Comparison of commercially available media for detection and isolation of Ureaplasma urealyticum and Mycoplasma hominis.

The Mycotrim Triphasic flask system (Irvine Scientific, Irvine, Calif.) was compared with a system composed of Mycotrim GU broth (Irvine Scientific) and A7 or A8 agar (Remel, Lenexa, Kans.) for the ability to detect Ureaplasma urealyticum and Mycoplasma hominis from 129 genital specimens. Of the 64 specimens positive for U. urealyticum, 25, 98, and 100% were detected on Mycotrim Triphasic agar and A7 and A8 agars, respectively. All 18 specimens that grew M. hominis were detected by A7 and A8 agars, and 94% grew on Mycotrim Triphasic agar. Mycotrim GU broth detected all of the positive specimens, and Mycotrim Triphasic broth detected all but one. Mycotrim GU broth inoculated simultaneously with either A7 or A8 agar was found to be more sensitive and cost-effective than the Mycotrim Triphasic flask system.     

Detection of Neisseria gonorrhoeae and Chlamydia trachomatis in genitourinary specimens from men and women by a coamplification PCR assay.

A coamplification PCR test for the direct detection of Neisseria gonorrhoeae and Chlamydia trachomatis in urethral and endocervical swabs and urine samples from men and women was compared to standard culture techniques. Processed specimens were amplified in single reaction tubes containing primers for both organisms, and PCR products were detected by a colorimetric microwell plate hybridization assay specific for each pathogen. Of 344 specimens from men, 45 (13.1%) urine specimens were PCR positive for C. trachomatis, 51 (14.8%) urethral swab specimens were PCR positive, and 29 urethral swab specimens (8.4%) were culture positive. After analysis of discrepancies, the resolved sensitivity and specificity of PCR for C. trachomatis were 96.2 and 99.3%, respectively, in urethral swab specimens, compared to 88.2 and 98.6% for urine specimens. Of the 192 specimens from women, 28 (14.6%) urine specimens were PCR positive for C. trachomatis, 32 (16.7%) endocervical specimens were PCR positive, and 19 (9.9%) endocervical specimens were culture positive. After analysis of discrepancies, the resolved sensitivity and specificity of PCR for C. trachomatis for endocervical specimens were both 100% compared to 100 and 99.4%, respectively, for urine specimens from women. In men, 68 (19.8%) urine specimens were PCR positive for N. gonorrhoeae, 73 (21.2%) urethral swabs were PCR positive, and 59 (17.2%) urethral swabs were culture positive. After analysis of discrepancies, the resolved sensitivity and specificity of PCR for N. gonorrhoeae were 97.3 and 97.0%, respectively, for urethral specimens compared to 94.4 and 98.5% for urine specimens. In women, 18 (9.4%) urine specimens were PCR positive for N. gonorrhoeae, 23 (12.0%) were endocervical swab PCR positive, and 15 (7.8%) endocervical specimens were culture positive. After analysis of discrepancies, the resolved sensitivity and specificity of PCR for N. gonorrhoeae were 100 and 99.4%, respectively, for endocervical specimens compared to 90.0 and 95.9% for female urine specimens. These results indicate that a multiplex PCR is highly sensitive for detecting both C. trachomatis and N. gonorrhoeae from a single urine or genital swab, providing a more cost-effective way of screening multiple pathogens.     

Comparison of media for isolation of Ureaplasma urealyticum and genital Mycoplasma species

A total of 484 frozen patient specimens originally positive for Ureaplasma urealyticum or Mycoplasma spp. or both were recultured, and the results were compared on the following media: Shepard's A7 agar, modified phenol red-urea, SP-4-urea, modified phenol red-arginine, and SP-4-arginine broths. Of 351 specimens positive for U. urealyticum, 30 (8.5%) were detected only in one or more of the broth media, whereas 117 (33%) were positive only on A7 agar. Separate use of the SP-4-urea broth or modified phenol red-urea broth isolated all but 1 and 2, respectively, of the negative A7 agar cultures. Of the 76 specimens positive for large colony Mycoplasma spp., 58 (76.3%) were not grown on the primary plating (A7) agar. Of 73 broth isolates, 73 (100%) grew in SP-4-arginine broth, and 64 (87.7%) grew in modified phenol red-arginine broth. Modified SP-4 broth appears to be a useful supplement to the A7 plating medium commonly used in the diagnostic laboratory for the isolation of U. urealyticum and Mycoplasma spp.     

