Comparison of two methods for detection of Chlamydia trachomatis and Ureaplasma urealyticum in male reproductive tract / 北京大学学报(医学版)

OBJECTIVE@#To investigate the value of clinical application of simultaneous amplification and testing of RNA (SAT-RNA) for detecting Chlamydia trachomatis (CT) and Ureaplasma urealyticum (UU) by comparing with the polymerase chain reaction testing of DNA (PCR-DNA) method.@*METHODS@#Specimens from both urethra swab and the first avoid urine which should be at least one hour after the previous urination were collected from 163 men who were scheduled for in vitro fertilization and embryo transfer (IVF-ET) treatment due to female factors at Center for Reproductive Medicine, Shengjing Hospital of China Medical University during the period of April 2016 to April 2017. Among the 163 men, 109 simultaneously provided semen that was collected after 3-7 days of sexual abstinence for the testing. Urine and semen specimens were detected for CT and UU with SAT-RNA, while urethra swab specimens were detected for CT and UU with standard PCR-DNA. Detection results of the SAT-RNA were compared with those of the PCR-DNA method.@*RESULTS@#The positive rate of UU in the urethra swab detected with PCR-DNA and that of UU in the urine with SAT-RNA were 47.24% and 47.85%, respectively, and the coincidence rate was 93.25%. In addition, the positive and negative coincidence rates were 93.51% and 93.02%, respectively, and the concordance between the two methods was very good (Kappa=0.865). On the other hand, the positive rate of CT in the swab specimen tested with PCR-DNA was 3.07% and that of CT in urine with SAT-RNA was 4.29%, and the coincidence rate was 97.55%. Moreover, the positive and negative coincidence rates were 80.00% and 98.10%, respectively, and the concordance between the two methods was good (Kappa=0.654). Regarding SAT-RNA detection of UU in the urine and semen specimen of the 109 patients, the positive rates of UU in the urine and semen specimens were 50.46% and 44.95%, respectively; and the coincidence rate between the two specimens was 88.99%. In addition, the positive coincidence rate and the negative coincidence rate was 93.88% and 85.00%, respectively, and the concordance between the two specimens was good (Kappa=0.780). Similarly, SAT-RNA detection of CT in the urine and semen specimens showed the positive rate was 5.50% and 3.67%, respectively; and the two specimens showed 98.17% coincidence rate. The positive and negative coincidence rates were 100.00% and 98.10%, respectively, and the concordance was also good (Kappa=0.791).@*CONCLUSION@#SAT-RNA detection of CT and UU in the urine specimen showed good concordance with the PCR-DNA detection of CT and UU in the urethra swab specimen. In addition, the concordance was also good between the urine and semen specimens detected with SAT-RNA. These results indicate that, as a less invasive and equally accurate procedure, SAT-RNA may be more suitable for clinical application.

Influence of storage time on DNA of Chlamydia trachomatis, Ureaplasma urealyticum, and Neisseria gonorrhoeae for accurate detection by quantitative real-time polymerase chain reaction

The shipment and storage conditions of clinical samples pose a major challenge to the detection accuracy of Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), and Ureaplasma urealyticum (UU) when using quantitative real-time polymerase chain reaction (qRT-PCR). The aim of the present study was to explore the influence of storage time at 4°C on the DNA of these pathogens and its effect on their detection by qRT-PCR. CT, NG, and UU positive genital swabs from 70 patients were collected, and DNA of all samples were extracted and divided into eight aliquots. One aliquot was immediately analyzed with qRT-PCR to assess the initial pathogen load, whereas the remaining samples were stored at 4°C and analyzed after 1, 2, 3, 7, 14, 21, and 28 days. No significant differences in CT, NG, and UU DNA loads were observed between baseline (day 0) and the subsequent time points (days 1, 2, 3, 7, 14, 21, and 28) in any of the 70 samples. Although a slight increase in DNA levels was observed at day 28 compared to day 0, paired sample t-test results revealed no significant differences between the mean DNA levels at different time points following storage at 4°C (all P>0.05). Overall, the CT, UU, and NG DNA loads from all genital swab samples were stable at 4°C over a 28-day period.

