UNLABELLED: The aim of this study was to identify parameters influencing DNA extraction and PCR amplification efficiencies in an attempt to standardize Mucorales qPCR. The Fungal PCR Initiative Mucorales Laboratory Working Group distributed two panels of simulated samples to 26 laboratories: Panel A (six sera spiked with Mucorales DNA and one negative control serum) and Panel B (six Mucorales DNA extracts). Panel A underwent DNA extraction in each laboratory according to the local procedure and were sent to a central laboratory for testing using three different qPCR techniques: one in-house qPCR assay and two commercial assays (MucorGenius and Fungiplex). Panel B DNA extracts were PCR amplified in each laboratory using local procedures: nine in-house qPCR assays and two commercial kits (MucorGenius and MycoGENIE). All data were compiled and anonymously analyzed at the central laboratory. For Panel A, a total of six different automated platforms and five manual extraction methods were used. Positive rates were 64%, 70%, and 89%, for the MucorGenius, Fungiplex, and the in-house qPCR assay, respectively. Using a large volume of serum for DNA extraction provided the highest analytical sensitivity (82.5% for 1 mL compared with 62.7% for smaller volumes, P < 0.01). For Panel B, five in-house qPCR assays and two commercial kits had >78% positivity. Using larger PCR input volumes (≥7 μL) was associated with the highest sensitivity at 95.5% compared to 58.3% when lower input volumes were used (P < 0.01). Using larger sample volumes for nucleic acid extraction and DNA template volumes for PCR amplification significantly improves the performance of Mucorales qPCR when testing serum. IMPORTANCE: Mucormycosis is a life-threatening mold infection affecting immunosuppressed patients but also other patients with diabetes or trauma. Better survival is linked to shorter delays in diagnosis and treatment initiation. Detection of Mucorales-free DNA in serum or plasma using quantitative PCR allows a prompt diagnosis and earlier treatment. Several techniques and protocols of quantitative Mucorales PCR are used in Europe, and improving performance remains a common objective of laboratories participating in the fungal PCR Initiative Working Group. This study, which combined results from 26 laboratories in Europe, showed that the main parameters underpinning sensitivity are the preanalytical variables (volume of serum used for DNA extraction and DNA template volume), irrespective of the extraction platforms and qPCR assay/platform.
- MeSH
- Molecular Diagnostic Techniques standards methods MeSH
- DNA, Fungal * blood genetics MeSH
- Real-Time Polymerase Chain Reaction * standards methods MeSH
- Humans MeSH
- Mucorales * genetics isolation & purification MeSH
- Mucormycosis * diagnosis microbiology blood MeSH
- Sensitivity and Specificity * MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Evaluation Study MeSH
Mycoplasma spp. contamination is a major concern in laboratories handling cell cultures, and routine detection methods are usually time-consuming, laborious and lack sensitivity. This study presents a streamlined workflow integrating rapid thermal DNA extraction (99 °C-1 min) with a SYBR Green-based qPCR for Mycoplasma detection. High-coverage primers targeting an 86-bp region of the 16S rDNA were designed using 109 Mycoplasma spp. sequences from GeneBank. In silico analysis confirmed full primer annealing to major cell culture contaminants (M. arginini, M. hominis, M. orale, and M. hyorhinis). Upon thermal lysis and qPCR optimization, the yield of the protocol was equivalent to that of phenol-chloroform extraction plus qPCR, with a detection limit of 64 bacterial cells. Finally, the performance of the protocol was confirmed in cell cultures with known Mycoplasma spp. contamination, accurately reproducing the contamination status. Thus, the developed protocol provides a simple, rapid, cost-effective, and sensitive method for monitoring Mycoplasma spp. in cell cultures.