Systematic screening tests for Chlamydia trachomatis, Mycoplasma hominis and Ureaplasma urealyticum in urogenital specimens of infertile couples

We conducted a prospective study on 100 couples consulting for infertility at the teaching Hospital of Tours, with the scope to determine if there is a benefit for systematic screening of Chlamydia trachomatis, Mycoplasma hominis and Ureaplasma urealyticum among genito-urinary specimen when exploring couples infertility. C. trachomatis was detected by PCR on sperm, endocervix and urine specimen. M. hominis and U. urealyticum were detected by culture on A7 agar medium and with minigaleries on sperm and endocervix specimen. Standard cultures were also performed on sperm, endocervix, vaginal and urine specimen. Only one specimen (sperm) was positive for C. trachomatis. Three percent of the specimen were positive for U. urealyticum (from which 2,5% of the sperm specimen). No specimen was positive for M. hominis. Our results show that screening of C. trachomatis, M. hominis and U. urealyticum is not systematically required for among check up of infertile couples, given the prevalence of chlamydiosis among the population studied. However, it would be interesting to perform it on a targeted population, according to anamnestic or clinical criteria. In addition, an important modification of vaginal flora was observed in 12% of cases, and 2 vaginosis were diagnosed; the putative consequences of this disequilibrium has to be further investigated.     

Liquid-based urine cytology as a tool for detection of human papillomavirus, Mycoplasma spp., and Ureaplasma spp. in men

Liquid-based urine cytology (LB-URC) was evaluated for cytological diagnosis and detection of human papillomavirus (HPV), Mycoplasma, and Ureaplasma. Midstream urine samples were collected from 141 male patients with urethritis and 154 controls without urethritis, and sediment cells were preserved in liquid-based cytology solution. Urethral swabs from urethritis patients were tested for the presence of Neisseria gonorrhoeae and Chlamydia trachomatis. Papanicolaou tests were performed for cytological evaluation. HPV, Mycoplasma, and Ureaplasma genomes were determined by PCR-based methods, and localization of HPV DNA in urothelial cells was examined by in situ hybridization (ISH). The β-globin gene was positive in 97.9% of LB-URC samples from urethritis patients and in 97.4% of control samples, suggesting that high-quality cellular DNA was obtained from the LB-URC samples. HPV DNA was detected in 29 (21.0%) urethritis cases and in five (3.3%) controls (P < 0.05). HPV type 16 (HPV 16) was most commonly found in urethritis patients. Cytological evaluations could be performed for 92.1% of urethritis patients and 64.3% of controls. Morphological changes suggestive of HPV infection were seen in 20.7% of the HPV-positive samples, and ISH demonstrated the presence of HPV DNA in both squamous and urothelial cells in HPV-positive samples. Mycoplasma genitalium, Mycoplasma hominis, Ureaplasma parvum, and Ureaplasma urealyticum were detected in 14.5%, 10.9%, 6.5%, and 12.3% of urethritis patients, respectively. The prevalence rates of these microorganisms (except Ureaplasma parvum) were significantly higher in urethritis cases than controls (P < 0.05). LB-URC is applicable for detection of HPV, Mycoplasma, and Ureaplasma. HPV infection occurs in urothelial cells, especially in gonococcal urethritis.     

Detection of Mycoplasma hominis septicemia by radiometric blood culture.

The ease with which Mycoplasma hominis can be recovered and the frequency of its occurrence in septicemia may not be fully appreciated. We detected the growth of M. hominis radiometrically with an automated blood culture instrument. The organism grew in both aerobic and anaerobic culture media, but the cultures were not visibly positive. It was necessary to stain the cultures with acridine orange to visualize M. hominis and to subculture them on Columbia base sheep blood agar to confirm the positive growth index indicated by the instrument. Sodium polyanetholesulfonate inhibited the growth of M. hominis and is not recommended for use as the anticoagulant when blood is cultured for Mycoplasma spp.     

Rapid detection of Mycoplasma genitalium,Mycoplasma hominis,Ureaplasma parvum,and Ureaplasma urealyticum organisms in genitourinary samples by PCR-microtiter plate hybridization assay

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.     

