Search

Mycoplasma genitalium is an important sexually transmitted pathogen affecting both men and women. Its extremely slow growth in vitro and very demanding culture requirements necessitate the use of molecular-based diagnostic tests for its detection in clinical specimens. The recent availability of U.S. Food and Drug Administration (FDA)-cleared commercial molecular-based assays has enabled diagnostic testing to become more widely available in the United States and no longer limited to specialized reference laboratories. Advances in the knowledge of the epidemiology and clinical significance of M. genitalium as a human pathogen made possible by the availability of molecular-based testing have led to updated guidelines for diagnostic testing and treatment that have been published in various countries. This review summarizes the importance of M. genitalium as an agent of human disease, explains the necessity of obtaining a microbiological diagnosis, describes currently available diagnostic methods, and discusses how the emergence of antimicrobial resistance has complicated treatment alternatives and influenced the development of diagnostic tests for resistance detection, with an emphasis on developments over the past few years.

Neisseria meningitidis is a common commensal bacterium found in the respiratory tract, but it can also cause severe, invasive disease. Vaccines have been employed which have been successful in helping to prevent invasive disease caused by encapsulated N. meningitidis from the A, C, W, Y, and B serogroups. Currently, nonencapsulated N. meningitidis groups are more common commensals in the population than in the prevaccine era. One emerging nonencapsulated group of bacteria is the U.S. N. meningitidis urethritis clade (US_NmUC), which can cause meningococcal urethritis in men. US_NmUC has unique genotypic and phenotypic features that may increase its fitness in the male urethra. It is diagnostically challenging to identify and distinguish meningococcal urethritis from Neisseria gonorrhoeae, as the clinical presentation and microbiological findings are overlapping. In this review, the history of meningococcal urethritis, emergence of US_NmUC, laboratory diagnosis, and clinical treatment are all explored.

Neisseria meningitidis , typically a resident of the oro- or nasopharynx and the causative agent of meningococcal meningitis and meningococcemia, is capable of invading and colonizing the urogenital tract. This can result in urethritis, akin to the syndrome caused by its sister species, N. gonorrhoeae , the etiologic agent of gonorrhea. Recently, meningococcal strains associated with outbreaks of urethritis were reported to share genetic characteristics with the gonococcus, raising the question of the extent to which these strains contain features that promote adaptation to the genitourinary niche, making them gonococcus-like and distinguishing them from other N. meningitidis strains. Here, we analyzed the genomes of 39 diverse N. meningitidis isolates associated with urethritis, collected independently over a decade and across three continents. In particular, we characterized the diversity of the nitrite reductase gene ( aniA ), the factor H-binding protein gene ( fHbp ), and the capsule biosynthetic locus, all of which are loci previously suggested to be associated with urogenital colonization. We observed notable diversity, including frameshift variants, in aniA and fHbp and the presence of intact, disrupted, and absent capsule biosynthetic genes, indicating that urogenital colonization and urethritis caused by N. meningitidis are possible across a range of meningococcal genotypes. Previously identified allelic patterns in urethritis-associated N. meningitidis strains may reflect genetic diversity in the underlying meningococcal population rather than novel adaptation to the urogenital tract., (Copyright © 2017 American Society for Microbiology.)

Mycoplasma genitalium is an important and emerging agent of sexually transmitted infection in females and males, carrying the potential for postinfection genital tract sequelae. Past efforts to identify this organism on a routine basis, which were problematic due to the fastidious nature of the bacterium and its antigenic intricacies, have recently become supplemented by molecular diagnostics. A number of these assays are available commercially. This minireview describes the format and performance indices of a number of M. genitalium DNA- and RNA-based amplification assays; many of these assays have contributed to an improved clinical and epidemiologic understanding of this organism., (Copyright © 2017 American Society for Microbiology.)

