;22:57-66. 8. Quint KD, van Doorn LJ, Kleter B, et al. A highly sensitive, multiplex broad-spectrum PCR-DNA- enzyme immunoassay and reverse hybridization assay for rapid detection and identiﬁcation of Chlamydia trachomatis serovars. J Mol Diagn. 2007;9:631-8. 9. Kuo C-C, Wang S-P, Holmes KK, et al. Immunotypes of Chlamydia trachomatis isolates in Seattle, Washington. Infect Immun. 1983;41:865-8. 10. Lan J, Meijer CJ, van den Hoek AR, et al. Genotyping of Chlamydia trachomatis serovars derived from heterosexual partners and a detailed genomic analysis of serovar F. Genitourin Med. 1995;71;299-303. 11. Naher H, Petzoldt D. Serovar distribution of urogenital C. trachomatis in Germany. Genitourin Med. 1991;67:114-6. 12. Batteiger BE, Lennington W, Newhall WJ, et al. Correlation of infecting serovar and local inﬂammation in genital chlamydial infections. J Infect Dis. 1989;160:332-6. 13. 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. J Infect Dis. 1995;172:1013-22. 14. Lampe MF, Wong KG, Stamm WE. Sequence conservation in the major outer membrane protein gene among Chlamydia trachomatis strains isolated from the upper and lower genital tract. J. Infect Dis. 1995;172:589-92. 15. Workowski KA, Stevens CE, Suchland RJ, et al. Clinical manifestations of genital infection due to Chlamydia trachomatis in women: differences related to serovars. Clin Infect Dis. 1994;19:756-60. 16. Ito JI, Jr., Lyons JM, Airo-Brown LP. Variation in virulence among oculogenital serovars of Chlamydia trachomatis in experimental genital tract infection. Infect Immun. 1990;58:2012-3. 17. Lyons JM, Ito JI, Jr., Penã AS, et al. Differences in growth characteristics and elementary body associated cytotoxicity between Chlamydia trachomatis oculogenital serovars D and H and Chlamydia muridarum. J Clin Pathol. 2005;58:397-401. 18. Morré SA, Munk C, Persson K, et al. Comparison of three commercially available peptide-based immunoglobulin G (IgG) and IgA assays to microimmunoﬂuorescence assay for detection of Chlamydia trachomatis antibodies. J Clin Microbiol. 2002;40:584-7. 19. Ortiz L, Angevine M, Kim SK, et al. T-cell epitopes in variable segments of Chlamydia trachomatis major outer membrane protein elicit serovar-speciﬁc immune responses in infected humans. Infect Immunol. 2000;68:1719-23. 20. Srivastava P, Gupta R, Jha HC, et al. Serovar-speciﬁc immune responses to peptides of variable regions of Chlamydia trachomatis major outer membrane protein in serovar D-infected women. Clin Exp Med. 2008;8:207-15. Caroline Bax BW.indd 94 26-08-10 12:17 Differences in serological responses of serogroups 95 21. Morré SA, van den Brule AJ, Rozendaal L, et al. The natural course of asymptomatic Chlamydia trachomatis infections: 45% clearance and no development of clinical PID after one-year follow-up. Int J STD AIDS. 2002;13 Suppl 2:12-8. 22. van de Snoek EM, Ossewaarde JM, van der Meijden WI, et al. The use of serological titres of IgA and IgG in (early) discrimination between rectal infection with non-lymphogranuloma venereum and lymphogranuloma venereum serovars of Chlamydia trachomatis. Sex Transm Infect 2007;83:330-4. 23. Anttila T, Saikku P, Koskela P, et al. Serotypes of Chlamydia trachomatis and risk for development of cervical squamous cell carcinoma. JAMA. 2001;285:47-51. Caroline Bax BW.indd 95 26-08-10 12:17 Caroline Bax BW.indd 96 26-08-10 12:17 Part IV General Discussion and Summary Caroline Bax BW.indd 97 26-08-10 12:17 Caroline Bax BW.indd 98 26-08-10 12:17 part IV | Chapter 8 General Discussion Caroline Bax BW.indd 99 26-08-10 12:17 Introduction Uterine instrumentation, such as curettage, intrauterine device (IUD) insertion, or hysterosalpingography (HSG), may lead to upper genital tract infections in women with Chlamydia trachomatis (CT) infections. There are no general policies in the Netherlands with regard to the prevention of complications of CT infections as a result of uterine instrumentation. To develop a guideline we determined the prevalence of CT infections in a general outpatient department (OPD) of Obstetrics and Gynaecology (O&G), and tried to identify risk factors in subpopulations. A second objective was to determine what sample site would be most effective in detecting CT infections in women. Further, we studied the distribution of serovars over various sample sites. The results of our prospective cohort studies will be discussed in PART I. Serum Chlamydia IgG antibody testing (CAT) has been widely introduced in the fertility work-up, as a screenings method for tubal pathology. The microimmunoﬂuorescence assay (MIF) is considered the ‘gold standard’. However, this assay is very laborious, requires experienced laboratory technician, and cross-reactivity may occur. Therefore, new serological assays have been developed. We evaluated two new available enzyme immunoassays (EIAs) in various populations. The role of serology will be discussed in part IV | Chapter 8 PART II. 100 Data about speciﬁc serovars and the clinical course of infection, the rate of upper genital tract progression, and the clearance-persistence rate are inconclusive. We need to get more epidemiological information about the CT serovars and serogroups. Therefore, in PART III we focus on the different CT serogroups and serovars, both to get insight in the serovar distribution within different ethnical groups, and to get insight in the relations between the serological responses and serovars/serogroups. In addition, recommendations and suggestions for future research are provided. Caroline Bax BW.indd 100 26-08-10 12:17 General Discussion 101 Part I Clinical manifestations Prevalence and risk factors - Conclusion Uterine instrumentation - Risk for upper genital tract infection - RCOG recommendations - Screen-and-treat or antibiotic prophylaxis - Conclusion Test characteristics and sample sites - DNA probe or urine analysis - Sample sites female patients - Sample sites male patients - The pharyngeal site - Multiple site testing - Conclusion Serovar - Concurrent serovar infections - Rectal versus urogenital serovars - Conclusion Prevalence and risk factors The overall prevalence of CT infections in our OPD of Obstetrics and Gynaecology was 4.5%, comparable to other European studies1,2. Risk factors, i.e. age and postcoital bleeding, as identiﬁed in other populations were conﬁrmed1,3 (Chapter 2). We found signiﬁcantly higher prevalences in younger women (15.8% < 20 years of age and 6.9% < 30 years of age). Approximately 75% of our CT infections occurred in women under 30 years of age, and nearly 92% in women under 40 years of age. In the United States population screening has been advised for women 15–24 years of age, in other countries age-based screening for women under 30 years of age has been advised2,4. We conclude that to prevent complications of uterine instrumentation it might be useful to screen all women in the fertile age (<40) or give prophylactic antibiotics. Our study conﬁrms the association between CT infections and postcoital bleeding. However, since all patients with the complaint of postcoital bleeding and a CT infection were under 30 years of age, this did not help to reveal more CT positive patients than age-based screening alone. We could not conﬁrm the relation between CT infections and ethnic origin as described in other studies, in which a signiﬁcantly higher prevalence was found in women from Suriname and the Netherlands Antilles, although we observed a trend in a similar direction5. Caroline Bax BW.indd 101 26-08-10 12:17 Conclusion The prevalence and risk factors we found were comparable to other studies (European, inner city, OPD Obstetrics and Gynaecology population). Uterine instrumentation Risk for upper genital tract infection Women undergoing uterine instrumentation are at risk for developing upper genital tract infection as a result of either ascending cervical infections, or as a result of reactivation of microorganisms persisting in the genital tract6. However, there are no randomised controlled trials in which the protective effects of prophylactic antibiotics for transcervical intrauterine procedures were studied7. Prevalence of pelvic inﬂammatory disease (PID) after uterine instrumentation is difﬁcult to assess. Forseyl et al. describe clinical pelvic infection in up to 4% of the patients after HSG, and in 10% of the patients after HSG with tubal disease present6. There is limited information available on complications of part IV | Chapter 8 IUD insertion or abortion curettage. Grimes et al. found after IUD insertion with or without prophylactic 102 antibiotics an OR of 0.70 (95% CI 0.36-1.38) in high risk populations, and therefore no signiﬁcant difference in subsequent PID8. There are no recent studies and in the older ones usually no differentiation between CT PID and PID caused by other bacteria is made. In many studies the criteria for PID are unclear. In some studies PID is conﬁrmed by laparoscopy, while in other studies symptoms of abdominal pain, sometimes combined with fever or ultrasound abnormalities are used as criteria for PID. Especially for women attending fertility clinics the issue of prophylactic antibiotics, or the screen-andtreat approach prior to uterine instrumentation, is under discussion. Both methods have their pros and cons which will be discussed in the following section. Cervical sampling by NAAT (nucleic acid ampliﬁcation test) is a sensitive test to detect CT infections9. IgG antibodies to CT are considered as evidence for a past CT infection; however they do not reﬂect an actual cervical infection10. Women with CT antibodies could be harbouring a (dormant) CT infection, and are therefore at risk for reactivation of the infection. The characteristics of the serological assays will be discussed in Part II. Chlamydial heat shock protein (cHSP60) antibodies appear to have a high correlation with tubal occlusion11, Chapter 5. However, in women without antibodies, the presence of Chlamydia in the upper genital tract can not be excluded. Therefore, Land et al. and Thomas et al. suggest that all subfertile women should receive prophylactic antibiotics before uterine instrumentation12,13. Ng et al. and Macmillan conclude otherwise14,15. They conclude that routine antibiotic prophylaxis may be associated with increased risk of persistent or recurrent infection16, and could lead to antibiotic resistance17,18. In case of routine antibiotic prophylaxis there is no screening of the partner nor screening for other bacteria, which may cause PID (Mycoplasma, anaerobe and aerobe bacteria, and gonorrhoeae). Witkin et al. suggest an alternative approach; both cervical IgA antibodies to CT and serum anti-chlamydial HSP60 screening, to provide the best indication as to which women may be harbouring CT19. Caroline Bax BW.indd 102 26-08-10 12:17 General Discussion 103 RCOG recommendations The Royal College of Obstetricians and Gynaecologists (RCOG) recommend in their 1998 guidelines that all women undergoing uterine instrumentation should be screened for Chlamydia, or should receive prophylactic antibiotics20. The 2000 guidelines are more ambiguous: screening for Chlamydia prior to uterine instrumentation should be considered only in patients at risk, i.e. women <25 years, and protection against upper genital tract infections should be considered in all women presenting for fertility investigation21. In their 2004 guidelines they state that before undergoing uterine instrumentation women should be offered screening for CT using an appropriately sensitive technique, and prophylactic antibiotics should be considered before uterine instrumentation if screening has not been carried out22. They do recommend antibiotic prophylaxis for termination of pregnancy. Screen-and-treat or antibiotic prophylaxis Penny et al. found screen-and-treat in preventing PID in women undergoing an induced abortion to be more cost-effective than prophylactic antibiotics23. Ng et al. and Macmillan made several side marks, as mentioned above14,15. We conducted a cost-effectiveness analysis for preventing PID after uterine instrumentation in a general O&G population (data not published). It turned out to be very difﬁcult to establish the prevalence of PID after uterine instrumentation. Prophylactic antibiotics for all seemed most cost-effective. We also sent a questionnaire to gynaecologist in the urban areas in the western part of the Netherlands in which we asked for the preferred strategy to prevent PID after uterine instrumentation (ﬁgure 1). Although the majority considered prophylactic antibiotics for all to be most effective, they preferred a screen-and-treat policy in daily practice (data not published). Figure 1. Tree for the disease progression with seven strategies to prevent CT PID after uterine instrumentation (© J.Kievit) Caroline Bax BW.indd 103 26-08-10 12:17 Conclusion In conclusion, policy for prevention of CT PID after uterine instrumentation in (sub)fertile women is still subject of debate. We would prefer a screen-and-treat policy, for both a PCR of a cervical sample as well as CAT. Test characteristics and sample sites (summarised in table 1.) DNA probe or urine analysis In our CT prevalence study (Chapter 2) nearly half of the CT infected patients (44%) were detected by only one of the two diagnostic tests (DNA probe (cervix, urethra and rectum) or urine analysis). Since no discrepant analysis was performed some false positive or negative results cannot be excluded. With respect to the sampling site of the DNA probe, we found 70.8% of the CT infected patients to be positive on the cervical sampling site. A remarkable high percentage of 12.5% of the CT infected patients was found positive at the urethral sampling site only, clearly indicating that sampling the cervical/vaginal part IV | Chapter 8 site only results in a signiﬁcant number of false negative patients. Multiple site infections were found in 104 18.8% of the patients. Six patients were found positive at the rectal sampling site, of which in 3 patients this was the only positive site. For women it is advised to test urine or a urethral specimen in combination with a cervical specimen24. Other studies found the vaginal introitus also a representative site to detect CT infections, with the advantage of being noninvasive25. Rectal swabs should only be obtained in women at high risk of being infected. We would have missed three CTI patients if we had used only the cervical swab and the urine analysis. These three patients were found positive by anorectal swab only. If we had used only the cervical and urethral swab we would have missed 15 CT patients (24.2%) (12 patients (19.4%) only positive by urine analysis and three patients only positive by rectal swab with negative urine analysis). Sample sites female patients In our second cohort of OPD O&G population, all patients (n=71) were tested at both the cervical and urethral sampling site (Chapter 3). We found 91.5% of the CT infected patients to be positive on the cervical sampling site, and 8.5% was positive on the urethral sampling site only. Multiple site infections were found in 53.5% of these women. These results are similar to our previous results described in Chapter 2. Mahto et al. found similar results, 91.1% of the CT infected patients to be diagnosed by cervical swab only, and 8.9% to be positive on the urethral site only26. In 85.6% multiple site infections were found. In all female patients (OPD O&G and STD clinic), cervix or vagina sampling revealed 94.9% of the CT infected patients. A questionnaire of the history of sexual behaviour would help to reveal the others (rectum and pharynx). Sample sites male patients In male patients, urethral site sampling or urine revealed 85.9% of the CT infected patients. In menwho-have-sex-with-men (MSM) rectal samples should be taken as well, to detect more patients with a Caroline Bax BW.indd 104 26-08-10 12:17 General Discussion 105 CT infection. In our study all, but one patient were found using these sample sites. In the literature the prevalence of rectal CT infections is 8.5% in a MSM population27. In our study, 28 out of 40 patients tested at the rectal site were positive for CT (70%). The pharyngeal site The pharyngeal sampling site does not seem to contribute much to the detection of CT infected patients. It is not known whether this site has clinical implications, if standard treatment is effective and of the infection can be transmitted via oral sex or kissing. We found 11 out of 141 (7.8%) tested female patients to be positive at this site, and 6 out of 43 (13.6%) tested male patients. Over 70% of the positive patients were positive on other sites too. In only ﬁve of the tested patients (2.7%) this was the only positive site The same positive rate was found by others (6-18%, in various populations)28,29. Multiple site testing The prevalence of multiple site infection in our studies was somewhat lower than described by others (table 1). Michel et al. found that over 80% of 73 women tested positive for CT on 4 sample sites (cervix, urethra, vaginal secretions and ﬁrst-void urine (FVU))30. The cervical swab had the highest sensitivity (98.6%) (CT organism load), followed by vaginal secretions (94.5%), urethra (93.2%) and FVU (84.9%). Vaginal swabs