scholarly journals The use of real-time PCR for evaluation of endometrial microbiota

2020 ◽  
pp. 14-20
Author(s):  
E.S. Voroshilina ◽  
D.L. Zornikov ◽  
O.V. Koposova ◽  
D.K. Islamidi ◽  
K.Yu. Ignatova ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Bacterial Load ◽  
Negative Control ◽  
Reproductive Age ◽  
Morphological Pattern ◽  
Chronic Endometritis ◽  
Healthy Women ◽  
Reproductive Age Women ◽  
Lactobacillus Spp

Chronic endometritis (CE) in women of the reproductive age is associated with infertility and recurrent pregnancy loss. The aim of this study was to evaluate the endometrial microbiota by means of real-time PCR in reproductive-age women depending on the morphological pattern of the endometrium. Using the Androflor real-time PCR kit, we analyzed endometrial aspirate collected from 23 patients with chronic endometritis, 30 patients with endometrial hyperplasia, and 19 healthy women. DNA of up to 9 groups of microorganisms was detected in all the analyzed samples in the amounts exceeding negative control. The total bacterial load (TBL) of the detected microorganisms was 10<sup>3</sup>–10<sup>6,4</sup> (median 10<sup>3,8</sup>) GE/ml. Lactobacillus spp. were detected the most often (86.1% of all samples). Opportunistic microorganisms (OM) were identified in 36.1% of all samples, including 22.2% of samples with lactobacilli and 13.9% — without lactobacilli. The variant of microbiota composition with Lactobacillus-dominance (more than 90%. in the TBL) was detected significantly less often in women with chronic endometritis compared to healthy women. Real-time PCR could be used for assessment of endometrial microbiota and allows us to determine its characteristics depending on the morphological pattern.

O-091 Semen microbiota in patients with astenozoospermia and healthy controls: cluster analysis of real-time PCR data

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
E Panacheva ◽  
D Pochernikov ◽  
E Voroshilina
Keyword(s):  
Cluster Analysis ◽  
Real Time ◽  
Real Time Pcr ◽  
Detection Rate ◽  
Bacterial Load ◽  
Rrna Gene ◽  
Rt Pcr ◽  
Silhouette Index ◽  
Enterococcus Spp ◽  
Lactobacillus Spp

Abstract Study question What are the differences in the semen microbiota composition of patients with asthenozoospermia and normospermia according to cluster analysis of PCR data? Summary answer The detection rate of 4 stable semen microbiota clusters and the dominant bacteria groups varied in patients with asthenozoospermia and normospermia. What is known already Most of the research dedicated to analyzing normal and pathological semen microbiota is based on 16S rRNA gene specific Next generation sequencing (NGS). It has shown that microbiota is represented by polymicrobial communities (clusters) that consist of microorganisms from different genera and bacteria phyla. Despite it being highly informative, NGS has several weaknesses: complex sample preparation, difficult sample intake control, long analysis process, complicated results interpretation, high cost of equipment and reagents. These factors make it virtually impossible to use this approach in routine medical practice. Quantitative real-time PCR (RT-PCR) is far more suitable for this. Study design, size, duration Patients included in the study (n = 301) came to the “Garmonia” Medical Center (Yekaterinburg, Russia) either seeking preconception care or for infertility treatment. Depending on the spermiogram results, they were divided into two groups. Group 1 (n = 171) — asthenozoospermia, Group 2 (n = 130) — normospermia. Participants/materials, setting, methods Semen microbiota was analyzed using RT-PCR kit Androflor (DNA-Technology, Russia). Cluster analysis was performed for 201 samples with the total bacterial load (TBL) of at least 103 GE/ml (asthenozoospermia = 96, normospermia = 105). Cluster analysis was conducted using the k-means ++ algorithm, scikit-learn. The Silhouette index and the Davies–Bouldin index (DBI) were used to confirm the stability of clusters. Main results and the role of chance Both in the samples with normospermia and asthenozoospermia, four stable microbiota clusters were distinguished. Cluster I was characterized by the prevalence of obligate anaerobes, Lactobacillus spp. were prevalent in Cluster II, Gram-positive facultative anaerobes were prevalent in Cluster III, Enterobacteriaceae/Enterococcus spp. were prevalent in Cluster IV. Cluster I was detected the most often in both groups. However, in normospermia it was represented by various obligate anaerobes without pronounced quantitative predominance of any bacteria group. In samples with asthenozoospermia one of the bacteria groups were prevalent in Cluster I: Bacteroides spp./Porphyromonas spp./Prevotella spp., Peptostreptococcus spp./Parvimonas spp. or Eubacterium spp. In samples with asthenozoospermia Cluster II was characterized by the prevalence of Lactobacillus spp., while in samples with normospermia other bacteria groups were present along with lactobacilli, mainly obligate anaerobes. In samples with normospermia Corynebacterium spp. and Streptococcus spp., typical of normal microbiota of male UGT, were prevalent in Cluster III. In samples with asthenozoospermia Cluster III were characterized by the prevalence of Staphylococcus spp. In samples with asthenozoospermia Lactobacillus spp was present in Cluster IV along with Enterobacteriaceae/Enterococcus spp., which was not typical of the samples with normospermia. Limitations, reasons for caution Cluster analysis was not conducted for the samples with TBL lower than 103 GE/ml, since their results were incompatible with the data received for the negative control samples. Wider implications of the findings Further research could determine the detection rate of the described bacterial clusters in semen with other pathologies. Establishing the relationship between the characteristics of semen microbiota and infertility in men might allow the development of new algorithms for treating patients with reproductive disorders, depending on the composition of semen microbiota. Trial registration number not applicable

