Chlamydia trachomatis is an obligate intracellular bacterium commonly causing sexually transmitted infection in men who have sex with men (MSM), being a significant source of morbidity.1 There are 15 serovars of C. trachomatis characterized based on antigenic variations of the major outer membrane protein, and are associated with factors such as sexuality, race, exhibition of symptoms, histopathology, and spontaneous resolution of infection.2–8 C. trachomatis serovars A–C are primarily associated with ocular disease such as trachoma; serovars D–J with sexually transmitted urogenital disease as well as conjunctivitis,9 and serovars L1–L3 are responsible for a painful and often serious condition known as lymphogranuloma venereum (LGV).10,11 The gene that encodes for major outer membrane protein, omp1, consists of 4 variable domains (VDI-VDIV) that enable serovar prediction based on sequence variations,12 enabling rapid molecular screening of populations.
The estimated prevalence of rectal C. trachomatis infection in MSM in the United Kingdom and United States ranges from 6.0% to 8.5%, being primarily asymptomatic (52%–86%). Data from studies of MSM in the United Kingdom suggest that urethral infections are less prevalent (3.3%–5.4%) and largely symptomatic (67.7%–68.5%).13–18 Based on studies conducted in the United States and Sweden, the most common serovars identified in MSM are G and D (45.2%–47.9% and 26.9%–29.6%, respectively), followed by serovar J.14,19 Little data exists regarding C. trachomatis infection in Australian MSM, though a study of HIV-negative MSM reported comparable incidence rates of urethral and anal C. trachomatis infection of 7.43 and 4.98 per 100 person-years, respectively.20 The only Australian data on C. trachomatis serovars among MSM was derived from 39 samples collected from men who frequented sex-on-premises (SOP) venues with the predominant serovars being D (53.8%), G (25.6%), and J (10.2%).5 This article presents results from the largest study conducted on C. trachomatis serovars and genotypic variants found in Australian MSM.
MATERIALS AND METHODS
Study Population and Sample Collection
A total of 612 C. trachomatis positive specimens from MSM visiting the Melbourne Sexual Health Centre (MSHC) and Sydney Sexual Health Centre (SSHC) between July 2004 and August 2008 were analyzed. Samples were deidentified; so matching of sites from the same patient was not possible. MSHC collected samples from 2004 to 2006, while SSHC collected samples from 2005 to 2008, with no samples obtained by this centre during 2006. Ethical approval for this study was obtained from the South East Sydney Illawarra Area Human Research Ethics Committee and The Alfred Human Research Ethics Committee. Demographic information including age, place of residence, reported overseas sexual contact during the previous 12 months, whether site specific symptoms were present, HIV status, Neisseria gonorrhoeae coinfection, prior C. trachomatis infection (self-reported), and self-reported number of sexual partners during the previous 3 and 12 months was collected.
Serovar and Genotype Determination of C. trachomatis Infections
The methodology used for the initial C. trachomatis testing, quantitation and subsequent determination of C. trachomatis serotypes via omp1 gene sequencing and qPCR screening have been described previously.21 To determine C. trachomatis genotype, partial omp1 gene sequences used for serovar determination were aligned using ClustalW22 with the SeaView and MEGA 4 programs.23,24 Sequences with ≥1 bp difference were binned together as a genotype of that particular serovar, with multiple genotypes of a serovar given an arbitrary number (e.g., Di, Dii). The Genbank accession numbers for all sequences generated from this study, as well as reference sequences, are presented in Table 3.
