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

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 Real-Time PCR for the Detection and Quantification of Geodermatophilaceae from Stone Samples and Identification of New Members of the Genus Blastococcus

2006 ◽  
Vol 72 (1) ◽  
pp. 346-352 ◽  
Author(s):  
Oscar Salazar ◽  
Aranzazu Valverde ◽  
Olga Genilloud
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Rrna Gene ◽  
Rt Pcr ◽  
New Members ◽  
The Family ◽  
Specific Amplification ◽  
Pcr Technology ◽  
Type Strains ◽  
Detection And Quantification

ABSTRACT Real-time PCR (RT-PCR) technology was used for the specific detection and quantification of members of the family Geodermatophilaceae in stone samples. Differences in the nucleotide sequences of the 16S rRNA gene region were used to design a pair of family-specific primers that were used to detect and quantify by RT-PCR DNA from members of this family in stone samples from different geographical origins in Spain. These primers were applied later to identify by PCR-specific amplification new members of the family Geodermatophilaceae isolated from the same stone samples. The diversity and taxonomic position of the wild-type strains identified from ribosomal sequence analysis suggest the presence of a new lineage within the genus Blastococcus.

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.

scholarly journals Gallbladder microbiota in patients with gallstone disease

2020 ◽  
Vol 12 (1) ◽  
pp. 37-44
Author(s):  
Vladimir I. Simanenkov ◽  
Alexandr N. Suvorov ◽  
Sergey V. Tikhonov ◽  
Elena I. Ermolenko ◽  
Viktoria D. Dekkanova ◽  
...  
Keyword(s):  
Laparoscopic Cholecystectomy ◽  
Real Time ◽  
Real Time Pcr ◽  
Gallstone Disease ◽  
Taxonomic Structure ◽  
E Coli ◽  
Cultural Method ◽  
Enterococcus Spp ◽  
Genes Encoding ◽  
Lactobacillus Spp

The article presents the results of investigating 20 patients with cholelithiasis. The bile and a piece of the gallbladder were taken to analyze microbiota during a scheduled laparoscopic cholecystectomy. The study of microbiota was carried out with the cultural method and real-time PCR. Bifidobacterium spp., Bacteroides spp., Lactobacillus spp., E. coli prevailed in the taxonomic structure of isolated bacteria. Isolated Enterococcus spp. had a lot of genes encoding various factors of pathogenicity.

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 Real-Time PCR Assay for Detection and Enumeration of Dekkera bruxellensis in Wine

2003 ◽  
Vol 69 (12) ◽  
pp. 7430-7434 ◽  
Author(s):  
Trevor G. Phister ◽  
David A. Mills
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Pcr Primers ◽  
Rrna Gene ◽  
Dekkera Bruxellensis ◽  
Pcr Assay ◽  
Specific Pcr ◽  
Spoilage Yeast ◽  
Pcr Method ◽  
26S Rrna

ABSTRACT Traditional methods to detect the spoilage yeast Dekkera bruxellensis from wine involve lengthy enrichments. To overcome this difficulty, we developed a quantitative real-time PCR method to directly detect and enumerate D. bruxellensis in wine. Specific PCR primers to D. bruxellensis were designed to the 26S rRNA gene, and nontarget yeast and bacteria common to the winery environment were not amplified. The assay was linear over a range of cell concentrations (6 log units) and could detect as little as 1 cell per ml in wine. The addition of large amounts of nontarget yeasts did not impact the efficiency of the assay. This method will be helpful to identify possible routes of D. bruxellensis infection in winery environments. Moreover, the time involved in performing the assay (3 h) should enable winemakers to more quickly make wine processing decisions in order to reduce the threat of spoilage by D. bruxellensis.

Complement C3 Genotyping of Slow and Fast Variants by Real Time PCR-High Resolution Melting

2012 ◽  
Vol 10 (3) ◽  
pp. 329-334 ◽  
Author(s):  
D.M. Valero-Hervás ◽  
P. Morales ◽  
M.J. Castro ◽  
P. Varela ◽  
M. Castillo-Rama ◽  
...  
Keyword(s):  
High Resolution ◽  
Real Time ◽  
Real Time Pcr ◽  
High Resolution Melting ◽  
Low Cost ◽  
Complement C3 ◽  
Amplification Refractory Mutation System ◽  
Rt Pcr ◽  
Dna Amount ◽  
Mutation System

“Slow” and “Fast” C3 complement variants (C3S and C3F) result from a g.304C>G polymorphism that changes arginine to glycine at position 102. C3 variants are associated with complement-mediated diseases and outcome in transplantation. In this work C3 genotyping is achieved by a Real Time PCR - High Resolution Melting (RT-PCR-HRM) optimized method. In an analysis of 49 subjects, 10.2% were C3FF, 36.7% were C3SF and 53.1% were C3SS. Allelic frequencies (70% for C3S and 30% for C3F) were in Hardy-Weinberg equilibrium and similar to those published previously. When comparing RT-PCR-HRM with the currently used Tetraprimer-Amplification Refractory Mutation System PCR (T-ARMS-PCR), coincidence was 93.8%. The procedure shown here includes a single primer pair and low DNA amount per reaction. Detection of C3 variants by RT-PCR-HRM is accurate, easy, fast and low cost, and it may be the method of choice for C3 genotyping.

