The Prevalence of HSV, HHV-6, HPV and Mycoplasma genitalium in Chlamydia trachomatis positive and Chlamydia trachomatis Negative Urogenital Samples among Young Women in Finland

Chlamydia trachomatis, Mycoplasma genitalium, herpes simplex virus (HSV) and human papillomavirus (HPV) cause sexually transmitted infections. In addition, human herpesvirus 6 (HHV-6) may be a genital co-pathogen. The prevalence rates of HSV, HHV-6, HPV, M. genitalium, and the C. trachomatis ompA genotypes were investigated by PCR in urogenital samples of the C. trachomatis nucleic acid amplification test positive (n = 157) and age-, community- and time-matched negative (n = 157) women. The prevalence of HPV DNA was significantly higher among the C. trachomatis positives than the C. trachomatis negatives (66% vs. 25%, p < 0.001). The prevalence of HSV (1.9% vs. 0%), HHV-6 (11% vs. 14%), and M. genitalium DNA (4.5% vs. 1.9%) was not significantly different between the C. trachomatis-positive and -negative women. Thirteen per cent of test-of-cure specimens tested positive for C. trachomatis. The prevalence of HSV, HHV-6, HPV, M. genitalium, and the C. trachomatis ompA genotypes did not significantly differ between those who cleared the C. trachomatis infection (n = 105) and those who did not (n = 16). The higher prevalence of HPV DNA among the C. trachomatis positives suggests greater sexual activity and increased risk for sexually transmitted pathogens.


Introduction
The global disease burden due to sexually transmitted infections (STIs) is substantial [1]. Chlamydia trachomatis urogenital infection is the most common sexually transmitted bacterial infection with 127 million cases annually [2]. Most chlamydial infections remain asymptomatic, but can result in pelvic inflammatory disease and severe reproductive complications [3]. Based on the ompA gene encoding the major outer membrane protein (MOMP), C. trachomatis can be classified into different genotypes, of which genotypes D-K cause most of the urogenital infections [4]. Mycoplasma genitalium infection is associated with non-gonococcal and non-chlamydial urethritis in men, and with urethritis, cervicitis, endometritis and pelvic inflammatory disease in women [5]. In addition, asymptomatic infections are frequent [5]. The increasing antimicrobial resistance of M. genitalium to macrolides and fluoroquinolones causes concern [6].
Genital herpes caused by herpes simplex virus (HSV-2 or HSV-1) affects over 500 million people worldwide [7,8]. After initial infection, herpesviruses establish lifelong latency in which the viral gene expression is limited. Upon reactivation, infectious viral particles and viral DNA are shed, and clinical disease can recur [9]. Human herpesvirus 6 (HHV-6) now includes two different viruses, HHV-6A and HHV-6B, with different epidemiological and biological characteristics and disease associations [10]. While less is known about the epidemiology of HHV-6A, HHV-6B is a ubiquitous virus and the seroprevalence of HHV-6 among adults is approximately 98% [11]. HHV-6B infection usually occurs early in childhood and can cause fever and rash (exanthema subitum), but HHV-6 infections including primary infections and reactivation can be asymptomatic [12]. Some individuals (less than 1%) have HHV-6 DNA integrated into telomeric ends of their chromosomes which could result in PCR positivity [12]. HHV-6 is known to be transmitted vertically, through saliva [12], and in addition, sexual transmission is possible [13]. Genital human papillomavirus (HPV) infections are common and the disease burden associated with HPV is enormous. The prevalence in women with normal cervical cytology is approximately 12% [14]. Many HPV infections clear spontaneously, but infections caused by the high-risk HPV genotypes can lead to cervical cancer, one of the leading cancers in women worldwide [15].
In Finland, C. trachomatis infections are notified by laboratories to the Finnish Infectious Disease Register maintained by the Finnish Institute for Health and Welfare since 1995. Genital HSV, HHV-6, HPV and M. genitalium infections are not notifiable and their prevalence is not known. Here, we wanted to study the prevalence of HSV, HHV-6, HPV and M. genitalium among C. trachomatis nucleic acid amplification test (NAAT) positive, and age-, community-and time-matched C. trachomatis NAAT-negative young women participating in a community-randomized trial on the effectiveness of C. trachomatis screening in Finland [16]. As genital coinfections can play a role in development of cancer (HPV) [17,18] or drive C. trachomatis into a persistent state (herpesviruses) [19,20], we studied whether the co-infections affect clearance of C. trachomatis.