All analyses were restricted to individuals with a successfully assigned C. trachomatis serovar classification. Demographic and sexual health variables including C. trachomatis serovar (pooled and by year) were stratified by city, and compared using a chi square test (categorical variables), or a Wilcoxon-Mann Whitney test (non-normal continuous variables). City, age, number of sexual partners, concurrent gonorrhea, HIV status, prior C. trachomatis infection, overseas sexual contact, and the presence of symptoms were assessed as predictors of the predominant serovars using logistic regression. An additional logistic regression analysis was performed to identify predictors of the presence of symptoms for all samples, and stratified by site of infection. Results were reported as odds ratios (OR) with 95% confidence intervals (CI). The mean log concentrations of C. trachomatis for each serovar/site were compared using an ANOVA test. All analyses were performed using Stata 11.25
A total of 612 C. trachomatis positive samples (344 anal swabs, 265 urine samples) were evaluated from MSM patients aged 18 to 73 at MSHC (Melbourne) and SSHC (Sydney) between 2004 and 2008. Of these samples, 571 (93.3%) were able to be successfully assigned a C. trachomatis serovar classification, 521 by omp1 gene sequencing, 528 by qPCR, and 482 by both methods. Anal swabs comprised 323 of these samples (Melbourne n = 158, Sydney n = 165) and there were 249 urine samples (Melbourne n = 147, Sydney n = 102). Overall, participants in Melbourne (n = 304) and Sydney (n = 267) were similar in age (range, 18–73 years), HIV status, overseas sexual partners during the previous 12 months, and number of partners in the previous year (Table 1). MSM in Melbourne were more likely to present with symptoms, and have concurrent gonorrhea, while their Sydney counterparts were more likely to have reported a prior chlamydial infection (P < 0.05).
C. trachomatis Serovar Distribution
The most common serovars detected were of the B complex group (serovars B, D, E, L2: 43.9%) followed by the Intermediate group (F, G: 36.7%) and C complex group (H, I, J, K: 19.4%), which are based on phylogenetic divisions of the omp1 gene.11 The most prevalent serovar was D (35.2%), followed by G (32.7%) and J (17.7%) (Table 1). The distribution of serovars was similar in Melbourne and Sydney, with the exception of serovar D (40% vs. 30%, P = 0.02) and serovar E (6% vs. 14%, P < 0.001) Only 4 cases of serovar H were identified in samples from Sydney, and the single case of serovars B, I, and L2 (L2b) were from Melbourne. C. trachomatis serovars A, C, L1, and L3 were not detected in this study. The proportion of each C. trachomatis serovar was similar irrespective of anatomical sampling site (data not shown), with the exception of serovar F, which was more common in urine samples compared to anal swabs (7.3% vs. 2.8%, respectively, P = 0.01).
There was a marked shift in the distribution of C. trachomatis serovar positivity between 2004 and 2008, with only serovar G remaining constant over this time period irrespective of city. For Melbourne, the proportion of serovar D samples increased (P < 0.01) in each consecutive year samples were collected, while the proportion of serovar J decreased (P = 0.02). In Sydney, a similar trend was observed for serovar D (P < 0.01); however, there was evidence of decreasing prevalence for serovar E and F (P < 0.01). No change was observed for serovar J (Table 2).
Quantification of C. trachomatis Load
Of the 528 samples assigned to serovars by qPCR, 429 (81.3%) C. trachomatis infections were quantified (including 4 mixed infections) with an average of 1.48 × 104 genome copies/anal swab (log10 = 4.17) and 3.72 × 103 copies/mL (log10 = 3.57) in urine samples. The mean log concentrations and variance were similar across serovar for each site (ANOVA, P = 0.17), with the detectable levels in anal swabs were consistently higher than samples of 1 mL of urine (Fig. 1). The mean and median concentrations shown for serovar F varied due to the low number of samples. The C. trachomatis load was not associated with a prior C. trachomatis infection, the presence of symptoms or any demographic information pertaining to the individual, including age (data not shown).
We identified 11 cases (2.6% of total quantified) of mixed C. trachomatis infection, 9 from Sydney (4.6% of total quantified for Sydney: 2 D + J, 2 G + J, 2 J + K, and 1 each of D + E, E + J, and H + K) and 2 from Melbourne (0.9% of total quantified for Melbourne: E + J and F + D). Mixed infections were equally distributed amongst urine and anal swab samples. Four of these infections had quantifiable C. trachomatis load; 2 were anal swab samples from Sydney (5.8 × 105 genome copies/swab serovar G and 2.3 × 104 copies/swab serovar J; 3.5 × 105 copies/swab serovar E and 2.1 × 105 copies/swab serovar J), 1 was a urine sample from Sydney (2.0 × 103 copies/mL serovar D and 1.3 × 105 copies/mL serovar J), and 1 was a urine sample from Melbourne (3.1 × 105 copies/mL serovar F and 5.0 × 105 copies/mL serovar D).
