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Epidemiology
Original article
Urogenital Chlamydia trachomatis strain types, defined by high-resolution multilocus sequence typing, in relation to ethnicity and urogenital symptoms among a young screening population in Amsterdam, The Netherlands
  1. Bart Versteeg1,
  2. Michelle Himschoot1,
  3. Ingrid V F van den Broek2,
  4. Reinier J M Bom1,3,
  5. Arjen G C L Speksnijder1,4,
  6. Maarten F Schim van der Loeff5,6,
  7. Sylvia M Bruisten1,6
  1. 1Public Health Laboratory, Cluster Infectious Diseases, Public Health Service Amsterdam, Amsterdam, The Netherlands
  2. 2Epidemiology and Surveillance Unit, National Institute of Public Health and the Environment, Bilthoven, The Netherlands
  3. 3Condomerie, Amsterdam, The Netherlands
  4. 4Department of Research and Education, Naturalis Biodiversity Center, Leiden, The Netherlands
  5. 5Department of Research, Cluster Infectious Diseases, Public Health Service Amsterdam, Amsterdam, The Netherlands
  6. 6Center for Infections and Immunity Amsterdam (CINIMA), Academic Medical Center, University of Amsterdam, Amsterdam, The etherlands
  1. Correspondence to Dr Sylvia Bruisten, Public Health Service Amsterdam, Cluster of Infectious Diseases, Public Health Laboratory, Nieuwe Achtergracht 100, Amsterdam 1018 WT, The Netherlands; sbruisten{at}ggd.amsterdam.nl

Abstract

Introduction Previous studies found conflicting results regarding associations between urogenital Chlamydia trachomatis infections and ethnicity or urogenital symptoms among at-risk populations using either ompA-based genotyping or high-resolution multilocus sequence typing (MLST). This study applied high-resolution MLST on samples of individuals from a selected young urban screening population to assess the relationship of C. trachomatis strain types with ethnicity and self-reported urogenital symptoms. Demographic and sexual risk behaviour characteristics of the identified clusters were also analysed.

Methods We selected C. trachomatis-positive samples from the Dutch Chlamydia Screening Implementation study among young individuals in Amsterdam, the Netherlands. All samples were typed using high-resolution MLST. Clusters were assigned using minimum spanning tree analysis and were combined with epidemiological data of the participants.

Results We obtained full MLST data for C. trachomatis-positive samples from 439 participants and detected nine ompA genovars. MLST analysis identified 175 sequence types and six large clusters; in one cluster, participants with Surinamese/Antillean ethnicity were over-represented (58.8%) and this cluster predominantly consisted of genovar I. In addition, we found one cluster with an over-representation of participants with Dutch ethnicity (90.0%) and which solely consisted of genovar G. No association was observed between C. trachomatis clusters and urogenital symptoms.

Conclusions We found an association between urogenital C. trachomatis clusters and ethnicity among young screening participants in Amsterdam, the Netherlands. However, no association was found between C. trachomatis clusters and self-reported urogenital symptoms.

  • CHLAMYDIA TRACHOMATIS
  • CHLAMYDIA INFECTION
  • BACTERIAL TYPING

https://doi.org/10.1136/sextrans-2014-051790

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    Introduction

    Chlamydia trachomatis is the most common bacterial sexually transmitted infection (STI) worldwide and often causes asymptomatic infections.1 If not properly treated, these may result in severe complications including epididymitis in men and pelvic inflammatory disease in women leading to ectopic pregnancy and tubal factor infertility.2–4

    Strain typing is important to understand the epidemiology of C. trachomatis. Traditional strain typing of C. trachomatis was based on serotyping of the major outer membrane protein or analysis of its coding gene ompA and revealed 15 main genovars.5 Using either serotyping or ompA genotyping, variations in ethnicity and clinical manifestations by C. trachomatis serovar types have been studied, but results were inconclusive.6–12 These inconclusive results may partly be explained by extensive recombination in the ompA gene making it an unstable target for strain typing, as was recently demonstrated by whole genome analysis of C. trachomatis.5 In the last decade, genetic typing methods using multiple loci were developed, such as multilocus sequence typing (MLST) and multilocus variable number of tandem repeat analysis (MLVA).13–15 Both our and other studies reported that ompA genotyping was far less discriminatory compared with high-resolution MLST.16–19 High-resolution MLST may therefore provide more insight into the association between C. trachomatis infections and urogenital symptoms. Moreover, when combined with epidemiological data such as demographic characteristics and sexual risk behaviour, high-resolution MLST can help to identify transmission pathways of certain strains.17 To date, only two studies investigated the association between C. trachomatis infections and urogenital symptoms and neither found a statistically significant association.18 ,19 One study was performed in a small at-risk population with limited statistical power to detect a possible association, given the large variety in chlamydia sequence types (STs).18 The other study was performed among senior high school students in Norway, but no sexual risk behaviour was reported, and thus no insight was obtained into the transmission pathways of certain strains.19

