Chlamydia trachomatis: clinical, bacterial, and host aspects of a silent love bug

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1 clinical, bacterial, and host aspects of a silent love bug Chlamydia trachomatis: clinical, bacterial, and host aspects of a silent love bug Chlamydia trachomatis: Esmée Lanjouw Esmée Lanjouw

2 Chlamydia trachomatis: Clinical, bacterial, and host aspects of a silent love bug Esmée Lanjouw

3 Chlamydia trachomatis: Clinical, bacterial, and host aspects of a silent love bug Author: Esmée Lanjouw Cover: Veelkleurige vrouw by Willeke Stubenitsky Lay Out: Jasper Koning koningjj@gmail.com Printed by: Gildeprint B.V. ISBN: Copyright 2017, Esmée Lanjouw, Rotterdam, The Netherlands. All rights reserved. No part of this publication may be reproduced, stored or transmitted in any form or by any means, without prior permission of the author. This thesis was kindly supported by: Polikliniek de Blaak and La Roche-Posay

4 VRIJE UNIVERSITEIT Chlamydia trachomatis: Clinical, bacterial, and host aspects of a silent love bug ACADEMISCH PROEFSCHRIFT ter verkrijging van de graad Doctor aan de Vrije Universiteit Amsterdam, op gezag van de rector magnificus prof.dr. V. Subramaniam in het openbaar te verdedigen ten overstaan van de promotiecommissie van de faculteit der Geneeskunde op vrijdag 17 maart 2017 om uur in de aula van de universiteit, De Boelelaan 1105 door Esmée Lanjouw geboren te Meppel

5 promotor: copromotoren: prof.dr. S.A. Morré dr. S. Ouburg dr. J. Spaargaren

6 CONTENTS Chapter 1 Chapter 2 Chapter 3 Chapter 4 Chapter 5 Chapter 6 Chapter 7 Chapter 8 Appendices Introduction Partially based on: 2010 European guideline for the management of Chlamydia trachomatis International Journal of STD and AIDS, 2010 Nov, 21(11): Background review for the 2015 European guideline on the management of Chlamydia trachomatis infections International Journal of STD and AIDS, 2015 Nov 24 Consecutively acquired sexually transmitted infections mimicking Crohn s disease American Journal of Gastroenterology, 2009 Feb; 104(2):532 3 The Dutch Chlamydia trachomatis Reference Laboratory : identification and surveillance of clinical samples for plasmid free and other Chlamydia trachomatis variants In progress Serovar D and E of serogroup B induce highest serological responses in urogenital Chlamydia trachomatis infections BMC Infectious Diseases, 2014 Jan 2;14:3 Functional polymorphisms in the Vitamin D metabolism pathway are not associated with susceptibility to Chlamydia trachomatis infection in humans Pathogens and Disease, 2016 Apr;74(3) General discussion Summary Samenvatting Acknowledgements - Dankwoord Curriculum Vitae List of publications

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8 CHAPTER 1 Introduction

9 INTRODUCTION TO THE THESIS In recent years the understanding and acceptance of the importance of Chlamydia trachomatis (CT) infections as an agent of upper genital tract infections, with pelvic inflammatory disease (PID), ectopic pregnancy, and tubal infertility as potential consequences has increased largely. Most, and probably all, observed differences in the clinical course of CT infections are influenced by host factors, environmental factors, and microbial factors. These factors can also interact with each other, for example infections with different micro-organisms at the same time may increase susceptibility to CT infections (1, 2). Advanced knowledge in this complex field of susceptibility and severity of disease has led to the development of new research tools to provide and stimulate an integrated approach in researching CT infections. Successful management of CT infections depends upon a cooperative interaction between public health, venereology and epidemiology with attention for monitoring and quality control. The development of international clinical guidelines with up to date literature in peer reviewed journals has largely increased evidence based management of CT infections, making guidelines relevant to the practicing clinician. 8Chapter 1 EPIDEMIOLOGY 1.1 Incidence, prevalence and risk groups Over 1,4 million new chlamydial infections were reported to the Centers of Disease Control in 50 states and the District of Columbia in 2013 and numbers are still increasing each year (3). In 2014, there were reported cases of chlamydia from 26 EU/EEA Member States, with 83% of all cases reported in four countries (Denmark, Norway, Sweden and the United Kingdom). This resulted in a total of 182 notified cases per population with young people between 15 and 24 years of age accounting for 63% of all reported cases (4, 5). The prevalence of CT in population-based studies ranges from 0.1% to 12.1% in men and from 1.1% to 10.6% in women depending upon country, urban areas, gender, age group, national or sub-national coverage, and inclusion of all or only sexually experienced participants (6). Ethnicity is also a determinant of CT prevalence with the highest rate (8-10%) in the Netherlands among people of Surinamese and Antillean descent (7). Young age and prior CT infections are the main risk factors to acquire a CT infection (8). Women under the age of 25 and especially under the age of 20 are at highest risk, due to increased sexual activity, a variety of partners, and the presence of cervical ectropion and friability of the cervix. An increase in reported chlamydial infections during the last 20 years especially in young people has been observed. It reflects the expansion of CT screening activities, the use of increasingly sensitive diagnostic tests, an increased emphasis on case reporting from

10 Introduction 9 health care providers and laboratories, increased sexual risk behavior especially among men who have sex with men (MSM), and improvements in the information systems used for reporting. It is not clear whether this can be interpreted as a true increase of the number of infections in the population. To supplement case report data, CT positivity and prevalence among people screened in a variety of settings are monitored (9, 10). In 2015, 18,585 chlamydial infections were diagnosed at STI clinics in the Netherlands (Figure 1). The number of chlamydial infections increased by 4.6 percent compared to 2014, despite a decrease of 3.4 percent in the number of consultations. This resulted in a rise in positivity rate from 12.6 percent in 2014 to 13.7 percent in 2015 (Public summary Sexual transmitted infections in the Netherlands in 2015) (11). CT prevalence for the general Dutch population has been estimated at 2%, and 2-6% among young people (<30 years of age) with highest prevalence (6%) in urban areas (12). The percentage of MSM diagnosed with chlamydia has been stable for years at around 10 percent (11, 13). Comparison between countries is challenging due to differences in the surveillance systems, the study population, diagnostic methods used, the number of samples tested, screening for chlamydia, and the proportion of underreporting. Since 2009, the European Centre for Disease Prevention and Control (ECDC) has published STI surveillance reports every year. However, as the majority of infections are asymptomatic, the true incidence of infection is likely to be significantly higher. Two thirds (63%) of all reported cases in Europe in 2014 were in young adults aged years; the highest rate of reported infection was in women aged years (1144 cases per ). These patterns are likely to reflect current testing practices in asymptomatic people as well as the true underlying disease incidence and prevalence (5). Figure 1: Total number of tests and positivity rate of CT infections by gender and sexual preference, (from: Center for Sexual health, RIVM, the Netherlands)

11 Differences in prevalence among European countries, as monitored by the ECDC, is presented in figure 2 (14). The overall rate of reported chlamydial diagnoses across Europe remains stable but at a high level. Country-specific reported rates, however, continue to show a large variation. The large difference in reported notification rates is likely related to the variation in the availability of diagnostics, surveillance strategies, the degree of underreporting, testing policies and the degree of their effective implementation across Europe. These differences are highlighted by the fact that close to 90% of cases are reported by four countries (Denmark, Norway, Sweden and the United Kingdom). All of these countries have implemented either a chlamydia screening programme (e.g. the United Kingdom) or have implemented national opportunistic testing programmes (15). The lack of data on testing rates limits the utility of surveillance data to understand the epidemiology of chlamydia infection at European level. The large majority of cases are diagnosed among heterosexuals, while MSM account for 7% of cases reported with known transmission route (5). Chapter 1 10 Figure 2. Rate of chlamydia diagnosis per population, by level of chlamydia control activity, EU/EEA Member States in 2013 Source: Adapted from Chlamydia control in Europe: a survey of Member States (15) and updated with 2013 surveillance data (14). Note: Countries not included: Poland, did not participate in the survey; Czech Republic, Germany, Liechtenstein and Portugal: no surveillance data: available; Austria, Belgium, France, Greece, Hungary, Italy, Netherlands and Spain: surveillance systems are not comprehensive (more recent data can be found at:

12 Introduction CT control programme Many countries have implemented STI control programmes including control of CT infections. Different control programmes are used within Europe varying from case management, partner notification, opportunistic screening in the general population to active screening programmes (16). The STI control programme in the Netherlands is monitored by the RIVM and the municipal health service (GGD STI centers). It is also possible to consult a general practitioner (GP) for STIs, but this is not fully compensated anymore. As a consequence, motivation to visit the GP is lacking and thus CT infections can be missed, leading to late complications and ongoing transmission. Free-of-charge screening is only offered to certain risk groups, including MSM. Current guidelines are based on sexual history: if an individual indicates to have performed fellatio or he or she was engaged in receptive anal intercourse, a sample is taken from that body site for diagnosis. If the patient practices genital intercourse, a urine sample or vaginal swab is obtained. Active case management and contact tracing is performed after a confirmed infection. Compared to other countries in Europe, the Netherlands have a highly extensive system to detect, report, and perform surveillance (16). Unfortunately this is not clear from figure 2, because the Netherlands are classified as sentinel surveillance system (only STI centers at public health services or (academic) hospitals are obliged to report STI infection rates to the RIVM) (17). 2. CLINICAL MANIFESTATIONS 2.1 Urogenital CT infections Up to 70% of women and up to 50% of men undergo an asymptomatic infection (71). The most reported symptoms by women include abnormal vaginal discharge, dysuria, and postcoital bleeding. Furthermore, a possible complication of a CT infection is an upper genital tract infection, with pelvic inflammatory disease (PID), ectopic pregnancy, and tubal infertility as potential consequences. The symptoms reported by men are generally dysuria and urethral discharge, and epididymitis and proctitis as late complications. Proctitis and pharyngitis are possible symptoms for an anorectal or oropharyngeal CT infection, respectively. In subfertile women with tubal pathology, serological markers of persistent CT infections are significantly more common compared to women without tubal pathology (18). Therefore it is thought that persistent infection of the urogenital tract in women could be an important cause of infertility. Persistent infection of CT is difficult to diagnose and treat because it is often culture-negative.

13 Chapter LGV infections Lymphogranuloma venereum (LGV) is a disease caused specifically by CT serovars L1- L3. LGV is a more invasive infection than those caused by the other serovars. LGV serovars predominantly infect monocytes and macrophages that pass through epithelial cell layers to regional lymph nodes. The infection may disseminate via the lymphatic system. Severe scarring and fibrosis of lymph nodes and destruction of (extra)genital sites may occur if not treated adequately (19). An LGV epidemic has been reported in the Netherlands and other European countries since The epidemic is caused by CT serovariant L2b. This L2b variant was also present in the 1980s in San Francisco with exactly the same mutations in the complete ompa gene and suggest that we are dealing with the same LGV variant >25 years later. The current LGV outbreak in industrialized countries has most likely been a slowly evolving epidemic with an organism that has gone unnoticed in the community for many years and is again being detected by new technologies (20). Primary infection is characterized by a genital ulcer or a mucosal inflammatory reaction at the site of inoculation. The incubation period is 3 to 12 days (21). These lesions spontaneously heal within a few days. This stage of disease is often missed. It is present in the MSM community and very uncommon in the heterosexual population (20, 22, 23). Approximately 2-27% of LGV infections can be asymptomatic and form an easily missed reservoir (21, 22, 70). The majority of reported infections in MSM are found anorectally. Given the increasing trend in several large European cities, the LGV epidemic is clearly not under control (24). 2.3 Non-urogenital infections Ocular infections Ocular infections can result in conjunctivitis in neonates and adults. In adults, this can be caused by auto-inoculation, genito-ocular or ocular-ocular contact (25, 26) and can lead to chronic conjunctivitis and persist for several months if left untreated. Neonatal infections Infants born to mothers through an infected birth canal may become colonised and may develop conjunctivitis and/or pneumonia (27). The vertical transmission risk for a newborn is 50 75% (28). 3. STRUCTURAL CHARACTERISTICS OF CT 3.1 Structural components CT is an obligate intracellular Gram-negative bacterium and infects predominantly mucosal epithelial cells for intracellular survival and subsequent growth (29, 30). Its life cycle has two distinct stages, called elementary bodies (EB) and reticulate bodies (EB),

14 Introduction 13 and requires the host cell for replication (figure 3). Many transmembrane proteins have been identified, such as the major outer membrane protein (MOMP, Omp2, Omp3), which is a protein that is involved in maintaining rigidity of the chlamydial membrane, attachment to the human epithelial cell and functions as a pore to provide the chlamydial body from nutrients once the bacteria have invaded the human cell (31, 32). Variations within MOMP are used to classify the bacterium. 3.2 Typing: serovars and genovars Serological classification of the bacterium was conventionally based on mono- and polyclonal antibodies against the major outer membrane protein (MOMP) of CT. Based on this, the different strains are called serovars. Single nucleotide polymorphisms (SNPs) are single bases within a gene sequence that differ from that gene s consensus sequence, and they are present in a subset of the population. Resulting gene expression changes can, in some cases, result in disease, or in differences in susceptibility to disease. Currently, classification is based on genetic determination of SNPs within the Omp1 gene coding for MOMP (33, 34), known as genovars. In literature, the term serovars is often used for both serovars and genovars. CT counts as many as 19 different serovars or genovars. Based on phylogenetic mapping, the serovars/genovars can be divided into three serogroups: B, C, and I (Intermediate) (35). The serovar/genovar distribution is Figure 3. CT infection and life-cycle. EB: elementary body. RB: reticulate body. Once inside a host cell the EBs are converted to metabolically active reticulate bodies (RB), which replicate by binary fission inside an inclusion body in the host cell. Fusion with a lysosome is evaded and after 36 to 48 hours RBs reorganize into EBs, followed by exocytosis and infection of new epithelial cells. Courtesy of Dr. S. Ouburg.

15 similar worldwide: overall, the most prevalent serovars are E, D, and F (36-38). In core groups, such as MSM and swingers, the distribution may be different (34, 39, 40). It is hypothesized that serovar clustering occurs within such a core group, and due to cultural, social, or sexual behaviour limited new serovars are introduced to the group. Many variants of CT have been identified. These variants are based on single SNPs in the variable regions of the Omp1 gene. These variants include D-, D*, I-, J-, Jv, L2b and the new variant (nvct), also known as the Swedish variant (41-43). These genovariants also can infect the urogenital tract, as well as the anorectal tract, and the oropharynx. The most important is genovariant L2b, which is prevalent among MSM, but not in the heterosexual population (20, 22). Typing, based on serovars, is still an important tool for the study of CT clinical presentations, epidemiology, and vaccine development. Chapter Biovars CT can be divided into serovars/genovars, but it is also possible to make a clinical subdivision into biovars, based on the sites of infection. These biovars are: the trachoma biovar (serovars A-C), urogenital biovar (serovars D-K, sporadically B and Ba) and the lymphogranuloma venereum biovar (serovars L1-L3). The main sites of inflammation associated with these respective biovars are the conjunctivae, urogenital tract, and inguinal lymph nodes. However, other infection sites such as the anorectal tract and oropharynx are also possible. 3.5 New classifications New classifications, e.g. based on multi-locus sequence typing (MLST), multi-locus variable number tandem repeat analysis (MLVA), or whole-genome sequencing, are presently proposed to further develop epidemiological studies and transmission studies (44-47). Besides the current use of MLST and MLVA in epidemiological transmission studies, both MLST and MLVA may be used to better identify patients at risk of late complications compared to the current serovars/genovars. MLST/MLVA typing can be used to distinguish new and recurring/persisting infections, and may be used for shortterm epidemiology and outbreak investigations (48, 49). 4. MICROBIOLOGICAL DETECTION The first clinical studies examining the association between CT and sequelae used culture of inclusion bodies from infected cells for diagnosis amongst women in hospital or attending STI clinics. These labour-intensive tests have been replaced with the Nucleic Acid Amplification Tests (NAATs). NAAT detects 10 30% more CT positive specimens than culture in studies comparing the two methods (50, 51). These NAATs are based

16 Introduction 15 on specific DNA or RNA amplification of CT, making these tests highly specific and sensitive, and less labour intensive than the other tests (52, 53). These diagnostic tests generate results quickly and are currently the gold standard for CT detection. 5. TREATMENT Although the natural course of infection has not been studied in great detail, it is assumed that many CT infections will clear spontaneously over time (54). Some infections may proceed to a chronic persistent state (55). Since sequelae might be severe and treatment of infected patients prevents further sexual transmission, treatment with antibiotics is recommended. Resistance, although infrequently reported to date, is thought to occur in CT and is associated with treatment failure (56, 57). A meta-analysis revealed that a single dose of azithromycin and a seven-day course of doxycycline are equally effective (58). The rate of compliance is of major concern and has been shown to be substantially higher in case of single dose azithromycin, in both patients (59) and their partners (60, 61). 6. GUIDELINES Healthcare has become very complex. Due to the continually expanding scientific literature, it has become very time-consuming and complex for the individual health care professional to keep up with medical literature and more importantly to synthesise it all into the best clinical practice. Guidelines aim to provide a state-of-the-art summary of current best clinical practice. They have become increasingly important in: (1) standardising clinical practice (i.e. reducing the variability in the treatments given to patients with the same diagnosis); (2) reassuring the funders of healthcare that clinicians are mainly using evidence-based interventions; 3) informing medical education; (4) research (identifying gaps in current knowledge which should be topics for future research). Clinical practice guidelines are systematically developed statements to assist practitioner and patient decisions about appropriate health care for specific clinical circumstances (62). In addition, guidelines can play an important role in health policy formation (63) and have evolved to cover topics across the health care continuum (i.e. health promotion, screening, and diagnosis). The people who develop guidelines attempt to cooperate nationally and internationally, striving for uniformity in the way guidelines are developed and publicized. There are many different guidelines featuring STIs in general or regarding one specific STI agent in particular. They are often characterized by a multidisciplinary approach and are written by a team of experts in this topic. It offers recommendations on the appropriate

17 diagnostic tests, treatment regimens, and health promotion principles needed for an effective management of CT urogenital infections and covers the management of the initial presentation, as well as the prevention of transmission and future infection. There are different organisations with different aims or different perspectives regarding guidelines worldwide. They are widely accessible, making adherence easier in different countries. Guidelines have also been translated into many European languages and are legalised as national STI diagnostic standards in many countries. Implementation can be extremely hard in many countries because of specific laws, economic and political changes, geographical isolation, language barriers, and financial limitations. 7. PATHOGENESIS AND IMMUNE RESPONSE Chapter 1 16 The immune response against CT infection is triggered after the attachment to, and invasion of, columnar epithelial cells of the urogenital/anorectal or oropharyngeal tract by infectious CT elementary bodies (EB) (29). Accessible extracellular CT particles are phagocytosed by macrophages and other antigen-presenting cells (APC), such as dendritic cells (DCs), present at the site of infection. Once located inside a cell, CT evades recognition by the immune system as it is not accessible for extracellular located pathogen receptors. In vitro and murine studies have shown that the T H 1-pathway is predominant in CT infections. Figure 4 shows, in a simplified manner, the intracellular pathway of an APC after recognition of CT. 8. IMMUNOGENETICS Striking differences have been observed regarding influencing the clinical course of CT infection. These differences include the mode of transmission, symptoms, persistence, and development of late complications. These differences could partially be explained by virulence factors of the bacterium itself, and attributed to (risk) behaviour of the individual, but no definitive answer can be given yet. This led to the hypothesis that aspects of the host immune system are also involved. Bailey et al. performed a twin study estimating the hereditability of cellular responses to ocular CT infection. They estimated that approximately 40% of variation in lymphoproliferative responses to CT antigens were due to host genetics (64). All elements responsible for an adequate immune response are encoded in the hosts DNA, and hence their functions may be altered by genetic variation within their respective genes. Multiple studies have linked genetics to pathogenesis of a variety of diseases, such as malaria, celiac disease, and HIV (65-67). Immune responses show many inter-individual differences and can partially be explained by host genetic factors.

18 Introduction 17 Figure 4. Simplified representation of TLR2, TLR4, TLR9, NOD1, and NOD2 signalling in response to CT in an antigen presenting cell. Courtesy of Dr. S. Verweij (from his thesis CT in the Dutch population: epidemiology, serological responses, and host genetics, 2014) Pathogen Recognition Receptors (PRRs) play a fundamental role in the first recognition of CT by the innate immune system and in the initiation of the subsequent immune response. As an example, our group has shown earlier that Single Nucleotide Polymorphisms (SNPs) in PRRs TLR4 and TLR9 individually increase the risk to develop tubal pathology after CT infection (see figure 5) (18). With the current knowledge of the immune system, both candidate gene approaches as well as Genome Wide Association Studies (GWAS) can be applied to find genes of interest involved in the different courses of CT infections and its outcome. SNPs used in our studies are most often functional SNPs linked to amino acid changes in the protein affecting function, but also SNPs within non-protein coding regions like the promotor region and introns. These SNPs may alter the expression or splicing and may also be responsible for an altered susceptibility to and severity of CT infection.

19 Figure 5. Examples of increasing risk of tubal pathology (TP) in CT IgG-positive subfertile women in relation to genotype of PRR genes (18). 9. PUBLIC HEALTH GENOMICS Chapter 1 18 Lately healthcare systems are facing considerable changes due to rapid advances in different branches of basic sciences including genomics, epigenetics and systems biology (computational and mathematical modeling of complex biological systems). Public Health Genomics is the use of genomics information to benefit public health. This is visualized as more effective personalized preventive care and disease treatments with better specificity, targeted to the genetic makeup of each patient. According to the CDC, Public Health genomics is an emerging field of study that assesses the impact of genes and their interaction with behavior, diet and the environment on the population s health (68). Successful implementation of this precision medicine carries the potential to change diagnostics and therapeutics of diseases (69). One of our purposes is to determine genetic susceptibility to urogenital CT infections and its complications like tubal pathology and infertility and ways public health officials can prevent and test for subfertility to enhance the concept of personalized medicine. For example, a biomarker containing host response profiles and host genetic traits involved in tubal pathology after urogenital CT infections is one of the higher purposes of studying host immune responses and host genetic traits influencing the course of CT infections. To implement a diagnostic tool predicting subfertility is a main future perspective to protect women for unnecessary invasive investigations like laparoscopies or enforcing IVF-procedures sooner. AIMS AND OUTLINE OF THIS THESIS Aims: The aim of the thesis is to provide insight in the treatment and management of Chlamydia trachomatis infections, look at epidemiological and clinical aspects of Chlamydia trachomatis infections and finally investigate aspects on the host serological response to Chlamydia trachomatis infections on the level of serovars and study host genetic factors which determine in a major part the susceptibility to and severity of Chlamydia trachomatis infections. The introduction of the thesis, Chapter 1, provides a general overview of Chlamydia trachomatis infections from both a clinical, an epidemiological and a basic point of view

20 Introduction 19 and is partially based on the 2010 European guideline for the management of Chlamydia trachomatis infections. Chapter 2 shows the updated 2015 European background review for the 2015 guideline for the management of Chlamydia trachomatis. It summarizes the literature according to the protocol of the IUSTI Guidelines editorial board. In Chapter 3 a rare clinical case of rectal LGV is presented, mimicking Crohn s disease. The difficulty of differentiation between LGV and inflammatory bowel disease (IBD) in patients with proctitis or IBD-related symptoms is shown. The role of LGV genotyping is discussed. In Chapter 4 surveillance, monitoring and quality control from a diagnostic point of view is presented focusing on the emergence of potential Chlamydia trachomatis variants which could result in diagnostically escape strains like the earlier identified new Chlamydia trachomatis variant in Sweden. Here we describe the results obtained by the Dutch Chlamydia trachomatis Reference Laboratory in the period In Chapter 5 serovar distributions and host IgG responses to Chlamydia trachomatis infections are analyzed in a large and diverse male and female study population and associations between Chlamydia trachomatis serovars and IgG titres are studied. Humoral IgG antibody responses are of interest since they are used in for instance tubal factor infertility diagnosis and they could play a role in the pathogenesis of a severe or prolonged infection. In Chapter 6 the role of host genetic variation in the susceptibility to and severity of Chlamydia trachomatis infection has been analyzed. The role of a set of defined polymorphisms in genes in the Vitamin D metabolic pathway is studied to investigate whether they might influence Chlamydia trachomatis host immunological responses. There are a number of studies that already indicated that specific polymorphisms in genes influences the immune responses and are important in defining the course of Chlamydia trachomatis infections specifically and other infections in general. Finally, Chapter 7 provides the general discussion. This synthesis chapter outlines the major topics which have been described under the aims and are placed in a broader perspective. This includes, amongst others, the clinical guidelines and the effect and implementation of such guidelines. Our studies analyzing serology (i.e. host responses to a Chlamydia trachomatis infection) and polymorphisms (host genetic factors influencing the course of infection) are translated to potential practical implications like determining a risk profile for identifying women who are more or less prone to develop tubal pathology after Chlamydia trachomatis infection. Visions on the future for this important clinical field and recommendations for further research are described in this last chapter.

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22 Introduction ;78(2): J. Spaargaren, J. Schachter, J. Moncada, H. J. de Vries, H. S. Fennema, A. S. Pena, et al. Slow epidemic of lymphogranuloma venereum L2b strain. Emerging infectious diseases. 2005;11(11): A. K. Van der Bij, J. Spaargaren, S. A. Morre, H. S. Fennema, A. Mindel, R. A. Coutinho, et al. Diagnostic and clinical implications of anorectal lymphogranuloma venereum in men who have sex with men: a retrospective case-control study. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America. 2006;42(2): J. Spaargaren, H. S. Fennema, S. A. Morre, H. J. de Vries, R. A. Coutinho. New lymphogranuloma venereum Chlamydia trachomatis variant, Amsterdam. Emerging infectious diseases. 2005;11(7): S. A. Morre, J. Spaargaren, J. S. Fennema, H. J. de Vries, R. A. Coutinho, A. S. Pena. Real-time polymerase chain reaction to diagnose lymphogranuloma venereum. Emerging infectious diseases. 2005;11(8): H. J. de Vries, A. Zingoni, A. Kreuter, H. Moi, J. A. White European guideline on the management of lymphogranuloma venereum. Journal of the European Academy of Dermatology and Venereology : JEADV L. A. Barbee, J. C. Dombrowski, R. Kerani, M. R. Golden. Effect of nucleic acid amplification testing on detection of extragenital gonorrhea and chlamydial infections in men who have sex with men sexually transmitted disease clinic patients. Sexually transmitted diseases. 2014;41(3): V. H. Hu, E. M. Harding-Esch, M. J. Burton, R. L. Bailey, J. Kadimpeul, D. C. W. Mabey. Epidemiology and control of trachoma: Systematic review. Trop Med Int Health. 2010;15(6): T. Darville. Chlamydia trachomatis infections in neonates and young children. Semin Pediatr Infect Dis. 2005;16(4): M. R. Hammerschlag. Chlamydial infections. J Pediatr. 1989;114(5): M. T. Mascellino, P. Boccia, A. Oliva. Immunopathogenesis in Chlamydia trachomatis Infected Women. ISRN obstetrics and gynecology. 2011;2011: C. Bebear, B. de Barbeyrac. Genital Chlamydia trachomatis infections. Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases. 2009;15(1): H. Su, N. G. Watkins, Y. X. Zhang, H. D. Caldwell. Chlamydia trachomatis-host cell interactions: role of the chlamydial major outer membrane protein as an adhesin. Infection and immunity. 1990;58(4): H. Su, L. Raymond, D. D. Rockey, E. Fischer, T. Hackstadt, H. D. Caldwell. A recombinant Chlamydia trachomatis major outer membrane protein binds to heparan sulfate receptors on epithelial cells. Proceedings of the National Academy of Sciences of the United States of America. 1996;93(20): M. Molano, C. J. Meijer, S. A. Morre, R. Pol, A. J. van den Brule. Combination of PCR targeting the VD2 of omp1 and reverse line blot analysis for typing of urogenital Chlamydia trachomatis serovars in cervical scrape specimens. Journal of clinical microbiology. 2004;42(7): K. D. Quint, L. J. van Doorn, B. Kleter, M. N. de Koning, H. A. van den Munckhof, S. A. Morre, 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. The Journal of molecular diagnostics : JMD. 2007;9(5): B. W. Brunelle, G. F. Sensabaugh. The ompa gene in Chlamydia trachomatis differs in phylogeny and rate of evolution from other regions of the genome. Infection and immunity. 2006;74(1): J. Spaargaren, I. Verhaest, S. Mooij, C. Smit, H. S. Fennema, R. A. Coutinho, et al. Analysis of Chlamydia trachomatis serovar distribution changes in the Netherlands ( ).