- MeSH
- Cell Culture Techniques * methods MeSH
- DNA, Bacterial genetics isolation & purification MeSH
- DNA Primers genetics MeSH
- Real-Time Polymerase Chain Reaction * methods MeSH
- Humans MeSH
- Mycoplasma * genetics isolation & purification classification MeSH
- Workflow MeSH
- DNA, Ribosomal genetics MeSH
- RNA, Ribosomal, 16S genetics MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
Male infertility is a multifactorial condition contributing to approximately 50% of all cases of couple infertility. In recent years, significant advances have been made in both diagnostics and treatment. This review summarizes key developments from 2019 to 2024 with direct relevance to routine clinical practice in Czech urology and andrology. Particular attention is paid to the updated semen analysis standards (World Health Organisation 6th edition, 2021), sperm DNA fragmentation testing, genetic evaluation (karyotyping, Y chromosome microdeletions, and exome sequencing), surgical management of varicocele, and sperm retrieval techniques for azoospermia, including microdissection testicular sperm extraction (micro-TESE). The article also discusses pharmacological options (gonadotropins, selective estrogen receptor modulators, antioxidants), the impact of lifestyle factors, and the importance of interdisciplinary collaboration with assisted reproduction centers. Future perspectives, including the role of preventive strategies in male reproductive health, are also addressed. The aim is to provide a comprehensive and clinically applicable overview of current recommendations and therapeutic approaches in andrology, with a focus on their implementation in the Czech urological setting.
- MeSH
- Semen Analysis methods MeSH
- Antioxidants pharmacology therapeutic use MeSH
- Reproductive Techniques, Assisted MeSH
- Genetic Testing methods MeSH
- Gonadotropins therapeutic use MeSH
- Humans MeSH
- Infertility, Male * diagnosis etiology therapy MeSH
- Sperm Retrieval MeSH
- Selective Estrogen Receptor Modulators pharmacology therapeutic use MeSH
- Varicocele surgery MeSH
- Life Style MeSH
- Check Tag
- Humans MeSH
- Publication type
- Systematic Review MeSH
Although the understanding of the causes of infertility is the key to its successful treatment, recent studies have shown that as many as 50% of male-caused infertility cases are considered idiopathic. The microbial colonization of the male reproductive system was shown to be associated with reduced male reproductive fitness. Investigation of the seminal microbiome, however, remains challenging. This article aimed to improve this situation by creating the first comprehensive review of literature on the metagenomic methods (including the pre-analytical and analytical approaches) used in the research on human seminal bacteriome (total bacterial DNA in the matrix), published in 2018-2024. A total of 29 studies addressing the analysis of the human seminal bacteriome were identified. The analysis typically involved DNA extraction from the supernatant using commercial kits, amplification of the gene for 16S rRNA, and sequencing of amplicons. Where the separation of seminal plasma was performed, centrifugation was the dominant method used for this purpose. The significant heterogeneity in individual steps of methodological approaches in the analysis of the human seminal bacteriome complicates the comparison of results among studies and the establishment of standard procedures, hindering clinical advancements. For this reason, a protocol for the analysis of the human seminal plasma bacteriome is proposed here, which could lead to improved comparability of results among studies and make future research more efficient. This protocol is founded on rigorous quality control measures, compliance with the WHO laboratory manual for sample collection, extensive pretreatment involving mechanical and enzymatic lysis, DNA extraction using the QIAamp DNA Mini Kit (Qiagen), and short-read sequencing conducted on the MiSeq platform (Illumina).
