Prevalence of Mycoplasma genitalium in men with urethritis and in high risk asymptomatic males in Tel Aviv: a prospective study

Abstract

We sought to investigate the prevalence of Mycoplasma genitalium, Chlamydia trachomatis, Neisseria gonorrhoeae, and Trichomonas vaginalis in men presenting to a sexually transmitted infection (STI) clinic, with special attention to M. genitalium, its occurrence in Israeli patients, coinfections, and risk factors. In a cross-sectional study, 259 men were successively enrolled in the Tel Aviv Levinsky Clinic for STIs between November 2008 and November 2010. There were 118 men with urethritis and 141 high-risk men without symptoms. M. genitalium, C. trachomatis, N. gonorrhoeae, and T. vaginalis were detected using nucleic acid amplification tests. Demographic characteristics and risk factors were documented. The overall prevalence of infection with M. genitalium, C. trachomatis, N. gonorrhoeae, and T. vaginalis, were 6.6%, 12.7%, 23.1%, and 0%, respectively. Prevalences in men with urethritis were 11.9%, 22%, and 49%, for M. genitalium, C. trachomatis, and N. gonorrhoeae, respectively. Prevalences in men without symptoms were 2.1%, 5.0%, and 1.4%, for M. genitalium, C. trachomatis, and N. gonorrhoeae, respectively. Co-infections were found only in symptomatic individuals, in whom 5.9% were infected concomitantly with C. trachomatis and N. gonorrhoeae, and 2.5% had dual infection with M. genitalium and N. gonorrhoeae. N. gonorrhoeae, C. trachomatis, and M. genitalium were significantly more prevalent in patients with urethritis. M. genitalium was significantly more prevalent in the heterosexual population than in homosexual males. To conclude, we have found that M. genitalium infection is associated with urethritis in Israeli men, and more so in the heterosexual population. Testing men for M. genitalium as a cause of non-gonococcal urethritis is warranted, particularly because of its poor response to doxycycline and possible failure of azithromycin.

Keywords

Urethritis sexually transmitted infection nucleic acid amplification tests homosexual men

Introduction

Urethritis is the most common clinical condition affecting the male reproductive tract.¹ It has traditionally been associated with sexually transmitted organisms such as Neisseria gonorrhoeae and Chlamydia trachomatis and, less frequently, with Trichomonas vaginalis.¹ More recently it has also been linked with Mycoplasma genitalium.² That M. genitalium is sexually transmitted and is a cause of urethritis in men has been systematically shown by experimental inoculation of primates,³ by studies of concordance rates among partners,⁴ and by studies of prevalence in men with and without urethritis.^2,5

Advances in highly sensitive and specific nucleic acid amplification tests that allow better detection of organisms broadened our knowledge on the effects of M. genitalium infection: there is evidence that this microorganism may enhance the transmission of HIV.⁶ M. genitalium infection was significantly associated with increased risk of cervicitis, pelvic inflammatory disease, preterm birth, spontaneous abortion, and infertility.⁷ Also, M. genitalium has been detected in the knee joint of patients with sexually acquired reactive arthritis.^2,8

M. genitalium has been found in subjects of different race and in various parts of the world. The prevalence of M. genitalium varies by country and by patient population, ranging from 1% in the general population to 42% in African men with non-gonococcal urethritis (NGU).⁹

The epidemiology of sexually transmitted infections (STIs) in Israel is similar to that in Europe; yet there is no information about the prevalence and the characteristics of M. genitalium infections among Israelis. This led us to study the prevalence of M. genitalium in men with urethritis and in high risk asymptomatic men attending an STI clinic in Tel Aviv, and, at the same time, to determine the clinical and epidemiologic characteristics of M. genitalium-associated urethritis in this population.

Patients and methods

Study population

In a cross-sectional study, male patients attending the Tel Aviv Levinsky Clinic for STIs were successively and prospectively enrolled between November 2008 and November 2010. Sociodemographic information, symptoms, sexual preference (homosexual or heterosexual), number of sexual partners in the previous three months, condom use, and STI history were obtained. All subjects received a clinical standard of care. Patients who had clinical symptoms (i.e. discharge, dysuria, or penile irritation) and whose urethral Gram stain smear demonstrated ≥5 polymorphonuclear leukocytes per oil-immersion field were classified as having urethritis.¹ The asymptomatic group consisted of patients free of symptoms who attended the clinic for the purpose of STI screening because of a high risk of STI (>20 lifetime sexual partners, new sexual partner, or history of prior STI). A urethral Gram stain was not performed in these patients, as the Levinsky Clinic's policy is to avoid the unpleasant urethral smear in asymptomatic men.

