Abstract
Urethral specimens from 172 men who attended sexually transmitted disease clinics in the Moscow Oblast were examined for Neisseria gonorrhoeae, Chlamydia trachomatis and Mycoplasma genitalium by nucleic acid amplification tests. N. gonorrhoeae was detected in the urethra of 41 (24%) of the 172 men and C. trachomatis in 57 (33%). The latter occurred in 15 (36%) of the 41 men who were infected by N. gonorrhoeae and in 42 (32%) of 131 uninfected by gonococci. Of the 42 men uninfected by gonococci but chlamydia infected, 39 (93%) had symptoms and/or signs of urethritis. M. genitalium was detected in 45 (26%) of the 172 men, in nine (22%) of the 41 men infected with N. gonorrhoeae and in 12 (21%) infected with C. trachomatis. M. genitalium was detected alone in 25 (28%) of the 89 men uninfected by either gonococci or C. trachomatis. Of these 25 men, 24 (96%) had urethral symptoms and signs of inflammation, a proportion significantly more than experienced by the 64 men uninfected by any of the microorganisms. Of the 31 men who apparently had no symptoms or signs of urethritis, only three (10%) were infected by M. genitalium. The data provide evidence for the pathogenicity and frequent occurrence of M. genitalium in men in Moscow and presumably elsewhere in Russia.
Keywords
INTRODUCTION
Mycoplasma genitalium was first isolated more than 25 years ago from the urethra of two of 13 men with acute non-gonococcal urethritis (NGU). 1 Subsequently, detection by culture proved almost impossible and it was only with the advent and use of the polymerase chain reaction (PCR) for detection that progress in understanding the prevalence and pathogenicity of this mycoplasma began to be made. Because of the source of the first isolates, and the relative ease of diagnosing urethritis and of obtaining specimens, it is not surprising that most attention was first paid to the relation between M. genitalium and acute NGU in men. During the course of at least 19 studies, it became apparent that the mycoplasma had a highly significant association not only with NGU but also with non-chlamydial NGU. 2 This, together with its ability to cause urethritis experimentally in male chimpanzees 3,4 and to fulfil other criteria recognized as important when considering causality, has clearly indicated that M. genitalium is one of the causes of non-chlamydial NGU. 5
M. genitalium has been reported to exist and to be associated with disease in various developed and developing countries in the world, but there have been no reports of its existence in Russia. As a consequence of a study designed to examine gonococcal and chlamydial infections in patients attending three clinics in Moscow, urethral specimens were stored frozen. We have subsequently taken this opportunity to test the extent to which those obtained from men might contain M. genitalium. We report here the results of this investigation together with an analysis of the association of M. genitalium with NGU and with non-chlamydial NGU.
METHODS
Patients
Male subjects were 18 years or older, assessed to be at high risk for chlamydial and gonococcal infections and attending one of three sexually transmitted disease clinics in the Moscow Oblast between March 2000 and March 2001. Men who tested negative for gonorrhoea on an initial Gram-stained urethral smear and who had an abnormal clinical examination, were the sexual partners of patients with gonorrhoea or, in a smear, had evidence of leucocytosis, suspicious Gram-negative intracellular organisms or extracellular diplococci were included. They were recruited as part of a wider trial of the performance of gonococcal diagnostic tests.
Two hundred and thirty-two men were tested for gonococcal and chlamydial infections by molecular techniques. The protocol was reviewed and ratified by the Ethics Review Board of the Central Institute of Skin and Venereal Diseases, Moscow, and by a District Health Authority Ethics Review Board in London. Permission was also given by the latter in 2004 to test stored specimens for M. genitalium as there was no possibility of linking to patient identity and, in addition, the interval between collection and testing was sufficiently long for the results not to influence patient management. Sufficient residual material was available, after gonococcal and chlamydial testing, for M. gentialium testing of urethral specimens from 172 of the 232 men originally recruited.
Procedure
Patients were provided with a written information sheet and asked to sign a consent form indicating their willingness to participate. Consenting patients were then allocated a study number. All patients were interviewed by a physician using a standard proforma to obtain subject characteristics and medical and sexual histories before a full clinical examination was undertaken and the findings recorded on the proforma.