- Publication type
- Journal Article MeSH
- Review MeSH
BACKGROUND: Human milk harbors diverse bacterial communities that contribute to infant health. Although pumping and storing milk is a common practice, the viable bacterial composition of pumped milk and the impact of storage practice on these bacteria remains under-explored. This metagenomic observational study aimed to characterize viable bacterial communities in freshly pumped human milk and its changes under different storage conditions. METHODS: In 2023, twelve lactating mothers from the CELSPAC: TNG cohort (Czech Republic) provided freshly pumped milk samples. These samples were stored under various conditions (refrigeration for 24 h, 48 h, or freezing for six weeks) and treated with propidium monoazide (PMA) to selectively identify viable cells. The DNA extracted from individual samples was subsequently analyzed using 16S rRNA amplicon sequencing on the Illumina platform. RESULTS: The genera Streptococcus, Staphylococcus, Diaphorobacter, Cutibacterium, and Corynebacterium were the most common viable bacteria in fresh human milk. The median sequencing depth and Shannon index of fresh human milk samples treated with PMA (+ PMA) were significantly lower than in untreated (-PMA) samples (p < 0.05 for all), which was true also for each time point. Also, significant changes in these parameters were observed between fresh human milk samples and their paired frozen samples (p < 0.05), while no differences were found between fresh human milk samples and those refrigerated for up to 48 h (p > 0.05). Of specific genera, only + PMA frozen human milk samples showed a significant decrease in the central log-ratio transformed relative abundances of the genera Diaphorobacter and Cutibacterium (p < 0.05) in comparison to + PMA fresh human milk samples. CONCLUSIONS: The study demonstrated that the bacterial profiles significantly differed between human milk samples treated with PMA, which represent only viable bacteria, and those untreated. While storage at 4 °C for up to 48 h did not significantly alter the overall diversity and composition of viable bacteria in human milk, freezing notably affected both the viability and relative abundances of some bacterial genera.
- MeSH
- Azides MeSH
- Bacteria * isolation & purification genetics classification MeSH
- Refrigeration MeSH
- Adult MeSH
- Humans MeSH
- Milk, Human * microbiology MeSH
- Microbiota * MeSH
- Propidium analogs & derivatives MeSH
- RNA, Ribosomal, 16S MeSH
- Food Storage * methods MeSH
- Freezing MeSH
- Check Tag
- Adult MeSH
- Humans MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Observational Study MeSH
INTRODUCTION: The objective of this systematic review is to evaluate the efficacy and safety of antigen-specific tolerance-inducing therapeutic approaches (products based on peptides, DNA and T cells) versus placebo or other comparators, where possible, in adult multiple sclerosis (MS) patients. METHODS: PubMed, CINAHL, Web of Science, Cochrane and International Clinical Trials Registry Platform, ClinicalTrials.gov were searched for published and unpublished studies. Screening, critical appraisal, and data extraction for included studies were carried out by two independent reviewers. For efficacy, phase I, II and III clinical trials (randomized/non-randomized; double blind/single blind/unblinded; single-center/multicenter; single-arm/two-arm) and for safety, phase I, II and III clinical trials (randomized/non-randomized; double blind/single blind/unblinded; controlled/uncontrolled; single-center/multicenter; single-arm/two-arm) were included. Observational studies (cross-sectional studies, cohort studies, case studies/reports etc), review articles, systematic reviews, meta-analysis, preclinical and pilot studies were excluded. All included studies were critically appraised using standardized JBI tools, with no exclusions based on methodological quality. Where possible, studies were pooled in statistical meta-analysis, presented in tabular format, and accompanied by narrative synthesis. The Grading of Recommendations, Assessment, Development and Evaluation (GRADE) approach for grading the certainty of evidence. RESULTS: Search yielded 2644 results and in total 26 studies were included in the final analysis. Twelve studies were RCTs and 14 were quasi-experimental. In total, there were 1427 subjects from the RCTs, and 314 from non-RCTs. Sample size of studies ranged from 10 to 612 patients. All studies included adult patients, principally aged 18-55/65 years. Critical appraisal scores for the RCTs were in the range 31% to 92%. For quasi-experimental studies, critical appraisal scores were in the range 45% to 78%. Due to high heterogeneity of the studies, efficacy of all antigen-specific treatment remained ambiguous. For all three types of treatment, there was no statistical difference in occurrence of adverse effects (AEs) between the treatment- and placebo-related AEs (for DNA-based treatment RR was 1.06 (0.94-1.10), p = 0.334; for peptides-base treatments RR was 1.04 (0.90-1.08), p = 0.115; for T-cells-based treatments RR was 1.31 (0.97-1.76), p = 0.08). There were no differences in RR for serious AEs (SAEs) between the treatments either for DNA-based treatment (RR was 0.63 (0.25-1.58), p = 0.322) or peptide-based treatment (RR was 0.86 (0.62-1.19), p = 0.361). There were no reported SAEs for T cell-based treatments, so meta-analysis for these therapies was not performed. The most frequent AEs were local reactions to injection, such as redness, erythema, pain. DISCUSSION: Antigen-specific tolerance-inducing therapeutic approaches appeared to be safe. However, the certainty for these results was very low for SAEs in peptide- and DNA-based therapies, whereas it was low for AEs in DNA- and T cells-based therapies and moderate for AEs in peptide-based therapies. The efficacy of antigen-specific therapies remains ambiguous. Larger, well-designed studies with high level quality are needed to ensure ultimate conclusions. REGISTRATION: The registration number provided following registration of the protocol in PROSPERO is 'CRD42021236776'.
