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Department of Urology, Gifu University School of Medicine, Gifu 500-8705,1 Research and Development Department, Mitsubishi Kagaku Bio-Clinical Laboratories, Inc., Tokyo 174-8555,2 Department of Urology, Toyota Memorial Hospital, Toyota 471-8513, Japan3
Received 1 April 2002/ Returned for modification 21 June 2002/ Accepted 14 July 2002
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For chronic NGU, it has been suggested that persistence of M. genitalium in the urethra after antimicrobial chemotherapy might be associated with this condition (5, 7, 10, 15). However, quantitative analysis of M. genitalium has been not performed in cases of chronic NGU. In the present study, therefore, we used the TaqMan assay to monitor longitudinal changes in M. genitalium loads in patients with recurrent NGU and to examine those cases for association of the M. genitalium load with clinical findings and inflammatory responses.
Five men with recurrent NGU, who had been included in a previous study (10), were enrolled in the present study. They attended the Department of Urology, Toyota Memorial Hospital, Toyota, Japan, between July 1999 and December 2001. Each of these patients had symptoms and signs consistent with acute urethritis at the first visit. For each patient, Gram-stained urethral smears showed five or more polymorphonuclear leucocytes (PMNLs) per high-power (x1,000) microscopic field in at least three fields. Urine specimens were subjected to AMPLICOR STD-1 assay (Roche Diagnostics, Indianapolis, Ind.) for detecting Neisseria gonorrhoeae and C. trachomatis and to a PCR- and phylogeny-based assay for detecting mycoplasmas and ureaplasmas. The AMPLICOR STD-1 assay was done according to the manufacturer's instructions. The PCR- and phylogeny-based assay, which could detect at least 10 copies of the 16S rRNA gene of M. genitalium, was done as described in our previous report (18). Two patients were positive for both C. trachomatis and M. genitalium, and three were positive only for M. genitalium. We treated all five patients with levofloxacin at 100 mg three times daily for 14 days. We asked them to practice sexual abstinence during treatment and to return for reexamination 7 days after the start of treatment and at the end of treatment, irrespective of the presence or absence of symptoms. With successive visits, we reexamined urethral smears and retested urine for pathogens.
DNA samples, which had been prepared from the first-pass urine for the phylogeny-based assay and stored at -70°C, were subjected to the TaqMan assay for quantification of M. genitalium DNA (17, 18). Briefly, precipitate from 1 ml of the first-pass urine was obtained by centrifugation and treated with digestion solution containing proteinase K (18). The DNA was purified by a classic phenol-chloroform procedure followed by ethanol precipitation and was then dissolved in 50 µl of TE buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA). A portion of the DNA solution was used for the PCR- and phylogeny-based assay, and the remaining solution was frozen and stored.
Sequences of primers and a probe for the TaqMan assay were derived from the 16S rRNA gene of M. genitalium (17). In our previous study this assay was confirmed to be highly specific for M. genitalium (17). For first-pass urine specimens the working range of this assay was from 5 x 107 to 5 x 101 copies of the M. genitalium 16S rRNA gene per 1 ml of urine (17). In this study, after thawing the DNA solutions we used 10 µl of each sample as template DNA for the TaqMan assay. All standard dilutions of M. genitalium DNA, negative controls including no DNAs, and clinical samples were run simultaneously (17). Amplification, data acquisition, and analyses were performed with the ABI Prism 7700 Sequence Detection System (Applied Biosystems, Foster City, Calif.). The number of targets initially present was determined according to a standard curve (4).
Bacterial load in the five patients ranged from 1.1 x 105 to 3.3 x 106 copies of the M. genitalium 16S rRNA gene per 1 ml of urine (mean, 1.54 x 106 copies/ml) before treatment (Fig. 1). Four patients visited the clinic 7 days after the start of treatment. There were no urethritis symptoms, and the urethral smears were negative for PMNLs. The PCR- and phylogeny-based assay results were positive for the mycoplasma, but M. genitalium loads were suppressed to below the detection level of 50 copies/ml. The four patients returned again for follow-up examination within 7 days after completing the 14-day levofloxacin treatment. They complained of no urethritis symptoms, the smears were still negative for PMNLs, and two of them, who had been positive for C. trachomatis at the first visit, were negative for chlamydia; thus, their NGU was judged clinically cured. In these four patients, however, the M. genitalium load was higher than it was 7 days after the start of the treatment and ranged from 2.0 x 103 to 2.8 x 104 copies/ml (mean, 1.35 x 104 copies/ml) (Fig. 1). From 4 to 28 days after completion of the 14-day levofloxacin treatment, each of these four patients noticed urethral symptoms and returned to the clinic. Urethral smears were positive for PMNLs. We considered the recurrent NGU to have occurred by M. genitalium persisting in the urethra, since all four patients reported abstinence or protected sexual intercourse with the use of condoms after completing the treatment. The first-pass urine samples were positive for M. genitalium by the PCR- and phylogeny-based assay, and the M. genitalium load ranged from 6.0 x 103 to 5.5 x 106 copies/ml (mean, 2.01 x 106 copies/ml) according to the TaqMan assay (Fig. 1). The load at the time of recurrence of NGU was as high as it was at the first visit when definite NGU was observed.
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Presently, guidelines or recommendations are not available for the treatment of M. genitalium-positive urethritis. The in vitro antimicrobial susceptibility profile of M. genitalium is similar to that of Mycoplasma pneumoniae (14). Tetracyclines, macrolides, and some of the newer quinolones are active against M. genitalium (1, 14). However, clinical data on treating M. genitalium-positive NGU with such anitmicrobial agents are extremely limited. In managing M. pneumoniae-induced disease, the mycoplasma is not easily eliminated from the respiratory tract, even when individuals are given adequate doses of antibiotics to which the mycoplasma is known to be sensitive in vitro (3, 12). Antibiotic treatments decrease numbers of the mycoplasma to undetectable levels, but in some cases the mycoplasma reappears and persists in the respiratory tract without clinical signs (3, 12). In this study, M. genitalium loads from men with M. genitalium-positive NGU were suppressed to below the detection level of 50 copies/ml during levofloxacin therapy, though the PCR- and phylogeny-based assay results were positive for the mycoplasma and urethritis symptoms and signs disappeared. At the end of the treatment the mycoplasma loads became detectable by the TaqMan assay, but there were still no symptoms or signs. Such persistent M. genitalium in the urethra for NGU might be analogous to persistent M. pneumoniae in the respiratory tract. Although the number of cases was quite limited in our present study, we observed regrowth of M. genitalium persisting in hosts and a possible association of an increase in the M. genitalium load with the emergence of symptoms and signs of NGU. In our previous study we observed no recurrence of NGU in seven patients with M. genitalium-positive NGU who became negative for the mycoplasma by the PCR and phylogeny assay after antimicrobial chemotherapy. Further studies are required to establish a new treatment algorithm for NGU, including M. genitalium-positive NGU. However, our results suggest that the antimicrobial chemotherapy that is capable of eliminating the mycoplasma from the urethra may be recommended in the management of patients with M. genitalium-positive NGU to prevent the recurrence of NGU.
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FOOTNOTES |
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) of the five patients, the load at the time of recurrence of NGU was as high as it was at the time of the first visit when definite NGU was observed. For the remaining patient (
), no increase in the number of M. genitalium organisms was observed at the time of NGU recurrence.