23 Chapter 1 22 Sexually transmitted infections. 2004;80(2): R. J. Suchland, L. O. Eckert, S. E. Hawes, W. E. Stamm. Longitudinal assessment of infecting serovars of Chlamydia trachomatis in Seattle public health clinics: Sexually transmitted diseases. 2003;30(4): X. Gao, X. S. Chen, Y. P. Yin, M. Y. Zhong, M. Q. Shi, W. H. Wei, et al. Distribution study of Chlamydia trachomatis serovars among high-risk women in China performed using PCRrestriction fragment length polymorphism genotyping. Journal of clinical microbiology. 2007;45(4): J. A. White. Manifestations and management of lymphogranuloma venereum. Current opinion in infectious diseases. 2009;22(1): L. O. Eckert, R. J. Suchland, S. E. Hawes, W. E. Stamm. Quantitative Chlamydia trachomatis cultures: correlation of chlamydial inclusion-forming units with serovar, age, sex, and race. The Journal of infectious diseases. 2000;182(2): S. A. Morre, J. M. Ossewaarde, J. Lan, G. J. van Doornum, J. M. Walboomers, D. M. MacLaren, 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. Journal of clinical microbiology. 1998;36(2): M. F. Lampe, R. J. Suchland, W. E. Stamm. Nucleotide sequence of the variable domains within the major outer membrane protein gene from serovariants of Chlamydia trachomatis. Infection and immunity. 1993;61(1): T. Ripa, P. Nilsson. A variant of Chlamydia trachomatis with deletion in cryptic plasmid: implications for use of PCR diagnostic tests. Euro Surveill. 2006;11(11):E M. Klint, H. H. Fuxelius, R. R. Goldkuhl, H. Skarin, C. Rutemark, S. G. Andersson, et al. High-resolution genotyping of Chlamydia trachomatis strains by multilocus sequence analysis. Journal of clinical microbiology. 2007;45(5): Y. Pannekoek, G. Morelli, B. Kusecek, S. A. Morre, J. M. Ossewaarde, A. A. Langerak, et al. Multi locus sequence typing of Chlamydiales: clonal groupings within the obligate intracellular bacteria Chlamydia trachomatis. BMC microbiology. 2008;8: L. N. Ikryannikova, M. M. Shkarupeta, E. A. Shitikov, E. N. Il ina, V. M. Govorun. Comparative evaluation of new typing schemes for urogenital Chlamydia trachomatis isolates. FEMS immunology and medical microbiology. 2010;59(2): C. Labiran, I. N. Clarke, L. T. Cutcliffe, Y. Wang, R. J. Skilton, K. Persson, et al. Genotyping markers used for multi locus VNTR analysis with ompa (MLVA-ompA) and multi sequence typing (MST) retain stability in Chlamydia trachomatis. Frontiers in cellular and infection microbiology. 2012;2: L. Christerson, B. Herrmann. Guidelines for high-resolution genotyping of chlamydia trachomatis using multilocus sequence analysis. Methods Mol Biol. 2012;903: S. Paauw. Chlamydia, gonorroe and syfilis flink gestegen. Medisch Contact T. C. Quinn, C. Gaydos, M. Shepherd, L. Bobo, E. W. Hook, 3rd, R. Viscidi, et al. Epidemiologic and microbiologic correlates of Chlamydia trachomatis infection in sexual partnerships. Jama. 1996;276(21): M. Puolakkainen, E. Hiltunen-Back, T. Reunala, S. Suhonen, P. Lahteenmaki, M. Lehtinen, 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. Journal of clinical microbiology. 1998;36(6): K. M. Doing, K. Curtis, J. W. Long, M. L. Volock. Prospective comparison of the Gen-probe PACE 2 assay and the Abbott ligase chain reaction for the direct detection of Chlamydia trachomatis in a low prevalence population. Journal of medical microbiology. 1999;48(5): D. J. Jespersen, K. S. Flatten, M. F. Jones, T. F. Smith. Prospective comparison of cell cultures and nucleic acid amplification tests for laboratory diagnosis of Chlamydia trachomatis Infections. Journal of clinical microbiology. 2005;43(10):

24 Introduction K. S. Parks, P. B. Dixon, C. M. Richey, E. W. Hook, 3rd. Spontaneous clearance of Chlamydia trachomatis infection in untreated patients. Sexually transmitted diseases. 1997;24(4): J. L. Joyner, J. M. Douglas, Jr., M. Foster, F. N. Judson. Persistence of Chlamydia trachomatis infection detected by polymerase chain reaction in untreated patients. Sexually transmitted diseases. 2002;29(4): J. Somani, V. B. Bhullar, K. A. Workowski, C. E. Farshy, C. M. Black. Multiple drug-resistant Chlamydia trachomatis associated with clinical treatment failure. The Journal of infectious diseases. 2000;181(4): S. A. Wang, J. R. Papp, W. E. Stamm, R. W. Peeling, D. H. Martin, K. K. Holmes. Evaluation of antimicrobial resistance and treatment failures for Chlamydia trachomatis: a meeting report. The Journal of infectious diseases. 2005;191(6): C. Y. Lau, A. K. Qureshi. Azithromycin versus doxycycline for genital chlamydial infections: a meta-analysis of randomized clinical trials. Sexually transmitted diseases. 2002;29(9): A. A. Adimora. Treatment of uncomplicated genital Chlamydia trachomatis infections in adults. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America. 2002;35(Suppl 2):S J. A. Schillinger, P. Kissinger, H. Calvet. Patient-delivered partner treatment with azithromycin to prevent repeated Chlamydia trachomatis infection among women: a randomized, controlled trial. Sexually transmitted M. R. Golden. Expedited partner therapy for sexually transmitted diseases: Editorial commentary. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America. 2005;41(5): S. H. Woolf, R. Grol, A. Hutchinson, M. Eccles, J. Grimshaw. Clinical guidelines: potential benefits, limitations, and harms of clinical guidelines. Bmj. 1999;318(7182): G. P. Browman, A. Snider, P. Ellis. Negotiating for change. The healthcare manager as catalyst for evidence-based practice: changing the healthcare environment and sharing experience. HealthcarePapers. 2003;3(3): R. L. Bailey, A. Natividad-Sancho, A. Fowler, R. W. Peeling, D. C. Mabey, H. C. Whittle, et al. Host genetic contribution to the cellular immune response to Chlamydia trachomatis: Heritability estimate from a Gambian twin study. Drugs of today. 2009;45 Suppl B: C. Timmann, T. Thye, M. Vens, J. Evans, J. May, C. Ehmen, et al. Genome-wide association study indicates two novel resistance loci for severe malaria. Nature. 2012;489(7416): B. Meresse, G. Malamut, N. Cerf-Bensussan. Celiac disease: an immunological jigsaw. Immunity. 2012;36(6): T. Kawamura, F. O. Gulden, M. Sugaya, D. T. McNamara, D. L. Borris, M. M. Lederman, et al. R5 HIV productively infects Langerhans cells, and infection levels are regulated by compound CCR5 polymorphisms. Proceedings of the National Academy of Sciences of the United States of America. 2003;100(14): A. Brand, P. Schroder, H. Brand, R. Zimmern. Getting ready for the future: integration of genomics into public health research, policy and practice in Europe and globally. Community Genet. 2006;9(1): J. A. Lal, J. Malogajski, S. P. Verweij, P. de Boer, E. Ambrosino, A. Brand, et al. Chlamydia trachomatis infections and subfertility: opportunities to translate host pathogen genomic data into public health. Public health genomics. 2013;16(1-2): N. H. de Vrieze, M. van Rooijen, M. F. Schim van der Loeff, H. J. de Vries. Anorectal and inguinal lymphogranuloma venereum among men who have sex with men in Amsterdam, The Netherlands: trends over time, symptomatology and concurrent infections. Sexually transmitted infections. 2013;89(7): Zimmerman HC, Potterat JJ, et al., Epidemiologic differences between chlamydia and gonorrhea. Am J Public Health Nov;80(11):

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26 CHAPTER 2 Background review for the 2015 European guideline on the management of Chlamydia trachomatis infections E. Lanjouw, S. Ouburg, H.J. de Vries, A. Stary, K. Radcliffe and M. Unemo International Journal of STD & AIDS Nov 24

27 SUMMARY Chapter 2 Chlamydia trachomatis (CT) infections are major public health concerns globally. Of particular grave concern is that the majority of persons with anogenital CT infections are asymptomatic and accordingly not aware of their infection, and this silent infection can subsequently result in severe reproductive tract complications and sequelae. The current review paper provides all background, evidence base and discussions for the 2015 European guideline on the management of CT infections (Lanjouw E, et al. Int J STD AIDS 2015). Comprehensive information and recommendations are included regarding the diagnosis, treatment and prevention of anogenital, pharyngeal and conjunctival CT infections in European countries. However, CT also causes the eye infection trachoma, which is not a sexually transmitted infection. The 2015 European CT guideline provides up-to-date guidance regarding broader indications for testing and treatment of CT infections; clearer recommendation of using validated nucleic acid amplification tests only for diagnosis; advice on (repeated) CT testing; recommendation of increased testing to reduce the incidence of pelvic inflammatory disease and prevent exposure to infection and recommendations to identify, verify and report CT variants. Improvement of access to testing, test performance, diagnostics, antimicrobial treatment and follow-up of CT patients are crucial to control its spread. 26

28 2015 European guideline on CT management review 27 AETIOLOGY, TRANSMISSION AND EPIDEMIOLOGY Chlamydia trachomatis (CT) is an obligate intracellular bacterium that is estimated to infect over 100 million people each year worldwide by sexual transmission (1). CT most frequently infects the lower urogenital tract in men and women, and it is the aetiologic agent of several common genital tract syndromes such as urethritis, cervicitis and pelvic inflammatory disease (PID) in women. However, CT can also cause extra-genital infections by sexual transmission, such as rectal, pharyngeal and ocular infections. Moreover, CT causes trachoma and pneumonia in newborns and the elderly, but these infections lie beyond the scope of this guideline. Of particularly grave concern is that the majority of persons with anogenital CT infection are not aware of their infection because it might frequently result in minimal or no symptoms. Urogenital chlamydial infection is usually an easily treatable sexually transmitted infection (STI) but, if not successfully detected and/or treated, it can lead to serious adverse outcomes in women, e.g. ascend to the upper genital tract to cause PID that can result in tubal factor infertility, ectopic pregnancy and chronic pelvic pain. Reinfection after treatment is common, and there might be long delays until some reproductive tract complications in women become apparent. Urogenital chlamydial infections do not result in any sustained immunity. As a result, reinfection and possibly persistent infection are common (2). Since the 1990s, an increase of urogenital CT infections has been reported from several countries, e.g. the USA, Canada, United Kingdom (UK) and the Scandinavian countries (3-5). The prevalence of CT in population-based studies has ranged from 0.1% to 12.1% in men and from 1.1% to 10.6% in women depending upon country, age group, national or sub-national coverage, and inclusion of all or only sexually experienced participants. The prevalence estimates in nationally representative samples of sexually experienced year olds in Europe have been relatively similar in women and men (estimated ranging between 3 5.3% and %, respectively) and statistically consistent with those in other high-income countries (4-7). Selection bias in CT prevalence surveys is likely, with over-estimation of prevalence being more likely than under-estimation. The incidence of diagnosed CT cases reported to the European Centre for Disease Prevention and Control (ECDC) from 26 European Union (EU) and European Economic Area (EEA) countries in 2013 was 182 per 100,000 population (384,555 cases). Accordingly, CT is the most commonly reported bacterial STI in Europe, especially among young adults (5). Young age (usually below 25 years of age) and behavioural risk factors such as prior CT infection, lack of consistent condom use and new or multiple partners per year are the main risk factors for acquisition of CT infection (8). Nevertheless, there was substantial variation across the EU/EEA countries in the incidence of reported CT cases, with rates ranging from below 1 to more than 600 cases per 100,000 population (5). Comparison between countries is considerably

29 challenged by differences in the surveillance systems, the diagnostic methods used, the access to and amount of testing and screening (general screening programme or opportunistic testing) for chlamydial infection and the proportion of underreporting (4). Chapter 2 28 Transmission of CT usually takes place by direct mucosal contact between two individuals during sexual intercourse (vaginal, anal or oral sex) or at birth through an infected cervical canal. It is difficult to estimate the risk of sexual transmission and there is also a lack of an agreed methodology on how to estimate transmissibility of CT from cross-sectional sexual partnership studies. It has been estimated using data from heterosexual couples attending an STD clinic in the USA that the transmission probability per vaginal coitus was 39.5% from men to women and 32.3% from women to men (9). However, the infection status of the couples was observed during the partnership and not at the end, and so the estimated transmission probabilities do not represent the per partnership transmission probability. Furthermore, the natural history of chlamydial infection where spontaneous clearance and reinfection within sexual partnerships can occur was not taken into account. One transmission dynamic mathematical modelling study provided estimates (10), based on data from a cross-sectional heterosexual partnership study in clinical attendees (11). The model estimated a median transmission probability of around 10% for a single act of vaginal coitus and around 55% over the course of a partnership in a population that has two partnerships in a six-month period. Partners of people with CT infection are very likely to be infected themselves (11), so contact notification and subsequent treatment are very important. Cervical ectopy, especially in young women, has been reported to increase the susceptibility to chlamydial infection. Cervical ectopy can also be more common in women using oral contraceptives (12), and hormonal contraceptives are associated with an increased risk of chlamydial infection (12-14). However, many confounding factors are involved and a strong evidence base is difficult to obtain. Vaginal douching is associated with bacterial vaginosis and HIV, both increasing the risk for other STIs including chlamydial infection (15); however, a direct association between douching and chlamydial infection is less consistent (16). As for HIV, the positive impact of circumcision on the risk of acquiring CT and thus prevention of CT by circumcision is more and more supported by the evidence in medical literature (17). CT belongs to the genus Chlamydia (phylum Chlamydiae, order Chlamydiales, family Chlamydiaceae) together with Chlamydia muridarum and Chlamydia suis. Other chlamydiae infecting humans, Chlamydophila pneumoniae and Chlamydophila psittaci, are currently classified in a separate genus (18). However, this subdivision of the family into the two genera Chlamydia and Chlamydophila has been discussed controversially during the past decade. Recently, in the light of recent genomic data and in the context of the unique biological properties of these microorganisms, it has been proposed to classify all the 11 currently recognised Chlamydiaceae species in a single Chlamydia genus (19). Three CT biovars comprising all 15 classical serovars and several

30 2015 European guideline on CT management review 29 additional serovars and genovars are recognized within the CT species: the trachoma biovar (serovars A C), the urogenital biovar (serovars D K), and the LGV biovar (serovars L1 L3). This guideline only covers the urogenital and LGV biovars of CT. CT preferably infects stratified columnar epithelium and transitional cells of nonkeratinised stratified squamous epithelium (epithelium of non-stratified columnar epithelium) of the cervix, urethra, pharynx and the rectum. Sometimes migration to endometrium, salpinx, epididymis, or colon occurs. The bacterium multiplies itself using a unique replication cycle in the host cell, and by means of cytolysis and exocytosis newly formed bacteria will be emitted. These new bacteria can subsequently infect other cells, and the whole life cycle of CT is repeated (20,21). CLINICAL FEATURES, COMPLICATIONS AND SEQUELAE A primary urogenital chlamydial infection is mostly asymptomatic, and accordingly infected individuals are not prompted to seek medical care (6,22). Frequently referred figures for the proportion of asymptomatic infections, ±70% in women and ±50% in men, are from historical contact tracing studies which used tests with suboptimal sensitivity (23). In a recent review by Davies et al. (22), it was concluded that asymptomatic infections can be even more common, i.e. estimated from 70% to 95.5% in women (24-29) and from 25% to 100% in men (25,26,28,29). To measure the true range in duration of infection in untreated CT infected patients in observational studies is unfortunately basically impossible, because the timing of infection is required. However, Molano et al. (30) described a CT clearance (from the point of detection of the infection) in 54% of untreated asymptomatic women at one year of follow-up, 82% at two years, and 94% at four years. In another study examining untreated asymptomatic women, the clearance rate was similar (45%) during the first year (31). A significant proportion of the used parameter values regarding natural course of infection is also referenced back to the early chlamydial literature, before the introduction of nucleic acid-based methods for diagnosis and the widespread testing of asymptomatic individuals. This means that earlier reports of quick clearance of infections can be based on insensitive culture test on the one hand, while on the other hand culture is the ultimate proof of viability. The long duration of undetected and untreated infection in women can result in that the bacteria cross the cervix and uterus, ascend into the upper genital tract, adhere, and ultimately result in associated complications and sequelae such as PID, ectopic pregnancy and tubal factor infertility. Appropriate testing of symptomatic and asymptomatic sexually active individuals is accordingly recommended to identify and treat the CT infections. The direct medical costs of CT infections were estimated at $516.7 million in 2008 in the USA, among the non-viral STIs the most costly infection (32,33). The direct costs associated with CT infections and their sequelae have decreased in recent years

31 as sequelae have been managed in a less costly manner, and ectopic pregnancies have been increasingly managed medically (34). Also of importance are the tangible costs, including the lost labour productivity, and the intangible costs, including psychological and emotional injury caused by infertility and ectopic pregnancy (35). Urogenital infections Symptoms and signs in women (2,36,37) 70 95% asymptomatic Mucopurulent cervicitis with or without contactbleeding Cervical friability Cervical oedema Endocervical ulcers Urethritis Dysuria Vaginal discharge Postcoital bleeding and intermenstrual bleeding Poorly differentiated abdominal pain or lower abdominal pain Chapter 2 30 Symptoms and signs suggestive of PID(2,38-41) Lower abdominal tenderness and pain usually bilateral Cervical motion tenderness on bimanual vaginal examination Adnexal tenderness on bimanual vaginal examination Deep dyspareunia particularly of recent onset Abnormal bleeding intermenstrual bleeding, postcoital bleeding and menorrhagia can occur secondary to associated cervicitis and endometritis Abnormal vaginal or cervical discharge as a result of associated cervicitis, endometritis or bacterial vaginosis Fever (>38 C) in moderate to severe PID Complications in women PID (endometritis, salpingitis, parametritis, oophoritis Tubo-ovarian abscess and/or pelvic peritonitis) Chronic pelvic pain Tubal infertility Ectopic pregnancy Sexually acquired reactive arthritis (SARA) (<1%) Fitz-Hugh-Curtis syndrome (PID and perihepatitis)

32 2015 European guideline on CT management review 31 Symptoms and signs in men (may be so mild that they are not noticed)(2,39,42,43) Usually more than 50% (25 100%) asymptomatic Urethritis Dysuria Urethral discharge Epididymitis Testicular pain Complications in men SARA (<1%) Epididymitis, epididymo-orchitis Rectal and pharyngeal infections CT infections of the rectum in men and women can result from unprotected anal intercourse and are typically asymptomatic. However, the infections may cause anal discharge and anorectal discomfort and also progress to proctocolitis (44,45). The rates of rectal chlamydial infection in men who have sex with men (MSM) have been reported to be between 3% and 10.5% in some sexual settings (46,47). Recent studies have shown an 8.4% prevalence of anorectal CT in women and almost all (94.5%) of these women also had urogenital CT (48,49). Pharyngeal chlamydial infections are also usually asymptomatic, but symptoms of a mild sore throat can occur (50). The rates of CT detection in the pharynx in MSM can range from 0.5% to 2.3% (47,51,52). Ocular infections Ocular infections can result in conjunctivitis in neonates and adults. In adults, this can be caused by auto-inoculation, genito-ocular or ocular-ocular contact (2,3,53-56) and can lead to chronic conjunctivitis and persist for several months if left untreated. Neonatal infections Infants born to mothers through an infected birth canal may become colonised and may develop conjunctivitis and/or pneumonia (55). The vertical transmission risk for a newborn is 50 75% (56). Lymphogranuloma venereum (LGV) Lymphogranuloma venereum (LGV) is an invasive ulcerative disease caused by the serovars L1, L2, or L3 of CT (57). LGV was rare in Western Europe and the USA for many years. However, since the detection of an outbreak in 2003 in Rotterdam, the Netherlands, LGV outbreaks have been verified amongst MSM in also several other European countries (58,59). Most cases have occurred in HIV-positive MSM (59-61). Most patients have presented with proctitis (2,62) or tenesmus, anorectal discharge

33 (often bloody) and discomfort, diarrhoea or altered bowel habits. Due to similarities between LGV and inflammatory bowel disease (IBD), LGV should be considered as a differential diagnosis in patients with proctitis or IBD-related symptoms, especially among HIV-positive men. Long-lasting examination, mistreatment and surgery can then be reduced (63,64). In contrast to the early reports, it has now been shown that approximately 25% of LGV infections can be asymptomatic and form an easily missed undetected reservoir (65). The majority of reported infections in MSM are found in the anorectal canal and not urogenital, which leaves the mode of transmission within the MSM network unclear (66). Given the increasing trend in several larger European cities, the LGV endemic is clearly not under control. Therefore, directed testing of LGV must be intensified (67). For additional information on LGV, see the latest version of the European Guideline on the Management of Lymphogranuloma Venereum (68) ( Chapter 2 32 Complications and sequelae Women Untreated CT infections can lead to serious complications. In older observational treatment studies, up to 30% of women with untreated urogenital CT infections developed PID (69,70). The reported incidence of PID has fallen in several countries over the last decades (3,71-74), and the risk of complications has been reported to be lower than previously estimated (75-77). PID is assumed to be the necessary intermediate condition between lower genital tract chlamydia infection and late sequelae. PID is a clinical syndrome, which results from ascending infection from the vagina and endocervix (78). Criteria for PID diagnosis are neither sensitive nor specific (79). Even when present, clinical symptoms and signs lack sensitivity and specificity (the positive predictive value of a clinical diagnosis is frequently around 65 90% compared to laparoscopic diagnosis) (38-40). A diagnosis of chlamydial PID is usually inferred from the findings of a positive CT test result in the lower genital tract in the presence of a compatible clinical picture. Lower abdominal pain and adnexal tenderness, which form the basis of the clinical diagnosis of PID, are non-specific (80). Laparoscopy is considered the gold standard diagnostic tool, but this is an invasive investigation that requires general anesthetics. It is rarely used for routine diagnosis of mild or moderate symptoms and signs. Indications for laparoscopy should be a symptomatic patient with suspicion of pelvic or tubo-ovarian abscess, whenever differential diagnoses cannot be excluded, and whenever there is no improvement of clinical symptoms and infect parameters within 72h despite adequate antibiotic therapy. Laparoscopy also helps to confirm the correct diagnosis and also offers the chance to perform adequate therapies such as rinsing and suctioning, adhesiolysis, salpingostomy or salpingectomy, or placing drainages for subsequent irrigation (40,81-83). Regardless of symptom intensity, the consequences of PID are severe. Of those

34 2015 European guideline on CT management review 33 with symptomatic PID, about 20% are subsequently infertile; 18 42% will experience debilitating, chronic pelvic pain; and 1 9% will have a life-threatening tubal pregnancy (84-88). The importance of subclinical PID became apparent with observations that most of the women with tubal factor infertility or ectopic pregnancy who had serologic evidence of chlamydial infection apparently had no history of PID (89,90). CT infection during pregnancy might lead to infant conjunctivitis and pneumonia and maternal postpartum endometritis (91). Furthermore, Fitz-Hugh-Curtis syndrome (PID and perihepatitis) could develop (92). Men Among men, urethritis is the most common syndrome resulting from CT infection. Complications (e.g. epididymitis, epididymo-orchitis) affect a minority of infected men and rarely result in reproductive health sequelae (93). There is no strong evidence base that CT causes infertility in men. However, CT has been indirectly associated with male subfertility or infertility as a result of a direct effect on sperm production, maturation, motility and viability (94-96). This could at least partly explain poor in vitro fertilisation outcome or fertilisation failure, and therefore all patients should be screened and treated for CT prior to assisted conception (97). Sexually acquired reactive arthritis (SARA) SARA has also been reported as a possible consequence of CT infection (30 40/100,000 infections) (98). There is much in favour of a causal relationship between CT and SARA, although true evidence is still lacking (99). SARA is a multisystem disease, which predominantly occurs in human leukocyte antigen B27-positive young males, and includes a combination of urethritis, conjunctivitis and arthritis. The fact that the causative agents are found in the synovial membrane or synovial fluid is indicative of infectious rather than reactive arthritis (100). To diagnose and treat non-gonococcal urethritis as soon as possible should be the aim in order to prevent the development of reactive arthritis. For additional information regarding the management of SARA, see the latest version of the European guideline for the management of sexually acquired reactive arthritis (101) ( Indications for laboratory testing (level of evidence IV; Grade C recommendation) Risk factor(s) for CT infection and/or other STIs (age<25 years, new sexual contact in the last year, more than one partner in the last year) Symptoms or signs of urethritis in men Cervical or vaginal discharge with risk factor for STI Acute epididymo-orchitis in a male aged <40 years or with risk factor for STI Acute pelvic pain and/or symptoms or signs of PID

35 Proctitis/proctocolitis according to risk Purulent conjunctivitis in a neonate or adult Atypical neonatal pneumonia Persons diagnosed with other STI Sexual contact of persons with an STI or PID Termination of pregnancy Any intrauterine interventions or manipulations LABORATORY DIAGNOSTICS Chapter 2 34 Recommended diagnostic assays Nucleic acid amplification tests (NAATs), identifying CT-specific nucleic acid (DNA or RNA) in clinical specimens, are recommended to be used for diagnostics, due to their superior sensitivity, specificity and speed (I; A) (97,102). Only if CT NAATs are not available or affordable, isolation of CT in cell culture or identification of CT by direct fluorescence assays (DFA) can be used for diagnosis of acute CT infection. NAATs have detected 10 30% more CT positive specimens than culture in studies comparing the two methods (11,103). It is not known whether the natural history of NAAT-positive CT infections in people without symptoms and at low CT loads is the same as that of culture-positive chlamydial infections (31). Evidence on the minimum period necessary before testing that can be recommended is lacking, although clinical experience suggests that positive NAAT results may be observed within 1 3 days of CT exposure. Nevertheless, in these situations mainly the infectious inoculum is detected. Thus, patients should be tested when they first present, however, if there is concern about a sexual exposure within the last two weeks they should have a repeat NAAT test two weeks after the exposure (IV; C). For adequate performance characteristics of all NAATs and other diagnostic methods, it is crucial to follow precisely the recommendations from the manufacturer concerning collection, transportation and storage of samples, as well as performance of the specific assay, including quality controls and participation in an appropriate external quality assessment (EQA) scheme. Furthermore, all diagnostic laboratories should have a quality assurance system and strive towards accreditation. NAAT validated and quality assured NAATs are recommended due to their superior sensitivity, specificity and speed of diagnosis of both symptomatic and asymptomatic chlamydial infections compared to all other diagnostic techniques (I; A) (97, ). Due to the high specificity of the appropriately validated CT NAATs (resulting in a high positive predictive value) and risk of losing low positive results in repeated testing, confirmatory testing of positive specimens is not recommended (78,111). Many

36 2015 European guideline on CT management review 35 commercially available NAATs can detect CT and Neisseria gonorrhoeae simultaneously, the NAATs can use less invasively collected specimens such as urine samples in men or vulvo-vaginal swabs in women and anorectal swabs in both genders (112), and the assays are well suited to automation, which results in increased standardization and quality assurance of extraction and detection, as well as significantly increased throughput (113). Nevertheless, it is crucial to realise that the performance characteristics of individual commercial as well as laboratory-developed (in house) CT NAATs differ. Given the rigorous evaluation required before approval of a diagnostic test by the United States of America Food and Drug Administration (FDA), which includes multisite clinical trials with comparisons against appropriate standards, FDA-approved CT NAATs are primarily recommended for diagnosis. However, globally, including in Europe, there are many additional commercially available or laboratory-developed CT NAATs in use ( ). If non-fda-approved NAATs are used, regional (such as EU), and/or other national validation and regulatory processes should provide safeguards on the quality and performance of the diagnostic NAAT. If validated and approved NAATs cannot be used, it is strongly recommended that the effectiveness of the proposed NAAT for the local settings is validated and quality assured before use against at least one internationally approved NAAT and subsequently used with appropriate positive, negative, and inhibition controls; participation in an appropriate EQA system is strongly recommended as well. It is important to also be aware that substances in the biological sample may inhibit the NAAT ( ). It is therefore advisable to include an inhibition control to prevent false-negative results. Some commercial NAATs include internal controls from DNA extraction to amplification, other assays have no or only extraction controls. External inhibition controls are recommended to be added to the sample to solve this lack of inhibition control. Most modern nucleic acid extraction assays will remove most inhibitors (120); however, users should still be aware of potential inhibition and invalid test results. Point-of-care tests (POCTs) Rapid point-of-care tests (POCTs) provide a quick and easy test result, and diagnosis and subsequent treatment can be provided at the same visit at clinic or even in remote settings. However, compared to NAATs the sensitivity of the current, mostly immunochromatographic, tests is clearly insufficient ( ), POCT with increased sensitivity has been developed, and newer POC NAATs are under development (122, ). These new POCT might be suitable for genital and extra-genital samples and evaluations are on-going (129). Currently available rapid POCT cannot be recommended in Europe, unless other more sensitive tests are unavailable and results are interpreted with caution.

