Cervical Cytology of Samples with Ureaplasma urealyticum, Ureaplasma parvum, Chlamydia trachomatis, Trichomonas vaginalis, Mycoplasma hominis, and Neisseria gonorrhoeae Detected by Multiplex PCR.

INTRODUCTION: Despite increasing application of molecular diagnostic methods for the detection of sexually transmitted infections, the cytological findings in pap smears of patients with pathogens that can be identified only by PCR are not yet well described. The aim of this study was to describe the most common cytological features in cervical pap smears of patients with Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, Trichomonas vaginalis, Mycoplasma hominis, Ureaplasma urealyticum, and Ureaplasma parvum detected by multiplex PCR. METHODS: Cervical samples for conventional and liquid-based cytology and for multiplex PCR were collected from women ranging from 23 to 54 years old, who underwent routine screening at a gynecological Unit. RESULTS: Multiplex PCR was positive in 36.2% of the samples: Ureaplasma parvum 14.9%, Chlamydia trachomatis 10.6%, Trichomonas vaginalis 10.6%, Mycoplasma hominis 8.5%, Ureaplasma urealyticum 4.2%, Neisseria gonorrhoeae 2.1%, and Mycoplasma genitalium (0). Multiple pathogens were observed in 12.8% of samples. Microscopic cervicitis (≥10 polymorphonuclear leukocytes/epithelial cell) and normal (predominantly lactobacillary) microbiota were the most frequent findings in the samples in which the pathogens were detected alone or in multiple infections, except for samples with Trichomonas vaginalis in which the coccobacillary microbiota was the most common. In samples with microscopic cervicitis and normal microbiota, those with at least one pathogen identified by multiplex PCR were significantly more frequent than those with no pathogen, 66.6% versus 33.3%. CONCLUSION: Failure to identify an inflammatory agent in pap smear with intense neutrophil exudate may suggest the presence of Ureaplasma parvum, Ureaplasma urealyticum, Chlamydia trachomatis, or Trichomonas vaginalis. A remark on the intensity of inflammation should be made in the reports of cervical pap smears so that this cytological finding can be correlated with clinical and PCR results.

Co-infection of sexually transmitted pathogens and Human Papillomavirus in cervical samples of women of Brazil.

BACKGROUND: Some sexually transmitted infectious agents, such as Chlamydia trachomatis and Herpes simplex, cause local inflammation, and could contribute to Human Papillomavirus (HPV) and cervical lesion progression. Thus, the aim of this study was to determine any association between the presence of microorganisms of gynecological importance, sexual behavior, clinical and demographical variables to the development and progress of cervical lesions. METHODS: One hundred and thirty-two women between 14 and 78 years and living at Vitória da Conquista, Bahia, Brazil, were included (62 individuals with cervical lesions and 70 without lesions). They answered a questionnaire to provide data for a socioeconomic and sexual activity profile. Samples of cervical swabs were collected and analyzed by PCR to detect genital microorganisms and HPV. Quantitative PCR was used to detect and quantify Ureaplasma urealyticum and Ureaplasma parvum. Univariate and multiple logistic regression were performed to measure the association with the cervical lesions, and an odds ratio (OR) with 95% confidence intervals (95%CI) were calculated. The Mann-Whitney U test was also used to compare the microorganism load in the case and control groups. The significance level was 5% in all hypotheses tested. RESULTS: Cervical lesions were associated with: women in a stable sexual relationship (OR = 14.21, 95%CI = 3.67-55.018), positive PCR for HPV (OR = 16.81, 95%CI = 4.19-67.42), Trichomonas vaginalis (OR = 8.566, 95%CI = 2.04-35.94) and Gardnerella vaginalis (OR = 6.13, 95%CI = 1.53-24.61), adjusted by age and qPCR for U. parvum. U. parvum load showed a statistical difference between the case and control groups (p-value = 0.002). CONCLUSION: Variables such as stable relationship, HPV, T. vaginalis, G. vaginalis were associated with cervical lesions in epidemiological studies. U. parvum load was higher in woman with cervical lesions compared with women without lesions. Additional studies are needed to better understand the role of these factors in cervical lesion development.

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.

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.

Vaginal microbiota of healthy pregnant Mexican women is constituted by four Lactobacillus species and several vaginosis-associated bacteria.

OBJECTIVE: To identify the microbiota communities in the vaginal tracts of healthy Mexican women across the pregnancy. METHODS: Vaginal swabs were obtained during the prenatal visit of women from all trimesters (n = 64) of healthy pregnant women of Mexico City. DNA was isolated from each sample, and PCR-DGGE and sequencing of 16S rRNA gene fragments were used to identify the bacterial communities. RESULTS: 21 different microorganisms were identified in the vaginal samples. Lactobacillus genus was present in 98% of women studied. Four lactobacilli species were identified in vaginal samples. L. acidophilus was the predominant (78%) followed by L. iners (54%), L. gasseri (20%), and L. delbrueckii (6%). 17 different microorganisms related to bacterial vaginosis conditions were identified. Ureaplasma urealyticum was the predominant (21%) followed by BVAB1 (17%) and Gemella bergeriae (7.8%). CONCLUSIONS: Lactobacillus genus predominates in the vaginal samples of Mexican pregnant women associated with different microorganisms related to bacterial vaginosis conditions.