The exclusion criteria included antibiotic use, such as fluoroquinolones, macrolides, and tetracyclines, within the past six months, and duration of symptoms of more than three months.

Specimen collection

First-voided urine specimens were collected from all men, ≥2 h post-urination.

Urine samples were stored and transported at 2–8°C, and processed within seven days of collection.

Gram staining

The patient's urethral discharge was smeared and spread immediately on a slide in the clinic. Slides were transported to microbiology laboratory where they were heat fixed and Gram staining was performed according to the usual technique.

Molecular testing

Genomic DNA was extracted from pretreated urine specimens by using the AccuPrep Genomic DNA Extraction Kit (Bioneer, Seoul, Korea), according to manufacturer's instructions.

PCR amplification was performed with the Seeplex® STD6 ACE Detection kit (Seegene, Inc., Seoul, Korea) according to manufacturer’s protocol.¹⁰ The kit contains sets of primers designed from highly conserved regions of genetic sequences for six pathogens (C. trachomatis, N. gonorrhoeae, M. genitalium, M. hominis, Ureaplasma and T. vaginalis) using Seegene DPO technology. An internal control is present in the mix for the detection of PCR inhibiting conditions (981 bp). For the negative control, sterile deionized water was used in place of DNA as the PCR template. To check the integrity of primers used in the PCR assay, positive DNA controls from the kit were assayed in the presence of all primer pairs. Amplified PCR products were separated and detected by automated gel electrophoresis using tee TapeStation detection system (Lab901, Bilson Glen, Loanhead, UK).¹⁰

Statistical analysis

The statistical analysis was performed in the R statistical software (R Foundation for Statistical Computing, Vienna, Austria). Patient characteristics and risk factors were summarized by descriptive statistics. Prevalence ratio estimates and 95% confidence intervals comparing STI prevalence between symptomatic and asymptomatic patients, and comparison of homosexuality prevalence for different STIs in symptomatic patients were computed using the epi.2by2 function from the epiR package. Fisher's exact test was used to compute the p values comparing dichotomous patient characteristics and risk factors in Tables 1 and 2. The Wilcoxon Rank Sum test was used to compute the p values for comparing continuous variables in Tables 1 and 2. p value was considered significant when p < 0.05.

Table 1.

Patient characteristics and risk factors for STI in symptomatic and asymptomatic patients.

Characteristic	Symptomatic patients Total number = 118 N (%)	Asymptomatic patients Total number = 141 N (%)	p value*
Age: median [IQR]	31 [25–35]	30 [26–38]	0.43
Homosexual men	60 (50.8%)	96 (68.1%)	<0.01
>20 lifetime sexual partners	83 (70.3%)	134 (95%)	<0.01
New sexual partner	102 (86.4%)	139 (98.5%)	<0.01
No. of sexual partners in the past three months. Median [IQR]	3 [1–6]	4 [2–19]	<0.01
Inconsistent condom use	114 (96.6%)	129 (91.5%)	0.9
Prior STI	60 (50.8%)	53 (37.6%)	0.02
Commercial sex worker	4 (3.38%)	11 (7.8%)	0.22
Use of sex services	20 (16.9%)	28 (19.8%)	0.8

p value was calculated using Fisher's exact test and Wilcoxon test for dichotomic and continuous variables, respectively. p value was considered significant when p < 0.05.

Table 2.

Epidemiological and clinical features according to STI agent in symptomatic patients.*

	MG (n = 14)^† N (%)	NG (n = 58)^† N (%)	CT (n = 26)^† N (%)	p value MG (n = 11) vs. NG (n = 55)^‡	p value MG (n = 14) vs. CT (n = 26)
Age: median [IQR]	31 [27–34]	30 [25–43]	31 [25–37]	0.73	0.64
MSM	5 (35.7%)	36 (63.2%)**	13 (50%)	0.02	0.51
>20 lifetime sexual partners	6 (42.8%)	43 (75.4%)**	22 (84.6%)	0.005	0.01
New sexual partner	11 (78.5%)	53 (91.3%)	25 (100%)^††	0.12	0.07
No. of sexual partners in the past three months: median [IQR]	2 [1–3]	4 [2–10]	3 [1–10]	0.001	0.09
Inconsistent condom use	14 (100.0%)	58 (100.0%)	23 (92.0%)^††	1	0.53
Prior STI	8 (57.1%)	27 (47.4%)**	16 (64.0%)^††	0.51	0.74
Commercial sex worker	0	4 (6.8%)	1 (3.8%)	1	1
Sex services consumption	1 (7.1%)	11 (20.0%)**	3 (12.0%)^††	0.67	1
Symptoms duration (days): median [IQR]	5.5 [2.2–21]	4.5 [3–7]	5 [3–10]	0.33	0.93

p value was calculated using Fisher's exact test and Wilcoxon test for dichotomic and continuous variables, respectively. p value was considered significant when p < 0.05.