The Prevalence of HSV, HHV-6, HPV and M. genitalium
The prevalence of HSV, HHV-6, HPV and M. genitalium DNA was analysed in the urogenital samples of 314 young women (157 C. trachomatis NAAT positive individuals and their 157 age-, community-and time-matched C. trachomatis NAAT negative controls). Among young women, HPV DNA was frequently (46%) detected and it was more common among the C. trachomatis positives than the C. trachomatis negatives (66% vs. 25%, p < 0.001) ( Table 1). HHV-6 DNA was present in 12%, M. genitalium DNA in 3.2% and HSV DNA in 1.0% of the samples. Of the ten samples positive for M. genitalium DNA, two samples (20%) contained a mutation associated with macrolide resistance. A slightly higher prevalence of M. genitalium and HSV DNA in the C. trachomatis-positive samples was observed, but due to the small numbers, these differences were not statistically significant. Among the 314 samples, 22 (21 among C. trachomatis NAAT positive and one among NAAT negative) were positive for three of the microbes analysed in this study. Only one sample tested positive for Neisseria gonorrhoeae. Among the 157 C. trachomatis-positive women, 166 infection episodes were evaluated (nine women had two C. trachomatis infection episodes during the study period). A test-of-cure (TOC) sample for C. trachomatis detection was taken approximately one month after the treatment as recommended by the Finnish national guideline [21], and it was available from 121 (73%) cases. The infection episodes were classified into not cleared (C. trachomatis TOC-positive) and cleared (C. trachomatis TOC-negative). Of the 121 TOC specimens, sixteen (13%) tested positive for C. trachomatis. The prevalence of HSV, HHV-6, HPV and M. genitalium DNA was similar between those who cleared C. trachomatis infection and those who did not ( Table 2). HHV-6 DNA seemed more frequent among those who gave a C. trachomatis positive TOC sample (19% vs. 10%). However, the difference was not significant. Table 2. The prevalence (% and 95% confidence interval, CI) of HSV, HHV-6, HPV and M. genitalium DNA in the urogenital samples of women who later gave a C. trachomatis test-of-cure positive (n = 16) and a C. trachomatis test-of-cure negative (n = 105) sample.

The C. trachomatis ompA Genotypes
Of the C. trachomatis NAAT-positive urogenital samples from 166 infection episodes, 146 (88%) samples could be genotyped by ompA PCR. The three most prevalent C. trachomatis ompA genotypes were E (n = 69, 47%), F (n = 39, 27%) and G (n = 13, 9%) ( Table 3). No statistically significant differences were observed in the genotype distribution between those who cleared the infection and those who did not.  1 Fisher's exact test was used to calculate the statistical significance.