Significance of Gardnerella vaginalis genotyping in diagnosis of recurrent bacterial vaginosis

2021 ◽  
Vol 1 (30) ◽  
pp. 48-52
Author(s):  
A. A. Krysanova ◽  
A. E. Gushchin ◽  
A. M. Savicheva
Keyword(s):  
Bacterial Vaginosis ◽  
Real Time ◽  
Real Time Pcr ◽  
Test System ◽  
Reproductive Age ◽  
Gardnerella Vaginalis ◽  
First Episode ◽  
Genotype 4 ◽  
Dna Concentration ◽  
Healthy Women

Objective. To assess the importance of identifying different genotypes of Gardnerella vaginalis in the diagnosis of recurrent bacterial vaginosis.Materials and methods. The study involved 299 women of reproductive age. All patients were divided into three groups (healthy women, women with the first episode of bacterial vaginosis, and women with recurrent bacterial vaginosis). DNA of Gardnerella vaginalis in vaginal discharge was detected by real-time PCR. The detection of four genotypes of G. vaginalis was performed using real-time multiplex PCR. To quantify the amplified PCR fragments, quantitative standard samples were constructed. Statistical analysis of the results was carried out using the statistical package NCSS 11 (NCSS, LCC).Results. In 38.2 % of healthy women, any one genotype of G. vaginalis was identified in the vaginal biotope, most often it was genotype 4 (35.2 %), while the concentration of G. vaginalis DNA was low (102–103 geqs/ml). When several genotypes of gardnerella were detected simultaneously in healthy women, the DNA concentration did not exceed 104 geqs/ml. A completely different picture was observed among women with bacterial vaginosis (BV). In the first episode of BV, genotype 4 of G. vaginalis prevailed, both as a single genotype and in combination with 1 or 2, or 3 genotypes. In the recurrent course of BV, only 3–4 genotypes of G. vaginalis were detected at once, and in 78 % of cases it had place is a combination of 1, 2 and 4 genotypes, and the DNA concentration was 107–108 geqs/ml.Conclusion. To diagnose recurrent forms of BV, it is necessary to develop and introduce into practice laboratory diagnostics a test system for detecting different genotypes of G. vaginalis by real-time PCR.

scholarly journals Semen microbiota: cluster analysis of real-time PCR data

2020 ◽  
Author(s):  
ES Voroshilina ◽  
DL Zornikov ◽  
AV Ivanov ◽  
DG Pochernikov ◽  
EA Panacheva
Keyword(s):  
Cluster Analysis ◽  
Species Diversity ◽  
Real Time ◽  
Real Time Pcr ◽  
Detection Rate ◽  
Reproductive Age ◽  
Threshold Values ◽  
Microbial Dna ◽  
Cluster 2 ◽  
Lactobacillus Spp

