Introduction
Sexually transmitted infections (STIs) have a significant impact on quality of life and reproductive health. The rates of STIs have increased substantially worldwide, including in high-income countries during the 21st century [Reference Williamson and Chen1]. There are more than 30 different bacteria, viruses, and parasites that can be transmitted through sexual contact [Reference Agacfidan and Kohl2]. While surveillance data are widely available for Chlamydia trachomatis and Neisseria gonorrhoeae, the epidemiology of Mycoplasma genitalium and Trichomonas vaginalis is less known [Reference Van Der Veer, Van Rooijen, Himschoot, De Vries and Bruisten3, Reference Kissinger4]. A meta-analysis published in 2018 included only two studies from Europe on the prevalence of M. genitalium in the general population, and three studies on men who have sex with men (MSM) who visited a community-based clinic in Europe. [Reference Baumann, Cina, Egli-Gany, Goutaki, Halbeisen, Lohrer, Ali, Scott and Low5]. Arguably, the prevalence of STI pathogens can be affected by a variety of reasons such as the study setting, the structure of the health care system, and whether the individuals with STI complaints are advised to go to primary care or to an STI clinic. In the general population of British individuals who did not exhibit symptoms of STI, the prevalence of M. genitalium was 1.2% in men and 1.3% in women [Reference Sonnenberg, Ison, Clifton, Field, Tanton, Soldan, Beddows, Alexander, Khanom, Saunders, Copas, Wellings, Mercer and Johnson6]. In patients who underwent STI screening in France, the prevalence was comparable, that is 1.7% [Reference Pereyre, Laurier Nadalié, Bébéar, Arfeuille, Beby-Defaux, Berçot, Boisset, Bourgeois, Carles, Decré, Garand, Gibaud, Grob, Jeannot, Kempf, Moreau, Petitjean-Lecherbonnier, Prère, Salord and Verhoeven7]. In the same French study, the prevalence of T. vaginalis was 3.4%. In the USA, the T. vaginalis positivity rate in a population-based study was reported at 2.3% among adolescents [Reference Miller, Swygard, Hobbs, Ford, Handcock, Morris, Schmitz, Cohen, Harris and Udry8] and 3.1% among reproductive-age women [Reference Sutton, Sternberg, Koumans, McQuillan, Berman and Markowitz9]. There is also paucity of data on the co-occurrence of M. genitalium and T. vaginalis with C. trachomatis and N. gonorrhoeae.
In the Netherlands, there is limited data on the prevalence of M. genitalium and. T. vaginalis, and the available data come from a mixed population. In a study of samples taken from primary care and hospital care settings, the prevalence of M. genitalium was 1.9% and T. vaginalis was 0.6% [Reference Jenniskens, Veerbeek, Deurloo, van Hannen and Thijsen10]. Another study conducted in the Netherlands that collected samples from STI clinics, general practitioners (GPs), and hospitals found M. genitalium and T. vaginalis prevalence of 1.4% and 4.5%, respectively [Reference de Jong, Rahamat-Langendoen, van Alphen, Hilt, van Herk, Pont, Melchers and van de Bovenkamp11]. Additional data are required on the epidemiology of M. genitalium and T. vaginalis in the primary care population of the Netherlands.
Therefore, the aim of this study is to describe the epidemiology of M. genitalium and T. vaginalis, especially in light of their co-occurrence with STI pathogens C. trachomatis and N. gonorrhoeae in patients who visited GPs in the Netherlands. As a second objective, we sought to describe the prevalence of M. genitalium genotypically resistant to azithromycin and fluoroquinolones.
Materials and methods
Study setting and population
The data used in this study were derived from specimens (pharyngeal-, urethral- (only in men), and vaginal swabs (only in women) and urine) submitted to the Star-shl Medical Laboratory during a four-year period (between January 2018 and December 2021). The specimens were obtained from patients who visited GPs due to urethral symptoms (that is dysuria or discharge) or vaginal symptoms (that is dysuria, discharge, or vaginal blood loss), or from their sexual contacts. The Star-shl Medical Laboratory provides services for 1,500 GPs, mostly from the southwestern region of the Netherlands. This region has an estimated population of 3.5 million inhabitants. In the whole country, there are around 12,000 GPs [12], and people can go to either their GP or the Municipal Public Health Service (GGD in Dutch) for an STI test. STI testing offered by the GGD is anonymous and frequently sought by high-risk patients, such as men who have sex with men (MSM) and sex workers. The present study relied solely on laboratory data, so approval from the ethical committee was not required.