Discussion
In this study, we explored the prevalence rates of HSV, HHV-6, HPV and M. genitalium by PCR among young women (18 and 22 years old) participating in a community-randomized C. trachomatis screening trial in Finland. HPV DNA was significantly more frequently detected among the C. trachomatis positives than the C. trachomatis negatives (66% vs. 25%). This finding is in an agreement with results of a large Italian study showing an HPV co-infection in 68% of C. trachomatis-positive ≤25-year-old women [22]. Globally, there is a relationship between HPV prevalence and age as the highest rates are observed in younger women of less than 25 years old, with a decline at older ages [14].
The high HPV prevalence suggests greater sexual activity and exposure to sexually transmissible microbes among those positive for C. trachomatis. This is of importance, as concomitant C. trachomatis and HPV infection is associated with high risk of cervical precancer [23], supporting the active role of C. trachomatis in the development of cervical precancer [17,18].
In contrast to HPV, the prevalence of HSV, HHV-6 and M. genitalium did not significantly differ among the C. trachomatis-positive and -negative young women. In this study, HSV DNA, indicating most likely asymptomatic shedding and including both HSV-1 and HSV-2, was rarely detected in the urogenital specimens (1%) among young women. This is in line with the previous Finnish data: genital herpes was clinically diagnosed in 3% of women attending twelve outpatient clinics in 1995-2006 [24], and approximately 2% of the STI clinic patients were culture-positive for HSV in 1994-2012 [25,26]. Among Finnish pregnant women, the seroprevalence for HSV-1 was 45% and for HSV-2 was 11% in 2012 [27]. The global estimates for genital HSV infections suggest higher prevalence: 0.8% (15-19 years old) and 2% (20-24 years old) for HSV1 [7], and 4.6% (15-19 years old) and 7.8% (20-24 years old) for HSV2 [8]. In our study, HHV-6 DNA was detected in 12% of the specimens. Similarly, an HHV-6 prevalence of up to 10% was reported in the genital tract of non-pregnant women in the USA [13], whereas up to 26% of Japanese pregnant women were shown to have HHV-6 DNA in their genital tract [28]. As HHV-6 induces a lifelong latent infection in humans and is capable of reactivation, either reactivation or (re)infection can explain HHV-6 DNA positivity.
The global estimate for the prevalence of M. genitalium is 1.7% in under 25-year-old women [29], that is slightly lower than the global prevalence of 3.8% for C. trachomatis in 15-49-year-old-women [2]. In our study, M. genitalium DNA was detected in 3.2% of the specimens, approximately as often as C. trachomatis [16]. Earlier, a somewhat higher prevalence of 5.6% for M. genitalium was reported among STI clinic patients in Finland [30]. In the other Nordic countries, the prevalence of 9.8% in Sweden, 4.9% in Norway and 9.0% in Denmark has been reported for M. genitalium [31]. In Norway, the prevalence of M. genitalium is suggested to be lower due to enhanced testing, treatment and partner notification [31]. In this study, two samples (20%) contained mutations in the 23S rRNA gene, leading to macrolide resistance in M. genitalium. Previously, such mutations were detected in 31% of M. genitalium-positive samples among STI clinic patients in Finland [30]. In the other Nordic countries, the prevalence of M. genitalium macrolide resistance is 18% in Sweden, 56% in Norway and 57% in Denmark [31]. This obviously reflects differences in the national treatment guidelines, as doxycycline is recommended in Sweden and azithromycin in Norway and Denmark in the treatment of C. trachomatis infection.
In addition, we studied whether co-infections affect clearance of C. trachomatis. The prevalence of HSV, HHV-6, HPV and M. genitalium did not significantly differ between those who cleared the C. trachomatis infection and those who did not, although in vitro studies suggest that herpesviruses and C. trachomatis might interact during human infection. Indeed, it has been shown that HSV and HHV-6 co-infections promote C. trachomatis persistence in vitro [19,20], and persistent forms of C. trachomatis are more resistant to antimicrobial therapy [32]. As 13% of TOC samples were positive for C. trachomatis, the impact of co-infections, especially those due to herpesviruses, warrants further studies.
Overall, the ompA genotype distribution in Finland has remained fairly stable during the last ten years, with E, F and G representing the most predominant genotypes [33,34]. Here, samples collected from asymptomatic women during a screening trial were studied, whereas the earlier studies included mainly samples from symptomatic patients. Previous studies have attempted to link certain C. trachomatis ompA genotypes to different clinical outcomes of the infection [35]. In this study, the ompA genotype distribution was similar among those who cleared the infection and those who did not suggesting that the C. trachomatis ompA genotype is not a decisive factor in the clearance of infection. Multiple factors, including host immunity [36] and infectious load [37] probably play a role in clearance.
This was the first study to explore the prevalence rates of HSV and HHV-6 DNA in urogenital samples among young women in Finland. Although the study was rather small, the participants and their controls were carefully matched to avoid confounding factors related to age, local epidemiology of these agents, and sampling time. The samples were collected from females volunteering in an HPV vaccination trial and simultaneously in a community-randomized C. trachomatis screening trial and the risk-taking behaviour among the participants may have been higher. Due to this, the true population-based prevalence rates and the prevalence of C. trachomatis and HPV co-infection might be lower than the ones reported here.
The interactions and consequences of co-infections with C. trachomatis, M. genitalium, HSV, HHV-6 and HPV can be numerous but are not well studied. In addition, STIs increase the risk of human immunodeficiency virus (HIV) acquisition and transmission [38]. Thus, it is important to understand the local and global epidemiology of the sexually transmitted microbes and also co-infections. This information can be used to prevent and control infections, to investigate the impact of screening interventions, and to develop guidelines for management. In addition, prevalence data can be used for monitoring purposes if drug resistance promotes further spread of infections. Ultimately, the development of vaccines against these sexually transmitted agents will result in permanent decline of the prevalence rates, as has already been observed with the implementation of HPV vaccination programs [39].