Infertilidad tubárica e infección genital por Chlamydia trachomatis-Ureaplasma urealyticum / Genital infection and tubal infertility by Chlamydia tra-chomatis or Ureaplasma urealyticum

Objetivo: establecer la asociación entre la infertilidad tubarica y la infección cervical por Chlamydia trachomatis (CT) o Ureaplasma urealiticum (UU), en mujeres infértiles. Métodos: investigación comparativa y aplicada, con diseño de tipo no experimental, de casos y controles, contemporáneo transeccional y de campo, que incluyó 60 mujeres, separadas en dos grupos pareados de acuerdo si eran infértiles (casos) o fértiles (controles), a las cuales se les tomó una muestra de hisopado endocervical para el diagnóstico molecular de CT o UU y se les realizó una histerosalpingografía para evaluar la permeabilidad de las trompas uterinas. Resultados: se detectó una prevalencia en mujeres infértiles y fértiles de infección por CT o UU del 18 por ciento y 35 por ciento, respectivamente; siendo mayor entre las mujeres infértiles, diferencia significativa solo para UU (p<0,05). Se detectó una mayor permeabilidad tubárica en las pacientes fértiles que en las infértiles (80 por ciento vs. 40 por ciento), siendo el compromiso tubárico mayor en las pacientes infértiles (p<0,05). Al asociar el diagnóstico de CT o UU con los resultados de la histerosalpingografía se constató que la detección de uno de estos microorganismos aumentaba casi 3 o 5 veces más la probabilidad de presentar obstrucción tubárica, respectivamente, diferencias no significativas (p>0,05). Conclusión: una gran parte de las mujeres infértiles presentan infección por CT o UU, patógenos de transmisión sexual que pudiesen tener responsabilidad en el daño tubárico.

Objective: to establish the association between tubal infertility and cervical infection by Chlamydia tra-chomatis (CT) or Ureaplasma urealyticum (UU) in infertile women. Methods: a comparative, and applied research with a non-experimental, case-control, contemporary-transactional and field design, including 60 women, separated into two groups matched according whether they were infertile (cases) or fertile (controls), in which was took a sample of endocervical swabs for molecular diagnosis of cT or UU and underwent hysterosalpingography to assess the permeability of the fallopian tubes. Results: it was detected in infertile and fertile women a prevalence of CT or UU infection of 18 percent and 35 percent, respectively; being higher detection among infertile women, although this difference was significant only for UU (p <0.05). Also detected more tubal permeability in fertile patients that in infertile (80 percent vs. 40 percent), being higher in engagement tubal in infertility patients (p<0.05). By associating the diagnosis of both CT and UU with hysterosalpingography'sresults found that the diagnosis of one of these microorganisms increased almost 3 to 5 times more likely to have obstruction of the fallopian tubes, respectively; although this higher risk doesn't showed significance (p>0.05). Conclusion: a large proportion of infertile women have CT or UU infection, sexually transmitted pathogens that might have tubal damage liability.

Investigation of Ureaplasma urealyticum biovars and their relationship with antimicrobial resistance.

Purpose: To develop Taqman fluorescence quantitative polymerase chain reaction (PCR) method for investigating the characteristics of the distributions of Ureaplasma urealyticum (UU) biovars and to explore the relationship between UU biovars and antimicrobial resistance. Materials and Methods: By the method of culture, Ureaplasma species were detected. Taqman fluorescence quantitative PCR for detecting UU biovars were developed and UU clinical isolates were detected to distinguish biovars. The broth micro-dilution susceptibility testing methods were used to determine UU susceptibility. Results: By Taqman PCR method, UU biovars was successfully detected. Of 126 samples, biovar 1 was found in 73 (57.94%). There was a statistical difference between genital-urinary tract infection group and asymptomatic group (P<0.05). In the region, UU biovar 1 to 9 kinds of agents kept higher susceptibility rates, but biovar 2 maintained higher susceptibility rates only to tetracyclines. Compared with biovar 1, UU biovar 2 resistance rates to 7 kinds of agents were higher (P<0.05). Conclusions: (1) Our new established Taqman PCR method is a useful tool for screening UU biovars. (2) UU biovar 1 predominated in asymptomatic population; whereas in genital-urinary tract infection population UU biovar 2 had a higher proportion. (3) The characteristics of drug resistance were different between UU biovars. Overall, both two biovars remained higher susceptibility rates to tetracyclines. A majority of biovor 1 strains were sensitive to macrolides and quinolones; while only a small number of biovar 2 strains kept sensitive to roxithromycin and quinolones, a large proportion of biovar 2 strains were found in intermediate ranges.