The detection of Mycoplasma genitalium was evaluated on 1,080 urine samples by the use of a Panther instrument. Overall sensitivity, specificity, positive predictive values, and negative predictive values were 100%, 99.4%, 93.6%, and 100%, respectively. Detection of M. genitalium by the use of the Panther transcription-mediated amplification assay offers a simple, accurate, and sensitive platform for diagnostic laboratories., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Nongonococcal urethritis (NGU) is a common clinical syndrome, but no etiological agent is identified in a significant proportion of cases. Whether the spectrum of pathogens differs between heterosexual men (MSW) and men who have sex with men (MSM) is largely unstudied but of considerable clinical relevance. A retrospective review was done using the electronic medical record database of Melbourne Sexual Health Centre, Australia. Cases were first presentations of symptomatic acute NGU with ≥ 5 polymorphonuclear leukocytes (PMNL)/high-powered field (HPF) on urethral Gram stain between January 2006 and December 2011. First-stream urine was tested for Chlamydia trachomatis and Mycoplasma genitalium by PCR. Demographic, laboratory, and behavioral characteristics of cases were examined by univariate and multivariable analyses. Of 1,295 first presentations of acute NGU, 401 (32%; 95% confidence interval [CI] of 29 to 34%) had C. trachomatis and 134 (11%; 95% CI of 9 to 13%) had M. genitalium detected. MSM with acute NGU were less likely to have C. trachomatis (adjusted odds ratio [AOR] = 0.4; 95% CI of 0.3 to 0.6) or M. genitalium (AOR = 0.5; 95% CI of 0.3 to 0.8) and more likely to have idiopathic NGU (AOR = 2.4; 95% CI of 1.8 to 3.3), to report 100% condom use for anal/vaginal sex (AOR = 3.6; 95% CI of 2.7 to 5.0), or to have engaged in sexual activities other than anal/vaginal sex (AOR = 8.0; 95% CI of 3.6 to 17.8). Even when C. trachomatis or M. genitalium was detected, MSM were more likely than MSW to report consistent condom use (OR = 4.7; 95% CI of 2.6 to 8.3). MSM with acute NGU are less likely to have the established bacterial sexually transmitted infections (STIs) and more likely to report protected anal sex or sexual activity other than anal sex prior to symptom onset than MSW. These data suggest that the etiologic spectrum of pathogens differs between MSM and MSW in acute NGU and that relatively low-risk practices are capable of inducing acute NGU., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

The distribution of Chlamydia trachomatis serovars and Neisseria gonorrhoeae coinfection was studied in a group of 100 C. trachomatis-positive males with urethritis in Greece. The serovar distribution revealed that apart from the predominant worldwide types E and F, the relatively uncommon type G is also prevalent. Gonococcal coinfection was frequent (30%) and was associated with genovariant Ja (75%, P = 0.008).

The aim of this study was to develop and evaluate a sensitive method for the simultaneous identification of 14 urogenital potential pathogens. A multiplex PCR-based reverse line blot (mPCR/RLB) assay was developed to detect 14 urogenital pathogens or putative pathogens, namely Trichomonas vaginalis, Streptococcus pneumoniae, Neisseria gonorrhoeae, Chlamydia trachomatis, Ureaplasma parvum, U. urealyticum, Gardnerella vaginalis, Haemophilus influenzae, herpes simplex virus type 1 (HSV1) and HSV2, N. meningitidis, Mycoplasma hominis, M. genitalium, and adenovirus, using two species-specific primer pairs and probes for each. The method was validated using a reference strain or a well-characterized clinical isolate of each target organism and was found to be both sensitive and specific. The limits of detection for the mPCR/RLB assay varied among the 14 target organisms from 4.2 x 10(-1) to 7.0 x 10(-11) ng/microl of genomic DNA. There were no cross-reactions among any of the probes. This method was used to test 529 first-voided urine specimens from male patients with and without urethritis attending two Sydney sexual health clinics. One or more target species were detected in 193 (36%) subjects. Of 233 positive results, overall 216 (93%) were concordant between mPCR/RLB and a comparator method (culture and/or species-specific PCR), 9 were positive only by mPCR/RLB, and 8 were positive only by the comparator method. The mPCR/RLB method was an accurate, convenient, and inexpensive method for the detection of multiple potential pathogens in first-voided urine specimens from men.