perform better in detecting CT infections than FVU, and nearly as well as cervical swabs, but are probably more acceptable than the latter in screening asymptomatic patients since no gynaecologic examination is necessary. In men over 96% of patients was found positive at two sites (urethra and FVU), with no signiﬁcant difference between the CT load, making FVU a more practical and acceptable screening method in asymptomatic patients. Tabel 1. Percentage of CT infected patients found per tested sampling site. Female Male(m)/Female(f ) Sample sites Authors cervix/vagina urethra multiple rectum pharynx Bax 2002 70.8% 37.5% 18.8% 12.5%(f ) - Bax 2010 (O&G) (STD) (*) 91.5% 94.9% (95.5%) 62.0% 2.8% (100%) 53.5% 18.1% (22.5%) 15.3%(f ) (84.4%)(f ) 16.5%(m) (70%)(m) 6.2%(f ) (7.8%)(f ) 3.5%(m) (14.0%)(m) - - (*) Matho (ref 26) 91.1% 94.4% 85.6% Ivens (ref 27) multiple 85.9% (86.4%) 4.1% (16.3%) 8.5%(m) Carré (ref 28) 3.8%(f ) 1.1%(m) Hamasuna† (ref 29) 44-61%(f ) 6-10%(m) Michel‡ (ref 30) Male urethra/urine 80.8% 96.6% (*) Percentage of tested patients (STD) † f=female sex worker, m=student ‡ For female patients positive on 4 samples sites, for male patients positive on 2 sample sites. Caroline Bax BW.indd 105 26-08-10 12:17 Conclusion Although DNA ampliﬁcation methods used on urine specimen are reported to be so sensitive that they may reﬂect positivity at the cervical sampling site, our study shows discrepancies. Therefore we conclude that, although population screening on CT infected patients with urine analysis is an easy method, cervical or vaginal specimens are complementary in women. The vaginal samples could also be self-obtained if preferred, as well as urine. In men FVU would probably be the best screening method. In both male and female patients rectal and/or pharyngeal samples should be obtained if rectal and/or oral sex is reported. Serovar Concurrent serovar infections Concurrent serovar infections in one sample site are rare. We found prevalence of concurrent serovar infections (2.6%) in the same (low) range as described in other studies31. Worldwide, serovars D, E, and F are most prevalent31,32. In our study, we found in most sampling sites part IV | Chapter 8 serovar G/Ga to be the third most prevalent serovar after D and E. Recent studies have demonstrated 106 an association between CT serovar G and squamous cell carcinoma33. Interestingly, the prevalence of serovar G/Ga was the lowest (5.7%) at the cervical sampling site in our study. Similar to our results, Lan et al. found serovar G as the third most prevalent serovar in young women visiting an OPD of Obstetrics and Gynaecology34. In some Asian countries higher prevalences of serovar G have been observed (7-15%), mostly in STD clinic populations, but also in obstetrical and gynaecological patients (14.9%)35. Rectal versus urogenital serovars In men, serovars D/Da and G/Ga were signiﬁcantly more prevalent in rectal than in urogenital swabs (28% vs. 7.9% and 40% vs. 13.6%, p=0.0081 and p=0.0033, respectively), suggesting tissue tropism, still based on unknown virulence factors. Also in women these serovars were more prevalent, although not signiﬁcant (33.3% vs. 9.6% and 22.2% vs. 13.8%). In men, serovar E was more prevalent in the urogenital swabs than in the rectal swabs (40.7% vs. 8%, p=0.0012), while in women it was approximately the same (35.3% vs. 44.4%), and in the normal range as compared to other Dutch studies. In female rectal specimens serovar E was most prevalent. The serovars B/Ba, H, I/Ia, and K were not found in the rectal swabs in both men and women. In women serovar J and F were also not found in rectal swabs. The same results were found by Barnes et al.36. The rectal swabs were obtained from MSM. They also tested the rectal swabs of 32 women and found two serovars B, one I/Ia, and one K. In our study those serovars were not found in men or women. It is suggested that these serovars are less viable in the rectum. The permeability to toxic substances could be inﬂuenced by the porin activity of the MOMP, therefore serotype might reﬂect organism permeability36,37. However, other still unknown virulence factors linked to serovars and CT strains in general might be responsible for the differences in sample site and infection rates. Caroline Bax BW.indd 106 26-08-10 12:17 General Discussion 107 Two explanations for the prevalence differences between rectal en urogenital specimens, not mutually exclusive are present: 1) serovars G/Ga and D/Da have a higher afﬁnity to epithelial cells of the rectum compared to urogenital epithelial cells, potentially partially mediated by the environment and 2) the high incidence of serovars G/Ga and D/Da in rectal specimens of MSM ﬁnds its origin in differences in sexual behaviour and group dynamics compared to heterosexuals. However, since in the heterosexual women included in our study the same trend was found, serovar distribution linked to core groups is less likely as an explanation. Other studies ﬁnd similar results; most rectal Chlamydia infections were caused by serovar G/Ga (47.9%) in MSM, while in the same population the prevalence of urogenital serovar G/ Ga for men and women was much lower (16% vs. 11% resp.)38,39. In San Francisco, rectal specimens of MSM were tested in two populations40. The prevalence of CT infections was 8.8% and 5.7% in patients visiting a STD clinic or a Gay men’s health centre, respectively. Unfortunately, no serovar analysis was performed. Barnes et al. describe signiﬁcant higher prevalences of serovar G/Ga in cervical isolates of heterosexual women and rectal isolates of MSM36. The prevalence of serovar G/Ga (13%) in the rectal isolates is however signiﬁcantly lower than in our study (40%) (p=0.0026). Conclusion In conclusion: the prevalence of multiple serovar infections at different sites of the same individual is relative low. Therefore serovar analysis could be performed on one positive sample site. Signiﬁcant differences in serovar distribution are found in rectal specimens of MSM with serovar G/Ga as the most prominent, with a same trend in women which did however not reach statistical signiﬁcance. Caroline Bax BW.indd 107 26-08-10 12:17 Part II Serology Chlamydia IgG antibodies Comparison of serological assays - Prevalence of antibodies - Test characteristics - Detection of tubal pathology - Conclusion Chlamydia IgA antibodies - Conclusion Chlamydia HSP60 antibodies - Seroprevalences in women with different degrees of tubal pathology - Conclusion part IV | Chapter 8 The role of serology 108 Chlamydia IgG antibodies Since many cases of tubal pathology are due to CT infections, serum Chlamydia IgG antibody testing (CAT) has been introduced in the fertility work-up, as a screenings method for tubal pathology. Chlamydia IgG antibodies remain detectable for years, even after antibiotic treatment, and are therefore a marker of a recent or past infection41,42. The microimmunoﬂuorescence assay (MIF) is considered to be the gold standard for the serological diagnosis of CT infections. However, this test has several limitations; cross-reactivity with Chlamydia pneumoniae (CP) in the existing assays should be taken into account43, MIF is laborious and reading subjective, and therefore, not suitable for daily routine. Recently new commercially available species-speciﬁc (peptide-based) enzyme immunoassays (EIAs) have been developed for the detection of CT antibodies. These EIAs provide objective reading and allow handling of more samples at the same time. So far they miss clinical evaluation. Comparison of serological assays Prevalence of antibodies (see table 2) Comparison was made of two new assays, CT-pELISA (Medac, Germany) and BAG-Chlamydia-EIA (Biologische Analysensystem GmbH, Germany) with MIF as gold standard, in various groups of obstetrical and gynaecological patients (patients with subfertility, pregnant women, control group and CT positive patients) (Chapter 4). The seroprevalence rates of IgG and IgA antibodies (using pELISA) found in our study were comparable to results found by others, except for our subfertility group, in which we found a lower prevalence44-47. Caroline Bax BW.indd 108 26-08-10 12:17 General Discussion 109 A possible explanation for the lower prevalence rate in our subfertility group might be that this group included only a small number of patients with tubal factor infertility (TFI) (n=11), where in other studies larger numbers of TFI patients were found. Therefore, comparison with non-TFI patients might be more applicable. The prevalence rates of CT-IgG-antibodies using the MIF were lower than described in other studies43,44,48. However, in other studies the titer at which the MIF is considered positive is often not mentioned, or lower. A lower titer will give more ‘false’ positive results. Table 2. Prevalence of Chlamydia trachomatis antibodies (IgG and IgA) using different detection assays in different gynaecological patient groups. Group Chapter 4 Morré (44) Maass (45) Petersen (46) Persson (47) Chapter 4 Gijsen (43) Morré (44) Clad (48) pELISA pELISA pELISA pELISA pELISA MIF (1:64) MIF (1:32) MIF (1:32) MIF (1:8) 31.6 32-58 Subfertility IgG 21.1 - 60 33-60 65 IgA 1.3 - 15.3 11-15 28 22-84 Pregnant IgG 17.3 - 12 30 IgA 3.3 - 3 10 23.3 16 Control* IgG 19.1 35 14.4 14 IgA 5 3 6.4 4 31.4 39 11 62.5 74 85 CT positive IgG 65 47 60.5 IgA 17.5 - 24.6 * Control group varies in studies: gynaecology patients, blood donors. Test characteristics (see table 3) By using the MIF assay as the gold standard, the test characteristics of the Chlamydia-EIA and pELISA for the determination of serological evidence of a recent or past CT infection therefore depended on the patient group tested. Both tests have reasonably high speciﬁcity and negative predictive value (NPV) and would therefore match the criteria of a screening test. We have to consider that cross-reactivity with other Chlamydia species may occur as in the MIF assay43,49. Morré et al. compare three ELISAs (including pELISA) with MIF as the gold standard44. They conclude that the ELISAs perform as well as the MIF, with the advantage of being less laborious, less expensive and easier to perform. The speciﬁcity and NPV are comparable to our results. Sensitivity and PPV in our study were somewhat lower. A possible explanation could be that Morré et al. found for their ‘in-house’ MIF assay a titer of ≥ 1:16 diagnostically signiﬁcant, while we used a titer of 1:6444. Both the ‘in-house’ character and different cut-off levels make it hard to compare results. We did not ﬁnd other studies describing the performance of the Chlamydia-EIA. In our study test characteristics for both tests were comparable, although the Chlamydia-EIA had a better sensitivity than pELISA and the pELISA a slightly better speciﬁcity than the Chlamydia-EIA. Caroline Bax BW.indd 109 26-08-10 12:17 Table 3. Test characteristics of the Chlamydia-EIA and pELISA in relation to MIF in different groupsa Chapter 4 Subfertility Pregnant Parameterb Chlamydia-EIA pELISA ChlamydiaEIA pELISA ChlamydiaEIA Control pELISA pELISA Morré (44) Sensitivity 66.7 58.3 77.1 62.9 76.8 47.8 71.4 Speciﬁcity 84.6 96.2 91.3 96.5 89.4 94 97.3 PPV 66.7 87.5 73 84.6 76.8 78.6 96.2 NPV 84.6 83.3 92.9 89.5 89.4 79.8 78.3 a MIF (IgG) was used as the gold standard. b For all parameters, values are percentages. Detection of tubal pathology (see table 4) When serology is used to detect tubal pathology, high speciﬁcity is important. However, when we used tubal pathology as the gold standard, speciﬁcity and NPV are somewhat lower. We have to consider that these results are based on a small number of patients (n = 32), and even a smaller number of patients with tubal pathology (n = 11). Further, in our study we evaluated tubal pathology by reviewing the laparoscopy part IV | Chapter 8 report. In 33% of patients with patent tubes, CT IgG antibodies were found by one or more assays, and 110 in 27% of patients with tubal pathology no CT IgG antibodies were found by any of the assays. The latter could be explained by other causes of tubal pathology. Further, not in all patients CT antibodies are formed. It is clear that no assay can predict the presence of tubal pathology with a very high sensitivity and speciﬁcity. Gijsen et al. described no signiﬁcant differences between two peptide-based EIAs and the MIF in predicting tubal pathology50. Our test results were comparable with theirs, with the exception of the pELISA, which showed a lower sensitivity but higher speciﬁcity. Land et al. compared CAT and tubal pathology at laparoscopy, using ﬁve serological tests51. They found a better NPV for the MIF (93%) (using the same titer as we did, 1:64) and pELISA (91%). Mouton et al. found a similar NPV (as Land et al.) for the pELISA (93%)52. A stricter use of deﬁnition for tubal pathology might be an explanation for different results. Land et al. suggested cross-reactivity with CP for all CAT tests, except pELISA. Verkooyen et al. however, found no suggestion for cross-reactions with CP53. Again, the use of different cut-off levels and different deﬁnitions of tubal pathology have to be taken into account. In the Netherlands tubal pathology is often deﬁned as extensive peri-adnexal adhesions Table 4. Test characteristics of the Chlamydia-EIA, pELISA and MIF in relation to tubal pathology (IgG). Gijsen (50) Land (51) Parametera Chapter 4 ChlamydiaEIA pELISA MIF (1:64) MIF (1:32) MIF (1:64) pELISA Mouton (52) pELISA Sensitivity 54.6 36.4 63.6 61 71 55 66.7 Speciﬁcity 71.4 85.7 81 68 74 87 75 PPV 50 57.1 63.6 LR+ 1.9 35 45 30.3 NPV 75 72 81 LR- 0.6 93 91 93.2 a For all parameters, values are percentages. Caroline Bax BW.indd 110 26-08-10 12:17 General Discussion 111 and/or distal occlusion of at least one tube. While in Finland the severity of tubal damage is described following the classiﬁcation of Hull and Rutherford. The appropriate cut-off level (number of false positive or false negative results) depends on patient characteristics, the available facilities and costs. CAT does seem more accurate in predicting severe distal tubal pathology than unspeciﬁed tuboperitoneal abnormalities54. Conclusion A screening test needs high speciﬁcity and a high NPV. pELISA seems to be a good alternative for MIF for the detection of CT antibodies. pELISA is more species-speciﬁc than the Chlamydia-EIA. It is less laborious and less expensive than MIF. When the pELISA and Chlamydia-EIA are compared with MIF as the gold standard, pELISA has the highest speciﬁcity, and in the subfertility group, it has an NPV comparable to that of Chlamydia-EIA. A higher NPV would help to better eliminate patients without tubal pathology. Chlamydia IgA antibodies For CP IgA antibodies have been associated with chronic inﬂammation and persistent infection55,56. The prevalence of IgA antibodies in the CT negative patients in our study were comparable to those found in blood donors, but the prevalence in CT positive patients we found was lower than found by Verkooyen et al. (pELISA: 17.5 vs. 45%, respectively)53. Again, differences in deﬁnition of tubal pathology could explain this. Signiﬁcantly more CT IgA antibodies were found in women with tubal pathology, compared to women without tubal pathology52,57. However, the OR of IgA antibodies was signiﬁcantly lower than the OR of IgG antibodies to CT (6.1 vs. 13.9, resp.)57. Mouton et al. however, found for IgA antibodies a higher PPV for tubal pathology compared to IgG antibodies (pELISA: 47.1% vs. 30.3%, respectively)52. Conclusion The role of IgA antibodies in the serodiagnosis of CT with the currently available assays remains further to be determined. Chlamydia HSP60 antibodies IgG antibodies to Chlamydia heat shock protein 60 (cHSP60) have been suggested as markers of chronic inﬂammation, and may therefore be good predictors of tubal pathology. These antibodies were found in over 70% of women with occluded tubes and in <20% of women with patent tubes58. Results of many studies are based on ‘in-house’ assays, which lack standardisation. Although comparison of the different ‘in-house’ assays is difﬁcult, there is consensus that the anti-cHSP60 responses in women increase with the severity of CT associated disease58,59. Due to the signiﬁcance of the possible association of the response to cHSP60 and progressive disease, a commercially produced assay that employs deﬁned Caroline Bax BW.indd 111 26-08-10 12:17 cHSP60 epitopes should allow for the comparison of results obtained in different laboratories, as well as forward the use of cHSP60 as a diagnostic tool, if the assay proves to be relevant in predicting pathology or clinical outcome of a urogenital chlamydial infection. Seroprevalences in women with different degrees of tubal pathology We evaluate a recently introduced commercially available cHSP60 serologic assay and determined the anti-cHSP60 responses in three groups women (Chapter 5). This is the ﬁrst study evaluating the commercially available cHSP60 assay in women with different degrees of tubal pathology (1. women with patent tubes, 2. pregnant women and 3. women with tubal pathology). We found the expected clear difference in IgG seroprevalence between women with and without (procedure conﬁrmed) tubal pathology, while an intermediate prevalence was