Do NAAT-Based Methods Increase the Diagnostic Sensitivity of Streptococcus agalactiae Carriage Detection in Pregnant Women?

The aim of the study was to evaluate particular polymerase chain reaction primers targeting selected representative genes and the influence of a preincubation step in a selective broth on the sensitivity of group B Streptococcus (GBS) detection by nucleic acid amplification techniques (NAAT). Research samples were vaginal and rectal swabs collected in duplicate from 97 pregnant women. They were used for enrichment broth culture-based diagnostics, bacterial DNA isolation, and amplification, using primers based on species-specific 16S rRNA, atr and cfb genes. To assess the sensitivity of GBS detection, additional isolation of samples preincubated in Todd-Hewitt broth with colistin and nalidixic acid was performed and then subjected to amplification again. The introduction of the preincubation step increased the sensitivity of GBS detection by about 33-63%. Moreover, NAAT made it possible to identify GBS DNA in an additional six samples that were negative in culture. The highest number of true positive results compared to the culture was obtained with the atr gene primers, as compared to cfb and 16S rRNA primers. Isolation of bacterial DNA after preincubation in enrichment broth significantly increases the sensitivity of NAAT-based methods applied for the detection of GBS from vaginal and rectal swabs. In the case of the cfb gene, the use of an additional gene to ensure the appropriate results should be considered.

Effects of exposure to air pollution on acute cardiovascular and respiratory admissions to the hospital and early mortality at emergency department.

BACKGROUND: Particulate matter (PM) and NO2 induce pathophysiological changes which contribute to an increased incidence of acute cardiovascular (CV) and respiratory (Rp) events. OBJECTIVES: To analyze the relationship between air quality and the frequency of admissions to the emergency department (ED) due to the CV diseases and Rp causes. MATERIAL AND METHODS: The study analyzed the reasons for admissions to the ED during the cold periods from January 2017 to January 2020. These data were combined with the average daily concentrations of NO2, PM2.5 and PM10, and the individual air quality indexes (IAQIs) for these pollutants. RESULTS: Our analyses have shown that 3468 (11.4%) and 1053 (3.46%) of all 30,419 analyzed patients were admitted to the ED for CV and Rp reasons, respectively. Cardiovascular patients were significantly more often admitted to the ED when the IAQI for NO2 was worse than very good, and the IAQI for PM2.5 or PM10 was worse than good. In such periods, diagnoses such as ischemic heart disease (IHD) or syncope were statistically more common and the risk of admission of a patient with a diagnosis such as IHD, heart failure (HF), syncope, stroke, or transient ischemic attack (TIA) was increased. Registered deaths occurred significantly more often among patients admitted on days with moderate or worse than moderate air quality determined in relation to PM10 in comparison to days with very good or good air quality (0.35% and 0.23%, respectively, p = 0.04). CONCLUSIONS: Air quality significantly affects the admissions to the ED for CV and Rp reasons and has an impact on mortality.

Updating the recommendations of the Working Group for the Preservation of Fertility in Oncological and Hematological Patients and Other Patients Treating Gonadier Therapies "ONCOFERTILITY" (GROF) of the Polish Society of Oncological Gynecology regarding cryopreserves and autologous transplant.

Update of the recommendations of the Fertility Preservation Working Group in Oncological, Hematological and Other Patients Treated with Gonadotoxic Therapies "ONCOFERTILITY" (GROF) of the Polish Society of Oncological Gynecology regarding cryopreservation and autologous ovarian tissue transplantation. The Fertility Preservation Working Group in Oncological, Hematological and Other Patients Treated with Gonadotoxic Therapies "ONCOFERTILITY" (GROF) of the Polish Society of Oncological Gynecology has developed current clinical guidelines and recommendations to improve the quality of healthcare provision in the area of reproductive health in patients undergoing therapy that may impair their reproductive potential. The guidelines are based on current scientific evidence available at the time of writing this document. In the absence of scientific evidence on some aspects, a consensus was reached among GROF stakeholders. The purpose of the guidelines is to assist healthcare professionals in making decisions in specific clinical situations regarding the selection of an appropriate and effective diagnostic and therapeutic process. The document provides practical guidelines for the management of cryopreservation and autologous ovarian tissue transplantation.