Predictors of Specific C. trachomatis Serovar Positivity
After adjusting for other demographic and sexual health factors, living in Sydney and having a previous C. trachomatis infection were predictors for serovar E infection (city OR: 2.75, 95% CI: 1.41–5.36; previous CT infection OR: 2.32, 95% CI: 1.19–4.52). Conversely, individuals with serovar J infection were less likely to have had a prior C. trachomatis infection (OR: 0.50, 95% CI: 0.26–0.95), and presenting with symptoms was significantly associated with serovar J positivity (OR: 1.90, 95% CI: 1.19–3.05). Interestingly, the only significant predictor of serovar G was previous sexual contact overseas (OR: 0.52, 95% CI: 0.32–0.85), suggesting that this exposure might have a protective effect against infection with this serovar. There were no significant predictors of serovar D or F positivity (data not shown).
Predictors of Symptoms
Symptoms were more common in MSM with urethral C. trachomatis infection compared to anal infection (68% vs. 28%, respectively, P < 0.001), and from Melbourne compared to Sydney (53% vs. 37%, respectively, P < 0.01) (data not shown). In a multivariable model, the significant risk factors for the presentation of symptoms were increasing age, gonorrheal coinfection, city, and a history of prior C. trachomatis infection (Table 3). When restricting the analysis to anal infections, the associations with city and history of prior C. trachomatis infection remained the same (city OR: 0.42, 95% CI: 0.23–0.74; prior C. trachomatis OR: 2.21, 95% CI: 1.09–4.45), while the association with a concurrent gonorrheal infection strengthened (OR: 5.95, 95% CI: 2.97–11.92). Age was no longer a significant risk factor. When the analysis was restricted to urethral infections, age remained a significant risk factor (18–25 years = reference; 26–30 years = OR: 1.74, 95% CI: 0.81–3.72; 31–35 years = OR: 2.88, 95% CI: 1.13–7.39; 36–40 years = OR: 2.59, 95% CI: 1.07–6.25; 40+ years = OR: 5.10, 95% CI: 1.67–15.65), as did gonorrheal coinfection (OR: 3.79, 95% CI: 1.06–13.55). City and prior C. trachomatis infection were no longer significant risk factors.
The frequency of reporting a history of C. trachomatis infection was most common in MSM between 26 and 40 years of age (25.51%), and less common in those over 40 years of age (16.92%), and those 18 to 25 years of age (11.95%) (P = 0.008). There was no association between age group and gonorrheal coinfection. In addition, there was no association between the presence of symptoms and the concentration of C. trachomatis detected, and equally no association was found comparing symptoms to those samples where no concentration was attainable.
C. trachomatis Genotype Distribution
Of the 22 genotypes identified in this study, 14 were able to be assigned a 100% match to sequences on Genbank, corresponding to 96.9% of the infections genotyped (n = 521) (Table 4). In comparison to the most abundant genotype for each serovar, there were 13 variant serovars, comprising 7.5% (n = 39) of the total number of samples, and 4 of these were solely silent mutations. The majority of variations identified in this study occurred in the conserved regions of the omp1 gene. Phylogenetic analysis of the partial amino acid coding sequences of the omp1 gene shows the largely homogenous distribution of genotypes in this study that group within their predicted clades.11 There was no significant difference between these genetic variants found in urine versus rectal samples and given the overall low level of genetic variability, there was no discernable difference between the C. trachomatis serovars and genotypic variants when compared to the demographic factors in this study (data not shown).
This was the largest study of its kind in MSM to date and depicts a high self-reported rate of prior C. trachomatis infection and median number of sexual partners over 12 months. The rate of HIV infection in this population was consistent with previous studies for the Sydney MSM community (8.0%), although was nearly double for Melbourne (9.2% vs. 5.0%).26 The proportion of individuals who exhibited symptoms for urethral and rectal C. trachomatis infection in our study was consistent with UK clinic-based MSM data,13,18 but was higher than findings from a community-based cohort study in Sydney.20 Interestingly, more anal infections from Melbourne showed symptoms than those from Sydney, as were those with a prior C. trachomatis infection. The age of the patient and prior C. trachomatis infections were associated, and both correlated with the likelihood of symptoms being present. This correlates with previous research on C. trachomatis infection in Australian heterosexual men showing symptoms were more likely to occur in those who were older with a history of C. trachomatis infection.27 This suggests that increasing age may be a surrogate marker for a prior C. trachomatis infection, and symptom recognition improves with recurrent infections, although further work is warranted to explore the possibilities of persistence and/or an immune mediated response. The age of the patient was more predictive of symptoms when looking at urethral infections, although symptoms declined for this group over the age of 40.