37 Chapter 2 36 Detection of CT variants A new variant of CT was detected in Sweden (nvct or swct) in This variant has a 377 bp deletion in the cryptic plasmid, which is used by some commercial assays as amplification target ( ). The nvct resulted in thousands of false-negative CT tests in Sweden. The nvct has mainly been reported in the Scandinavian countries and rarely in other countries. All major commercial assays used in Europe have now been redesigned to ensure detection of the new variant (133). Plasmid-free CT variants have been found, but they are rare (134). It is believed that the plasmid-free variants are less virulent (IIb) ( ). Laboratories are advised to use NAATs capable of detecting all known CT variants and to further investigate any unexplained significant increases or declines in the local incidence or positivity rate (I; A). Since anorectal LGV needs different management as opposed to non-lgv infections, it is recommended to identify LGV patients by testing all MSM who report receptive anal sex in the previous six months for anorectal CT infection with a commercially available NAAT (139) Subsequently, MSM who are anorectal CT positive are recommended to be tested for LGV proctitis using a genovar L-specific NAAT (68). For additional information on LGV, see the latest version of the European Guideline on the Management of Lymphogranuloma Venereum (68) ( europe/euroguidelines. htm#current). It is recommended that laboratories participate in quality assurance programmes, including an appropriate EQA programme, to identify genetic variants and unusual clinical presentations (IA) (140,141). Expert networks, such as the ECDC European STI Network or International Union Against Sexually Transmitted Infections (IUSTI), can help to quickly assess new CT variants or clinical presentations, and can be used to rapidly disseminate information to other professionals worldwide. It is recommended that laboratories participate in international STI expert networks. Specimens Urogenital specimens The recommended first-choice specimens for diagnosis of urogenital chlamydial infections with NAATs are first-void urine for men and (self-collected) vulvovaginal swabs for women (I; A). First-void urine (up to 20 ml sampled>1 h after previous micturition) is highly acceptable for men as this is easy to collect and does not cause discomfort. In men, first-void urine also contains the highest CT load ( ). Specialised swabs, collection units and transportation units have been developed to optimize the yield of CT, either by collection in the clinic or self-collection at home (106, ). First-void urine should be used to diagnose CT infections with NAATs in men (I; A). Sensitivity of first-void urine testing in women is less compared to in males (102). Women

38 2015 European guideline on CT management review 37 have more often a cervico-vaginal infection than an urethral infection and the CT load in urine samples is substantially lower than in men (144). In women, a vulvo-vaginal swab (healthcare worker or self-collected) is the preferred specimen for detection of CT (I; A) (104,147, ), and also well accepted by women (151,155). Patients can send dry vulvo-vaginal swabs by mail to the laboratory without significant loss of sensitivity (156). If clinical examination is performed, a cervical specimen can be sampled. However, according to recent data NAATs on a (self-collected) vulvo-vaginal specimen is at least as sensitive. First-void urine from women should only be used if other specimens are not available (II; B) (110,143). The use of Pap-smears is not recommended for screening, case finding or other diagnostic purposes, even though several methods to optimise detection in Pap-smears have been published (157,158). Penile skin swabs cannot currently be recommended (78,102,159). Pharyngeal, rectal and conjunctival specimens No manufacturer of CT NAATs has licensed extra-genital specimens for diagnosis. However, NAATs are the preferred test for all these specimens and some NAATs have been adequately validated for these specimens (IIa; B) ( ). These specimens should not replace appropriate urogenital specimens, but should be used additionally when indicated by risk or sexual practice. In general, collecting pharyngeal and rectal specimens should always be considered in MSM and in heterosexuals according to risk (78,165). Cell culture and DFAs are not suitable for rectal specimens, due to suboptimal sensitivity as well as cytotoxicity and possible cross-reactions, respectively. Accordingly, NAATs are recommended for testing of rectal specimens, although laboratories should be aware that sensitivity and specificity can be lower compared to urogenital specimens (162, ). Confirmation of the positive results with an independent assay may be appropriate (II) (162,166,167). With the increase (or persisting presence) of rectal LGV infections, especially in MSM (60,170,171), it is recommended that positive rectal specimens from MSM are genotyped for LGV, irrespective of the presence of anorectal symptoms (II; B), for instance by a reference laboratory. For additional information on LGV, see the latest versions of the European Guideline on the Management of Lymphogranuloma Venereum (68) and the European Guideline on the management of proctitis, proctocolitis and enteritis caused by sexually transmissible pathogens (139) ( europe/euroguidelines.htm#current). Semen specimens NAATs can detect CT in semen ( ); however, there is a good correlation between first-void urine positivity and semen positivity (173,175,176), first-void urine is easier to

39 obtain, and it is exceedingly difficult to exclude that the CT detected in semen is not only from the urethra that is passed during semen sampling. Accordingly, testing of semen specimens has not proven to be of any main value and is not recommended (II; B). Serology Serology is not recommended for screening or diagnosis of acute uncomplicated anogenital CT infections. In many patients, only invasive CT infections will lead to detectable levels of antibodies and antibody levels might also remain positive for years. However, when NAATs are not available detection of specific antibodies to CT may support the diagnosis of invasive disease, such as LGV involving the lymph nodes and neonatal pneumonia (I; A) (177,178). Chapter 2 38 CT serology key points Differences in serological responses between men and women have been described, as well different; CT serovars resulting in different serological responses (179); Duration of antibody levels is unknown and may differ between persons. Detectable antibody levels may persist for a long time after clearance of the infection (180), which may result in false-positive results in testing; No value in the diagnosis of uncomplicated lower urogenital tract infections (181); Limited value in the diagnosis of ascending infections ( ) and for infertility work-up (185); Chlamydia antibody titres may predict development of tubal pathology (77,186,187) but this is under debate (188); LGV serovars tend to infect draining lymph nodes, resulting in greater chances of a detectable systemic antibody response. All MSM with rectal LGV infections develop high antibody titres (177) High antibody titres may be used as a diagnostic tool, but NAATs are preferable (178) Detection of CT-specific IgM can be a diagnostic tool in neonatal chlamydial pneumonia (55,189); Reports on Chlamydia antibody testing are extremely difficult to assess. The microimmunofluorescence (MIF) is claimed to be the test of choice, but only few laboratories are capable of performing the test adequately (190). Specificity has been greatly enhanced by using peptide-based assays. They are very useful in detecting infections in the past, for instance as testing assays in infertility work-up. Quality assurance Quality assurance is a requirement for high-quality and valid laboratory results, and all diagnostic laboratories should have a quality assurance system and strive towards accreditation. Reference standards and internal controls (positive, negative and,

40 2015 European guideline on CT management review 39 if required in NAATs, inhibition controls) should be an integral part of all tests. Laboratories should participate in national and international EQA programmes (I; A) to assess proficiency and laboratory performance to assure quality of testing. Reevaluation of random samples by an independent laboratory with an independent test will help reduce false-positive and false-negative results. Assay performance problems might be missed when following the manufacturer s instructions, but are likely to be detected in a quality control programme (141,191). It should be noted that when specimens are self-collected, especially at home, quality assurance is limited before the specimens are received in the mail. Errors and manipulation before will remain unnoticed. Testing in STI and sexual health clinics and repeat testing Annual CT testing in STI and sexual health clinics is recommended for sexually active young women and men (<25 years of age), and should be considered for MSM (2a; B). Repeated testing in 3 6 months should be offered to young women and men (<25 years of age) who test positive for CT (III; C) (78, ). Clinical guidelines in many countries recommend annual CT testing for all sexually active young (<25 years of age) women (78,192,199) and extend to young men in some countries (200,201) However, in recent years, mathematical modelling studies have suggested that to achieve population level impact on CT transmission, screening programmes need to achieve very high testing coverage and also high rates of partner notification and repeated testing for reinfection after treatment ( ). Mathematical modelling also suggested that treating symptomatic men and women and screening 38% of women aged 15 to 24 years annually would significantly reduce the average number of secondary infections and that screening men and women aged up to 29 years may affect CT transmission (203). Modelling by Althaus et al. (204) also estimated that in a populationwide screening programme, the treatment of current partners is the most effective strategy for reducing CT transmission at the population level. A recent systematic review showed that CT/N. gonorrhoeae screening in educational settings is a feasible approach to screen large numbers of young people and to identify and treat new infections (205). The review demonstrated that screening has been conducted in a range of educational facilities in a number of countries and screened a large number of both male and female students, although some strategies seemed to reach a greater number of students than others. However, only a few programmes reported on important outcomes such as treatment, partner notification and retesting after treatment. Future evaluations of school-based programme should also focus on collection and reporting of these important programme outcomes. The main rationale for current CT screening or opportunistic testing is, however, that early detection and treatment will prevent or interrupt reproductive tract morbidity, particularly in women.

41 Chapter 2 40 Mathematical modelling studies in the USA have shown that repeat infection rates peak at 2 5 months after the initial infection (206), supporting the US CDC recommendation that any person diagnosed as having CT infection should be retested within 3 12 months of treatment (III; C) (78,193,195,196,207). The English National Chlamydia Screening Programme (NCSP) guidelines recommend retesting annually or on change of sexual partner for all sexually active under 25 years of age and, in 2013, began to include recommendations of retesting around three months after a positive test (208,209). This guidance is based on evidence that young adults who test positive for CT are 2 6 times more likely to have a subsequent positive test (210) and that repeated chlamydial infection is associated with an increased risk of complications such as PID and tubal infertility. Recently, the frequency and risk factors for incident and redetected CT infection in a community-based cohort of English women who provided follow-up samples in the Prevention of Pelvic Infection (POPI) Chlamydia screening trial have been examined. These results showed that the annual incidence and redetection rates of CT infection in women in the community were high, particularly among sexually active teenagers. They highlighted the need for targeted screening among those with a new sexual partner or recent history of infection (207). However, also the timing of progression from endocervical CT infection can affect the impact of a Chlamydia control programme. If CT ascends to infect fallopian tube cells immediately after endocervical infection and inflammation follows soon after, opportunistic testing and treatment would, in most cases, be too late to prevent tubal pathology (211). The reduction in the incidence of PID in randomized clinical trials (RCTs) comparing women receiving chlamydia screening interventions with control groups (86, ) suggests that there must be an interval after endocervical infection during which screening can prevent or limit clinical PID. In four RCTs performed in EU Member States (Denmark, The Netherlands and the UK) that examined the effect of Chlamydia screening on the incidence of PID (86, ), the Chlamydia screening intervention was done on a single occasion. All trials found lower number of PID cases in the intervention group compared to the control group. The pooled average risk ratio of PID in the intervention compared with the control group was 0.64 (95% CI 0.45, 0.90), and there was exceedingly small statistical heterogeneity between results of the different trials. Accordingly, these RCTs suggest there is a window of opportunity in which treatment for screening-detected CT can interrupt tubal pathology. For the outcome, PID the GRADE tool (215) found moderate quality evidence of efficacy from the same four RCTs that Chlamydia screening reduces the incidence of PID when compared to control groups receiving usual care (4). The estimated absolute risk reduction was four cases of PID from any cause per 1000 women screened. The level of evidence was downgraded from high to moderate because of the high risk of selection bias in the methods used in the earliest trials (212,213). For the outcome of

42 2015 European guideline on CT management review 41 a change in CT incidence, the GRADE tool (215) found low quality of evidence from two effectiveness trials (216,217). The quality of evidence from non-randomised study designs begins as low. The risk of selection bias, which was greatest for the trial by Cohen et al. (217) was a factor that downgraded the quality, but this was balanced by the finding of a dose response relationship in the effects of the two trials (4). Assumptions about model structure and about the probability of complications of CT in several studies tend to favour screening. MANAGEMENT OF PATIENTS Information, explanation and advice for the patient Patients with positive CT test should be advised to abstain from sexual contact for seven days after they and their partners have completed treatment and their possible symptoms have resolved (IV; C) Patients with positive CT test (and their sexual contacts) should be given information about their infection, including details about transmission, prevention and complications. It is recommended that both verbal and written information be provided (IV; C) Information for patients is available on the IUSTI Europe website for guidelines ( Patients with positive CT test should be considered for and encouraged to have testing for other STIs, including gonorrhoea, syphilis and HIV (IV; C) Indications for therapy (IV; C) Identification of CT or CTspecific nucleic acid (DNA or RNA) in a clinical specimen On epidemiological grounds, if a recent sexual contact has confirmed chlamydial infection (NAAT specimen should also be sampled for testing) On epidemiological grounds, mother of neonate with confirmed chlamydial infection (NAAT specimen should also be sampled for testing) On epidemiological grounds, treatment can be considered following sexual assault (NAAT specimen should also be sampled for testing) On demonstration of a purulent urethral discharge in men or mucopurulent cervicitis in women when diagnostic tests are not available and after specimen collection for laboratory testing. In this circumstance, dependent on local gonorrhoea incidence combined treatment for chlamydial infection and gonorrhoea should be considered.

43 THERAPY Chapter 2 42 Treatment of individuals with CT urogenital infection prevents sexual transmission and complications, including PID. Treatment of pregnant women will prevent the transmission of infection to infants during delivery. Furthermore, prompt treatment of persons with chlamydial infection is important; by decreasing the interval between CT diagnosis and treatment complications can be limited. CT is susceptible to many antibiotics such as tetracyclines, macrolides, penicillins and fluoroquinolones, which all are drugs that have been used for treatment of human chlamydial infection. Induced resistance to antimicrobials in CT has been demonstrated in vitro. However, there is still no evidence of any stable, homotypic genetic and phenotypic resistance to any therapeutic antimicrobial in clinical CT strains that affects the treatment in humans ( ). Nevertheless, in recent years concerns have been raised over clinical failures in CT-infected patients treated particularly with azithromycin 1 g single oral dose ( ). Some of these treatment failures can be explained by reinfection, poor compliance or tolerance of treatment or detection of nucleic acid from non-viable CT due to test-of-cure (TOC) performed too early (227). However, the reasons for the remaining treatment failures remain unclear (228), though a suboptimal duration of exposure to azithromycin after the 1 g single dose and a low-level absorption of azithromycin in some patients may be involved (219). Some earlier work suggested that a prolonged course of azithromycin is likely to be sufficiently bactericidal to CT229 and in respiratory tract infections azithromycin 1.5 g administered over 3 to 5 days has been reported to achieve therapeutic levels in target tissues for up to 10 days (230,231). Based on the half life (68 h) of azithromycin, it has been suggested that increasing the dose of azithromycin to 3 g (1 g single dose then 500mg once daily for four days) would likely maintain tissue levels for over 12 days (219). Furthermore, it has been suggested that use of azithromycin 1 g stat increases the risk of inducing macrolide resistance in Mycoplasma genitalium (M. genitalium) ( ). Accordingly, when a concomitant M. genitalium infection has been verified or can be suspected, treatment with azithromycin 500mg day 1, followed by azithromycin 250mg once a day for four days ( ) should be considered (III; C). Recently, it was shown that this five days azithromycin treatment regimen can effectively eradicate also CT, that is, the eradication rate for CT was 98.8% (79 of 80 patients infected with both M. genitalium and CT) (236). Nevertheless, appropriate large and well-designed RCTs using the five days azithromycin regimen to examine the eradication frequency of both M. genitalium and CT are crucial, and when using this regimen TOC for both bacteria should be considered.

44 2015 European guideline on CT management review 43 Recommended treatment for uncomplicated urogenital CT infections First-line (Ia; A) Doxycycline 100 mg twice a day for seven days (oral; contraindicated in pregnancy) or Azithromycin 1 g stat (oral) Second-line (II; B) (TOC should be subsequently performed) Erythromycin 500 mg twice a day for seven days(oral) or Levofloxacin 500 mg once a day for seven days (oral;contraindicated in pregnancy) or Ofloxacin 200 mg twice a day for seven days (oral; contraindicated in pregnancy) Third-line (II; B) (TOC should be subsequently performed) Josamycin 500 mg three times or 1000 mg twice a day for seven days (oral) For many years, the two accepted first-line oral antimicrobial therapies for uncomplicated urogenital chlamydial infections have been azithromycin 1 g stat and doxycycline 100 mg twice daily for seven days. A meta-analysis of 23 RCTs comparing these regimens for urogenital chlamydial infections showed a statistical superiority in favour of doxycycline (237). However, the difference in efficacy was small at 1.5% 2.6% (approximately 97% versus 95% efficacy). This difference is not clinically significant and both azithromycin and doxycycline can be recommended as first-line regimens (Ia; A). When a concomitant M. genitalium infection has been verified or is suspected, treatment with azithromycin 500 mg day 1, followed by azithromycin 250 mg once a day for four days (232,233,235,236), should be considered (III; C). The quinolones ofloxacin (238,239) and levofloxacin (240) and the macrolides erythromycin (238,241,242) and, where available, josamycin ( ), have also been shown to be effective. As there is substantially less published, particularly recent, data on the effectiveness of these agents they are only recommended second-line (ofloxacin, levofloxacin, and erythromycin) or third-line (josamycin), and if used a TOC should be performed (II, B). The rate of compliance is of major concern and has been shown to be substantially higher in the case of a single dose of azithromycin, in both patients (246) and their sexual contacts (247,248) (I). Adverse side effects affect compliance negatively as well, making erythromycin, which is well known for its gastrointestinal side effects, an unattractive alternative. Rifalazil 25 mg has been suggested to be a promising welltolerated single dose oral alternative in women. However, a recent small study (including 82 CT women) showed a suboptimal microbiologic cure rate of only 84.8% with rifalazil, versus 92.1% with azithromycin (Ib; A) (249). People living with HIV infection should be treated in the same way as HIV negative ones (IV; C).

45 Recommended treatment for uncomplicated CT non-lgv rectal and pharyngeal infections Doxycycline 100 mg twice a day for seven days (oral) (I; A) (preferred if rectal infection) or Azithromycin 1 g stat (oral) (IIa; A) (if rectal infection, a TOC should be subsequently performed) Chapter 2 44 No RCTs for the treatment of non-lgv rectal or pharyngeal chlamydial infections have been performed. The results of the treatment trials for urogenital infection have been extrapolated to support the recommendation that the same regimens be used for treating infections at other sites. For rectal infections four non-randomised studies have been published which showed higher efficacy rates for doxycycline (98 100%) than for azithromycin (74 87%) at this anatomical site ( ). Conversely, another study (also non-randomised) showed azithromycin to be 94% effective; a similar rate to that for urogenital infections (254). However, all these five studies had important limitations; none was randomised, only one was prospective, only two had information on both regimens and in three less than half the patients had a TOC performed (226). Because of the low quality of the data supporting the superiority of doxycycline over azithromycin for treating rectal infections both regimens continue to be recommended as first-line. However, pending further studies and ideally double-blinded, placebo-controlled RCTs, if rectal chlamydia is treated with azithromycin then a TOC should be performed (IIa; A). Recommended treatment for uncomplicated LGV infections First-line (IIb; B) Doxycycline 100mg twice a day for 21 days (oral) Second line (III; B). Erythromycin 500 mg four times a day for 21 days (oral) Azithromycin in single- or multiple-dose regimens has also been proposed ( ), but evidence is lacking to currently recommend azithromycin (IV; C). Doxycycline is contraindicated in pregnancy and breast feeding. Adjunctive therapy 1. If fluctuant buboes appear they should be aspirated promptly through healthy adjacent skin (IV; C) 2. Surgical incision of buboes is not usually recommended due to potential complications such as chronic sinus formation (IV; C) 3. Patients with residual fibrotic lesions or fistulae do not benefit from further courses of antibiotics so surgical repair, including reconstructive genital surgery, should be considered (IV; C).

46 2015 European guideline on CT management review 45 LGV is an invasive CT infection and can, if left untreated, have serious and permanent adverse sequelae. Most of these complications are preventable if treatment is initiated in the early stages (IV; C). Despite a paucity of robust evidence (258) regarding the efficacy of therapy for any rectal chlamydial infections (LGV or non-lgv), 3 weeks of oral doxycycline 100mg twice daily to treat LGV is recommended (78,259,260). The vast majority of recent MSM case reports have observed complete responses to this therapy; shorter courses may not eradicate the organism (261). For detailed and updated information regarding the management of LGV, see the latest versions of the European Guideline on the Management of Lymphogranuloma Venereum (68) and the European Guideline on the management of proctitis, proctocolitis and enteritis caused by sexually transmissible pathogens (139) ( Pregnancy CT infections also occur during pregnancy. Infection can be associated with premature labour, preterm birth and neonatal conjunctivitis and pneumonia ( ). The choice of drugs for treatment is important because of their possible adverse effects on foetal development and pregnancy outcome. Recommended treatment for uncomplicated urogenital CT infection in pregnancy and during breast feeding (TOC should be subsequently performed) First-line (I; A)(78,195,268,269) Azithromycin 1 g stat (oral) Second line Amoxicillin 500 mg three times a day for seven days (oral) or Erythromycin 500 mg 4 times a day for 7 days (oral) Third line Josamycin 500 mg three times or 1000 mg twice a day for 7 days (oral) Doxycycline and fluoroquinolones are contraindicated. Azithromycin has been considered safe and an effective antibiotic with a short period of treatment according to clinical experience and in some studies ( ), and azithromycin is also recommended by the WHO in pregnancy. Unfortunately, in some countries azithromycin is not permitted to be administered in pregnancy, and therefore erythromycin or amoxicillin is regarded as an option (269,272). In countries, where josamycin is available, it is regarded as an alternative drug with a well-tolerated regimen (243). In the first trimester, it is only recommended under strict conditions.

47 PID PID is usually the result of infection ascending from the endocervix causing endometritis, salpingitis, parametritis, oophoritis, tuboovarian abscess and/or pelvic peritonitis. CT and N. gonorrhoeae have been identified as causative agents (38) whilst M. genitalium and anaerobes can also be implicated. PID may be symptomatic or asymptomatic (38 40). Mild and moderate cases should be treated as outpatients with oral therapy (Ib; A): otherwise ceftriaxone 500 mg single dose intramuscularly (or cefoxitin 2 g single dose intramuscularly with probenecid 1 g orally) followed by doxycycline 100 mg twice daily orally plus metronidazole 400 mg twice daily for 14 days orally or Ofloxacin 400 mg twice daily orally plus metronidazole 500 mg twice daily for 14 days orally (2,39,85,273,274) (ofloxacin may be replaced by levofloxacin 500mg once daily orally (275)) (Ib; A) Chapter 2 46 For more complicated PID cases, inpatient or alternative treatment, follow-up and partner notification see the latest version of the European guideline for the management of pelvic inflammatory disease (276) ( europe/euroguidelines.htm#current). CT conjunctivitis (195) CT infection should be included in the differential diagnosis in sexually active individuals presenting with acute or chronic follicular conjunctivitis. Ocular manifestations associated with CT genital infection may be more common than currently reported; this condition is likely to be underdiagnosed due to a low awareness among care givers and patients (195,277,278). Topical therapy alone is not effective (279). Whenever diagnosed, CT conjunctivitis should prompt for testing for genital CT infection and other STIs such as HIV, gonorrhoea and syphilis. Recommended treatment for CT conjunctivitis Azithromycin 1 g stat (oral) (279) (IIa; A) or Doxycycline 100 mg twice a day for 7 days (oral)(i; A) Contact notification and management of sexual contact(s) Contact notification should be performed and documented by appropriately trained professionals at the time of diagnosis to improve outcome (Ib; A); Sexual contacts should be contacted and offered (and encouraged) testing together with treatment and, if infected, counseling (as index patient) for chlamydial infection and other STIs (IV; C) (78,218, );

48 2015 European guideline on CT management review 47 All sexual contacts within the preceding 6 months of onset of symptoms or diagnosis should ideally be evaluated, tested and treated (IV; C) (78,192,218,282,283); If sexual contact(s) does not attend for evaluation and testing, epidemiological treatment should ideally be offered (IV; C) (78,218,282) Infected and untreated sexual contacts may reinfect the index patient as well as be at risk of adverse outcome of the CT infection. Therefore, contact notification is recommended, as it is important to reduce onward transmission of infection and it may reduce persistent and recurring infections (284). Contact notification strategies, including the subsequent management of contacts, differ widely between countries (285). These differences are due to different socioeconomic, legal, ethical, privacy and human rights, cultural and religious frameworks (192,282,285). Patient notification (where the index patient notifies the sexual contact(s)) and provider notification (where the healthcare provider notifies the sexual partner(s)) are currently the most commonly used methods. The ease of traveling means that sexual contacts may not only have come from the local region, but also nationally and internationally. Efforts should be made to ideally trace all local, national, and international sexual partners. All sexual partners (as far as practically and legally possible) should be notified of a possible CT infection, and offered testing and treatment. Sexual health counselling during the clinic visits reduces sexual risk taking and increases health awareness (280,281). This counselling should be offered where possible. Studies on the duration of human urogenital CT infections have shown that CT clearance increases over time, with approximately half of infections spontaneously resolving one year after initial positive test (286). However, the evidence base regarding ideal look-back period is limited and legal and practical considerations have to be taken into account when deciding a feasible look-back period. In many countries, these considerations limit the look-back period to two to three months, reducing the overall impact of contact notification strategies. Where no regulatory barriers exist, expedited partner therapy or patient-delivered partner therapy can be an efficient way to treat partners and reduce the infection rates (284, ). However, local, practical and legal restrictions may prevent the introduction of such methods (192,287,294). The main concerns are the lack of supervision of administration of prescription drugs, lack of monitoring of therapeutic effect, side-effects and adverse outcomes, the lack of opportunity to clinically examine and test for CT or other STIs, as well as the lack of onwards contact notification, and safe sex education. It appears, however, well accepted by patients and partners ( ). Patient delivered therapy should therefore only be implemented as part of a larger system of contact notification strategies. Given the wide differences between countries, no definitive recommendation can be given regarding expedited partner therapy or patient-delivered partner therapy. For further information, see the latest version of the European guidelines for the management of partners of persons with sexually transmitted infection (282) ( regions/europe/euroguidelines.htm#current).

49 FOLLOW-UP AND TEST-OF-CURE (TOC) Chapter 2 48 A TOC is not recommended to be routinely performed in patients treated with recommended first-line regimens but should be performed in pregnancy, in complicated infections, if symptoms persist, if second-line or third-line regimens have been used, and if non-compliance to therapy or re-exposure of infection is suspected (IV; C). It should also be considered in extra-genital infections (250), particularly when azithromycin 1 g stat has been administered for treatment of rectal infections. When indicated, TOC using NAATs should be performed 4 weeks after completion of therapy (III; B) (78,194,218,250, ). Repeated testing in 3 6 months should ideally be offered to young women and men (<25 years of age) who test positive for CT (78, ). There is still no evidence of any homotypic genetic and phenotypic resistance to any therapeutic antimicrobial in CT that results in treatment failure in humans ( ). A positive TOC may be due to noncompliance to treatment, reinfection from an untreated or new contact, inadequacy of treatment, and a false positive result (mainly due to detection of nucleic acid from non-viable CT because retesting was performed too early) (227). The diagnostic NAATs cannot discriminate between viable and dead CT. Accordingly, NAATs can remain positive more than three weeks (and possibly up to eight weeks due to intermittent shedding of nucleic acid) after treatment; due to detection of residual, non-viable chlamydial DNA or RNA that have not been cleared by the host (78,218, ). Recent years, major concerns have been raised over clinical failures in >5% of individuals treated for chlamydial infection with azithromycin 1 g single oral dose ( ). The reasons for these treatment failures remain unclear (228), though a short duration of exposure may be responsible (219). Further work is crucial to establish whether routine TOC should be recommended as suggested by some authors (IV; C) (219,223) TOC should be differentiated from repeated testing for reinfection. Reinfection, particularly in young women and men (<25 years of age), is common, frequently occurs within two to five months of the previous infection, and repeated chlamydia infection is associated with an increased risk of complications such as PID and tubal infertility (194,198,205,210, ). Accordingly, repeated testing in 3 6 months should be offered to young women and men (<25 years of age) who test positive for CT (III; C) (78, ,196,198,200,276). Asymptomatic infections have also been increasingly identified in MSM (304). Accordingly, repeated testing for TOC of asymptomatic MSM with rectal chlamydia after treatment for uncomplicated chlamydial infection (azithromycin 1 g single oral dose or doxycycline 100 mg, 7 days) should be considered to ensure that any LGV infection is not missed. For further information, see the latest version of the European guidelines for the management of partners of persons with sexually transmitted infection (282) and the European Guideline on the Management of Lymphogranuloma Venereum (68) (

50 2015 European guideline on CT management review 49 Notification of CT cases CT infections should be notified to local, regional and national authorities as mandated by statute. The ECDC is responsible for the EU/EEA-wide surveillance of communicable diseases including CT infections. Acknowledgements The authors thank Wichor Bramer, biomedical information specialist of the Medical Library of the Erasmus MC for his help in organising the literature search in the databases and setting up a search algorithm. Declaration of conflicting interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Funding The author(s) received no financial support for the research, authorship, and/or publication of this article. Search strategy Evidence was provided by a thorough and systemic review of the literature in the databases Embase.com, Medline (OvidSP), PubMed (articles supplied by publishers not yet indexed in Medline), Web-of-science, Scopus, Cinahl, Cochrane DARE, and Google Scholar. Searches were performed on 18 March 2014 and on 28 November 2014, and the following broad search terms were used: CT, systematic review, meta-analysis, guideline, protocol. After deduplication, 3041 articles published from 1992 to 2014 were screened on title/ abstract, which resulted in 824 references considered for inclusion when the guideline was written. Relevant STI guidelines produced by the US Centers for Disease Control and Prevention ( std/treatment/2015/) and the British Association for Sexual Health and HIV ( were also reviewed. Levels of evidence and grading of recommendations Tables of levels of evidence and grading of recommendations that were used in the present guideline can be found at: Levels_of_Evidence.

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56 2015 European guideline on CT management review 55 and Neisseria gonorrhoeae. Ann Intern Med 2005; 142: 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 CT infection.j Clin Microbiol 1998; 36: Skidmore S, Horner P and Mallinson H. Testing specimens for CT. Sex Transm Infect 2006; 82: Skidmore S, Horner P, Herring A, et al. Vulvovaginalswab or first-catch urine specimen to detect Chlamydia trachomatis in women in a community setting? J Clin Microbiol 2006; 44: Choe HS, Lee DS, Lee SJ, et al. Performance of Anyplex II multiplex real-time PCR for the diagnosis of seven sexually transmitted infections: comparison with currently available methods. Int J Infect Dis 2013; 17:e1134 e Gimenes F, Medina FS, Abreu AL, et al. Sensitive simultaneous detection of seven sexually transmitted agents in semen by multiplex-pcr and of HPV by single PCR. PLoS One 2014; 9: e Kumamoto Y, Matsumoto T, Fujisawa M, et al. Detection of CT and Neisseria gonorrhoeae in urogenital and oral specimens using the Cobas_ 4800, APTIMA Combo 2_ TMA, and ProbeTec ET SDA assays. Eur J Microbiol Immunol (Bp) 2012; 2: Le Roy C, Le Hen I, Clerc M, et al. The first performance report for the Bio-Rad Dx CT/ NG/MG assay for simultaneous detection of CT, Neisseria gonorrhoeae and Mycoplasma genitalium in urogenital samples. J Microbiol Methods 2012; 89: Mushanski LM, Brandt K, Coffin N, et al. Comparison of the BD viper system with XTR technology to the Gen-Probe APTIMA COMBO 2 assay using the TIGRIS DTS system for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae in urine specimens. Sex Transm Dis 2012; 39: Schachter J, Chow JM, Howard H, et al. Detection of CT by nucleic acid amplification testing: our evaluation suggests that CDC-recommended approaches for confirmatory testing are illadvised. J ClinMicrobiol 2006; 44: van der Helm JJ, Hoebe CJ, van Rooijen MS, et al. High performance and acceptability of self-collected rectal swabs for diagnosis of CT and Neisseria gonorrhoeae in men who have sex with men and women. Sex Transm Dis 2009; 36: Cheng A, Qian QF and Kirby JE. Evaluation of the Abbott RealTime CT/NG assay in comparison to th Roche Cobas Amplicor CT/NG assay. J Clin Microbiol 2011; 49: Unemo M, Rossouw A, James V, et al. Can the Swedish new variant of CT (nvct) be detected by UK NEQAS participants from seventeen European countries and five additional countries/regions in 2009? Euro Surveill 2009; 14: pii: Reischl U, Straube E and Unemo M. The Swedish new variant of CT (nvct) remains undetected by many European laboratories as revealed in the recent PCR/NAT ring trial organised by INSTAND e.v., Germany. Euro Surveill 2009; 14: pii: Shipitsyna E, Zolotoverkhaya E, Agne-Stadling I, et al. First evaluation of six nucleic acid amplification tests widely used in the diagnosis of CT in Russia. J Eur Acad Dermatol Venereol 2009; 23: Horner P, Skidmore S, Herring A, et al. Enhanced enzyme immunoassay with negative-grayzone testing compared to a single nucleic acid amplification technique for community-based chlamydial screening of men. J Clin Microbiol 2005; 43: Crucitti T, Jespers V, Van Damme L, et al. Vaginal microbicides can interfere with nucleic acid amplification tests used for the diagnosis of CT and Neisseria gonorrhoeae infection. Diagn Microbiol Infect Dis 2007; 57: Mahony J, Chong S, Jang D, et al. Urine specimens from pregnant and nonpregnant women inhibitory to amplification of CT nucleic acid by PCR, ligase chain reaction, and transcription-

