Prevalence and significance of Mycoplasma genitalium in women living with HIV in Denmark

Objective Mycoplasma genitalium (M. genitalium) is a sexually transmitted pathogen associated with urethritis, cervicitis, and pelvic inflammatory disease. Previous studies have shown a strong association between M. genitalium and HIV infection, therefore screening and treatment for M. genitalium has been suggested as part of HIV prevention strategies. The objective of this study was to determine the prevalence of M. genitalium in women living with HIV (WLWH) in Denmark, and to compare the result with data on symptoms from the lower abdomen, sexual habits and immune status. 234 women, recruited from Danish HIV centres as part of a larger observational study on aspects of living with HIV as a woman (the SHADE study), were included. Results We tested cervical samples for M. genitalium by specific PCR. We found three samples positive (1.3%). The women were between 30 and 50 years old, all were of Asian origin, sexually active, and on antiretroviral treatment with supressed HIV RNA and CD4 count >350 cells/µL. None reported symptoms from the lower abdomen. The prevalence of M. genitalium infection in WLWH in Denmark is low, thus systematic screening for M. genitalium in this group does not seem relevant. Electronic supplementary material The online version of this article (doi:10.1186/s13104-017-2776-5) contains supplementary material, which is available to authorized users.


Introduction
Mycoplasma genitalium (M. genitalium) is a well-known sexually transmitted pathogen. It is an important cause of non-gonococcal urethritis in men, and in women it has been associated with cervicitis, urethritis and pelvic inflammatory disease [1]. Data from the last decade show a strong association between M. genitalium and HIV [2][3][4]. Women living with HIV (WLWH) infected with M. genitalium have been found to shed higher amounts of HIV in vaginal fluids, thereby possibly enhancing HIV transmission [5]. Infection with M. genitalium can lead to cervical inflammation, and it is possible that the infection also increases the risk of acquiring HIV infection, as is the case in other sexually transmitted diseases (STD) [6].
The prevalence of M. genitalium in women in the general population of high-income countries ranges from 1 to 4%, but is 10% or even higher among women attending STD clinics [7][8][9]. In the US, a survey of WLWH showed a 9.9% prevalence of M. genitalium infection [10]. Studies from Africa have documented a much higher prevalence in risk groups [11]. They also documented higher prevalence of M. genitalium infection among HIV positive women compared with HIV negative women, and it seems the infection persists longer in the HIV positive [12,13]. One longitudinal study showed a twofold increased risk of being infected with HIV when M. genitalium positive [14].
There are no data on M. genitalium infection in WLWH in Denmark or other parts of Scandinavia. We obtained cervical samples from WLWH participating in a larger observational study on aspects of living with HIV as a woman (the SHADE study). The aim of this study was to determine the prevalence of M. genitalium in WLWH in Denmark, and secondly to compare the result with data on symptoms from the lower abdomen, sexual habits and immune status.

Registries
The Civil Registration System (CRS) The CRS is a national registry of all Danish residents [16]. A 10-digit personal identification number (PIN) is assigned to everyone at birth or immigration. The PIN was used as a linkage to the Danish HIV Cohort Study (DHCS).
Danish HIV Cohort Study (DHCS) The DHCS is a prospective, observational, nationwide cohort study of all people living with HIV seen at the Danish HIV clinics since January 1995 [17]. From the DHCS we obtained data on baseline HIV characteristics.

Study design
We tested cervical samples collected from 234 WLWH. Women were recruited from six Danish HIV centres as part of the SHADE cohort; study on HIV, cervical abnormalities and infections in women in Denmark. The SHADE cohort is a multicenter, prospective, observational cohort study of WLWH in Denmark attending regular outpatient care for their HIV infection. The study focuses on STD, contraception, sexual activity, human papillomavirus (HPV) infection, cervical cytological abnormalities, and other aspects of living with HIV as a woman. Results from the SHADE study have been published elsewhere [18].
Women were asked to participate if they had a known HIV-1 infection and were ≥18 years of age. Exclusion criteria were pregnancy and alcohol and/or drug abuse impeding adherence to the protocol. Of 1392 eligible women in the DHCS, 334 were included in the SHADE cohort. Women were enrolled between February 2011 and February 2012. The 234 WLWH included in the present study were those attending the 2 years follow-up of the SHADE study from February 2013-February 2014. At enrolment, women were tested for Chlamydia trachomatis, Neisseria gonorrhoeae, syphilis, and herpes simplex (HSV-1 and HSV-2). Gynaecological examinations were performed at entry and at 6, 12 and 24 months' follow-up. An interview survey was performed to obtain background information. The questionnaire used has been described in detail elsewhere [18].
At entry, written and oral informed consent was obtained from all participants. The protocol has been approved by the Danish Data Protection Agency and by the Danish National Committee on Health Research Ethics.