There appeared to be no association between C. trachomatis serovar (or genotypic variant) and anatomical site of infection, with the exception of serovar F more common in urethral infections, albeit with low sample numbers. The single LGV case identified in this study was from an anal swab collected from a symptomatic HIV-infected Melbourne MSM, supporting findings indicating a low prevalence of LGV in Australian MSM, primarily in those who are HIV-positive with no evidence of a subclinical pool.28
Of the anal swab and urine samples collected and typed, the predominant serovars were D, G, and J, consistent with other international studies.14,19 Compared to the Australian MSM SOP study of 39 samples collected by Lister et al in 2001–2002,5 our proportionate yield of serovar D was lower (55.1% vs. 35.2%), possibly attributable to our larger sample size or a more concentrated pool of serovar D in SOP venues. The majority of isolates in our study were 100% identical to that of the SOP samples (genotypes B, Di, Dvii, Ei, Gi, and Ji), and the genotypic variations in both studies were primarily associated with conserved regions of the omp1 gene though our study revealed more amino acid substitutions (our study = 13/24 substitutions; SOP = 4/12 substitutions).
The predominant serovars from our study differed over time, with the proportion that were serovar D consecutively increasing from year to year in both Melbourne and Sydney while serovar G remained constant. Interestingly, serovar G was also inversely associated with sexual contact overseas, and it would be of interest to see the distribution of the dominant genotype of this serovar in MSM communities around the world. Patients infected with serovar J were more likely to exhibit symptoms and less likely to associated with a prior C. trachomatis infection. Serovar J was also present in the majority of mixed infections. The overall prevalence of mixed C. trachomatis serovar infections in this study was relatively low, 2.6%, with a far lower rate in samples from MSHC (0.9%) compared to SSHC (4.6%), although these figures do not include multiple infections of the same serovar, and most were near the detection limit of the assays used. The majority of cases required a nested step for qPCR detection, suggesting a low C. trachomatis load in mixed infections.
There was a higher load of C. trachomatis detected in anal swabs than urine samples, although we did not observe a relationship between copy number and the presence of symptoms. This highlights the possibility that many MSM may be asymptomatic and yet carry a high load of C. trachomatis, suggesting subclinical infectivity and low correlation of the presence of symptoms and infection, particularly with rectal C. trachomatis. This is further supported by Annan et al stating that there is a reservoir of undiagnosed rectal C. trachomatis infections in the MSM community,13 and by Lister et al emphasizing there is no clear association between rectal symptoms and presence of infection.29 The log mean and variance of the C. trachomatis concentrations detected in urine samples was comparable to that cited by Michel et al30 (P = 0.37), despite different methodologies used, and to our knowledge this is the first paper to report C. trachomatis loads for MSM rectal infections. The low level of genetic variability detected within the observed C. trachomatis serovars indicates a conserved genetic pool that is of interest particularly as nearly a quarter of the MSM involved in our study reported overseas sexual contact during the previous 12 months and the distribution of these serovars changed markedly from year to year. The changes in prevalence were not consistent between Melbourne and Sydney, and further differences were identified between these 2 cities, with Melbourne MSM more likely to be symptomatic and have a gonorrheal coinfection while Sydney MSM were more likely to report a prior C. trachomatis infection. Additionally, serovar E infections were more common in Sydney, particular in 2005. It is unclear whether these trends can be generalized to a wider population of MSM in each city, or if variations occur within subpopulations. Therefore, it is important to assess whether our findings are reproducible in other samples from MSM in Melbourne and Sydney. Further work is also warranted to investigate the host relationships between the detected serovars and anal/urethral infections, and to assess the distribution of C. trachomatis serovars over time among MSM in the community. In conclusion, this study gives the most comprehensive overview to date of the predominant genetic variants of C. trachomatis among MSM in Australia.
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