Participants and Samples
The samples studied here represent a subset of samples collected from women participating in an HPV vaccination trial [40,41] and simultaneously in a community-randomized trial on the effectiveness of C. trachomatis screening [16] at the age of 18 and 22 in Finland in 2010-2017. The self-collected vaginal swabs rinsed in first-void urine were sent to Fimlab, Tampere, Finland for C. trachomatis and N. gonorrhoeae testing with the Abbott RealTime CT/NG assay (Abbott Molecular, Des Plaines, IL, USA). All women with a C. trachomatis-positive sample and their partners received free-of-cost treatment (1 g single dose azithromycin) and were invited to give a test-of-cure sample one month later. The samples had previously been analysed for HPV DNA with methods described earlier at the Department of Clinical Microbiology, Skåne University Hospital, Malmö, Sweden [42,43].
The study material consisted of urogenital samples from 314 women (157 C. trachomatis NAAT positive individuals, and their 157 age-, community-and time-matched C. trachomatis NAAT negative controls). Altogether 166 C. trachomatis infection episodes were analysed among the 157 women. A test-of-cure sample was available from 121 (73%) women. These women were classified as C. trachomatis test-of-cure positive (infection not cleared) (n = 16) or C. trachomatis test-of-cure negative (infection cleared) (n = 105). A test-of-cure sample was not available from 45 women (27%). All participants gave their informed consent for inclusion before they participated in the C. trachomatis screening trial. The study was conducted in concordance with the Declaration of Helsinki and an ethics approval for the C. trachomatis screening trial was obtained from the Ethical Review Board of the North Ostrobothnia Hospital District (19.11.2012; §295/2012). HSV (HSV-1 and HSV-2 DNA polymerase gene) [44], HHV-6 (HHV-6A and HHV-6B U67 gene) [45] and M. genitalium (mgbB gene) [46] were detected with real-time PCRs developed earlier with modifications described below. The samples positive for M. genitalium DNA were analysed for mutations associated with macrolide resistance within the 23S rRNA gene with a method developed previously [47]. To evaluate the specimen quality (i.e., the presence of PCR inhibitors) and the performance of the nucleic acid extraction, the human beta-globin gene was amplified [47]. The ompA genotype of the C. trachomatis positive samples was determined with a method described earlier [48] with modifications [33].

DNA Extraction and Real-Time PCR
The primers and probes were purchased from Integrated DNA Technologies (IDT, Coralville, IA, USA) and Applied Biosystems (Thermo Fisher Scientific, Waltham, MA, USA). The real-time PCRs were performed in a 25 µL volume containing 12.5 µL Maxima Probe/ROX qPCR Master Mix (Thermo Fisher Scientific). The PCR for HSV contained 400 nM primers and 160 nM probes, for HHV-6 the PCR contained 100 nM forward primers, 600 nM reverse primers and 200 nM probes, for M. genitalium the PCR contained 100 nM primers and 100 nM probes, for M. genitalium macrolide resistance the PCR contained 50 nM of forward primers, 400 nM of reverse primers and 150 nM of probes, and for beta-globin the PCR contained 200 nM primers and 100 nM probes. All the probes included 6-FAM as a reporter dye and the HSV probe included a ZEN/Iowa Black FQ double-quencher.
The PCR analyses were performed on an ABI 7500 instrument and Sequence Detection Software version 1.3.1 (Applied Biosystems, Thermo Fisher Scientific). The thermal cycling conditions were two minutes at 50 • C, ten minutes at 95 • C, 45 cycles of 15 s at 95 • C and one minute at 60 • C. The template volume was 5 µL, and each sample was amplified in duplicate for HSV, M. genitalium and C. trachomatis ompA PCR, and in quadruplicate for HHV-6 PCR. The beta-globin gene PCR was performed in a single reaction and the gene was successfully amplified among all the samples analysed. No-template control was included in each run to assess for reagent contamination and it remained negative for all of the PCRs. As an amplification control for HSV, HHV-6 and M. genitalium PCR, the PCR target sequences in pIDTSMART-AMP plasmids were purchased from IDT. As an amplification control, DNA extracted from C. trachomatis reference strain types D to

Statistical Analysis
For comparison of the prevalence of HSV, HHV-6, HPV and M. genitalium DNA in C. trachomatis positives and negatives, the prevalence of HSV, HHV-6, HPV and M. genitalium DNA in C. trachomatis TOC positives and negatives, and the prevalence of C. trachomatis ompA genotypes D-K in C. trachomatis TOC positives and negatives, Fisher's exact test was used. A p-value less than 0.05 was considered statistically significant. Statistical analyses were performed with the IBM SPSS Statistics v24 (IBM, Armonk, NY, USA). The 95% confidence intervals for the proportion of positives for HSV, HHV-6, HPV and M. genitalium were also calculated [49].