Mycoplasma genitalium was isolated originally from the urethra of two men with non-gonococcal urethritis (NGU) in 1980.^1,2 Isolation of this bacterium is very difficult but the use of polymerase chain reaction (PCR) technology has consistently shown M genitalium to be a major cause of sexually transmitted non-chlamydial non-gonococcal urethritis (NCNGU) among men.^3–9 There is also increasing evidence suggesting that M genitalium causes mucopurulent cervicitis in women¹⁰ and that it may cause endometritis¹¹ and possibly tubal infection with sequelae in the form of ectopic pregnancy or tubal infertility.¹² Thus, the role of M genitalium may not significantly differ from that of genital Chlamydia trachomatis infection. Most M genitalium studies in STD clinic outpatients have focused on symptomatic patients with urethritis and have used non-symptomatic patients as controls. These studies demonstrate that M genitalium is detected significantly more frequently among symptomatic patients than among asymptomatic controls, thus indicating that this bacterium is a pathogen of the genital tract.^3,6,13 The role of C trachomatis and Neisseria gonorrhoeae as pathogens is well established.^14–17 In contrast with M genitalium, the role of Ureaplasma urealyticum appears to be less clearly defined.^7,18,19

The aim of this cross sectional study was to compare C trachomatis and M genitalium infections in terms of signs and symptoms in male STD clinic attendees and to study the prevalence of the bacteria and the rate of infection among sexual partners. A secondary aim was to study the benefit of microscopic examination of urethral smears.

METHODS

Patients

During a 6 month study period from 1 February 2000 to 31 July 2000, all male attendees at the Örebro University Hospital STD clinic were included. Data were collected on a standard questionnaire regarding the reasons for attendance, age, symptoms of urethritis (dysuria and discharge), number of sexual partners within the past 6 months, condom use, sexual intercourse with men, history of STIs, probable STIs among the partners, recent or current antibiotic treatment, and other diseases.

Sampling

A total of 519 men between 16 and 67 years of age (median 27 years) were included. Smears were taken with a blunt curette from the distal urethra and stained with methylene blue. The amount of exudate recovered was estimated, using profuse as a definition of discharge (>≈1 cm² of a single cell layer smear on the slide), moderate and poor as a graduation within the normal amount of secretion. During the study period, the patients were seen by seven clinicians, but four of those examined 95% of all patients. All smears were examined microscopically (1000×) with a Nikon Labophot microscope. The definition of urethritis is generally >4 polymorphonuclear leucocytes (PMNL) per high power field (HPF) in more than four high power fields,¹⁷ but in the current study, smears with 5–10 PMNL per HPF were defined as “grey zone urethritis” and >10 PMNL per HPF as urethritis. After urethral smear was sampled the first void urine (FVU) was collected for C trachomatis and M genitalium tests and distributed in two screw capped 13 ml polypropylene tubes (Sarstedt, Nümbrecht, Germany). Partners, women and men, attending the STD clinic during the study were all examined for C trachomatis and M.genitalium.

Microbiological analysis

One of the tubes containing 5–10 ml of urine was sent the same day to the department of clinical microbiology, Örebro University Hospital, and stored at 2–8°C. The urine samples were tested by the Cobas Amplicor Chlamydia trachomatis Test (Roche Diagnostics Systems, Inc, Branchburg, NJ, USA) as described by the manufacturer.

The other tube containing 5–10 ml FVU was sent the same day by express mail to Statens Serum Institut, Copenhagen, Denmark for M genitalium PCR test. M genitalium was detected by an inhibitor controlled PCR using primers detecting the M genitalium 16S rRNA gene.²⁰ All positive results were confirmed by a PCR detecting the MgPa adhesin gene.²¹ Samples for Neisseria gonorrhoeae (culture) were taken from 88 men. Samples were taken selectively on certain indications—that is, unprotected sexual contacts abroad, purulent discharge, unprotected sexual contacts between men, and partner notification because of gonorrhoea, and not as a screening test because of the current low incidence in Sweden (0.7/100 000 inhabitants—that is, 588 cases in 2000).²²