Next-Generation Sequencing as a Tool to Detect Vaginal Microbiota Disturbances during Pregnancy.

The physiological microbiota of the vagina is responsible for providing a protective barrier, but Some factors can disturb the balance in its composition. At that time, the amounts of the genus Lactobacillus decrease, which may lead to the development of infection and severe complications during pregnancy. The aim of the study was the analysis of the bacterial composition of the vagina in 32 Caucasian women at each trimester of pregnancy using the next-generation sequencing method and primers targeting V3-V4 regions. In the studied group, the dominant species were Lactobacillus iners, Lactobacillus gasseri, and Lactobacillusplantarum. Statistically significant differences in the quantitative composition between trimesters were observed in relation to Lactobacillus jensenii,Streptococcus agalactiae, Lactobacillus iners, Gardnerella spp. Out of the 32 patients, 20 demonstrated fluctuations within the genus Lactobacillus, and 9 of them, at different stages of pregnancy, exhibited the presence of potentially pathogenic microbiota, among others: Streptococcus agalactiae, Gardnerella spp., Atopobium vaginae, and Enterococcus faecalis. The composition of the vaginal microbiota during pregnancy was subject to partial changes over trimesters. Although in one-third of the studied patients, both the qualitative and quantitative composition of microbiota was relatively constant, in the remaining patients, physiological and potentially pathogenic fluctuations were distinguished.

The dynamics of vaginal and rectal Lactobacillus spp. flora in subsequent trimesters of pregnancy in healthy Polish women, assessed using the Sanger sequencing method.

BACKGROUND: Lactobacilli play an important role in maintaining vaginal health and protection against bacterial infections in the genital tract. The aim of this study is to show the dynamics of changes of the vaginal and rectal Lactobacillus flora during pregnancy by using the Sanger sequencing method. METHOD: The study included 31 healthy pregnant women without clinical signs of genitourinary infections. The material was taken in the three trimesters of pregnancy by vaginal and rectal swabs and grown on the MRS agar quantitatively to estimate the number of Lactobacillus spp. [CFU/ml]. Afterwards, 3 to 8 morphologically different lactobacilli colonies were taken for identification. Bacterial species identification was performed by 16 s rDNA sequence fragment analyses using the Sanger method. RESULTS: Among the patients tested, the most common species colonizing the vagina in the first trimester were: L. crispatus 29%, L. gasseri 19.4% and L. rhamnosus 16.1%, in the second trimester: L. crispatus 51.6%, L. gasseri 25.8%, L. rhamnosus 19.4% and L. amylovorus 16.1%, and in the third trimester the most common Lactobacillus species were: L. crispatus 25.8%, L. gasseri 25.8% and L. johnsonii 19.4%. In rectal species, the number decreased in the second and third trimesters in comparison to the first trimester (p = 0.003). An analysis of rectal dynamics showed that in the first trimester, the most common species were: L. johnsonii 19.4%, and L. plantarum 9.7%, in the second trimester: L. crispatus 9.7% and L. mucosae 6.5%, and in the third trimester: L. casei 9.7% and L. rhamnosus 9.7%. Individual dynamics of the Lactobacillus species composition showed variability, characterized by continuous, intermittent, or periodic colonization. The patients examined were mostly colonized by three Lactobacillus species in vagina (32.3%), whereas for the rectum, one Lactobacillus species during the whole pregnancy duration was common (32.3%). CONCLUSION: This study showed that in the examined group of healthy, pregnant Polish women, the vaginal Lactobacillus flora, both qualitative and quantitative, was stable during the three subsequent trimesters. In contrast, the number of rectal Lactobacillus species dramatically decreased after the first trimester.

A study of the effects of therapeutic doses of ionizing radiation in vitro on Lactobacillus isolates originating from the vagina - a pilot study.

