Longitudinal quantitative detection by real-time PCR of Mycoplasma genitalium in first-pass urine of men with recurrent nongonococcal urethritis

By using a TaqMan assay we monitored longitudinal changes in Mycoplasma genitalium loads in five men with recurrent M. genitalium-positive nongonococcal urethritis. We observed regrowth of M. genitalium persisting in hosts after treatment and a possible association of the increase in the M. genitalium load with emergence of symptoms and signs of nongonococcal urethritis in four of these patients.     

Development of a quantitative real-time PCR assay for detection of Mycoplasma genitalium

Mycoplasma genitalium is known to cause nonchlamydial, nongonococcal urethritis in men and to be associated with pelvic inflammatory disease in women. Specific and sensitive PCR methods are needed for diagnosis of this bacterium because it is very difficult to culture from patient samples. To determine the bacterial load in patients' specimens, a quantitative real-time LightCycler PCR was developed. The housekeeping gene gap encoding glyceraldehyde-3-phosphate dehydrogenase was chosen as the target gene. The assay could consistently detect five genome copies per reaction. To evaluate the PCR, we tested 246 selected urethral swab samples from men attending a clinic for sexually transmitted diseases. Eighty-two of the samples were found positive for M. genitalium by a conventional 16S rRNA gene PCR assay, whereas 164 samples were randomly chosen among those tested negative. Of the positive samples, 78 (95.1%) were found positive, whereas 6 (3.7%) of the negatives were found positive by the LightCycler assay. The patient samples were also tested with a quantitative TaqMan assay, and the bacterial load was compared to the LightCycler results. A good linear correlation between the LightCycler and the TaqMan assays was found with a correlation coefficient of 0.89 and a slope of 0.99. Significantly more M. genitalium-positive men had urethritis, discharge, and dysuria than had M. genitalium-negative men. The M. genitalium DNA load in samples from patients with urethritis was significantly higher than in samples from those without (61 and 2.9 copies/microl, respectively [P = 0.0005]). This assay may prove useful in the monitoring of treatment and for optimizing sample preparation methods.     

Isolation of Mycoplasma genitalium strains from the male urethra.

Mycoplasma genitalium is a human mycoplasma species which, on the basis of detection by PCR, has been incriminated as a cause of nongonococcal urethritis. Previously, only two strains from the urogenital tract and five strains from extragenital sites have been isolated. We have developed a method for the isolation of this fastidious microbe. M. genitalium from PCR-positive urethral specimens was initially propagated in Vero cell cultures grown in serum-free medium supplemented with Ultroser HY serum substitute. Growth was monitored by PCR. The M. genitalium strains grown in cell cultures could subsequently be subcultured in modified Friis's FF broth medium. Several passages in broth medium were required before growth on agar medium was attained. A total of 11 urethral specimens positive for M. genitalium by PCR from male patients with urethritis were investigated. Six strains were adapted to growth in broth medium, and four of these strains were cloned. Three specimens were overgrown by other mycoplasmas during propagation in the cell cultures. In only two PCR-positive specimens was propagation of M. genitalium unsuccessful. The use of cell culture combined with PCR monitoring of mycoplasmal growth may prove to be more widely applicable for the isolation of other fastidious mollicutes.     

Identification and characterization of immunogenic proteins of mycoplasma genitalium

Mycoplasma genitalium causes nonchlamydial nongonococcal urethritis. M. genitalium was detected by PCR in 17 urethral swabs obtained from 99 men with and without urethritis (J. S. Jensen, R. Orsum, B. Dohn, S. Uldum, A. M. Worm, and K. Lind, Genitourin. Med. 69:265-269, 1993), and later, four M. genitalium strains were isolated (J. S. Jensen, H. T. Hansen, and K. Lind, J. Clin. Microbiol. 34:286-291, 1996). The objective of this study was to characterize immunogenic proteins of M. genitalium by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting by using a hyperimmune rabbit serum against M. genitalium G37, determine their identity by mass spectrometry, and develop an M. genitalium-specific enzyme-linked immunosorbent assay (ELISA) free from cross-reactivity with M. pneumoniae antibodies. Using recombinant fragments of the C-terminal part of MgPa (rMgPa), we developed a specific ELISA for detection of M. genitalium antibodies. This antigen did not bind M. pneumoniae antibodies. Using serum samples from the 99 men with and without urethritis, we found that 26 had immunoglobulin G (IgG) antibodies to M. genitalium. There was a strong statistically significant correlation between PCR and IgG antibodies to M. genitalium (odds ratio [OR], 5.9; 95% confidence interval [CI], 2.3 to 21.5; P = 0.002). Furthermore, men with recurrent urethritis were more likely to have antibodies to M. genitalium than were those without recurrent urethritis (OR, 4.0; 95% CI, 1.1 to 14.5; P = 0.0383) and they had significantly higher antibody titers. By use of the rMgPa ELISA, this study further substantiates the importance of M. genitalium as a cause of male urethritis.     