Follow up

All patients infected with C trachomatis and/or M genitalium were asked to re-attend for a follow up visit 4–5 weeks after commencing antibiotic treatment. All recent partners of C trachomatis and M genitalium infected patients were notified and asked to attend the STD clinic for C trachomatis and M genitalium testing and genital examination. Recent partners were defined as all partners during the past 6 months before attendance or at least the two latest partners. As a part of this study the treatment efficacy in M genitalium infected patients was evaluated in an open pilot study.²³ The results from that study suggest that tetracyclines are not sufficient to eradicate M genitalium, but that azithromycin might be effective.

Statistical analysis

The χ² test and Fisher’s exact test were used to test for differences in proportions and Mann-Whitney U test and Kruskal-Wallis test for non-parametric comparison of groups. Stata statistical software version 8.0 was used for calculating confidence intervals.

RESULTS

Neisseria gonorrhoeae was isolated from two men. No patient was smear positive and culture negative for N gonorrhoeae. Non-gonococcal urethritis (NGU) was detected in 271 men, comprising 61 patients between 16 and 56 years of age (median 23 years) with C trachomatis infection and 34 patients between 20 and 55 years of age (median 28 years) with M genitalium infection. Four patients with chlamydia had a concurrent M genitalium infection and were excluded in the comparison of signs and symptoms between M genitalium and C trachomatis. Initially, 41 men had positive PCR tests for M genitalium, but seven, of which three had a microscopic urethritis and four not, had tests that were not confirmed and hence they were excluded from the study. Among these seven patients one had symptoms—that is, symptoms of epididymitis, but the remaining six were asymptomatic. The remaining 180 men with M genitalium negative NCNGU were between 16 and 54 years old (median 25.5 years). These patients were considered as having non-specific urethritis (NSU). Among the 180 men 42 had a microscopic urethritis, where the physician diagnosed 11 as prostatitis, 22 as genital papillomavirus infection, and nine with genital herpes simplex infection; these diseases themselves might be the cause of urethritis.

The overall prevalence of M genitalium was 7% (34/512), and that of C trachomatis 12% (61/512) and of NSU 35% (180/512). M genitalium infected men had symptoms of urethritis significantly more often than those with chlamydial infection—that is, 73% (22/30) versus 40% (23/57) and with a relative risk (RR) of 1.8 (95% CI 1.2 to 2.7). The M genitalium and C trachomatis groups were also compared with the NSU group where the C trachomatis group was indexed as 1 (table 1). The rate of microscopic signs of urethritis was high in both infections reaching about 90% and no significant difference was found (RR 0.8; 95% CI 0.3 to 2.0).

The number of partners in the M genitalium positive NGU, C trachomatis positive NGU, and NSU groups was significantly different (p = 0.03). The patients with C trachomatis NGU reported significantly more partners in the previous 6 months.

There were no significant differences regarding history of previous STI (urethritis) between the different groups (table 1).

The mean duration of symptoms was 4.2 weeks (median 3, range 1–25) for the 21/23 (data missing from two) men with symptomatic C trachomatis NGU, 5.7 weeks (median 2, range 1–60) for the 22 men with symptomatic M genitalium positive NGU (p = 0.42), and 6.5 weeks (median 2.5, range 1–12) for the three men with symptoms and verified infection with both bacteria. Only 27% (49/180) of the men with NSU had symptoms correlated to urethritis compared to 23 (40%) of 57 with chlamydial infection (p = 0.068) and 22 (73%) of 30 with M genitalium infection (p<0.0001). Of the men with NSU 49% (75/152) had neither symptoms nor visible discharge compared to 20% (10/51) (p<0.001) and 7% (2/27) (p<0.001), respectively, among C trachomatis and M genitalium positive men (table 2). Men with C trachomatis positive or M genitalium positive NGU were more likely to have discharge (as a sign) than were those with NSU (p<0.001).