scholarly journals Increased Expression of the Pro-Protein Convertase Furin Predicts Decreased Survival in Ovarian Cancer

2007 ◽  
Vol 29 (4) ◽  
pp. 289-299
Author(s):  
Robert E. Page ◽  
Andrés J. P. Klein-Szanto ◽  
Samuel Litwin ◽  
Emmanuelle Nicolas ◽  
Raid Al-Jumaily ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Tumor Progression ◽  
Real Time ◽  
Cell Lines ◽  
Cancer Cell ◽  
Real Time Pcr ◽  
Ovarian Tumor ◽  
Cancer Cell Lines ◽  
Rt Pcr ◽  
Primary Tumors

Background: Proprotein convertases (PCs) are serine proteases that after restricted proteolysis activate many proteins that play a crucial role in cancer such as metalloproteinases, growth factors and growth factor receptors, adhesion molecules, and angiogenic factors. Although the expression of several PCs is increased in many tumors, their expression in primary ovarian tumors has not been studied in detail. We sought to determine if there was an association between the expression of the ubiquitously expressed PCs, furin, PACE-4, PC-5 and PC-7, and ovarian tumor progression. Methods: We assessed their expression by RT-PCR, Real-time PCR, Western blot, and immunohistochemistry using cells derived from normal human ovarian surface epithelium (HOSE) and cancer cell lines as well as ovarian epithelial cancer specimens (45 RT-PCR/Real-time PCR, and 120 archival specimens for Immunohistochemistry). Results: We found that furin expression was restricted to the cancer cell lines. In contrast, PACE-4 and PC-7 showed expression only in normal HOSE cells lines. Furthermore, furin was predominantly expressed in primary tumors from patients who survived for less than five years. The other PCs are either expressed in the group of survivors (PC-7 and PACE4) or expressed in low amounts (PC-5). Conclusions: Our studies point to a clear relationship between furin and ovarian cancer. In addition, these results show that furin exhibits the closest association with ovarian cancer among the ubiquitously expressed PCs, arguing against the redundancy of these proteases. In summary, furin may constitute a marker for ovarian tumor progression and could contribute to predict the outcome of this disease.

scholarly journals Evaluation of the performance of the COVID-19 rapid antigen tests in a tertiary level hospital in Bangladesh.

2021 ◽  
Author(s):  
Mohammad Jahidur Rahman Khan ◽  
Md. Shahadat Hossain ◽  
Samshad Jahan Shumu ◽  
Md. Selim Reza ◽  
Farzana Mim ◽  
...  
Keyword(s):  
Viral Load ◽  
Real Time ◽  
Sensitivity And Specificity ◽  
Detection Rate ◽  
High Sensitivity ◽  
Rt Pcr ◽  
College Hospital ◽  
Medical College ◽  
Qrt Pcr ◽  
Antigen Tests

Abstract Background: While the COVID-19 pandemic is a worldwide crisis, tests with high sensitivity and specificity are essential for identifying and managing COVID-19 patients. Globally, several rapid antigen tests RATs for COVID-19 have been developed, but their clinical efficacy has not been well established. This study aimed to evaluate the performance of several rapid antigen tests (RATs) to diagnose SARS-CoV-2 infection.Methods: This prospective observational study was conducted at Shaheed Suhrawardy Medical College hospital from February 2021 to April 2021 in Dhaka, Bangladesh. This study included the patients admitted in this hospital at the COVID-19 isolation unit or referred from the triage facility of the outdoor department of this hospital suspected as COVID-19 case. Two nasopharyngeal samples were collected simultaneously. one sample was used on the spot for the RAT. The other was sent to the adjacent Shaheed Suhrawardy Medical College COVID-19 RT-PCR laboratory for real-time reverse transcription-polymerase chain reaction (qRT-PCR). The performance of the RAT was evaluated using the results of qRT-PCR as a reference.Results: A total of 223 patients were included in this study, and the real-time RT-PCR detected SARS-CoV-2 in 84 (37.7%) patients. Of these 84 patients, 9 (10.7%) were asymptomatic. The overall sensitivity and specificity of RATs were 78.6% and 99.3%, respectively. The sensitivity was 81.3% in symptomatic cases and 55.6% in asymptomatic cases. False-negatives were observed in 18 patients, 3 of whom were asymptomatic and had a low viral load (cycle threshold (Ct) > 30). The detection rate of RATs was 100% when the Ct value was up to 24. The detection rate was 42.3% when the Ct was >29. The detection rate of RATs was 92.3% when the onset of symptoms was within three days. The detection rate was 33.3% when the onset of symptoms was >7 days.Conclusions: RATs for COVID-19 used in this study delivered an acceptable performance in patients with high viral load and within the first week of the onset of symptoms. They can be used as a supplementary method to RT-PCR for the diagnosis of COVID-19 patients.