Head-to-head multicenter comparison of DNA probe and nucleic acid amplification tests for Chlamydia trachomatis infection in women performed with an improved reference standard

Few evaluations of tests for Chlamydia trachomatis have compared nucleic acid amplification tests (NAATs) with diagnostic tests other than those by culture. In a five-city study of 3,551 women, we compared the results of commercial ligase chain reaction (LCR) and PCR tests performed on cervical swabs and urine with the results of PACE 2 tests performed on cervical swabs, using independent reference standards that included both cervical swabs and urethral swab-urine specimens. Using cervical culture as a standard, the sensitivities of PACE 2, LCR, and PCR tests with cervical specimens were 78.1, 96.9, and 89.9%, respectively, and the specificities were 99.3, 97.5, and 98.2%, respectively. Using either cervical swab or urine LCR-positive tests as the standard decreased sensitivities to 60.8% for PACE 2 and to 75.8 and 74.9% for PCR with cervical swabs and urine, respectively. Specificities increased to 99.7% for PACE 2 and to 99.7 and 99.4% for PCR with cervical swabs and urine, respectively. Sensitivities with a cervical swab-urine PCR standard were 61.9% for PACE 2 and 85.5 and 80.8% for LCR with cervical swabs and urine, respectively. Specificities were 99.6% for PACE 2 and 99.0 and 98.9% for LCR with cervical swabs and urine, respectively. Cervical swab versus urine differences were significant only for PCR specificities (P = 0.034). Overall, LCR sensitivity exceeded that of PCR, and sensitivities obtained with cervical swabs exceeded those obtained with urine specimens by small amounts. These data have substantiated, using a large multicenter sample and a patient standard, that LCR and PCR tests performed on endocervical swabs and urine are superior to PACE 2 tests for screening C. trachomatis infections in women. In our study, NAATs improved the detection of infected women by 17 to 38% compared to PACE 2.     

Rapid detection of vanA and vanB genes directly from clinical specimens and enrichment broths by real-time multiplex PCR assay

A real-time PCR assay previously developed for use on the Roche LightCycler platform was investigated as an alternative to culture for the direct detection of vancomycin-resistant enterococci (VRE) in clinical specimens. PCR primers and fluorescence resonance energy transfer hybridization probes specific for the vanA and vanB genes were combined in a multiplex real-time PCR assay performed directly with fecal material obtained by rectal swabbing and with enrichment broth samples. DNA was prepared from the rectal swabs and enrichment broths with a commercially available DNA preparation column designed specifically for use with fecal specimens. One hundred eighty duplicate rectal swabs were obtained from 42 patients who were previously found to be positive for VRE and who were being monitored for carriage of VRE. Direct and enrichment broth cultures were performed with one swab, while PCR was performed with the other swab as well as any corresponding presumptive positive enrichment broth. In total, 100 specimens from 30 patients remained positive for VRE by at least one method. The multiplex real-time PCR was positive for 88 enrichment broths of rectal swabs from 27 patients but for only 45 rectal swabs from 15 patients. Direct culture was positive for VRE for only 43 specimens from 11 patients, while enrichment broth culture was positive for VRE for 75 specimens from 22 patients. Inhibition studies for the multiplex real-time PCR assay, performed by spiking the DNA extracts from 50 negative rectal swabs and the corresponding enrichment broths with between 1 and 10 CFU of a VanB Enterococcus faecium strain, detected inhibition rates of 55.1 and 10%, respectively. PCR performed directly with enrichment broths was found to be significantly more sensitive than enrichment broth culture (P < 0.025). Negative samples were identified significantly earlier by PCR than by culture alone.     

实时荧光定量PCR检测泌尿生殖道患者尿液解脲脲原体

目的应用实时荧光核酸恒温扩增检测技术（SAT）检测泌尿生殖道患者尿液解脲脲原体（UU）,并评价其敏感性和特异性。方法对140例疑似泌尿生殖道感染患者的拭子和尿液样本,分别采用培养法、SAT进行解脲脲原体检测,检测结果有差异的标本用实时荧光定量PCR法对拭子进行复测,根据实验结果评估SAT检测尿液UU的敏感性和特异性。结果 140例疑似患者试子培养和尿液SAT检测阳性率均为60%,两者比较差异无统计学意义（P〉0.05）,其中16例培养结果与SAT检测结果不一致,8例培养法阳性、SAT阴性的拭子PCR结果复测阳性;8例培养法阴性、SAT阳性的拭子标本PCR结果 4例阴性、4例阳性,结合培养法结果和PCR结果作为＂扩大金标准＂,得出SAT对尿液检测的敏感性为95.5%、特异性为100%。结论 SAT检测泌尿生殖道患者尿液UU具有取样方便,检测快速、准确等优点,适用于临床实验室泌尿生殖道UU的检测。     