Eno powder (GlaxoSmithKline), an antacid preparation readily available over the counter, was used instead of a CO(2) generator for the growth of 15 strains of Neisseria gonorrhoeae obtained from men with urethritis. Due to its easy accessibility and low cost, Eno powder can be useful in developing countries for transporting clinical specimens from resource-poor peripheral labs to reference laboratories.

Isolation of Mycoplasma genitalium from clinical specimens remains difficult. We describe an improvement of the Vero cell coculture method in which the growth of M. genitalium was monitored by quantitative real-time PCR. Four new M. genitalium strains were isolated from six first-void urine specimens of male Japanese patients with urethritis. In two of them, only M. genitalium was detected: one also contained Ureaplasma urealyticum, and one contained Chlamydia trachomatis, Neisseria gonorrhoeae, U. urealyticum, and Ureaplasma parvum. In the specimens yielding isolates of M. genitalium, growth was documented by quantitative PCR after two to five passages in Vero cells. The complete isolation procedure from the initial inoculation to completion of single-colony cloning took about 1 year. Isolation of M. genitalium from urine specimens proved to be more difficult than from swab specimens. Due to the cytotoxic effect of urine, a procedure involving washing of the urinary sediment was introduced. Furthermore, prolonged storage of the urine specimens before culture was shown to be detrimental to the success of isolation, as shown by the lack of success in attempts to isolate M. genitalium from mailed urine specimens as well as by simulation experiments. High concentrations of penicillin G and amphotericin B were surprisingly inhibitory to the growth of wild-type M. genitalium strains, but penicillin G at 200 IU/ml and polymyxin B at 500 microg/ml could be used as selective antibiotics to avoid bacterial overgrowth in the Vero cell cultures.

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.

Sexually active young adults in the small college town of La Crosse, Wisconsin, were evaluated for conventional sexually transmitted pathogens and tested for infections with mycoplasmas. The prevalence in 65 symptomatic men or women and 137 healthy volunteers (67 men and 70 women) was compared. Urine specimens from both cohorts were tested by ligase chain reaction for Chlamydia trachomatis or Neisseria gonorrhoeae. In addition, the urethral or cervical swabs from the symptomatic subjects were tested by PCR for Mycoplasma genitalium and cultured for Mycoplasma hominis and the ureaplasmas. The results confirmed a relatively low prevalence of gonorrhea among symptomatic men (12%) and chlamydia among symptomatic men (15%) and normal women (3%). In contrast, infections with mycoplasmas, especially the ureaplasmas (57%), were common and the organisms were the only potential sexually transmitted pathogen detected in 40 (62%) symptomatic subjects. Because of the high prevalence, we also evaluated urethral swabs from an additional 25 normal female volunteers and recovered ureaplasmas from 4 (16%) subjects. Additionally, the participants rarely used protection during sexual intercourse and some symptomatic subjects apparently acquired their infections despite using condoms regularly. The findings demonstrate a strong association between abnormal urogenital findings and detection of myoplasmas, particularly ureaplasmas, and suggest the infections will remain common.

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.

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.

Nonchlamydial, nongonococcal urethritis (NCNGU) is suggested to be a sexually transmitted disease in men. NCNGU patients were compared to control subjects with regard to the presence of potentially infectious bacteria in the first void urine. Patients' pre- and post-antibiotic-treatment urine samples and two samples obtained 2 weeks apart from healthy volunteers, who did not receive antibiotic therapy, were analyzed with broad-spectrum PCR tests aiming at eubacterial small subunit rRNA genes. Restriction fragment length polymorphism analysis of the amplicons cloned from the mixtures of PCR products revealed that many different species of microorganisms were found to be colonizing the male urethra. We document here clear differences in the composition of the resident urethral flora between samples obtained from various individuals and between samples obtained at various points in time for a single individual. No major changes in population complexity were found upon antimicrobial treatment. In two of five patients a previously suggested pathogen (Mycoplasma genitalium or Haemophilus parainfluenzae) was accurately identified on the basis of DNA sequencing. No ubiquitous, azithromycin-sensitive organism was identified as a common pathogen in all patients, but up to 40% of all clones represented as-yet-unclassified bacterial species. Relatively often Pseudomonas spp. or Pseudomonas-like organisms were identified in the bacterial flora of patients. Interestingly, an as-yet-uncharacterized microbial species was identified as a negative predictor of NCNGU. This species was identified in all control subjects and was absent from all of the patient' samples (5 of 5 versus 0 of 5, P = 0.0079). This suggests that NCNGU might also be diagnosed by assessing the absence rather than the presence of certain bacterial species.