    In a previous study, we investigated the association between C. trachomatis infections and ethnic groups, with a focus on sexual mixing between Surinamese migrants and native Dutch and Surinamese residents to identify transmission networks between Surinam and the Netherlands.20 However, as yet, no studies have been performed including individuals from a general urban population to investigate associations between C. trachomatis infections and specific ethnic groups.

    In the Netherlands, a Chlamydia Screening Implementation (CSI) programme was conducted aiming to investigate the additional effect of annual, systematic chlamydia screening in existing STI testing facilities.21 Participants were aged 16–29 years old and invited on an annual basis. Most participants lived in Amsterdam, a city with a large diverse ethnic population. The CSI study, however, was characterised by a low response rate and, unfortunately, data on demographic and sexual risk behaviour of those who refused to participate are lacking. Therefore, the CSI population may not be truly representative of the general population.

    As the CSI study had samples and data of a selected young, sexually active population living in Amsterdam, it provided a unique opportunity to: (1) investigate the relationship of C. trachomatis clusters, defined by MLST, with ethnicity and urogenital symptoms in a selected urban population of young screening participants and (2) identify epidemiological characteristics of C. trachomatis MLST clusters within this population.

    Methods

    Study population and data collection

    Participants of the CSI programme were recruited among residents in Amsterdam, Rotterdam, and South Limburg from 2008 to 2010, and three screening rounds were conducted.21–23 All individuals aged 16–29 years, identified by municipal registries, received postal invitations. The CSI programme had a participation rate of 16% after the first invitation, which declined to 10% after the third invitation.21 Chlamydia prevalence among participants was around 4.5% and did not substantially decrease during the study period.21 Data and samples from the Amsterdam part of the CSI programme in the Netherlands were used and included samples from all three screening rounds. All participants received a test package and were requested to complete an online questionnaire on a voluntary basis. Data collection included age, gender, ethnicity, residential district, sexual risk behaviour, history of STI and self-reported urogenital symptoms. Male participants were requested to provide a urine sample for testing, whereas female participants were requested to provide a vaginal swab or urine sample for testing. All collected urine samples and swabs were tested for the presence of C. trachomatis RNA using the Aptima CT-single assay (Hologic/Gen-Probe, San Diego, California, USA) at the Public Health Laboratory in Amsterdam. Positive results were confirmed with the Aptima combo assay. Positive C. trachomatis samples were stored at −80°C. Participants were asked their consent to store and use their samples for further research.21 For this study, we only included the first provided sample from each participant who gave informed consent. The medical ethics committee of the Free University of Amsterdam approved the CSI study.

    DNA amplification

    DNA from all Aptima combo positive samples was extracted by isopropanol precipitation and retested for the presence of chlamydial DNA using an inhouse pmpH real time PCR.24 ,25 For DNA samples that tested negative, DNA was re-extracted from the original samples and retested. All samples that repeatedly tested negative were excluded. All isolates with a cycling threshold ≤35 in the pmpH real time PCR were regarded suitable for further typing.

    Nested PCR and sequencing of MLST regions

    DNA isolates were amplified by a nested PCR for the regions ompA, CT046 (hctB), CT058, CT144, CT172 and CT682 (pbpB) as described previously.16 ,17 The inner PCR was performed with M13-tagged primers as described previously.17 ,20 The M13-tagged amplified DNA was processed at a central sequence facility using primers that specifically target the M13-amplified region.

    MLST data analysis

    The obtained sequences were assembled and trimmed using BioNumerics 7 (Applied Maths, Sint-Martens-Latem, Belgium) and checked against the C. trachomatis MLST database (http://mlstdb.bmc.uu.se). Samples in which all six loci were successfully amplified, sequenced and identified obtained an MLST ST. Incomplete and low quality samples were reamplified and resequenced. Minimum spanning trees were generated with BioNumerics 7 using the MLST STs. A cluster was defined as a group of STs in which each ST differed by not more than one locus from another ST. Clusters containing 10 or more samples were defined as large clusters, which were considered unique strain types. We combined small clusters (n<10) and singletons into a residual group for statistical analysis.