57 Chapter 2 56 mediated amplification: identification of urinary substances associated with inhibition and removal of inhibitory activity. J Clin Microbiol 1998; 36: Chong S, Jang D, Song X, et al. Specimen processing and concentration of CT added can influence false-negative rates in the LCx assay but not in the APTIMA Combo 2 assay when testing for inhibitors. J Clin Microbiol 2003; 41: Van Dommelen L, Van Tiel FH, Ouburg S, et al. Alarmingly poor performance in CT pointof-care testing. Clin Microbiol Infect 2010; 16: S van der Helm JJ, Sabajo LO, Grunberg AW, et al. Point-of-care test for detection of urogenital chlamydia in women shows low sensitivity. A performance evaluation study in two clinics in Suriname. PLoS One 2012; 7: e Libbus MK. Chlamydia rapid test was moderately accurate for diagnosing Chlamydia infection in women. Evid Based Nurs 2008; 11: Hislop J, Quayyum Z, Flett G, et al. Systematic review of the clinical effectiveness and costeffectiveness of rapid point-of-care tests for the detection of genital chlamydia infection in women and men Review. Health Technol Assess 2010; 14: 1 97; iii iv Skidmore S. Poorly performing point-of-care tests for chlamydia: what can be done? Sex Transm Infect 2010; 86: Mahilum-Tapay L, Laitila V, Wawrzyniak JJ, et al. New point of care Chlamydia rapid test bridging the gap between diagnosis and treatment: performance evaluation study. BMJ 2007; 335: Nadala EC, Goh BT, Magbanua JP, et al. Performance evaluation of a new rapid urine test for chlamydia in men: prospective cohort study. BMJ 2009; 339: b Huang W, Gaydos CA, Barnes MR, et al. Comparative effectiveness of a rapid point-of-care test for detection of CT among women in a clinical setting. Sex Transm Infect 2013; 89: Gaydos CA, Van Der Pol B, Jett-Goheen M, et al. Performance of the Cepheid CT/NG Xpert rapid PCR test for detection of CT and Neisseria gonorrhoeae. J Clin Microbiol 2013; 51: Herrmann B. A new genetic variant of CT. Sex Transm Infect 2007; 83: Ripa T and Nilsson P. A variant of CT with deletion in cryptic plasmid: implications for use of PCR diagnostic tests. Euro Surveill 2006; 11: E Unemo M, Seth-Smith HM, Cutcliffe LT, et al. The Swedish new variant of CT: genome sequence, morphology, cell tropism and phenotypic characterization. Microbiology 2010; 156: Unemo M and Clarke IN. The Swedish new variant of CT. Curr Opin Infect Dis 2011; 24: An Q and Olive DM. Molecular cloning and nucleic acid sequencing of CT 16S rrna genes from patient samples lacking the cryptic plasmid. Mol Cell Probes 1994; 8: Clarke IN. Evolution of CT. Ann N Y Acad Sci 2011; 1230: E11 E Lei L, Chen J, Hou S, et al. Reduced live organism recovery and lack of hydrosalpinx in mice infected with plasmid-free Chlamydia muridarum. Infect Immun 2014; 82: Sigar IM, Schripsema JH, Wang Y, et al. Plasmid deficiency in urogenital isolates of CT reduces infectivity and virulence in a mouse model. Pathog Dis 2014; 70: Magbanua JP, Goh BT, Michel CE, et al. CT variant not detected by plasmid based nucleic acid amplification tests: molecular characterization and failure of single dose azithromycin. Sex Transm Infect 2007; 83: de Vries HJ, Zingoni A, White JA, et al European Guideline on the management of proctitis, proctocolitis and enteritis caused by sexually transmissible pathogens. Int J STD AIDS 2013; 25: Ratcliff RM, Chang G, Kok T, et al. Molecular diagnosis of medical viruses. Curr Issues Mol Biol 2007; 9:

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59 Chapter 2 58 Pediatr Infect Dis J 1997; 16: Ota KV, Tamari IE, Smieja M, et al. Detection of Neisseria gonorrhoeae and CT in pharyngeal and rectal specimens using the BD Probetec ET system, the Gen-Probe Aptima Combo 2 assay and culture. Sex Transm Infect 2009; 85: Peters RPH, Verweij SP, Nijsten N, et al. Evaluation of sexual history-based screening of anatomic sites for CT and Neisseria gonorrhoeae infection in men having sex with men in routine practice.bmc Infect Dis 2011; 11: Tipple C, Hill SC and Smith A. Is screening for pharyngeal CT warranted in high-risk groups? Int J STD AIDS 2010; 21: Marcus JL, Bernstein KT, Kohn RP, et al. Infections missed by urethral-only screening for Chlamydia or Gonorrhea detection among men who have sex with men. Sex Transm Dis 2011; 38: Jebakumar SP, Storey C, Lusher M, et al. Value of screening for oro-pharyngeal CT infection. J Clin Path 1995; 48: Schachter J, Moncada J, Liska S, et al. Nucleic acid amplification tests in the diagnosis of chlamydial and gonococcal infections of the oropharynx and rectum in men who have sex with men. Sex Transm Dis 2008; 35: Alexander S, Martin I and Ison C. Confirming the CT status of referred rectal specimens.sex Transm Infect 2007; 83: Mimiaga MJ, Mayer KH, Reisner SL, et al. Asymptomatic gonorrhea and chlamydial infections detected by nucleic acid amplification tests among Boston area men who have sex with men. Sex Transm Dis 2008; 35: Bachmann LH, Johnson RE, Cheng H, et al. Nucleic acid amplification tests for diagnosis of Neisseria gonorrhoeae and CT rectal infections. J Clin Microbiol 2010; 48: Ward H, de Vries HJC and van de Laar M. Re-emergence of lymphogranuloma venereum in Europe and the public health response. Sex Transm Infect 2011; 87: A19 A Ward H, Martin I, Macdonald N, et al. Lymphogranuloma venereum in the United Kingdom. Clin Infect Dis 2007; 44: le Roy C, Papaxanthos A, Liesenfeld O, et al. Swabs (dry or collected in universal transport medium) and semen can be used for the detection of CT using the Cobas 4800 system. J Med Microbiol 2013; 62: Gdoura R, Kchaou W, Ammar-Keskes L, et al. Assessment of CT, Ureaplasma urealyticum, Ureaplasma parvum, Mycoplasma hominis, and Mycoplasma genitalium in semen and first void urine specimens of asymptomatic male partners of infertile couples. J Androl 2008; 29: de Barbeyrac B, Mehats V, Papaxanthos A, et al. Establishment of a protocol for the detection of CT in semen specimens using the Cobas 4800 CT/NG test. Sex Transm Infect 2011; 87: A Hamdad-Daoudi F, Petit J and Eb F. Assessment of CT infection in asymptomatic male partners of infertile couples. J Med Microbiol 2004; 53: Pannekoek Y, Westenberg SM, Eijk PP, et al. Assessment of CT infection of semen specimens by ligase chain reaction. J Med Microbiol 2003; 52: van der 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 nonlymphogranuloma venereum and lymphogranuloma venereum serovars of CT. Seks Transm Infect 2007; 83: de Vries HJ, Smelov V, Ouburg S, et al. Anal lymphogranuloma venereum infection screening with IgA anti CT-specific major outer membrane protein serology. Sex Transm Dis 2010; 37: Verweij SP, Lanjouw E, Bax CJ, et al. Serovar D and E of serogroup B induce highest serological responses in urogenital CT infections. BMC Infect Dis 2014; 14: 3.

60 2015 European guideline on CT management review 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: Black CM. Current methods of laboratory diagnosis of CT infections. Clin Microbiol Rev 1997; 10: Clad A, Freidank HM, Kunze M, et al. Detection of seroconversion and persistence of CT antibodies in five different serological tests. Eur J Clin Microbiol Infect Dis 2000; 19: Mouton JW, Peeters MF, van Rijssort-Vos JH, et al. Tubal factor pathology caused by CT: the role of serology. Int J STD AIDS 2002; 13(Suppl 2): Verkooyen RP, Peeters MF, van Rijsoort-Vos JH, et al. Sensitivity and specificity of three new commercially available CT tests. Int J STD AIDS 2002; 13(Suppl 2): Land JA and Evers JL. Chlamydia infection and subfertility.best Pract Res Clin Obstet Gynaecol 2002; 16: Broeze KA, Opmeer BC, Coppus SFPJ, et al. Chlamydia antibody testing and diagnosing tubal pathology in subfertile women: an individual patient data meta-analysis. Hum Reprod Update 2011; 17: Dabekausen YAJM, Evers JLH, Land JA, et al. CT antibody testing is more accurate than hysterosalpingography in predicting tubal factor infertility. Fertil Steril 1994; 61: Mol BW, Broeze KA, Bhattacharya S, et al. Estimation of the accuracy of the Chlamydia antibody test based on individual data of subfertile women. Mol Hum Reprod 2009; 24: i She RC, Welch R, Wilson AR, et al. Correlation of Chlamydia and Chlamydophila spp. IgG and IgM antibodies by microimmunofluorescence with antigen detection methods. J Clin Lab Anal 2011; 25: Peeling RW, Wang SP, Grayston JT, et al. Chlamydia pneumoniae serology: interlaboratory variation in microimmunofluorescence assay results. J Infect Dis 2000; 181(Suppl 3): S426 S Gronowski AM, Copper S, Baorto D, et al. Reproducibility problems with the Abbott laboratories LCx assay for CT and Neisseria gonorrhoeae. J Clin Microbiol 2000; 38: Lanjouw E, Ossewaarde JM, Stary A, et al European guideline for the management of Chlamydia trachomatis infections. Int J STD AIDS 2010; 21: Ministry of Health, Wellington, New Zealand. Chlamydia management guidelines 2008, www. health.govt.nz/system/files/documents/publications/chlamydiamanagement-guidelines. pdf (2008, accessed 30 August 2015) Fung M, Scott KC, Kent CK, et al. Chlamydial and gonococcal reinfection among men: a systematic review of data to evaluate the need for retesting. Sex Transm Infect 2007; 83: Scottish Intercollegiate Guidelines Network (SIGN). Management of genital CT infection. A national clinical guideline, (2009, accessed 30 August 2015) Public Health Agency of Canada. Canadian guidelines on sexually transmitted infections, (accessed 30 August 2015) Health Protection Agency. National Chlamydia screening programme. England: Author, Hosenfeld CB, Workowski KA, Berman S, et al. Repeat infection with chlamydia and gonorrhea among females: a systematic review of the literature. Sex Transm Dis 2009; 36: Royal Australian College of General Practitioners. Guidelines for preventive activities in general practice. 8th ed. redbook8.pdf (accessed 30 August 2015).

61 Chapter Public Health England. National Chlamydia screening programme, nhs.uk/ps/news.asp (accessed 30 August 2015) van de Laar MJ and Fontaine J. ECDC guidance on chlamydia control in Europe: next steps. Euro Surveill 2009; 14: pii: Regan DG, Wilson DP and Hocking JS. Coverage is the key for effective screening of CT in Australia. J Infect Dis 2008; 198: Glasser JW, Owusu-Edusei K, Glick SN, et al. Controlling chlamydia: population modeling to assess promising interventions. Sex Transm Infect 2013; 89(suppl 1): A Althaus CL, Heijne JC, Herzog SA, et al. Individual and population level effects of partner notification for CT. PLoS One 2012; 7: e Jamil MS, Bauer HM, Hocking JS, et al. Chlamydia screening strategies and outcomes in educational settings: a systematic review. Sex Transm Dis 2014; 41: Heijne JC, Herzog SA, Althaus CL, et al. Insights into the timing of repeated testing after treatment for CT: data and modelling study. Sex Transm Infect 2013; 89: Aghaizu A, Reid F, Kerry S, et al. Frequency and risk factors for incident and redetected CT infection in sexually active, young, multi-ethnic women: a community based cohort study. Sex Transm Infect 2014; 90: British Association for Sexual Health and HIV. CT UK testing guidelines, bashh.org/documents/3352.pdf (2010, accessed 30 August 2015) Public Health England. Position statement. NCSP recommended case management change: routine offer of re-test to young adults testing positive for Chlamydia, www. chlamydiascreening.nhs.uk (2013, accessed 15 August 2015) Scott Lamontagne D, Baster K, Emmett L, et al. Incidence and reinfection rates of genital chlamydial infection among women aged years attending general practice, family planning and genitourinary medicine clinics in England: a prospective cohort study by the Chlamydia Recall Study Advisory Group. Sex Transm Infect 2007; 83: Smith KJ, Cook RL and Roberts MS. Time from sexually transmitted infection acquisition to pelvic inflammatory disease development: influence on the cost-effectiveness of different screening intervals. Value Health 2007; 10: Scholes D, Stergachis A, Heidrich FE, et al. Prevention of pelvic inflammatory disease by screening for cervical chlamydial infection. New Engl J Med 1996; 334: Ostergaard L, Andersen B, Moller JK, et al. Home sampling versus conventional swab sampling for screening of CT in women: a cluster-randomized 1-year follow-up study. Clin Infect Dis 2000; 31: Andersen B, van Valkengoed I, Sokolowski I, et al. Impact of intensified testing for urogenital Chlamydia trachomatis infections: a randomised study with 9-year follow-up. Sex Transm Infect 2011; 87: Atkins D, Best D, Briss PA, et al. Grading quality of evidence and strength of recommendations. BMJ 2004; 328: van den Broek IV, van Bergen JE, Brouwers EE, et al. Effectiveness of yearly, register based screening for chlamydia in the Netherlands: controlled trial with randomized stepped wedge implementation. BMJ 2012; 345: e Cohen DA, Nsuami M, Martin DH, et al. Repeated school-based screening for sexually transmitted diseases:a feasible strategy for reaching adolescents. Pediatrics 1999; 104: British Association of Sexual Health and HIV Draft UK national guideline for the management of genital infection with CT, (2013, accessed 30 August 2015) Horner PJ. Azithromycin antimicrobial resistance and genital CT infection: duration of therapy may be the key to improving efficacy. Sex Transm Infect 2012; 88: Sandoz KM and Rockey DD. Antibiotic resistance in Chlamydiae. Future Microbiol 2010; 5:

62 2015 European guideline on CT management review Wang SA, Papp JR, Stamm WE, et al. Evaluation of antimicrobial resistance and treatment failures for CT: a meeting report. J Infect Dis 2005; 191: O Neill CE, Seth-Smith HM, Van Der Pol B, et al. CT clinical isolates identified as tetracycline resistant do not exhibit resistance in vitro: whole-genome sequencing reveals a mutation in porb but no evidence for tetracycline resistance genes. Microbiology 2013; 159: Handsfield HH. Questioning azithromycin for chlamydial infection. Sex Transm Dis 2011; 38: Schwebke JR, Rompalo A, Taylor S, et al. Re-evaluating the treatment of nongonococcal urethritis: emphasizing emerging pathogens a randomized clinical trial. Clin Infect Dis 2011; 52: Sena AC, Lensing S, Rompalo A, et al. CT, Mycoplasma genitalium, and Trichomonas vaginalis infections in men with nongonococcal urethritis: predictors and persistence after therapy. J Infect Dis 2012; 206: Kong FY and Hocking JS. Treatment challenges for urogenital and anorectal CT. BMC Infect Dis 2015; 15: Somani J, Bhullar VB, Workowski KA, et al. Multiple drug-resistant CT associated with clinical treatment failure. J Infect Dis 2000; 181: Bhengraj AR, Srivastava P and Mittal A. Lack of mutation in macrolide resistance genes in CT clinical isolates with decreased susceptibility to azithromycin. Int J Antimicrob Agents 2011; 38: Mpiga P and Ravaoarinoro M. Effects of sustained antibiotic bactericidal treatment on CT infected epithelial-like cells (HeLa) and monocyte-like cells (THP-1 and U-937). Int J Antimicrob Agents 2006; 27: Amsden GW. Erythromycin, clarithromycin, and azithromycin: are the differences real? Clin Ther 1996; 18: 56 72; discussion Lode H, Borner K, Koeppe P, et al. Azithromycin review of key chemical, pharmacokinetic and microbiological features. J Antimicrob Chemother 1996; 37(Suppl C): Bjornelius E, Anagrius C, Bojs G, et al. Antibiotic treatment of symptomatic Mycoplasma genitalium infection in Scandinavia: a controlled clinical trial. Sex Transm Infect 2008; 84: Anagrius C, Lore B and Jensen JS. Treatment of Mycoplasma genitalium. Observations from a Swedish STD clinic. PloS One 2013; 8: e Horner P, Blee K and Adams E. Time to manage Mycoplasma genitalium as an STI: but not with azithromycin 1 g Curr Opin Infect Dis 2014; 27: Taylor-Robinson D and Jensen JS. Mycoplasma genitalium: from Chrysalis to multicolored butterfly. Clin Microbiol Rev 2011; 24: Unemo M, Endre KMA and Moi H. The five days azithromycin treatment regimen (500mg_1, 250mg_4) used for Mycoplasma genitalium infections also effectively eradicates CT. Acta Derm Venereol 2015; 95: Kong FY, Tabrizi SN, Law M, et al. Azithromycin versus doxycycline for the treatment of genital Chlamydia infection: a meta-analysis of randomized controlled trials. Clin Infect Dis 2014; 59: Ibsen HH, Moller BR, Halkier-Sorensen L, et al. Treatment of nongonococcal urethritis: comparison of ofloxacin and erythromycin. Sex Transm Dis 1989; 16: Maiti H, Chowdhury FH, Richmond SJ, et al. Ofloxacin in the treatment of uncomplicated gonorrhea and chlamydial genital infection. Clin Ther 1991; 13: Takahashi S, Ichihara K, Hashimoto J, et al. Clinical efficacy of levofloxacin 500 mg once daily for 7 days for patients with non-gonococcal urethritis. J Infect Chemother 2011; 17: Cramers M, Kaspersen P, From E, et al. Pivampicillin compared with erythromycin for

63 Chapter 2 62 treating women withgenital CT infection. Genitourin Med 1988; 64: Worm AM, Hoff G, Kroon S, et al. Roxithromycin compared with erythromycin against genitourinary chlamydial infections. Genitourin Med 1989; 65: Khrianin AA and Reshetnikov OV. Is it safe to use josamycin in the obstetrics practice in Russia? Antibiot Khimioter 2007; 52: 32 36; [in Russian] Primiero FM, Caruso G, Grottanelli F, et al. Josamycin in the treatment of CT cervicitis. J Chemother 1989; 1: Lucisano A, Vitale AM, Cinque B, et al. Josamycin in the treatment of chlamydial genital infections in infertile women. J Chemother 1989; 1: Adimora AA. Treatment of uncomplicated genital CT infections in adults. Clin Infect Dis 2002; 35: S183 S Schillinger JA, Kissinger P and Calvet H. Patient-delivered partner treatment with azithromycin to prevent repeated CT infection among women: a randomized, controlled trial. Sex Transm Dis 2003; 30: Golden MR, Whittington WL, Handsfield HH, et al. Effect of expedited treatment of sex partners on recurrent or persistent gonorrhea or chlamydial infection. New Engl J Med 2005; 352: Geisler WM, Pascual ML, Mathew J, et al. Randomized, double-blind, multicenter safety and efficacy study of rifalazil compared with azithromycin for treatment of uncomplicated genital Chlamydia trachomatis infection in women. Antimicrob Agents Chemother 2014; 58: Steedman NM and McMillan A. Treatment of asymptomatic rectal CT: is single-dose azithromycin effective? Int J STD AIDS 2009; 20: Elgalib A, Alexander S, Tong CYW, et al. Seven days of doxycycline is an effective treatment for asymptomatic rectal CT infection. Int J STD AIDS 2011; 22: Hathorn E, Opie C and Goold P. What is the appropriate treatment for the management of rectal Chlamydia trachomatis in men and women? Sex Transm Infect 2012; 88: Khosropour CM, Dombrowski JC, Barbee LA, et al. Comparing azithromycin and doxycycline for the treatment of rectal chlamydial infection: a retrospective cohort study. Sex Transm Dis 2014; 41: Drummond F, Ryder N, Wand H, et al. Is azithromycin adequate treatment for asymptomatic rectal chlamydia? Int J STD AIDS 2011; 22: Hill SC, Hodson L and Smith A. An audit on the management of lymphogranuloma venereum in a sexual health clinic in London, UK. Int J STD AIDS 2010; 21: Nieuwenhuis RF, Ossewaarde JM, van der Meijden WI, et al. Unusual presentation of early lymphogranuloma venereum in an HIV-1 infected patient: effective treatment with 1 g azithromycin. Sex Transm Infect 2003; 79: van Nieuwkoop C, Gooskens J, Smit VT, et al. Lymphogranuloma venereum proctocolitis: mucosal T cell immunity of the rectum associated with chlamydial clearance and clinical recovery. Gut 2007; 56: McLean CA, Stoner BP and Workowski KA. Treatment of lymphogranuloma venereum. Clin Infect Dis 2007; 44(Suppl 3): S147 S White J, O Farrell N and Daniels D UK National Guideline for the management of lymphogranuloma venereum: clinical Effectiveness Group of the British Association for Sexual Health and HIV (CEG/BASHH) Guideline development group. Int J STD AIDS 2013; 24: Centers for Disease Control and Prevention (CDC). Lymphogranuloma venereum among men who have sex with men Netherlands, MMWR Morb Mortal Wkly Rep 2004; 53: de Vries HJ, Smelov V, Middelburg JG, et al. Delayed microbial cure of lymphogranuloma venereum proctitis with doxycycline treatment. Clin Infect Dis 2009; 48: e53 e56.

64 2015 European guideline on CT management review Baud D, Regan L and Greub G. Emerging role of Chlamydia and Chlamydia-like organisms in adverse pregnancy outcomes. Curr Opin Infect Dis 2008; 21: Blas MM, Canchihuaman FA, Alva IE, et al. Pregnancy outcomes in women infected with CT:a population-based cohort study in Washington State. Sex Transm Infect 2007; 83: Rustveld LO, Kelsey SF and Sharma R. Association between maternal infections and preeclampsia: a systematic review of epidemiologic studies. Matern Child Health J 2008; 12: Chow JM, Kang MS, Samuel MC, et al. Assessment of the association of CT infection and adverse perinatal outcomes with the use of population based Chlamydia case report registries and birth records.public Health Rep 2009; 124: Matthew Chico R, Hack BB, Newport MJ, et al. On the pathway to better birth outcomes? A systematic review of azithromycin and curable sexually transmitted infections. Expert Rev Anti Infect Ther 2013; 11: de Attayde Silva MJPM, Dantas Florencio GL, Erbolato Gabiatti JR, et al. Perinatal morbidity and mortality associated with chlamydial infection: a metaanalysis study. Braz J Infect Dis 2011; 15: British Association for Sexual Health and HIV. UK national guideline for the management of genital tract infection with CT, (2006, accessed 30 August 2015) Pitsouni E, Iavazzo C, Athanasiou S, et al. Single-dose azithromycin versus erythromycin or amoxicillin for CT infection during pregnancy: a meta-analysis of randomised controlled trials. Int J Antimicrob Agents 2007; 30: Rahangdale L, Guerry S, Bauer HM, et al. An observational cohort study of CT treatment in pregnancy. Sex Transm Dis 2006; 33: Sarkar M, Woodland C, Koren G, et al. Pregnancy outcome following gestational exposure to azithromycin. BMC Pregnancy Childbirth 2006; 6: Meyer T. Diagnosis and treatment of CT infections. Hautarzt 2012; 63: Hemsell DL, Little BB, Faro S, et al. Comparison of three regimens recommended by the Centers for Disease Control and Prevention for the treatment of women hospitalized with acute pelvic inflammatory disease. Clin Infect Dis 1994; 19: Martens MG, Gordon S, Yarborough DR, et al. Multicenter randomized trial of ofloxacin versus cefoxitin and doxycycline in outpatient treatment of pelvic inflammatory disease. Ambulatory PID Research Group. South Med J 1993; 86: Judlin P, Liao Q, Liu Z, et al. Efficacy and safety of moxifloxacin in uncomplicated pelvic inflammatory disease: the MONALISA study. BJOG 2010; 117: Ross J, Judlin P and Jensen J European guideline for the management of pelvic inflammatory disease. Int J STD AIDS 2014; 25: Postema EJ, Remeijer L and van der Meijden WI. Epidemiology of genital chlamydial infections in patients with chlamydial conjunctivitis; a retrospective study. Genitourin Med 1996; 72: Sulis G, Urbinati L, Franzoni A, et al. CT conjunctivitis in a male teenager: a case report. Le infezioni in medicina: rivista periodica di eziologia, epidemiologia, diagnostica, clinica e terapia delle patologie infettive 2014; 22: Members AAoO-CEDP. Preferred practice pattern guidelines: conjunctivitis limited revision, 2011, Wilson TE, Hogben M, Malka ES, et al. A randomized controlled trial for reducing risks for sexually transmitted infections through enhanced patient-based partner notification. Am J Public Health 2009; 99(Suppl 1): S104 S Hogben M and Kissinger P. A review of partner notification for sex partners of men infected with chlamydia. Sex Transm Dis 2008; 35: S34 S39.

65 Chapter Tiplica GS, Evans C, Gomberg M, et al European guidelines for the management of partners of persons with sexually transmitted infections, europe/word_docs/17.1_european_guide line_on_pn.doc (2013, accessed 30 August 2015) McClean H, Carne CA, Sullivan AK, et al. Chlamydial partner notification in the British Association for Sexual Health and HIV (BASHH) 2011 UK national audit against the BASHH Medical Foundation for AIDS and Sexual Health Sexually Transmitted Infections Management Standards. Int J STD AIDS 2012; 23: McIlveen H. Review: partner notification interventions can reduce persistent or recurrent sexually transmitted infections. Evid Based Nurs 2007; 10: Arthur G, Lowndes CM, Blackham J, et al. Divergent approaches to partner notification for sexually transmitted infections across the European Union. Sex Transm Dis 2005; 32: Geisler WM. Duration of untreated, uncomplicated CT genital infection and factors associated with chlamydia resolution: a review of human studies. J Infect Dis 2010; 201(Suppl 2): S104 S Geisler WM. Management of uncomplicated CT infections in adolescents and adults: evidence reviewed for the 2006 Centers for Disease Control and Prevention sexually transmitted diseases treatment guidelines. Clin Infect Dis 2007; 44(Suppl 3): S77 S Bell G and Potterat J. Partner notification for sexually transmitted infections in the modern world: a practitioner perspective on challenges and opportunities. Seks Transm Infect 2011; 87: ii34 ii Ferreira A, Young T, Mathews C, et al. Strategies for partner notification for sexually transmitted infections, including HIV. [Update of Cochrane Database Syst Rev. 2001; 4: CD002843; PMID: ]. Cochrane Database Syst Rev 2013; 10: CD Althaus CL, Turner KME, Mercer CH, et al. Effectiveness and cost-effectiveness of traditional and new partner notification technologies for curable sexually transmitted infections: observational study, systematic reviews and mathematical modelling. Health Technol Assess 2014; 18: Pavlin NL, Parker RM, Piggin AK, et al. Better than nothing? Patient-delivered partner therapy and partner notification for chlamydia: the views of Australian general practitioners. BMC Infect Dis 2009; 10: Hogben M, Kidd S and Burstein GR. Expedited partner therapy for sexually transmitted infections. Curr Opin Obstet Gynecol 2012; 24: Kissinger P and Hogben M. Expedited partner treatment for sexually transmitted infections: an update. Curr Infect Dis Rep 2011; 13: Hodge JG Jr, Pulver A, HogbenM, et al. Expedited partner therapy for sexually transmitted diseases: assessing the legal environment. Am J Public Health 2008; 98: Shivasankar S and Challenor R. Patient-delivered partner therapy in the UK: what do the professionals think? Int J STD AIDS 2008; 19: Coyne KM, Cohen CE, Smith NA, et al. Patient-delivered partner medication in the UK: an unlawful but popular choice. Int J STD AIDS 2007; 18: Shivasankar S, Challenor R and Ekanayaka R. Patientdelivered partner therapy in the UK: what do patients think? Int J STD AIDS 2008; 19: Dukers-Muijrers NH, Morre SA, Speksnijder A, et al. CT test-of-cure cannot be based on a single highly sensitive laboratory test taken at least 3 weeks after treatment. PLoS One 2012; 7: e Renault CA, Israelski DM, Levy V, et al. Time to clearance of CT ribosomal RNA in women treated for chlamydial infection. Sex Health 2011; 8: Dukers-Muijrers NH, Speksnijder AG, Morre SA, et al. Detection of anorectal and cervicovaginal Chlamydia trachomatis infections following azithromycin treatment:

66 2015 European guideline on CT management review 65 prospective cohort study with multiple timesequential measures of rrna, DNA, quantitative load and symptoms. PLoS One 2013; 8: e Dunne EF, Chapin JB, Rietmeijer CA, et al. Rate and predictors of repeat CT infection among men. Sex Transm Dis 2008; 35: S40 S Kjaer HO, Dimcevski G, Hoff G, et al. Recurrence of urogenital CT infection evaluated by mailed samples obtained at home: 24 weeks prospective follow up study. Sex Transm Infect 2000; 76: Whittington WLH, Kent C, Kissinger P, et al. Determinants of persistent and recurrent Chlamydia trachomatis infection in young women: results of a multicenter cohort study. Sex Transm Dis 2001; 28: Van der Bij AK, Spaargaren J, Morre SA, et al. Diagnostic and clinical implications of anorectal lymphogranuloma venereum in men who have sex with men: a retrospective casecontrol study. Clin Infect Dis 2006; 42:

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68 CHAPTER 3 Consecutively acquired sexually transmitted infections mimicking Crohn s disease E. Lanjouw, P.L.A. van Daele, M.P.E. Raes and W.I. van der Meijden American Journal of Gastroenterology 2009; 104(2):

69 CASE REPORT A 28-year-old man presented with painful eyes and visual disturbances at the department of Ophthalmology of our hospital, which appeared to be caused by anterior uveitis. To exclude an underlying systemic disease, he was sent to the immunology department. Apart from his visual complaints, he reported to have constipation with blood-stained anal discharge since approximately 1 year. He reported to have unprotected receptive anal sexual contacts with male partners. His medical history revealed acute hepatitis B infection, urethral Chlamydia trachomatis (CT) infection, rectal Neisseria gonorrhoeae infection and rectal condylomata acuminata. Physical examination at the immunology department revealed an annular plaque at the soft palate and perianal skin defects, including perianal fistulas. The main differential diagnostic consideration was Crohn s disease, based on the co-occurrence of uveitis, gastro-intestinal complaints, an elevated sedimentation rate, and a positive family history. Chapter 3 68 Furthermore, routine venereological screening at the Sexual Health Department was carried out to exclude rectal lymphogranuloma venereum (LGV). On physical examination perianal fistulas were seen (Figure 1A). Rectal examination was painful and a circular, narrowed structure was palpated at a distance of approximately 5 cm. Proctoscopy showed both pus and blood, partly obscuring the mucosa. A striking feature was the small anal lumen and multiple condylomata acuminata. On account of clinical suspicion of atypical secondary syphilis (uveitis anterior and an annular structure at the palatum, resembling a condyloma latum (Figure 1B)), a rapid plasma reagin test was performed which was strongly positive (3+). A strand displacement amplification (SDA) test (BD ProbeTec3, BD Dignostic System, Aalst, Belgium) on a rectal swab was positive for N. gonorrhoeae and CT and serotyping demonstrated the L-serotype of CT. During a colonoscopy, performed by the gastro-enterontologist, 4 mm punch biopsies were taken from the rectal mucosa. Histology showed characteristics consistent with LGV. Laboratory results furthermore showed a treponema screening assay (TSA)-ratio of 6.9, a venereal disease research laboratory test (VDRL) titre of 1:64 and a positive fluorescent treponemal antibody absorption test (FTA). HIV and hepatitis C virus tests were negative. Magnetic resonance imaging demonstrated rectal A B Figure 1. A) Perianal fistula. B) condyloma latum at the palatum

70 LGV mimicking Crohn s disease 69 Figure 2. Magnetic Resonance Imaging (MRI) showing rectal infiltration and perianal fistulae with an internal fissure in the anal canal lumen in the dorsal midline. In the middle exterior a fissure with local dilatation of fistulae resembles a local abscess, found in the subcutis nearby the exterior sphincter. infiltration and perianal fistulas, with a fissure in the anal canal lumen (Figure 2). Treatment consisted of 2.4 million units benzathine penicillin (Penidural) intramuscularly for secondary syphilis and 21 days doxycyclin 100 mg twice daily for both LGV and rectal N. gonorrhoeae. Within 1 month he experienced a remission of his uveitis and proctitis symptoms. He reported a normal bowel habit, the daily rectal bleeding had ceased and the perianal fistulas had become smaller. The annular plaque at his palatum had also disappeared. However, the anal canal still showed a stricture. Laboratory results showed negative rectal smears for CT and N. gonorrhoeae and the VDRL titer had declined to 1:8. Cryotherapy was started for treatment of his intra-anal warts. LGV is endemic in areas of East and West Africa, India, parts of Southeast Asia and the Caribbean. In 2003, the first LGV case was reported in Rotterdam. Since then multiple LGV cases were described in Western Europe in especially white, HIV- and hepatitis C virus-positive men who have sex with men (MSM) (1-4). In this patient, the most striking features were the obvious narrowing of the anal lumen and a negative HIV test. If LGV is left untreated, chronic inflammation, scarring, the development of strictures and fistulas, genital elephantiasis, and infertility may occur (1). Treatment is necessary to prevent late sequelae and transmission. Owing to chronic inflammation, late sequelae and new infections, e.g., syphilis, may arise more easily. LGV can mimic Crohn s disease. On the basis of the combination of uveitis and prolonged gastro-intestinal symptoms, Crohn s disease was strongly suspected in this case. An adequate sexual history can be helpful in the differential diagnosis. In sexually active MSM with rectal complaints, LGV should always be considered even if the HIV-test is negative.