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.

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.

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.

Of the 331 Neisseria gonorrhoeae strains isolated in Greece from 1996 to 1999, 39 (11.8%) exhibited decreased susceptibility to quinolones due to gyrA and parC mutations. Conventional typing and pulsed-field gel electrophoresis showed that 34 of these isolates were clonally related. Epidemiological data indicated that the epidemic clone was sustained in a group of high-frequency transmitters.

To assess the antimicrobial resistance of Neisseria gonorrhoeae isolated from 1993 through 1998 in Japan, susceptibility testing was conducted on 502 isolates. Selected isolates were characterized by auxotype and analysis for mutations within the quinolone resistance-determining region (QRDR) in the gyrA and parC genes, which confer fluoroquinolone resistance on the organism. Plasmid-mediated penicillin resistance (penicillinase-producing N. gonorrhoeae) decreased significantly from 1993-1994 (7.9%) to 1997-1998 (2.0%). Chromosomally mediated penicillin resistance decreased from 1993-1994 (12.6%) to 1995-1996 (1.9%) and then increased in 1997-1998 (10.7%). Chromosomally mediated tetracycline resistance decreased from 1993-1994 (3.3%) to 1997-1998 (2.0%), and no plasmid-mediated high-level tetracycline resistance was found. Isolates with ciprofloxacin resistance (MIC >/= 1 microg/ml) increased significantly from 1993-1994 (6.6%) to 1997-1998 (24.4%). The proline-requiring isolates were less susceptible to ciprofloxacin than the prototrophic or arginine-requiring isolates. Ciprofloxacin-resistant isolates contained three or four amino acid substitutions within the QRDR in the GyrA and ParC proteins.

We evaluated the performances of a ligase chain reaction (LCR)-based assay and two enzyme immunoassays (Chlamydiazyme and IDEIA) in the detection of Chlamydia trachomatis in urine specimens. We compared the results of testing urine specimens by these assays with those of urethral swab culture by examining samples from 131 men with nongonococcal urethritis. Discrepant results were analyzed by testing urethral swab specimens for C. trachomatis by a PCR-based assay. After the resolution of discrepant results, the sensitivity of urethral swab culture was 85.3%, whereas those of the LCR assay, Chlamydiazyme, and IDEIA with urine specimens were 94.1, 82.4, and 94.1%, respectively. The LCR assay and IDEIA were more sensitive than was urethral swab culture. In addition, the LCR assay, with a sensitivity equal to that of IDEIA, was more specific. Overall, the LCR assay proved to be superior to the enzyme immunoassays in detecting C. trachomatis in urine specimens. Testing urine specimens by LCR assay should be a helpful alternative method for diagnosing C. trachomatis urethral infection in men with nongonococcal urethritis.

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.

In order to test the hypothesis that specimens blocking with a neutralizing reagent below the cutoff of the Chlamydiazyme enzyme immunoassay represent infected patients, we used direct fluorescent-antibody staining for elementary bodies (EBs) and PCR to confirm results for cervical swabs collected from 55,963 women and urethral swabs or first-void urine (FVU) samples collected from 5,781 men attending physicians' offices in the Toronto, Canada, area. Within a grey zone arbitrarily selected to represent values up to 40% below the positive threshold of the test run, 134 cervical swabs, 44 urethral swabs, and 39 FVU specimens exhibited a blocking response ( > 50% reduction in signal). Three or more EBs were observed in each of 98 cervical swabs (73.1%), 38 urethral swabs (86.4%), and 21 FVU specimens (53.8%). Of the 36 cervical swabs with fewer than three EBs, 33 were PCR positive; the positive PCR results for male specimens were 6 of 6 urethral swabs and 17 of 18 FVU samples. Application of the blocking test to specimens negative in the Chlamydiazyme enzyme immunoassay but having optical densities within 40% of the cutoff added 14.2% (217 of 1,531 specimens) more positive results to the survey. A total of 213 of 217 samples (98.2%) were reconfirmed as having EBs or DNA.