Mycoplasma genitalium attaches to human spermatozoa

BACKGROUND: Mycoplasma genitalium causes urogenital diseases in men and women and is presumed to be sexually transmitted. We wanted to investigate whether spermatozoa could serve as vectors for M.genitalium in order to cause upper genital diseases in women. METHODS: By use of Nomarski light microscopy and transmission X-ray microscopy, the attachment of M.genitalium to spermatozoa was studied. Semen was incubated in vitro with M.genitalium. Purified, motile spermatozoa were examined for attachment of M.genitalium by immunofluorescence microscopy. RESULTS: Mycoplasma genitalium was shown to adhere to the head, midpiece and tail of the spermatozoa. The spermatozoa became immotile when many M.genitalium were attached. However, the motile spermatozoa were demonstrated to carry M.genitalium and in this case the mycoplasmas were seen to attach mostly to the midpiece or neck region. Occasionally, M.genitalium was seen at the head but not at the tail. By X-ray microscopy, it was possible to observe the diffentiated structure of M.genitalium, and the attachment seemed to be mediated by the tip. CONCLUSIONS: Mycoplasma genitalium can bind to human spermatozoa and thus could be carried by motile sperm. This ability may be important in the process of causing female genital diseases and infertility.     

Use of TaqMan 5' nuclease real-time PCR for quantitative detection of Mycoplasma genitalium DNA in males with and without urethritis who were attendees at a sexually transmitted disease clinic

Mycoplasma genitalium is a cause of nongonococcal urethritis, particularly in patients not infected with Chlamydia trachomatis. A quantitative 5' nuclease assay (TaqMan PCR) was developed and validated. The assay detected a fragment of the MgPa adhesin gene by use of a TaqMan MGB (minor groove binder) probe and included an internal processing control to detect PCR inhibition. Urethral swab specimens and first-void urine samples from M. genitalium-positive men were examined, and the M. genitalium DNA load was correlated to symptoms and signs. The assay consistently detected <5 genome copies without cross-reactions with other mycoplasmas. Urine and urethral swab specimens from men with urethritis had higher M. genitalium DNA loads than specimens from men without urethritis. However, a very broad overlap of DNA loads between patients with and without urethritis was observed. Urethral swab specimens from patients with urethral discharge had a significantly higher DNA load than specimens from patients without discharge. This correlation was not found in first-void urine specimens.     

Diagnostic assessment of Mycoplasma genitalium in culture-positive women

Detection of Mycoplasma genitalium-mediated, chlamydia-negative nongonococcal urethritis and other M. genitalium-linked infectious etiologies has been very challenging. Although M. genitalium is considered a leading cause of genitourinary symptoms in men and women, extreme difficulties in its cultivation due to its highly fastidious nature and the lack of routine and effective diagnostic tests have slowed the generation of clinical data which directly implicate the presence of M. genitalium in disease pathogenesis. In this study, we compared enzyme-linked immunosorbent assays (ELISAs) and immunoblot and PCR assays in M. genitalium culture-positive women over 1 to 3 years of clinical visits to determine the usefulness of independent diagnostic strategies. Furthermore, the value of combinatorial diagnostic assessments is described, which provides insights into the dynamics of M. genitalium-host interactions. Overall, we show that neither ELISA nor PCR, alone or in combination, provides the sensitivity required to confidently predict the existence of viable M. genitalium organisms in cervical and vaginal samples. Additionally, culture-positive women exhibited a range of antibody responsiveness to M. genitalium based upon ELISA and immunoblot assessments, indicating immune diversity among this high-risk population.     

Isolation and characterization of Mycoplasma genitalium strains from the human respiratory tract.