observed in pregnant women. The same pattern but with lesser incidence was observed in the anti-cHSP60 responses. Finally, the median cHSP60 titers increased from group 1-3: 50, 100 and 200, respectively, suggesting an association between the level of cHSP60 response and tubal pathology. part IV | Chapter 8 Petersen et al. and Clad et al. found cHSP60 antibodies in women with pelvic inﬂammatory disease (85% 112 in patients with CT positive swabs and patients with occluded tubes, 20% in blood donors) and in subfertile women with open or occluded fallopian tubes (31% and 70% respectively)60, 61. The standardisation provided through this new commercially available assay will potentially enhance the comparability of cHSP60 results between laboratories. The results presented here, although obtained in small but well deﬁned groups, look promising. Indeed, power calculations (alpha =0.5, beta=0.1) show that doubling (1.7 times) the size of the (sub)groups would results in signiﬁcant p values instead of clear trends. Den Hartog et al. found a seroprevalence of cHSP antibodies of 15% (n=38) in women without tubal pathology and of 50.8% (n=30) in women with tubal pathology57. However, the OR of cHSP60 IgG antibodies was signiﬁcantly lower than the OR of CT IgG antibodies (5.9 vs. 13.9). Cross-reactivity with the very similar and highly present CP HSP60 is considered to be the reason for false positive results. Conclusion In conclusion, new commercially available EIAs, including cHSP60 assays, are good alternatives for the laborious ‘gold standard’ MIF. The concordance between CT IgG and cHSP60 positivity is high, almost 90%. An association between the level of cHSP60 response and tubal pathology has been suggested. However, further studies are needed in larger groups with different degrees of CT infection induced tubal pathology to further determine the diagnostic, prognostic, and clinical relevance of this new assay. Caroline Bax BW.indd 112 26-08-10 12:17 General Discussion 113 The role of serology Overall, differences in cut-off levels, differences in assays, differences in the deﬁnition of tubal pathology and different patients groups make it difﬁcult to compare results. Laparoscopy is considered the best test to diagnose tubal pathology, and it is the reference test in the evaluation of the diagnostic performance of other tests. However, it not suitable as a screenings test. For a long time, hysterosalpingography (HSG) has been the screenings test for tubal patency. Compared to laparoscopy, HSG has a speciﬁcity of 83%, and sensitivity of 65%62. For CAT to be used as a screenings test for tubal pathology it needs high speciﬁcity and high NPV. Recently, CAT has shown to perform just as well as HSG in predicting tubal factor infertility63-65. One of the limitations of CAT is the number of false positive results for the identiﬁcation of tubal pathology, which is reﬂected in the low PPV. In those patients laparoscopies would be performed in the absence of tubal pathology. Cross-reactivity with the prevalent CP is generally considered a cause43,51. The prevalence of CP antibodies in subfertile women with and without distal tubal pathology is over 70%43,66. However, despite the similarities between CT and CP, CP does not seem to contribute to the development of tubal pathology66. CAT is not useful in discriminating between clearance and persistence of CT infections. And persistence is an important risk factor for tubal pathology. Serological markers for persistent infection, i.e. CT IgA, cHSP60 and high-sensitivity C-reactive protein (hs-CRP), were signiﬁcantly more prevalent in women with tubal pathology57. As described before, CT IgA antibodies and cHSP60, do not always seem to perform superior to CAT. Combining CAT and hs-CRP, seems promising in identifying women with the highest risk of tubal pathology57,67. The combination of tests for humoral and cell-mediated immune response could also help to improve the tubal factor infertility prediction model68. Caroline Bax BW.indd 113 26-08-10 12:17 Part III Serovar Serovar distribution - Epidemiology - Ethnicity - Populations -- OPD Obstetrics and Gynaecology -- Female STD clinic population -- Male STD clinic population - Age - Conclusion Serologic responses - IgG responses in different serogroups part IV | Chapter 8 - Conclusion 114 Serovar distribution Currently, 19 different CT serovars and in addition many serovariants have been identiﬁed by sero- and genotyping69,70. The serovars can be divided into three serogroups: the B-group (serovars B, Ba, D, Da, E, L1, L2, L2a), the intermediate group (serovars F, G, Ga) and the C-group (serovars I, Ia, J, K, C, A, H, L3). The most prevalent CT strains worldwide are serovars D, E and F. They account for approximately 70% of the typed urogenital serovars. Conjunctivitis is mainly caused by serovars A-C. In urogenital infections serovars D-K are predominantly isolated. Serovars L1-L3 can be found in the inguinal lymph nodes31,32,71,72. Epidemiology An increasing number of isolates are typed worldwide and provide a wealth of information on the epidemiology of CT infections. Several studies have described inconclusive data about speciﬁc serovars and the clinical course of infection, the rate of upper genital tract progression, and the clearance-persistence rate. Information about the differences in serovar distribution could give information about the epidemiology of CT infections and might have clinical implications31,36,71,73-75. There is a clear difference in the treatment of a Lymphogranuloma venereum (LGV) infection or a CT infection with serovar D-K. Further, since 2003 serovariant L2b has been found, which has to be tested speciﬁcally with a recently developed new assay76. Therefore serovar determination could be clinically relevant. A recent study about serovar distribution in the Netherlands showed no signiﬁcant serovar distribution shift over time, but geographical differences were observed. This could be the result of different ethnic composition of the population77,78. Caroline Bax BW.indd 114 26-08-10 12:17 General Discussion 115 Therefore, we determined the CT serovar distribution among different ethnic populations in the region The Hague in two well deﬁned cohorts and compared the results to previous obtained Dutch serovars distribution studies (Chapter 6). In this study we determined for the ﬁrst time whether the geographical serovar distribution differences are ethnic-based. This study will obtain relevant epidemiological data on CT serovars distributions. Ethnicity The overall distribution of serovars we found closely resembles that of found in other studies in the Netherlands. Worldwide serovars D, E and F are most prevalent31,32. In our study serovar G/Ga was the third most prevalent serovar after E and F. The predominance of the B-group serovars in most studies conducted in different geographic locations and at different times suggests that these serovars have a biological advantage over the other serovars. When we compared our serovar distribution to the study of Spaargaren et al. we found signiﬁcant differences in serovar H and I/Ia. Geisler et al. found an association between serovar Ia and black race in a STD clinic population79. The overall serovar distribution in the predominantly black population was similar to that reported elsewhere. But serovar Ia was only found in a (high-risk) black population, an association also found by others78,80. These racial differences in serovar distribution might be due to behavioural, geographical or biological factors. It has been suggested that one of the reasons could be that there is less mixing with partners from a different race (relatively closed populations)78. And geographic distribution could inﬂuence the distribution of serovar Ia. Also intrinsic biological differences among persons of different race or among chlamydial serovars could inﬂuence acquisition, transmission and duration of infection (host susceptibility or immune response)39,78. In only one other Dutch study serogroup distribution was described between Dutch Caucasian (DC) and Surinam patients in a STD clinic population75. They found that, for both men and women, serovars F and G/Ga were less common among (high-risk) Surinam patients. Surinam men were more often infected with serovar I and E, Surinam women more often with serovar J. We could not conﬁrm these ﬁndings. In our study the serovars for Surinam men were 40% in the B-group, 40% in the intermediate group, and 20% in the C-group. For Surinam women the serovars were 43.8% in the B-group, 50% in the intermediate group, and were 6.3% in the C-group (1 serovar J). In the Netherlands higher prevalences of CT infections are found in Surinam and Netherlands Antilles (NA) patients5. One might compare these high risk groups with high risk black patients. However, also in our NA patients the prevalence of CT serovars is lowest in the C-group (prevalence serovar I/Ia in NA men 7.7% and in NA women 8%). A previous study on ethnicity in The Hague suggested a different spreading in ethnicity, i.e. less DC and more Turkish/Moroccan patients81. Our ethnic groups were smaller than expected and therefore slightly underpowered. Our power analysis was based in the ﬁrst cohort. However, compared to our ﬁrst cohort we included less non-DC patients. Since one comparison between DC and Netherlands Antilles patients was borderline signiﬁcant, larger studies might reveal clearer differences. Caroline Bax BW.indd 115 26-08-10 12:17 Populations To make comparisons with other studies concerning demographic, clinical and behavioural parameters it is important to take the population into account. We looked at 2 well deﬁned populations and will describe them separately. OPD Obstetrics and Gynaecology In the OPD O&G patients we found no signiﬁcant difference in the serogroup distribution between symptomatic and asymptomatic patients. Persson et al. found in an OPD O&G population in Sweden a serogroup distribution compared to our study73. They found that symptoms were not associated with any serovar. Lan et al. found an association between serovar G and symptomatic infection in OPD O&G patients in the Netherlands34. We found the highest prevalence in the intermediate group for symptomatic patients, but only 3 out of 19 were serovar G/Ga. Female STD clinic population part IV | Chapter 8 In the female STD clinic population no signiﬁcant differences were found. An earlier Dutch study showed 116 a different serogroup distribution compared to our study; B-group similar (54.1% vs. 51.8%), intermediate group lower prevalence (14.8% vs. 36.7%), and C-group higher prevalence (29.6% vs. 11.3%)75. There might be an increase of serovar G over time, as found by Suchland et al. in Seattle (over a 9-year period signiﬁcant increase of serovar G)80. We found a prevalence of serovar G/Ga of 15.3% vs. 6.7% found by van Duynhoven et al. in 199875. A similar tendency was found by Geisler et al.82. Others found that serovars F and G (intermediate group) were associated with fewer signs of cervical infection. No differences in serovar distribution were found between patients with PID and those with lower genital tract infections74. Lower abdominal pain (referring to possible PID) in women was more often associated with serovars F (30%) and G (33%) (Intermediate group overall 32%, B-group 6% and C-group 13%; OR 5.1). A similar observation was made for serovar K. Also a tendency was found towards fewer clinical signs of cervical infection with serovar F and G (not signiﬁcant)71,75. Reason could be that serovar F produces less signs of cervical infection and is therefore more often unrecognised and may lead to upper genital tract infection74. However, van der Laar et al. found in women no association between infecting serovar and clinical signs of cervical or urethral infection31. Two other Dutch studies found for women an association between asymptomatic infections and serovar Ia32,34. We could not conﬁrm these ﬁndings. Male STD clinic population For the male STD clinic populations we found no signiﬁcant differences. As was found in the female STD clinic population the prevalence of the intermediate serogroup seems to increase over time75,82. In men studies on speciﬁc serovar and clinical manifestations are contradictory31,32,75,83. Caroline Bax BW.indd 116 26-08-10 12:17 General Discussion 117 Age Age is an important risk factor for CT infections. It is known that the prevalence of CT infections declines with increasing age, in an OPD O&G population, as well as in a STD clinic population34. Although behavioural factors, such as age at ﬁrst sexual intercourse, frequency of partner change, and failure to use barrier contraception, clearly are important in contributing to the increased prevalence of chlamydial infections in younger women. The number of inclusion forming units (IFUs) in culture also has been shown to be higher in younger women78. Higher inclusion counts in younger patients may imply greater transmissibility of infection and may be a factor contributing to the very high age-speciﬁc prevalence of CT infection in adolescents. These infections may represent initial encounters with CT in immunologically naïve individuals, while priori immunity in older individuals may reduce the IFU counts in recurrent infection. In addition, if IFU counts decline with increasing duration of infection, older individuals would be expected to have lower counts. Eckert et al. found that women have signiﬁcantly higher IFU counts than men, black patients had signiﬁcantly lower IFU counts than whites, and C-group serovars had signiﬁcantly lower IFU counts than B-group serovars39. Suchland et al. found in Seattle, Public Health clinic, serovar B, Ia and mixed serovars to be associated with younger age. Serovar D, F, H and K were associated with older age, serovar G tended to be the associated with the oldest patients. C-group serovars might be more common in older patients because immunity against the more prevalent B-group serovars developed earlier in life80. This is consistent with the ﬁnding of lower IFUs in C-group serovars and in older patients39. In our study serovar E was the most prevalent serovar in all age-groups. In the age-group >40, serovar G was evenly present. We could not conﬁrm the ﬁndings that C-group serovars were more prevalent in older women (<20=16.7%, 20-29=13.8%, 30-39=12.3%, >40=15.8%). We did ﬁnd a higher prevalence of serovar J in women >40 years of age. The serovar distribution in different age-groups showed no signiﬁcant differences. A similar distribution was found by others80. Lan et al. found in asymptomatic women <30 years of age serovar E to be the most prevalent one (33.3%), and in OPD O&G patients <30 years of age serovar F to be the most prevalent one (30%)34. Conclusion In conclusion, this is the ﬁrst Dutch study taking ethnicity into account and the second largest study on serovar distribution in the Netherlands. No major differences were found between different ethnic groups. It did show clear differences in serovar distribution compared to previously published Dutch serovar studies. The clinical course of infection does not seem to be inﬂuenced much by the infecting serovar. Further studies on differences in clinical course should include analyses of genetic host factors. Host variation might play an important role in the development of clinical disease. Caroline Bax BW.indd 117 26-08-10 12:17 Serologic responses Relations between speciﬁc serovars and the clinical course of infection have been observed, although conﬂicting data have been reported, as we have described before37,71,74,83-86. Ito et al. demonstrated that serovars D and E (belonging to serogroup B) cause the longest duration of infection in a murine model. Furthermore, a comparison of the invasiveness of strains D and H demonstrated a much higher frequency of uterine horn infection with serovar D87. In addition, they studied the in vitro growth, and elementary body (EB)-associated cytotoxicity of CT serovars D and H, in order to identify the above mentioned differences. These differences correlate with virulence variations between these strains in the mouse model of human female genital tract infection, and with phenotypic characteristics that could explain human epidemiological data on serovar prevalence and levels of shedding during serovar D and H infection. They showed that serovar H EBs were signiﬁcantly more cytotoxic compared with serovar D EBs, which have a longer duration of infection in the murine model and are much more prevalent in humans 88. The data suggest a relation between the different serovars and the serologic responses in the murine model. This relation has not yet been studied in humans, but the murine and in vitro data suggest part IV | Chapter 8 different serologic responses could be observed. 