No abstract

Adhesion of Mycoplasma pneumoniae and the closely related M. genitalium to HEp-2 cells was investigated. The main surface proteins known to be involved in adhesion are P1 of M. pneumoniae and its homologue, MgPa, of M. genitalium. Both proteins are also immunodominant proteins. Protein P116 is another immunodominant protein of M. pneumoniae. These immunogenic proteins were investigated for their surface exposure and involvement in adhesion to host epithelial cells. Immunofluorescence microscopy (IFM) was used to detect M. pneumoniae and M. genitalium adhering to HEp-2 cells. Monospecific antibodies were produced against fragments of the surface proteins lacking tryptophan stop codons and were used for adhesion detection, surface exposure and adhesion inhibition IFM assays. Three monospecific antibodies were made against MgPa covering regions in the N-terminal, the middle and the C-terminal part; two monospecific antibodies were produced against P1 covering regions of the N-and the C-terminal part and one monospecific antibody was made against most of P116. Only the C-terminal parts of P1 and MgPa were surface exposed and blocking of these regions with the monospecific antibody resulted in inhibition of cytadsorption. Protein P116 was shown to be surface exposed and an essential protein involved in adhesion because the anti-P116 antibody prevented attachment of M. pneumoniae to the HEp-2 cells independently of P1. This study adds to the understanding of the molecular biology of M. genitalium and M. pneumoniae and presents a method to study the proteins involved in adhesion of these mycoplasmas.

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.

Mycoplasma genitalium is a pathogen of the human urogenital tract. It was isolated for the first time in 1981 from two men with urethritis. Transmission electron microscopy revealed the small size of M. genitalium and a flask-shaped body with an electron-dense tip structure (29, 41). M. genitalium belongs to the class Mollicutes, comprising small bacteria without a cell wall. Mycoplasmas, including M. genitalium, are characterized by the requirement for complex growth media and utilization of UGA as a tryptophan codon. The genome of M. genitalium is small (580 kbp) and was sequenced in 1995 (8).M. genitalium appears to be an important cause of nonchlamydial nongonococcal urethritis, with an average prevalence of more than 20% in men with this condition (12,15,18). M. genitalium is sexually transmitted (7, 20) and may cause genital tract diseases in women, such as cervicitis (7, 28), pelvic inflammatory disease (35), and endometritis (5). M. genitalium G37 has been shown to induce urethritis in chimpanzees, followed by a sustained antibody response (39). Experimentally infected monkeys developed moderate to severe salpingitis (30). In humans, a fourfold rise in antibody titers measured by microimmunofluorescence was observed for 29% of men with nongonococcal urethritis compared to 12% of men without urethritis (37). This suggests that the infection of the human male urethra by M. genitalium causes the stimulation of an antibody response. Furthermore, an association between antibodies to M. genitalium in women with tubal factor infertility has been demonstrated (4).M. genitalium is closely related to Mycoplasma pneumoniae, the cause of "walking pneumonia," and they share many features, like shape and cell and genome structures. The attachment protein MgPa of M. genitalium has been identified as a homologue to the P1 adhesin of M. pneumoniae (13). The electron-dense core of M. pneumoniae comprises a network of proteins that localize and maintain P1 at the tip during adhesion to host cells (21,22). Like P1 of M. pneumoniae, MgPa is the immunodominant protein of M. genitalium (14,27,31).In order to analyze human serum samples for antibodies to M. genitalium, different serological methods with different combinations of M. genitalium antigens have been attempted. However, serological cross-reactions to M. pneumoniae are reported to be a major problem in many of the tests that are based on whole-cell antigens of M. genitalium. Examples hereof are the enzyme-linked immunosorbent assay (ELISA) described by Jacobs et al. with whole M. genitalium cells as the antigen (14), the complement fixation test with chloroformmethanol-extracted M. genitalium cells, indirect immunofluorescence on M. genitalium microcolonies, and the indirect hemagglutination test using sonicated M. genitalium cells described by Lind (25) and Lind and Kristensen (26). The metabolism inhibition test was described to be less cross-reactive (25, 38), but it is also a more complicated method to use than the more modern serological methods. More specific s...

