Maeda S

Threat to Cefixime Treatment for Gonorrhea

To the Editor: From November 2002 through May 2003, 4 Japanese men, ranging in age from 23 to 45 years, visited the Department of Urology at Toyota Memorial Hospital, Toyota, Japan. Physical examinations showed urethral discharge and dysuria. Each had had sexual contact with sex workers in central Japan. Four strains of Neisseria gonorrhoeae were isolated from urethral specimens. Treatment comprised 200 mg cefixime, twice a day for 3 days. However, all 4 patients returned to the clinic with continuing symptoms, despite having completed the prescribed course of cefixime and abstaining from sexual activity. N. gonorrhoeae was again isolated from urethral swabs. Each patient was then treated with 1 g intravenous ceftriaxone. In the 3 patients who returned to the clinic for followup, the ceftriaxone treatment resulted in clinical and microbiologic cure. Pulsed-field gel electrophoresis (PFGE) analysis of the SpeI-digested DNAs of N. gonorrhoeae was performed to assess the relatedness of pre- and posttreatment isolates (1). For these 8 isolates, MICs of penicillin G, tetracycline, cefixime, cefdinir, cefodizime, ceftriaxone, levofloxacin, azithromycin, and spectinomycin were determined on chocolate agar (GC) medium base supplemented with 1% IsoVital X (Becton Dickinson, Franklin Lakes, NJ, USA) and containing serial 2-fold dilutions of each agent (2). Media were inoculated with 104 CFU and incubated at 35°C in 5% CO2 overnight. The MIC was defined as the lowest concentration inhibiting growth to <1 CFU. β-lactamase activity of the isolates was tested with a nitrocefin disk. The nucleotide sequences of the full-length penA gene encoding the penicillin-binding protein 2 (PBP 2) were identified in the isolates (1). Briefly, genomic DNAs from each isolate were subjected to PCR to amplify 3 fragments of the penA gene of N. gonorrrhoeae. PCR products were sequenced by the dye terminator method and with an automatic sequencer. In each of the 4 cases, the PFGE patterns of the pre- and posttreatment isolates had the same numbers of bands (12–16 fragments), and the corresponding bands were the same apparent size; the pre- and posttreatment isolates were indistinguishable (3). MICs of antimicrobial agents for the 8 isolates are shown in the Table. All isolates were enzymatically negative for β-lactamase and possessed identical mosaic alterations in PBP 2. The mosaic PBP 2 was composed of fragments of PBP 2 from N. cinerea and N. perflava and was identical to that identified in our previous study (1). Table Antimicrobial drug susceptibilities of clinical isolates of Neisseria gonorrhoeae from patients with gonococcal urethritis treated unsuccessfully with a 3-day cefixime regimen* Until recently, Japanese guidelines recommended oral administration of cefixime, 200 mg twice a day for 3 days to prolong the period of time for which the serum drug concentration remains above the MIC (4). However, treatment failure with this cefixime regimen was observed in our 4 cases of gonorrhea. The isolates showed cefixime MICs of 0.5–1 μg/mL and harbored mosaic alterations in PBP 2. Most recently, the emergence and spread of such strains in Japan (1,5,6) have led to the recommendation that ceftriaxone and spectinomycin should be used as the primary therapy for gonorrhea instead of oral cephalosporins (7). In 2001, treatment failure was reported in a Caucasian man residing in Hawaii who had been given a single 400-mg dose of cefixime for gonorrhea (8). Pre- and posttreatment strains of N. gonorrhoeae were recovered from this patient, and 1 strain was isolated from his Japanese female sex partner who had visited Hawaii from Japan. Another pretreatment strain was isolated from a Micronesian man with gonorrhea residing in Hawaii, who had had sex with a woman from Malaysia or the Marshall Islands. This man was successfully treated with a single 400-mg dose of cefixime. For these strains, MICs of penicillin, tetracycline, spectinomycin, cefixime, ceftriaxone, ciprofloxacin, and azithromycin were 8.0, 4.0–8.0, <32, 0.25–0.5, 0.125, 8–16, and 0.125–0.25 μg/mL, respectively. The antibiograms of these strains in Hawaii were similar to those of strains with mosaic PBP 2 found in our 4 patients. The introduction to Hawaii of such multidrug resistant strains might be related to sex partners from Asia. Although the strains were not analyzed for alterations in PBP 2, they could have been derived from strains with cefixime resistance-associated mosaic PBP 2. The strains with mosaic PBP 2 showed such decreased susceptibility to cefixime that they were not effectively eradicated by the 3-day treatment. Although it is not clear whether these strains are also resistant to the single 400-mg dose of cefixime (8, 9), their emergence and spread could threaten treatment for gonorrhea with cefixime (10). Global emergence and spread of such multidrug-resistant strains of N. gonorrhoeae would be a matter of serious concern. The antimicrobial susceptibilities of current gonococcal isolates must be monitored periodically. In particular, posttreatment isolates from patients treated unsuccessfully with cefixime should be surveyed.