118 Different commercial serologic assays to detect IgG against CT are currently available 44. Medac Diagnostika has recently developed a new CT IgG ELISA kit (Chlamydia trachomatis-IgG-ELISA plus) which allows quantitative measurement of IgG levels in serum, enabling to study the relation between host serologic responses and speciﬁc serovars. IgG responses in different serogroups We compared serologic IgG responses in urogenital CT infections based on serogroup (Chapter 7). The mean CT IgG response was signiﬁcantly the highest in serogroup B (including the most prevalent serovar E), compared with the other serogroups. No differences were observed in the mean CT IgG concentrations between serogroups C and I. This study clearly shows that serovar E (in the B serogroup) results in the highest serologic responses. This result is partly in line with a previous study showing that variable segments of the serovar E MOMP induce high serologic responses89. A similar increase in serologic responses to a speciﬁc serovar (D) has recently been described in an Indian population90. Furthermore, Morré et al. have shown that serovar E is more frequent in persistent infections compared with resolving infections91. In the study by van der Snoek et al. it was shown that rectally L2-infected men had signiﬁcantly higher serologic titers compared with men not infected rectally with LGV92. The authors concluded that these signiﬁcantly increased titers, which can slowly diminish over time, probably represent the severe, more invasive and more often chronic inﬂammation of the rectum caused by LGV serovars, compared with other serovars. Murine studies have shown that serovar D results in a longer and more invasive infection compared to serovar H, but resulted in less cytotoxicity88. Combining the results from this study with the published literature clearly shows that speciﬁc serovars may elicit stronger serologic responses compared with other serovars, and that this might be due to a more aggressive, or invasive course of infection. Speciﬁcally, serovars in the B serogroup (i.e., serovars D, E and L2) seem to result in more severe Caroline Bax BW.indd 118 26-08-10 12:17 General Discussion 119 infections. LGV infections have a more invasive character and therefore a stronger serological response. These results and those on the toxicity of CT serovars33,88 can be combined with data on development of complications and symptoms in order to gain more insight into the immunological responses underlying Chlamydia pathogenesis. Conclusion In conclusion, the present study demonstrates that serovars of serogroup B induce higher serologic responses than serovars of serogroups C and I. The results of this study will be included in a much larger European Union Framework study to conﬁrm these results, to further enhance the power of the study, and enable multivariate analyses to obtain further insight into the immunopathogenesis of CT infections. This will enable risk proﬁling of at-risk patients to prevent development of long-term complications. Caroline Bax BW.indd 119 26-08-10 12:17 Recommendations and suggestions for future research CT infection is the most prevalent sexually transmitted disease, which is strongly associated with PID, ectopic pregnancy and tubal factor subfertility. Prevalences are increasing worldwide with almost 100 million new infections each year. Striking differences between individuals have been observed in the clinical course of infection with CT. In the case of sexually transmitted infection with CT the following differences have been observed: i) transmission versus no transmission; ii) symptomatic versus asymptomatic course of infection; iii) persistence versus clearance of infection; and iv) development of late complications (e.g. tubal factor subfertility) versus no development of late complications. However, only a small portion of women develop secondary complications after infection. In general, the differences in the clinical course of infection can be explained by the interaction between the host (host factors) and the pathogen (virulence factors). This is an interaction which will be inﬂuenced by environmental factors such as co-infections (see ﬁgure 2) and the serological responses induced part IV | Chapter 8 by infection. 120 Figure 2. Integrated research approach for Chlamydia trachomatis infections as based on the clear differences in the clinical course of infection observed between individuals93,94. Clinical recommendations Young age, complaints of contact bleeding, being of Surinam or Netherlands Antilles origin, and living in urban areas are important risk factors for CT infections in the Netherlands95. Intrauterine instrumentation could lead to upper genital tract infection if an infection is present. However, it turned out to be difﬁcult to predict the increased risk of intrauterine instrumentation on the development of PID. Whether screen-and-treat or antibiotic prophylaxis for all is the best policy remains therefore still subject of debate, and the decision which policy to follow will remain up to the physician. However, we advise that when intrauterine instrumentation is performed one should screen for CT infections in women in their fertile years (both by PCR and CAT). Caroline Bax BW.indd 120 26-08-10 12:17 General Discussion 121 In women, taking cervical swabs and urine analysis were complementary. In a gynaecologic population, in which a gynaecologic examination is performed in most patients, taking cervical swabs would give the most accurate information (with regard to the intrauterine instrumentation), without extra effort. However, for screening purposes urine analysis alone of self-taken vaginal swabs may be more acceptable. Since the prevalence of multiple serovar infections is relatively low, serovar analysis could be performed on one positive sample site. CT serovars and strains In the Chapters 1 and 2 we described the demographic en epidemiological data in a O&G outpatient and STD population, while in Chapters 4, 5 and 7 we assessed serological responses to infection and its use for diagnostic and prognostic purposes in relation to susceptibility to and severity of infection. Besides obtaining new insight in the prevalence in our study groups our ﬁndings on sample site speciﬁc serovar distributions, potentially tissue tropism, is of major interest. Future research should focus on ﬁrst reproducing these results in a larger cohort and secondly if the ﬁndings will be conﬁrmed research looking more into the biological and molecular background should be initiated to identify the factors responsible for this proposed tissue tropism. In the Chapters 3, 6 and 7 we focussed speciﬁcally on the microbial factors, mainly the different strains, called serovars of CT. Serovar typing is used for epidemiological studies and transmission studies, but is also of clinical relevance for instance in identifying the speciﬁc serovar in rectal infections as LGV types versus other types, as the LGV types need a 3 week doxycycline instead on a one time azithromycin. Besides the tissue tropism and general epidemiological distribution of serovars identiﬁed in the studied populations, we showed for the ﬁrst time a relation between serovars and serological responses mimicking perfectly the incidence of the serogroups in the populations worldwide. We are currently already extending these studies to conﬁrm the results obtained. Further future research should focus at the virulence factors responsible. This will mean we have to look beyond the ompA gene which deﬁnes the serovars. Very recently, Multi Locus Sequence Typing (MLST) a technique in general used for phylogenetic analyses96 based on co-called housekeeping genes, has been adapted focussing on CT 6 genes97 with high variability, so we can look beyond the serovar level to strain variation. The ﬁrst results show this technique identiﬁes already 3-5 times more strains that typing on ompA only. The ultimate approach is sequencing the complete genome of 1Mb from CT for many different strains and serovars to identify on a genome level which genes are linked to tissue tropism, virulence, upper genital progression, persistence and many other factors of interest, to further unravel the differences in the clinical course of infection. Host genetics In this thesis 2 of the 3 factors inﬂuencing the clinical course have been studied, the third, host factors (see ﬁgure 2) were not addressed but are of major interest and importance in the course of infections. Since the cellular immune response to CT is subject to genetic inﬂuences, the degree and mechanisms of such genetic control will have important implications in understanding the immunopathogenesis of CT infection. Deeper knowledge in these areas will lead to therapeutic strategies and vaccine development. Caroline Bax BW.indd 121 26-08-10 12:17 Chlamydia twin studies published by Bailey et al. 98 describe the most relevant study in the ﬁeld of Chlamydia immunogenetics. They estimated the relative contribution of host genetics to the total variation in lymphoproliferative responses to CT antigen by analysing these responses in 64 Gambian twin pairs from trachoma-endemic areas. Proliferative responses to serovar A elementary body (EB) antigens were found to be stronger in monozygotic twins than in dizygotic twin pairs. Based on these observations, they calculated a heritability of 0.39, thus suggesting that host genetic factors contribute to almost 40% of the clinical presentation. Serology There seems to be more and more evidence that Chlamydia antibody testing (CAT) can replace HSG in predicting tubal factor subfertility. Laparoscopy remains the gold standard. However, CAT can help to identify those patients in which a laparoscopy is indicated. Serological markers for persistent infection, i.e. CT IgA, cHSP60 and high-sensitivity C-reactive protein (hs-CRP), are more prevalent in women with tubal pathology. Especially the combination of CAT and hs-CRP needs further exploration, but seems promising in identifying women at high risk of tubal pathology. The combination of test for humoral part IV | Chapter 8 and cell-mediated immune response could help to improve the tubal factor subfertility prediction model. 122 We were the ﬁrst to compare serologic IgG responses in urogenital CT infections based on serogroup. Our study demonstrates that serovars of serogroup B induce higher serologic responses than serovars of serogroups C and I. The results of this study will be included in a much larger European Union Framework99 study to conﬁrm these results, to further enhance the power of the study, and enable multivariate analyses to obtain further insight into the immunopathogenesis of CT infections. This will enable risk proﬁling of at-risk patients to prevent development of long-term complications. Integration of all results Integration of the three lines in ﬁgure 2 is needed on an international level. The European Union funded EpiGenChlamydia Consortium (www.EpiGenChlamydia.EU) 99 aims to structure such transnational research to such a degree that comparative genomics and genetic epidemiology can be performed in large numbers of unrelated individuals. Biobanking and data-warehouse building are the most central deliverables of the Coordination Action of the Consortium in Functional Genomics Research. In addition, the collective synergy acquired in this Coordination Action will allow for the generation of scientiﬁc knowledge on the CT–host interaction, knowledge on the genetic predisposition to CT infection and the development of tools for early detection of a predisposition to CT infection and its complications. Implementing strategies to promote a faster path for genetic knowledge from bench to bedside will be needed in the future. The various stakeholders in public health play a key role in translating the implications of genomics such as deriving from molecular epidemiology and host-pathogen genomics. This knowledge will not only enable clinical interventions but also health promotion messages and disease prevention programs to be targeted at susceptible individuals as well as subgroups of the population based on their genomic proﬁle (personalised healthcare). The ﬁeld involved in this translation is called Caroline Bax BW.indd 122 26-08-10 12:17 General Discussion 123 Public Health Genomics100,101 which has as major task “the responsible and effective translation of genome-based knowledge and technologies into public policy and health services for the beneﬁt of population health” (Bellagio statement, 2005: see www.graphint.org for details). Caroline Bax BW.indd 123 26-08-10 12:17 part IV | Chapter 8 References 124 1. Warszawski J, Meyer L, Weber P. Criteria for selective screening of cervical Chlamydia trachomatis infections in women attending private gynaecologic practices. Eur J Obs Gyn Reprod Biol. 1999;86:5-10. 2. Macmillan S, McKenzie H, Flett G, et al. Which women should be tested for Chlamydia trachomatis? Br J Obstet Gynaecol. 2000;107:1088-93. 3. Stergachis A, Scholes D, Heidrich FE, et al. Selective screening for Chlamydia trachomatis infections in a primary care population on women. Am J Epidemiol. 1993;138:143-53. 4. Centers for Disease Control and Prevention. Recommendations for the prevention and management of Chlamydia trachomatis infections, 1993. MMWR Morb Mortal Wkly Rep. 1993;42(RR-12):1-39. 5. van der Hoek JAR, Mulder-Folkerts DKF, Courtinho RA, et al. Opportunistisch screening op genitale infecties met Chlamydia trachomatis onder de seksueel actieve bevolking van Amsterdam. Meer dan 90% deelname en bijna 5% prevalentie. Ned Tijdschr Geneeskd. 1999;143:668-72. 6. Forseyl JP, Caul EO, Paul ID, et al. Chlamydia trachomatis, tubal disease and the incidence of symptomatic and asymptomatic infection following hysterosalpingography. Hum Reprod. 1990;5:444-7. 7. Thinkhamrop J, Laopaiboon M, Lumbiganon P. Prophylactic antibiotics for transcervical intrauterine procedures. Cochrane Database of Systematic Review 2007, Issue 3. Edited: 2009, Issue 1. 8. Grimes DA, Lopez LM, Schultz KF. Antibiotic prophylaxis for interauterine contraceptive device insertion. Cochrane Database of Systematic Reviews 1999, Issue 3. Update in: The Cochrane Library 2010, Issue 1. 9. Robuffo I, Fazii P, Rulli A, et al. Upgraded diagnostic value of Gen-Probe PACE 2 assay for detection of Chlamydia trachomatis infection. J Biol Regul Homeost Agents. 2008;22:253-61. 10. Thomas K, Coughlin L, Mannion PT, et al. The value of Chlamydia trachomatis antibody testing as part of routine infertility investigations. Hum Reprod. 2000;15:1079-82. 11. Brunham RC, Maclean IW, Binns B, et al. Chlamydia trachomatis: its role in tubal infertility. J Infect Dis. 1985;152:1275-82. 12. Land JA, Gijsen AP, Evers JLH, et al. Chlamydia trachomatis in subfertile women undergoing uterine instrumentation. Hum Reprod. 2002;17:525-7. 13. Thomas K, Simms I. Chlamydia trachomatis in subfertile women undergoing uterine instrumentation. How can we help in the avoidance of iatrogenic pelvic inﬂammatory disease? Hum Reprod. 2002;17:14312. 14. Ng EH, Ngai CS, Ho PC. Chlamydia trachomatis in infertile women undergoing uterine instrumentation: Screen or treat? Hum Reprod. 2002;17:2215-6. 15. Macmillan S. Chlamydia trachomatis in subfertile women undergoing uterine instrumentation. The clinician’s role. Hum Reprod. 2002;17:1433-6. 16. Patton DL, Askienazy-Elbhar M, Henry-Suchet J, et al. Detection of Chlamydia trachomatis in fallopian tube tissue in women with postinfectious infertility. Am J Obstet Gynecol. 1994;171:95-101. 17. Wise R, Hart T, Cars O, et al. Antimicrobial resistance. Is a major threat to public health. Br Med J. 1998;317:609-10. 18. Somani J, Bhullar VB, Workowski KA, et al. Multiple drug-resistant Chlamydia trachomatis associated with clinical treatment failure. J Infect Dis. 2000;181:1421-7. 19. Witkin SS, Linhares IM. Chlamydia trachomatis in subfertile women undergoing uterine instrumentation: an alternative to direct microbial testing or prophylactic antibiotic treatment. Hum Reprod. 2002;17:1938-41. 20. Royal College of Obstetricians and Gynaecologists. The initial investigation and management of the infertile couple. RCOG Press, London, UK, 1998. 21. Royal College of Obstetricians and Gynaecologists. The management of infertility in the tertiary care. RCOG Press, London, UK, 2000. 22. Royal College of Obstetricians and Gynaecologists. Fertility: assessment and treatment for people with fertility problems. RCOG Press, London, UK, 2004. Caroline Bax BW.indd 124 26-08-10 12:17 General Discussion 125 23. Penney GC, Thomson M, Norman J, et al. A randomised comparison of strategies for reducing infective complications of induced abortion. Br J Obstet Gynaecol. 1998;105:599-604. 24. Brokenshire MK, Say PJ, van Vonno AH, et al. Evaluation of the microparticle enzyme immunoassay Abbott IMx Select Chlamydia and the importance of urethral site sampling to detect Chlamydia trachomatis in women. Genitourin Med. 1997;73:498-502. 25. Wiesenfeld HC, Heine RP, Rideout A, et al. The vaginal introitus, a novel site for Chlamydia trachomatis testing in women. Am J Obstet Gynecol. 1996;174:1542-6. 26. Mahto M, Mallison H. Should we consider alternatives to combined cervical and urethral swabs for detection of Chlamydia trachomatis in females. Sex Transm Infect. 2007;83:335-6. 27. Ivens D, MacDonald K, Bansi L, et al. Screening for rectal chlamydia infection in a genitourinary medicine clinic. Int J STD AIDS. 2007;18:404-6. 28. Carré H, Edman AC, Boman J, et al. Chlamydia trachomatis in the troat: is testing necessary? Acta Derm Venereol. 