Mycoplasma genitalium, an organism first isolated from the urethras of two men with nongonococcal urethritis, has been found in throat specimens from military recruits participating in an inactivated Mycoplasma pneumoniae vaccine field trial in 1974-1975. Four of 16 preserved throat isolates, previously identified as strains of M. pneumoniae, have now been shown to be mixtures of M. pneumoniae and M. genitalium. Purification of these mixed mycoplasmas by selection of single colonies confirmed the presence of M. genitalium. Identification of M. genitalium was based upon the occurrence of a species-specific 140-kilodalton protein adhesin in these isolates and their serologic reactivity to an M. genitalium antiserum. The frequent occurrence of both M. pneumoniae and M. genitalium in a number of these throat specimens, in combination with their shared antigenic cross-reactivities, suggests the likelihood that M. genitalium strains are easily missed in the usual laboratory identification procedures. What role M. genitalium may play in human respiratory disease remains to be determined.     

A novel polymerase chain reaction assay to detect Mycoplasma genitalium.

AIMS: To design and validate a polymerase chain reaction (PCR) assay targeting the 16S rRNA gene of Mycoplasma genitalium. METHODS: Primers were designed that were complementary to the 16S rRNA gene sequence of M genitalium. After optimisation of the reaction conditions, the PCR was tested against nine M genitalium strains, a dilution series of M genitalium DNA, and a panel of common microorganisms. The PCR was also challenged in parallel with a published assay against 54 urine specimens from men with urethritis. RESULTS: The expected 341 bp product was produced on amplification of material from all M genitalium strains and from none of the other microorganisms tested. The lower limit of detection was 50 genome copies. The new assay detected M genitalium DNA in nine of 54 men with urethritis, in comparison with eight positive specimens detected with the alternative PCR. CONCLUSIONS: This novel PCR targeting the M genitalium 16S rRNA gene has been optimised and now provides a sensitive and specific alternative or addition to the available MgPa gene targeting assays.     

Quantitative detection of Mycoplasma genitalium from first-pass urine of men with urethritis and asymptomatic men by real-time PCR

We developed a TaqMan-based real-time PCR assay for quantifying Mycoplasma genitalium. This assay is able to specifically quantify concentrations of the M. genitalium 16S rRNA gene ranging from 10(7) to 10 copies/reaction. Using the TaqMan assay, we quantified the M. genitalium 16S rRNA gene in first-pass urine of men with urethritis and asymptomatic men who were positive for M. genitalium by PCR- and phylogeny-based assay. Of 130 men with gonococcal urethritis (GU), five were positive for M. genitalium. The mycoplasma load for each specimen was <5 x 10 copies/ml. Of 84 men with chlamydial non-GU (CNGU), seven were positive for M. genitalium. One man had an M. genitalium load of <5 x 10 copies/ml, and six men had loads ranging from 1.1 x 10(7) to 2.7 x 10(2) copies/ml. Of 86 men with nonchlamydial NGU (NCNGU), 17 were positive for M. genitalium. The mycoplasma loads for these men ranged from 3.3 x 10(6) to 2.3 x 10(2) copies/ml. Of 76 asymptomatic men, only two were positive for M. genitalium. For these men, the loads were 2 x 10(2) and <5 x 10 copies/ml. The patients with NGU had significantly higher concentrations of M. genitalium in their first-pass urine than did men with GU (P < 0.01) or asymptomatic men (P < 0.05). In addition, M. genitalium loads were significantly higher in men with NCNGU than those in asymptomatic men (P < 0.05). The quantitative assessment of M. genitalium loads by the TaqMan assay will provide useful information for understanding the pathogenicity of this mycoplasma in the urogenital tract.     

Sequence-based typing of Mycoplasma genitalium reveals sexual transmission

Mycoplasma genitalium causes male nonchlamydial, nongonococcal urethritis and is associated with cervicitis and pelvic inflammatory disease in women. Epidemiological studies indicate that M. genitalium is sexually transmitted, and the aim of the present study was to further substantiate this by means of a DNA typing system. A typing assay based on a diagnostic mgpB gene PCR was developed, evaluated, and applied directly to urogenital specimens. The assay had a low limit of detection and hence a high typeability. Sequences of isolates from 52 unrelated patients were divided into 29 different sequence types, giving a discriminatory index of 0.95. Two to six M. genitalium-positive specimens were collected from each of 44 patients over a median interval of 56 days (range, 11 to 1,395). Forty had the same sequence type in consecutive specimens. Specimens collected from two men were repeatedly positive at intervals of 472 and 1,395 days, respectively, but the sequence types had changed. A new strain was introduced in one sexual dyad, and the sequence types changed subsequently. Seventy-nine M. genitalium-positive specimens from 19 couples were investigated, and all partners initially had concordant sequence types, but one couple had discordant types at one time point before a newly introduced strain took over. The present typing system is simple and reproducible and has an excellent discriminatory capacity which might prove useful in studies of sexual networks and for evaluation of treatment failures. In the laboratory, this system may document the uniqueness of newly isolated M. genitalium strains.     

