The prevalence of T. vaginalis among men attending an STD clinic ranged from 2.8% to 17% [11,12], with the highest prevalence in those aged >40 years [13,14], and was as high as 73% in male partners of women diagnosed with vaginal trichomoniasis [15]. Recently, T. vaginalis was detected in only 1 of 435 men who visited a health examination center in South Korea [10]. However, the prevalence of T. vaginalis significantly increased to 21.2% in male patients with chronic recurrent prostatitis and urethritis [16]. Few recent studies examined the prevalence of T. vaginalis infection in men. Here, we examined 201 urine specimens from a primary care urology clinic in South Korea using PCR assays to determine the prevalence of T. vaginalis and associated clinical conditions in men.

From May 2013 to November 2013, 201 male patients from a primary care urology clinic (Top Urology Clinic) in Daegu, South Korea, were screened for trichomoniasis by PCR and for bacterial STDs by multiplex PCR. As is required by the Declaration of Helsinki, donor confidentiality was maintained throughout, and the study was approved by the Institutional Review Board with written consent (KNUH 2013-04-051). The bivariate analysis included 201 male patients who completed questionnaires. Demographic characteristics and urogenital symptoms of trichomoniasis were assessed by self-administered questionnaires.

In this study, first-voided urine (VB1) specimens, which were reported to be sufficient for the detection of T. vaginalis in men, were used in the PCR assays [16]. VB1 (30-40 ml) samples from male patients were provided in sterile 50-ml screw-cap plastic tubes, immediately frozen at -20℃, and transported on ice to a laboratory where they were stored at -70℃. The diagnosis of T. vaginalis infection was made using PCR targeting Tvk [17] and the β-tubulin gene [18], while the diagnosis of bacterial sexually-transmitted infections (STIs) was made using multiplex PCR (STD6 ACE Detection kit, SeeGene, Seoul, Korea) [19]. After thawing, the frozen urine specimens were divided into 2 tubes (10 ml each), equilibrated at room temperature, centrifuged at 5,000 g for 15 min, and resuspended in 1 ml of sterile saline.

DNA was prepared from the suspension by 2 different methods; the QIAamp DNA Mini kit (Qiagen, Valencia, California, USA) for multiplex PCR according to the manufacturer's instructions or heat-treating with a chelating resin for PCR [18,20]. Briefly, 1 ml of suspended urine precipitate was centrifuged at 10,000 g for 5 min, suspended in 200 µl of a 5% suspension of chelating resin (Chelex 100; Sigma, St. Louis, Missouri, USA) in 0.01 M Tris buffer (pH 8.0), and incubated at 56℃ for 30 min. Preparations were mixed gently, boiled for 10 min, and centrifuged at 12,000 g for 1 min in a microcentrifuge, and 1 µl of supernatant was directly used as the template for PCR. The PCR conditions were as follows: an initiation step at 95℃ for 5 min; 40 cycles at 90℃ for 1 min, 60℃ for 30 sec, and 72℃ for 2 min; and a final extension step at 72℃ for 10 min. The PCR primer set Tvk 3/7 (Tvk 3, 5'-ATTGTCGAACATTGGTCTTACCCTC-3'; Tvk 7, 5'-TCTGTGCCGTCTTCAAGTATGC-3') [17] amplified the 261 bp products and BTUB 9/2 (BTUB 9, 5'-CATTGATAACGAAGCTCTTTACGA-3'; BTUB 2, 5'-GCATGTTGTGCCGGACATAACCAT-3') [18] amplified a DNA product of 112 bp from T. vaginalis genomic DNA. The lower limit of T. vaginalis detection by PCR using Tvk 3/7 and BTUB 9/2 was found to be 1 organism per reaction, consistent with a previous report [18]. Multiplex PCR based on the dual priming oligonucleotide (DPO) system (Seegene, Seoul, Korea) was used for the diagnosis of STIs, including Trichomonas. This method enables the simultaneous detection of Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, Ureaplasma urealyticum, Mycoplasma hominis, and T. vaginalis [19]. In cases of discrepancies between PCR and multiplex PCR results, PCR results (both Tvk 3/7 and BTUB 9/2 PCR-positive) were considered as true positive for trichomoniasis.

The mean age of the 201 male participants was 53.6 years (range; 17-87 years). Though the group was age-diverse (Table 1), 81.6% (164/201) of the participants were older than 40 years. In this cohort of male patients who visited a primary care urology clinic, the prevalence of T. vaginalis infection was 4.0% (8/201) by PCR assay with 100% concordance between the Tvk and BTUB primer sets (Table 1). The median age of 8 men with Trichomonas-positive specimens was 52 years (range; 28-74 years). As shown in Table 1, the prevalence of T. vaginalis infection was 11.1% (1/9) in those aged 20-29 years, 5.7% (2/35) in those aged 40-49 years, 5.7% (3/53) in those aged 50-59 years, 2.1% (1/48) in those aged 60-69 years, and 3.8% (1/26) in those aged 70-79 years, with male patients in the 20-29 age group showing the highest rate of T. vaginalis infection (Table 1). However, if we consider the relative proportion of each age group to the total number of Trichomonas-infected patients, 12.5% (1/8) of infected patients were <30 years, 25.0% (2/8) were 40-49 years, 37.5% (3/8) were 50-59 years, 12.5% (1/8) were 60-69 years, and 12.5% (1/8) were 70-79 years, indicating that more than 60% (62.5%) of T. vaginalis infections occurred in men older than 50 years.