To this day semen microbiota is still poorly understood, and clinical significance of detecting specific microorganism groups has not been clearly determined. The aim of this work was to conduct cluster analysis of semen microbiota detected using real-time PCR. 634 semen samples of reproductive age men were analyzed using the Androflor kit. Microbial DNA in the quantity of no less than 103 GE/ml was detected in 460 samples (72.5%). From 1 to 14 microorganism groups were detected in 350 samples (55.2%) in the quantities that exceeded the threshold values (the detection rate of specific groups: 3.3–21.0%). In these 350 samples 4 stable microbiota clusters were determined. Each of the clusters was characterized by the prevalence of a specific microorganism group: obligate anaerobes (cluster 1; n = 172; detection rate — 49.1%), Lactobacillus spp. (cluster 2; n = 78; detection rate — 22.3%), gram-positive facultative anaerobes (cluster 3; n = 62; detection rate — 17.7%), Enterobacteriaceae / Enterococcoccus (cluster 4; n = 62; detection rate — 10.9%). Cluster 1 was less stable and was characterized by the larger species diversity compared to other clusters.

scholarly journals Validation of a Novel Diagnostic Approach Combining the VersaTREK™ System for Recovery and Real-Time PCR for the Identification of Mycobacterium chimaera in Water Samples

2021 ◽  
Vol 9 (5) ◽  
pp. 1031
Author(s):  
Roberto Zoccola ◽  
Alessia Di Blasio ◽  
Tiziana Bossotto ◽  
Angela Pontei ◽  
Maria Angelillo ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Detection System ◽  
Bacterial Load ◽  
Microbial Control ◽  
Hospital Setting ◽  
Sample Volume ◽  
Analytical Sensitivity ◽  
Initial Water

Mycobacterium chimaera is an emerging pathogen associated with endocarditis and vasculitis following cardiac surgery. Although it can take up to 6–8 weeks to culture on selective solid media, culture-based detection remains the gold standard for diagnosis, so more rapid methods are urgently needed. For the present study, we processed environmental M. chimaera infected simulates at volumes defined in international guidelines. Each preparation underwent real-time PCR; inoculates were placed in a VersaTREK™ automated microbial detection system and onto selective Middlebrook 7H11 agar plates. The validation tests showed that real-time PCR detected DNA up to a concentration of 10 ng/µL. A comparison of the isolation tests showed that the PCR method detected DNA in a dilution of ×102 CFU/mL in the bacterial suspensions, whereas the limit of detection in the VersaTREK™ was <10 CFU/mL. Within less than 3 days, the VersaTREK™ detected an initial bacterial load of 100 CFU. The detection limit did not seem to be influenced by NaOH decontamination or the initial water sample volume; analytical sensitivity was 1.5 × 102 CFU/mL; positivity was determined in under 15 days. VersaTREK™ can expedite mycobacterial growth in a culture. When combined with PCR, it can increase the overall recovery of mycobacteria in environmental samples, making it potentially applicable for microbial control in the hospital setting and also in environments with low levels of contamination by viable mycobacteria.

scholarly journals The Relation of Diabetes Type 2 with Sexual Function among Reproductive Age Women in Iran, a Case-Control Study

2017 ◽  
Vol 2017 ◽  
pp. 1-5 ◽  
Author(s):  
Poorandokht Afshari ◽  
Shiva Yazdizadeh ◽  
Parvin Abedi ◽  
Homayra Rashidi
Keyword(s):  
Type 2 Diabetes ◽  
Sexual Function ◽  
Case Control Study ◽  
Case Control ◽  
Reproductive Age ◽  
Diabetic Patients ◽  
Healthy Women ◽  
Reproductive Age Women ◽  
Control Study