†

Seven patients had Neisseria gonorrhoeae (NG) and Chlamydia trachomatis (CT) co-infection and appear in both NG and CT columns; three patients had NG and Mycoplasma genitalium (MG) co-infection and appear in both NG and MG columns

‡

The comparison between MG vs. NG excluded the three NG–MG co-infected patients, thus resulted in 11 MG patients and 55 NG patients.

Only 57 NG patients with available data.

††

Only 25 CT patients with available data.

Ethical approval

The study was approved by the E. Wolfson Hospital Review Board (number1062).

Results

Patients characteristics and risk factors for STD

Of a total of 259 men who were enrolled in the study, 118 (45.5%) had symptoms of dysuria and macroscopic urethral discharge (and urethral Gram stain smear demonstrated ≥5 polymorphonuclear leucocytes per oil-immersion field), and thus are referred to as symptomatic patients whereas 141 (54.4%) were asymptomatic patients at high risk of STI.

Patient characteristics and risk factors for STI in symptomatic and asymptomatic patients are presented in Table 1.

Mean age of the study participants was 32.4 years, the median was 30 years and range was 17–65 years, interquartile range (IQR) 26–37. Symptomatic patients and asymptomatic patients had similar age distribution (Table 1). Criteria used for the selection of the asymptomatic patients at high risk of STI were, as expected, significantly more prevalent among the asymptomatic group of patients than among the symptomatic group of patients. An exception was prior STI, which was significantly higher in the symptomatic group of patients, 60 out of 115 symptomatic patients (50.8%) vs. 53 out of 141 asymptomatic patients (37.6%). Rates of inconsistent condom use, commercial sex work, and use of sex services were not significantly different between symptomatic and asymptomatic patients.

STI prevalence

The overall rate of M. genitalium infection, C. trachomatis infection, and N. gonorrhoeae infection in the study population was 17 of 259 patients (6.6%), 33 of 259 patients (12.7%), and 60 of 259 patients (23.1%), respectively. Not a single case of trichomoniasis was detected. At least one STI pathogen was detected in 88 of 118 (74.6%) symptomatic patients as opposed to 12 of 141 (8.5%) asymptomatic patients (prevalence ratio [PR] = 8.8, confidence interval [CI] = 5.1–15.2).

A strong association between STI and symptoms was demonstrated: M. genitalium was detected in 14 of 118 (11.9%) symptomatic patients compared to only 3 of 141 (2.1%) asymptomatic patients (PR = 5.6, CI [1.6–19]), C. trachomatis in 26 of 118 (22%) vs. 7 of 141 (5%) (PR = 4.4, CI [2–9.9]) and N. gonorrhoeae in 58 of 118 (49%) vs. 2 of 141 (1.4%) (PR = 34.7, CI [8.7–139]), respectively.

Co-infection was found only among symptomatic patients: 5.9% of symptomatic patients (seven patients) had gonorrhea and chlamydia concomitant infection; 2.5% of symptomatic patients (three patients) had N. gonorrhoeae and M. genitalium dual infection.

Epidemiological and clinical features according to STI pathogen in symptomatic patients

Most (9/14, 64.3%) symptomatic patients with M. genitalium were heterosexual men, in contrast to symptomatic gonorrhea and chlamydia patients among whom only about a half or less were heterosexual men (Table 2). Moreover, all asymptomatic M. genitalium patients were heterosexual men (three out of three). There was an insignificant association between M. genitalium and male heterosexuality among symptomatic patients (PR = 1.5, CI [0.7–3.1]). Analysis of the whole cohort found a significant association between M. genitalium and male heterosexuality (PR = 2.2, CI [1–4.5]). A significant association between gonorrhea and male homosexuality (PR = 1.6, CI [1.1–2.4]) was found among symptomatic patients. No association was found between C. trachomatis and sexual preference among symptomatic patients (PR = 1, CI [0.7–1.6]).

Comparison of the number of sexual partners in the previous three months between symptomatic patients according to the STI pathogen was done using Wilcoxon test; a significant lower number of sexual partners in the previous three months was found among M. genitalium patients compared to gonorrhea patients (p value = 0.001) but not compared to chlamydia patients (p value = 0.09). Symptomatic M. genitalium patients tended to have a more ‘conservative’ sexual behavior than other symptomatic patients. They had a lower rate of >20 lifetime sexual partners compared to both gonorrhea and chlamydia patients (p value = 0.005 and p value = 0.01, respectively) and a lower rate of new sexual partner compared to chlamydia patients, with a tendency toward statistical significance (p value = 0.07) but not compared to gonorrhea patients (p value = 0.12). Rate of use of commercial sex services was insignificantly lower among M. genitalium patients. Symptom duration was insignificantly longer in M. genitalium patients vs. gonococcal patients and chlamydia patients.

