Mycoplasma genitalium Infections in Asymptomatic Men and Men with Urethritis Attending a Sexually Transmitted Diseases Clinic in New Orleans
- Louisiana State University Health Sciences Center and the Delgado Clinic, New Orleans Health Department, New Orleans, Louisiana
- Reprints or correspondence: Dr. David H. Martin, Section of Infectious Diseases, Louisiana State University Health Sciences Center, 1542 Tulane Ave., New Orleans, LA 70112 (dhmartin{at}lsuhsc.edu).
Abstract
We report the results of a study of Mycoplasma genitalium (detected with a modified polymerase chain reaction [PCR] assay) in men with urethritis and in asymptomatic control subjects at a sexually transmitted diseases clinic in New Orleans. Data for 97 men with urethritis and 184 asymptomatic men were available for analysis. M. genitalium infection rates in symptomatic and asymptomatic men who were negative for Chlamydia trachomatis and Neisseria gonorrhoeae were 25% and 7%, respectively (P = .006). M. genitalium coinfection rates among men with chlamydial and gonococcal urethritis were 35% and 14%, respectively. Men with M. genitalium urethritis resembled those with C. trachomatis in that both groups were younger and more likely to experience milder urethral symptoms. Among men with urethritis, the sensitivities of PCR of urine and swab specimens for the detection of M. genitalium were 87% and 91%, respectively. M. genitalium is associated with nongonococcal urethritis in this population.
Nongonococcal urethritis (NGU) is one of the most common sexually transmitted diseases (STDs) diagnosed in men [1]. Men with NGU can be categorized into those with and those without Chlamydia trachomatis infection. Studies performed in the late 1970s and early 1980s reported that 35%–50% of cases of NGU were due to C. trachomatis infection [2]. More recently, the proportion of cases of NGU caused by C. trachomatis has been observed to be as low as 15%, possibly because of the implementation of and improvement in screening and treatment programs for C. trachomatis during the 1990s [3]. Ureaplasma urealyticum appears to cause some of these cases of nonchlamydial NGU. In fact, at one time, this organism was thought to cause the majority of cases of NGU, but recent studies have not shown differences in the prevalences of U. urealyticum between patients and controls [4–7]. Trichomonas vaginalis causes a small proportion of cases of NGU, as does herpes simplex virus. The roles of a variety of other organisms in NGU have been investigated without conclusive results [1].
M. genitalium was first isolated from the urethras of 2 of 13 men with urethritis [8], but studies that attempted to assess its association with disease were inhibited by the difficulty of growing the organism in culture. In more recent years, more reliable detection became possible after the development of specific PCR assays [9, 10]. Subsequent studies that used these assays have provided strong evidence of an association between acute urethritis in heterosexual men and M. genitalium infection [6, 11–18]. These new findings suggest that the association is likely to be causal, a notion consistent with the known virulence characteristics of this microorganism and its ability to cause urethritis in nonhuman primates [19, 20].
We undertook this study to test the hypothesis that M. genitalium is associated with urethritis in men who attended an urban STD clinic in New Orleans, Louisiana, and, at the same time, to determine the clinical and epidemiologic characteristics of M. genitalium-associated disease.
Patients and Methods
Patients. We studied 101 men aged 16–54 years who had clinical symptoms or signs of urethritis and who attended the City of New Orleans' STD clinic. A diagnosis of urethritis was made if there were ⩾4 polymorphonuclear leukocytes per oil-power microscopic field in the urethral smear and if the patient complained of experiencing discharge, dysuria, or penile irritation. Subjects were excluded from the analysis if they had urinated <1 h before testing or had received antibiotic treatment within the 3 months before the study. Four men were subsequently excluded because of failure to meet all study criteria; therefore, data from 97 patients were available for analysis.
After written informed consent was obtained, patients were interviewed and examined. Demographic, clinical, and STD risk factor data were recorded on a standardized questionnaire. Two samples were obtained from the urethra via urethral swabbing. The first was obtained by insertion of the swab just within the urethral meatus and was used to prepare a smear on a glass slide for quantification of polymorphonuclear leukocytes and was then submitted to clinic's laboratory for chlamydial and gonococcal testing by use of the Gen-Probe Pace 2 assay. The second, a thin wire Dacron-tipped swab, was inserted 1–2 cm within the urethra and was rotated several times to obtain a specimen for the M. genitalium PCR assay. This swab was placed in dry tube and transported to the laboratory at room temperature. Each participant then provided ∼30 mL of the first voided urine that was stored at 4°C until it was transported to the laboratory on the day of collection. Patients were treated with doxycycline at the completion of the study visit.