71 REFERENCES 1. Nieuwenhuis RF, Ossewaarde JM, Götz HM et al. Resurgence of lymphogranuloma venereum in Western Europe: an outbreak of CT serovar L2 proctitis in the Netherlands among men who have sex with men. Clin Infect Dis 2004;39: Scieux C, Barnes R, Bianchi A et al. Lymphogranuloma venereum: 27 cases in Paris. J Infect Dis 1989;160: Van de Laar MJ, Koedijk FD, Gotz HM et al. A slow epidemic of LGV in the Netherlands in 2004 and Euro Surveill 2006;11: Jebbari H, Alexander S, Ward H, et al., UK LGV Incident Group. Update on lymphogranuloma venereum in the United Kingdom. Sex Transm Infect 2007;83:324 6; e-pub 25 June Chapter 3 70

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74 CHAPTER 4 The Dutch Chlamydia trachomatis Reference Laboratory : identification and surveillance of clinical samples for plasmid free and other Chlamydia trachomatis variants E. Lanjouw*, D.J. de Waaij*, S. Ouburg, J. Pleijster, S.M. Bruisten, M.A.B. van der Sande, B.H. van Benthem, C.J.G. Kampman, Variant Analyses Group, A.G.M. van der Zanden, S.A. Morré in progress * Shared first authorship

75 ABSTRACT Background: The emergence of the Swedish variant Chlamydia trachomatis (CT) strain showed the relevance of good monitoring. Since CT plasmid free strains and other variants can be missed by specific NAAT assays the Dutch Chlamydia trachomatis Reference Laboratory developed a surveillance programme to monitor potential variant emergences in the Netherlands by two approaches. Methods: Six laboratories from STI centers, Municiple Health centers and a University center participated in a monitoring study. Each center was asked to provide randomly 25 CT negative samples and 10 CT positive samples quarterly so that samples each year were collected to generate enough power to be able to detect a 2% emergence of unique strains like plasmid free variants without being the result of change. CT was detected using 3 plasmid targets and the ompa chromosomal target. Secondly, the Dutch Chlamydia trachomatis Reference Laboratory functioned as an expertise center for questions on CT strain related issues. Chapter 4 74 Results: In the period , 3554 samples were collected from the different participating centers. In total we identified 7 plasmid free variants. The variants were detected in both males and females and at different anatomical sites. 7 plasmid free variants resulted in an incidence of 0.20%. All except one were detected in the routinely tested CT positive samples provided by the different centers. This means that 86% (6/7) of the plasmid free samples was already detected and treated. From one specimen additional material could be collected (male urine) which was used for MLST typing and which was typed as serovar E. As expertise center we were involved in detecting the first Swedish variant (swct) in the Netherlands and the first heterosexual female infected with the LGV L2b strain (bubonic). Conclusion: This is the first study in the Netherlands monitoring the emergence of potential CT variants. The incidence of plasmid free CT strains was very low, only 0.20% based on analyses of 3554 samples from different geographical regions in the Netherlands. Over the last 3 years no plasmid free variants were detected, for which now no further action is needed nor is there a potential health care issue. In addition to this study, two other variants were identified.

76 Dutch CT Reference Laboratory 75 INTRODUCTION The micro-organism Chlamydia trachomatis (CT) is the most commonly reported cause of bacterial sexually transmitted infections (STI) in developed countries. Untreated urogenital CT infections can lead to complications such as pelvic inflammatory disease and infertility in women and epididymitis in men (1). Up to 80% of infected women and 50% of infected men are asymptomatic (2), most of these individuals thus remain undiagnosed. Targeted screening of high risk populations has been recommended to control CT infections (3), and several countries including the United Kingdom have embarked on screening programmes and are currently all based on nucleic acid amplification tests (NAATs) (4). Most commercial NAATs for the detection of CT rely either on conserved regions of 16S ribosomal RNA or on the 7.5 kb cryptic plasmid of the organism as the target for amplification (5, 6). Mutations in the primer and probe sequences and plasmid free variants could have devastating effects on control of CT infections. Indeed, a CT variant with a deletion in the plasmid was identified in Sweden, after an apparent 25% decrease in CT infections was noted with major effects on the control of the STI (7). This variant had a 377 bp deletion in the region of the plasmid targeted by two commercially NAATs, the M2000 (Abbott Laboratories, Abbott Park, IL, USA) and Amplicor CT/NG PCR (Roche Diagnostic Systems, Branchburg, NJ, USA) tests, and it therefore yielded false negative results in both of these tests. However, this strain remained detectable by another plasmid based NAAT, the ProbeTec ET (BD Biosciences, Sparks, MD, USA), because the deletion did not affect the target region of that test. Naturally occurring plasmid free clinical isolates are rare, indicating the potential plasmids importance in chlamydial pathogenesis (8). Plasmid-deficient CT strains and the murine pathogen Chlamydia muridarum have been studied in both nonhuman primate and murine infection models. A common theme emerging from these investigations is that in vivo infections with plasmid-deficient organisms either are often asymptomatic (9, 10) or exhibit significantly reduced pathology (11), providing evidence that the plasmid plays an essential role in chlamydial pathogenesis. The molecular basis of plasmid-mediated virulence is poorly understood, but is linked to enhanced pro-inflammatory cytokine stimulation by engagement of Toll-like receptors (TLR) in murine models (12 14). Since plasmid free strains can still be undetected and thus be left untreated, the Dutch Chlamydia trachomatis Reference Laboratory developed a surveillance programme from 2010 onwards to monitor potential plasmid free variants and other variant emergences in the Netherlands.

77 MATERIAL & METHODS Chapter 4 76 Sample collection Laboratories in STI centers, Municipal Health Centers and a University center (Maastricht) are participating in this ongoing study. We started with 5 centers; University center Maastricht-Heerlen, CERTE Groningen, Municipal Health Center Amsterdam, Municipal Health Center Rotterdam, STI center the Hague and in 2011 LabMicTA (Hengelo) was added to also include the Eastern part of the Netherlands in the surveillance programme. Each center was asked to provide randomly 25 CT negative samples and 10 CT positive samples quarterly such that Amsterdam Den Haag Rotterdam Maastricht Groningen Hengelo Heerlen samples per year were collected to generate a power to detect a 2% emergence of unique strains like plasmid free variants. Samples were sent frozen to our Laboratory in the VU University Medical Center, Amsterdam, the Netherlands. The results were analysed according the site of collection (vaginal, urine, oral, anal) and to sexes. DNA isolation and CT detection From 200µl patient material DNA was isolated with the High Pure Template Preparation Kit (HPPTP, Roche Diagnostics, USA). For the detection of CT a real time PCR machine (LC480 Roche) was used. CT was detected by two separate PCRs, one based on the CT cryptic plasmid DNA and one based on the CT chromosomal DNA. The CT chromosomal DNA positive but CT cryptic plasmid DNA negative samples were retested with two new developed cryptic plasmid DNA ORF based PCRs i.e. cryptic plasmid ORF2 and ORF7. The PCR negative samples for cryptic plasmid ORF2 and 7 but positive for the chromosomal PCR were called cryptic plasmid negative (plasmid free CT strain). Serovar typing by Multi Locus Sequence Typing (MLST) MLST was performed as described previously (15) to determine serovars. For short, DNA isolates were amplified by a nested PCR and sequenced for the regions ompa, CT046 (hctb), CT058, CT144, CT172, and CT682 (pbpb). The cleaned primer-toprimer sequences were checked against the CT MLST database (mlstdb.bmc.uu.se).

78 Dutch CT Reference Laboratory 77 Non-monitorinig study variant identification The Dutch Chlamydia trachomatis Reference Laboratory functioned as an expertise center for questions on CT strain related issues. Variants identified by this approach were described separately. RESULTS Sample collection In the period we collected 3554 samples from the different participating centers (see table 1). Table 1: Collection of samples in the period Total C. trachomatis C. trachomatis Total: = C. trachomatis positive by NAAT, - = C. trachomatis negative by NAAT Surveillance: identification of a plasmid free variant In total we identified 7 plasmid free variants (negative for all three plasmid PCRs and positive for the chromosomal ompa target)) from which 5 were confirmed by an independent sample (see table 2). In the last 3 years, we did not identify any plasmid free variant. The variants were detected in both males and females and at different anatomical sites. 7 plasmid free variants correspond to an incidence of 0.20%. Six variants were detected in the CT positive samples provided by the different centers, and one variant was detected in the CT negative samples. This coincides with 86% (6/7) of the plasmid free samples being already detected and treated. It also reflects that in the CT negative samples collected 0.04% is potentially plasmid free (1/2531). In the total set of the CT positive sample study 1.5% of the samples were discrepant as compared to the original provided results. Table 2: Characteristics on the identified C. trachomatis plasmid free variants Year No Anatomical site/gender Confirmation on independent sample VS/F No P/M, A/F yes, yes U/M, VS/F, O/M, U/M yes, yes, no, yes VS= vaginal swab, P= proctum, A= anal, U= urine, O= oral, F= female, M= male

79 In the CT negative samples we detected 9 CT+ samples (0.36%). From one specimen (CT plasmid negative and CT chromosomal positive) additional material could be collected (male urine) which was used for MLST typing and a serovar E was detected. Chapter 4 78 Non-monitoring study variant identification Variant 1 A woman presented with a bubo in her right groin. CT PCR from pus of the abscess tested positive. Serum taken from the patient at the same date turned out to be strongly positive (OD>2.0) for anti- CT IgG antibodies (CT-IgG plus, Medac Diagnostika, Germany). PCR analyses of urine for Neisseria gonorrhoeae and CT were negative. Given the strong suspicion of Lymphogranuloma venereum (LGV), the pus sample was sent to the Dutch Chlamydia trachomatis Reference Laboratory (located at the Laboratory of Immunogenetics, VU Medical Center, Amsterdam, the Netherlands). This sample tested positive for LGV; additional epidemiological LGV genotyping resulted in subtype L2b. Therefore, the diagnosis of inguinal LGV was assessed and the patient was treated with doxycycline 100 mg twice daily orally for 3 weeks. During the 2 months following the start of the treatment with doxycycline, the initial bubo in the groin completely resolved without any sequelae up to the last visit. We showed, by real-time PCR, that the woman was infected with CT, serovariant L2b. This is the first reported case study of a female patient with bubonic LGV caused by serovariant L2b, which was probably contracted via her bisexual male partner. This case has been described previously in more detail (16). Variant 2 A patient (A) attended the STI outpatient clinic from the Municipal Health Center in Zeeland, the Netherlands in January Since December 2006 he had a LAT relation with a Swedish woman (B) living in Sweden. Before his LAT relationship he was tested for CT and found negative. Female B had an ex-partner (C) who notified her in June 2007 (over half a year after ending the relationship) that he had encountered a CT infection. Patient B did not undergo a STI test since she had an antibiotic course for a throat infection, which was also effective against CT as stated by her physician. Patient A had dysuria but tested negatively in July 2007 for CT and Neisseria gonorrhea and was thus not treated and complaints disappeared in a few months. In December 2007 his girlfriend (B) was tested for CT and HPV in a cervical cancer screening study and tested positive for CT, presenting asymptomatic for the infection. The samples of patient A who was tested in January 2008 without STI symptoms, who tested positive for CT and because his girlfriend B was from Sweden, were sent to the Dutch Chlamydia trachomatis Reference Laboratory for further testing. As a result the Swedish variant (swct), also called the new variant CT (nvct), was identified for the first time in the Netherlands. This case has been described previously in more detail in a Dutch scientific bulletin (17).

80 Dutch CT Reference Laboratory 79 DISCUSSION This is the first study in the Netherlands monitoring the emergence of potential CT variants. The incidence of plasmid free variants was very low, only 0.20%, based on analyses of 3554 samples from different geographical regions in the Netherlands during 2010 and Among the original CT negative provided samples only 1 plasmid free variant was detected, corresponding with 0.04% that could have been missed. Since the last 3 years, no plasmid free variants have been detected, it can be concluded that for now, based on the results, no action is needed nor is there a potential health care issue. Collaboration was established with the Dutch Chlamydia trachomatis Reference Laboratory leading to the identification of two other variants, the first Swedish CT variant in the Netherlands and the first case worldwide of a heterosexual woman with a bubonic infection with the LGV L2b strain. The participation rate of the different STI centers was adequate enough to obtain the required amount of samples ensuring the power of the study. The main reason why most plasmid free samples were detected by the original providers is that after the emergence of the Swedish variant most plasmid based CT detection assays have been transferred to a so-called dual assay, targeting both the plasmid and the chromosome of CT. Systems targeting chromosomal targets like ribosomaal 16S RNA based NAATS (Hologic) did not need to adapt their assays. The setting in which the first plasmid-free strain was detected, incorporated a system to monitor all their samples. That center detected only one additional sample in 2015, again emphasizing the limited spread and the fact that it is probably not an escape-variant to treatment. The number of discrepant samples is limited and falls within the sensitivity and specificity ranges of the NAAT assays used. From most samples with a plasmid free variant the amount of DNA and the quality was not enough for serovar determination. Except for one sample, from which it was known ahead based on additional testing (RNA target) in the original setting, an additional urine sample could be collected; this strain was typed as serovar E by MLST and ompa sequencing. This is also the most frequent identified plasmid free CT serovar found. For future research it would be of interest to sequence the obtained plasmid free strain to verify whether it shows differences at chromosomal level distinct from the other identified and genome sequences plasmid free serovar E strains. The presented case with the swct variant shows the importance of both monitoring the emergence of new CT variants, the importance of adapting diagnostic assays to identify clinically important variants (Roche and Abbott) and it shows the utmost importance of a good clinical intake of patients to assess upfront positivity for CT variants (17). Also

81 the identified first female worldwide with a bubonic infection with the LGV L2b strain, circulating among men having sex with men (MSM) predominantly, shows the successful reference task of the Laboratory (16). Since 2003, outbreaks of LGV have been reported in European countries, North America, and Australia. The current outbreak has been almost exclusively attributed to a new variant, designated L2b. Although urogenital cases of LGV have been described in the heterosexual population, the first report on a CT L2b infection in a female was in 2011 but this was a case of proctitis (18). Very recently it has been described that there is a chance in the molecular epidemiology of CT strains involved in an outbreak of rectal LGV in France during January April Until 2012, the CT L2b strain predominated, however, starting in 2013, most cases involved the L2 strain and in addition 4 genetic L2b ompa variants were identified. (19) Chapter 4 In conclusion, the number of CT plasmid free variants in the Dutch population is very low, and in general they are detected by the current NAAT assays used, meaning that there is no need for active intervention based on the results from this current study. Also the incidence of other variants was low. The current surveillance setting functions well; rare cases are presented to the Dutch Chlamydia trachomatis Reference Laboratory emphasizing the relevance of the founded Reference laboratory by the Centre for Infectious Disease Control from the National Institute for Public Health and the Environment (Bilthoven, the Netherlands). 80

82 Dutch CT Reference Laboratory 81 ACKNOWLEDGEMENTS Since 2009 Prof.dr. S.A. Morré is coordinating the Dutch Chlamydia trachomatis Reference Laboratory (DCTRL) on behalf of the RIVM thatis funding the work performed in the DCTRL. Since 2014 the DCTRL tasks are in part performed in collaboration with Prof. dr. Christian Hoebe. We would like to thank all persons from the STI clinics participating in the Chlamydia trachomatis variant screening by the Dutch Chlamydia trachomatis Reference Laboratory. Variant Analyses group: 1) MUMC Maastricht, Department of medical Microbiology, Maastricht University medical center (MUMC+): Christian Hoebe, Petra Wolffs and Judith Nelissen, 2) Infection Laboratory Groningen (now CERTE) and the Department of Medical Microbiology, Groningen, The Netherlands Antoine Beerens and Dirk Luijt, 3) Department of Infectious Diseases, Public Health ServFice (GGD) of Amsterdam: Arken Speksnijder, Sylvia Bruisten and Raisa Brunst, 4) Den Hague STD centre, Municipal Public Health Service Haaglanden, The Hague: David Spitaels, Saskia van Leeuwen-Voerman and Johan Mutsears, 5) LabMicTA, Laboratory for Microbiology and Public Health, Enschede: Adri van der Zanden and Maria de Boer, and 6) Municipal Public Health Service Rotterdam-Rijnmond and ErasmusMC, Rotterdam: Hannelore Götz and Willemien Zandijk. The authors thank dr. Joke Spaargaren for critical reading the manuscript.

83 REFERENCES Chapter Centers for Disease Control and Prevention. Sexually transmitted diseases treatment guidelines. Morb Mortal Wkly Rep 2002;51: Gaydos CA, Theodore M, Dalesio N, et al. Comparison of three nucleic acid amplification tests for detection of Chlamydia trachomatis in urine specimens. J Clin Microbiol 2004;42: Centers for Disease Control and Prevention. Sexually transmitted disease surveillance Atlanta, GA: Department of Health and Human Services, September, LaMontagne DS, Fenton KA, Randall S, et al. Establishing the National Chlamydia Screening Programme in England: results from the first full year of screening. Sex Transm Infect 2004;80: * Jensen JS, Bradshaw CS, Tabrizi SN, Fairley CK, Hamasuna R (2008) Clin Infect Dis 47: Chernesky M, Jang D, Luinstra K, et al. High analytical sensitivity and low rates of inhibition may contribute to detection of Chlamydia trachomatis in significantly more women by the APTIMA Combo 2 assay. J Clin Microbiol 2006;44: Van Der Pol B, Ferrero DV, Buck-Barrington L, et al. Multicenter evaluation of the BDProbeTec ET System for detection of Chlamydia trachomatis and Neisseria gonorrhoeae in urine specimens, female endocervical swabs, and male urethral swabs. J Clin Microbiol 2001;39: Ripa T, Nilsson P. A variant of Chlamydia trachomatis with deletion in cryptic plasmid: implications for use of PCR diagnostic tests. Euro Surveill. 2006;11: E , Comanducci M, Ricci S, Cevenini R, Ratti G Diversity of the Chlamydia trachomatis common plasmid in biovars with different pathogenicity. Plasmid 23: Kari L, Whitmire WM, Olivares-Zavaleta N, Goheen MM, Taylor LD, Carlson JH, Sturdevant GL, Lu C, Bakios LE, Randall LB, Parnell MJ, Zhong G, Caldwell HD A live-attenuated chlamydial vaccine protects against trachoma in nonhuman primates. J. Exp. Med. 208: Olivares-Zavaleta N, Whitmire W, Gardner D, Caldwell HD Immunization with the attenuated plasmidless Chlamydia trachomatis L2(25667R) strain provides partial protection in a murine model of female genitourinary tract infection. Vaccine 28: O Connell CM, Ingalls RR, Andrews CW, Jr, Scurlock AM, Darville T Plasmiddeficient Chlamydia muridarum fail to induce immune pathology and protect against oviduct disease. J. Immunol. 179: O Connell CM, AbdelRahman YM, Green E, Darville HK, Saira K, Smith B, Darville T, Scurlock AM, Meyer CR, Belland RJ Tolllike receptor 2 activation by Chlamydia trachomatis is plasmid dependent, and plasmid-responsive chromosomal loci are coordinately regulated in response to glucose limitation by C. trachomatis but not by C. muridarum. Infect. Immun. 79: Li Z, Chen D, Zhong Y, Wang S, Zhong G The chlamydial plasmid-encoded protein Pgp3 is secreted into the cytosol of Chlamydia infected cells. Infect. Immun. 76: Rockey DD Unraveling the basic biology and clinical significance of the chlamydial plasmid. J. Exp. Med. 208: Bom RJ, van der Helm JJ, Schim van der Loeff MF et al. Distinct transmission networks of Chlamydia trachomatis in men who have sex with men and heterosexual adults in Amsterdam, The Netherlands. PLoS One 2013;8:e Verweij SP, Ouburg S, de Vries H, Morré SA, van Ginkel CJ, Bos H, Sebens FW. The first case record of a female patient with bubonic lymphogranuloma venereum (LGV), serovariant L2b. Sex Transm Infect Aug;88(5):346-7.

84 Dutch CT Reference Laboratory E. van Dijk, L. Meijaard, S.A. Morré, P.H.M. Savelkoul, A. Catsburg. Zweedse Chlamydia trachomatis-variant in Zeeland (The Netherlands). INFECTIEZIEKTEN Bulletin, 2008 (2); 19: Peuchant O, Baldit C, Le Roy C, Trombert-Paolantoni S, Clerc M, Bébéar C, de Barbeyrac B. First case of Chlamydia trachomatis L2b proctitis in a woman. Clin Microbiol Infect Dec;17(12):E Peuchant O, Touati A, Sperandio C, Hénin N, Laurier-Nadalié C, Bébéar C, de Barbeyrac B. Changing Pattern of Chlamydia trachomatis Strains in Lymphogranuloma Venereum Outbreak, France, Emerg Infect Dis Nov;22(11):

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86 CHAPTER 5 Serovar D and E of serogroup B induce highest serological responses in urogenital Chlamydia trachomatis infections E. Lanjouw *, S.P. Verweij *, C.J. Bax, K.D. Quint, P.M. Oostvogel, P.J. Dörr,J. Pleijster, H.J.C. de Vries, R.P.H. Peters, S. Ouburg, and S.A. Morré BMC Infectious Diseases :3 * Contributed equally

87 ABSTRACT Background Chlamydia trachomatis is the most prevalent bacterial sexually transmitted infection (STI) worldwide. A strong link between C. trachomatis serogroup/serovar and serological response has been suggested in a previous preliminary study. The aim of the current study was to confirm and strengthen those findings about serological IgG responses in relation to C. trachomatis serogroups and serovars. Methods The study population (n=718) consisted of two patient groups with similar characteristics of Dutch STI clinic visitors. We performed genotyping of serovars and used titre based and quantitative commercially available ELISA kits (medac Diagnostika) to determine specific serum IgG levels. Optical density (OD) values generated by both tests were used to calculate the IgG titres (cut-off 1:50). Analyses were conducted stratified by gender. Chapter 5 86 Results We observed very significant differences when comparing the median IgG titres of three serogroups, B, C and I: in women for B vs. C: p < (median titres B 200 vs. C <50); B vs. I: p < (200 vs. 50), and in men for B vs. C: p = (150 vs. <50); B vs. I: p = (150 vs. <50); C vs. I was not significant for both sexes. Serovars D and E of serogroup B had the highest median IgG titres compared to the other serovars in both men and women: 200 and 200 vs. 100 for women and 100 and 200 vs. 75 for men, respectively. Conclusions This study shows that B group serovars induce higher serological responses compared to the C and I group serovars in vivo in both men and women.

88 Serological responses in CT infections 87 BACKGROUND Chlamydia trachomatis is the most prevalent bacterial sexually transmitted infection (STI) worldwide. The clinical course of infection is frequently asymptomatic resulting in patients not seeking treatment. Untreated urogenital C. trachomatis infections may give rise to late complications, including pelvic inflammatory disease, ectopic pregnancy, and sub- or infertility (1). Assessment of serological responses to the bacterium resulted in a classification in serovars (2). Nowadays, 19 different serovars are known, usually causing conjunctival and urogenital infections. The serovars are subdivided in three serogroups based on phylogenetic comparisons of the ompa gene. Serogroup B contains serovars B, Ba, D, Da, E, L1, L2, L2a; serogroup C contains: A, C, H, I, Ia, J, K, L3; serogroup I contains: F, G, Ga (3,4). Identification and typing of serovars is currently done by rapid PCR-based techniques; (5-7). Geographical distribution of the serogroups is very similar worldwide on a national level, but there are variations in distribution within cities (8,9). The most prevalent serovars worldwide are D, E, and F (10,11). Several studies have shown associations between urogenital serovars and severity of clinical symptoms and the course of C. trachomatis infections (11,12), although conflicting data have also been reported (13,14). It has been shown that serovar K is associated with abnormal vaginal discharge, and serovar Ga is associated with dysuria in men (12). Van Duynhoven et al. (11) reported that infections with serovars D/D, H, and K appeared to be associated with inflammation, as shown by the presence of 10 or more leukocytes per microscopic field (magnification, x400). This was determined in first-void urine samples from men and in gram-stained cervical smears from women. However, this was not statistically significant. A comparison study of serovars D and H in a murine model shows that serovar H elementary bodies (EB) are more cytotoxic than EBs of serovar D, but the serovar D EBs have a longer duration of infection (15). In all studies however, the B group serovars are most prevalent and the C group serovars least common, suggesting different immunological responses by the host to serovars/serogroups. This may be of clinical interest since an association was observed between higher antibody responses and severity of tubal pathology (16). In a previous study (17) we measured immunoglobulin (Ig) G titres in 235 C. trachomatis infected patients. For these measurements we used a quantitative ELISA kit developed by medac Diagnostika. We observed that the most prevalent serogroup B induced significantly higher serological IgG responses than serogroups C and I (17). These results suggest a relation between serovars and the serological responses in human and potentially offer a partial explanation of the geographical prevalence of specific serovars. These results, however, have not been verified in different patient populations, nor have they been confirmed using other serological tests. The aim of this study was to confirm and expand our previous study concerning

89 serological IgG responses in relation to C. trachomatis serogroups and serovars. We used a study population consisting of two different patient groups and for our experiments we made use of titre-based and quantitative serological ELISA assays. We explored whether or not the most prevalent serovars (D, E, and F) induced the highest serological responses, in addition to analysing at serogroup level only as has been done previously (17). METHODS Chapter 5 88 Patient populations and C. trachomatis detection The study included samples from 718 C. trachomatis infected patients, derived from two different patient groups. The first group (Study 1) was obtained from 357 microbiologically confirmed C. trachomatis patients (as determined by PACE2 assay, Gen Probe) obtained from the STI outpatient clinic (n = 312) and the department of obstetrics and gynaecology (n = 45) in The Hague, the Netherlands. The samples include cervical and/or vaginal swabs, urethral swabs, and urine specimens. Serum samples were collected from all patients. These samples were stored in the period January to October Reasons for patients visiting either the STI clinic or gynaecology department and the collection of demographic data are described elsewhere (17). To prevent possible bias in serological responses we excluded patients with a multiple site infection or mixed infection, and patients with an infection other than the urethral tract or cervicovaginal tract (n = 70). A total number of 287 patients (149 women and 138 men) were used for analyses, of which 82 were unique compared to the previous study (17). The second patient group (Study 2) included 361 C. trachomatis infected women (as determined by COBAS AMPLICOR, Hoffman La Roche, Basel, Switzerland). Characteristics are described elsewhere (18). In short, cervical swabs were taken and serum was collected from women attending an STI outpatient clinic in Amsterdam, the Netherlands. Demographical and clinical data were available and informed consent was obtained (18). Ethnicity in both patient groups was self-reported, which has been shown a highly valid and representative marker of ethnicity in this context (19). Figure 1 shows a flowchart summarising the in- and exclusion criteria and the groups analysed. Ethical approval The Medical Research Involving Human Subjects Act (WMO, Dutch Law) stating official approval of the study by the Medical Ethical Committee does not apply to our anonymous human material collected (MEC Letter reference: # ) C. trachomatis genotyping Serogroup and serovar determination for Study 1 was performed with the C. trachomatis- DT assay (Labo Biomedical Products BV, Rijswijk, the Netherlands) as described

90 Serological responses in CT infections 89 elsewhere (8). Shortly, DNA was extracted from the swabs and urine samples to detect C. trachomatis for confirmation of the PACE2 assay. After confirmation, genotyping was performed using a reverse hybridization probe line blot with a probe for the cryptic plasmid and a probe for the three serogroups and all serovars. C. trachomatis genotyping in Study 2 was performed by means of PCR based RFLP analyses as described previously or, for the second half by sequencing the VD2 region of the ompa gene (20). C. trachomatis serology Study 1: determination of C. trachomatis IgG levels in serum of all patients was done by pelisa (Chlamydia trachomatis-igg-elisa plus kit (medac Diagnostika, Germany)) and was performed according to the manufacturer s protocols. This is an assay generating quantitative results (Arbitrary Units/ml), based on OD values. These OD values can also be used to calculate the titres. For this study, we calculated the titres to make the Figure 1. The figure shows a flowchart summarising the in- and exclusion criteria and the groups analysed. *: reference [17]. NAAT 1: PACE2 assay. NAAT 2: COBAS AMPLICOR. Typing 1: C. trachomatis-dt assay. Typing 2: sequencing VD2.