TThe Syva MicroTrak Chlamydia enzyme immunoassay (EIA; Syva Company, San Jose, Calif.) with cytospin and direct fluorescent-antibody assay (DFA) confirmation was evaluated on 43,630 urogenital specimens over a 1-year period in the Provincial Laboratory in Regina, Saskatchewan, Canada. This was a two-phase study intended to define a testing algorithm for Chlamydia trachomatis that would be both highly accurate and cost-effective in our high-volume (> 3,000 tests per month) laboratory. The prevalence of C. trachomatis infection in our population is moderate (8 to 9%). In phase 1, we tested 6,022 male and female urogenital specimens by EIA. All specimens with optical densities above the cutoff value and those within 30% below the cutoff value were retested by DFA. This was 648 specimens (10.8% of the total). A total of 100% (211 of 211) of the specimens with optical densities equal to or greater than 1.00 absorbance unit (AU) above the cutoff value, 98.2% (175 of 178) of the specimens with optical densities of between 0.500 and 0.999 AU above the cutoff value, and 83% (167 of 201) of the specimens with optical densities within 0.499 AU above the cutoff value were confirmed to be positive. A total of 12% (7 of 58) of the specimens with optical densities within 30% below the cutoff value were positive by DFA. In phase 2, we tested 37,608 specimens (32,495 from females; 5,113 from males) by EIA. Only those specimens with optical densities of between 0.499 AU above and 30% below the cutoff value required confirmation on the basis of data from phase 1 of the study. This was 4.5% of all specimens tested. This decrease in the proportion of specimens requiring confirmation provides a significant cost savings to the laboratory. The testing algorithm gives us a 1-day turnaround time to the final confirmed test results. The MicroTrak EIA performed very well in both phases of the study, with a sensitivity, specificity, positive predictive value, and negative predictive value of 96.1, 99.1, 90.3, and 99.7%, respectively, in phase 2. We suggest that for laboratories that use EIA for Chlamydia testing, a study such as this one will identify an appropriate optical density range for confirmatory testing for samples from that particular population.

First-void urine specimens from 683 men (592 without symptoms) were tested for Chlamydia trachomatis by a polymerase chain reaction (PCR) with KL1 and KL2 plasmid primers and by a Chlamydiazyme enzyme immunoassay (EIA). Thirty-seven specimens were confirmed to be positive by using the EIA blocking reagent and a second set of plasmid primers (T1 and T2). By comparing unconfirmed PCR results (KL1 and KL2 primers only) with the blocked Chlamydiazyme EIA results, the sensitivity and specificity of PCR were 100% (37 of 37 specimens) and 99.5% (643 of 646 specimens), respectively. Three additional specimens were negative by EIA but positive by PCR and were confirmed to be positive with primers T1 and T2. Two of the three specimens were from men with symptoms. The confirmatory PCR assay performed equally well in detecting positive specimens from symptomatic (31 of 31) and asymptomatic (9 of 9) men. Comparison of confirmatory testing of first-void urine specimens by PCR and EIA showed that PCR was 100% sensitive (40 of 40 specimens) and that the EIA was 92.5% sensitive (37 of 40 specimens) but that the assays were equally specific (100%).