    Statistics

    Ethnicity was established based on the country of birth of the participant and that of his/her parents (from the municipal register) following definitions used by the National Bureau of Statistics.21 When the person and his/her parents were born in the Netherlands, the ethnicity was Dutch. When the mother was born in another country, the person had the ethnicity of the mother. When both the mother and the person himself/herself were born in the Netherlands, or the country of birth of the mother was unknown, the country of birth of the father was decisive for the ethnicity of the participant. Because the numbers of participants in all ethnic groups except the Dutch and Surinamese/Antillean were small, we grouped African, Asian, Western European, Eastern European, and Central and South American ethnicities together into one remaining group of ethnicity for statistical analyses. For women, urogenital symptoms included: vaginal discharge, blood loss between menses, blood loss after or during sex, dysuria, recurrent need to urinate, and lower abdominal pain. For men, urogenital symptoms included: dysuria, recurrent need to urinate and penile discharge. Due to the low number of people who reported urogenital symptoms, these were all considered one group. History of STI included a history of chlamydia, gonorrhoea, syphilis or genital warts. Due to the low number of people who reported a history of STI, these were all combined into one group.

    Differences between groups and clusters were tested using the Pearson χ2 test for categorical data. The Fisher exact test was used when an expected cell count was <1. For continuous variables, Mann–Whitney U tests and Kruskall–Wallis tests were used. A p value <0.05 was considered statistically significant. Analyses were performed with SPSS package V.21.0 (SPSS, Chicago, Illinois, USA).

    Results

    Study population

    From April 2008 until November 2010, a total of 2217 participants from the Amsterdam part of the CSI study had a positive C. trachomatis sample at enrolment, of whom 638 (28.8%) provided informed consent. Of these, 572 samples (25.8%) were available for typing analysis (see online supplementary figure S1). In 514 of 572 samples (89.9%), sufficient chlamydial DNA could be demonstrated using real time PCR and for 474 (82.9%) samples full MLST profiles could be obtained. Of these, 34 samples (7.2%) had to partly be reamplified and resequenced in order to obtain a complete profile. We excluded 26 participants who lived outside of Amsterdam, and nine participants for whom no information regarding ethnicity was available in the municipal population register. Overall, no significant differences were observed for age, gender, ethnicity or residential district between participants with fully typed samples (n=439, see online supplementary figure S1; Group A) and those excluded (n=133). A subgroup of 342 (59.8%) participants also completed the online questionnaire (see online supplementary figure S1; Group B).

    Genovar distribution

    Since ompA is part of the MLST scheme, genovars could be assigned to all fully typed samples (table 1). Among the 439 participants, we found nine different genovars: B, 0.2% (n=1); D, 12.3% (n=54); E, 40.5% (n=178); F, 20.7% (n=91); G, 9.1% (n=40); H, 2.5% (n=11); I, 3.9% (n=17); J, 5.9% (n=26); and K, 4.8% (n=21).

    View this table:
    Table 1

    Demographic characteristics and sexual risk behaviour characteristics of CSI participants with Chlamydia trachomatis infections per ethnicity, Amsterdam, 2008–2010

    Comparison of demographic and sexual risk behaviour characteristics between ethnic groups

    Demographic characteristics and sexual risk behaviour characteristics of participants are shown in table 1. The majority of these were female participants (72.7%) and more than half of the participants were of Dutch ethnicity (53.5%). Furthermore, a majority of participants had no history of STI (86.3%), reported no same-sex experience (96.9%) and had no current urogenital symptoms related to STI infection (75.4%). Participants from Surinamese/Antillean ethnicity (median age of 20 years (IQR 18–23)) were often younger than participants of Dutch (median 23 years (IQR 18–23)) or other ethnicities (median age 24 years (IQR 22–27)). Moreover, many participants from Surinamese/Antillean ethnicity lived in the South-East district of Amsterdam, whereas participants with Dutch or other ethnicities were more equally distributed over the residential districts. In addition, we found that participants with Dutch or Surinamese/Antillean ethnicity more often had a history of STI compared with other ethnicities (table 1).

    MLST analysis

    Data for 439 participants were available for analysis, and using their complete MLST profile, 175 STs were identified. Of these, 67 (38.3%) had multiple representatives ranging from 2 to 59 samples comprising 75.4% of all samples. The remaining 108 STs were found in only one isolate each (singletons). A minimum spanning tree was generated in which six large clusters could be identified (figure 1). These clusters ranged from 10 to 197 samples comprising 79.5% of all samples. The remaining 90 samples were distributed over 49 singletons and nine small clusters.