Determination of infectious load of Mycoplasma genitalium in clinical samples of human vaginal cells

Mycoplasma genitalium is a leading cause of chlamydia-negative, nongonoccocal urethritis and has been directly implicated in numerous other genitourinary as well as extragenitourinary tract pathologies. Detection of M. genitalium has relied almost entirely on PCR amplification of clinical specimens and evidence of seroconversion since these mycoplasmas are highly fastidious and culture isolation by microbiological techniques is very rare. We have established a combinatorial strategy using confocal immunoanalysis (CIA) and real-time PCR to qualitatively and quantitatively assess patterns of M. genitalium infection in women attending a sexually transmitted disease-related health clinic in San Antonio, Tex. CIA allows spatial examination of mycoplasmas on surfaces and inside human target cells, plus the ability to evaluate cell-to-cell patterns and variances within samples. Real-time PCR permits determination of genome copy numbers of mycoplasmas and human cells by multiplex amplification using mycoplasma gyrA and human RNase P gene sequences, which indicates overall levels of mycoplasma infection and degree of parasitism. These assays are strongly correlated and, in combination, permit detection and elucidation of heretofore-unrecognized patterns of M. genitalium infections in clinical and experimental samples.     

Increased Frequency of Detection of Ureaplasma urealyticum and Mycoplasma genitalium in AIDS Patients without Urethral Symptoms

The roles of Mycoplasma genitalium and Ureaplasma urealyticum in nongonococcal urethritis are not yet well established. The aim of this study was to determine the presence of these microorganisms in the urethral tracts of 187 human immunodeficiency virus type 1 (HIV-1)-infected male patients with no clinical signs of urethritis. The results indicate that the prevalence of M. genitalium and U. urealyticum was higher in AIDS patients than in asymptomatic, HIV-1-infected patients and in healthy individuals. The high rate of mycoplasmas and ureaplasmas detected in AIDS patients, in the absence of urethritis, argues against major roles in causing disease at the urethral mucosal level for these microorganisms.     

ELISA法检测江门地区性病门诊患者生殖支原体抗原

目的应用酶联免疫吸附试验（ELISA）检测性病门诊患者泌尿生殖道生殖支原体（Mg）抗原，探讨江门地区性传播疾病（STD）门诊患者Mg感染状况。方法采用Mg多克隆抗体，标记酶结合物，建立检测Mg抗原的双抗体夹心ELISA法，对性病门诊患者泌尿生殖道分泌物进行MIg抗原检测。结果双抗体夹心ELISA法可检测到5μg／ml蛋白浓度，除肺炎支原体外与其他泌尿生殖道常见支原体和细菌无交叉反应；共检测了174例标本，Mg抗原阳性33例，阳性率为18．97％，其中男性和女性阳性率分别为19．88％和7．69％；男性患者中，非淋菌性尿道炎（NGU）、淋病、前列腺炎患者的Mg抗原阳性率分别为13．43％、62．5％和4．84％。结论STD门诊患者存在Mg感染，应用双抗体夹心ELISA法检测Mg抗原具有一定的灵敏度，且具有快速、简便的特点，适合临床筛查Mg抗原。     

Amplified-Fragment Length Polymorphism Fingerprinting of Mycoplasma Species

Amplified-fragment length polymorphism (AFLP) is a whole-genome fingerprinting method based on selective amplification of restriction fragments. The potential of the method for the characterization of mycoplasmas was investigated in a total of 50 strains of human and animal origin, including Mycoplasma genitalium (n = 11), Mycoplasma pneumoniae (n = 5), Mycoplasma hominis (n = 5), Mycoplasma hyopneumoniae (n = 9), Myco plasma flocculare (n = 5), Mycoplasma hyosynoviae (n = 10), and Mycoplasma dispar (n = 5). AFLP templates were prepared by the digestion of mycoplasmal DNA with BglII and MfeI restriction endonucleases and subsequent ligation of corresponding site-specific adapters. The amplification of AFLP templates with a single set of nonselective primers resulted in reproducible fingerprints of approximately 60 to 80 fragments in the size range of 50 to 500 bp. The method was able to discriminate the analyzed strains at species and intraspecies levels as well. Each of the tested Mycoplasma species developed a banding pattern entirely different from those obtained from other species under analysis. Subtle intraspecies genomic differences were detected among strains of all of the Mycoplasma species analyzed. The extent of polymorphism varied markedly between the analyzed mycoplasmas, comprising pattern similarity levels from 61.7% detected among M. dispar strains to 95.9% detected among M. genitalium strains. The results of the present study provide evidence of the high discriminatory power of AFLP analysis, suggesting the possible applicability of this method to the molecular characterization of mycoplasmas.     