Sampling and molecular testing
Swabs and first-void urine were collected and transferred to appropriate sample collection media, and transported to the laboratory within the same day. For the detection of M. genitalium and T. vaginalis, Aptima™ M. genitalium [Reference Gaydos, Manhart, Taylor, Lillis, Hook, Klausner, Remillard, Love, McKinney and Getman13] and Aptima™ Trichomonas vaginalis assay [Reference Chapin and Andrea14] (both Hologic Inc, San Diego, USA), respectively, were used. The Aptima™ Combo 2 (Hologic Inc, San Diego, USA) was used for the detection of C. trachomatis and N. gonorrhoeae [Reference Gaydos, Quinn, Willis, Weissfeld, Hook, Martin, Ferrero and Schachter15]. These assays were performed in Panther systems (Hologic Inc, San Diego, USA), and are in-vitro diagnostics that involve the capture of rRNA target molecules and transcription-mediated amplification of specific regions.
Among samples from patients who tested positive for M. genitalium, 202 samples were randomly selected and tested for macrolide resistance using the Allplex™ MG & AziR assay that allowed detection of any of six point mutations in region V of the 23S rRNA gene (A2058C, A2058G, A2058T, A2509C, A2509G, A2509T) that may be responsible for azithromycin resistance [Reference Machalek, Tao, Shilling, Jensen, Unemo, Murray, EPF, Low, Garland, Vodstrcil, Fairley, Hocking, Zhang and Bradshaw16]. In the same samples, fluoroquinolone resistance was tested using the Allplex MG™ & MoxiR assay that detects single nucleotide polymorphisms (SNPs) in the parC gene: A247C, G248A, G248T, G259A, G259C, G259T [Reference Machalek, Tao, Shilling, Jensen, Unemo, Murray, EPF, Low, Garland, Vodstrcil, Fairley, Hocking, Zhang and Bradshaw16]. The mutation detection assays were from Seegene (Seoul, South Korea) and performed according to the manufacturer’s instructions. If more than one sample was positive for M. genitalium, only one sample per patient was randomly chosen for these antibiotic resistance tests.
Statistical analysis
Statistical analyses were conducted in Rstudio (version 2022.07.1), using R version 4.2.1. We calculated and presented the proportion of patients who tested positive for the STI pathogens investigated in this study (including the percentage and their 95% confidence interval, 95% CI). We also calculated the proportion of patients with a co-occurrence of M. genitalium or T. vaginalis with C. trachomatis and N. gonorrhoeae. No formal statistical testing was performed since this study did not pose any particular hypothesis.
Results
Prevalence
During the study period, GPs ordered tests for all four pathogens in 7,411 female patients (median age 29.4 years (IQR 23.4 to 38.5)), and for C. trachomatis, N. gonorrhoeae, and M. genitalium in 5,732 male patients (30.4 years (24.5 to 40.2)).
In female patients, 7.0% (95%CI: 6.4 to 7.6) were positive for C. trachomatis and 0.8% (0.6 to 1.0) were positive for N. gonorrhoea. M. genitalium was positive in 6.7% (6.2 to 7.4) and T. vaginalis was positive in 1.9% (1.6 to 2.2).
In male patients, 5.5% (95%CI: 4.9 to 6.1) were positive for C. trachomatis and 1.7% (1.4 to 2.1) were positive for N. gonorrhoea. M. genitalium was positive in 3.7% (3.3 to 4.3).
M. genitalium co-occurred with C. trachomatis in 1.4% (0.3 to 0.6) of female patients and in 0.7% (0.5 to 0.9) of male patients. T. vaginalis co-occurred with C. trachomatis only in 0.2% (0.1 to 0.3) of female patients. The co-occurrence of M. genitalium and N. gonorrhoea was rarely observed, that is in 0.1% (0.04 to 0.2) of female patients, and in 0.2% (0.1 to 0.4) of male patients.
Macrolide and fluoroquinolone resistance in M. genitalium
Macrolide resistance–associated gene mutations were detected in 149 out of 202 (73.8.2%) randomly selected M. genitalium positive samples. The frequency of fluoroquinolone resistance–associated gene was much lower than macrolide resistance, that is 20 out of 202 (9.9%) randomly selected M. genitalium positive samples. Both macrolide and fluoroquinolone gene mutations were detected in five patients. The mutations associated with macrolide and fluoroquinolone resistance found in this study are presented in Table 1.