BACKGROUND: Ionizing radiation is used as a therapeutic option in the treatment of certain neoplastic lesions located, among others, in the pelvic region. The therapeutic doses of radiation employed often result in adverse effects manifesting themselves primarily in the form of genital tract infections in patients or diarrhea. The data available in the literature indicate disorders in the microbial ecosystem caused by ionizing radiation, which leads to the problems mentioned above. In the present study, we examined the influence of ionizing radiation on 52 selected strains of bacteria: Lactobacillus crispatus, L. fermentum, L. plantarum, L. reuteri, L. acidophilus L. amylovorus, L. casei, L. helveticus, L. paracasei, L. rhamnosus, L. salivarius and L. gasseri. This collection of Lactobacillus bacteria isolates of various species, obtained from the genital tract and gastrointestinal tract of healthy women, was tested for resistance to therapeutic doses of ionizing radiation. RESULTS: The species studied, were isolated from the genital tract (n = 30) and from the anus (n = 22) of healthy pregnant women. Three doses of 3 Gy (fractionated dose) and 50 Gy (total dose of the whole radiotherapy cycle) were applied. The greatest differences in survival of the tested strains in comparison to the control group (not subjected to radiation) were observed at the dose of 50 Gy. However, the results were not statistically significant. Survival decrease to zero was not demonstrated for any of the tested strains. CONCLUSIONS: Therapeutic doses of radiation do not affect the Lactobacillus bacteria significantly.

Dynamics of colonization with group B streptococci in relation to normal flora in women during subsequent trimesters of pregnancy.

The main objective of the study was to compare the qualitative and quantitative composition of vaginal and rectal flora in GBS-positive (n=15) and GBS-negative (n=27) pregnant women examined in three subsequent trimesters of their pregnancy. Study samples consisted of vaginal and rectal smears and urine samples. GBS numbers were determined by the quantitatively cultured method [cfu/ml] and with the use of qPCR. Five GBS colonies were isolated per each positive sample and genotyped by PFGE and serotyping. The normal flora components: Lactobacillus, Bifidobacterium and Candida were quantitatively cultured. Carriage of GBS in subsequent trimesters in vagina/anus was variable and fluctuated between 17% and 28%. Quantitative GBS analyses showed that the vaginal population was at a constant level with the mean value equal to 3.94×104 cfu/ml, in contrast to the rectal population where the highest values appeared in the third trimester 4.37×105. The use of qPCR gave 7% more positive results for vaginal/rectal swabs. Genetic similarity analysis showed that one GBS clone was present in 73% of carriers during pregnancy, while in 27% of patients, 2 clones were found. H2O2-positive vaginal lactobacilli were detected in all women, while H2O2-negative lactobacilli and Bifidobacterium occurred more frequently in the anus in about 50% of women. Candida was present in the vagina in 30% of women. The analysis of women in three consecutive trimesters of pregnancy on the basis of a study group and control group showed no statistically significant differences in either the species (qualitative) or quantitative composition in vaginal and rectal flora in both of the groups. Therefore, GBS should be considered as a component of the microbiota and an opportunistic microorganism rather than a typical pathogen, because it does not distort the composition of women's normal genital tract flora.

Adult stem cells therapy for urine incontinence in women.

The past few years brought high development in obtaining and culturing autologous adult stem cells. In this paper we review publications of experimental investigations and clinical trials of the muscle-derived cells and the application in the treatment of stress urinary incontinence among women. Mesenchymal stem cells (MSCs) can be obtained from bone marrow but it is associated with a painful biopsy procedure. Collection of muscle-derived stem cells (MDSCs) is less harmful because the skeletal muscle biopsy is performed with a small caliber needle in local anesthesia. The stem-based therapy could be the next step in the treatment of urinary incontinence. There are still many elements of therapy such as effectiveness or long-term side effects which need to be researched.

Genetic characterization and diversity of Streptococcus agalactiae isolates with macrolide resistance.