Cervicitis and genitourinary symptoms in women culture positive for Mycoplasma genitalium

PROBLEM: Mycoplasma genitalium has been associated with male urethritis. We sought to relate M. genitalium to genitourinary signs and symptoms in women. METHOD OF STUDY: We compared 26 culture-positive women (group 1), 257 additional polymerase chain reaction-positive women (group 2), and 107 negative control women. We used logistic regression to evaluate signs and symptoms, controlling for co-infections, pregnancy, age, and intervention group assignment. RESULTS: Comparing group 1 with controls, we found significantly elevated odds ratios (ORs) for intermediate vaginal discharge (OR = 5.4; 95% confidence interval 1.01, 29.2) and action in response to discharge [3.9 (1.1, 13.5)]. Non-significant increases were observed for pathologic vaginal discharge [3.8 (0.78, 18.2)], pathologic dyspareunia [1.5 (0.25, 9.0)], vaginal odor [2.1 (0.75, 5.7)], and cervical mucopus [4.1 (0.74, 22.4)]. Group 2 results were similar, but showed no increase in cervical mucopus relative to controls. CONCLUSION: Infection with M. genitalium in women is independently related to increased genitourinary symptomatology.     

Mycoplasma pneumoniae and Mycoplasma genitalium mixture in synovial fluid isolate.

A mycoplasma cultured from synovial fluid specimens from a patient with pneumonia and subsequent polyarthritis was identified initially as Mycoplasma pneumoniae. In retrospective studies, the culture was shown also to contain Mycoplasma genitalium. In this paper, the laboratory techniques employed in the identification and separation of the two species are presented, and evidence to implicate postinfectious autoimmunity is provided. An increasing number of reports of M. genitalium in human tissue sites and difficulties in isolation and identification of the organism in the clinical laboratory suggest the need for more extensive application of rapid and specific detection systems for both M. genitalium and M. pneumoniae in the clinical laboratory.     

Serological investigation of Mycoplasma genitalium in infertile women

BACKGROUND: The role of Mycoplasma genitalium in the pathogenesis of pelvic inflammatory disease has not been characterized. METHODS: Sera from 308 infertile women were investigated for antibodies to M. genitalium by immunoblotting. Women with tubal factor infertility (TFI) made up 132 of the patients, 67 of the women had an infertile male partner and 109 were infertile for unknown reasons. RESULTS: Of the TFI patients 29 (22.0%) were seropositive to the major adhesin, MgPa, of M. genitalium versus 11 (6.3%) in the group of women with normal tubes. No cross-reactions between MgPa and P1 of the related Mycoplasma pneumoniae were found. Besides, MgPa positive sera were confirmed by immunoblotting using a cloned fragment of the C-terminal part of MgPa specific to M. genitalium. Chlamydia trachomatis is known to be able to cause infertility as a result of salpingitis. Therefore, the sera were tested against C. trachomatis using a commercial ELISA test. Seventy-five (56.8%) of the TFI patients were seropositive to C. trachomatis. Eight (27.6%) TFI patients seropositive to MgPa were negative to C. trachomatis. CONCLUSIONS: This study indicates that M. genitalium may be an independent risk factor in the development of an inflammatory process leading to scarring of the uterine tubes in women and thereby causing infertility.     

Expression and functional identification of the hypothetical adhesin P32 from Mycoplasma genitalium

Mycoplasma genitalium is the main causative agent for non-gonococcal and non-chlamydial urethritis. P32 is the putative surface-exposed membrane protein of M. genitalium and it has substaintial identity in amino acid sequence with adhesin protein P30 from M. pnewnoniae. Since M. pneumoniae mutants lacking P30 protein is defective in cytadherence, P32 protein has been proposed to be an essential adhesin implicated in the adherence of M. genitalium to host cells. The prokaryotic expression vector pET-30 ( )/p32 was constructed in the present study, and the recombinant protein was expressed in E. coli and purified under denaturing condition. As demonstrated by the immunoblotting analysis, the recombinant protein could react with rabbit antisera against M. genitalium, and adherence inhibition assays were petformed with antisera against this recombinant protein. It was demonstrated that P32 protein apperared to be an adhesion protein of M. genitalium, thus providing the experimental basis for better understanding of the pathogenesis of M. genitalium infection and for the development of the related vaccines against the infection.