Chlamydia trachomatis Serovar Distribution and Neisseria gonorrhoeae Coinfection in Male Patients with Urethritis in Greece

Chlamydia trachomatis is the leading cause of bacterial sexually transmitted diseases (STDs) in industrialized countries, producing infections of the upper and lower genital tract in males and females, including urethritis, epididymitis, and proctitis in males (7, 9, 14, 19). Currently, more than 18 different serovars of the organism have been identified based on conventional serotyping, while more than 29 variants have been recognized by employing monoclonal antibodies or genotypic methods (1, 15).

The knowledge of the distribution profile of C. trachomatis urogenital serovars has been the focus of several studies in different regions worldwide, since it provides valuable information about their epidemiology and pathogenicity that contributes to the implementation of sufficient STD control measures. The most common serovar detected worldwide is E (up to 22 to 49% of cases) followed by serovars F and D (17 to 22% and 9 to 19%, respectively), while other serovars are less frequently identified (1, 10, 12, 15, 16, 17).

No data are available in Southern European countries, such as Greece, about the circulating C. trachomatis serovars among either general or specific populations, apart from a previous study in Italy that examined the C. trachomatis serovar distribution in a group of male patients with urethritis (4). The C. trachomatis serovars D through K, including the serovars Da and Ia and the genovariant Ja, are related to genital tract disease (2). Moreover, on a worldwide basis, although concomitant infection with Neisseria gonorrhoeae and C. trachomatis is well established and frequent according to epidemiological data (3, 5, 11, 20), very little is known about the molecular biology-based identification of N. gonorrhoeae coexisting infection and its association with C. trachomatis serovars.

The objectives of this study were to provide novel data regarding the C. trachomatis serovar distribution in this specific geographical area, by examining a specific group of symptomatic male patients with C. trachomatis urethritis living in Greece, and also to investigate the presence of N. gonorrhoeae coinfection by employing molecular methods and to discover any possible links of N. gonorrhoeae coinfection with particular C. trachomatis serovars.

(This study was presented in part at the 18th European Congress of Clinical Microbiology and Infectious Diseases, Barcelona, Spain, 19 to 22 April 2008.)

The first 100 C. trachomatis-infected males who consecutively presented at the outpatient clinic of the Andreas Sygros Hospital for Skin and Venereal Diseases in Athens during the period 2006-2007 were included in the study. All of them presented clinical signs and symptoms of acute urethritis defined as follows: presence of urethral discharge and/or dysuria in combination with the detection of five or more polymorphonuclear leukocytes per high-power field on the intraurethral swab specimen. Primary demographic, epidemiological, and clinical data were recorded for each patient. The protocol of the study was reviewed and approved by the ethics committee of the hospital.

Conventional detection of N. gonorrhoeae on intraurethral swabs was performed using Gram staining and culture on Thayer-Martin medium. Molecular detection of both C. trachomatis and N. gonorrhoeae on intraurethral swabs was performed using the C. trachomatis/N. gonorrhoeae Cobas Amplicor system (Roche Inc., Branchburg, NJ).

Chlamydial serovar assignment was performed after PCR amplification and DNA sequencing of the C. trachomatis omp1 gene, as previously described (2). PCR products were purified using the GFX PCR DNA and gel band purification kit (Amersham Biosciences UK Ltd., Little Chalfont, Buckinghamshire, United Kingdom), and sequencing was performed with the internal primers in an ABI 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA). Partial sequences were aligned using the DAMBE 4.2 software (21); a consensus sequence for the whole omp1 gene of each strain was obtained and compared with available sequences in GenBank.

Statistical analysis was performed using the SPSS 13.0 statistical software. Chi-square and Fisher's exact tests were used for the comparisons of proportions. A P value of ≤0.05 was considered to be statistically significant. A multiple logistic regression analysis was conducted in order to estimate the independent risk factors associated with N. gonorrhoeae coinfection.