Point mutations associated with azithromycin and fluoroquinolone resistance in positive M. genitalium samples
Discussion
Dutch guidelines do not recommend routine screening for asymptomatic patients for M. genitalium and T. vaginalis [17, Reference Hetem, Kuizenga Wessel, Bruisten, Braam, van Rooijen, Vergunst, Nijhuis, Berns, Brand and van Dam18], and perhaps this is the reason why knowledge on the co-occurrence of these pathogens with other STIs in the Netherlands is scarce. The difference in the prevalence of STI pathogens is determined largely by the population and the test that is used. The comparison should therefore be made, for example between GP populations and mixed-study populations (e.g. hospitals, patients visiting STI clinics). Plausibly, our GP patient population will have a lower prevalence of STIs including M. genitalium than the population of patients who visited the GGD. Due to anonymous testing, the GGD is frequently sought by high-risk patients, such as MSM and sex workers. The prevalence of M. genitalium in our study population (5.5% combined male and female) is three times higher than the prevalence of M. genitalium in a mixed GP and hospital population in a bordering region in the Netherlands (2.1%) [Reference Jenniskens, Veerbeek, Deurloo, van Hannen and Thijsen10]. The prevalence of T. vaginalis in our study was also slightly higher than in that study (1% (combined male and female) versus 0.7%). An explanation for this could be the high sensitivity of the TMA-based Aptima assay [Reference Braam, Hetem, Vergunst, Wessel, Van Rooijen, Nijhuis, Schim van der Loeff, van Dam and Bruisten19] used in our study. Another study from the Netherlands using a mixed-study population (STI clinics, GPs, and hospitals), where no separate data could be derived for the GP population, showed a prevalence of M. genitalium and T. vaginalis of 4.5% and 1.4%, respectively [Reference de Jong, Rahamat-Langendoen, van Alphen, Hilt, van Herk, Pont, Melchers and van de Bovenkamp11]. In that study, an in-house PCR was used.
Our study confirmed a very high percentage of a genotypic macrolide resistance as also found in the study from Hetem and colleagues from the Netherlands. It was shown in that study that 66% of positive M. genitalium samples showed macrolide resistance–associated mutations [Reference Hetem, Kuizenga Wessel, Bruisten, Braam, van Rooijen, Vergunst, Nijhuis, Berns, Brand and van Dam18]. This study was published after a systematic review and meta-analysis that showed macrolide resistance proportions of 27.5% in non-Nordic European countries. The range is however wide, from 6.5% in Belgium to 74.3% in the UK [Reference Machalek, Tao, Shilling, Jensen, Unemo, Murray, EPF, Low, Garland, Vodstrcil, Fairley, Hocking, Zhang and Bradshaw16]. In this systematic review, the proportion of M. genitalium that was resistant to fluoroquinolone in non-Nordic countries was 3.2%, lower than the 9.9% found in our study.
Our findings present several new recommendations for practice. Since M. genitalium is occasionally found, it is recommended that it should be tested when test results for C. trachomatis or N. gonorrhoeae turn out to be negative but the patient still experiences STI symptoms. Another recommendation is that macrolide resistance should be performed for patients who retain symptoms after macrolide treatment. The number of macrolide resistance gene mutations may be high, but a discrepancy between genotypic and phenotypic resistance may occur.
Our study has several limitations. It does not collect specific clinical data, but our study was not designed to link the clinical data to STI pathogens. Another limitation is that we only performed macrolide or fluoroquinolone resistance testing in 30% of the positive M. genitalium samples due to limited funding.
In conclusion, we revealed the relatively low prevalence of M. genitalium in a large GP population in the Netherlands, but, co-occurrence with C. trachomatis and resistance to azithromycin may be observed. Therefore, when treating STIs, these prevalence and resistance data should be taken into account.
Data availability statement
The data that support the findings of this study are available on request from the corresponding author.
Author contribution
Conceptualisation and study design: E.Y., K.M., K.T.D.T.; Data curation: E.Y., K.M., N.v.L., C.H., K.T.D.T.; Data analysis: E.Y., K.M., K.T.D.T.; Interpretation of data: E.Y., K.T.D.T.; Drafting the original manuscript: E.Y., K.T.D.T.; Review & editing manuscript: all authors. All authors approved this manuscript version to be submitted.
Funding statement
The authors received no specific funding for this work.
Competing interest
The authors declare no competing interests.
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