Quantitative Detection of Mycoplasma genitalium from First-Pass Urine of Men with Urethritis and Asymptomatic Men by Real-Time PCR

ABSTRACT 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 107 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 × 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 × 10 copies/ml, and six men had loads ranging from 1.1 × 107 to 2.7 × 102 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 × 106 to 2.3 × 102 copies/ml. Of 76 asymptomatic men, only two were positive for M. genitalium. For these men, the loads were 2 × 102 and <5 × 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.

Association of Ureaplasma urealyticum (biovar 2) with nongonococcal urethritis.

Background The tiny (T)-strain mycoplasmas, designated in 1974 as Ureaplasma urealyticum, have been divided into 2 species, Ureaplasma parvum (biovar 1) and U. urealyticum (biovar 2), but association of each of these species with nongonococcal urethritis (NGU) remains unclear. Goal The goal of this study was to determine whether U. parvum (biovar 1) or U. urealyticum (biovar 2) is associated with NGU. Study Design The prevalences of U. parvum (biovar 1) and U. urealyticum (biovar 2) in 572 patients with urethritis were compared with those in 141 men without urethritis. Results The prevalence of U. urealyticum (biovar 2) in men with NGU (15.8%) or with nonchlamydial NGU (18.0%) was significantly higher than that in men without urethritis (7.8%). The prevalence of U. parvum (biovar 1) in men with NGU (8.5%) or with nonchlamydial NGU (11.1%) did not differ significantly from that in men without urethritis (13.5%). Conclusion Our results showed a significant association between U. urealyticum (biovar 2) and NGU. They also suggest that the presence of U. parvum (biovar 1) in the male urethra might be the result of colonization.

Remarkable Increase in Central Japan in 2001-2002 of Neisseria gonorrhoeae Isolates with Decreased Susceptibility to Penicillin, Tetracycline, Oral Cephalosporins, and Fluoroquinolones

ABSTRACT Four hundred sixty-two clinical isolates of Neisseria gonorrhoeae recovered from 1999 through 2002 in central Japan were examined for MICs of antimicrobial agents. The majority was sensitive to ceftriaxone and spectinomycin, but a remarkable increase in isolates with decreased susceptibility to penicillin, tetracycline, oral cephalosporins, and fluoroquinolones was observed from 2001 through 2002.

Rapid Detection of Mycoplasma genitalium, Mycoplasma hominis, Ureaplasma parvum, and Ureaplasma urealyticum Organisms in Genitourinary Samples by PCR-Microtiter Plate Hybridization Assay

ABSTRACT We present a method for detecting the presence of Mycoplasma genitalium, Mycoplasma hominis, Ureaplasma parvum, and Ureaplasma urealyticum organisms, which are thought to be associated with nongonococcal urethritis (NGU) and other genitourinary infections, in clinical samples. This method consists of PCR amplification of a part of the 16S rRNA gene followed by 96-well microtiter plate hybridization assay using four species-specific capture probes to detect the targets. To test the efficacy of this method, we applied it to the detection of the four species in the urine of patients with NGU. There were no cross-reactions with other human mycoplasmas or ureaplasmas, and the PCR-microtiter plate hybridization assay detected as few as 10 copies of the 16S rRNA gene of each of the four species. Based on these results, this PCR-microtiter plate hybridization assay can be considered an effective tool for the diagnosis of genitourinary infections with mycoplasmas or ureaplasmas.