Simultaneous and rapid differential diagnosis of Mycoplasma genitalium and Ureaplasma urealyticum based on a polymerase chain reaction-restriction fragment length polymorphism.

Objectives: The aim of this investigation was to simultaneously detect and differentiate Mycoplasma genitalium and Ureaplasma urealyticum in female patients suffering from genital complications by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP). Materials and Methods : Genital swabs were taken from 210 patients. They were transported to the laboratory in phosphate-buffered saline. For PCR, samples were analysed with genus-specific MyUu-R and MyUu-F primers. This primer set, which was originally designed in our laboratory, amplified a 465 bp fragment (M. genitalium) and a 559 bp fragment (U. urealyticum). Samples containing a band of the expected sizes for the Mycoplasma strains were subjected to digestion with a restriction endonuclease enzyme of TaqI and Cac8I. Results: Of the 210 samples, a total of 100 (47.6%) samples were found to be positive for Mycoplasmas (seven M. genitalium isolates, 3.3%; and 89 U. urealyticum isolates, 42.4%), and coinfections with both species were detected in four samples (1.9%). The PCR-RFLP results showed that M. genitalium and U. urealyticum are different by enzyme patterns. Conclusion: PCR-RFLP offers a rapid and easily applicable protocol to simultaneous detection and differentiation of M. genitalium and U. urealyticum from clinical samples when specific primers and restriction enzymes are used.

Occurrence of Ureaplasma parvum and Ureaplasma urealyticum in Women with Cervical Dysplasia in Katowice, Poland

The aim of this study was to evaluate the occurrence of genital mycoplasmas, especially Ureaplasma parvum and Ureaplasma urealyticum, in women with atypical squamous cells of undetermined significance (ASCUS), low grade squamous intraepithelial lesions (LSIL) and high grade squamous intraepithelial lesions (HSIL), compared to women with normal cytology living in Katowice, Poland. Two sterile swabs were used to obtain material from the posterior vaginal fornix of 143 women with squamous intraepithelial lesions and 39 healthy women: first for general bacteriology, second for detection of urogenital mycoplasmas using Mycoplasma IST2 kit. From each positive Mycoplasma IST2 culture DNA was isolated and PCR was performed for identification of U. parvum and U. urealyticum. Mycoplasma IST was positive in 34.1% cases. Urogenital mycoplasmas were demonstrated in women with HSIL significantly more often compared to women with LSIL, ASCUS, and with normal cytology. DNA of U. parvum was demonstrated in majority of Mycoplasma IST2-positive cases, U. urealyticum DNA-only in 9 (4.9%). Predominance of 3/14 serovars of U. parvum was demonstrated. U. urealyticum biovar 2 was present more often in women with squamous intraepithelial lesions.

Clastogenic effects of different Ureaplasma urealyticum serovars on human chromosomes

The possibility that Ureaplasma urealyticum might play an important role in human infertility was first raised more than 20 years ago, but this association remains speculative. Considering the hypothesis that the pathogenicity of Ureaplasma urealyticum may depend on its serotypes, the clastogenic effcts of different strains of Ureaplasma urealyticum, at concentrations of 10(3) CCU (color changing units)/ml, 10(4) CCU/ml and 10(5) CCU/ml, were evaluated in vitro in short-term cultures of human lyphocytes. Total or partial mitotic inhibition was produced by Ureaplasma urealyticum serotypes 2,3 and 10 independent of the concentration (10(3) CCU/ml, 10(4) CCU/ml or 10 (5) CCU/ml) of the microorganisms employed. In contrast, the clastogenic effects observed with serotypes 1,7 and 12 varied according to the concentration employed in the test. Mitotic alterations were observed in Ureaplasma urealyticum serotypes 5,6,7,8,9,11 and 12. Chromatid gaps (53.0 percent) and chromatid breaks (13.9 percent) were the most frequent types of alterations observed. The results of this in vitro assay demonstrated that the clastogenic effects varied with the Ureaplasma urealyticum serotypes evaluated.