Urethral specimens were obtained from each subject at recruitment and on the next two days. A disposable loop specimen was applied to a slide for Gram staining. Specimens to be tested for Chlamydia trachomatis and Neisseria gonorrhoeae by the ligase chain reaction (LCR) assay (Abbott Laboratories Ltd, Maidenhead, UK) were obtained using a thin LCR swab, placed in LCR transport medium and refrigerated immediately at −7°C. These specimens were taken on ice on the same day to the Microbiology Laboratory of the Central Institute of Skin and Venereal Diseases, Moscow, where they were frozen immediately at −70°C. Before transportation to London, all specimens were thawed, mixed thoroughly and the swab squeezed against the side of the tube and discarded. The sample was divided into two equal volumes in 2 mL screw-capped vials and the aliquots refrozen at −70°C. One of these aliquots was transported to London. During this procedure, although the vials were kept cold, the contents thawed, but were refrozen at −70°C within six hours of leaving Moscow.
The specimens were tested for N. gonorrhoeae and C. trachomatis by an LCR assay using the manufacturer's instructions and the results were categorized as positive, negative or equivocal according to the manufacturer's cut-off value. In the case of equivocal results, the assay was repeated, the specimens being tested at a dilution of 1:4. Any remaining equivocal results were considered negative.
Three years after the tests mentioned above, the remaining materials from the specimens were transported, frozen in cardice, to the Mycoplasma Laboratory of the Statens Serum Institut, Copenhagen, where they were kept at −70°C until tested for M. genitalium and again for C. trachomatis. Specimens taken at recruitment were tested; if such a specimen was no longer available or there was insufficient material, a specimen taken on the first or second (rarely) day after recruitment was tested. The detection of M. genitalium was by a PCR assay targeting the16S rRNA gene and using an internal control for inhibition, as described before. 6 Positive results were confirmed with a PCR targeting the mgp adhesin gene. 7,8 C. trachomatis was sought again, this time by a PCR, as described before, 7 to assess whether storage, freezing and thawing, and transportation of specimens had adversely affected the materials.
Gram-stained smears were used to detect polymorphonuclear leukocytes (PMNLs). Urethritis was diagnosed when there were ≥5 PMNLs per high-power (1000 × ) microscope field.
Statistical analysis
Data were analysed in SAS version 9.1. Confidence intervals on proportions were calculated using standard errors derived from normal approximation to the binomial distribution and P values for difference in proportions were obtained from Fisher's exact test (FET). One-sided tests were used to examine the null hypothesis that infection gave higher rates of symptoms and signs.
RESULTS
Patient characteristics (Table 1
)
Men attended three clinics, but for the purpose of analysis the numbers have been combined. Specimens from 172 men were available for testing in the M. genitalium assay. Of these men, 24 (14%) were 18–19 years of age, 48 (26%) were 20–24 years and 100 (58%) were 25 years or more. Reasons for suspicion of genital infection were distributed as follows. Twenty-four men (14%) were contacts of known cases of gonorrhoea, 67 (39%) men had a suspicious urethral smear, 15 (9%) had clinical signs and 66 (39%) had multiple reasons for inclusion.
Detection of N. gonorrhoeae
N. gonorrhoeae was detected in the urethra of 41 (24%) of the 172 men (Table 1). All these men had symptoms and/or signs of urethritis and were subsequently treated for gonococcal urethritis. Potentially, therefore, there were 131 (76%) men who had NGU.
Univariate characteristics of subjects and detection of microorganisms
Detection of C. trachomatis
Three specimens were shown to be positive by LCR testing but negative by PCR and three were negative by LCR but positive by PCR. Otherwise, all results were concordant, indicating that previous procedures had not materially caused DNA damage. The LCR results were used as the gold standard for the current study. As a consequence, C. trachomatis was detected in the urethra of 57 (33%) of the 172 men (Table 1). It occurred in 15 (36%) of the 41 men infected with N. gonorrhoeae and in 42 (32%) of 131 men without gonococcal infection.
Detection of M. genitalium
M. genitalium was detected in the urethra of 45 (26%) of the 172 men (Table 1). It was also found in nine (22%) of the 41 men infected with N. gonorrhoeae and in 12 (21%) of the 57 men infected with C. trachomatis. M. genitalium was detected alone in 25 (28%) of the 89 men who were not infected by the two aforementioned microorganisms.
Prevalence of clinical features among subjects infected with M. genitalium or C. trachomatis alone
As shown in Table 2, of the 25 men who were infected by M. genitalium only, 24 (96%) had urethral symptoms and 24 (96%) had urethral signs of inflammation compared with 47 (74%) and 56 (87%) of the 64 men who were negative for all three microorganisms. The difference between the two groups in terms of the prevalence of symptoms was statistically significant (FET; P = 0.011). Of the 31 men infected by C. trachomatis alone, 28 (90%) had symptoms and 29 (94%) had signs of urethritis. The prevalence of signs among these groups was also significantly greater than among subjects who were negative for all three microorganisms (FET; P = 0.036).