MYCOPLASMA GENITALIUM: ANOTHER IMPORTANT PATHOGEN OF NONGONOCOCCAL URETHRITIS

PURPOSE We reviewed findings on the pathogenic role of Mycoplasma genitalium in nongonococcal urethritis and the treatment of men with M. genitalium positive nongonococcal urethritis. MATERIALS AND METHODS We reviewed literature selected from peer reviewed journals listed in MEDLINE and from resources cited in those articles from 1967 to January 2001. RESULTS M. genitalium was first isolated from 2 men with nongonococcal urethritis and thereafter it was shown to cause urethritis in subhuman primates inoculated intraurethrally. This mycoplasma has been detected significantly more often in patients with acute nongonococcal urethritis, particularly in those with nonchlamydial nongonococcal urethritis, than in those without urethritis. The prevalence of M. genitalium positive nonchlamydial nongonococcal urethritis is 18.4% to 45.5% of all nonchlamydial nongonococcal urethritis cases. In addition, the persistence of M. genitalium in the urethra after antimicrobial chemotherapy is associated with persistent or recurrent nongonococcal urethritis. M. genitalium is highly susceptible to tetracycline, macrolide and some new fluoroquinolones. The regimen of 100 mg. doxycycline orally twice daily for 7 days, which is recommended for chlamydial nongonococcal urethritis, seems to be effective for M. genitalium positive nongonococcal urethritis, although clinical data to substantiate this regimen are limited. CONCLUSIONS The various results reported to date tend to support the proposition that M. genitalium is a pathogen of nongonococcal urethritis. However, currently diagnostic methods for this important mycoplasma are not available in clinical practice. Because of the possible association of the posttreatment presence of M. genitalium in the urethra with persistent or recurrent nongonococcal urethritis, eradication of this mycoplasma from the urethra is essential for managing M. genitalium positive disease. However, clinical data on treating M. genitalium positive nongonococcal urethritis are extremely limited. Thus, further studies are required to develop new diagnostic methods that would be available in clinical settings and establish a new treatment algorithm for nongonococcal urethritis, including M. genitalium positive disease.

Phylogeny-Based Rapid Identification of Mycoplasmas and Ureaplasmas from Urethritis Patients

ABSTRACT Some strains of mycoplasmas and ureaplasmas (family Mycoplasmataceae) are associated with nongonococcal urethritis (NGU) or other genitourinary infections. We have developed a rapid and reliable method of identifying the presence and prevalence of mycoplasmas and ureaplasmas in men with NGU. This method is based on the amplification of a part of the 16S rRNA gene by PCR and phylogenetic analysis. A portion of the 16S rRNA gene from 15 prototype strains was amplified with a set of common primers, and their nucleotides were sequenced. The nucleotide sequence of the V4 and V5 regions was analyzed by the neighbor-joining method. The 15 prototype strains were grouped into three distinct clusters, allowing us to clearly segregate the strains into distinct lineages. To determine the prevalence of these pathogens among patients with NGU, this protocol was tested with 148 urine samples. Amplifications were observed for 42 samples, and their nucleotide sequences were analyzed along with those of the 15 prototype strains. The phylogenetic tree thus constructed indicated that 15 of the 42 formed a cluster with Mycoplasma genitalium. Among the remaining specimens, 2 formed a cluster with Mycoplasma hominis, 19 with Ureaplasma urealyticum, and 5 with Ureaplasma parvum; the remaining sample contained both M. genitalium and U. urealyticum. This phylogeny-based identification of mycoplasmas and ureaplasmas provides not only a powerful tool for rapid diagnosis but also the basis for etiological studies of these pathogens.

