Clinical characteristics, serology and serovar studies on Chlamydia trachomatis

Transcription

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

Clinical characteristics, serology and serovar studies on Chlamydia trachomatis

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