    Figure 1
    Figure 1

    Minimum spanning tree showing the multilocus sequence typing pattern of 439 Chlamydia trachomatis-positive samples of CSI participants. Each circle represents one sequence type (ST). The size of the circles is proportional to the number of samples with identical ST profiles. Bold lines connect types that differ for one single locus. Halos indicate the distinct clusters. Colours indicate the ethnic groups; orange: Dutch ethnicity (n=235); green: Surinamese/Antillean ethnicity (n=88); and brown: other ethnicities (n=116).

    Cluster analysis

    Cluster analysis was performed for 439 participants with demographic data (figure 1) of whom 342 provided complete questionnaire data on sexual risk behaviour (figure 2). Among all 439 participants, no significant differences between clusters were observed regarding gender and residential district (table 2A). We did, however, observe a significant difference in age between clusters (p=0.005). Participants of cluster III were older with a median age of 25 (IQR 23–28) compared with a median age of 22–24 years among participants of all other clusters. In addition, significant differences were observed in the proportion of ethnic groups between clusters (p=0.036). Cluster V consisted of genovar I and genovar J, and contained a larger proportion of participants with Surinamese/Antillean ethnicity (58.8%). Moreover, more cluster V participants (41.2%) lived in the South-East of Amsterdam compared with all other clusters (varying between 9% and 20%). In contrast, cluster VI consisted only of genovar G and had a larger proportion of participants with Dutch ethnicity. These participants with Dutch ethnicity were equally distributed over the residential districts of Amsterdam. Among the subgroup of 342 participants, no significant differences between clusters were observed regarding sexual risk behaviour (table 2B). Also, no significant differences were observed in the proportions of participants with current STI-related urogenital symptoms (figure 2).

    View this table:
    Table 2

    Demographic characteristics and sexual risk behaviour characteristics of CSI participants per Chlamydia trachomatis MLST cluster, Amsterdam, 2008–2010

    Figure 2
    Figure 2

    Minimum spanning tree showing the multilocus sequence typing pattern of Chlamydia trachomatis-positive samples from CSI participants who provided complete questionnaire data. The total number of participants was 439, of whom 342 reported symptoms. Each circle represents one sequence type (ST). The size of the circles is proportional to the number of samples with identical ST profiles. Bold lines connect types that differ for one single locus. Halos indicate the distinct clusters. Colours indicate symptomatology; white: unknown symptomatology (n=97); pink: self-reported urogenital symptoms (n=84); and blue: no self-reported urogenital symptoms (n=258).

    Discussion

    Using high-resolution typing, we identified an association between C. trachomatis clusters and ethnicity in a selected young sexually active population living in Amsterdam, the Netherlands. We observed that one C. trachomatis cluster (cluster V, figure 1) consisted mainly of participants of non-Dutch ethnicity, of whom the majority were of Surinamese/Antillean ethnicity. In addition, in another C. trachomatis cluster (cluster VI, figure 1), participants with Dutch ethnicity were over-represented. However, we did not observe an association between the identified C. trachomatis clusters and urogenital symptoms.

    The majority of infections among participants involved genovars D, E and F, which are known to be the most prevalent genovars among heterosexual populations worldwide.8 ,26 Interestingly, we also found a high prevalence of genovar G, whereas other studies among heterosexuals in the Netherlands reported a high prevalence of genovar I.17 ,20 Considering the small number of participants reporting same-sex experiences (3.1%), it is unlikely that the increased genovar G prevalence is related to the men who have sex with men culture and population in Amsterdam. This difference in genovar prevalence may be explained by the fact that other studies in the Netherlands recruited participants at an STI clinic, whereas the CSI participants were recruited among the general population. However, as it cannot be ascertained that the CSI population is representative of the general population, it remains unknown whether the distribution of C. trachomatis genovars and STs truly differs between the STI clinic population and the general population.

    We observed differences between the identified C. trachomatis clusters and corresponding ompA genovars among participants with Dutch ethnicity and those with Surinamese/Antillean or other ethnicities. Participants with Dutch ethnicity were less often infected with genovars I and J compared with participants with Surinamese/Antillean or other ethnicities. In addition, participants of Dutch ethnicity were more often infected with genovar G. Our results therefore support previous studies, which reported associations between ethnicity and C. trachomatis serogroup distributions.9 ,12 Moreover, a greater proportion of cluster V participants lived in the South-East of Amsterdam, which is known for its high number of Surinamese and Antillean migrants and low socio-economic status. We previously reported that sexual mixing between Surinamese migrants and native Dutch and Surinamese residents occurs frequently. However, this sexual mixing may not be sufficient for chlamydia transmission. Therefore, strain types may not effectively mix between both populations and thus prevent infections from spreading to other risk populations, allowing differences in prevalence to be sustained.20 In this previous study, we observed a similar cluster containing both genovars I and J that was over-represented among Surinamese migrant and native Surinamese individuals. In accordance, these STs coincide with the STs from cluster V predominantly consisting of samples from Surinamese/Antillean participants. Further research is needed, as it is unknown whether the association between ethnicity and C. trachomatis clusters is restricted to the selected population of young screening participants or whether this association also exists among the general population.