Multiplex PCR-ELISA for direct detection of MRSA in nasal swabs advantageous for rapid identification of non-MRSA carriers

In the study presented here 251 nasal swabs obtained from medical staff were directly investigated for MRSA using a commercial multiplex PCR system in parallel with conventional culture methods to determine the usefulness of PCR for rapid screening. Both methods identified 3.2% (8/251) of specimens as MRSA-positive; one sample was culture-positive only, and three were PCR-positive only. PCR correctly identified 215 of 239 (90%) negative swab samples, but one sample with weak cultural growth was not detected and was therefore considered false negative. The comparative sensitivity of culture versus PCR was 75% (9/12) versus 91.6% (11/12). Although PCR had a low positive predictive value (31.4%) its negative predictive value was high (99.5%). The results of this study indicate the multiplex PCR is suitable for the rapid identification of MRSA-negative individuals directly from nasal swabs in populations with a low MRSA prevalence, but positive results need to be confirmed by culture.     

Comparison of the Mycoplasma Duo test with PCR for detection of ureaplasma species in endotracheal aspirates from premature infants

We compared the Mycoplasma Duo kit (Sanofi Diagnostics Pasteur) with PCR for detection of Ureaplasma spp. in endotracheal aspirates from 60 premature neonates. The overall agreement between the two tests was 96%. The Mycoplasma Duo assay is a useful alternative to culture and PCR for detection of neonatal Ureaplasma infection.     

桑拿女工人型支原体宫颈感染率及其可能的临床意义

目的调查分析桑拿女工人型支原体宫颈感染率及其可能的临床意义。方法采用女性阴道分泌物革兰染色方法检测阴道毛滴虫、念珠菌和线索细胞,酶联免疫法检测沙眼衣原体,培养法鉴定淋球菌和支原体。结果1030例样本中。人型支原体阳性52例（5％）,其中5例为单独人型支原体定植,而47例（90．4％）为混合菌感染,以解脲脲原体为主（30／52,57．7％）。宫颈炎组与非宫颈炎组人型支原体的感染率差异无统计学意义（χ2=0．51,P〉0．05）。7例人型支原体阳性的宫颈炎病例同时混合感染沙眼衣原体或淋球菌,10例人型支原体阳性的非宫颈炎病例同时伴有念珠菌或阴道毛滴虫感染,或细菌性阴道病。结论无论女性是否患宫颈炎,人型支原体均可以定植于宫颈管,但并不是单独引起宫颈炎的病原体。     

Comparison of the effectiveness of polymerase chain reaction and enzyme immunoassay in detecting Chlamydia trachomatis in different female genitourinary specimens

BACKGROUND: In high-volume laboratories, enzyme immunoassay (EIA) is the most commonly used method of detecting Chlamydia trachomatis. The optimal specimen for detecting C trachomatis is a combined urethral and cervical swab. OBJECTIVE: To compare EIA with the combined urethral and cervical swab with polymerase chain reaction (PCR) on urine alone and urine mixed with cervical cells. PATIENTS AND METHODS: Phase 1 of the study included 752 sets of specimens used for comparison. In phase 2, another 212 samples of urine and urine plus cervical cells were added to the study for comparison of the 2 specimen types using PCR. RESULTS: In phase 1, 648 samples were negative and 76 were positive by all 3 methods and specimen combinations. Enzyme immunoassay was able to detect 81 positive samples (10.8%), whereas PCR on urine alone detected 97 positive samples (12.9%) and PCR on urine plus cervical cells detected 102 positive samples (13.6%), giving a sensitivity of 75%, 93.3%, and 98. 1% respectively. In phase 2, PCR on urine alone detected 119 positive samples (12.3%) and PCR on urine plus cervical cells detected 127 positive samples (13.1%), with a sensitivity of 92.2% and 98.5%, respectively. CONCLUSION: Polymerase chain reaction on urine alone or urine plus cervical cells is superior to EIA on combined cervical and urethral swabs. There is a slight advantage of adding cervical cells to the urine compared with the urine specimen alone when PCR is used as the assay for detection. The total inhibition rate in our female population is only 3.1% when PCR is used.     