- MeSH
- Antigens * immunology therapeutic use MeSH
- Humans MeSH
- Multiple Sclerosis * therapy immunology MeSH
- Treatment Outcome MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Review MeSH
- Systematic Review MeSH
BACKGROUND: Understanding the temporal variability of the microbiome is critical for translating associations of the microbiome with health and disease into clinical practice. The aim of this study is to assess the extent of temporal variability of the human urinary microbiota. A pair of urine samples were collected from study participants at 3-40-month interval. DNA was extracted and the bacterial V4 hypervariable region of the 16S rRNA gene was sequenced on the Illumina MiSeq platform. The alpha diversity of paired samples was analyzed using Chao1 and Shannon indices and PERMANOVA was used to test the factors influencing beta diversity. RESULTS: A total of 63 participants (43 men and 20 women with a mean age of 63.0 and 57.1 years, respectively) were included in the final analysis. An average of 152 ± 128 bacterial operational taxonomic units (OTUs) were identified in each urine sample from the entire cohort. There was an average of 41 ± 32 overlapping OTUs in each sample pair, accounting for 66.3 ± 29.4% of the relative abundance. There was a clear correlation between the number of overlapping OTUs and the relative abundance covered. The difference in Chao1 index between paired samples was statistically significant; the difference in Shannon index was not. Beta diversity did not differ significantly within the paired samples. Neither age nor sex of the participants influenced the variation in community composition. With a longer interval between the collections, the relative abundance covered by the overlapping OTUs changed significantly but not the number of OTUs. CONCLUSION: Our findings demonstrated that, while the relative abundance of dominant bacteria varied, repeated collections generally shared more than 60% of the bacterial community. Furthermore, we observed little variation in the alpha and beta diversity of the microbial community in human urine. These results help to understand the dynamics of human urinary microbiota and enable interpretation of future studies.
- MeSH
- Bacteria * classification genetics isolation & purification MeSH
- Biodiversity MeSH
- Time Factors MeSH
- DNA, Bacterial genetics MeSH
- Middle Aged MeSH
- Humans MeSH
- Microbiota * genetics MeSH
- Urine * microbiology MeSH
- Prospective Studies MeSH
- RNA, Ribosomal, 16S genetics MeSH
- Sequence Analysis, DNA MeSH
- Check Tag
- Middle Aged MeSH
- Humans MeSH
- Male MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Observational Study MeSH
Úvod: V důsledku metabolických dějů dochází v živých strukturách k endogenní produkci chemiluminiscence, kterou také označujeme jako biologickou autochemiluminiscenci (BAL). Generování BAL je úzce spojeno s oxidačními procesy, tvorbou volných radikálů a obecně oxidačně-redukční homeostázou zkoumaného biologického materiálu. BAL byla již dříve studována v savčích buněčných modelech a tkáních. Doposud ovšem nebyl tento jev popsán v případě struktur zubní tkáně. Kromě endogenně generované BAL lze BAL indukovat i exogenně, a to jak fyzikálními (UV záření, mechanické poškození, teplo), tak i chemickými (oxidační činidla, např. H2O2) a biotickými (patogeny) faktory. Metodika: V předložené práci byla zkoumána endogenně produkovaná i exogenně indukovaná BAL v povrchových a vnitřních strukturách semiretinovaných a retinovaných třetích molárů, které byly indikovány k extrakci zubním lékařem pro jejich nevhodné uložení v čelisti u dvou pacientů (žena, 21 let, muž, 22 let). Detekce BAL byla provedena po mechanickém odstranění zubního plaku rotačním kartáčkem. Pomocí piezoelektrické pily byly připraveny