91 results in accordance with the results of Study 2. Cut-off levels for negative, grey zone, and positive C. trachomatis IgG serology were used as described by the manufacturer (cut-off 1:50). Study 2: determination of IgG levels was done previously by Chlamydia trachomatis-igg- ELISA (medac Diagnostika, Germany) according to the manufacturer s protocol. Titres > 1:50 were considered positive. Similar titre calculations for both Medac assays were performed as described by the manufacturer s instructions. Statistical analyses Since different antibody levels against C. trachomatis have been described in men and women (21), we analysed men and women separately, except when stated differently. Mann-Whitney U tests were used to compare medium titres between the serogroups (B vs. C; B vs. I; C vs. I). Mann-Whitney U tests were used to compare medium titres of serovars within serogrous. Analyses were performed using Prof. Avery s utest program (22). P values < 0.05 were considered statistically significant. Chapter 5 90 RESULTS Characteristics and serovar distribution between the two patient groups (Study 1 and 2) were compared. We compared serological distribution of the serogroups and serovars, and we compared age between the two individual patient groups. We did not observe any significant differences in distribution of serological titres between both patient groups in total, or between ethnic subgroups (general distribution: 57.3% Dutch Caucasian; 8.0% Surinam; 8.5% Dutch Antilles; 3.8% North African; 2.4% western European (non Dutch); 2.0% eastern European; 18.0% other nationalities). We observed no differences in mean age and distribution of the serological titres per serovar and serogroup between both patient groups in total, or between the ethnic subgroups. Therefore we combined both groups and analysed them as one. Serogroup analyses The serological titres per serogroup and per serovar are shown in table 1 and figure 2 for both men and women. Serogroup B had significantly higher median IgG titres compared to the C and I serogroups analysed in both groups. Female group: B vs. C: p < (p: 2.0x10-6 ; 200 vs. <50); B vs. I: p < (p: 2.0x10-6 ; 200 vs. 50); male group: B vs. C: p = (150 vs. <50); B vs. I: p = (150 vs. <50). No significant differences were observed between serogroups C and I. For women C vs. I: p = 0.14 (<50 vs. 50), for men C vs. I: p = 0.65 (<50 vs. <50).

92 Serological responses in CT infections 91 Table 1. Median serological IgG titres in men and women subdivided by serogroup and serovar Women (n = 510) Serogroup n % Median Range Serovar n % Median Range titre IgG titre IgG B * <50 D/Da/D < E < C <50 < H < I/Ia/I <50 < J <50 < K < I < F < G/Ga < Men (n = 138) Serogroup n % Median Range Serovar n % Median Range titre IgG titre IgG B < D/Da/D < E < C <50 < H n/a n/a I/Ia/I <50 50 J <50 < K < I <50 < F G/Ga <50 < *: B-group significantly different from C and I group (p < and p < ) respectively. : B-group significantly different from C and I group (p = and p = ) respectively. Serovar analyses Table 1 shows the median IgG titres per serovar for both women and men. At serovar level in the female group, serovars D and E demonstrated the highest median IgG, followed by serovars K, H, G, and F. Serovars J and I showed the lowest titres. In the Dutch male group serovars D and E induced the highest median IgG titres, followed by serovar G, K, F, J, and I. Serovar H was not present in this group. In-group analyses Within the serogroups, no difference was observed between serovars D and E in both men and women. For females, median IgG titres in serovar J infected patients were significantly lower compared to serovar K infected patients (p = 0.029; <50 vs. 100). No differences were observed between the other serovars of group C. In serogroup I (serovars F and G) no significant differences were observed in median IgG titres. No differences in serological responses were observed within the serogroups for the male group.

93 DISCUSSION This study shows that the most prevalent urogenital C. trachomatis serogroup B causes the highest serological IgG responses. More specifically, both serovars D and E of the B serogroup show higher IgG serological responses than the other serovars. Within serogroup C of the combined Dutch female patient group, we found a small difference between serovar K and J. The results generated in this study may be of clinical interest since an association was observed between antibody response and severity of tubal pathology in an earlier study (16). We observed higher serological responses in women compared to men for all serovars, but this did not reach statistic al significance. Eighty-two samples from Study 1were unique compared to the previous study after exclusion. These samples showed similar results as compared to the earlier samples (17). Compared to our previous study (17) of 235 patients, we increased our total study group to 648 patients and differentiated between men and women and confirmed our preliminary results. Both of our studies were P = P < Chapter 5 P = P < B C I B C I Figure 2. Median titres for three serogroups in men and women. The figure shows the median titres for three serogroups in men and women, depicted as a boxplot. The upper and lower limit of the box represents the first and third quartile. The cross represents the median of the group. The upper and lower limit of the range is presented as horizontal bars on a vertical axis. The large range between upper and lower limits are mostly caused by single samples that might be considered outliers. The distribution of the titres is bet represented by the medians, and the first and third quartile limits.

94 Serological responses in CT infections 93 performed with the medac ELISA assays. Interestingly, we observed similar results in a group of C. trachomatis infected Danish women (n = 91) using a different C. trachomatisspecific serological IgG test (SeroCT IgG ELISA; Savyon Diagnostics Ltd., Ashdod, Israel) (23); serogroup B serovars induced the highest serological response compared to the other serovars (unpublished data). In general, serological C. trachomatis IgG assays perform equally in sensitivity and specificity for these STI cohorts as we have previously shown (23). Recently a potentially new target for C. trachomatis serology has been evaluated, but this assay is not yet commercially available (24). This pgp3 assay showed highest relevance for titre positivity over time as compared to other commercially available tests. In our study population no follow-up samples were obtained. It would be of interest to compare the pgp3 assay with our used medac assay in the future since about 30% of the samples had C. trachomatis DNA present without a detectable serological response. Current detection methods for C. trachomatis are based on PCR techniques for DNA or RNA amplification. We used two different methods for C. trachomatis detection in this study and combined the results. However, the sensitivities and specificities of the current detection methods are very comparable, so no potential bias was introduced (25). We also used two different serovar determination techniques. Quint et al. compared the C. trachomatis-dt assay to sequencing of the VD2 region for C. trachomatis genotyping. They found equal performances for both methods, so no potential bias was introduced in this study by making use of two different genotyping methods (26). To gain more knowledge about differences in serological IgG responses between serovars in humans, studies in murine models and at cellular level may give more insight in this biological mechanism. Ito et al. (27) suggested in an earlier study that high serological responses in mice induced by the B serogroup (D and E) may be caused by longer antigenic stimulation, implicating that these high responses just reflect duration of infection. Lyons et al. (15) compared the cytotoxicity between the serovars D and H. They found that serovar H is much more cytotoxic than serovar D. Results of these studies suggested that serovars D and E (of the B serogroup) induce higher serological IgG responses in mice and have a longer duration than the C and I serogroup serovars. In contrast, serovar H is more cytotoxic than serovar D, but induces a lower IgG titre in the murine model. Eckert et al. (28) studied median IFU counts, a measure for virulence, in McCoy cells inoculated with C. trachomatis infected human urethral and cervical specimens. They observed that serovars B, D, and E induced the highest amount of IFU, and serovars I and J induced the smallest amount of IFU. This is a similar trend to what we find with regard to the serological responses in our study population. By using a large group of patients, this study confirmed our previous results regarding serological responses and based on the size of the study population we were also able to link back to the serovar level (17). Our results bring us one step closer to understanding the immunological response against a C. trachomatis infection at both serogroup and

95 serovar level, and the variation in serological responses between women and men. From a clinical point of view it would be interesting to identify a relation between serological responses and C. trachomatis serovars/serogroups. However, none of the limited human studies performed on C. trachomatis serovars and the clinical course of infection included serological responses, making it currently impossible to translate our finding into a direct application for the clinic (29-31). The knowledge generated in this study may aid in the development of a vaccine against C. trachomatis infection for which immunological responses to specific serovars linked to the natural duration of infection might be useful. CONCLUSIONS In conclusion, this study shows that serogroup B induces the highest serological responses compared to the other serogroups. More specifically, serovars D and E show higher serological responses than the other serovars. Chapter 5 ACKNOWLEDGEMENTS This work was in line with the European Commission within the Sixth Framework Programme through the EpiGenChlamydia project (contract no. LSHG-CT ). See for more details. 94

96 Serological responses in CT infections 95 REFERENCE LIST 1. Morré SA, Spaargaren J, Ossewaarde JM, Land JA, Bax CJ, Dorr PJ, Oostvogel PM, Vanrompay D, Savelkoul PH, Pannekoek Y et al.: Description of the ICTI consortium: an integrated approach to the study of Chlamydia trachomatis infection. Drugs Today (Barc ) 2006, 42 Suppl A: Brunelle 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: Dean D, Millman K: Molecular and mutation trends analyses of omp1 alleles for serovar E of Chlamydia trachomatis. Implications for the immunopathogenesis of disease. J Clin Invest 1997, 99: Morré SA, Ossewaarde JM, Lan J, van Doornum GJ, Walboomers JM, MacLaren DM, Meijer CJ, van den Brule AJ: 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: Meijer A, Morré SA, van den Brule AJ, Savelkoul PH, Ossewaarde JM: Genomic relatedness of Chlamydia isolates determined by amplified fragment length polymorphism analysis. J Bacteriol 1999, 181: Morré SA, Moes R, van Valkengoed IG, Boeke JP, van Eijk JT, Meijer CJ, van den Brule AJ: Genotyping of Chlamydia trachomatis in urine specimens will facilitate large epidemiological studies. J Clin Microbiol 1998, 36: Molano M, Meijer CJ, Morré SA, Pol R, van den Brule AJ: Combination of PCR targeting the VD2 of omp1 and reverse line blot analysis for typing of urogenital Chlamydia trachomatis serovars in cervical scrape specimens. J Clin Microbiol 2004, 42: Quint KD, van Doorn LJ, Kleter B, de Koning MN, van den Munckhof HA, Morré SA, ter HB, Weiderpass E, Harbers G, Melchers WJ 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: White JA: Manifestations and management of lymphogranuloma venereum. Curr Opin Infect Dis 2009, 22: Byrne GI: Chlamydia trachomatis strains and virulence: rethinking links to infection prevalence and disease severity. J Infect Dis 2010, 201 Suppl 2:S126-S van Duynhoven YT, Ossewaarde JM, Derksen-Nawrocki RP, van der Meijden WI, van de Laar MJ: Chlamydia trachomatis genotypes: correlation with clinical manifestations of infection and patients characteristics. Clin Infect Dis 1998, 26: Morré SA, Rozendaal L, van Valkengoed IG, Boeke AJ, van Voorst Vader PC, Schirm J, de Blok S, van den Hoek JA, van Doornum GJ, Meijer CJ et al.: Urogenital Chlamydia trachomatis serovars in men and women with a symptomatic or asymptomatic infection: an association with clinical manifestations? J Clin Microbiol 2000, 38: 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 urogenital tract. J Infect Dis 1995, 172: Workowski KA, Stevens CE, Suchland RJ, Holmes KK, Eschenbach DA, Pettinger MB, Stamm WE: Clinical manifestations of genital infection due to Chlamydia trachomatis in women: differences related to serovar. Clin Infect Dis 1994, 19: Lyons JM, Ito JI, Jr., Pena AS, Morré SA: 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: Akande VA, Hunt LP, Cahill DJ, Caul EO, Ford WC, Jenkins JM: Tubal damage in infertile women: prediction using chlamydia serology. Hum Reprod 2003, 18: Verweij SP, Bax CJ, Quint KD, Quint WG, van Leeuwen AP, Peters RP, Oostvogel PM, Mutsaers

97 Chapter 5 96 JA, Dorr PJ, Pleijster J et al.: Significantly higher serologic responses of Chlamydia trachomatis B group serovars versus C and I serogroups. Drugs Today (Barc ) 2009, 45 Suppl B: Ouburg S, Spaargaren J, den Hartog JE, Land JA, Fennema JS, Pleijster J, Pena AS, Morré SA: The CD14 functional gene polymorphism -260 C>T is not involved in either the susceptibility to Chlamydia trachomatis infection or the development of tubal pathology. BMC Infect Dis 2005, 5: Haasnoot A, Koedijk FD, Op de Coul ELM, Gotz HM, van der Sande M.A., VAN DEN Broek IV: Comparing two definitions of ethnicity for identifying young persons at risk for chlamydia. Epidemiol Infect 2012, 140: Spaargaren J, Verhaest I, Mooij S, Smit C, Fennema HS, Coutinho RA, Salvador PA, Morré SA: Analysis of Chlamydia trachomatis serovar distribution changes in the Netherlands ( ). Sex Transm Infect 2004, 80: Mardh P-A, Paavonen J, Puolakkainen M: Chlamydia. New York: Plenum Medical Book Company; Mann-Whitney U Test [ 23. Morré SA, Munk C, Persson K, Kruger-Kjaer S, van Dijk R, Meijer CJ, van den Brule AJ: 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: Horner PJ, Wills GS, Reynolds R, Johnson AM, Muir DA, Winston A, Broadbent AJ, Parker D, McClure MO: Effect of time since exposure to Chlamydia trachomatis on chlamydia antibody detection in women: a cross-sectional study. Sex Transm Infect 2013, 89: Land JA, van Bergen JE, Morre SA, Postma MJ: Epidemiology of Chlamydia trachomatis infection in women and the cost-effectiveness of screening. Hum Reprod Update 2010, 16: Quint KD, Bom RJ, Bruisten SM, van Doorn LJ, Nassir HN, Melchers WJ, de Vries HJ, Morre SA, Quint WG: Comparison of three genotyping methods to identify Chlamydia trachomatis genotypes in positive men and women. Mol Cell Probes 2010, 24: 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: Eckert LO, Suchland RJ, Hawes SE, Stamm WE: Quantitative Chlamydia trachomatis cultures: correlation of chlamydial inclusion-forming units with serovar, age, sex, and race. J Infect Dis 2000, 182: Morré SA, van den Brule AJ, Rozendaal L, Boeke AJ, Voorhorst FJ, de Blok S., Meijer CJ: 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: Molano M, Meijer CJ, Weiderpass E, Arslan A, Posso H, Franceschi S, Ronderos M, Munoz N, van den Brule AJ: The natural course of Chlamydia trachomatis infection in asymptomatic Colombian women: a 5-year follow-up study. J Infect Dis 2005, 191: Dean D, Suchland RJ, Stamm WE: Evidence for long-term cervical persistence of Chlamydia trachomatis by omp1 genotyping. J Infect Dis 2000, 182:

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100 CHAPTER 6 Functional polymorphisms in vitamin D metabolism genes are not associated with susceptibility to Chlamydia trachomatis infection E. Lanjouw*, I. Branković*, S. Ouburg, J. Pleijster, J. Spaargaren, C.J. Hoebe, S.A. Morré Pathogens and Disease, 2016 Apr;74(3) *Contributed equally

101 ABSTRACT Chapter Chlamydia trachomatis (CT) is the most common sexually transmitted bacterium worldwide. Its often asymptomatic course of infection increases chances of transmission, but also increases risk of late complications, including tubal pathology. Genetic variations in the host immune system are known to impact the course of infections, including that of CT. Recent studies have shown the impact of vitamin D on the regulation of the immune system. In a recent mouse model it was shown that vitamin D knockout mice had a more prolonged and severe urogenital CT infection. This indicates a potential role for the vitamin D pathway in CT infections. In this study we assessed the impact of eight polymorphisms in five genes (VDR (rs G>A, rs C>T), CYP27B1 (rs G>T), DHCR7 (rs G>A, rs G>A), GC (rs ), and CYP2R1 (rs G>A, rs G>A)) on susceptibility to CT infections. In our cohort of 67 CTpositive and 507 CTnegative women we did not observe statistically significant differences between the genotype distributions of the four polymorphisms. This suggests that VDR, CYP27B1, DHCR7, GC, and CYP2R1 do not play a major role in susceptibility to CT infections. However, due to its pleiotropic nature in the immune system a role for the vitamin D pathway may not be excluded from the whole clinical course of CT infections (e.g. late complications), and further research is therefore required.

102 Vitamin D polymorphisms and susceptibility to CT infections 101 INTRODUCTION Chlamydia trachomatis (CT) infects approximately 100 million people each year and is therefore the most prevalent bacterial sexually transmitted infection in the world (1). The infection is often asymptomatic and frequently remains untreated due to late diagnosis, thereby resulting in late complications, such as ectopic pregnancy and tubal factor infertility (TFI)(2). The clinical course of a CT infection varies greatly between individuals, and is in part mediated by diverse host immunogenetic factors (3-8). These factors appear at this point to be the most promising biological indicators of complicated infection with CT (9-12). Investigations of the role of the active form of vitamin D (1,25-(OH)2D3) within different types of cells of the immune system attest to its immunomodulatory properties (13-16). Aside from its well-established roles in calcium homeostasis and regulation of bone density, interactions of vitamin D with the immune system have been detected several decades ago. However, it was only in recent years that the precise nature of these interactions became clearer (15, 17). All types of immune cells can respond to the stimulation by 1,25-(OH)2D3. In monocytes and macrophages the synthesis of vitamin D active form from its circulating form by 25-hydroxyvitamin-D-1α-hydroxylase (also referred to as 1-OHase, encoded by CYP27B1) is regulated via mechanisms entirely independent of calcium metabolism (13). In that respect, we can distinguish two distinct groups of functions of vitamin D: one as a circulating hormone with skeletal homeostasis as its primary role; and the other one akin to a cytokine with localised production and activity (18). A growing body of studies from recent years has been published on the nature of its functions as the regulator of the immune system in responses to infections. Exposure to Mycobacterium tuberculosis affects local expression of genes within vitamin D biopathway (19, 20). The initial event involves sensing of specific bacterial components by pattern-recognition-receptors (PRRs), such as Toll-like receptors (TLRs). This event triggers changes in the local vitamin D metabolism in these cells, upregulating the expression of the aforementioned 1-OHase, but also of vitamin D receptor (VDR)(17, 19, 20). Specifically, the activation of the TLR2/1 heterodimer leads to the induction of 1-OHase and VDR, which in turn upregulate antimicrobial peptides cathelicidins (19). It has been shown that VDR, upon activation by 1,25-(OH)2D3, acts as a transcription factor for genes for cathelicidins and other host defence peptides, such as defensins (3). Moreover, the role of the active form of vitamin D in regulating the expression of cathelicidin is not restricted to monocytes and macrophages, but also epithelial cells on barrier surfaces such as mucosa, skin and the gut (20). Treating cell cultures with vitamin D s active form leads to upregulation of cathelicidins and defensins in response to Pseudomonas aeruginosa and Escherichia coli (3, 21). These peptides were also demonstrably bactericidal when applied in vitro to CT (22-25), hence we assume that depletion of antimicrobial peptides in response to insufficient levels

103 Chapter of vitamin D s active form could potentially lead to less effective eradication of this pathogen. Other notable functions of vitamin D in antimicrobial responses involve promotion of autophagy. He and colleagues (26) observed in their murine model study that genital chlamydial infection was more severe and prolonged in VDR-/- knock-out mice compared to VDR+/+ mice. Inflammatory response also lasted longer in the knock-out mice, which was likely due to the observed down-regulation of leukocyte elastase inhibitor (LEI), an anti-inflammatory protein. Lack of vitamin D may therefore lead to heightened risk of developing an infection due to the compromised antibacterial actions of the immune system and barrier tissues (15). In recent years, a re-evaluation of what should be considered by vitamin D deficiency is taking place. Instead, the concept of suboptimal vitamin D levels is now being commonly distinguished. Suboptimal levels are not associated with deficiency-related diseases such as rickets, but nevertheless exert their negative effect on health. Therefore, it is not only deficiency, but insufficiency that is now considered to negatively affect the response to diseases (15). As a great proportion of the global population appears to have insufficient serum levels of vitamin D (17, 27) this undoubtedly raises various issues from the perspective of public health and prevention. If the circulating form plasma levels are too low, as is the case in vitamin D insufficiency, 1-OHase in monocytes and macrophages lacks substrate for synthesising the active form. The outcome is insufficient levels of 1,25-(OH)2D3, resulting in less binding to and activation of VDR. Hence, the VDR-regulated expression of antimicrobial peptide genes would be diminished, leading to a less effective defence against the pathogen (18). The availability of the circulating form, however, may also depend on the functionality of enzymes and transport molecules constituting the vitamin D metabolic pathway. Furthermore, the conversion into the active form and the rate of reverse conversion into the circulating form can also modulate the amount and activity of vitamin D in target cells. Therefore, different polymorphisms in genes for conversion enzymes, transport molecules, and the VDR can affect host defence via the change in bioavailability of 1,25-(OH)2D3 and its activation of transcription-regulating functions of the VDR (28). Genetic differences in genes involved in biosynthesis, serum transport, and cellular conversion of vitamin D and its precursors and metabolites have been shown to influence bioavailability of vitamin D in humans, as well as health status for various infections (29-32). To date, no studies examining polymorphisms within genes of the vitamin D biopathway and CT have been published. Therefore, we conduct an analysis of polymorphisms with reported functional effect on vitamin D metabolism. We investigated whether there were differences in susceptibility to urogenital CT infection in humans, based on polymorphisms within the following genes of the vitamin D pathway: VDR, CYP2R1, DHCR7, GC, and CYP27B1. Their function is briefly outlined in Table 1.

104 Vitamin D polymorphisms and susceptibility to CT infections 103 Table 1. Genes in the vitamin D biopathway researched in this study. Gene Protein Function CYP2R1 vitamin D 25-hydroxylase Converts the pre-vitamin D (synthesised in the skin) into its main circulating form, 25-OHD 3, in the liver. CYP27B1 25-hydroxyvitamin D 3 1-alpha hydroxylase (1-OHase) Catalyses the hydroxylation of 25-OHD 3 (circulating form) to 1,25-(OH) 2D 3 (bioactive form of Vitamin D) VDR vitamin D receptor Upon activation by vitamin D active form, binds to retinoid-x receptor (RXR-α) and acts as a transcription factor for vitamin D response element genes GC Group specific Component Vitamin D binding protein and precursor to macrophage-activating factor DHCR7 7-DeHydroCholesterol Reductase Regulates vitamin D by regulating it s precursor 7- dehydrocholesterol METHODS Study population In the study, we used samples obtained from a STD outpatient clinic in South Limburg. We selected Dutch Caucasian women for this study and all participating women were between years of age. All women were asked to sign an informed consent. CTstatus was assessed by the outpatient clinic using the available NAAT assay. The Medical Research Involving Human Subjects Act (WMO, Dutch Law) stated that official approval of the study by the Medical Ethical Committee does not apply to our anonymous human material collected (MEC Letter reference: # ). The local medical ethical committee also approved this study, based on the fact that in the Netherlands ethical approval is not required for a retrospective use of de-identified clinical samples. Nevertheless, since we performed host genetic marker studies in relation to Chlamydial infection, we made sure all participants signed informed consent forms. Selection of polymorphisms and laboratory analyses We selected polymorphisms for analysis based on the literature featured in Genetic Associations database, as well as PubMed searches using terms vitamin D, polymorphism OR SNP, and infection. Only polymorphisms with minor allele frequencies higher than 5% were considered (based on the data in NCBI SNP database, dbsnp). We included the SNPs from the following genes: VDR (rs G>A, rs C>T), CYP27B1 (rs G>T), DHCR7 (rs G>A, rs G>A), GC (rs ), and CYP2R1 (rs G>A, rs G>A). All polymorphisms were genotyped using Roche LightCycler 480 Real-Time PCR under standard conditions.

105 Analyses Groups were tested for Hardy Weinberg Equilibrium to assess Mendelian Inheritance. Fisher Exact and χ2 tests were used where appropriate. A p-value <0.05 was considered statistically significant. RESULTS The genotype frequencies of the eight studied SNPs were in Hardy Weinberg equilibrium. Differences in genotype distribution between CT positive and negative women as assessed by the presence / absence of CT DNA detected by real-time PCR were tested with Fisher Exact and/or χ2 tests, and are presented in table 2. We did not observe statistically significant differences in genotype distributions of these eight SNPs between CT positive and negative women. Furthermore we did not observe statistically significant differences in genotype distributions of these eight SNPs between CT symptomatic and asymtomatic women Chapter DISCUSSION We report on our case-control study in which we assessed potential associations between eight polymorphisms in the vitamin D biopathway genes (VDR (rs G>A, rs C>T), CYP27B1 (rs G>T), DHCR7 (rs G>A, rs G>A), GC (rs ), and CYP2R1 (rs G>A, rs G>A)) and susceptibility to urogenital CT infection. We did not detect any statistically significant associations of these polymorphisms with the infection. VDR encodes the intracellular vitamin D receptor which is involved in the regulation of transcription for a number of genes and can form dimers with RXR-α, including those genes whose products are involved in antimicrobial defense. The effects of mutant allele of rs may thus be modulated by particular variations in the gene encoding the RXR-α protein, given their potential for interaction and gene expression alterations. Successive studies should take into account potential interactions between polymorphisms in these two genes and investigate the nature of the interaction. Some near-trend values were observed, however these were lost when corrections for multiple testing were taken into account. These results may indicate a lack of statistical power. Carriertraits, or combinations of polymorphisms, may influence the susceptibility to CT infections, however due to lack of sufficient sample numbers, carriertrait analyses were not performed. Polymorphism rs is a the start codon polymorphism (33). Two protein variants can exist corresponding to the two available start sites: a long version of the VDR

106 Vitamin D polymorphisms and susceptibility to CT infections 105 Table 2. Overview of analysed polymorphisms with genotype frequencies for controls and cases Gene rs Genotype CT + CT - CT+ CT+ Symp Asymp n: 67 n: 507 n: 10 n: 57 VDR rs GG 29,9% 35,5% 40,0% 28,1% GA 61,2% 47,9% 40,0% 64,9% AA 9,0% 16,6% 20,0% 7,0% MAF 39,6% 40,5% 40,0% 39,5% VDR rs CC 43,3% 40,2% 50,0% 42,1% CT 41,8% 45,6% 40,0% 42,1% TT 14,9% 14,2% 10,0% 15,8% MAF 35,8% 37,0% 30,0% 36,8% CYP27B1 rs GG 49,3% 47,1% 30,0% 52,6% GT 43,3% 41,8% 50,0% 42,1% TT 7,5% 11,0% 20,0% 5,3% MAF 29,1% 32,0% 45,0% 26,3% DHCR7 rs GG 55,2% 56,0% 60,0% 54,4% GA 40,3% 36,7% 30,0% 42,1% AA 4,5% 7,3% 10,0% 3,5% MAF 24,6% 25,6% 25,0% 24,6% DHCR7 rs GG 73,1% 71,8% 80,0% 71,9% GA 25,4% 25,8% 10,0% 28,1% AA 1,5% 2,4% 10,0% 0,0% MAF 14,2% 15,3% 15,0% 14,0% CYP2R1 rs GG 44,8% 42,0% 50,0% 43,9% GA 44,8% 43,0% 40,0% 45,6% AA 10,4% 15,0% 10,0% 10,5% MAF 32,8% 36,5% 30,0% 33,3% CYP2R1 rs GG 44,8% 42,0% 50,0% 43,9% GA 44,8% 43,0% 40,0% 45,6% AA 10,4% 15,0% 10,0% 10,5% MAF 32,8% 36,5% 30,0% 33,3% GC rs GG 43,3% 56,4% 30,0% 45,6% GA 50,7% 37,3% 50,0% 50,9% AA 6,0% 6,3% 20,0% 3,5% MAF 31,3% 25,0% 45,0% 28,9% (CT = CT; MAF = minor allele frequency; Symp = symptomatic; Asymp = asymptomatic). protein is the T-allele (minor allele), also referred to as the M1 form, i.e., the methionine at first position. A protein variant shortened by three amino acids is the C-allele (major), also referred to as the M4 form, i.e., the methionine at fourth position (34). Findings of other studies (35, 36) suggest a risk effect for the minor allele.. In human monocytes and dendritic cells with a homozygous short (CC) VDR genotype, expression of IL-12

107 (mrna and protein) was higher than in cells with a long (TT) VDR genotype (36). This polymorphism therefore affects immune cell behavior, with a more active immune system for the short version of VDR. Enzyme coded by CYP2R1 converts vitamin D into the circulating form, thereby enabling its transport to target cells where they can be converted into physiologically active (hormonal) form. Chapter Vitamin D polymorphisms and other diseases Our researched four polymorphisms have so far been investigated in regards to susceptibility for a small number of other infectious diseases, namely tuberculosis, acute lower respiratory infection (ALRI), and chronic hepatitis C (HCV) (30, 37, 38). VDR polymorphism rs was particularly researched. There is more evidence for their roles in non-communicable diseases, such as cancer, diabetes and autoimmune disorders (39-42). These susceptibilities were in cases of some diseases ethnicityspecific (40). The rs TT genotype was associated with the risk of ALRI that was approximately seven times that of the CC genotype (30). In the study of Wilkinson et al., (37) when tuberculosis patients were stratified according to localised or severe disease, there was a significant excess of patients with anatomically localised (extrapulmonary) tuberculosis bearing the T allele. Therefore, it appears that the minor (T) allele that confers the longer protein variant of VDR does lead to a heightened risk of these infections. Stimulation with bioactive vitamin D leads to decreased expression of MHC class II molecules and co-stimulators in B cells, as well as to decreased proliferation, differentiation, and cytokine production and immune response in T cells (43, 44). A theory is that the production of CYP27B1, as well as activation of vitamin D by T cells and dendritic cells in extralymphoid tissues, might increase the T-cell programming of antigen-specific response, the so-called T-cell homing (36). Overall, the findings support a suppressive role of vitamin D in the adaptive immune system (45). Low levels of vitamin D in serum are associated with a higher risk of developing MS (46), and several studies show that MS patients have lower levels of 25-OHD3 compared to controls (43). A recent study showed that high circulating 25-OHD3 levels among MS patients were associated with improved regulatory T-cell function. It also had an impact on T-helper (Th) cells, shifting the balance in favour of Th2 cells (43). Currently, the CYP27B1 rs promoter polymorphism is regarded to be more important than polymorphisms within VDR genes at least in the development of endocrine autoimmune disorders. Additionally, in chronic hepatitis C (HCV) patients with genotype AA of the 1-OHase promoter polymorphism, higher serum concentrations of 1,25-(OH)2D3 were observed compared to patients with genotype AC or CC (38). This was unexpected since other studies suggested that the CC genotype constitutes a pathogenic co-factor in immune dysfunction by impairing intracellular

108 Vitamin D polymorphisms and susceptibility to CT infections 107 1,25-(OH)2D3 levels in mononuclear cells and T cells (47, 48). The adverse genotype CC was more frequent in patients with chronic hepatitis C compared to healthy populations, which may be a hint that resolution of acute hepatitis C is supported by the beneficial genotype AA. 1,25-(OH)2D3 is the bioactive and, therefore, relevant form of vitamin D, which is a key player in innate and adaptive immunity. Active vitamin D has the potency to significantly enhance T cell receptor signalling, which might play an important role in establishing a sufficient adaptive immune response during acute hepatitis C or to overcome T cell exhaustion during chronic hepatitis C (36). It is possible that vitamin D-induced antibacterial peptides are not crucial for susceptibility to CT infection in humans the same way they appear to be in cases of several other diseases. Although their bactericidal capacity was demonstrated in several in vitro studies of CT infections (22-25), more studies are required to elucidate whether these peptides are effective against CT within host cells. Their effect might also vary depending on the stage of the chlamydial biological cycle. Aside from upregulating antibacterial peptides upon successful PRR sensing, vitamin D is required for IFN-γ-mediated antimicrobial functions in macrophages (49). By releasing IFN-γ, T cells enhance microbicidal capabilities of macrophages, stimulating antimicrobial peptide production, endolysosomal killing and autophagy. This effect is achieved through IL-15 induction by IFN-γ, which is also capable of up-regulating VDR and CYP27B1 (49). In that respect, vitamin D is a mediator of both the innate and adaptive immune response to pathogens. IFN-γ is an important cytokine in specific responses to CT and the clearance of the infection (50). It would be recommended for that reason to additionally examine polymorphisms in IFNG gene and perform trait analyses. In conclusion, our case-control study did not register any associations between functional polymorphisms in the vitamin D biocascade and susceptibility to CT infections. This does not exclude a potential role for the vitamin D pathway in the severity of CT infections (e.g. development of tubal pathology). Other studies point to a more complex relationship between circulating levels of vitamin D, its intracellular conversion rate into the active form, expression of vitamin D response genes, and their regulation of the innate and adaptive immunity. CT infection requires the involvement of different immune mechanisms, and vitamin D regulates these mechanisms in specific manners. It would be important to analyse how its effect on the adaptive immunity (especially IFN-γ-associated Th1 response) versus innate mechanisms is achieved and how it relates to urogenital CT infection. Further research is needed to elucidate these processes and explain how these actions exert their effect and whether there are disease-specific properties differ from their previously researched pleiotropic involvements in inflammatory conditions underlying for instance cancer, tissue injury, and atherosclerosis.