To compare the prevalences of infections with M. genitalium, C. trachomatis, and Neisseria gonorrhoeae in symptomatic men, as control subjects, we used 215 men who attended the same clinic during the period of September 2000 through January 2001, who denied having symptoms of urethritis, and who were enrolled in a study of the ProbeTec DNA amplification assay (Becton-Dickinson) for detection of C. trachomatis and N. gonorrhoeae. These men attended the clinic because they had contact with women who had STDs (44%), for a screening examination (45%), or because of symptoms of other STDs, such as genital warts (12%). Comparisons of asymptomatic men with men who had urethritis were based on urine assay results only, because swab specimens for M. genitalium were not obtained from asymptomatic men. The urine volume obtained was inadequate for study for 31 men, which left 184 men for analysis.
These studies were approved by the Louisiana State University Health Sciences Center Institutional Review Board in accordance with the Helsinki Declaration of 1975, as revised in 1983.
PCR assay for detection of M. genitalium. Upon arrival in the laboratory, urine specimens were mixed thoroughly and formed into 1-mL aliquots for storage at -70°C. After thawing, aliquots were processed first by centrifugation for 15 min at 13,000 g in a microcentrifuge. All urine was carefully removed from the pellet before it was resuspended in 200 μL of lysis buffer and 200 μL of binding buffer (High Pure PCR Template Preparation Kit; Boehringer Mannheim). After addition of 40 μL of proteinase K, the mixture was incubated for 1 h at 72°C and then processed according to the manufacturer's instructions. The samples were suspended in 45 μL of elution buffer.
Swabs were gently mixed for 5 min in 400 μL of binding buffer and 400 μL of lysis buffer (total, 800 μL) upon arrival at the laboratory. The swabs were then wrung out on the sides of the tube and the binding buffer/lysis buffer suspension was equally divided into 2 vials and stored at -70°C. Swab samples were processed as described above for the resuspended urine pellets.
PCR was performed on the swab and urine DNA lysates using the AmpliTaq Gold PCR Kit (Perkin-Elmer Applied Biosystems). The PCR target was a 495-bp fragment beginning 85 bp upstream from the M. genitalium adhesion gene start codon. The primer designations were MgPaW1 (5′-AAGTGGAGCGATCATTACTAAC-3′) and MgPaWR1 (5′-CCGTTGTTATCATACCTTCTGA-3′). These primers were designed in our laboratory on the basis of a computer analysis of optimal primers for this region of the genome. The 25-μL PCR reaction mixture contained 5 μL of lysate, 25 pM of each primer, 0.25 mM of each dNTP (New England Biolab), 3.5 mM of MgCl2, and 2.5 U of Gold Taq polymerase. PCR amplification conditions were 95°C for 10 min, followed by 35 cycles at 94°C for 40 s, 41°C for 40 s, 72°C for 40 s, and a final extension period of 15 min at 72°C.
Ten microliters of the PCR product was electrophoresed through 1.2% agarose gel, stained with ethidium bromide, and photographed with a Polaroid camera. The presence of appropriately sized bands were determined from the photograph. PCR products were then transferred from the gel to Nytran Plus membranes (Schleicher & Schuell) overnight by means of Southern blot test. The membrane was then baked at 80°C for 2 h followed by prehybridization for 1 h at 55°C in a buffer containing 5× standard saline citrate (SSC), 0.1% SDS, 1 mM EDTA, and 5% blocking buffer (Boehringer Mannheim). After prehybridization, 100 pM of biotinylated M. genitalium-specific internal DNA probe (5′-TTTGCTTACCAACCCAAGCAGTTAAG-3′; Biotin) was added and the membrane was incubated overnight at 55°C. After washing 3 times in 5× SSC/0.1% SDS at 55°C, the blots were incubated in 2× SSC/0.1% SDS with 1 μg of streptavidin-horseradish peroxidase (Vector Laboratories) for 45 min at room temperature and then washed 3 times in 2× SSC/0.1% SDS solution. The membrane was removed from the washing solution and placed face-up on polyethylene wrap. Retained wash solution was carefully removed with absorbent tissue.
The membrane was then covered for 2 min with chemiluminescent detection reagent (ECL Western blot test detection reagents; Amersham Pharmacia Biotech) at room temperature. The reagents were again removed carefully with absorbent tissues. The membrane was then completely wrapped in a sheet of polyethylene and placed in a X-ray film cassette together with Hyperfilm MP (Amersham Pharmacia Biotech) in a darkroom. The film was exposed for 30–90 s, developed, and air dried at room temperature. Positive samples were identified by the presence of a ∼495-bp band, compared with a molecular weight control.