Prevalence of clinical features among men with M. genitalium only and with C. trachomatis only infections and among those with no detected microorganism
1 P = 0.003; 2 P = 0.007; 3 P = 0.011; 4 P = 0.036; 5 P = 0.017; 6 P = 0.043. All values based on use of the one-sided Fisher's exact test
Prevalence of microorganisms among subjects with different clinical features
As shown in Table 3, overall, of 141 men with urethral symptoms, 32 (23%) were infected with N. gonorrhoeae, 51 (36%) with C. trachomatis and 35 (30%) with M. genitalium. Of 152 men with clinical signs of urethritis, 34 (24%) were infected with N. gonorrhoeae, 53 (36%) with C. trachomatis and 43 (29%) with M. genitalium. In men with either symptoms or signs of urethritis, single infections with M. genitalium were as common as were single infections with C. trachomatis. Of 31 men who apparently had no symptoms or signs of urethritis, only six (19%) were infected by C. trachomatis and three (10%) by M. genitalium.
Prevalence of detected microorganisms in subjects with different clinical features
NG = Neisseria gonorrhoeae; CT = Chlamydia trachomatis; MG = Mycoplasma genitalium
In summary, the data presented in Tables 2 and 3 indicate that M. genitalium was detected in the urethra almost as frequently as C. trachomatis and that symptoms and signs associated with the mycoplasma were almost as intense as those caused by C. trachomatis.
DISCUSSION
M. genitalium has been found in subjects of different race and in many parts of the world. It has been shown to fulfil the criteria, in an extension of Henle-Koch's 9 postulates, for being regarded as one of the causes of NGU, particularly non-chlamydial NGU2 and has been shown to be more closely associated with symptomatic than with asymptomatic urethritis. 10,11 In such previous studies concerned with the relation between M. genitalium and urethritis, men with urethritis were compared with control groups. The current study was different in that the opportunity was taken to search for M. genitalium in valuable urethral specimens that otherwise would have been discarded, but in the knowledge that a group of subjects without urethritis had not been sought. Despite this, it was apparent on analysis that groups of men acted as ‘controls’. Thus, a group in whom neither N. gonorrhoeae nor C. trachomatis had been detected could be used to determine the prevalence of M. genitalium, and a small group (18%) who had no symptoms or signs could be compared with those who did. It is notable that men who were infected by M. genitalium had symptoms and/or signs of urethritis significantly more frequently than men who were not infected by any of the microorganisms under consideration. Thus, the data presented here provide convincing evidence that M. genitalium occurs frequently in men in Moscow and presumably, therefore, in other parts of Russia and thus, like C. trachomatis, is a potent cause of urethritis in men. With regard to disease caused by N. gonorrhoeae, the design of the study was such that men who had obvious gonococcal infections (as judged by Gram-stained smears) were excluded. This may then account for those men diagnosed as N. gonorrhoeae positive in the study having less severe disease than might otherwise have been expected.
The advent of M. genitalium has made the treatment of NGU less straightforward than once thought. It is important to realize that the use of a tetracycline to treat men who have NGU is inappropriate in many cases because it has been shown that such treatment often fails to eliminate M. genitalium and leads to the development of chronic disease. 12,13 Persistence of the organism is seen less when patients are treated with azithromycin 12 and a prolonged dosage schedule may be particularly beneficial. 14,15
Whether M. genitalium should be sought routinely in men with NGU is an obvious question. Russian-developed kits for M. genitalium now exist and, in the future, perhaps those combined with testing for other microorganisms may become plentiful and foster such an approach. However, in the current absence of a widespread and easily available PCR test or other nucleic acid amplification tests, the only justification would seem to be in cases of NGU that are persistent and have not responded to antimicrobial therapy. The same must be said for Ureaplasma urealyticum, the role of which we have not addressed in this study. In the case of this microorganism, we feel that routine testing is misplaced. 16 Furthermore, when financial restrictions impinge on health care, attention should be focused more on M. genitalium than on U. urealyticum, because the former not only is an undoubted cause of male urethritis but also seems to have a greater potential than U. urealyticum for causing severe disease in women. 5