Clinical assessment of women with pelvic pain was a poor indicator of disease seen at laparoscopy. Thus, of 109 women, 22 at laparoscopy had salpingitis, 19 had adhesions without salpingitis, 20 had endometriosis or ovarian pathology and 48 no observable abnormality. In all laparoscopic categories, Ureaplasma spp. and Mycoplasma hominis, but not Mycoplasma genitalium, were at least as common in the cervix/vagina as Chlamydia trachomatis and equally frequent in the endometrium. However, C. trachomatis had the greatest propensity for spread to the Fallopian tubes. Thus, of 28 women who had C. trachomatis organisms in the vagina/cervix, 13 had them in a Fallopian tube (ratio 2.2:1); the ratio was 6:1 for Neisseria gonorrhoeae, 8:1 for M. genitalium, 21:1 for M. hominis and 31:1 for Ureaplasma spp. M. hominis organisms in a large number were detected most often in women with salpingitis. The likelihood of spread of Ureaplasma urealyticum and U. parvum from the lower to the upper genital tract was about the same and they were detected only once each in a tube, which was not inflamed in either case. Multiple bacteria were often detected at a single site, making it difficult to establish the exact cause of disease. However N. gonorrhoeae was considered to be the sole cause of salpingitis in one woman and the primary or equal primary contributor in four others; C. trachomatis was involved in at least 11 women, mostly as the sole cause or as the primary contributor; M. genitalium was considered the cause in one woman and had possible involvement in three others; and M. hominis was a questionable sole cause in one woman and the primary or equal primary contributor in three. Serologically, C. trachomatis was related to adhesions, without salpingitis, more often (63%) than any other micro-organism. M. genitalium may have been implicated in one case. Serologically, a previous C. trachomatis infection was indicated in 40% of women without an observable laparoscopic abnormality. C. trachomatis in the endometrium and tubes of women without any laparoscopic abnormality suggests subclinical disease, endometritis or endosalpingitis. There was evidence for a smaller proportion (19%) of women without an abnormality having been infected previously with M. genitalium. To some extent this is consistent with the infrequency of acute M. genitalium infections in this cohort of women.

In Chlamydia antibody testing, MIF is superior in the assessment of tubal pathology. In the initial screen for tubal pathology MIF should therefore be the test of first choice.

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/l, respectively [P ‫؍‬ 0.0005]). This assay may prove useful in the monitoring of treatment and for optimizing sample preparation methods.Mycoplasma genitalium is a human mycoplasma species, and there is accumulating evidence that it causes nonchlamydial nongonococcal urethritis (NCNGU). In a recent review, the prevalence of M. genitalium in male patients with NCNGU was extracted from studies published from 1993 to 2002 (10). The average prevalence of M. genitalium was 21.7% of 1,233 patients with NCNGU compared to 6% of 1,537 patients without urethritis. M. genitalium is also found in the female genital tract, although the diseases it causes in women have been less thoroughly studied. In women, M. genitalium has been associated with cervicitis (16, 26), endometritis (5), and tubal factor infertility (4). The concordance of M. genitalium infection among sexual partners implies that M. genitalium is sexually transmitted (1, 20).M. genitalium belongs to the class Mollicutes-bacteria without a cell wall. It is the smallest known bacterium, having a genome size of 580 kbp, and is closely related to Mycoplasma pneumoniae. Both organisms are flask-shaped with a terminal tip structure that is used for attachment to host cells, and they share several antigens impeding diagnostic serology. It is well established that M. pneumoniae causes atypical pneumonia, but occasionally it has been isolated from the urogenital tract (9,18).Only a few laboratories have been successful in culturing...

Molecular diagnostic measurements are currently underpinned by the polymerase chain reaction (PCR). There are also a number of alternative nucleic acid amplification technologies, which unlike PCR, work at a single temperature. These ‘isothermal’ methods, reportedly offer potential advantages over PCR such as simplicity, speed and resistance to inhibitors and could also be used for quantitative molecular analysis. However there are currently limited mechanisms to evaluate their quantitative performance, which would assist assay development and study comparisons. This study uses a sexually transmitted infection diagnostic model in combination with an adapted metric termed isothermal doubling time (IDT), akin to PCR efficiency, to compare quantitative PCR and quantitative loop-mediated isothermal amplification (qLAMP) assays, and to quantify the impact of matrix interference. The performance metric described here facilitates the comparison of qLAMP assays that could assist assay development and validation activities.