podélné řezy vedené tak, aby došlo k odhalení všech vnitřních částí zubu. Takto připravené vzorky – celého vnitřního řezu a vnější části celého zubu – byly podrobeny detekci BAL ve světlotěsné komoře za použití fotonásobičového modulu. Následně byly vzorky ošetřeny roztokem oxidačního činidla 3% H2O2 a redukčního činidla 10 mM TCEP (tris(karboxyethyl)fosfin). Výsledky: U obou vzorků zubu bylo prokázáno, že produkují BAL. Produkce endogenní chemiluminiscence byla pozorována ve vnitřních strukturách zubu (18 600 pulzů/600 s), která byla přibližně 2,7krát vyšší než BAL detekovaná na povrchových strukturách zubu (6 900 pulzů/600 s). Po ošetření H2O2 došlo k významnému (až 14násobnému) nárůstu BAL pro vnitřní struktury zubu ve srovnání s bazální intenzitou endogenně produkované BAL. Aplikace TCEP (negativní kontrola) vedla k mírnému potlačení produkce BAL. Závěr: Výsledky této pilotní studie ukazují, že BAL může být produkována nejenom měkkými tkáněmi, ale i tvrdou zubní tkání. Získané výsledky by mohly být využity k výzkumu metabolické aktivity a reaktivity vnitřních i vnějších částí zubu, a to především v kontextu výzkumu oxidačněredukční homeostázy. Detekce BAL by také mohla být aplikována pro vývoj nových zobrazovacích technik.
Introduction: As a result of metabolic processes, the endogenous production of chemiluminescence occurs in living biological structures, which we also refer to as biological autochemiluminescence (BAL). The generation of BAL is closely connected with oxidation processes, the formation of free radicals, and in general the redox homeostasis of the investigated biological material. BAL has previously been studied in mammalian cells and tissues. So far, however, this phenomenon has not been described in dental tissue structures. In addition to endogenously generated BAL, BAL can be exogenously induced by physical (UV radiation, mechanical damage, heat), chemical (oxidizing agents, e.g. H2O2) or biotic (pathogens) factors. Methods: Endogenously and exogenously induced BAL were investigated on the surface and internal structures of semi-impacted and impacted third molars, which were indicated for extraction by a dentist due to their inappropriate placement in the jaw in two patients (a 21-year-old woman and a 22-year-old man). BAL detection was performed with samples after dental plaque was mechanically removed with a rotating brush. Using a piezosurgery unit with a saw headpiece, longitudinal sections were made to reveal all internal parts of the tooth. The samples prepared in this way – the entire internal section and the external part of the entire tooth – were subjected to BAL detection in a dark chamber using H7360-01 PMT photomultiplier. Subsequently, the samples were treated with a solution of the oxidizing agent 3% H2O2 or the reducing agent 10 mM TCEP (tris(carboxyethyl)phosphine). Results: Both tooth samples were shown to produce BAL. Endogenous chemiluminescence production was observed in the internal structures of the tooth (18,600 counts/600 s), which was 2.7-fold higher than the BAL detected on the tooth outer surfaces (6,900 counts/600 s). After H2O2 treatment, there was a significant (up to 14-fold) increase in BAL for internal tooth structures compared to the basal intensity of endogenously produced BAL. The application of TCEP (negative control) resulted in a residual suppression of BAL production. Conclusion: The results of this pilot study show that BAL can be produced not only by soft tissues but also by hard dental tissue. The obtained results could be used for further research of the metabolic activity and reactivity of the inner and outer parts of the tooth, especially in the context of redox biology research. BAL detection could also be applied in the development of new imaging techniques.