2008;88:187-8. 29. Hamasuna R, Hoshina S, Imai H, et al. Usefulness of oral wash specimens for detecting Chlamydia trachomatis from high-risk groups in Japan. Int J Urol. 2007;14:473-5. 30. Michel CE, Sonnex C, Carne CA, et al. Chlamydia trachomatis load at matched anatomic sites: implications for screening strategies. J Clin Microbiol. 2007;45:1395-1402. 31. van der Laar MJ, van Duynhoven YT, Fennema JS, et al. Differences in clinical manifestations of genital chlamydial infections related to serovars. Genitourin Med. 1996;72:261-5. 32. Morré SA, Rozendaal L, van Valkengoed IGM, et al. Urogenital Chlamydia trachomatis serovars in men and women with symptomatic or asymptomatic infection: an association with clinical manifestation? J Clin Microbiol. 2000;38:2292-6. 33. Anttila T, Saikku P, Koskela P, et al. Serotypes of Chlamydia trachomatis and risk for development of cervical squamous cell carcinoma. JAMA. 2001;285:47–51. 34. Lan J, Melchers I, Meijer CJLM, et al. Prevalence and serovar distribution of asymptomatical cervical Chlamydia trachomatis infections as determined by highly sensitive PCR. J Clin Microbiol. 1995;33:3194-7. 35. Gao X, Chen X-S, Yin Y-P, et al. Distribution of Chlamydia trachomatis serovars among high-risk women in China performed using PCR-restriction fragment length polymorphism genotyping. J Clin Microbiol. 2007;45:1185-9. 36. Barnes RC, Rompalo AM, Stamm WE. Comparison of Chlamydia trachomatis serovars causing rectal and cervical infections. J Infect Dis. 1987;156:953-8. 37. Kuo C-C, Wang S-P, Holmes KK, et al. Immunotypes of Chlamydia trachomatis isolates in Seattle, Washington. Infect Immun. 1983;41:865-8. 38. Geisler WM, Whittington WLH, Suchland RJ, et al. Epidemiology of anorectal chlamydial and gonococcal infections among men having sex with men in Seattle: utelizing serovar and auxotype strain typing. Sex Transm Dis. 2002;29:189-95. 39. Eckert LO, Suchland RJ, Hawes SE, et al. Quantitative Chlamydia trachomatis cultures: correlation of chlamydial inclusion-forming units with serovar, gender and race. J Infect Dis. 2000;182:540-4. 40. Kent CK, Chaw JK, Wong W, et al. Prevalence of rectal, urethral, and pharyngeal Chlamydia and Gonorrhoea detected in 2 clinical settings among men who have sex with men: San Francisco, California, 2003. Clin Infect Dis. 2005;41:67-74. 41. Clad A, Freidank HM, Kunze M, et al. Detection of seroconversion and persistence of Chlamydia trachomatis antibodies in ﬁve different serological tests. Eur J Clin Microbiol Infect Dis. 2000;19:932-7. 42. Gijsen AP, Land JA, Goossens VJ, et al. Chlamydia antibody testing in screening for tubal factor subfertility: the signiﬁcance of IgG antibody decline over time. Hum Reprod. 2002;17:699-703. 43. Gijsen AP, Land JA, Goossens VJ, et al. Chlamydia pneumoniae and screening for tubal factor subfertility. Hum Reprod. 2001;16:487-91. 44. Morré SA, Munk C, Persson K, et al. Comparison of three commercially available peptide-based immunoglobulin G (IgG) and IgA assays to microimmunoﬂuorescence assay for detection of Chlamydia trachomatis antibodies. J Clin Microbiol. 2002;40:584-7. Caroline Bax BW.indd 125 26-08-10 12:17 part IV | Chapter 8 126 45. Maass M, Franke D, Birkelund S, et al. Evaluation of pELISA medac for the detection of Chlamydia trachomatis-speciﬁc IgG and IgA, p.112. In P. Saikku (ed.), Proceedings Fourth Meeting of the European Society for Chlamydia Research, Helsinki, Finland, 2000. 46. Petersen EE, Bätz O, Böttcher M. Chlamydia trachomatis serology: prevalence of antibodies in women attending IVF centers, p.133. In P. Saikku (ed.), Proceedings Fourth Meeting of the European Society for Chlamydia Research, Helsinki, Finland, 2000. 47. Persson K, Osser S. A new peptide-based ELISA for antibodies to Chlamydia trachomatis assessed in patients with tubal factor infertility, p.132. In P. Saikku (ed.), Proceedings Fourth Meeting of the European Society for Chlamydia Research, Helsinki, Finland, 2000. 48. Clad A, Freidank H, Plünnecke J, et al. Chlamydia trachomatis species-speciﬁc serology: ImmunoComb Chlamydia Bivalent versus Microimmunoﬂuorescence (MIF). Infect. 1994;22:165-73. 49. Wagenvoort JH, Koumans D, van de Cruijs M. How useful is the Chlamydia micro-immunoﬂuorescence (MIF) test for the gynaecologist? Eur J Obstet Gynaecol Reprod Biol. 1999;84:13-5. 50. Gijsen AP, Goossens VJ, Land JA, et al. The predictive value of chlamydia antibody testing (CAT) in screening for tubal factor subfertility: micro-immunoﬂuorescence (MIF) versus ELISA, p.101. In P. Saikku (ed.), Proceedings Fourth Meeting of the European Society for Chlamydia Research, Helsinki, Finland, 2000. 51. Land JA, Gijsen AP, Kessels AG, et al. Performance of ﬁve serological chlamydia antibody tests in subfertile women. Hum Reprod. 2003;18:2621-7. 52. Mouton JW, Peeters MF, van Rijsoort-Vos JH, et al. Tubal factor pathology caused by Chlamydia trachomatis: the role of serology. Int J STD AIDS. 2002;13(Suppl.2):26-9. 53. Verkooyen RP, Peeters MF, Rijsoort-Vos JH, et al. Sensitivity and speciﬁcity of three new commercially available Chlamydia trachomatis tests. Int J STD AIDS. 2002;13(Suppl.2):23-5. 54. Land JA, Evers JL, Goossens VJ. How to use Chlamydia antibody testing in subfertility patients. Hum Reprod. 1998;13:1094-8. 55. Falck G, Gnarpe J, Hansson LO, et al. Comparison of individuals with and without speciﬁc IgA antibodies to Chlamydia pneumoniae. Respiratory morbidity and the metabolic syndrome. Chest. 2002;122:1587-93. 56. Saikku P. Epidemiology of Chlamydia pneumoniae in atherosclerosis. Am Heart J. 1999;138:S500-3. 57. den Hartog JE, Land JA, Stassen FR, et al. Serological markers of persistent C. trachomatis infections in women with tubal pathology subfertility. Hum Reprod. 2005;20:986-90. 58. Freidank HM, Clad A, Herr AS, et al. Immune response to Chlamydia trachomatis heat-shock protein in infertile female patients and inﬂuence of Chlamydia pneumoniae antibodies. Eur J Clin Microbiol Infect Dis. 1995;14:1063-9. 59. Claman P, Honey L, Peeling RW, et al. The presence of serum antibody to the chlamydial heat shock protein (CHSP60) as a diagnostic test for tubal factor infertility. Fertil Steril. 1997;67:501-4. 60. Petersen EE, Clad A, Pichlmeier U, et al. The extended Chlamydia trachomatis diagnosis in patients with pelvic inﬂammatory disease - a better approach for the diagnosis of upper genital tract infections. Clin Lab. 2003;49:277-81. 61. Clad A, Petersen EE, Dettlaff S. Antibodies to Chlamydia trachomatis heat shock protein 60 (CHSP60) and Chlamydia trachomatis major outer membrane protein (MOMP) in women with different tubal status. Clin Lab. 2003;49:269-71. 62. Swart P, Mol BW, van der Veen F, et al. The accuracy of hysterosalpingography in the diagnosis of tubal pathology: a meta-analysis. Fertil Steril. 1995;64:486-91. 63. Perquin DA, Beersma MF, de Craen AJ, et al. The value of Chlamydia trachomatis-speciﬁc IgG antibody testing and hysterosalpingography for predicting tubal pathology and occurence of pregnancy. Fertil Steril. 2007;88:224-6. 64. Veenemans LM, van der Linden PJ. The value of Chlamydia trachomatis antibody testing in predicting tubal factor infertility. Hum Reprod. 2002;17:695-8. 65. Mol BW, Dijkman B, Wertheim P, et al. The accuracy of serum chlamydial antibodies in the diagnosis of tubal pathology: a meta-analysis. Fertil Steril. 1997;67:1031-7. Caroline Bax BW.indd 126 26-08-10 12:17 General Discussion 127 66. den Hartog JE, Land JA, Stassen FR, et al. The role of chlamydia genus-speciﬁc and species-speciﬁc IgG antibody testing in predicting tubal disease in subfertile women. Hum Reprod. 2004;19:1380-4. 67. den Hartog JE, Lardenoije CM, Severens JL, et al. Screening strategies for tubal factor subfertility. Hum Reprod. 2008;23:1840-8. 68. Tiitinen A, Surcel HM, Halttunen M, et al. Chlamydia trachomatis and chlamydial heat shock protein 60-speciﬁc antibody and cell-mediated responses predict tubal factor infertility. Hum Reprod 2006;21:1533-8. 69. Morré SA, Ossewaarde JM, Lan J, et al. Serotyping and genotyping of genital Chlamydia trachomatis isolates reveal variants of serovars Ba, G, and J as conﬁrmed by omp1 nucleotide sequence analysis. J Clin Microbiol. 1998;36:345-51. 70. Dean D and Miller K. Molecular and mutation trend analysis of omp1 alleles for serovar E of Chlamydia trachomatis. Implications for the immunopathogenesis of disease. J Clin Invest.1997;99:475-83. 71. 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. J Infect Dis. 1995;172:1013-22. 72. Yang CL, Zhang Y-X, Watkins NG, et al. DNA sequence polymorphism of the Chlamydia trachomatis omp1 gene. J Infect Dis. 1993;168:1225-30. 73. Persson K and Osser S. Lack of evidence of a relationship between genital symptoms, cervicitis and salpingitis and different serovars of Chlamydia trachomatis. Eur J Microbiol Infect Dis. 1993;12:195-9. 74. Workowski KA, Stevens CE, Suchland RJ, et al. Clinical manifestations of genital infection due to Chlamydia trachomatis in women: differences related to serovars. Clin Infect Dis. 1994;19:756-60. 75. van Duynhoven YTHP, Ossewaarde JM, Derksen-Nawrocki RP, et al. Chlamydia trachomatis genotypes: correlation with clinical manifestations of infections and patients’ characteristics. Clin Infect Dis. 1998;26:314-22. 76. Spaargaren J, Schachter J, Moncada J, et al. Slow epidemic of lymphogranuloma venereum L2b strain. Emerg Infect Dis. 2005;11:1787-8. 77. Spaargaren J, Verhaest I, Mooij S, et al. Analysis of Chlamydia trachomatis serovar distribution changes in the Netherlands (1986-2002). Sex Transm Inf. 2004;80:151-2. 78. Workowski KA, Suchland RJ, Pettinger MB, et al. Association of genital infection with speciﬁc Chlamydia trachomatis serovars and race. J Infect Dis. 1992;166:1445-9. 79. Geisler WM, Suchland RJ and Stamm WE. Association of Chlamydia trachomatis serovar Ia infection with black race in a sexually transmitted disease clinic patient population in Birmingham, Alabama. Sex Transm Dis. 2006;33:621-4. 80. Suchland RJ, Eckert LO, Hawes SE, et al. Longitudinal assessment of infecting serovars of Chlamydia trachomatis in Seattle public health clinics: 1988-1996. Sex Transm Dis. 2003;30:357-61. 81. Bax CJ, Oostvogel PM, Mutsaers JAEM, et al. Clinical characteristics of Chlamydia trachomatis infections in a general outpatient department of obstetrics and gynaecology in the Netherlands. Sex Transm Inf. 2002;78(6):E6. 82. Geisler WM, Suchland RJ, Whittington WLH, et al. The relationship of serovar to clinical manifestations of urogenital Chlamydia trachomatis infection. Sex Transm Dis. 2003;30:160-5. 83. Batteiger BE, Lennington W, Newhall WJ, et al. Correlation of infecting serovar and local inﬂammation in genital chlamydial infections. J Infect Dis. 1989;160:332-6. 84. Lan J, Meijer CJ, van den Hoek AR, et al. Genotyping of Chlamydia trachomatis serovars derived from heterosexual partners and a detailed genomic analysis of serovar F. Genitourin Med. 1995;71;299-303. 85. Naher H, Petzoldt D. Serovar distribution of urogenital C. trachomatis in Germany. Genitourin Med. 1991;67:114-6. 86. Lampe MF, Wong KG, Stamm WE. Sequence conservation in the major outer membrane protein gene among Chlamydia trachomatis strains isolated from the upper and lower genital tract. J. Infect Dis. 1995;172:589-92. 87. Ito JI, Jr., Lyons JM, Airo-Brown LP. Variation in virulence among oculogenital serovars of Chlamydia trachomatis in experimental genital tract infection. Infect Immun. 1990;58:2012-3. Caroline Bax BW.indd 127 26-08-10 12:17 part IV | Chapter 8 128 88. Lyons JM, Ito JI, Jr., Penã AS, et al. Differences in growth characteristics and elementary body associated cytotoxicity between Chlamydia trachomatis oculogenital serovars D and H and Chlamydia muridarum. J Clin Pathol. 2005;58:397-401. 89. Ortiz L, Angevine M, Kim SK, et al. T-cell epitopes in variable segments of Chlamydia trachomatis major outer membrane protein elicit serovar-speciﬁc immune responses in infected humans. Infect Immunol. 2000;68:1719-23. 90. Srivastava P, Gupta R, Jha HC, et al. Serovar-speciﬁc immune responses to peptides of variable regions of Chlamydia trachomatis major outer membrane protein in serovar D-infected women. Clin Exp Med. 2008;8:207-15. 91. Morré SA, van den Brule AJ, Rozendaal L, et al. The natural course of asymptomatic Chlamydia trachomatis infections: 45% clearance and no development of clinical PID after one-year follow-up. Int J STD AIDS. 2002;13 Suppl 2:12-8. 92. van de Snoek EM, Ossewaarde JM, van der Meijden WI, et al. The use of serological titres of IgA and IgG in (early) discrimination between rectal infection with non-lymphogranuloma venereum and lymphogranuloma venereum serovars of Chlamydia trachomatis. Sex Transm Infect. 2007;83:330-4. 93. Morré SA, Spaargaren J, Ossewaarde JM, et al. Description of the ICTI consortium: an integrated approach to the study of Chlamydia trachomatis infection. Drugs Today. 2006;42:Suppl.A:107-14. 94. Lyons JM, Ouburg S, Morré SA. An integrated approach to Chlamydia trachomatis infection: the ICTI Consortium, an update. Drugs Today (Barc). 2009 Nov;45: Suppl.B:15-23. 95. van Bergen J, Götz HM, Richardus JH, et al. and PILOT CT study-group. Prevalence of urogenital Chlamydia trachomatis increases signiﬁcantly with level of urbanisation and suggests targeted screening approaches: results from the ﬁrst national population based study in the Netherlands. Sex Transm Infect. 2005;81:17-23. 96. Dean D, Bruno W, Wan R, et al. Single nucleotide polymorphisms predictive of disease phenotype and diverse and emerging strains of Chlamydia trachomatis from geographic ocular and sexually transmitted infections discovered by multi-locus typing using genes conserved across Chlamydiaceae species. Emerg Infect Dis. 2009:15(9);1385-94. 97. Klint M, Fuxelius HH, Goldkuhl RR, et al. High-resolution genotyping of Chlamydia trachomatis strains by multilocus sequence analysis. J Clin Microbiol. 2007;45:1410-4. 98. Bailey RL, Natividad-Sancho A, Fowler A, et al. Host genetic contribution to the cellular immune response to Chlamydia trachomatis: Heritability estimate from a Gambian twin study. Drugs Today. 2009;45 Suppl B:45-50. 99. Morré SA, Ouburg S, Peña AS, et al. The EU FP6 EpiGenChlamydia Consortium: contribution of molecular epidemiology and host-pathogen genomics to understanding Chlamydia trachomatis-related disease. Drugs Today (Barc). 2009;45 Suppl B:7-13. 100. Brand A, Brand H, Schulte in den BT. The impact of genetics and genomics on public health. Eur J Hum Genet. 2008;16:5-13 101. Brand A. Integrative genomics, personal-genome tests and personalized healthcare: the future is being built today. Eur J Hum Genet. 2009;17:977-8. Caroline Bax BW.indd 128 26-08-10 12:17 Caroline Bax BW.indd 129 26-08-10 12:17 Caroline Bax BW.indd 130 26-08-10 12:17 part IV | Chapter 9 Summary en Nederlandse Samenvatting Caroline Bax BW.indd 131 26-08-10 12:17 Caroline Bax BW.indd 132 26-08-10 12:17 Summary en Nederlandse Samenvatting 133 Summary In search of epidemiological risk factors for Chlamydia trachomatis (CT) and prevention of tubal pathology we performed two prospective-cohort studies. The results are presented in this thesis. The ﬁrst study was reported in 2002-2004 using a well deﬁned cohort of patients visiting an outpatient department (OPD) of Obstetrics and Gynaecology (O&G) located in the central urban area of The Hague, and describes the epidemiological data of patients with CT infections. In the same population we compared two different serological assays for detection of CT antibodies, and evaluated the characteristics of a newly developed assay detecting heat shock protein 60 (HSP60) antibodies and its role in predicting tubal pathology. In 2007 a second, sequential study was started at the same OPD of O&G and at the nearby regional sexually transmitted disease clinic to ﬁnalise some of the objectives of our study. CT infection is the most prevalent sexual transmitted disease in the Netherlands and worldwide. In the Netherlands some 60.000 people are infected each year of which 35.000 are women. In women approximately 70% of the infections are asymptomatic. However, infections caused by CT may lead to serious complications such as pelvic inﬂammatory disease (PID). Damage to the tubes may result in (tubal factor) subfertility and ectopic pregnancy. Infections in pregnant women may lead to neonatal conjunctivitis and pneumonia. The highest prevalence is found in young women, who are especially at risk for complications. It is unknown why some women have asymptomatic infections and others develop symptomatic upper genital tract infections. It has been suggested that different serovars and/or host-factors may be involved in the clinical course of infection, the rate of upper genital tract progression, and the clearance or persistence of infection. Treatment of CT infections is simple and effective and when given in time can prevent late complications. With the development of techniques to detect CT in urine samples screening programs became possible and large scale screening programs for asymptomatic infections have been performed. Uterine instrumentation such as intrauterine device (IUD) insertions, hysterosalpingography (HSG) and abortion curettage may cause upper genital tract infection if an