109 Acknowledgements We give our gratitude to Dr. Caroline Bax (formerly GGD Den Haag, The Hague, The Netherlands, currently VU University Medical Center, Amsterdam The Netherlands) for supplying the STD cohort also used in the study. Also, we wish to thank the collaborators at GGD Amsterdam, The Netherlands, for the cohort they provided for use in the study. Partial funding was obtained from, or the work was in line with, the following grants: Dutch NGI Life Sciences Pre-Seed Grant Reg.Nr ; USA NIH R21 Grant Prime award number 1R21AI with subaward number ( ) and the European EuroTransBio Grant, Reference number ETB. REFERENCES Chapter WHO. Global incidence and prevalence of selected curable sexually transmitted infections World Health Organization Mabey D. Epidemiology of sexually transmitted infections: worldwide. Medicine. 2014;42(6): Wang C, Tang J, Geisler WM, Crowley-Nowick PA, Wilson CM, Kaslow RA. Human Leukocyte Antigen and Cytokine Gene Variants as Predictors of Recurrent Chlamydia trachomatis Infection in High-Risk Adolescents. Journal of Infectious Diseases. 2005;191(7): den Hartog JE, Ouburg S, Land JA, Lyons JM, Ito JI, Peña AS, et al. Do host genetic traits in the bacterial sensing system play a role in the development of Chlamydia trachomatis-associated tubal pathology in subfertile women? BMC Infectious Diseases. 2006;6: Bailey RL, Natividad-Sancho A, Fowler A, Peeling RW, Mabey DC, Whittle HC, et al. Host genetic contribution to the cellular immune response to Chlamydia trachomatis: Heritability estimate from a Gambian twin study. Drugs Today (Barc). 2009;45 Suppl B: Jiang J, Karimi O, Ouburg S, Champion CI, Khurana A, Liu G, et al. Interruption of CXCL13- CXCR5 axis increases upper genital tract pathology and activation of NKT cells following chlamydial genital infection. PLoS One. 2012;7(11):e Al-Kuhlani M, Rothchild J, Pal S, de la Maza LM, Ouburg S, Morré SA, et al. TRAIL-R1 Is a Negative Regulator of Pro-Inflammatory Responses and Modulates Long-Term Sequelae Resulting from Chlamydia trachomatis Infections in Humans. PLoS ONE. 2014;9(4):e Morre SA, Karimi O, Ouburg S. Chlamydia trachomatis: identification of susceptibility markers for ocular and sexually transmitted infection by immunogenetics. FEMS Immunol Med Microbiol. 2009;55(2): Ouburg S, Lyons JM, Land JA, den Hartog JE, Fennema JS, de Vries HJ, et al. TLR9 KO mice, haplotypes and CPG indices in Chlamydia trachomatis infection. Drugs Today (Barc). 2009;45 Suppl B: Lal JA, Malogajski J, Verweij SP, de Boer P, Ambrosino E, Brand A, et al. Chlamydia trachomatis infections and subfertility: opportunities to translate host pathogen genomic data into public health. Public Health Genomics. 2013;16(1-2): Malogajski J, Brankovic I, Verweij SP, Ambrosino E, van Agtmael MA, Brand A, et al. Translational potential into health care of basic genomic and genetic findings for human immunodeficiency virus, Chlamydia trachomatis, and human papilloma virus. Biomed Res Int. 2013;2013: Brankovic I, Malogajski J, Morre SA. Biobanking and translation of human genetics and genomics for infectious diseases. Appl Transl Genom. 2014;3(2): Stoffels K, Overbergh L, Giulietti A, Verlinden L, Bouillon R, Mathieu C. Immune regulation of 25-hydroxyvitamin-D3-1alpha-hydroxylase in human monocytes. J Bone Miner Res. 2006;21(1):37-47.

110 Vitamin D polymorphisms and susceptibility to CT infections White JH. Vitamin D signaling, infectious diseases, and regulation of innate immunity. Infect Immun. 2008;76(9): Hewison M. Vitamin D and innate and adaptive immunity. Vitam Horm. 2011;86: Hewison M. An update on vitamin D and human immunity. Clin Endocrinol (Oxf). 2012;76(3): Holick MF. Vitamin D deficiency. N Engl J Med. 2007;357(3): Hewison M. Vitamin D and the immune system: new perspectives on an old theme. Endocrinol Metab Clin North Am. 2010;39(2):365-79, table of contents. 19. Liu PT, Stenger S, Li H, Wenzel L, Tan BH, Krutzik SR, et al. Toll-like receptor triggering of a vitamin D-mediated human antimicrobial response. Science. 2006;311(5768): Liu N, Nguyen L, Chun RF, Lagishetty V, Ren S, Wu S, et al. Altered endocrine and autocrine metabolism of vitamin D in a mouse model of gastrointestinal inflammation. Endocrinology. 2008;149(10): Hewison M. Antibacterial effects of vitamin D. Nat Rev Endocrinol. 2011;7(6): Yasin B, Harwig SS, Lehrer RI, Wagar EA. Susceptibility of Chlamydia trachomatis to protegrins and defensins. Infect Immun. 1996;64(3): Yasin B, Lehrer RI, Harwig SS, Wagar EA. Protegrins: structural requirements for inactivating elementary bodies of Chlamydia trachomatis. Infect Immun. 1996;64(11): Ramanathan B, Davis EG, Ross CR, Blecha F. Cathelicidins: microbicidal activity, mechanisms of action, and roles in innate immunity. Microbes Infect. 2002;4(3): Donati M, Di Francesco A, Di Paolo M, Fiani N, Benincasa M, Gennaro R, et al. Activity of Cathelicidin Peptides against Simkania negevensis. Int J Pept. 2011;2011: He Q, Ananaba GA, Patrickson J, Pitts S, Yi Y, Yan F, et al. Chlamydial infection in vitamin D receptor knockout mice is more intense and prolonged than in wild-type mice. J Steroid Biochem Mol Biol. 2013;135: Mithal A, Wahl DA, Bonjour JP, Burckhardt P, Dawson-Hughes B, Eisman JA, et al. Global vitamin D status and determinants of hypovitaminosis D. Osteoporos Int. 2009;20(11): Jurutka PW, Remus LS, Whitfield GK, Thompson PD, Hsieh JC, Zitzer H, et al. The polymorphic N terminus in human vitamin D receptor isoforms influences transcriptional activity by modulating interaction with transcription factor IIB. Mol Endocrinol. 2000;14(3): Hansdottir S, Monick MM, Hinde SL, Lovan N, Look DC, Hunninghake GW. Respiratory epithelial cells convert inactive vitamin D to its active form: potential effects on host defense. J Immunol. 2008;181(10): Roth DE, Jones AB, Prosser C, Robinson JL, Vohra S. Vitamin D receptor polymorphisms and the risk of acute lower respiratory tract infection in early childhood. J Infect Dis. 2008;197(5): Segaert S. Vitamin D regulation of cathelicidin in the skin: toward a renaissance of vitamin D in dermatology? J Invest Dermatol. 2008;128(4): Moodley A, Qin M, Singh KK, Spector SA. Vitamin D-related host genetic variants alter HIV disease progression in children. Pediatr Infect Dis J. 2013;32(11): Arai H, Miyamoto K, Taketani Y, Yamamoto H, Iemori Y, Morita K, et al. A vitamin D receptor gene polymorphism in the translation initiation codon: effect on protein activity and relation to bone mineral density in Japanese women. J Bone Miner Res. 1997;12(6): Uitterlinden AG, Fang Y, Van Meurs JB, Pols HA, Van Leeuwen JP. Genetics and biology of vitamin D receptor polymorphisms. Gene. 2004;338(2): Colin EM, Weel AE, Uitterlinden AG, Buurman CJ, Birkenhager JC, Pols HA, et al. Consequences of vitamin D receptor gene polymorphisms for growth inhibition of cultured human peripheral blood mononuclear cells by 1, 25-dihydroxyvitamin D3. Clin Endocrinol (Oxf). 2000;52(2): van Etten E, Verlinden L, Giulietti A, Ramos-Lopez E, Branisteanu DD, Ferreira GB, et al. The vitamin D receptor gene FokI polymorphism: functional impact on the immune system. Eur J Immunol. 2007;37(2):

111 Chapter Wilkinson RJ, Llewelyn M, Toossi Z, Patel P, Pasvol G, Lalvani A, et al. Influence of vitamin D deficiency and vitamin D receptor polymorphisms on tuberculosis among Gujarati Asians in west London: a case-control study. Lancet. 2000;355(9204): Lange CM, Bojunga J, Ramos-Lopez E, von Wagner M, Hassler A, Vermehren J, et al. Vitamin D deficiency and a CYP27B promoter polymorphism are associated with chronic hepatitis C and poor response to interferon-alfa based therapy. J Hepatol. 2011;54(5): Simmons JD, Mullighan C, Welsh KI, Jewell DP. Vitamin D receptor gene polymorphism: association with Crohn s disease susceptibility. Gut. 2000;47(2): Lee YH, Bae SC, Choi SJ, Ji JD, Song GG. Associations between vitamin D receptor polymorphisms and susceptibility to rheumatoid arthritis and systemic lupus erythematosus: a meta-analysis. Mol Biol Rep. 2011;38(6): Lee YH, Choi SJ, Ji JD, Song GG. Vitamin D receptor ApaI, TaqI, BsmI, and FokI polymorphisms and psoriasis susceptibility: a meta-analysis. Mol Biol Rep. 2012;39(6): Li C, Qi H, Wei S, Wang L, Fan X, Duan S, et al. Vitamin D receptor gene polymorphisms and the risk of Parkinson s disease. Neurol Sci. 2015;36(2): Smolders J, Thewissen M, Peelen E, Menheere P, Tervaert JW, Damoiseaux J, et al. Vitamin D status is positively correlated with regulatory T cell function in patients with multiple sclerosis. PLoS One. 2009;4(8):e Gorman S, Kuritzky LA, Judge MA, Dixon KM, McGlade JP, Mason RS, et al. Topically applied 1,25-dihydroxyvitamin D3 enhances the suppressive activity of CD4+CD25+ cells in the draining lymph nodes. J Immunol. 2007;179(9): Sundqvist E, Baarnhielm M, Alfredsson L, Hillert J, Olsson T, Kockum I. Confirmation of association between multiple sclerosis and CYP27B1. Eur J Hum Genet. 2010;18(12): Munger KL, Levin LI, Hollis BW, Howard NS, Ascherio A. Serum 25-hydroxyvitamin D levels and risk of multiple sclerosis. JAMA. 2006;296(23): Lopez ER, Zwermann O, Segni M, Meyer G, Reincke M, Seissler J, et al. A promoter polymorphism of the CYP27B1 gene is associated with Addison s disease, Hashimoto s thyroiditis, Graves disease and type 1 diabetes mellitus in Germans. Eur J Endocrinol. 2004;151(2): Bailey R, Cooper JD, Zeitels L, Smyth DJ, Yang JH, Walker NM, et al. Association of the vitamin D metabolism gene CYP27B1 with type 1 diabetes. Diabetes. 2007;56(10): Fabri M, Stenger S, Shin DM, Yuk JM, Liu PT, Realegeno S, et al. Vitamin D is required for IFN-gamma-mediated antimicrobial activity of human macrophages. Sci Transl Med. 2011;3(104):104ra Gottlieb SL, Martin DH, Xu F, Byrne GI, Brunham RC. Summary: The natural history and immunobiology of Chlamydia trachomatis genital infection and implications for Chlamydia control. J Infect Dis. 2010;201 Suppl 2:S

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114 CHAPTER 7 General Discussion

115 GENERAL DISCUSSION Chapter Chlamydia trachomatis (CT) is the most prevalent sexually transmitted bacterial infection worldwide. The number of cases is still increasing. This is partly explained by the increased sensitivity of diagnostic tests and the increase in the numbers of tests performed (1). In order to control CT infections it is essential to identify CT positive patients. This process is hampered by the often asymptomatic character of the infection in both women (estimated from 70% to 95.5%) and men (estimated from 25% to 100%) (2-6). Consequently, unknowingly infected individuals are not prompted to seek medical care and provide an ongoing reservoir for infection (6, 7). When symptoms do occur, the cervix is the most commonly infected anatomic site in women. They include classic findings of cervicitis: mucopurulent endo-cervical discharge, easily induced endocervical bleeding, or edematous ectopy. Untreated, cervical infection can ascend to cause pelvic inflammatory disease and its sequelae of chronic pain and infertility (8, 9). Urethritis is most common in men, and proctitis in men who have sex with men (MSM). Controlling CT transmission in order to prevent reproductive tract complications is a major public health concern and can be achieved by effective intervention strategies. The 2015 European CT guideline on the management of Chlamydia trachomatis infections is an important resource because it contains extensive information with a broad approach regarding all issues involved in clinical management of urogenital CT infections based on most recent literature. Despite the possibility of spontaneous clearance of a CT infection without any damage to the reproductive functions (10), some women develop severe late complications. The key problem is that the natural course of a CT infection is still partly unclear: why do some women develop symptoms or an upper genital tract infection and others do not? It is likely that this is due to a complex interaction between bacterial, environmental and host (genetic) factors. In this thesis we address to understand female susceptibility to acquire a CT infection resulting in tuba pathology. Our studies are based on serology and host genetics. Defining host risk profiles prone for tuba pathology, offers the possibility to predict which women are at risk for subfertility. A serology test as part of routine subfertility diagnosis is the next step. This in turn could prevent unnecessary laparoscopies, save time patients unsuccessfully spend trying to get pregnant and avoid psychological burden. CLINICAL ASPECTS OF CT INFECTIONS Guidelines should be acknowledged but not blindly followed To provide up-to-date guidance regarding the management of urogenital CT infections, we undertook a literature search to identify the main issues concerning testing, treatment,

116 General Discussion and Summary 115 follow up, and prevention. The results of this literature review are merged in our 2015 European guideline (11, 12). Differences between the earlier 2010 guideline and the new 2015 one consist of broader indications for testing and treatment of CT infections; clearer recommendation of using NAATs for diagnosis; advice on (repeated) CT testing; recommendation of increased testing to reduce the incidence of pelvic inflammatory disease; and recommendations to identify, verify and report CT variants (chapter 1 and 2). Implementation of guidelines is challenging Guidelines are produced to create a (inter)national evidence-based gold standard that is up to date to be used amongst others by clinicians and policy makers. Moreover it often functions as a quality assessment tool in medical health care. Unfortunately, some research studies on the implementation of clinical practice guidelines often reveal only modest effects (13). A recent study (2016) has shown that these studies could have greater relevance if a number of pitfalls were to be considered in advance (14). To start with, implementation research has shown that a guideline needs to be experienced as personal and as an extension of the already available toolbox of knowledge and expertise by the professional offering the concerned care. All concerned disciplines like physicians, nurses, paramedics and management need to be motivated to incorporate the new guideline (15). These recommendations are very useful and can function as a guide to improve implementation of our 2015 guideline as well. The most important message resulting from our guideline involves good cooperation between all these different disciplines including medical microbiological laboratories. Those laboratories should participate in quality assurance programs to identify for instance CT variants potentially linked to unusual clinical presentations or actually escaping NAAT diagnostics. If all involved professionals communicate effectively with each other and report their cases to national reference centers in order to keep up to date with incidence and prevalence, sudden CT variants or different clinical presentations can be noticed and reported. Established expert networks such as ECDC and IUSTI can help to disseminate appropriate information to other professionals in Europe. Secondly, there are many implementation strategies, like (patient) education, intervision, visitation, national campaigns, or financial stimuli (16). A combination of these tools can contribute to a successful implementation. One of the recommendations of the 2015 guideline mentions that treatment of current partners is the most effective strategy for reducing CT transmission at population level. This can be accomplished by evaluating, testing, treating, and offering counseling for CT and other STIs to all sexual contacts within the preceding three and ideally six months of onset of symptoms or diagnosis. This advice can be brought into practice by patient education after intervision of professionals working at an outpatient STI clinic who have agreed on and made arrangements to act to this new recommendation. A financial injection to the STI outpatient clinic contributes positively even more: it takes more time to educate patients regarding

117 prevention, and testing and treatment of sexual contacts also takes time and costs more. Furthermore, as health professionals and patients are moving towards shared models of decision making, there is a growing need for integrated decision support tools that facilitate uptake of best evidence in routine clinical practice in a patient-centered manner. Each recommendation of a guideline requests a specific clinical approach or a change of routine to realize the desired change (17). The recommendation in our 2015 guideline that pharyngeal and rectal swabs should be considered to be taken in MSM and in heterosexuals according to risk, comprises extra questions during history taking and thus changing professional routine. If the adaptations can be made clearer in advance, incorporation into practice will become easier. In conclusion, guidelines are indispensable in present medical care, but recommendations are population based. Future challenges for research into guideline implementation is to study how guidelines really could assist patients best in delivered personalized care. Professionals can than perhaps balance their decisions more and relate it to an individual based approach. Chapter LGV epidemic is still not under control CT positive patients are mostly low-risk patients without symptoms. Recent RIVM reports show a decline of the amount of patients tested at Public Health services in the Netherlands, but at the same time there is an increase in the percentage of patients positive for STIs at these Public Health services (Report from the Rijksinstituut voor Volksgezondheid en Milieu, RIVM, 2015). Finding and defining high risk populations is a great step forward in order to start diagnostic detection and treatment. The case report in chapter 3 illustrates that a differential diagnosis based on history taking, physical examination and further testing could be limited. Only when considering the patient as part of a sexual high risk group with the LGV epidemic in mind, further risk analysis can be made and relevant tests performed. Repeat testing should be considered for MSM. This advice is partly based on mathematical modelling studies in the USA, demonstrating that repeat infection rates peak at 2 5 months after the initial infection (18). Detecting and identifying risk groups or inner core groups like young persons under the age of 25, MSM or swingers can contribute to case finding. Universally testing urogenital, anorectal and oropharyngeal CT infection should be recommended especially in MSM. Recent reports from France have demonstrated that the lymphogranuloma venereum outbreak is no longer dominated by the variant L2b. Many L-positive specimens were misidentified as L2b in France. For routine detection to differentiate between L strains and variant L2b they use two real-time PCRs. The first one exploits the unique 36-bp deletion present in all LGV genovars (19), and the second one allows the identification of L2b variant by targeting a 9-bp insertion unique for L2b within the pmph gene of CT (20). From an

118 General Discussion and Summary 117 epidemiological point of view these real-time PCRs were compared with sequencing of the OmpA gene, to investigate geographical distribution. 25% of CT-positive specimens were not confirmed as L2b by sequencing, which suggests a recent shift in the population to different LGV strains by mutations. European studies are now initiated to investigate this further (21). Adequate detection is a major first step to increase case finding, to denote prevalence and incidence, and to detect CT variants. The importance of monitoring routine diagnostics with more extensive diagnostics at strain level combined with up to date epidemiological data is well-illustrated by the present LGV-epidemic. Underreporting of CT cases to national connected STI reference laboratories could be a serious danger to public health The widely available (commercial) FDA-approved CT automated nucleic acid amplification (NAAT) tests are primarily recommended for diagnostics nowadays. These tests are very sensitive and specific and can detect symptomatic and asymptomatic CT infections at an early stage (22). More and more non-accredited commercial laboratories are emerging, offering cheap STI tests to be performed at home (home test) or by sending self- taken samples to a testing site (laboratory test). Patients are interested in the availability of self-tests so they can stay anonymous, avoid a visit to their family doctor and save costs. A recent report (2016) from Public Health services has shown that the availability of self-tests on the internet varies greatly and that there is wide variety in products and prices, but also in content and quality of information (23). The results of this evaluation show that the reliability of home tests for CT is not demonstrated yet and that the information on the websites is not complete. Laboratory tests are often more reliable and the information provided on the internet is better than for home tests, but can still be improved. Apart from the advantages of home based testing however, participation of commercial laboratories in (inter)national quality assurance programs is often lacking, as well as reporting the number of CT infections to national STI monitoring institutions. This could lead to misinterpretation of the actual national prevalence (underdiagnosing) and variants could be missed. Therefore it is important that these laboratories communicate to STI monitoring institutions to stimulate surveillance. Furthermore the assurance of quality of testing of these commercial laboratories is difficult to measure. The route of indication for testing, risk analysis, detection, and management of the patient and his or her partner can become blurred. Provision of information about the test products and clear online advice in case of STI diagnosis are two essential elements of a good, reliable self-test (23). Ideally, all professionals involved in performing CT tests should report the total amount of performed tests and the results to national reference centers. Secondly they need to participate in quality assurance programs to increase quality in general and control national prevalence and incidence specifically.

119 Repeat testing should be offered to high risk groups The main rationale for current CT screening or opportunistic testing is that early detection and treatment will prevent or interrupt reproductive tract morbidity, particularly in women. Moderate quality evidence of efficacy of screening has been demonstrated by four RCTs (9). In general, CT screening reduces the incidence of PID when compared to control groups receiving usual care. However, recently repeated screening rounds have not shown to be cost-effective in the Netherlands and are therefore not recommended (24). Repeat testing, however, should be offered after 3-6 months to young persons (<25 years of age) who have been tested positive for CT. Chapter Detection remains the cornerstone to control CT infection As recommended by the 2015 IUSTI European guideline for the management of CT infections it is very important to maintain the link between clinical, diagnostic, and epidemiological data in a national database to keep up to date with actual prevalence, monitoring different clinical outcomes, and CT variants. This is well illustrated by the sudden drop in CT prevalence in Sweden of nearly 80% in some areas and an overall drop of 25% in 2006 (25, 26), where eventually a CT variant with a deletion in the plasmid was identified that could not be detected by two former NAAT tests frequently used at the time (Roche and Abbott) in many regions. However, this strain remained detectable by another plasmid based NAAT, the ProbeTec ET (BD Biosciences, Sparks, MD, USA), because the deletion did not affect the target region of that test. This variant is now known as the Swedish variant (swct) or new variant (nvct). This observation led to quick improvements in the other two diagnostic NAATs to detect this new variant as well. Prevalence rates of nvct have decreased significantly over the last eight years and possibly converged into a steady state of 5-7% irrespectively of initial county rates. Genotyping indicates that nvct is clonal and genetically stable (27). Since plasmid free strains can still be undetected and thus untreated, from 2010 onwards we developed a screening programme in The Netherlands to monitor potential plasmid free variant emergences in The Netherlands i.e. the Dutch Chlamydia trachomatis Reference Laboratory (chapter 4). To investigate whether CT variants could have a predictive value on future development of tubal factor infertility, we undertook a study to examine the extent. In the period we collected 3554 samples from different participating centers. In total 7 plasmid free variants (negative for all three plasmid PCRs and positive for the chromosomal ompa traget) were identified of which 5 were confirmed by an independent sample. The variants were detected in both males and females and at different anatomical sites. In conclusion, the number of plasmid free variants in the Dutch population is very low, and in general is detected by the current NAAT assays used, meaning that there is no need for action based on the results from the current study presented.

120 General Discussion and Summary 119 Discongruent samples should be retested to detect variations at the DNA level. Therefore it is important to incorporate different NAAT tests targeting different areas in the CT DNA at specialized reference laboratories, to detect different or new strains. Furthermore the discovery of the LGV-serovar L2b made clear that monitoring new emerging variants or the reappearance of known ones is very relevant. NAATs can then be adapted when new variants emerge as was done previously with the swct variant and if clinically relevant. The clinical course of infection is thought to be the result of an interplay between the bacterium, the host immune system, and this is influenced by environmental factors This thesis also aimed to contribute to a future diagnostic approach to distinguish women at risk for a complicated course of CT infection leading to, amongst others, tubal factor infertility This challenge was addressed in the following chapters of this thesis (chapter 5 and 6). Most infected women clear the CT bacteria without any damage to their reproductive functions, but some develop severe late complications, like endometritis, salpingitis, tubal occlusion, and periadnexal adhesions, leading to tubal factor infertility. Understanding the causal link between CT lower genital tract infection and inflammation of the upper genital tract leading to tubal infertility is challenging due to the fact that this can be a silent complication. It is usually diagnosed years after the initial infection (28). The clinical course of CT infections is heterogeneous i.e. transmission, symptoms, clearance, and development of late complications differ per patient (29, 30). Environmental factors (e.g. sexual (risk) behavior, smoking, medication), microbial factors (e.g. virulence factors) and host factors (e.g. host genetic variations) (figure 1) together can affect the chance to acquire an infection (susceptibility) and can influence the severity of the infection (31). Advanced knowledge in this complex field of susceptibility to and severity of disease can improve the development of new research tools and stimulate an integrated approach in investigating CT infections. Host response to infection e.g. adaptive immunity as measured by Ig responses Assessment of serological responses to the CT bacterium resulted in a classification in serovars. Nowadays, 19 different serovars are known, usually causing conjunctival and urogenital infections. Differences in the incidence and prevalence between serovars and serogroups in men, in women and in different patient groups have been found worldwide. This is linked to transmission networks and clinical presentation. To acquire more insight in the immune response of serovars related to clearance and persistence of infection is a challenging issue (32, 33). The most prevalent serovar E from the serogroup B induces significantly higher serological IgG responses than serogroups C and I in urogenital CT infections (34).