The main demographic and epidemiological data of the total participants are presented in Table 1. Types E and G were the most common, being detected in 37% and 23% of the participants, respectively. Types F, Ja, and D were identified in lower percentages of the patients (14%, 12%, and 10%, respectively), while 4% of patients had an infection due to type B. Mixed C. trachomatis infections were not identified. No differences in demographic, epidemiological, and clinical characteristics were established among patients infected by different C. trachomatis serovars.

The overall N. gonorrhoeae coinfection rate was 30%. The N. gonorrhoeae coinfection rates among the distinct C. trachomatis serovars detected in our study are presented in Table 2. Patients with an infection due to genovariant Ja had a significantly higher coinfection rate (75%, P = 0.008), whereas coinfection rates among patients with serovars D, F, G, and E were 40%, 28.6%, 21.7%, and 21.6%, respectively. Multiple analyses revealed that independent predictors for N. gonorrhoeae coinfection were genovariant Ja, duration of symptoms, and family status (Table 3).

Several of the most frequently detected serovars in urogenital infections worldwide were also identified in the present study. Serovar E, which is the predominant C. trachomatis type in most studies, comprising up to 50% of all urogenital chlamydial infections (1, 8, 10, 12), was also the prevalent serovar in our study sample, accounting for 37% of all cases. It is also of note that the relatively uncommon serovar G was found to prevail in Greece, exceeding the rates detected previously in other studies from Europe or elsewhere (4, 10, 12, 13, 15). However, serovar D, which in most epidemiological surveys is among the three most frequent C. trachomatis serovars (4, 8, 10, 12, 14, 18), had a relatively low occurrence in our sample. These inconsistencies may possibly reflect the differences between our study population and those examined by relevant studies, in terms of demographic and clinical characteristics, as well as the different laboratory methods used for the diagnosis of the infections. An intriguing finding regarding the C. trachomatis serovar distribution is the presence of four cases with infection due to serovar B, which is mainly associated with trachoma (2). The detection of trachoma-associated serovars in genital tract samples has been also reported in a previous study (7).

Gonococcal coinfection was diagnosed using conventional and molecular methods in 30% of the C. trachomatis-infected patients. The rate of concurrent N. gonorrhoeae infection estimated by our study was relatively high, similarly to the reports of the previous study from Italy that also examined heterosexual C. trachomatis-infected males with urethritis (4). However, in the latter study, the detection of N. gonorrhoeae was based on culture, while the main proportion of males exhibited high-risk sexual behavior, by means of having sex with prostitutes, resulting in a greater exposure to STDs than that of our study population. In the present study, a PCR assay was used for the simultaneous detection of C. trachomatis and N. gonorrhoeae, in order to avoid an underestimation due to the lower sensitivity of conventional diagnostic methods (6). To the best of our knowledge, no studies focused on C. trachomatis serovar distribution have employed PCR for N. gonorrhoeae diagnosis.

A noteworthy observation is that a significantly higher proportion of N. gonorrhoeae coinfections were indicated among patients with genovariant Ja than among other patients. No data are reported in the literature with regard to the association of N. gonorrhoeae coinfection with particular C. trachomatis serovars, apart from a nonsignificant association of N. gonorrhoeae coinfection with serovar D (4). The Ja genovariant is usually infrequent or totally absent in the worldwide serovar distribution patterns (13, 14, 16, 18). This association can be possibly interpreted by the fact that the sexual network for the two microorganisms is the same, promoting in this way the concomitant infection. An interesting issue would be to further examine whether all or most of the N. gonorrhoeae isolates from the Ja-coinfected patients were of the same strain, so as to elucidate if certain N. gonorrhoeae strains are correlated with C. trachomatis infection. This would help shed light on whether the Ja coinfection cluster may have been a more generalized phenomenon or a coincidental localized outbreak. In our study, the latter assumption might be a stronger possibility, due to the relatively small sample size.

In conclusion, the present study gave indications that in Greece, apart from the predominant worldwide serovars E and F, the relatively uncommon urogenital serovar G is also prevalent. Furthermore, N. gonorrhoeae coinfection in our population was associated with the globally infrequent genovariant Ja.

• Copyright © 2010 American Society for Microbiology