asymptomatic cervical infection is present, by ascending infection or by reactivation of viable microorganisms in the upper genital tract. Therefore, in a population of obstetrical and gynaecological patients it is important to determine risk factors for infection To optimise prevention strategies of such complications we wanted to use these risk factors for CT infections in our obstetrical and gynaecological population. Little is known about the dynamics of CT serovars and their speciﬁc role in pathogenicity. Insight in changes of serovar distribution in different ethnic groups over time, in relation to clinical data, could explain changes in the epidemiology of CT infections and have clinical implications. CHAPTER 1 outlines the pathogenesis, clinical course and detection methods of CT infections and describes the aims of this thesis. Caroline Bax BW.indd 133 26-08-10 12:17 Part I Clinical characteristics In CHAPTER 2 we describe the prevalence and risk factors of CT infections in a general OPD of Obstetrics and Gynaecology in the Netherlands. Age was the most important risk factor in our population (overall prevalence of CT infections 4.5%, in women under 20 years of age 15.8%). Another risk factor we found was post coital bleeding. We could not conﬁrm signiﬁcant differences between women from different ethnic origin or between women using different kinds of contraceptives. Twelve (19.4%) patients with CT infections were found positive by urine test only, and 15 (24.2%) only by DNA probe. In our cohort the analyses of urine and of cervical specimens are complementary for the determination of the prevalence of CT infections in women. A guideline for the preventive use of antibiotics in uterine instrumentation needs a cost effectiveness analysis to assess the tools of age based screening and/or antibiotic prophylaxis. part IV | Chapter 9 In CHAPTER 3 we take another look at the sampling sites and also take the infecting serovar into con- 134 sideration. The aims of the study were to determine the incidence of concurrent infections on serovar level, to determine the incidence of multiple anatomical infected sites on CT detection and serovar level, and analysis of site speciﬁc serovar distribution, to identify tissue tropism, with special attention to serovar G/Ga for urogenital vs. rectal specimens. In all female patients (OPD O&G and STD clinic), cervix or vagina sampling revealed 94.9% of the CT infected patients. Other sites are explained by sexual behaviour (rectum and pharynx). In male patients, urethral site sampling or urine revealed 85.9% of the CT infected patients. In menwho-have-sex-with-men (MSM) the advice is to take rectal samples as well, to detect more patients. The pharyngeal sampling site does not seem to contribute much to the detection of CT infected patients. Samples of 433 patients (256 female and 177 male) were collected sequentially and were used for CT serovar detection and typing. In 11 patients (2.6%) we found concurrent serovars in one sample site. In 62 (34.1%) female (38 from the OPD O&G) and four (9.3%) male patients multiple sample site infections were found. A high percentage of women tested on the cervical/vaginal and rectal site were found positive on both sites (36.1%, 22 out of 61). In men, for serovar D/Da, E and G/Ga signiﬁcant differences were found in serovar distribution between urogenital and rectal specimens (D/Da: p= 0.0081, E: p=0.0012, and G/Ga: p=0.0033). The prevalence of multiple serovar infections at different sites of the same individual is relative low. Therefore serovar analysis could be performed on one positive sample site. Signiﬁcant differences in Caroline Bax BW.indd 134 26-08-10 12:17 Summary en Nederlandse Samenvatting 135 serovar distribution are found in rectal specimens of MSM with serovar G/Ga as the most prominent, suggesting tissue tropism. A same trend was found in women which, however, did not reach statistical signiﬁcance. Part II Serology Continuously new commercially available species-speciﬁc (peptide-based) enzyme immunoassays (EIAs) are being developed for the detection of CT antibodies. Serological assays have been used to detect antichlamydial antibodies in the fertility workup, predicting tubal pathology. Their value for fertility evaluation remains an ongoing subject of debate. There is wide variation between various tests in the correlation of antichlamydial antibodies with both current CT infections and tubal pathology. Therefore, in CHAPTER 4 we compare two new serological EIAs (pELISA (Medac) and Chlamydia-EIA (Biologische Analysensystem GmbH)) with microimmunoﬂuorescence (MIF) as a ‘gold standard’ to detect CT antibodies in pregnant women, subfertility patients, a control group of various gynaecological patients and CT positive patients. There were no signiﬁcant differences in seroprevalence rates, except for the group of CT-positive patients (p < 0.01). When we looked further in our subfertility group, we found that in 67% of women with patent tubes no CT IgG antibodies were detected by any of the assays, while in 27% of the women with tubal pathology no CT IgG antibodies were detected. A possible explanation could be that not in all patients with CT infections IgG antibodies are formed. Test characteristics were calculated for pELISA and Chlamydia-EIA. The diagnostic accuracy of all assays showed relative poor performance in predicting tubal pathology. pELISA seems to be a good alternative to MIF. It has a high speciﬁcity, is easier to perform and less expensive than MIF, and could therefore be used as a screenings test. CHAPTER 5 focuses on CT heat shock protein 60 (cHSP60) antibodies in women without and with tubal pathology using a new commercially available assay. cHSP60 assays are used in research setting in assessing serological responses to urogenital CT infection. Although comparison of the different ‘in-house’ assays is difﬁcult owing to a lack of standardisation, there is a consensus among the users of these assays that the anti-cHSP60 responses in women increase with the severity of CT associated disease. A commercially produced assay that employs deﬁned cHSP60 epitopes should allow for the comparison of results obtained in different laboratories, as well as forward the use of cHSP60 as a diagnostic tool, if the assay proves to be relevant in predicting pathology or clinical outcome of a urogenital chlamydial infection. Caroline Bax BW.indd 135 26-08-10 12:17 We evaluated a recently introduced commercially available cHSP60 serological assay (Medac, Wedel, Germany) and determined the anti-cHSP60 responses in three well deﬁned groups of women (1. women without tubal pathology as assessed by either HSG or laparoscopy, 2. pregnant women (unknown tubal status, proved fertility), and 3. women with conﬁrmed tubal pathology). CT IgG positivity was previously determined to be 19% for group 1, 40% for group 2, and 64% for group 3, showing the expected clear difference in IgG seroprevalence between women with and without procedure conﬁrmed tubal pathology, while an intermediate prevalence was observed in pregnant women. The same pattern but with lesser incidence was observed in the anti-cHSP60 responses being 4.8%, 16%, and 27%, for groups 1–3, respectively (χ2 test for trend: χ2=3.1, p=0.079, group 1 vs. group 3: p=0.096, OR 10.6). The incidences of anti-cHSP60 were increased in the CT IgG positive subgroups to 25%, 35%, and 43%, for groups 1–3, respectively, while only 3.8% anti-cHSP60 titres were observed in the CT IgG negative subgroups, all in subgroup 2 (unknown tubal status, proved fertility). This indicates that the concordance between CT IgG and cHSP60 positivity is high, almost 90%; however, clearly a different subgroup of women is identiﬁed by the cHSP60 assay since only 40% of the CT IgG positive women has a cHSP60 response (measurement of agreement: kappa 0.371). Finally, the median cHSP60 part IV | Chapter 9 titres increased from groups 1–3: 50, 100, and 200, respectively, suggesting an association between the 136 level of cHSP60 response and tubal pathology. The results presented here, although obtained in small but well deﬁned groups, look suggestively promising. Indeed, power calculations (alpha=0.5, beta=0.1) show that doubling (1.7 times) the size of the (sub)groups would result in signiﬁcant p values instead of clear trends. Therefore, further studies are needed in larger groups with different degrees of pathology due to CT infections to further determine the diagnostic, prognostic, and clinical relevance of this new assay. Part III Serovar Currently, 19 different CT serovars and serovariants have been identiﬁed by sero- and genotyping. The serovars can be divided into three serogroups: the B-group (serovars B, Ba, D, Da, E, L1, L2, L2a), the intermediate group (serovars F, G, Ga) and the C-group (serovars I, Ia, J, K, C, A, H, L3). The most prevalent CT strains worldwide are serovars D, E and F. They account for approximately 70% of the typed urogenital serovars. Conjunctivitis is mainly caused by serovars A-C. In urogenital infections serovars D-K are predominantly isolated. Serovars L1-L3 can be found in the inguinal lymph nodes. Several studies have described inconclusive data about speciﬁc serovars and the clinical course of infection, the rate of upper genital tract progression, and the clearance-persistence rate. Information about the differences in serovar distribution could give information about the epidemiology of CT infections and might have clinical implications. A recent study about serovar distribution in the Netherlands showed no signiﬁcant serovar distribution shift over time, but geographical differences were observed. This could be the result of different ethnic composition. Caroline Bax BW.indd 136 26-08-10 12:17 Summary en Nederlandse Samenvatting 137 CHAPTER 6 relates to serovar distribution. The objective of this study is to determine the CT serovar distribution among different ethnic populations in the region The Hague in two well deﬁned cohorts and to compare the results to previous obtained Dutch serovars distribution studies. The overall distribution of serovars closely resembles that of found in other studies in the Netherlands. The predominance of the B-group serovars in most studies conducted in different geographic location and at different times suggests that these serovars have a biological advantage over the other serovars. Compared to the serovar distribution in the study of Spaargaren et al. we found signiﬁcant differences in serovar H and I/Ia. An association has been found between serovar Ia and (high-risk) black race. These racial differences in serovar distribution might be due to behavioural, geographical or biological factors. It has been suggested that one of the reasons could be that there is less mixing with partners from a different race (relatively closed populations). And geographic distribution could inﬂuence the distribution of serovar Ia. Also intrinsic biological differences among persons of different race or among chlamydial serovars could inﬂuence acquisition, transmission and duration of infection (host susceptibility or immune response). In the Netherlands higher prevalences of CT infections are found in Surinam and Netherlands Antilles (NA) patients. One might compare these high risk groups with high risk black patients in American studies. We compared serogroup distributions in different ethnic groups (Dutch Caucasian (DC), Surinam, NA and Turkish/Moroccan), but no signiﬁcant differences were found. Therefore, when ethnicity was taken into account no major differences were found. Various demographic, clinical and behavioural parameters were analysed for a possible association with the infecting serogroup. For nearly all variables the serogroup distribution showed that serogroup B was most prevalent. The clinical course of infection does not seem to be inﬂuenced much by the infecting serovar. Further studies on the differences in clinical course should include analyses of host genetic factors. Host variation might play an important role in the development of clinical disease. CHAPTER 7 presents the serologic responses of CT B-group serovars versus C- and I-groups. Worldwide, serogroup B is the most prevalent followed by serogroup I. Clear differences have been observed in the duration of infection and growth kinetics between serovars from different serogroups in murine and cell culture models. Reasons for these observed differences are bacterial and host related, and are not well understood. We determined the differences in immunoglobulin (Ig) G responses between the three serogroups in a group of patients infected with different serovars. Serovars were assessed from 235 CT positive patients and quantitative IgG responses were determined. Analyses of variance were used to compare the IgG responses between the three serogroups. Of the serovars, 46% were B group (with serovar E the most prevalent: 35.3%), 39.6% were I group and 14.3% were C group. A highly signiﬁcant difference Caroline Bax BW.indd 137 26-08-10 12:17 in serologic response was shown when comparing the mean IgG concentrations (AU/mL) of patients having serovars in the most prevalent serogroup compared to the other serogroups: B = 135, C = 46 and I = 60 (B vs. C and B vs. I, p< 0.001). This is the ﬁrst study to compare serologic IgG responses in urogenital CT infections based on serogroup. The present study demonstrates that serovars of serogroup B induce higher serologic responses than serovars of serogroups C and I. The results of this study will be included in a much larger European Union Framework study to conﬁrm these results, to further enhance the power of the study, and enable multivariate analyses to obtain further insight into the immunopathogenesis of CT infections. This will enable risk proﬁling of at-risk patients to prevent development of long-term complications. Part IV General Discussion and Summary In CHAPTER 8, the results of our two prospective-cohort studies are summarised and discussed in a part IV | Chapter 9 broader spectrum. 138 Which women are at what risk for upper genital tract infection and forthcoming sequelae of CT infections? We performed epidemiological studies to identify some base-line characteristics and hopefully future studies will contribute to the identiﬁcation of those at risk. Uterine instrumentation could lead to upper genital tract infection if an asymptomatic cervical infection is present, by ascending infection or by reactivation of viable microorganisms in the upper genital tract. Especially in a gynaecologic population, where these procedures are frequently performed, it is important to identify these women. Young age, complaints of contact bleeding, being of Surinam or Netherlands Antilles origin, and living in urban areas are important risk factors for CT infections. However, it turned out to be difﬁcult to predict the increased risk of uterine instrumentation on the development of PID. Studies describing this are often not clear on the criteria of the diagnosis of PID or the causing agent. Whether screen-and-treat or antibiotic prophylaxis for all is the best policy remains therefore still subject of debate. Since it is not ethical to do a randomised-controlled trial on this subject, the decision which policy to follow will remain up to the physician. However, we advise that when intrauterine instrumentation is performed one should screen for CT infections in women in their fertile years (both by PCR and CAT). It is important to realise that pregnancy is not a guarantee for no infection. Nowadays, several assays can detect a CT and gonorrhoea in one sample. The role of other bacteria in the development of PID remains unclear. In women, taking cervical swabs and urine analysis were complementary in the identiﬁcation of CT positive patients. In a gynaecologic population, in which a gynaecologic examination is performed in most patients, taking cervical swabs would give the most accurate information (with regard to the uterine instrumentation), without extra effort. However, for screening purposes urine analysis alone of self-taken vaginal swabs may be more acceptable. Caroline Bax BW.indd 138 26-08-10 12:17 Summary en Nederlandse Samenvatting 139 There seems to be more and more evidence that Chlamydia antibody testing (CAT) can replace HSG in predicting tubal factor subfertility. Laparoscopy remains however the gold standard. But CAT can help to identify those patients in which a laparoscopy is indicated. Serological markers for persistent infection, i.e. CT IgA, cHSP60 and high-sensitivity C-reactive protein (hs-CRP), are more prevalent in women with tubal pathology. Especially the combination of CAT and hs-CRP needs further exploration, but seems promising in identifying women at high risk of tubal pathology. The combination of tests for humoral and cell-mediated immune response could help to improve the tubal factor subfertility prediction model . Although we did ﬁnd some clear differences in serovar distribution as compared to previous Dutch published serovar studies, the clinical course of infection did not seem to be inﬂuenced much by the infecting serovar. Our ethnic groups were smaller than expected and therefore slightly