Background.Diabetic patients are at the greater risk of retinopathy, nephropathy, neuropathy, and sexual dysfunction compared to the general population.Objective.The aim of this study was to evaluate the sexual dysfunction in type 2 diabetes reproductive age women in Iran.Method.This was a case-control study carried out on 130 women with type 2 diabetes and 130 healthy women. The type 2 diabetes diagnosis was confirmed with abnormal fasting blood sugar, abnormal random blood sugar test, and abnormal level of HbA1C. Eligible women were requested to complete a demographic questionnaire and female sexual function index (FSFI). The chi-square test, independentt-test, and Multivariate Analysis of Covariance (MANCOVA) were used for analyzing data.Results.Results of this study showed that diabetic women had significantly lower sexual desire, arousal, lubrication, and orgasm and more pain compared to the healthy women (p<0.05). Also diabetic women had lower sexual satisfaction compared to the healthy women (p=0.002). The total score of sexual function was significantly lower in the diabetic women compared to the healthy women (21.25±7.04versus22.43±7.6,p=0.004).Conclusion.Results of this study showed that the score of all dimensions of sexual function in diabetic patients was lower than that in healthy women. Education and counseling about controlling diabetes and sexual function among diabetic women in reproductive age are recommended.

scholarly journals Effects of sample handling on the detection of porcine reproductive and respiratory syndrome virus in oral fluids by reverse-transcription real-time PCR

2018 ◽  
Vol 30 (6) ◽  
pp. 807-812 ◽  
Author(s):  
Ashley C. Weiser ◽  
Korakrit Poonsuk ◽  
Sarah A. Bade ◽  
Phillip C. Gauger ◽  
Marisa Rotolo ◽  
...  
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Real Time Pcr ◽  
False Negative ◽  
Rna Extraction ◽  
Free Water ◽  
Negative Control ◽  
Respiratory Syndrome Virus ◽  
Sample Handling ◽  
Oral Fluids

We evaluated effects of handling procedures on detection of porcine reproductive and respiratory syndrome virus (PRRSV) in oral fluids (OFs) by reverse-transcription real-time PCR (RT-rtPCR). The experiments were conducted using a composite sample of PRRSV-positive OF collected from 5-wk-old pigs vaccinated 15 d earlier with a modified-live PRRSV vaccine. Five pre-extraction sample-handling steps and all combinations thereof were evaluated: 1) thaw temperature (4°C or 25°C); 2) sample diluent (1:1 dilution with nuclease-free water or guanidinium thiocyanate–phenol); 3a) sonication of the sample (yes or no); 3b) temperature (4°C or 25°C) at which step 3a was conducted; and 4) temperature at which the sample was maintained after step 3b and until RNA extraction was initiated (4°C or 25°C). All combinations of the 5 sample-handling steps (i.e., 32 unique treatments) were tested in a completely randomized factorial design with 4 replicates and 1 negative control for each treatment. The entire experiment was repeated on 5 separate days to produce a total of 800 PRRSV RT-rtPCR results. Binary (positive or negative) data were analyzed by logistic regression and results (Ct) were analyzed using a generalized linear model. Overall, 1 false-positive result was observed among 160 negative controls (99.4% specificity), and 85 false-negative results were observed among the 640 known-positive samples (86.7% sensitivity). The most significant factor affecting test outcome was thaw temperature (4°C or 25°C); samples thawed at 4°C had higher positivity rate (94% vs. 80%, p < 0.0001) and lower Ct (36.2 vs. 37.5, p < 0.0001).

scholarly journals Detection and quantification of Campylobacter in foods: New analytic approaches to detect and quantify Campylobacter spp. in food samples

2020 ◽  
Vol 9 (2) ◽  
Author(s):  
Maria Francesca Peruzy ◽  
Yolande Thérèse Rose Proroga ◽  
Federico Capuano ◽  
Federica Corrado ◽  
Serena Santonicola ◽  
...  
Keyword(s):  
Real Time ◽  
Campylobacter Jejuni ◽  
Internal Control ◽  
Real Time Pcr ◽  
Quantitative Methods ◽  
Bacterial Load ◽  
Digital Pcr ◽  
Food Samples ◽  
Meat Samples ◽  
Campylobacter Spp