The polymerase chain reaction for Mycoplasma gallisepticum detection

On the basis of the aligned 16S rRNA sequences of Mollicutes, a pair of primers was chosen for the detection of Mycoplasma gallisepticum. When used in the polymerase chain reaction (PCR), the primers detected a specific amplification of all Mg strains tested, yielding an expected 330 bp product. Amplification was not detected when other Mollicutes or E. coli were used as PCR templates. SPF chickens were experimentally inoculated with two strains of M. gallisepticum or Mycoplasma iowae. Tracheal swabs were collected 8, 15, 20 and 28 days after inoculation, and cultured for mycoplasma or tested by PCR. PCR products were detected by hybridization with a digoxigenin-labeled probe and by chemilumines-cence. The results showed that culture was positive for 49/73 swabs while PCR detected 70/72 positive samples. Thus, PCR can provide the basis of a sensitive, specific, rapid and non-radio-active method for detecting M. gallisepticum.     

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

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.     

High rates of genital mycoplasma infection in the highlands of Papua New Guinea determined both by culture and by a commercial detection kit.

Duplicate vaginal swabs were collected from 100 women, and comparisons were made between an in-house broth-agar culture system and a commercially available kit, the Mycoplasma IST kit (bioMérieux), for the detection of Mycoplasma hominis and Ureaplasma urealyticum. There was good agreement between the two systems for detection of the genital mycoplasmas in terms of sensitivity, with values of > 92% being obtained. In terms of specificity, the mutual comparisons were less favorable, though specificity values of > 72% were obtained. Statistically there was no significant difference in the performance of the two tests (P < 0.1 for both M. hominis and U. urealyticum). While the broth-agar culture system was considerably less expensive than the kit, the Mycoplasma IST kit provided additional information on antibiotic susceptibilities and had the advantages of a shelf life of up to 12 months and not requiring the preparation of culture media. The prevalences of colonization obtained for M. hominis and U. urealyticum were extremely high in this randomly selected group of women from periurban and rural settlements in the Eastern Highlands of Papua New Guinea, being > or = 70% for M. hominis and > or = 78% for U. urealyticum. colonization with both genital mycoplasmas simultaneously was also very common, with > or = 60% of women being colonized by both M. hominis and U. urealyticum.     

Polymerase chain reaction versus culture for detection ofUreaplasma urealyticum andMycoplasma hominis in the urogenital tract of adults and the respiratory tract of newborns

The efficiency of the polymerase chain reaction (PCR) was compared with that of culture for detection ofUreaplasma urealyticum andMycoplasma hominis in 726 clinical specimens comprising 189 gynecological samples, 362 urological samples, and 175 samples from newborn infants. The sensitivity of PCR versus culture was 95% for both organisms, while the sensitivity of culture versus PCR was 91% forUreaplasma urealyticum and 84% forMycoplasma hominis. Furthermore, PCR tests were faster than culture tests, allowing the time to diagnosis to be reduced from two to five days to 24 h.     

Diagnosis of Chlamydia trachomatis urethritis in men by polymerase chain reaction assay of first-catch urine.

To determine the accuracy of a recently developed polymerase chain reaction (PCR) urine assay to detect Chlamydia trachomatis urethral infection in men, we obtained urethral swabs and first-catch urine from 365 men attending a sexually transmitted diseases clinic. Thirty-three (9%) of the 365 men were infected with C. trachomatis as defined by urethral culture. Thirty-two of the 33 men with culture-positive urethral swabs also had PCR-positive urine assays. Of 332 patients with culture-negative urethral swabs, 325 had PCR-negative urine. Compared with chlamydia culture of urethral specimens, PCR assay of urine samples thus had a sensitivity of 97% and a specificity of 98%. The positive predictive value of the urine PCR assay was 82%, and the negative predictive value was 99%. Analysis of discrepant results indicated that six of seven PCR-positive, urethral culture-negative patients probably had chlamydial urethritis. All six patients had symptoms of urethritis and had either a positive urethral swab PCR or a positive urine PCR with a different amplification target. After resolution of discrepant results, (defining true positives as the 33 culture-positive patients and the 6 PCR-positive, culture-negative patients just described), the sensitivity and specificity of culture were 85% (33 of 39) and 100% (326 of 326), respectively. The revised sensitivity and specificity of PCR were 97% (38 of 39) and 99.7% (325 of 326), respectively. We conclude that this urine PCR assay provides a highly sensitive, noninvasive alternative method for the detection of C. trachomatis urethral infection in high-risk men attending a sexually transmitted diseases clinic. This assay could greatly facilitate the testing of larger numbers of male patients for chlamydial infection and should be studied in other settings.