Thirty-three clinical isolates from male nongonococcal urethritis and 28 isolates from soft tissue infections and ulcers were identified as Bacteroides ureolyticus by conventional bacteriological tests and were compared with five reference strains of the species. Whole-cell proteins from these clinical isolates and the reference strains were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The majority of the strains from the two sources could be divided into five different groups, named phenons I to V; phenons I to IV have been described previously by others, while phenon V has been described recently by us. Digestion of chromosomal DNA from 16 of the clinical isolates (including strains representative of each of the five SDS-PAGE phenons) and the five reference strains was attempted with restriction endonucleases EcoRI, PstI, SmaI, and HindIII. After electrophoresis in agarose gels, good digestion was observed with HindIII only, and 12 different banding patterns (restriction endonuclease analysis [REA] profiles) were obtained for the 19 strains digested; one nongonococcal urethritis isolate and one reference strain did not show any digestion. From the agarose gels, HindIII-digested fragments of DNA were transferred to nylon membranes by use of vacuum blotting and subjected to hybridization with 32P-labelled 16S-23S rRNA from Escherichia coli. The resultant pattern of bands (ribotypes), which depends on the restriction fragment length polymorphisms in the rRNA genes, was used as a measure of genomic variation within the species. In total, 13 different ribotypes were obtained for the 19 strains. For some strains, good correlation was achieved among the SDS-PAGE phenons, REA profiles, and ribotypes. However, for others, REA analysis and ribotyping were able to discriminate between strains which shared the same SDS-PAGE phenon. Interestingly, these two techniques of DNA characterization were able to differentiate between isolates from the genital tract and those associated with soft tissue infections and ulcers.

First-void urine (FVU) sediments of 240 men were tested for Chlamydia trachomatis antigens by two enzyme immunoassays, TestPack Chlamydia (15 min) and Chlamydiazyme (3.5 h), and the results were compared with urethral swab culture results. The sensitivity and specificity on FVU sediment for TestPack Chlamydia were 76.2% (32 of 42 specimens) and 95.5% (189 of 198 specimens) versus 81.0% (34 of 42 specimens) and 96.5% (191 of 198 specimens) for Chlamydiazyme, respectively. Rapid, on-site detection of chlamydial antigen in male FVU would shorten the infectious period by hastening diagnosis and treatment.

A differential agar medium for the identification of Ureaplasma urealyticum in primary cultures of clinical specimens is described. The differential medium (no. A7) is specific for the identification of U. urealyticum and other members of the genus Ureaplasma. Large-colony, classical Mycoplasma and Acholeplasma species and Proteus L colonies are unreactive on this differential medium. The medium incorporates the biochemical principle of the direct spot test for urease in colonies of Ureaplasma and contains added urea and a sensitive indicator of ammonia, manganous sulfate. Ureaplasma colonies on this medium are identified as dark golden-brown or rich deep-brown colonies, in sharp contrast to the light background of the medium, when viewed by direct transmitted illumination under the low power of the microscope.

A simple method is presented for producing large numbers of inclusions of Chlamydia trachomatis serotype L2 in cell cultures on slides for immunofluorescence antibody staining. Preliminary results with a total of 1,241 human sera from different groups were consistent with findings by earlier methods: 82% of chlamydia-positive men with nongonococcal urethritis had antibodies at titers of greater than or equal to 8, with a geometric mean titer (GMT) of 44.9; 68.5% of chlamydia-negative men with nongonococcal urethritis had titers of greater than or equal to 8, with a GMT of 38.6; 27% of male blood donors had titers of greater than or equal to 8, with a GMT of 19.6; 95.0% of chlamydia-positive women had titers of greater than or equal to 8, with a GMT of 80.3; 67.0% of chlamydia-negative partners of men with nongonococcal urethritis had titers of greater than or equal to 8, with a GMT of 47.0; 50.2% of control women had titers of greater than or equal to 8, with a GMT of 27.7% and 8.1% of children (aged 1 to 15 years) had titers of greater than or equal to 8, with a GMT of 17.8.

A total of 194 male urethral and 402 cervical specimens were obtained from patients at the venereal disease outpatient clinic of University Hospital, Rotterdam, The Netherlands, to evaluate the IDEIA test (Boots Celltech) for the detection of chlamydial infections. The prevalences of culture-positive males and females were 17.5 and 8.2%, respectively. The respective overall sensitivities and specificities found were 67.6 and 93.7% for the males and 63.6 and 93.8% for the females. The highest sensitivity (83.3%) was found in male patients with more than 20 leukocytes per field in the sediment of the first-voided urine (magnification, X250) and in women with more than 10 leukocytes per field in a cervical Gram stain (magnification, X800). However, in men without urethritis and in women with fewer than 10 leukocytes per field in the Gram stain, sensitivities of 44.4 and 40%, respectively, were found. Culture-positive, IDEIA-negative results were predominantly observed in samples with few inclusions in the culture.