Study population
The median age at inclusion in the study was 44 years (range 25-78 years). Treatment status was generally good, with 94% of the women on antiretroviral treatment (ART), and 86% on ART with an undetectable HIV RNA. CD4 count was 350 cells/µL or above in 80% of the cases. Symptoms from the lower abdomen were reported in 21% of the cases, most frequently vaginal discharge and abnormal menstrual bleeding. When asked about the number of lifetime sexual partners, 67% of the women reported less than 15 partners, 33% reported more than 15 partners, and of those, 18% had more than 25 lifetime sexual partners. When asked about sexual activity in the previous 6 months up to the follow up visit, 71% were sexually active. Furthermore, 56% were married or cohabitating and 32% reported to be single. Baseline characteristics of the WLWH are listed in Table 1.

Analyses
Samples were obtained by the treating physician performing a gynecological examination. Flocked swabs were collected in UTM transport medium, and M. genitalium was detected by TaqMan ™ PCR amplifying a conserved region of the MgPa adhesin gene [19]. Positive results were confirmed by conventional PCR amplifying the 23S rRNA gene, and the resulting PCR products were subsequently sequenced to detect macrolide resistance mediating mutations [20]. The women who tested positive for M. genitalium were offered treatment with oral azithromycin for 5 days (500 mg day one, followed by 250 mg daily day 2-5). Per protocol treatment in case of macrolide resistance was oral moxifloxacin 400 mg daily for 7 days (Additional file 1).

Statistical analyses
Continuous variables were summarized as median and interquartile ranges (IQR) and compared using Wilcoxon rank sum test. Categorical variables were reported as counts and percentages and compared with Chi square test or Fisher's exact test as appropriate. Further details on the statistical analyses on the SHADE cohort have been published previously [18].

Results
Of the 234 women, three (1.3%) were PCR positive for M. genitalium. All carried a macrolide susceptible strain. The prevalence was too low to perform adjusted analyses aiming at predicting associated factors.
Of the WLWH in this study, 45% were white, 40% were black and 14% were Asian. Interestingly, all three M. genitalium positive women were from Asia (Thailand); thus, the prevalence among women from Asia was 10% (95% CI 2.0-27.0%) which was significantly higher than the 0% prevalence found in both white and black women (p = 0.01 and p = 0.02, respectively). Characteristics of the M. genitalium positive women are shown in Table 2.

Discussion
We found a relatively low prevalence of 1.3% of M. genitalium infections in this population. The result is comparable to the prevalence of 1-4% reported in general populations in high-income countries [7][8][9], but much lower than in studies of African and American WLWH, where the prevalence generally has been much higher [11,21]. Interestingly, all M. genitalium infected women were from Thailand. Although numbers were small, all three were infected with macrolide susceptible strains of M. genitalium, which may be surprising as the level of macrolide resistance in Thailand is expected to be high, although no precise figures exist. If the women were infected in Denmark, we would expect nearly half to carry resistance mediating mutations [20]. The low prevalence of M. genitalium could be explained by several factors. The median age is relatively high in the present study compared to other studies of both WLWH and general populations. Furthermore, the women in this population seemed to be less sexually active than WLWH in other study populations. The majority of the women were in a relationship or had a regular partner. A recent study from France showed a similar low prevalence of M. genitalium (3.8%) among WLWH [22]. The study population was comparable to the present study population with respect to the median age (41.3 and 44 years respectively), race (42 and 55% non-white respectively), and

Conclusions
The prevalence of M. genitalium was relatively low in this study population. The result is comparable to the prevalence reported in the general populations in highincome countries. Screening for M. genitalium as part of HIV prevention strategies does not seem relevant in this setting.

Limitations
With only three positive results, there was no basis for a statistical analysis of risk factors associated with M. genitalium infection. The women who participated in this study were generally older and less sexually active than the women in most other studies of HIV and M. genitalium, and most of them were successfully treated with fully supressed HIV RNA.