    It is known that high-risk sexual behaviour is more common among people with Antillean and Surinamese ethnicity; they more often have multiple partners in the preceding 6 months, have their first sexual experience at a younger age and are less likely to use a condom.12 ,20 ,23 Besides higher sexual risk behaviour, a recent study found that higher C. trachomatis prevalence among Surinamese/Antillean people could also be explained by differences in education and neighbourhood, which are both markers for socio-economic status.27 Therefore, it is most likely that the association between ethnicity and the type or strain of C. trachomatis reflects differences in socio-economic status, sexual risk behaviour and relative scarcity of mixing of sexual partners between various ethnic groups.12 ,20 ,23 ,27 ,28

    In accordance with previous studies, no relationship was found between C. trachomatis clusters and the presence of urogenital symptoms suggesting that no C. trachomatis strains exist that specifically cause symptomatic or asymptomatic urogenital infections.18 ,19 MLST is limited by the fact that only a small fraction of the genome is used for typing, and so samples that are indistinguishable with respect to the MLST target regions may still have (considerable) variation in the remaining DNA. Whole genome analysis may therefore provide more insight into the possible association between C. trachomatis strain types and urogenital symptoms. In addition, human host factors and genetics may also play an important role in the development of symptomatic infections.

    Some limitations of this study should be noted. A majority were female participants which may have introduced a bias, causing the identified relationships to be more associated with C. trachomatis infections in women compared with men. The uptake for screening is generally higher among women compared with men, which was also observed in the CSI programme.21 Furthermore, the urogenital symptoms were not specific for C. trachomatis infections. Infectious agents that were not tested for could have caused these and reported symptoms may therefore have both low sensitivity and low specificity. This may have introduced a bias and limited our capacity to detect associations between reported urogenital symptoms and C. trachomatis infections. Another limitation is that, due to the sample size, various ethnicities were grouped together in one ‘other’ group, meaning that the study could not investigate associations between clusters and other ethnic groups, besides Surinamese/Antilleans and Dutch. New studies should therefore be carefully designed to investigate associations between clusters and other ethnicities.

    In conclusion, we found an association between C. trachomatis clusters and ethnicity in a selected young sexually active population in Amsterdam, the Netherlands. However, no association between C. trachomatis clusters and urogenital symptoms could be observed. Further research, using whole genome analysis, may provide more insight into the possible associations between C. trachomatis infections and urogenital symptoms. This knowledge is needed to gain more insight into the causes of symptomatic and asymptomatic infections, as well as improving current screening and prevention programmes aiming to reduce C. trachomatis transmission.

    Key messages

    • Previous studies found conflicting results regarding associations between urogenital Chlamydia trachomatis infections and ethnicity or urogenital symptoms among at-risk populations.

    • High-resolution multilocus sequence typing enables more accurate distinction between C. trachomatis strain types than ompA typing in relation to ethnicity.

    • Among a selected population of young screening participants in Amsterdam, urogenital C. trachomatis clusters were associated with ethnicity, but not with reported symptoms.

    Acknowledgments

    The authors thank the CSI study project group consisting of Jan van Bergen, Han Fennema, Hannelore Götz, Christian Hoebe, Eelco Over, Marianne van der Sande, Boris Schmid and Eline Op de Coul.

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    Supplementary materials

    • Supplementary Data

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    • Abstract in Dutch

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    Footnotes

    • Handling editor Jackie A Cassell

    • Contributors AGCLS and SMB contributed to the design of this study to investigate the relation between Chlamydia trachomatis strain types and both ethnicity and symptomatology. AGCLS was involved in the testing of samples for C. trachomatis positivity. MH carried out the MLST typing under supervision of SMB and RJMB. IVFvdB collected and analysed the CSI data. BV, SMB and MFSvdL performed the data analyses. BV and SMB drafted the paper. All authors contributed to the interpretation of the results, writing the manuscript and gave intellectual feedback. All authors have contributed to, have seen, and approved the final, submitted version of the manuscript.

    • Funding This work was supported by the Public Health Service of Amsterdam (GGD Amsterdam), the Netherlands.

    • Competing interests None.

    • Ethics approval Medical Ethics Committee of the Free University of Amsterdam.

    • Provenance and peer review Not commissioned; externally peer reviewed.