underpowered. Since one comparison between DC and Netherlands Antilles patients was borderline signiﬁcant, larger studies might reveal clearer differences. Further studies on the differences in clinical course should include analyses of host genetic factors. Host variation might play an important role in the development of clinical disease. Serovars of serogroup B induce higher serologic responses than serovars of serogroups C and I. The results of this study will be included in a much larger European Union Framework study to conﬁrm these results, to further enhance the power of the study, and enable multivariate analyses to obtain further insight into the immunopathogenesis of CT infections. This will enable risk proﬁling of at-risk patients to prevent development of long-term complications. Finally, some recommendations and suggestions for future research are provided. Caroline Bax BW.indd 139 26-08-10 12:17 Nederlandse Samenvatting Om epidemiologische risicofactoren voor Chlamydia trachomatis (CT) infecties te vinden en ter preventie van tubapathologie hebben wij twee prospectieve-cohort studies verricht. De resultaten worden in dit proefschrift gepresenteerd. De eerste studie werd beschreven tussen 2002 en 2004, en maakte gebruik van een goed gedeﬁnieerd cohort van patiënten die de polikliniek Obstetrie en Gynaecologie (O&G) bezochten in het centrum van Den Haag, en beschrijft de epidemiologische gegevens van patiënten met een CT infectie. In dezelfde populatie hebben we twee serologische tests voor het aantonen van CT antistoffen vergeleken, en hebben we de karakteristieken van een nieuw ontwikkelde test voor het aantonen van heat shock protein 60 (HSP60) antistoffen geëvalueerd en hun rol in het voorspellen van tubapathologie. In 2007 zijn we een tweede sequentiële studie gestart op dezelfde polikliniek Obstetrie en Gynaecologie en tevens op de dichtbij gelegen regionale GGD (SOA polikliniek), om zo de doelstellingen van onze studie af te ronden. part IV | Chapter 9 CT infecties behoren tot de meest voorkomende seksueel overdraagbare aandoeningen in Nederland 140 en ook wereldwijd. In Nederland lopen ca. 60.000 mensen jaarlijks een CT infectie op, waarvan 35.000 vrouwen. Bij vrouwen verloopt 70% van de infecties asymptomatisch. Echter, infecties veroorzaakt door CT kunnen tot ernstige complicaties leiden, zoals pelvic inﬂammatory disease (PID). Schade aan de tubae kan resulteren in (tuba factor) subfertiliteit en het krijgen van een extra-uteriene graviditeit. Bij zwangere vrouwen kan de infectie leiden tot neonatale conjunctivitis en pneumonie. De hoogste prevalentie wordt gevonden bij jonge vrouwen, en zij lopen vooral risico op complicaties. Het is onbekend waarom sommige vrouwen een asymptomatische infectie doormaken en waarom er bij anderen sprake is van een symptomatisch hogere genitale infectie. Er wordt gesuggereerd dat verschillende serovars en/of gastheerfactoren betrokken kunnen zijn bij het klinisch beloop van de infectie, de mate van opstijgende infecties en het klaren of persisteren van de infectie. De behandeling van CT infecties is simpel en effectief, en kan late complicaties voorkomen als deze op tijd wordt gegeven. Met de ontwikkeling van technieken om CT aan te tonen in urinemonsters werden screeningsprogramma’s mogelijk en screeningsprogramma’s voor asymptomatische infecties worden nu op grote schaal toegepast. Intra-uteriene ingrepen, zoals het plaatsten van een intrauterine device (IUD), hysterosalpingograﬁe (HSG) en abortus curettage kunnen leiden tot een hogere genitale infectie als er sprake is van een asymptomatische cervicale infectie, door opstijgende infectie of door reactivatie van levensvatbare microorganismen in het hogere genitaal. Daarom is het belangrijk om in een populatie van obstetrische en gynaecologische patiënten risicofactoren voor een infectie te bepalen. Om de preventiestrategieën voor deze complicaties te optimaliseren, wilden we deze risicofactoren gebruiken voor onze obstetrische en gynaecologische populatie. Er is weinig bekend van de dynamiek van CT serovars en hun speciﬁeke rol in pathogeniciteit. Inzicht in de veranderingen van serovar distributie in Caroline Bax BW.indd 140 26-08-10 12:17 Summary en Nederlandse Samenvatting 141 verschillende etnische groepen in het verloop van tijd, in relatie tot klinische gegevens, zou verschillen in de epidemiologie kunnen verklaren en zou klinische implicatie kunnen hebben. HOOFDSTUK 1 geeft een overzicht van de pathogenesis, het klinisch beloop en detectie methoden van CT infecties en beschrijft de doelstellingen van dit proefschrift. Deel I Klinische karakteristieken In HOOFDSTUK 2 beschrijven we de prevalentie en risicofactoren van CT infecties in een algemene polikliniek Obstetrie en Gynaecologie in Nederland. Leeftijd was de meest belangrijke risicofactor in onze populatie (totale prevalentie van CT infecties 4.5%, bij vrouwen jonger dan 20 jaar 15.8%). Een andere risicofactor was postcoïtaal bloedverlies. De signiﬁcante verschillen tussen vrouwen van verschillende etniciteit of tussen vrouwen die verschillende anticonceptiva gebruikten konden wij niet bevestigen. Twaalf (19.4%) patiënten met een CT infectie werden gevonden met alleen een positieve urinetest, en 15 (24.2%) met alleen de DNA-probe. In ons cohort vulden analyse van urine en van cervicale probes elkaar aan in het bepalen van de prevalentie van CT infectie bij vrouwen. Voor een richtlijn voor het preventieve gebruik van antibiotica bij intra-uteriene ingrepen is een kosten-baten analyse nodig om de mogelijkheden van leeftijdsgebonden screening en antibiotica profylaxe te beoordelen. In HOOFDSTUK 3 werpen we een nieuwe blik op de afnameplaatsen en we nemen tevens het serovar dat de infectie veroorzaakt mee in de overwegingen. De doelstellingen van deze studie waren het bepalen van de incidentie van dubbele infectie op serovarniveau, het bepalen van de incidentie van meerdere geïnfecteerde anatomische plaatsen CT detectie en serovar-niveau, en analyse van lokatie-speciﬁeke serovar-distributie, om de reactie op verschillen weefsels te identiﬁceren, met speciale aandacht voor serovar G/Ga in urogenitale versus rectale afnamen. Bij alle vrouwelijke patiënten (polikliniek O&G en SOA polikliniek), werd door middel van de cervicale en vaginale afnamen 94.4% van de met CT geïnfecteerde patiënten gedetecteerd. Andere locaties kunnen worden verklaard door seksueel gedrag (rectum en keel). Bij mannelijke patiënten openbaarde de urethrale afname of urine 85.9% van de met CT geïnfecteerde patiënten. Bij homo- of biseksuele mannen is het advies om ook rectale afnamen te doen om zo meer patiënten te detecteren. De keelafname lijkt niet veel bij te dragen aan het detecteren van CT geïnfecteerde patiënten. Afnamen van 433 patiënten (256 vrouw en 177 man) werden sequentieel verzameld en gebruikt voor CT serovar detectie en typering. Bij 11 patiënten (2.6%) vonden we dubbele serovars op één afname locatie. Caroline Bax BW.indd 141 26-08-10 12:17 Bij 62 (34.1%) vrouwen (38 van de polikliniek O&G) en bij vier (9.3%) mannen werden CT infecties op meerdere afnamelocaties gevonden. Een hoog percentage van de vrouwen die zowel op de cervicale/ vaginale als rectale locatie was getest werd op beide locaties positief bevonden (36.1%, 22 van de 61). Bij mannen werd voor serovar D/Da, E, en G/Ga een signiﬁcant verschil gevonden tussen urogenitale en rectale afnamen (D/Da: p= 0.0081, E: p=0.0012, en G/Ga: p=0.0033). De prevalentie van infecties met meerdere serovars op verschillende locaties in één individu is relatief laag. Daarom is serovar analyse slechts op één van de positieve locaties voldoende. Er werden signiﬁcante verschillen gevonden in serovar distributie van de rectale afnamen van homo- of biseksuele mannen met serovar G/Ga als meest prominente, wat verschil in gevoeligheid voor verschillende weefsels suggereert. Eenzelfde trend werd gevonden bij vrouwen, echter, dit haalde geen statistische signiﬁcatie. Deel II Serologie part IV | Chapter 9 Er worden continu nieuwe commercieel beschikbare species-speciﬁeke (peptide-gebaseerde) enzyme 142 immunoassays (EIAs) ontwikkeld voor de detectie van CT antistoffen. Serologische testen zijn gebruikt voor het detecteren van antichlamydia antistoffen in het fertiliteitonderzoek, om tubapathologie te voorspellen. De waarde voor fertiliteitevaluatie blijft een onderwerp van discussie. Er bestaat een grote variatie tussen de verschillende testen in de correlatie tussen antichlamydia antistoffen en zowel een huidige CT infectie als tubapathologie. Daarom vergelijken wij in HOOFDSTUK 4 twee nieuwe serologische EIAs, pELISA (Medac) en Chlamydia-EIA (Biologische Analysensystem GmbH)) met de microimmunoﬂuorescentie (MIF) als de gouden standaard, om CT antistoffen te detecteren bij zwangere vrouwen, subfertiele patiënten, een controlegroep van patiënten met verschillende gynaecologische klachten en CT positieve patiënten. Er waren geen signiﬁcante verschillen in de seroprevalentiecijfers, behoudens voor de groep CT positieve patiënten (p<0.01). Wanneer we de subfertiliteitgroep verder bekeken, vonden we dat bij 67% van de vrouwen met doorgankelijke tubae, door geen van de testen CT IgG antistoffen werden aangetoond, terwijl bij 27% van de vrouwen met tubapathologie, geen CT IgG antistoffen werden aangetoond. Een mogelijke verklaring zou kunnen zijn dat niet bij alle patiënten met een CT infectie IgG antistoffen worden aangemaakt. De testkarakteristieken voor de pELISA en Chlamydia-EIA werden berekend. De diagnostische nauwkeurigheid van alle testen voor het voorspellen van tubapathologie was matig. De pELISA lijkt een goed alternatief voor de MIF. Het heeft een hoge speciﬁciteit, is makkelijker uit te voeren en goedkoper dan de MIF, en zou daarom gebruikt kunnen worden als een screeningstest. In HOOFDSTUK 5 richten we ons op CT heat shock protein 60 (cHSP60) antistoffen bij vrouwen met en zonder tubapathologie, gebruikmakend van een nieuwe commercieel beschikbare test. Caroline Bax BW.indd 142 26-08-10 12:17 Summary en Nederlandse Samenvatting 143 cHSP60 testen worden in onderzoekssetting gebruikt bij het beoordelen van de serologische respons op een urogenitale CT infectie. Hoewel vergelijking tussen de verschillende ‘in-house’ testen moeilijk is door gebrek aan standaardisatie, is er wel consensus tussen de gebruikers van deze testen dat de anti-cHSP60 respons bij vrouwen toeneemt met de ernst van de met CT geassocieerde ziekte. Met een commercieel geproduceerde test die gebruik maakt van gedeﬁnieerde cHSP60 epitopen zouden resultaten verkregen in verschillende laboratoria met elkaar vergeleken kunnen worden, en zou ook het gebruik van cHSP60 als een diagnostisch hulpmiddel gestimuleerd kunnen worden, als de test relevant blijkt in het voorspellen van pathologie of klinische uitkomst van een urogenitale Chlamydia infectie. Wij evalueerden een recent geïntroduceerde commercieel beschikbare CHSP60 test (Medac, Wedel, Duitsland) en we bepaalden de anti-cHSP60 respons in drie goed gedeﬁnieerde groepen vrouwen (1. vrouwen zonder tubapathologie bij HSG of laparoscopie, 2. zwangere vrouwen (onbekende tuba status, bewezen fertiel), en 3. vrouwen met bewezen tubapathologie). De eerder bepaalde CT IgG positiviteit, 19% voor groep 1, 40% voor groep 2 en 64% voor groep 3, liet zoals verwacht een duidelijk verschil zien in IgG seroprevalentie tussen vrouwen met en zonder bewezen tuba pathologie, terwijl een intermediaire prevalentie werd opgemerkt bij zwangere vrouwen. Eenzelfde patroon, maar met een lagere incidentie, werd gevonden voor de anti-cHSP60 respons, te weten 4.8%, 16%, en 27%, voor groep 1–3, respectievelijk (χ2 test voor trend: χ2=3.1, p=0.079, groep 1 vs. groep 3: p=0.096, OR 10.6). De incidentie van anti-cHSP60 was toegenomen in de CT IgG positieve subgroepen tot 25%, 35%, en 43%, voor groepen 1-3, respectievelijk, terwijl slechts 3.8% anti-CHSP60 titers werd gevonden in de CT IgG negatieve subgroepen, allen in subgroep 2 (onbekende tuba status, bewezen fertiel). Dit geeft aan dat de concordantie tussen CT IgG en cHSP60 positiviteit hoog is, bijna 90%; echter, er wordt duidelijk een andere subgroep van vrouwen geïdentiﬁceerd door de cHSP60 test omdat slechts 40% van de CT IgG positieve vrouwen een cHSP60 respons heeft (measurement of agreement: kappa 0.371). Als laatste, de mediaan cHSP60 titer neemt toe van groep 1-3: 50, 100, en 200, respectievelijk, wat een associatie tussen het niveau van de cHSP60 respons en tubapathologie suggereert. De resultaten hier gepresenteerd, hoewel verkregen in kleine maar goed gedeﬁnieerde groepen, lijken veelbelovend. Powerberekeningen (alpha=0.5, beta=0.1) laten zien dat het verdubbelen (1.7 keer) van de grootte van de (sub)groepen zou resulteren in signiﬁcante p waarden in plaats van duidelijke trends. Verdere studies zijn nodig in grotere groepen met verschillende ernst van tubapathologie door CT infecties om de diagnostische, prognostische en klinische relevantie van deze test vast te stellen. Deel III Serovar Op dit moment zijn 19 verschillende CT serovars en serovarianten geïdentiﬁceerd door middel van seroen genotypering. De serovars kunnen in drie serogroepen onderverdeeld worden: de B-groep (serovars B, Ba, D, Da, E, L1, L2, L2a), the intermediaire groep (serovars F, G, Ga) en the C-groep (serovars I, Ia, Caroline Bax BW.indd 143 26-08-10 12:17 J, K, C, A, H, L3). Wereldwijd komen serovars D, E en F het meest voor. Zij nemen ongeveer 70% van de getypeerde serovars voor hun rekening. Conjunctivitis wordt vooral veroorzaakt door de serovars A-C. Bij urogenitale infecties worden vooral serovars D-K geïsoleerd. De serovars L1-L3 worden vooral in de inguinale lymfeklieren gevonden. Er worden door verschillende studies niet overtuigende gegevens beschreven over speciﬁeke serovars en het klinisch beloop van de infectie, de mate van opstijgende infectie en het klaren of persisteren van de infectie. Verschillen in serovar-distributie zou informatie kunnen geven over de epidemiologie van CT infecties en kan klinische implicaties hebben. Een recente studie over de serovar-distributie in Nederland toonde geen distributie verschuiving in de tijd, maar er werden wel geograﬁsche verschillen gezien. Dit zou verklaard kunnen worden door verschillende etnische samenstelling. HOOFDSTUK 6 heeft betrekking op de serovar-distributie. De doelstelling van deze studie is om de CT serovar-distributie te bepalen in verschillende etnische populaties in de regio Den Haag, in twee goed gedeﬁnieerde cohorten, en om deze resultaten te vergelijken met eerder verrichtte Nederlandse serovar- part IV | Chapter 9 distributie studies. 144 De totale serovar-distributie lijkt erg op wat ook in andere studies is gevonden in Nederland. Het overheersen van de B-groep serovars, zoals gevonden wordt in de meeste studies, op verschillende locaties en in verschillende tijdsperioden suggereert dat deze serovars een biologisch voordeel hebben ten opzichte van andere serovars. Vergeleken met de serovar-distributie studie van Spaargaren et al. vonden wij signiﬁcante verschillen voor serovar H en I/Ia. Er is een associatie gevonden tussen serovar Ia en (hoog risico) negroïde personen. Deze raciale verschillen zouden veroorzaakt kunnen worden door gedrags-, geograﬁsche- of biologische factoren. Er wordt gesuggereerd dat één van de redenen zou kunnen zijn dat er minder contact is met partners van een andere etniciteit (relatief gesloten gemeenschap). En de geograﬁsche verdeling zou de distributie van serovar Ia kunnen beïnvloeden. Ook intrinsieke biologische verschillen tussen personen van verschillende etniciteit of tussen verschillende serovars zouden het verkrijgen, de transmissie en de duur van de infectie (gastheer gevoeligheid of immuun respons) kunnen beïnvloeden. In Nederland worden hogere prevalenties van CT infecties gevonden bij patiënten van zowel Surinaamse als Antilliaanse herkomst. Deze groepen kunnen vergeleken worden met de hoog risico negroïde patiënten in Amerikaanse studies. Wij hebben serogroep-distributies vergeleken in verschillende etnische groepen (Dutch Caucasian (DC), Surinaams, Nederlandse Antillen (NA) en Turks/ Marokkaans), maar er werden geen signiﬁcante verschillen gevonden. Dus, wanneer er rekening werd gehouden met de etniciteit werden er geen grote verschillen gevonden. Om een mogelijke associatie met een serogroep aan te tonen werden verschillende demograﬁsche-, klinische- en gedrags-parameters geanalyseerd. Voor vrijwel alle variabelen gold dat serogroep B het meest voorkomend was in de serogroep-distributie. Het klinisch beloop van de infectie lijkt niet erg beïnvloed te worden door het infecterende serovar. Nieuwe studies over verschillen in het klinisch beloop zouden analyse van gastheer-genetische factoren Caroline Bax BW.indd 144 26-08-10 12:17 Summary en Nederlandse Samenvatting 145 mee moeten nemen. Gastheer-variatie zou een belangrijke rol kunnen spelen in de ontwikkeling van klinische ziekte. In HOOFDSTUK 7 wordt de serologische respons weergegeven van de CT B-groep serovars versus de C- en I-groep. Wereldwijd zijn de B-groep serovars het meest voorkomend, gevolgd door serogroep I. In muis en cel cultuur onderzoek worden duidelijke verschillen gevonden tussen serovars van verschillende