121 Figure 1: The complex nature of CT infections. Host factors (e.g. genetic variation and immunological factors), environmental factors (e.g. (sexual risk) behavior, diet, smoking, coinfections with other microorganisms, medication) and bacterial factors (e.g. virulence factors) influence the observed differences in the clinical course of infection. The different factors can also interact with each other, e.g. infection with other microorganisms may increase the susceptibility to CT infection. Figure modified from Morré et al.(31) Chapter These results suggest a relation between serovars and the serological responses in humans and potentially offer a partial explanation of the geographical prevalence of specific serovars. These results, however, have not been verified in different patient populations for the IgG responses induced by different serovars, nor have they been confirmed using other IgG based serological tests. Therefore we investigated two different patient groups (n=718) in different geographical regions (The Hague and Amsterdam) with similar characteristics of Dutch STI clinic visitors (Chapter 5). We performed genotyping to identify serovars and used titer based and quantitative commercially available ELISA kits (Medac Diagnostika) to determine specific serum IgG levels. The results showed very significant differences when comparing the median IgG titers of three serogroups, B, C, and I: in women for B vs. C: p < (median titers B 200 vs. C <50); B vs. I: p < (200 vs. 50), and in men for B vs. C: p = (150 vs. <50); B vs. I: p = (150 vs. <50); C vs. I was not significant for both sexes. Serovars D and E of serogroup B had the highest median IgG titers compared to the other serovars in both men and women: 200 and 200 vs. 100 for women and 100 and 200 vs. 75 for men, respectively. Also the serovar/serogroup prevalence in both cohorts was similar but serological response for all serovars was higher in women than in men. Serovar D is the most virulent strain and establishes a longer and more invasive infection, which is characterized by more shedding during the acute phase of CT infection (35, 36). We propose, based on our results and findings in the literature that the variation in serological responses in human populations induced by serogroup B is associated with

122 General Discussion and Summary 121 their high number of EBs. An indirect link between serovars and prolonged and severe inflammation could exist and can be associated with tubal pathology (37, 38). This may be useful knowledge for the improvement of serology markers in the future that can be relevant for the detection of infertility due to STIs like CT. Recently, seroprevalence of anti-pgp3 antibodies (an immunodominant antigen and putative virulence factor) have demonstrated to be an indicator of current and prior CT exposure. It may be higher in women with repeat CT infection and may decrease with time (39). This and other targets like TARP and CPAF (Mikrogen) might be interesting ones to investigate on the level of serovar induced IgG responses. It was demonstrated that a multidisciplinary approach is needed to develop a kind of risk profile, which includes the determination of the serovar involved, for women who are prone of developing late complications whereas others do not. The risk of tubal pathology Worldwide 15% of couples trying to conceive, are suffering from subfertility (40, 41). One of the major causes of female subfertility is tubal pathology (TP) (40), and CT is the single most common cause for tubal infertility. Most women with tubal factor infertility do not have a history of (acute) PID despite serological evidence of a previous infection with CT or Neisseria gonorrhoeae (NG) (42-44) suggesting a history of subclinical PID and infection. Subclinical PID can influence women s fertility despite provision of treatment for sexually transmitted diseases. These findings suggest that a proportion of female infertility is attributable to subclinical PID (45). Recent mathematical modelling studies show that the proportion of PID attributable to CT infection is on average 35% for women aged years. Since CT infection is a disease of young people, PID incidence decreases strongly with age by a factor of around 5-fold (46). Various immunological markers have been studied for their ability to indicate an individual s history of CT infection and increased probability for late complications (47-50). In infertile women, the presence of CT IgG antibodies in serum is associated with tubal pathology and lower natural conception rates, even in case of tubal patency. Elevated levels of anti-ct antibodies can be detected in 30 70% of women with tubal pathology (47, 51), compared to around 10 20% in the general female population of reproductive age (52, 53). In fertility clinics in the Netherlands, CT IgG antibody testing (IgG-CAT) in serum is used as a screening test for tubal pathology and for selecting high-risk patients for laparoscopy (54, 55). A noninvasive biomarker enabling selection of women at high risk of late complications of CT infection would be useful for targeted prevention at the individual level but also facilitate natural history studies and provide an outcome marker for screening intervention studies and candidate vaccine trials (54). Recently a concise exploratory study has been performed investigating the presence of IgA in vaginal mucosa: local IgA antibodies were associated with tuba pathology and

123 Chapter 7 positive history of CT infection in women attending a fertility clinic (56). This study suggests that local IgA antibodies might have a stronger discriminative value than local IgG and could be of value in the future as a biomarker to indicate which women have a higher chance to develop late sequelae from CT infections. Further prospective cohort studies are needed to what extent and when IgA in self-taken vaginal swabs could be of value as a biomarker. Laparoscopy is widely used to assess the risk of TP in women positive for CT IgG serology. This procedure is invasive and expensive (on average 3000 Euros including additional costs) and requires general anesthesia. Although CT serology is used, as advised in the guidelines in the fertility work-up of infertile women, the predictive value for tubal factor infertility is far from perfect. Many CT IgG positive women undergo laparoscopy while not needed (40 45%) or do not get intervention while needed (19%) (figure 2). A meta-analysis by Broeze et al. has shown that the combination of integration of patient characteristics, results of CAT (CT IgG) and hysterosalpingography (HSG) in order to predict diagnostic performance of tuba pathology is promising: Area under the curve AUC is 0.74 compared to previous 0.63 (57). Cost-efficiency could therefore increase by adding this tuba patency test into practice. Unfortunately they do not discuss a cut off value for laparoscopy or IVF either. Future studies should be performed to investigate this in order to make a decision strategy for IVF or laparoscopy. 122 HOST GENETICS Host genetic factors play a role in the susceptibility to and severity of CT infection It is clear from a large body of evidence that host genetic factors play a role in the susceptibility to and severity of CT infections, as shown by twin studies (59) and many candidate gene studies. To bring this current host genetic work to the next level, large scale Single Nucleotide Polymorphism (SNP) typing and SNP identification in confirmation cohorts are essential and in progress. This work will provide insight into what type of host genetic profiles can help to improve subfertility diagnoses that are based on previous CT infection(s) and potentially other STIs. Besides single SNPs combination of SNPs in so-called carrier traits will significantly increase the ability to predict and identify the women at greatest risk of severe complications from CT infection (58). Susceptibility to CT infections in humans in association with polymorphisms in five genes related to the vitamin D pathway could not be supported yet Genetic variations in the host immune system are known to impact the course of infections, including that of CT. Recent studies indicate that vitamin D hormone regulates the immune system through induction of natural host defense peptides, activation of

124 General Discussion and Summary 123 Figure 2: Current serology protocol for subfertility resulting from CT infection. Women with negative C. trachomatis serology are advised to try to conceive for one year; however, 20% of those women actually have tubal pathology; triage of these women is incorrect due to incomplete serology tests.. Of the women with a positive CT serological test, 40 45% do not have tubal pathology after laparoscopic examination and triage has been incorrect again. Figure from Lal et al. (58). pattern recognition toll-like receptors, and anti-inflammatory activity (60, 61). Q. He et al. reported in 2013 on the use of vitamin D receptor knock-out (VDR / ) mouse and HeLa cells (human cervical epithelial cells) to test the hypothesis that the vitamin D endocrine system attenuates CT infection. They demonstrated that the vitamin D endocrine system reduces the risk for prolonged CT infections through regulation of several proteins and Leukocyte elastase inhibitor (LEI), an anti-inflammatory protein, which is involved in its anti-inflammatory activity (62). Vitamin D in vitro could play a role in the protection of the immune system against a prolonged course of a CT infection. In our study, we used vaginal samples obtained from a STI outpatient clinic in the southern part of Limburg, the Netherlands (chapter 6). We selected Dutch Caucasian women, all between years of age. CT status was assessed by the outpatient clinic using the available NAAT assay. We assessed the impact of eight polymorphisms in five genes (VDR (rs G>A, rs C>T), CYP27B1 (rs G>T), DHCR7 (rs G>A, rs G>A), GC (rs ), and CYP2R1 (rs G>A, rs G>A)) on susceptibility to CT infections. In our cohort of 67 CT positive and 507 CT negative

125 women we did not observe statistically significant differences between the genotype distributions of the eight polymorphisms studied. For that reason, we assume that VDR, CYP27B1, DHCR7, GC, and CYP2R1 do not seem to play a major role in susceptibility to CT infections. They demonstrably play an important role in other diseases, for example in killing the intracellular Mycobacterium tuberculosis (63). A limitation of our study is the potential lack of statistical power and the relative small sample size. Unfortunately we have only examined susceptibility. In the future we would like to study more host genetic factors that could be involved in the Vitamin D pathway and CT infections, and find more associations that we could link to late complications. Chapter 7 Host genetic markers could potentially increase the positive or negative prediction of TF infertility resulting in more targeted use of invasive procedures like laparoscopies A genetic test as part of routine subfertility diagnosis could potentially save time and money by decreasing the number of unnecessary laparoscopies. Likewise it could save time patients unsuccessfully spend trying to get pregnant. This host genetic diagnostic test should potentially be combined with a CT serology test [47]. The genetic trait should contain a series of markers in specific pathways and could contain also so-called SNP load or gene load linked to decision making to predict the presence of TP. 124 Figure 3. Prediction model for tubal pathology including the usage of the host genetic trait in development (adapted figure by S. Morré). As can been seen in both arms of the triage of subfertile women major percentages of miss-triage takes place. We argue that combining serology with a human genetic trait can reduce the number of misclassifications in the two triage arms. This means that CT+ (20%) with a good host genetic marker trait (green bar) do not need laparoscopy. CT+ with an adverse host genetic trait (red bar) means forwarding to laparoscopy. There will remain a host genetic marker trait range in which no clear advice can be given regarding the use of laparoscopy (grey bar).

126 General Discussion and Summary 125 What is envisioned for the near future is the addition of a host genetic marker profile in the subfertility workup (see figure 3). Currently we are further investigating and extending the set of host genetic markers with the aim to transfer these to a clinical application. This translation and implementation will be supported by approaches defined in the field of Public Health Genomics (PHG) ensuring 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). PHG promotes the implementation of strategies to promote a faster path for genetic knowledge from bench to bedside. This knowledge will not only enable clinical interventions but also health promotion messages and disease prevention programs. Customized advice at susceptible individuals as well as subgroups of the population based on their genomic profile enhances personalized healthcare. CONCLUSIONS AND FUTURE PERSPECTIVES In summary, this thesis contributes in optimizing intervention strategies for the management of Chlamydia trachomatis (CT) infections. Clinical guidelines are helpful for professionals in providing easily accessible (online) information for the management of urogenital CT infections. A uniform strategy based on adequate detection, reporting of CT cases by all authorities involved in CT testing to national reference laboratories, and adequate treatment is fundamental to control CT infections. This strategy should be evidence based. The European guidelines summarize best available evidence to date, but the scientific basis of the 2015 guideline would benefit from improvement in both quality and quantity of CT-related clinical trials. In the field of immunology, serological responses and host genetic factors are associated with the clinical course of CT infections. Ideally, the best predictive diagnostic for the triage of subfertile women with potential tubal pathology would be a dual assay composed of the most predictive serological and host genetic markers that are involved in the development of late complications in women after CT infection. Invasive and costly procedures like laparoscopies could then in part be prevented.

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130 General Discussion and Summary 129 by Chlamydia trachomatis: the role of serology. International journal of STD & AIDS. 2002;13 Suppl 2: Lyytikainen E, Kaasila M, Koskela P, Lehtinen M, Patama T, Pukkala E, et al. Chlamydia trachomatis seroprevalence atlas of Finland Sexually transmitted infections. 2008;84(1): van Aar F, de Moraes M, Morre SA, van Bergen JE, van der Klis FR, Land JA, et al. Chlamydia trachomatis IgG seroprevalence in the general population of the Netherlands in 1996 and in 2007: differential changes by gender and age. Sexually transmitted infections Gottlieb SL, Berman SM, Low N. Screening and treatment to prevent sequelae in women with Chlamydia trachomatis genital infection: how much do we know? Journal of Infectious Diseases. 2010;201:S Broeze KA, Opmeer BC, Coppus SFPJ, Van Geloven N, Alves MFC, Anestad G, et al. Chlamydia antibody testing and diagnosing tubal pathology in subfertile women: An individual patient data meta-analysis. Human reproduction update. 2011;17(3): van den Broek IV, Land JA, van Bergen JE, Morre SA, van der Sande MA. Chlamydia trachomatis Antibody Testing in Vaginal Mucosal Material versus Blood Samples of Women Attending a Fertility Clinic and an STI Clinic. Obstet Gynecol Int. 2014;2014: Broeze KA, Opmeer BC, Coppus SF, Van Geloven N, Den Hartog JE, Land JA, et al. Integration of patient characteristics and the results of Chlamydia antibody testing and hysterosalpingography in the diagnosis of tubal pathology: An individual patient data metaanalysis. Human reproduction. 2012;27(10): Lal JA, Malogajski J, Verweij SP, de Boer P, Ambrosino E, Brand A, et al. Chlamydia trachomatis infections and subfertility: opportunities to translate host pathogen genomic data into public health. Public health genomics. 2013;16(1-2): Bailey RL, Natividad-Sancho A, Fowler A, Peeling RW, Mabey DC, Whittle HC, et al. Host genetic contribution to the cellular immune response to Chlamydia trachomatis: Heritability estimate from a Gambian twin study. Drugs of today. 2009;45 Suppl B: White JH. Vitamin D signaling, infectious diseases, and regulation of innate immunity. Infection and immunity. 2008;76(9): Hewison M. Antibacterial effects of vitamin D. Nature reviews Endocrinology. 2011;7(6): He Q, Ananaba GA, Patrickson J, Pitts S, Yi Y, Yan F, et al. Chlamydial infection in vitamin D receptor knockout mice is more intense and prolonged than in wild-type mice. The Journal of steroid biochemistry and molecular biology. 2013;135: Liu PT, Stenger S, Li H, Wenzel L, Tan BH, Krutzik SR, et al. Toll-like receptor triggering of a vitamin D-mediated human antimicrobial response. Science. 2006;311(5768):

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132 CHAPTER 8 Summary Samenvatting

133 SUMMARY Chapter In this thesis factors related to the often asymptomatic character of Chlamydia trachomatis (CT) infections are presented and discussed with a focus on both clinical and more biological studies including host responses. The 2015 European background review for the management of CT infections acts as a clinical overview of where we stand today in the burden of CT infections and presents updated information about epidemiology and clinical management. As the natural course of a urogenital CT infection still remains partly unclear, we have focussed on two types of host responses that might contribute to the susceptibility of infections or to a prolonged, severe CT infection leading to tubal pathology in women. Firstly, we have investigated host serological responses induced by the pathogen CT during urogenital infections to elucidate serovar distributions in association with IgG serum concentrations. Secondly, we have investigated host genetic variation determining the host immunological responses which are influenced by genetic variation in the genes involved in recognition of CT. We studied the abovementioned aspects of CT infections in a number of target groups in the Netherlands. Below, we summarise the major findings and conclusions per chapter. In chapter 1, the introduction of this thesis, an overview of all major epidemiological and clinical aspects of CT infections are given. It is partly based on our European guideline for the management of urogenital CT infections. In chapter 2, the background review for the 2015 European guideline on the management of CT infections is presented. This is a detailed description of background, evidence base of recommendations and discussions regarding our 2015 European guideline on the management of urogenital CT infections. European guidelines are produced to stimulate evidence based management by all professionals involved in sexual health care. The aim is to decrease transmission of CT infections and consequently prevent the rate of complications like PID and infertility. The most important updates include: broader indications for testing and treatment of CT infections; clearer recommendation of using exclusively validated and quality assured highly sensitive and specific nucleic acid amplification tests for diagnosis; advice on (repeated) CT testing; recommendations of increased testing particularly at sexually transmitted infections and sexual health clinics to reduce the incidence of pelvic inflammatory disease and prevent exposure to infection; and recommendations to identify, verify and report CT variants. Details regarding the etiology, transmission, clearance, epidemiology and taxonomy of CT, clinical features, recommended diagnostics (including quality assurance), advice for CT infected patients, indications for therapy, recommended and alternative treatment regimens for urogenital and extragenital CT

134 Summary 133 infections, contact tracing and management, and the notification of CT cases are included. In chapter 3 we present a rare case of a young man with visual impairment and constipation with bloody anal discharge for 1 year. He reported to have had unprotected receptive anal sexual contacts with male partners. Physical examination revealed an annular plaque at the soft palate and perianal skin defects, including perianal fistulas. Further examination showed a positive NAAT on a rectal swab for N. gonorrhoeae and CT. CT genotyping demonstrated the L-serotype of CT confirming lymphogranuloma venereum (LGV). Further laboratory tests confirmed secondary syphilis which corresponded with the clinical picture of uveitis anterior and an annular structure at the palatum, resembling a condyloma latum. HIV and hepatitis C virus tests were negative. This advanced stage of LGV can mimic Crohn s disease. An adequate sexual history can be helpful in the differential diagnosis. In sexually active MSM with rectal complaints, LGV should always be considered, even if the HIV test is negative. With the increase of rectal LGV infections, especially in MSM, it is recommended that positive rectal specimens are subsequently genotyped for LGV. In chapter 4 we describe the results obtained by the Dutch Chlamydia trachomatis reference Laboratory in the period This Laboratory is an initiative of the Epidemiology & Surveillance Unit, Centre for Infectious Disease Control, from the National Institute of Public Health & the Environment (RIVM) and the VU University Medical Center, Department of Medical Microbiology and Infection Control. The two main tasks of the Laboratory are: 1) providing CT surveillance in different geographical regions in the Netherlands by collecting CT-positive and CT-negative samples of men and women of all representative anatomical sites. This task has been initiated to monitor CT variant emergences, like the Swedish variant, to safeguard Dutch CT detection reliability and 2) functioning as a central CT Laboratory for questions regarding potential CT diagnostic difficulties including potential CT variants. For the first task we screened 25 CT- and 10 CT+ samples from 6 geographical locations each quarter of the year with a yearly minimum of 500 samples. 1.9% of the samples were discrepant as compared to the initial reported results. Sample degradation in combination with very low titres was the main reason for this. Seven samples contained a plasmid free strain identified via newly developed PCRs, resulting in an incidence of 0.2%. As this is a low percentage and most commercial tests use either RNA as a target or have a dual target system (plasmid and chromosomal), this discrepancy does not have a major effect on the detection rate. For the second task, the laboratory has been approached by different parties resulting in identification of a Swedish variant in the Netherlands and also the first rectal lymphogranuloma strain L2b in a female, a strain which was till then only identified in men having sex with men (MSM).

135 In summary the core tasks of the CT reference laboratory have resulted in the identification of different CT variants in the Netherlands. Hence it has proven its merit. Chapter 8 We evaluated in chapter 5 the serological immunoglobulin (Ig) G serum concentrations in CT infected patients, since antibodies have been associated with clearing CT infections but also with the presence of tubal pathology in women. We have investigated urogenital swabs and serum samples from 718 Dutch positive CT patients attending a STI clinic in two major cities in the Netherlands (The Hague and Amsterdam). Detection of CT DNA in swabs was performed by two different PCR techniques. Genotyping of serovars was performed using the CT-DT assay (Labo Biomedical Products BV, Rijswijk, the Netherlands) which is based on the reverse hybridization assay (RHA) methodology and by means of PCR based Restriction Fragment Length Polymorphism (RFLP) analyses. Determination of CT IgG levels in serum of all patients was done by a specific Enzyme- Linked Immuno Sorbent Assay (pelisa) test generating quantitative results based on Optic Density (OD) values to further calculate IgG titres. Our results show that the most prevalent urogenital serovars from the B serogroup, serovars D and E, induce the highest IgG serum concentrations, and the least prevalent serovars from the C group induce the lowest concentrations in both men and women. These results provide more insight in understanding the immunological response against a CT infection at both serogroup and serovar level. 134 Genetic variations in genes encoding the host immune system are known to impact the course of infections in general. A single nucleotide polymorphism (SNP) is a genetic variation in a single nucleotide that occurs at a specific position in the genome, where each variation is present to some appreciable degree within a population (e.g. > 1%). Recent studies have shown a positive impact of vitamin D on the regulation of the immune system. The examination of polymorphisms within genes of the vitamin D biopathway in relation to CT infection has been described in chapter 6 to further elucidate differences in susceptibility to urogenital CT infection in humans. An analysis of polymorphisms with either a proven or theoretical functional impact (either amino acid change, or impact on protein expression) and/or haplotype tagging (finding a set of so-called tagging SNPs) was eligible for inclusion in this study. We included SNPs from the following genes: VDR (rs G > A, rs C > T), CYP27B1 (rs G > T), DHCR7 (rs G > A, rs G > A), GC (rs ) and CYP2R1 (rs G > A, rs G > A). All polymorphisms were genotyped by LGC Genomics using their own form of competitive allele-specific PCR (KASP). In our STI cohort of Dutch Caucasian women (n=500), we did not observe statistically significant differences between the genotype distributions of the eight polymorphisms. For that reason, we assume that VDR, CYP27B1, DHCR7, GC, and CYP2R1 do not seem to play a major role in susceptibility to CT infections as they apparently do in other

136 Summary 135 diseases. However, genes in the vitamin D pathway exhibit a pleiotropic role in the immune system, therefore the role of vitamin D should not be disregarded yet for the entire clinical course of CT infections and requires further research with a focus to late complications including tubal factor infertility. This can be of value for implementing host genetic markers into clinical applications in the future to distinguish women prone for tubal pathology. Chapter 7 is the discussion of the presented work. This thesis contributes amongst others in optimizing intervention strategies for the management of CT infections. Clinical guidelines are helpful for professionals in providing easily accessible (online) information for the management of urogenital CT infections. Improvement in both quality and quantity of CT-related clinical trials can further strengthen the evidence base. Secondly, the identification of women at risk for developing tubal pathology and thus infertility should be enhanced. Ideally, the best predictive tool for the triage of subfertile women with potential tubal pathology would be a dual assay composed of the most predictive serological and host genetic markers that are involved in the development of late complications in women after CT infection. Unnecessary, invasive and costly procedures like laparoscopies could in part be prevented.

137 SAMENVATTING Chapter 8 Het natuurlijk beloop van Chlamydia trachomatis (CT) infecties is tot op heden nog steeds niet voldoende in kaart gebracht om te voorspellen wie late complicaties krijgt na infectie en wie de infectie complicatieloos kan klaren. In dit proefschrift worden resultaten van verschillende studies over CT infecties gepresenteerd met zowel klinische als biologische aspecten waaronder gastheerreacties. Het achtergrondartikel over de Europese richtlijn voor 2015 voor de behandeling van CT-infecties fungeert als een klinisch overzicht van waar we nu staan in de ziektelast van CTinfecties en geeft actuele informatie over de epidemiologie en klinische presentatie. Aangezien het natuurlijk beloop van een urogenitale CT-infectie nog steeds gedeeltelijk onduidelijk is, hebben we ons gericht op twee typen gastheerreacties die bijdragen aan de gevoeligheid voor infecties of bijdragen aan het langdurige, ernstige verloop van CT-infecties, die kunnen leiden tot o.a. tubapathologie. Ten eerste hebben we de serologische responsen bestudeerd die door CT tijdens urogenitale infecties worden gegenereerd; daarbij is geprobeerd om een associatie te vinden tussen het type serovar en de IgG concentratie in het serum. Ten tweede hebben we gastheer-genetische variaties in genen die betrokken zijn bij de herkenning van CT onderzocht. We bestudeerden de bovengenoemde aspecten van CT-infecties in diverse doelgroepen in Nederland. Hieronder vatten we de belangrijkste bevindingen en conclusies per hoofdstuk samen. 136 In hoofdstuk 1, de introductie van dit proefschrift, wordt een overzicht van alle belangrijke epidemiologische en klinische aspecten van CT-infecties gegeven. Het is gedeeltelijk gebaseerd op onze Europese richtlijn voor de behandeling van urogenitale CT-infecties. In hoofdstuk 2 wordt het achtergrondartikel over de 2015 Europese richtlijnen voor de behandeling van CT-infecties gepresenteerd. Dit is een gedetailleerde beschrijving van de achtergrond en wordt onderbouwd door wetenschappelijk bewijs ten aanzien van de aanbevelingen en discussies aangaande de 2015 Europese richtlijn. Europese richtlijnen worden geproduceerd om medisch wetenschappelijk handelen door alle professionele partijen, die bij seksuele gezondheidszorg betrokken zijn, te stimuleren en te uniformeren. Het doel is om het overdragen van CT-infecties en daarmee de kans op complicaties zoals PID (een ontsteking in het kleine bekken ten gevolge van verspreiding van microorganismen vanuit de vagina en de baarmoederhals) en onvruchtbaarheid te verminderen. De belangrijkste updates zijn: ruimere indicaties voor het testen en behandelen van CT-infecties; duidelijkere aanbevelingen ten aanzien van het exclusief gebruiken van NAAT testen (specifieke nucleïnezuur amplificatie testen) die uitsluitend gevalideerd en kwalitatief hoog-sensitief en -specifiek zijn; advies over het beleid ten aanzien van hertesten; aanbevelingen voor uitgebreidere testafnames bij SOA-poli s en GGD en om de incidentie van eileiderontsteking te verminderen en opstijgende infectie te

138 Samenvatting 137 voorkomen; en aanbevelingen om mogelijke CT-varianten te identificeren, te controleren en te rapporteren. Details met betrekking tot de etiologie, de transmissie, de klaring, epidemiologie en taxonomie van CT, klinische kenmerken, aanbevolen diagnostiek (met inbegrip van kwaliteitswaarborging), advies voor patiënten geïnfecteerd met CT, indicaties voor behandeling, aanbevolen en alternatieve behandelingen voor urogenitale en extra-genitale CT-infecties, het opsporen en behandelen van seksuele contacten, en het rapporteren van nieuwe CT-infecties zijn inbegrepen. In hoofdstuk 3 beschrijven we een opmerkelijke casus van een jongeman met visus klachten en obstipatie met bloederige anale afscheiding sinds één jaar. Hij rapporteerde onbeschermde passieve anale seksuele contacten met mannelijke partners. Bij het lichamelijk onderzoek werd een annulaire plaque gezien op het zachte gehemelte, perianale huiddefecten en tevens perianale fistels. Aanvullend onderzoek toonde positieve NAAT resultaten op rectaal materiaal voor N. gonorrhoeae en C. trachomatis; serotypering bevestigde een L-serotype C. trachomatis oftewel een LGV infectie. Aanvullende laboratorium testen bevestigden secundaire syfilis, hetgeen correspondeerde met de klinische presentatie van uveitis anterior en een annulaire laesie op het gehemelte dat een condyloma latum voorstelde. HIV en Hepatitis C virus testen waren negatief. Dit gevorderde stadium van LGV infectie kan klinisch gelijkenis vertonen met de ziekte van Crohn. Een adequate seksuele anamnese is behulpzaam in het opstellen van de differentiële diagnose. In seksueel actieve MSM met rectale symptomen zou LGV altijd moeten worden overwogen, ook als HIV-testen negatief zijn. Omdat het aantal rectale LGV infecties met name in MSM nog steeds toeneemt, wordt aanbevolen om positieve rectale monsters altijd aanvullend te genotyperen op LGV. In hoofdstuk 4 worden de resultaten beschreven die zijn verkregen door het Nederlandse Chlamydia trachomatis referentie laboratorium in de periode Dit laboratorium is een initiatief van de Epidemiologie & Surveillance Unit, Centrum voor Infectieziektenbestrijding, van het Nationaal Instituut voor Volksgezondheid en Milieu (RIVM) en het VU medisch centrum, Afdeling Medische Microbiologie en Infectie Preventie. De twee belangrijkste taken van dit laboratorium zijn: 1) het verrichten van CT surveillance programma s in verschillende geografische regio s in Nederland. Dit wordt gedaan door CT-positieve en CT-negatieve monsters van mannen en vrouwen te verzamelen van alle representatieve anatomische lokalisaties. Het doel is surveillance; met andere woorden in een zo vroeg mogelijk stadium het optreden van CT-varianten opsporen, net zoals destijds de Zweedse variant, om de betrouwbaarheid van detectie van CT in Nederland te kunnen waarborgen, 2) te fungeren als een informatiecentrum voor het beantwoorden van vragen over o.a. diagnostische problemen met inbegrip van mogelijke CT-varianten. Voor de eerste taak worden ieder kwartaal 25 CT negatieve en 10 CT positieve monsters, met een jaarlijkse minimum van 500 monsters uit 6 geografische

139 locaties in Nederland verzameld. Het resultaat tot nu toe is dat binnen deze studie 1,9% afwijkende monsters zijn gevonden, ten opzichte van de bepalingen van de deelnemende centra. Degradatie van het materiaal in combinatie met zeer lage hoeveelheden CT (load) waren de belangrijkste redenen van dit resultaat. Zeven plasmide-vrije stammen werden geïdentificeerd met de nieuw ontwikkelde PCRs. Aangezien dit een laag percentage betreft en de meeste commerciële testen ofwel RNA als target hebben of een dubbel target systeem (plasmide én chromosoom), heeft deze discrepantie geen groot effect op de detectiegraad. Voor de tweede taak is het referentie laboratorium benaderd door diverse partijen dat heeft geresulteerd in de identificatie van een Zweedse variant in Nederland en ook de eerste rectale Lymphogranuloma stam L2b in een vrouw, een stam die tot dan toe alleen vastgesteld was bij mannen die seks hebben met mannen (MSM). Met andere woorden de referentietaken hebben geleid tot het identificeren van verschillende CT-varianten en heeft daarmee zijn opzet en waarde bewezen. Chapter We analyseerden in hoofdstuk 5 de serologische immunoglobuline (Ig) G serumconcentraties in CT-geïnfecteerde patiënten, aangezien antilichamen zijn geassocieerd met het klaren van CT-infecties maar ook met de aanwezigheid van tubapathologie bij vrouwen. We hebben urogenitale swabs en serum onderzocht van 718 Nederlandse positieve CT-patiënten die een soa-polikliniek bezochten in Den Haag of Amsterdam. Detectie van DNA van CT in swabs werd uitgevoerd met behulp van twee verschillende PCR technieken. Genotypering van serovars werd uitgevoerd met de CT-DT assay (Labo Biomedical Products BV, Rijswijk, Nederland) die gebaseerd is op de reverse hybridisatie assay (RHA) methodologie en via PCR gebaseerde Restriction Fragment Length Polymorphism (RFLP) analyse. Bepaling van CT IgG niveaus in het serum van alle patiënten werd gedaan middels een specifieke Enzyme-Linked Immuno Sorbent Assay (ELISA)-test die kwantitatieve resultaten genereert op basis van Optical Density (OD) waarden om zo IgG titers te kunnen berekenen. Onze resultaten tonen aan dat de meest voorkomende urogenitale serovars van serogroep B, serovars D en E, de hoogste IgG serumconcentraties induceren, en de minst voorkomende serovars uit groep C de laagste concentraties induceren in zowel mannen als vrouwen. Deze resultaten geven iets meer inzicht in de immunologische respons tegen een CT-infectie zowel op serogroep als op serovar niveau. Genetische variaties in genen die coderen voor het immuunsysteem van de gastheer zijn betrokken bij het verloop van infecties. Een single nucleotide polymorfisme (SNP) is een genetische variatie in één nucleotide die optreedt op een bepaalde plaats in het genoom. Recente studies hebben een positief effect van vitamine D op de regulering van het immuunsysteem aangetoond. Onderzoek naar polymorfismen in genen van de vitamine D biopathway in relatie tot CT-infecties is beschreven in hoofdstuk 6 om verschillen in gevoeligheid voor urogenitale CT-infecties in mensen te onderzoeken.

140 Samenvatting 139 Een analyse van polymorfismen met ofwel een bewezen ofwel theoretisch functioneel effect (ofwel aminozuur verandering, of invloed op eiwit expressie) en/of haplotype tagging (het vinden van een set van zogenaamde tagging SNPs) werd uitgevoerd. We onderzochten SNPs uit de volgende genen: VDR (rs G> A, rs C> T), CYP27B1 (rs G> T), DHCR7 (rs G> A, rs G> A), GC (rs ) en CYP2R1 (rs G> A, rs G> A). Alle polymorfismen werden gegenotypeerd door LGC Genomics met gebruikmaking van competitieve allel-specifieke PCR (KASP). In ons SOA cohort van Nederlandse blanke vrouwen (n = 574), werden geen statistisch significante verschillen gevonden tussen de genotype verdelingen van de acht polymorfismen. VDR, CYP27B1, DHCR7, GC, en CYP2R1 polymorfismen lijken dus geen belangrijke rol te spelen bij de gevoeligheid voor CTinfecties zoals ze blijkbaar wel doen bij bepaalde andere ziekten. Echter, genen in de vitamine D biopathway vertonen een pleiotrope rol in het immuunsysteem, dus de rol van vitamine D moet nog niet direct verworpen worden voor het gehele klinische beloop van CT-infecties en vereist verder onderzoek met focus op late complicaties zoals onvruchtbaarheid. Dit kan van waarde zijn voor het implementeren van gastheergenetische markers in klinische toepassingen in de toekomst om vrouwen die vatbaar zijn voor tubapathologie te onderscheiden. Hoofdstuk 7 is de discussie over het gepresenteerde werk. Dit proefschrift draagt onder andere bij aan het optimaliseren van interventiestrategieën om CT-infecties onder controle te krijgen. Klinische richtlijnen zijn nuttig voor professionals en tegenwoordig is deze informatie voor het behandelen van urogenitale CT-infecties gemakkelijk (online) toegankelijk. Verbetering van zowel de kwaliteit en kwantiteit van CT-gerelateerde klinische trials bevordert het niveau van wetenschappelijk bewijs. Ten tweede dient de identificatie van vrouwen die risico lopen op het ontwikkelen van tubapathologie en dus onvruchtbaarheid te worden vereenvoudigd. Idealiter wordt een niet invasieve voorspellende tool ontwikkeld voor de triage van subfertiele vrouwen met potentieel tubapathologie. Deze tool zou bestaan uit een tweeledige assay met serologische markers én gastheer-genetische markers die gezamenlijk kunnen bijdragen aan het beter voorspellen van het wel of niet optreden van late complicaties bij vrouwen na een CT-infectie. Onnodig invasieve en kostbare procedures, zoals laparoscopieën, zouden dan grotendeels kunnen worden voorkomen.

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142 APPENDICES Acknowledgements - Dankwoord Curriculum Vitae List of publications