Macrolide resistance in 169 Streptococcus agalactiae [group B streptococcus (GBS)] isolates originating from pregnant carriers was investigated. Using multiplex PCR the presence of genes encoding erythromycin resistance and capsular polysaccharides, as well as surface proteins, was determined. Random amplification of polymorphic DNA (RAPD) and PFGE were used to characterize specific clones among the isolates. In the examined population of women, erythromycin-resistant strains were found in 4.5 % of patients, whereas clindamycin-resistant strains were found in 3 % of patients, which was 16 % of strains resistant to erythromycin and 10 % of strains resistant to clindamycin among GBS isolates, respectively. Among the isolates, the largest percentage was represented by the constitutive macrolide-lincosamide-streptogramin B (cMLS(B)) phenotype (63 %), then the inductive macrolide-lincosamide-streptogramin B (iMLS(B)) phenotype (26 %) and the macrolide resistance (M) phenotype (11 %). The ermB gene was indicated in all isolates with the cMLS(B) phenotype and V serotype, whereas mefA/mefE genes were found in isolates with the M phenotype and Ia serotype. Among resistance isolates, serotype V was predominant (67 %), followed by serotypes II (15 %), Ia (11 %) and III (7 %). The most common surface protein encoding genes were alp3 (70 %), then rib (11 %), epsilon (7.5 %), bca (7.5 %) and alp2 (4 %). A statistically significant relationship between macrolide resistance, serotype V and the alp3 gene was demonstrated. PFGE, in comparison to the RAPD method, gave better genetic discrimination of GBS isolates. A relatively high genetic diversity among investigated strains was shown. In addition, the largest genetic homogeneity was found in serotype V.

[Analysis of serotypes distribution of group B streptococci origin from pregnant carriage using multiplex PCR]. / Rozklad serotypów paciorkowców grupy B, izolowanych z przypadków nosicielstwa, oznaczonych metoda multipleks PCR.

The aim of study was to evaluate the applicability of the PCR multiplex method for the identification of Streptococcus agalactiae (group B streptococci; GBS) serotypes and to indicate the participation of serotypes in the group of pregnant women. 1178 pregnant women at 35-37 weeks of pregnancy were studied between 2007-2009. Vaginal and rectal swabs collected from these women were tested for the presence of GBS. S. agalactiae identification was done basing on the culture method according to the CDC recommendations. For all isolated GBS strains the surface polysaccharides which conditioning serotypes were determined using commercially available serological latex kit and multiplex PCR method with two sets of specific primers. The frequency of GBS carriage among 1176 pregnant women was 30% (353 cases). Simultaneous colonization of the vagina and anus was demonstrated in 80% women, whereas GBS carriage only in the vagina or only in the anus was indicated in 9% and 11% cases, respectively. In pregnant women colonized by GBS serotype III (35%) dominated. The other serotypes were detected with the following frequency: Ia (20%), V (17%), II (15%), Ib (8%) and IV (5%). In the analyzed women population, GBS strains belonging to the VI, VII, VIII serotypes were not found. The results of our study show a great diagnostic usefulness of multiplex PCR method for identification of genes encoding GBS surface polysaccharides that are typical for individual serotypes.

Metallothionein expression and infiltration of cytotoxic lymphocytes in uterine and tubal implantation sites.

INTRODUCTION: In spite of increasing number of immune cells in the endometrium during the decidualization, the integrity of endometrial tissue in each menstrual cycle is maintained by adaptive changes in expression of several factors that regulate activity of immune cells and concomitant hormonal alterations during the menstrual cycle. This regulatory function of endometrium is also related to resistance to apoptosis, in which metallothionein (MT) may play a role. MATERIALS AND METHODS: Study group included 26 women with spontaneous abortion and 18 patients with tubal ectopic pregnancy. Control group included 17 women whose endometrial tissue samples were taken during the normal secretory cycle phase. Expression of metallothionein (MT), CD56 and CD69 were assessed in tissue samples by immunohistochemistry. RESULTS: The number of CD56-positive cells was significantly higher in women with ruptured than unruptured ectopics. MT expression was higher in tubal mucosa distant from the implantation site in ruptured compared to unruptured ectopics. It was found also to be significantly lower than in decidua taken from women with spontaneous abortion. CD69 expression was similar in women with spontaneous abortion as well as patients with ruptured ectopics compared to the control group. On the other hand, CD69 expression in unruptured ectopics was significantly lower than in women with spontaneous abortion and the control group. CONCLUSION: The concentration of immune cells and increase of their activity in tubal mucosa, with insufficient protection against immune-mediated apoptosis assessed by MT expression, might result in tubal rupture during ectopic pregnancy.