The optimum conditions for the detection of human immunoglobulin G (IgG), IgM, and IgA antibodies to Ureaplasma urealyticum by an enzyme-linked immunosorbent assay (ELISA) were established by using a cell lysate antigen and commercially available alkaline phosphatase conjugates. No significant cross-reactions were observed among rabbit antisera to a variety of mycoplasmas of human origin and ureaplasma antigen, thus demonstrating the specificity of the ELISA. All human sera were assayed at a 1:200 dilution. Antigen was used at 20 mug of protein/ml and conjugates were diluted 1:500. Presence of IgG antibody to U. urealyticum was significantly associated with isolation of U. urealyticum (P < 0.001) in 110 women. Seventeen acute-phase and 19 convalescent-phase sera from male nongonococcal urethritis (NGU) patients were tested for the presence of antibody by both the metabolism inhibition assay and by ELISA, with overall agreements of 82 and 95% for acute- and convalescent-phase sera, respectively. Serum antibody responses were demonstrated to selected serotypes in the metabolism inhibition test, but the response as measured by the ELISA was independent of the serotype of the antigen used. Serum antibody levels in NGU patients were significantly higher (P < 0.002) than the normal serum standard in the IgG, IgM, and IgA classes. Additionally, the magnitude of change between acute- and convalescent-phase sera was greater for NGU patients than for normal asymptomatic ureaplasma-positive male controls. A significant change in antibody levels of one or more antibody classes was detected for 12 of 18 (67%) NGU patients by ELISA. Ten of the 12 (83%) individuals had a change in the IgM class, which is suggestive of an active infectious process. The ELISA is advantageous in that it requires only a single serotype antigen, uses one serum dilution, is class specific, and allows quantitative detection of differences between acute- and convalescent-phase sera.

McCoy cells treated in six different ways, in addition to untreated cells, were compared to determine which gave rise to the largest number of Chlamydia trachomatis inclusions when tested with a laboratory-passaged strain. The same batch of cells was treated by irradiation, preinoculation exposure to cytochalasin B or 5-iodo-2'-deoxyuridine, and postinoculation exposure to cycloheximide, hydrocortisone, or emetine. Significantly more inclusions were always found in cells which had been treated with cycloheximide than in cells treated in any other way. Conversely, untreated McCoy cells always had significantly fewer inclusions than cells which had received some form of treatment. Similar results were obtained when cycloheximide-treated, irradiated, and untreated cells were inoculated with urethral specimens containing unpassaged organisms.

Isolates from five patients with genitourinary infections diagnosed over a 7-week period were identified as CDC group HB-5. The organisms caused clinical presentations of pelvic inflammatory disease in three women and urethritis in the only male in the series. The remaining patient received surgical treatment for a Bartholin gland abscess. Since the clinical and temporal presentations were remarkable and the questions of sexual mode of transmission and strain relatedness were of concern, the five strains were examined further by multilocus enzyme electrophoresis and ribosomal DNA typing. Overall, the five clinical isolates were more closely related to each other than to the control strains. This is the first report describing genitourinary infections caused by group HB-5. While the pathophysiology of group HB-5 infections remains to be fully elucidated, the possibility that this organism may be sexually transmitted deserves further study.

A cell wall-defective form of Neisseria gonorrhoeae from an exudate of an untreated patient with gonococcal urethritis was isolated on medium containing 7% polyvinylpyrrolidone. Initial attempts to grow the organism by standard microbiological methods had failed. This isolate was incapable of reversion to a normal gonococcus even after numerous subcultures and appeared to be a stable cell wall-defective form.

A new colony type of Neisseria gonorrhoeae was detected in the primary cultures from 8 of 180 men with gonococcal urethritis. This colony type contrasts with those previously described by having a rough and irregular surface. In six of the eight cases, the rough form predominated. The distinctive morphology of the rough colony variant could be maintained indefinitely by selective subculture. By electron microscopy, organisms taken from rough colonies of each of the eight isolates were piliated. Antimicrobial susceptibilities of type 1 and rough clones derived from the same patients were identical for ampicillin, penicillin, tetracycline, and spectinomycin. After inoculation of rough colonies into subcutaneous chambers in mice and guinea pigs, type 1 colonies predominated in cultures of material obtained from the chambers. This new piliated colony type of N. gonorrhoeae may provide an opportunity to investigate factors other than pili that contribute to gonococcal virulence.