Discussion

The prevalence of M. genitalium in our population of men with urethritis attending an STI clinic in Tel Aviv (∼12%) was lower than that reported in men with urethritis in Russia (26%),¹¹ in Baltimore, MD, USA (22%),¹² in New Orleans, LA, USA (24%)¹³ and in Paris, France (21%),¹⁴ similar to that reported in men with urethritis in Australia (9% of NGU),¹⁵ in Siberia (8–12%),¹⁶ Bristol, UK (10.9%),¹⁷ and Birmingham, UK (12%).¹⁸ Yet higher than that reported in men with arthritis in Sweden (6%),¹⁹ in Milwaukee, WI, USA (6.8%),²⁰ London, UK (2.7%),²¹ and Amsterdam, The Netherlands (5.2%).²² Unlike the findings of several similar studies,^12,14,23 in which C. trachomatis was the most common organism, we found that N. gonorrhoeae was the prevailing one, as did Mena et al.¹³ We have not documented a single case of T. vaginalis infection. The same finding (0/193) was observed by Dupin et al.¹⁴ in Paris, France. In the majority of studies of urethritis in males attending an STI clinic, the rates of detection of T. vaginalis were low (<5%).^{12,15,20,22–27}

The results of this study add further evidence of the association between male urethritis and M. genitalium, as documented by others.^{4,5,12–14,17,20,30–32} M. genitalium was significantly more common in the group with symptomatic urethritis compared with the asymptomatic group (11.9% vs. 2.1%). This was previously documented by several authors.^{5,12–14,19,25,26,28,29,30} Similar rates of M. genitalium and C. trachomatis in asymptomatic attendees of an STI clinic were reported by Moi et al.⁵

The co-infection rate was low and was documented only in symptomatic patients. The rate of co-infection was 5.9% for gonorrhea and chlamydia and 2.5% for gonorrhea and M. genitalium infection. Our co-infection rate was lower than that of Gaydos et al.,¹² Mena et al.,¹³ and Gambini et al.,³¹ but similar to that reported by others.^4,16,17,27

We have found a significant association between M. genitalium and male heterosexuality as opposed to gonorrhea, which was significantly associated with male homosexuality. Bing-jie Zheng et al.³² have shown that M. genitalium was more prevalent in bisexual men compared to those who engaged only in homosexual behaviors. Others^5,26 have found that homosexual men had significantly lower prevalence of M. genitalium urethritis compared with heterosexual men. In the same sense was the finding¹⁵ that M. genitalium was associated with sex with women and unprotected vaginal sex. Bradshaw et al.²³ have found that M. genitalium was less common than C. trachomatis and N. gonorrhoeae in homosexual men attending male-only saunas, and that it was most likely to be detected as an asymptomatic rectal or a urethral infection (out of 521 throat swabs, not a single one was positive for M. genitalium).²³

We suppose that the lower prevalence of M. genitalium urethritis in homosexual men compared with heterosexual men is explained by the extremely low prevalence of M. genitalium colonization in the pharynx of both men (including homosexual) and women.^23,33,34 Oral sex is a particularly common sexual practice in homosexual men,²³ and oral sex was reported much more frequently in homosexual than in heterosexual encounters.³⁵ Thus, this common sexual practice in homosexual men involves a site which is a poor reservoir of M. genitalium resulting in reduced transmission of the pathogen,²³ and consequently in lower rate of M. genitalium urethritis in this population.

The limitations of the study included the inability to enroll larger numbers of men with urethritis, and consequently the low number of M. genitalium infections, which weakens analyses of association. The low prevalence of M. genitalium in our asymptomatic group could be due to sampling bias as 95% of the recruits in this group were men with high sexual risk behavior (>20 lifetime sexual partners).

Strengths included prospective collection of data and specimen and using the nucleic acid amplification tests (NAATs) for four organisms concurrently, thereby identifying co-infections. Also, this is the first study to document the incidence and characteristics of M. genitalium urethritis in Israel.

We conclude that M. genitalium is an important cause of urethritis in Tel Aviv, Israel. Its clinical and epidemiological characteristics are similar to those in other western countries. Testing men for M. genitalium as a cause of NGU is warranted, particularly because of its poor response to doxycycline and possible failure of azithromycin.³⁶

Footnotes

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

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