Altogether, among all 512 attendees in the current study, eight men reported having sex with men, of which four had had only passive anal sex, three both active and passive anal sex, and one only reciprocal oro-genital sex (fellatio). The 34 M genitalium infected men were all heterosexual, except one who also had a concurrent C trachomatis infection and had had fellatio with a man a year before attendance, but who subsequently had only had sexual intercourse with women (>20 partners). Among the other C trachomatis positive men, there were two homosexual men, of which one was notified because of his partner’s chlamydial infection (tested at another clinic) and the other had had partners who could not be identified. Also, among the 180 men with a NSU there was one homosexual man, but his partners did not attend the clinic for testing.

Nineteen female partners of 18 men infected with M genitalium were examined; 12 (63%) of them were M genitalium positive. Four (21%) were C trachomatis positive; two of these patients had partners who were infected with both C trachomatis and M genitalium. One woman had negative tests but a mucopurulent cervicitis. Correspondingly, 39 partners of 28 C trachomatis positive men were examined; three men each had three partners and six men each had two partners. Twenty six (67%) of those partners were C trachomatis positive and two (5%) were M genitalium positive. The M genitalium positive women were partners of dual infected men. Eight (17%) of the 47 examined partners of men with NSU were C trachomatis positive, and three (6%) were M genitalium positive. Both C trachomatis and M genitalium were found significantly more often in partners of men with the corresponding infection, than in partners of men with NSU (p<0.0001 for both). The diagnosis and number of partners of each group are shown in table 3.

DISCUSSION

In this cross sectional study, the prevalence of C trachomatis infection was higher than that of M genitalium infection among male STD clinic attendees (12% versus 7%, respectively). Only four patients were infected with both bacteria. Only a few true cross sectional studies of STD clinic populations have been published.^5,24 Most investigations have been case-control studies. The inclusion criterion in the case group has been either symptoms or microscopic signs of urethritis; in the control group, asymptomatic men or men without microscopic signs of urethritis have been included.^3,4,6–8,25 In a recent Swedish report,²⁴ all attendees were tested for both M genitalium and C trachomatis and a similar prevalence for M genitalium (6%) was found; however, the prevalence of C trachomatis was surprisingly low (5%). In patients examined in another Swedish study⁵ 3 years earlier than those in the present study, a C trachomatis prevalence of 19% and an M genitalium prevalence of 7% were found. The reason for the differences in the C trachomatis prevalence in three Swedish STD clinics within a relatively limited time frame is not clear, but may reflect differences in the populations studied. It is surprising though that such differences were not found in the prevalence of M genitalium.

In a recent French study,¹⁸M genitalium was found significantly more frequently among a group of men with urethritis and symptoms than in a group of men with symptoms but no urethritis, which was also the case for C trachomatis but not for Ureaplasma urealyticum, where the same prevalence (26% v 22%) was found in both groups. The criteria for urethral symptoms were wider than in the present study. Also the criterion for urethritis was different, based on examination of FVU and not urethral smears.¹⁸

In the above mentioned case-control studies the proportion of M genitalium and C trachomatis in the NGU groups was similar to the results in the current study, although the number of patients with NSU in our study was higher than in some reports^4,8 and equal to others.^5,13,25,26

M genitalium infected men had symptoms of urethritis significantly more often than those infected with C trachomatis. This was not reflected in the microscopic signs of urethritis and the reason for this finding is not clear. It could be speculated that the production of hydrogen peroxide by M genitalium²⁷ may contribute to the symptoms. Whether the symptoms reflect also a potential for deeper invasion such as is seen in a chimpanzee model, where two of the 10 inoculated animals had M genitalium isolated from the blood stream²⁸ is not clear. The number of patients in the present study was limited and therefore our findings have to be verified in future studies. There is evidence that M genitalium may cause endometritis, PID, and sexually acquired reactive arthritis.^2,11,29 Most published studies have focused on symptomatic patients comparing signs and symptoms of infected people with the two organisms. In this study there was no difference in microscopic signs between the bacteria. The high proportion of urethritis in men infected with M genitalium and the low rate of mixed infections support the conclusion made by others that M genitalium is a pathogen.^{2–9,13,24–26,30} The present study also showed that M genitalium infection seems to have a high prevalence in the society and therefore might be considered for screening purposes at STD clinics.