scholarly journals Μελέτη του μεταβολισμού του σιδήρου στον σκελετικό μυ

2012 ◽  
Author(s):  
Αικατερίνη Πολονύφη
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
T Test ◽  
Rt Pcr ◽  
Densitometric Analysis ◽  
Paired T Test

Ο μεταβολισμός του σιδήρου δεν είναι επαρκώς μελετημένος στον σκελετικό ιστό σε αντίθεση με τον ηπατικό. Σκοπός της μελέτης είναι η σύγκριση της έκφρασης των γονιδίων του μεταβολισμού του σιδήρου στον ανθρώπινο σκελετικό και ηπατικό ιστό. Συλλέχτηκαν βιοψίες μυϊκού και ηπατικού ιστού από έξι φυσιολογικά άτομα. Επιλέχθηκαν να μελετηθούν12 γονίδια που εμπλέκονται στο μεταβολισμό του σιδήρου για την εισαγωγή σιδήρου στο κύτταρο, αποθήκευση και εξαγωγή του καθώς και δύο μόρια ρύθμισης της ομοιοστασίας του σιδήρου: εισαγωγή σιδήρου [οι υποδοχείς της τρανσφερρίνης (TfR1 και TfR2), το μόριο HFE, ο μεταφορέας δισθενών μετάλλων (DMT1,DMT1nonIRE), και το σιδεροφόρο μόριο λιποκαλίνη (NGAL)], αποθήκευση σιδήρου [η βαριά αλυσίδα της φερριτίνης (FTH1)] και εξαγωγή σιδήρου [η φερροπορτίνη (IREG1), η ηφαιστίνη (HEPH) και η σερουλοπλασμίνη (CP)] καθώς και δύο μόρια ρύθμισης της ομοιοστασίας του σιδήρου [η εψιδίνη (HAMP) και η αιμοτζουβελίνη (HJV)]. Ακολούθησαν αλυσιδωτές αντιδράσεις της πολυμεράσης, RT-PCR και ημιποσοτικοποίηση των επιπέδων έκφρασης των γονιδίων με τη μέθοδο της πυκνομετρίας (Densitometric Analysis). Τα αποτελέσματα εκφράζονται με βάση το ποσοστό επί τοις εκατό του γονιδίου της β-ακτίνης. Το γονίδιο της β-ακτίνης χρησιμοποιήθηκε για την κανονικοποίηση των επιπέδων έκφρασης, ως γονίδιο αναφοράς της μελέτης. Αναδεικνυόμενες διαφορές συγκριτικής έκφρασης μεγαλύτερες του 20%, των μελετημένων γονιδίων του μεταβολισμού του σιδήρου με ημιποσοτικοποίηση, αναλύθηκαν περαιτέρω με την μέθοδο της PCR σε αληθινό χρόνο (Real time PCR, qPCR) και ποσοτικοποιήθηκαν (LightCycler, Roche). Η στατιστική ανάλυση των αποτελεσμάτων ποσοτικοποίησης έγινε με το one paired t test και τα αποτελέσματα είναι στατιστικά σημαντικά (p<0,05). H συγκριτική μελέτη μεταξύ ανθρώπινου ηπατικού και σκελετικού ιστού στα επιλεγμένα 12 γονίδια έδειξε ότι: 1. Περισσότερα απο τα γονίδια: HJV, TFR1, HFE, DMT1, DMT1nonIRE, NGAL, HEPH, IREG1 ,DMT1(IRE) , DMT1nonIRE, FTH1 εκφράζονται και στους δύο ιστούς με ποσοστό έκφρασης >70% των επιπέδων έκφρασης της βακτίνης. 2. Εξαίρεση αποτελούν τα HAMP, CP και TfR2 που απουσιάζουν ή παρουσιάζουν ελάχιστη έκφραση (<10% των επιπέδων έκφρασης της βακτίνης) στο σκελετικό μυ αντίστοιχα. 3. Ενώ τα HJV και HEPH παρουσιάζουν μεγαλύτερη έκφραση των επιπέδων mRNA στο σκελετικό μύ συγκριτικά με το ήπαρ (SM/L=2,65±1,1(p<0,05) και SM/L=1,5±0,06(p<0,05 αντίστοιχα στην Q-PCR). (Εικόνα 1). Η εργασία αυτή αφορά φυσιολογικές καταστάσεις ανθρώπινων ιστών, δίνει όμως σημαντικά ερεθίσματα για αντίστοιχες μελέτες του μεταβολισμού του σιδήρου σε παθολογικές καταστάσεις. Υπογραμμίζει δε την σπουδαιότητα του σκελετικού μυϊκού ιστού και την ανάλογη συμμετοχή του στη ομοιοστασία του σιδήρου. Οι ποσοτικές διαφορές που παρατηρούνται στην έκφραση γονιδίων που εμπλέκονται σε διάφορα κυτταρικά μονοπάτια αναδεικνύουν την ανάγκη περαιτέρω έρευνας του κυτταρικού μεταβολισμού στο σκελετικό ιστό.

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