An examination of marital status showed that 87.5% (7/8) of T. vaginalis-infected patients were currently or previously married. According to these results, it is likely that T. vaginalis is more prevalent in older ages. However, owing to the small number of young participants (10-40 years), no statistical association (P=0.920) was obtained between Trichomonas infection and older ages. The analysis of urogenital symptoms reported via self-administered questionnaires indicated that 37.5% of T. vaginalis-infected male patients had no penile discharge, odor, or lower abdominal pain, while 12.5% and 50.0% reported penile tingling and pain during urination, respectively. Among the 8 Trichomonas-positive male patients, 50.0% (4/8) were diagnosed with prostatitis, 37.5% (3/8) were with benign prostatic hyperplasia (BPH), and 12.5% (1/8) were with urethritis (Table 2). As shown in Table 2, no Trichomonas-positive reaction was obtained by multiplex PCR (Table 2). In the 8 samples that were positive by PCR assay, 2 (25.0%) were positive for C. trachomatis and 1 (12.5%) was positive for M. genitalium by multiplex PCR.

Skerk et al. [21,22] reported that 71.0-74.2% of chronic prostatitis cases were attributable to infection, 10.5-19.0% of which were due to trichomoniasis. Recently, Lee et al. [16] detected T. vaginalis in 21.2% of Korean patients complaining of lower urinary tract symptoms, 71.4% of whom had chronic prostatitis. Among 8 Trichomonas-positive male patients in our study, 87.5% (7/8) were diagnosed with prostatitis or BPH (Table 2), showing that the vast majority of Trichomonas-positive male patients had prostatic diseases, similar to previous reports.

As shown in Table 2, no Trichomonas-positive reaction was obtained by multiplex PCR. Although the detection limit for purified T. vaginalis genomic DNA from in vitro culture samples was similar for PCR and multiplex PCR (data not shown), their diagnostic performance in urine specimens differed. Previous reports have suggested the possible limitations of multiplex PCR, which include PCR drift by stochastic fluctuation in the interaction of PCR reagents or competitive inhibition by PCR selection [10].

Concurrent STIs, such as C. trachomatis or N. gonorrhoeae, are frequently detected in persons with trichomoniasis [23]. In the United States, approximately 10% of male partners with trichomoniasis were coinfected with C. trachomatis or N. gonorrhoeae [15]. In our study, 25.0% and 12.5% of Trichomonas-positive male patients were coinfected with C. trachomatis and M. genitalium, respectively.

As mentioned above, 2 studies conducted in the United States in the early 2000s reported that 12.9% (47/363) [8] and 17.3% (52/300) [12] of men attending an STI clinic were positive for T. vaginalis by PCR assay. In Japan, a study conducted in the 2000s using PCR reported that no T. vaginalis was detected in 100 Japanese men with and without urethritis [24]. Recently, the prevalence of T. vaginalis was reported to be 1.4% (3/215) in men with urethritis and 1.0% (1/98) in men without urethritis [25]. In the present study, 4% (8/201) of male patients attending a primary care urology clinic were Trichomonas-positive, indicating that the male prevalence of T. vaginalis in South Korea appears to be lower than that in the United States and higher than that in Japan.

In contrast to women with Trichomonas infection, the association between Trichomonas infection and older ages in men is not fully understood. A United States study conducted in the late 1990s found that T. vaginalis infection was significantly associated with age (0.8%, <30 years versus 5.1%, ≥30 years). However, Wendel et al. [8] recently reported that the prevalence of T. vaginalis in men aged ≤28 years (13%, 23/178) was similar to that of men aged >28 years (24/185). In the present study, T. vaginalis was significantly more prevalent among older men who had been married (≥40 years, 87.5%) than among relatively younger single men (<40 years, 12.5%). During the study period, the number of patients aged <40 years was small compared to the number of patients aged ≥40 years. Although further studies are required to examine the prevalence of T. vaginalis in young men, it can be considered that STIs, including T. vaginalis infection are indeed rare in men younger than 40 years or that the majority of this subgroup is asymptomatic and thus seldom visits the Urology Clinic. It appears that >80% of T. vaginalis infections are asymptomatic, causing the infection to persist. Here, we found that the majority of T. vaginalis infections occurred in men older than 40 years with urethritis or prostatic diseases. Thus, it can be supposed that few men, especially those younger than 40 years, are diagnosed with and treated for trichomoniasis in South Korea. With the exception of male partners of women diagnosed with vaginal trichomoniasis, T. vaginalis infection has not been routinely considered in male patients attending primary care urology clinics in South Korea owing to the availability or cost of diagnostic testing. Thus, the early diagnosis and treatment of T. vaginalis infection, especially in young men, and rapid point-of-care tests are urgently needed to prevent the increase of this disease burden.