Ureaplasma urealyticum and Mycoplasma hominis sensitivity to bacteriocins produced by two Lactobacilli strains.

The purpose of the present study was to determine the inhibitory activities of two bacteriocins, produced by lactobacilli, against genital mycoplasmas. In this study, infections produced by genital mycoplasmas were studied; of these, 1.3% were caused by Mycoplasma hominis, 10.7% by Ureaplasma urealyticum and 5.6% by U. urealyticum + M. hominis. U. urealyticum was isolated from 75 out of 123 patients with genital mycoplasmas, while M. hominis was isolated from 9 patients (7.3%) and both U. urealyticum and M. hominis from 39 patients (31.7%). Bacteriocins, L23 and L60, produced by Lactobacillus fermentum and L. rhamnosus, respectively, appear to be two novel inhibitors of bacterial infection with potential antibacterial activity. Both bacteriocins proved to be active against 100% of strains tested; MICs of bacteriocin L23 ranged between 320 and 160 UA ml(-1) for 78% of the M. hominis strains and between 320 and 80 UA ml(-1) for 95% of the U. urealyticum strains. In addition, bacteriocin L60 was still active at 160 UA ml(-1) for a high percentage (56%) of M. hominis strains, and at 80 UA ml(-1) for 53% of the U. urealyticum strains. Interestingly, these antimicrobial substances produced by lactobacilli showed an inhibitory activity against genital mycoplasmas even when diluted. Altogether, our study indicates that the bacteriocins, L23 and L60, are good candidates for the treatment or prevention of genital infections in women.

[Development of a multiple PCR method for the identification of Ureaplasma parvum and Ureaplasma urealyticum]. / Desarrollo de un método de PCR-múltiple para la identificación de Ureaplasma parvum y Ureaplasma urealyticum.

Ureaplasma parvum and Ureaplasma urealyticum, also known as biovar parvum and biovar T960, respectively, could be associated with several disorders in men, women, and mainly, in newborn children with under weight. Several methods have been developed in order to identify the species or biovars of ureaplasmas. We developed a Multiplex-PCR method using the UPS-UPSA and UUS2-UUA2 primers, specific for U. parvum and U. urealyticum, respectively. This Multiplex-PCR method was used to identify cultures of clinical positive samples to Ureaplasma spp. by the "MYCOFAST Evolution-2" Kit. Of 56 positive cultures to Ureaplasma spp. from newborn children, 70% were U. parvum and 30% U. urealyticum; in 76 positive samples in women, 83% corresponded to U. parvum and 17% to U. urealyticum, while in 63 positive samples of men, 76% identified U. parvum and 24% U. urealyticum. The PCR-multiplex method showed specificity for the identification of the biovars or species of ureaplasmas of clinical interest.

[Detection using molecular biology techniques of Mycoplasma hominis and Ureaplasma urealyticum in urogenital samples]. / Detección por técnicas de biología molecular de Mycoplasma hominis y Ureaplasma urealyticum en muestras urogenitales.

Mycoplasma hominis and Ureaplasma urealyticum are species closely related to urogenital diseases such as pyelonephritis, nongonococcal urethritis, urinary calculi, epididymitis, pelvic inflammation, infertility, abortions and post-delivery fever. They can also cause pneumonia and meningitis in newborn infants. In this paper we used nucleic acid hybridization and polymerase chain reaction to analyze 22 samples from patients with different urogenital symptoms in order to detect mycoplasmas and ureaplasmas. We obtained 10 positive samples and 12 were negative. From positive samples we identified two with Mycoplasma hominis, two with Ureaplasma and six with both species. The results obtained by these molecular techniques were compared with reference methods and we found coincident results in 18 samples, while in four the results were discordant. These discordant findings were not statistically significant.

Detection of mycoplasmas in urethral swabs from HIV-1 infected patients and control individuals using culture techniques and polymerase chain reaction.

The objective of the present study was to determine the prevalence of certain mycoplasma species, i.e., Mycoplasma hominis, Ureaplasma urealyticum and Mycoplasma penetrans, in urethral swabs from HIV-1 infected patients compared to swabs from a control group. Mycoplasmas were detected by routine culture techniques and by the Polymerase Chain Reaction (PCR) technique, using 16SrRNA generic primers of conserved region and Mycoplasma penetrans specific primers. The positivity rates obtained with the two methods were comparable. Nevertheless, PCR was more sensitive, while the culture techniques allowed the quantification of the isolates. The results showed no significant difference (p < 0.05) in positivity rates between the methods used for mycoplasma detection.

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.