Association of Mycoplasma genitalium persistence in the urethra with recurrence of nongonococcal urethritis.

Background Most patients with recurrent symptomatic nongonococcal urethritis receive negative test results for Chlamydia trachomatis and Ureaplasma urealyticum, and the cause of such recurrence usually is unknown. Goal To assess the association of Mycoplasma genitalium with recurrent nongonococcal urethritis. Study Design In this study, 72 men with nongonococcal urethritis were treated with levofloxacin. Before and after treatment, symptoms and signs were assessed and first-pass urine was examined for C trachomatis, M genitalium, U urealyticum, and Mycoplasma hominis by polymerase chain reaction–based assays. Results In 6 of 45 men who had no symptoms and no evidence of inflammation after treatment, nongonococcal urethritis recurred. Of these 6 men, 5 had positive test results for M genitalium before levofloxacin treatment, which remained positive afterward. After the second treatment for recurrent nongonococcal urethritis, one man was still had a positive test result for the mycoplasma and experienced a subsequent recurrence. Conclusions This study suggests that the persistence of M genitalium in the urethra may be associated with recurrence of nongonococcal urethritis.

Detection of Mycoplasma genitalium, Mycoplasma hominis, Ureaplasma parvum (biovar 1) and Ureaplasma urealyticum (biovar 2) in patients with non‐gonococcal urethritis using polymerase chain reaction‐microtiter plate hybridization

Abstract Aim: To use polymerase chain reaction (PCR)‐microtiter plate hybridization assays to detect Mycoplasma genitalium, Mycoplasma hominis, Ureaplasma parvum (biovar 1) and Ureaplasma urealyticum (biovar 2) in first‐voided urine specimens from patients with non‐gonococcal urethritis (NGU).

DETECTION OF MYCOPLASMA GENITALIUM IN PATIENTS WITH URETHRITIS

PURPOSE We attempted to detect Mycoplasma genitalium in urethral swab specimens by a polymerase chain reaction based assay to determine the prevalence of M. genitalium in patients with urethritis. MATERIALS AND METHODS We examined a total of 171 Japanese men who presented to our hospital from February 1995 through January 1997. Of these men 150 had symptoms and signs compatible with acute urethritis and 21 had no symptoms or signs of urethritis. Urethral swab specimens were used to culture Neisseria gonorrhoeae, to detect Chlamydia trachomatis by an enzyme immunoassay and to detect M. genitalium by a polymerase chain reaction based assay. RESULTS Gonococcal urethritis was diagnosed in 74 symptomatic men, and nongonococcal urethritis was diagnosed in 76 symptomatic men. Of the 74 cases of gonococcal urethritis 3 (4.1%) were positive for M. genitalium, and 14 (18.9%) were positive for C. trachomatis. Of the 76 cases of nongonococcal urethritis 10 (13.2%) were positive for M. genitalium, and 42 (55.2%) were positive for C. trachomatis. While only 1 of the 42 cases with chlamydial nongonococcal urethritis (2.4%) was positive for M. genitalium, 9 of the 34 chlamydia negative nongonococcal urethritis cases (26.5%) were positive for the mycoplasma. In contrast, all 21 cases men were negative for N. gonorrhoeae, M. genitalium, and C. trachomatis. CONCLUSIONS The prevalences of M. genitalium in patients with gonococcal urethritis and nongonococcal urethritis who attended our clinic were 4.1 and 13.2%, respectively. M. genitalium was detected significantly more often in men with nongonococcal urethritis than in asymptomatic men. In addition, its prevalence in men with chlamydia negative nongonococcal urethritis (26.5%) was significantly greater than in those with chlamydia positive nongonococcal urethritis (2.4%). These findings suggest that M. genitalium may be associated with the development of nongonococcal urethritis independent of C. trachomatis.