For all positive urine specimens, a fresh aliquot of the specimen was obtained from the freezer and was reprocessed and amplified a second time to control for any possible PCR contamination. For all positive swab specimens, the second aliquot of the originally processed specimen was thawed and reamplified. Among symptomatic men, 6 had an initially positive specimen that was negative when the assay was repeated. Three of these were positive twice when the other specimen was used and therefore clearly represented false-negative results of repeated tests. Urine specimens from 5 asymptomatic men had positive results of initial tests but negative results of subsequent tests. It is not possible to know whether these nonrepeating results were false-negative findings or whether the initial test results were falsely positive. The fact that all but 1 of 8 specimens in question (from 3 symptomatic and 5 asymptomatic men) had positive results only on the Southern blot test suggests the presence of small numbers of organisms as the likely explanation. Nonetheless, to be as conservative as possible in estimating the association of M. genitalium with disease, specimens were not considered to contain the organism unless both the first and second aliquot of each tested positive.
C. trachomatis and N. gonorrhoeae testing. Urine specimens were processed ⩽24 h after they were obtained, and they were amplified according to the manufacturer's instructions for the ProbeTec strand displacement DNA amplification assay for both organisms. An internal amplification control was used for all specimens [21]. The Gen-Probe Pace 2 results were not used in this analysis.
Data analysis. Clinical and behavioral data were collected on standardized questionnaires and were entered into Microsoft Access. Statistical analysis was performed with χ2 and Fisher's exact tests for bivariate analyses by use of the SPSS software package (SPSS).
Results
The demographic and behavioral characteristics of the patients who constituted the study group are summarized in table 1. There were no significant demographic differences between the group of men with urethritis and the asymptomatic control group. African American patients accounted for >90% of the total population for both groups. The median age was 25 years, and the mean age was 26 years. As expected, asymptomatic men were more likely to have had a recent sex partner with an STD; this is a common reason that such men visit STD clinics. Symptomatic men were more likely to have had a recent new partner; this was also an expected finding because such women are the most common sources of a newly acquired STDs in men.
Table 1
Demographic, clinical, and behavioral characteristics of men with and men without symptomatic urethritis who were assessed for presence of Mycoplasma genitalium.
A total of 23 (24%) of 97 men with urethritis were infected with M. genitalium. Both the swab and urine specimens were positive for 18 men, whereas only the swab specimen was positive for 3 men, and 2 men had positive results of the urine test only. Coinfection rates were high (table 2). Seven (35%) of 20 men infected with C. trachomatis were also infected with M. genitalium. Among 29 men infected with N. gonorrhoeae, 4 (14%) also had M. genitalium infection. Three (19%) of the 16 men with both chlamydial and gonococcal infections also had M. genitalium.
Table 2
Mycoplasma genitalium infection rates in men with urethritis in relation to Chlamydia trachomatis and Neisseria gonorrhoeae infections.
Of the 41 urine and swab specimens that had positive Southern blot test results, we found that only 26 (63%) were unequivocally positive by gel electrophoresis alone. As summarized in table 3, we found that specimens obtained from men who were coinfected with both M. genitalium and chlamydia or gonococci accounted for all but 1 of the discrepant results between agarose gel electrophoresis and Southern blot test.
Table 3
Sensitivity of agarose gel electrophoresis for the detection of Mycoplasma genitalium infection in men with urethritis caused by M. genitalium alone and in men coinfected with Chlamydia trachomatis and/or Neisseria gonorrhoeae.
By eliminating men who were infected with >1 organism, we were able to compare the clinical and behavioral characteristics of men with urethritis caused by each of the 3 organisms alone (table 4). Men with gonococcal urethritis were older than were men infected with C. trachomatis or M. genitalium, but the difference was not significant. Both men with chlamydial urethritis and those with mycoplasmal urethritis were less likely than men with gonococcal urethritis to have a history of dysuria, to have a gross discharge on examination, and to have a yellow discharge. Men with M. genitalium infection more frequently acknowledged having had a sexual partner within the 30 days before the study who had a history of diagnosis or treatment of an STD than did men with either gonococcal or chlamydial disease.
Table 4
Characteristics of men with urethritis, according to organism detected.
We compared the 184 asymptomatic control subjects to men with urethritis by basing the definition of M. genitalium infection on urine specimen results only. Use of this definition reduced the total number of symptomatic M. genitalium-infected men to 20. As can be seen in table 5, M. genitalium, C. trachomatis, and N. gonorrhoeae infection rates in the group of men with urethritis were all significantly higher than in the asymptomatic control group. Of the 97 symptomatic men with urethritis, 74 (76%) had ⩾1 potential pathogen identified. When patients with other infections were excluded, M. genitalium was identified in 8 (25%) of the 32 men with symptomatic urethritis and only 10 (7%) of the 142 asymptomatic controls (P = .006, determined by Fisher's exact test).
Table 5
Organisms identified in men with urethritis and in asymptomatic control subjects.