The aim of the present study was to develop rapid qualitative and quantitative methods based on the use of Real-Time PCR and Droplet Digital PCR (ddPCR), in order to have reliable techniques to detect and quantify Campylobacter spp. in food samples. The gene 16S-rRNA was used as specific target for Campylobacter spp. Real- Time PCR evaluation assay and a not competitive internal control was ushered in it. To investigate the selectivity of the method, 26 Campylobacter strains and 40 non-Campylobacter strains were tested and in order to verify the application of Real-Time PCR method, 5 pork meat samples were experimentally inoculated with a Campylobacter jejuni strain. Subsequently, dilutions with a bacterial load of Campylobacter jejuni within 10-106 CFU/mL were chosen for the optimization of the ddPCR assay. Lastly, a total of 54 naturally contaminated foods samples were analyzed through molecular (Real-Time PCR and ddPCR) and traditional methods. The Real-Time PCR protocol demonstrated to amplify only the Campylobacter spp. strains and when Campylobacter jejuni was experimentally inoculated in meat samples the pathogen was always detected. The ddPCRs assay allowed to quantify a level of contamination of 10 CFU/mL, but it was unable to quantify levels of 105 – 106 CFU/mL. Lastly, Campylobacter spp. was never detected in the 54 samples tested. In conclusion, the novel analytic approach proposed, based on an initial screening of the samples with Real-Time PCR and then on quantification of Campylobacter spp. with a ddPCR on those positive, represents a quick monitoring tool and, if used correctly, it would allow the implementation of food safety.

Chronic endometritis with normal and thin endometrium during treatment of reproductive-age women: ultrasound and histological correlations

2017 ◽  
Vol 23 (1) ◽  
pp. 29
Author(s):  
I. O. Marinkin ◽  
N. V. Trunchenko ◽  
Yu. V. Seryapina ◽  
Е. V. Nikitenko ◽  
K. Yu. Makarov ◽  
...  
Keyword(s):  
Reproductive Age ◽  
Thin Endometrium ◽  
Chronic Endometritis ◽  
Reproductive Age Women

scholarly journals The skin microbiota in pregnant women suffering from atopic dermatitis

2016 ◽  
Vol 97 (2) ◽  
pp. 222-229
Author(s):  
A Yu Lonshakova-Medvedeva ◽  
K N Monakhov ◽  
A N Suvorov ◽  
O V Lavrova
Keyword(s):  
Staphylococcus Aureus ◽  
Atopic Dermatitis ◽  
Pregnant Women ◽  
Bacterial Load ◽  
Life Quality ◽  
Reproductive Age ◽  
Control Group ◽  
Skin Microbiota ◽  
Healthy Women ◽  
Basic Care

Aim. To study the skin microbiota of pregnant women suffering from atopic dermatitis.Methods. 53 women of reproductive age suffering from atopic dermatitis (28 pregnant and 25 non-pregnant) were examined. The control group included dermatologically healthy women (25 pregnant and 25 non-pregnant). Prior to treatment initiation and on 15-day of study pathological process spread, the SCORAD index (scoring of atopic dermatitis - atopic dermatitis severity assessment), dermatology life quality index determination were conducted. In addition, microbiological study of material taken from the forehead, elbow bend skin and visually unaltered forearm skin was performed.Results. In women (pregnant and non-pregnant), suffering from atopic dermatitis skin total bacterial load is increased. In all groups, the skin microbiota is presented mainly by staphylococci: in dermatologically healthy people - coagulase-negative, in atopic dermatitis - Staphylococcusя aureus. In atopic dermatitis Staphylococcus aureus is isolated from both lesions and visually unaltered skin. In pregnant women with atopic dermatitis skin bacterial load was higher, Staphylococcus aureus was found more commonly. The skin microbiota in dermatologically healthy women was more diverse in respect of species comparing with patients with atopic dermatitis. Basic care remedies use leads to clinical improvement and a decrease in the skin total bacterial load and Staphylococcus aureus load. Daily use of emollients has no effect on saprophytic microorganisms.Conclusion. In pregnant patients with atopic dermatitis higher skin total bacterial load and higher rate of skin colonization by Staphylococcus aureus are observed.

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