Consecutive clinical specimens of Chlamydia trachomatis (1,048) were inoculated in parallel on DEAE-dextran- and cycloheximide-treated HeLa 229 cells and cycloheximide-treated McCoy cells. HeLa 229 cell culture detected 113 positive specimens, and McCoy cell culture detected 103 positive specimens. This difference is not significant. However, HeLa 229 cell culture yielded significantly more inclusions than McCoy cell culture in the 95 specimens positive in both cell types (P = 0.042). For routine diagnostic purposes, a choice for one of the cell types may be determined by local preferences.

An agar growth inhibition method for serotyping Ureaplasma urealyticum is described, and the results of applying this method to serotyping 338 strains of the organism are presented. The serotyped strains consisted of cloned isolates from male patients with primary and recurrent nongonococcal urethritis (NGU), isolates from symptomatic patients with other genitourinary tract infections and disorders, and isolates from asymptomatic carriers of U. urealyticum in the genitourinary tract (controls). Among 122 male patients with NGU, serotype 4 was associated most frequently (52%) with this disease at Camp Lejeune, N.C. Seventeen percent of the isolates were type 2. The remaining isolates consisted of types 1, 3, 6, and 8 and accounted for 6 to 9% each of the serotypes isolated from the NGU group. Types 5 and 7 were not isolated. Among 91 symptomatic patients with other genitourinary tract infections and disorders, U. urealyticum type 4 also was associated most frequently (37%) with these disorders. The remaining isolates, represented by types 1, 2, 3, 6, 7, and 8, accounted for 9 to 15% each of the types isolated from this group. Type 5 was not isolated. Among 125 symptomfree carriers of U. urealyticum in the genitourinary tract, type 8 was recovered most frequently (30%), whereas type 4 was isolated next most frequently (24%). The remaining isolates consisted of types 1, 2, 3, 5, and 6 and accounted for 2 to 15% each in this asymptomatic control group. Type 7 was not isolated. Of the present eight serotypes of U. urealyticum studied in this investigation, type 4 was associated most frequently with disease (NGU) and certain other disorders of the genitourinary tract at Camp Lejeune. A previously unknown association of U. urealyticum with frequently abacteriuric, unexplained pyuria (with or without urethral pruritus and dysuria) is reported, suggesting the existence of asymptomatic Ureaplasma urethritis.

A maltose-negative strain of Neisseria meningitidis isolated from a male patient with urethritis was initially identified as a gonococcus on the basis of carbohydrate utilization tests. Additional investigations initiated because of atypical colony morphology and a negative superoxol reaction showed the isolate to be a meningococcus with defective maltose-phosphorylating capacity.

The existence of unusual colonies of Ureaplasma urealyticum (T mycoplasmas) in primary agar cultures of certain urine specimens is reported and their morphology is illustrated.

Sixty-nine Caucasian males without a previous history of urethritis and who developed nongonococcal urethritis (NGU) and 39 similar men without urethritis (NU) were cultured from the urethra for Chlamydia trachomatis, Mycoplasma hominis, Ureaplasma urealyticum, aerobes, and anaerobes. C. trachomatis infection was proven by culture of serology in 26 (38%) of the NGU group and 1 (3%) of the NU group; the C. trachomatis-negative NGU group had significantly more U. urealyticum (81%) than the C. trachomatis-positive NGU group (42%) or the NU group (59%). Aerobes were isolated from significantly more NU men (91%) than from men with NGU (66%). The aerobic and anaerobic flora of the two NGU groups were similar. The NU group had significantly more aerobic lactobacilli. Haemophilus vaginalis, alpha-hemolytic streptococci (not Streptococcus faecalis), and anaerobes, predominantly Bacteroides species. This study has provided information about the prevalence and the variety of the aerobic and anaerobic microbiological flora of the anterior urethra of sexually active males. It does not implicate any bacteria other than C. trachomatis and U. urealyticum as potential causes of NGU.