Discussion
Although a number of studies have reported that there is an association between M. genitalium and NGU, to our knowledge, ours is the first study performed in the southern United States and the first that involves a predominantly African American population. We found that urine specimens obtained from 25% of 32 men with nonchlamydial NGU were positive for M. genitalium compared with specimens obtained from only 7% of 142 asymptomatic men seen at the same clinic who tested negative for both C. trachomatis and N. gonorrhoeae infection. In reports published elsewhere, M. genitalium has been found in 11%–35% of men with nonchlamydial NGU [4–6, 11–16]. Reported rates of M. genitalium infection among asymptomatic control subjects have ranged from 0% to 9%. The consistency of the data plus the fulfillment of Koch's postulates in primates [19, 20] provide strong evidence for a causal role of M. genitalium in NGU.
Previous studies from Europe and the United States of the role of M. genitalium as a urethral pathogen have not included men with gonococcal urethritis. We found that 14% of symptomatic men with gonococcal urethritis also harbored M. genitalium. The most striking difference in our series compared with most of the previously published studies was the high rate of M. genitalium infection (35%) among symptomatic men with chlamydial infection. Only a single study, which was from Italy, has reported similarly high coinfection rates for these 2 organisms [14]. The only other published article from the United States reported that only 3 (8%) of 36 men with chlamydial urethritis had concomitant M. genitalium infection [6].
There are 2 possible explanations for the difference between our observation and the observations reported by most other investigators. Except for a recent study from Seattle [6], none of the previous studies of M. genitalium in men with urethritis used Southern blot testing. Our data indicate that, in the presence of a coinfecting organism, gel electrophoresis alone may lack sensitivity for the detection of the M. genitalium PCR amplicon (table 3); therefore, the prevalence of coinfection may have been underestimated in many of the previous studies. Another possible explanation may be that there were differences in the populations studied. Data from studies performed in Western Europe, England, Japan, and Seattle, Washington, have included populations that have much lower incidences and prevalences of STD than does the population of New Orleans [22]. Two studies that were recently published by investigators working in Africa presented data that were more similar to ours in that there were significant M. genitalium coinfection rates among patients with both N. gonorrhoeae (6%–11%) and C. trachomatis (8%–21%), even though the Southern blot test was not used for M. genitalium detection [4, 5]. In populations with high STD infection rates, men who acquire acute urethritis from a new partner would be more likely to be infected with >1 pathogen. For example, in our series, 35% of the men with gonorrhea were coinfected with C. trachomatis, whereas, in areas of the United States where chlamydia-control programs have been in place for some time, only 5%–10% would be expected to be coinfected [23]. Additional studies of populations with high endemic rates of gonococcal and chlamydial infection that use Southern blot tests or other specific probe-based detection methods are needed to confirm our findings. In addition, investigation of M. genitalium infection in the female partners of men with NGU in different populations would be helpful in determining the epidemiology of M. genitalium in relationship to other STD pathogens.
Our data confirmed those recently reported by Morency et al. [5], who suggested that the symptoms and signs of M. genitalium-associated urethritis are more similar to chlamydial urethritis than gonococcal urethritis. In a comparison of men infected with only a single pathogen, we found that both chlamydia- and M. genitalium-infected men were less likely than men with gonococcal urethritis to present with a complaint of dysuria and that the urethral discharge for the former was less copious and less likely to be purulent. This finding is not surprising in view of the fact that the early studies of NGU did not find clinical differences between the clinical presentation of chlamydial and nonchlamydial NGU [1]. The observation that men with M. genitalium-associated urethritis were more likely to have had a partner with an STD in the past 30 days than were men with chlamydial or gonococcal urethritis is of interest. The data suggest that perhaps M. genitalium causes symptomatic disease in women more frequently than does either C. trachomatis or N. gonorrhoeae. These observations require confirmation, given the small number of subjects in our series.
A unique aspect of our study was the use of both urethral swabs and urine specimens for detection of M. genitalium infection. Eighteen symptomatic men had positive results of both swab and urine examinations, 3 had positive results by swab alone, and 2 had positive results by urine alone. These data validate the use of urine specimens for detection of M. genitalium infection in epidemiologic studies, although the use of both specimens modestly increased the sensitivity of detection.
In conclusion, these are exciting times for persons who are interested in the role of M. genitalium in genital tract disease. Recent studies have suggested that M. genitalium urethritis responds adequately to neither the tetracycline class of antibiotics nor to quinolones [24]. Carefully performed randomized treatment trials are needed to verify these data, because the implications for current NGU treatment recommendations are important. Another issue that needs investigation is the role of M. genitalium in genital tract inflammatory disease in women. If M. genitalium causes pelvic inflammatory disease or enhances HIV shedding in women, it will have to be considered, along with N. gonorrhoeae and C. trachomatis, as an important sexually transmitted threat to human health.
- Received February 12, 2002.
- Accepted July 16, 2002.
- © 2002 Infectious Diseases Society of America
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