In this study, 12 of 19 (63%) female partners of men infected with M genitalium tested at the STD clinic also had a M genitalium infection compared with 26 of 39 (67%) chlamydial infected female partners of men with C trachomatis infection. These data emphasise the role of M genitalium as a sexually transmitted pathogen, since only three of 47 partners of men with NSU were M genitalium positive. C trachomatis is a notifiable infection including mandatory partner notification, but partners can attend any clinic, which might explain the rather low number of partners per man infected with C trachomatis (39/61) who were examined at our STD clinic. Since legislation regarding M genitalium infection does not exist, often only current partners attended the STD clinic. This might explain the rather low attendance rate among partners. For NSU cases some patients attended because of one of their female partners had C trachomatis infection, but had negative test results. This might explain the high prevalence of C trachomatis among the partners of men with NSU.

The men with chlamydial infection reported a higher number of recent partners than the other groups, including the M genitalium positive group. Such a difference has not been demonstrated in other studies.^4,8M genitalium positive men were older than those with C trachomatis infection. Whether the M genitalium positive men have carried the infection for a longer period of time is not known. Unfortunately, we do not have data on the number of lifetime partners, which may have provided an explanation for this difference; however, no difference in the duration of symptoms was seen. Only eight men reported sexual contact with other men, although three had a C trachomatis infection, and one of those was also infected with M genitalium. It is not possible from this study to draw any conclusions as to whether M genitalium is more or less prevalent among men who have sex with men than among heterosexual men.

In the large NSU group comprising 180 men with urethritis without recognised cause (66% of NGU), significantly more patients were asymptomatic compared with both the M genitalium and C trachomatis groups, 27% versus 73% and 40% respectively. This presents a serious dilemma in daily clinical work. Is the inflammation caused by a bacterial infection, and are these patients in need of treatment? Most C trachomatis infected men are asymptomatic and they should therefore not have been treated if both symptoms and microscopic signs were set as criteria for treatment. The sensitivity of C trachomatis PCR tests is high but less than 95% and possibly even lower for M genitalium PCR tests, so patients with false negative test results may benefit from treatment. Horner and co-workers³¹ have suggested that treatment guidelines should be revised, and propose that asymptomatic men without discharge should not receive antibiotic treatment. In the current study we attempted to set more objective criteria for the measurement of discharge, and experienced clinicians examined most patients (approximately >85%). The high rate of patients without discharge in combination with lack of symptoms in the NSU group (49%) and the corresponding high rate of discharge and symptoms in C trachomatis and M genitalium infected men support the proposal by Horner et al, although some patients would have been missed at the examination. In these patients, treatment would have been delayed with the risk for further transmission of the infections and possible risk for sequelae.

The most widely accepted and used criterion for urethritis is >4 PMNL/HPF which was established in the late 1970s.¹⁷ The result of this study calls for a reconsideration of this criterion, since none of the patients with C trachomatis or M genitalium infection had fewer than 10 PMNL/HPF. Only among the men with NSU was “grey zone urethritis” found—in 29 patients. Obviously, the interpretation of the smear depends on several variables: the instrument for sampling, the standard of the microscope and how it is used, the experience of the clinician, and the interpretation of discharge and smears. This subjective procedure can never be strictly scientifically standardised. We believe that more studies are required to examine the diagnostic tools for determining the clinically relevant definition of urethritis.

In summary, M genitalium was strongly associated with symptomatic urethritis and men infected with both C trachomatis and M genitalium transmitted the infections to a large proportion (two thirds) of their sexual partners. More studies are needed to determine the potential sequelae of M genitalium infection in men as well as in women.