serogroepen in de duur van de infectie en groei kinetiek. Redenen voor deze verschillen zijn bacterieel en gastheer gerelateerd, maar worden nog niet goed begrepen. Wij hebben de verschillen in immunoglobuline (Ig) G respons bepaald tussen de drie serogroepen bij patiënten die met verschillende serovars geïnfecteerd waren. Bij 235 CT positieve patiënten werden serovars getypeerd en en werd de kwantitatieve IgG respons bepaald. Variantie-analyse werd toegepast om de IgG responses tussen de drie serogroepen te vergelijken. Van alle serovars kwam 46% uit de B-groep (met serovar E als meest voorkomende:35.3%), 39.6% uit de I-groep en 14.3% uit de C-groep. Er werd een zeer signiﬁcant verschil in de serologische respons gezien wanneer de gemiddelde IgG concentraties (AU/ml) van patiënten met serovars uit de meest voorkomende serogroep werden vergeleken met andere serogroepen: B = 135, C = 46 en I = 60 (B vs. C en B vs. I, p< 0.001). Dit is de eerste studie die een vergelijking maakt van serologische IgG respons bij urogenitale CT infecties op basis van serogroepen. Deze studie laat zien dat de serovars van de B-groep een sterkere serologische respons induceren dan de serovars van de C- en I-groep. Het resultaat van deze studie zal geïncludeerd worden in een grotere studie van de Europese Unie om deze resultaten te bevestigen, om de power van de studie verder te verhogen, en om multivariate analyse mogelijk te maken, om zo meer inzicht te verkrijgen in de immunopathogenese van CT infecties. Dit zal risicoproﬁlering mogelijk maken van risico patiënten om zo langetermijn-complicaties te voorkomen. Deel IV Algemene Discussie en Samenvatting In HOOFDSTUK 8 worden de resultaten van onze twee prospectieve-cohort studies samengevat en en bediscussieerd in een breder spectrum. Welke vrouwen lopen welk risico op opstijgende infectie en de restverschijnselen die daardoor ontstaan? Wij hebben epidemiologische studies verricht om een aantal basiskarakteristieken te identiﬁceren en hopelijk kunnen toekomstige studies bijdragen aan de identiﬁcatie van risicopatiënten. Intra-uteriene ingrepen kunnen leiden tot een hogere genitale infectie als er sprake is van een asymptomatische cervicale infectie, door opstijgende infectie of door reactivatie van levensvatbare micro-organismen in het hogere Caroline Bax BW.indd 145 26-08-10 12:17 genitaal. Vooral in een gynaecologische populatie, waar deze ingrepen frequent worden uitgevoerd, is het van belang deze vrouwen te identiﬁceren. Jonge leeftijd, klachten van postcoitaal bloedverlies, Surinaamse of Antilliaanse etniciteit en het wonen in stedelijke gebieden zijn belangrijke risicofactoren voor CT infecties. Het bleek echter moeilijk om het toegenomen risico door intra-uteriene ingrepen op het ontwikkelen van een PID te voorspellen. In studies waarin dit onderwerp beschreven wordt zijn de criteria vaak niet duidelijk waarop de diagnose PID is gesteld, of over het micro-organisme dat de infectie veroorzaakt. Of screen-and-treat of antibiotica profylaxe de beste strategie is blijft nog onderwerp van discussie. Omdat het niet ethisch verantwoord is hier een randomized-controlled trial over te doen, zal de beslissing welke strategie te volgen bepaald worden door de behandelend arts. Wij adviseren echter, om bij intra-uteriene ingrepen te screenen op CT infecties bij vrouwen in de vruchtbare leeftijd (middels PCR en CAT).Het is belangrijk om te realiseren dat zwangerschap geen garantie is voor de afwezigheid van een infectie. Tegenwoordig kunnen door verschillende testen zowel CT als gonorroe in één afname bepaald worden. De rol van andere bacteriën bij de ontwikkeling van een PID blijft onduidelijk. part IV | Chapter 9 Bij vrouwen zijn cervix afname en urine-analyse aanvullend bij het identiﬁceren van CT positieve 146 patiënten. In een gynaecologische populatie, waar gynaecologisch onderzoek bij de meeste patiënten wordt verricht, geeft het afnemen van cervix kweken de meest nauwkeurige informatie (met betrekking tot intra-uteriene ingrepen), zonder extra moeite. Echter, voor screeningsdoeleinden zijn urine-analyse of zelf-afgenomen vaginale kweken meer acceptabel. Er komt steeds meer bewijs dat chlamydia antibody testing (CAT) het HSG kan vervangen bij het voorspellen van tubapathologie. Laparoscopie blijft echter de gouden standaard. Maar CAT kan helpen bij het identiﬁceren van die patiënten bij wie een laparoscopie geïndiceerd is. Serologische markers voor een persisterende infectie, zoals CT IgA, cHSP60 en high-sensitivity C-reactive protein (hs-CPR), zijn vaker aanwezig bij vrouwen met tubapathologie. Vooral de combinatie van CAT en hs-CRP dient verder onderzocht te worden, en lijkt veelbelovend bij het identiﬁceren van vrouwen met een hoog risico op tubapathologie. De combinatie van testen voor humorale en cel-gemedieerde immuun respons zouden kunnen helpen bij het verbeteren van het tuba factor subfertiliteit predictiemodel. Alhoewel wij enkele duidelijke verschillen vonden in serovar-distributie in vergelijking met andere Nederlandse studies, lijkt het klinisch beloop van de infectie niet erg beïnvloed te worden door het serovar dat de infectie veroorzaakt. Onze etnische groepen zijn wat kleiner dan verwacht en daardoor underpowered. Omdat één vergelijking tussen DC en Antilliaanse patiënten bijna signiﬁcant was, zouden grotere studies duidelijker verschillen kunnen laten zien. Nieuwe studies over verschillen in het klinisch beloop zouden analyse van gastheer-genetische factoren mee moeten nemen. Gastheer-variatie zou een belangrijke rol kunnen spelen in de ontwikkeling van klinische ziekte. Caroline Bax BW.indd 146 26-08-10 12:17 Summary en Nederlandse Samenvatting 147 De serovars van de B-groep induceren een sterkere serologische respons dan de serovars van de C- en I-groep. Het resultaat van deze studie zal geïncludeerd worden in een grotere studie van de Europese Unie om deze resultaten te bevestigen, om de power van de studie verder te verhogen, en om multivariate analyse mogelijk te maken, om zo meer inzicht te verkrijgen in de immunopathogenese van CT infecties. Dit zal risicoproﬁlering mogelijk maken van risicopatiënten om zo langetermijn-complicaties te voorkomen. Als laatste worden nog aanbevelingen en suggesties voor verder onderzoek gedaan. Caroline Bax BW.indd 147 26-08-10 12:17 Caroline Bax BW.indd 148 26-08-10 12:17 Abbreviations Caroline Bax BW.indd 149 26-08-10 12:17 Caroline Bax BW.indd 150 26-08-10 12:17 Abbreviations CT Chlamydia trachomatis CP Chlamydia pneumoniae LGV lymphogranuloma venereum EB elementary body RB reticulate body MOMP major outer membrane protein LPS lipopolysaccharide IUD intrauterine device HSG hysterosalpingography PID pelvic inﬂammatory disease TFI tubal factor infertility STI sexual transmitted infection STD sexual transmitted disease OPD outpatient department HPV human papillomavirus NG Neisseria gonorrhoea CAT Chlamydia antibody testing HSP heat shock protein hs-CRP high-sensitivity C-reactive protein EIA enzyme immunoassay DFA direct ﬂuorescent antibody assay NAAT nucleic acid ampliﬁcation test POC point of care (rapid test) MIF microimmunoﬂuorescence TMA template mediated ampliﬁcation PCR polymerase chain reaction LCR ligase chain reaction FVU ﬁrst-void urine DC Dutch Caucasian NA Netherlands Antilles RFLP restriction fragment length polymorphism MLST Multi Locus Sequence Typing Caroline Bax BW.indd 151 151 26-08-10 12:17 Caroline Bax BW.indd 152 26-08-10 12:17 Authors and afﬁliations Caroline Bax BW.indd 153 26-08-10 12:17 Caroline Bax BW.indd 154 26-08-10 12:17 Authors and afﬁliations 155 From the department of Obstetrics, Academic Medical Center, Amsterdam Caroline Bax From the department of Obstetrics and Gynaecology, MC Haaglanden, The Hague Joep Dörr, Machiel Craandijk From the department of Medical Microbiology, MC Haaglanden, The Hague Paul Oostvogel, Johan Mutsaers, Casper Jansen, Stella Schmidt From the department of Gynaecology, Leiden University Medical Center, Leiden Baptist Trimbos From the department of Statistics, Leiden University Medical Center, Leiden Ronald Brand From the Laboratory of Immunogenetics, Department of Pathology, VU Medical Center, Amsterdam Servaas Morré, Sander Ouburg, Stephan Verweij From the DDL Diagnostic Laboratory, Voorburg Koen Quint, Wim Quint From The Hague Municipal Health Service, The Hague Remco Peters, Petra van Leeuwen From the department of Pathology, VU Medical Center, Amsterdam Chris Meijer Caroline Bax BW.indd 155 26-08-10 12:17 Caroline Bax BW.indd 156 26-08-10 12:17 Acknowledgements/Dankwoord Caroline Bax BW.indd 157 26-08-10 12:17 Caroline Bax BW.indd 158 26-08-10 12:17 Acknowledgements/Dankwoord 159 Een promotietraject en een wereldreis verschillen niet zoveel van elkaar. Na elke bereikte bestemming lonkt de volgende alweer, je doet verwachte en onverwachte ontdekkingen en het contact met en de hulp van anderen is onontbeerlijk. Baptist, jouw betrokkenheid bij de brainstormsessies, op zoek naar nieuwe mogelijkheden, heb ik als zeer stimulerend ervaren. Jouw doortastendheid en enthousiasme in de laatste fase van de promotie heeft tot een vlotte afronding geleid. Joep, het is echt af! Er zijn momenten geweest dat wij beiden dachten dat het er niet meer in zat. Ik ben blij en trots dat het toch gelukt is. Servaas, ik ken niemand die zo enthousiast is over Chlamydia en zoveel Cola light drinkt als jij. Je bent een enorme motivator en ziet overal nieuwe mogelijkheden. Ik ben erg blij dat onze paden zich opnieuw gekruist hebben en ik weet zeker dat we in de toekomst nog veel zullen samenwerken. Paul, eigenlijk ben je mijn derde co-promotor. Altijd tijd voor een Nespressootje, tijd om te overleggen, kritisch over de waarde van onderzoek, bereid je kamer te delen, en aan één woord genoeg om te weten hoe het ervoor staat bij ons cluppie. Ik heb erg goede herinneringen aan ons bezoek aan de Champions League wedstrijden met Tim en Jelle. Arts-assistenten en gynaecologen in het Westeinde ziekenhuis, voor het vragen van patiënten en verzamelen van vele, vele, vele kweekstokken. In het bijzonder Machiel Craandijk en Claire Peters† voor de opzet en start van het onderzoek. Iske voor je hulp bij het bacteriële vaginose stuk. En de dokters assistenten van de poli en het secretariaat voor de logistieke ondersteuning en kopjes kofﬁe. Medewerkers van de Medische Microbiologie in het Westeinde ziekenhuis, Johan, Casper, Stella, Arno, Rob, en alle anderen, voor de uitleg en hulp bij de analyses, voor het verzamelen van de monsters, en voor de logistieke ondersteuning. Medewerkers van het laboratorium voor immunogenetica van het VUmc, voor de hulp bij het zoeken in de vriezers naar de monsters en het uitvoeren van de serovar typeringen en serologie. Sander, voor jouw hulp bij de database, statistiek en beoordeling van en suggesties voor de artikelen. Stephan en Koen voor de analyses en het medeauteurschap. GGD Den Haag, Remco Peters en Petra van Leeuwen, voor het aanleveren van vele data en monsters. Dick Oepkes, dankzij jouw telefoontje op mijn eerste ’werkloze’ dag na mijn opleiding zit ik waar ik nu zit. Je werd mijn klankbord in het nemen van beslissingen over mijn toekomst. Dank voor je vertrouwen in mij. Caroline Bax BW.indd 159 26-08-10 12:17 De staf Verloskunde van het LUMC, voor het warme nest waar ik als jonge klare in terecht kwam. Jullie stonden aan het begin van mijn ontwikkeling tot perinatoloog en gaven mij tevens de mogelijkheid en stimulatie om het onderzoek een nieuwe start te geven. En natuurlijk de poli en secretariaat verloskunde voor alles daaromheen. Christianne de Groot, van co-assistent tot gynaecoloog hebben onze paden in het Westeinde zich gekruist. Dank voor je steun en motivatie door de jaren heen, je visie op thuis en werk en je adviezen over de toekomst. Ik vind het fantastisch dat we nu beide in Amsterdam (gaan) werken en hoop dat we elkaar regelmatig zien. Mijn collega’s in het AMC, die al na zo’n korte tijd vertrouwen in mij hadden en mij de gelegenheid hebben gegeven mijn proefschrift af te ronden. Inge en Maria, mijn kamergenoten, dank voor alle gezel- Acknowledgements/Dankwoord ligheid en tips. Kees, dank voor het creëren van de omslag van dit boekje. 160 Sas en Zos, lieve vriendinnen, in goede en mindere tijden. Het is zó ﬁjn dat jullie er altijd zijn. Housesitter Arv, dank voor de goede zorgen tijdens onze afwezigheid en je altijd zo lieve attenties en aandacht. En de andere niet-huilen-schoenen-weekenders, Chris, Elles, Fem, Fré, Jan, Manyaan, San, en Tas. Tijd voor ontspanning met een vast programma in Kijkduin. Met hoeveel pas je eigenlijk in zo’n Albatros? Leo, wie anders dan jij begrijpt mijn grappen echt? De frequentie van elkaar zien is zeker niet rechtevenredig met de mate van onze vriendschap. Irene en Anoeska, we zijn samen ‘groot’ geworden. Verloskundige vriendinnen houden je als academisch gynaecoloog fysiologisch. Kitty, Suus en Marion, de vaste kern van onze collega-eet-date-club. Onbewust hebben we ons eigen intervisie clubje opgericht. Het is heerlijk om op wisselende locaties en met wisselende aanschuivers de ups-en downs van thuis en werk te kunnen bespreken. Kitty, wat super dat jij nu ook nog mijn paranimf wilt zijn. Arijaan, promo-buddie en paranimf. Wat had ik zonder jou moeten beginnen. Wetenschap in de periferie geeft obstakels waar we beiden veelvuldig tegenaan zijn gelopen. Wat is het dan goed om je hart te kunnen luchten bij iemand die het precies begrijpt. A, ik vind je een heel bijzonder mens, met altijd een berichtje met exact de juiste woorden op het juiste moment. Ik kijk uit naar jouw promotie. Ben, Truus, Anne, Ron, Ole en Sophie, voor jullie interesse en alle gezellige zondag-etentjes. Lieve mama, elk krantenknipsel over Chlamydia stuurde jij op. Ik vind het zo verdrietig dat jij er niet meer bij kunt zijn. Ik weet hoe trots je zou zijn geweest. Caroline Bax BW.indd 160 26-08-10 12:17 Acknowledgements/Dankwoord 161 Dirk, it’s just us now. Hopelijk vinden we de tijd om elkaar veel te zien. Samen, maar natuurlijk ook met Birgit en de kinderen. Pieter, jij bent er altijd! Ik ben je ontzettend dankbaar dat we onze droom hebben kunnen verwezenlijken en het hebben aangedurfd de zeilreis met onze prachtjongens te ondernemen. De Grande Blu is onze vaste thuishaven op nieuwe plaatsen met nieuwe mensen. Samen zijn, precies weten wat je aan elkaar hebt, ontspanning en avontuur. Ik hoop dat we dat gevoel de rest van ons leven kunnen vasthouden. Okke en Hugo, dit is nu het boekje van de computer! Lieve mannen, jullie zijn m’n alles, ik hou de hele wereld van jullie! Maar een reiziger treurt niet. Want er is namelijk allang een knagend nieuw verlangen naar de volgende dag. Hij weet dat elk eindpunt een illusie is. Het is niet meer dan een pauze in het zoeken naar nieuwe verdere grenzen en vers aan te snijden wegen. (Uit: De 10 geboden van een wereldreiziger) Caroline Bax BW.indd 161 26-08-10 12:17 Caroline Bax BW.indd 162 26-08-10 12:17 Curriculum Vitae Caroline Bax BW.indd 163 26-08-10 12:17 Caroline Bax BW.indd 164 26-08-10 12:17 Curriculum Vitae 165 Caroline Bax werd geboren op 4 oktober 1970 in Tilburg. Zij behaalde haar VWO diploma aan het Elshof College in Nijmegen in 1989, waarna zij in datzelfde jaar begon aan de studie Geneeskunde aan de Rijksuniversiteit Leiden. In 1994 behaalde zij haar doctoraal examen. In de wachttijd voor de co-schappen deed zij een keuze co-schap oncologie, o.l.v. dr. J. Ragaz, aan het British Columbia Cancer Agency, in Vancouver, Canada, en deed onderzoek naar ‘development of a therapy-plan for stage III and inﬂammatory breast cancer’. Aan het einde van haar co-schappen deed zij tijdens een tweede keuze co-schap bij de afdeling Gynaecologie en Verloskunde van het MC Haaglanden te Den Haag, o.l.v. dr. P.J. Dörr en dr. J. Lind, onderzoek naar de foetale, maternale en neonatale gevolgen van drugsgebruik in de zwangerschap. In 1997 behaalde zij haar artsexamen, waarna zij werkzaam was als ANIOS op de afdeling Gynaecologie en Verloskunde van het MC Haaglanden te Den Haag. In deze tijd nam zij het onderzoek beschreven in dit proefschrift over van Machiel Craandijk en Claire Peters†. In 2000 begon zij als AIOSKO o.l.v. dr. P.J. Dörr en prof. dr. J.B. Trimbos. In 2002 begon zij aan de opleiding tot gynaecoloog in het MC Haaglanden te Den Haag (opleider dr. P.J. Dörr) en Leids Universitair Medisch Centrum (opleider prof. dr. H.H.H. Kanhai). In deze tijd werd ook het contact gelegd met de onderzoekers van het Laboratorium van Immunogenetica, afdeling Pathologie, van het VUmc te Amsterdam (dr. S.A. Morré en dr. S. Ouburg). In augustus 2007 volgde inschrijving in het specialistenregister. Van september 2007 tot september 2008 was zij werkzaam als gynaecoloog bij de afdeling Verloskunde in het LUMC te Leiden en werkte zij samen met de onderzoekers van het VUmc en de GGD te Den Haag aan het tweede deel van het promotie onderzoek. De resultaten van deze samenwerking staan beschreven in dit proefschrift. Van oktober 2008 tot augustus 2009 zeilde zij met haar man Pieter Bakker en hun twee kinderen, Okke (2004) en Hugo (2006), op hun zeilboot Grande Blu door het Caribisch gebied en de Middellandse zee. Sinds oktober 2009 is zij werkzaam als fellow perinatologie in het AMC te Amsterdam. Caroline Bax BW.indd 165 26-08-10 12:17