Waterpipe smoking (WPS) has adverse health effects that include endothelial dysfunction with mechanisms involving oxidative stress and inflammation. Nonetheless, there is a scarcity of data on the direct impact of WPS on endothelial function. In this study, we assessed the in vitro effects of waterpipe smoke extract (WPSE) on aortic endothelial cell lines, namely the TeloHAEC. The WPSE markedly caused concentration- and time-dependent decreases in cellular viability. When compared with the control, at a concentration of 20 % and an incubation period of 48 h, the WPSE significantly increased the levels of lactate dehydrogenase, and markers of oxidative stress including thiobarbituric acid reactive substances, superoxide dismutase, catalase, and reduced glutathione. Moreover, the concentrations of proinflammatory cytokine (tumor necrosis factor alpha), and adhesion molecules (E-selectin and intercellular adhesion molecule-1) were also significantly augmented. Likewise, WPSE triggered mitochondrial dysfunction, DNA oxidative damage, as well as apoptosis in TeloHAEC cells. Similarly, cells cultured with WPSE have shown increased expression of phosphorylated nuclear factor-kappaB and hypoxia-inducible factor 1-alpha (HIF-1alpha). In conclusion, our study showed that WPSE triggers endothelial inflammation, oxidative stress, DNA damage, mitochondrial dysfunction, and apoptosis via mechanisms involving the activation of nuclear factor-kappaB and HIF-1alpha. Key words Waterpipe smoking, Aortic endothelial cells, Inflammation, Oxidative Stress.
- MeSH
- Aorta * drug effects metabolism MeSH
- Apoptosis drug effects MeSH
- Cell Line MeSH
- Endothelial Cells * drug effects metabolism MeSH
- Smoke * adverse effects MeSH
- Water Pipe Smoking * adverse effects metabolism MeSH
- Humans MeSH
- Oxidative Stress * drug effects MeSH
- DNA Damage drug effects MeSH
- Cell Survival drug effects MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
BACKGROUND: To validate the clinical utility of a previously identified circulating tumor DNA methylation marker (meth-ctDNA) panel for disease detection and survival outcomes, meth-ctDNA markers were compared to PSA levels and PSMA PET/CT findings in men with different stages of prostate cancer (PCa). METHODS: 122 PCa patients who underwent [68Ga]Ga-PSMA-11 PET/CT and plasma sampling (03/2019-08/2021) were analyzed. cfDNA was extracted, and a panel of 8 individual meth-ctDNA markers was queried. PET scans were qualitatively and quantitatively assessed. PSA and meth-ctDNA markers were compared to PET findings, and their relative prognostic value was evaluated. RESULTS: PSA discriminated best between negative and tumor-indicative PET scans in all (AUC 0.77) and hormone-sensitive (hsPC) patients (0.737). In castration-resistant PCa (CRPC), the meth-ctDNA marker KLF8 performed best (AUC 0.824). CHST11 differentiated best between non- and metastatic scans (AUC 0.705) overall, KLF8 best in hsPC and CRPC (AUC 0.662, 0.85). Several meth-ctDNA markers correlated low to moderate with the tumor volume in all (5/8) and CRPC patients (6/8), while PSA levels correlated moderately to strongly with the tumor volume in all groups (all p < 0.001). CRPC overall survival was independently associated with LDAH and PSA (p = 0.0168, p < 0.001). CONCLUSION: The studied meth-ctDNA markers are promising for the minimally-invasive detection and prognostication of CRPC but do not allow for clinical characterization of hsPC. Prospective studies are warranted for their use in therapy response and outcome prediction in CRPC and potential incremental value for PCa monitoring in PSA-low settings.
- MeSH
- Circulating Tumor DNA genetics blood MeSH
- Edetic Acid analogs & derivatives MeSH
- Gallium Isotopes * MeSH
- Middle Aged MeSH
- Humans MeSH
- DNA Methylation * genetics MeSH
- Biomarkers, Tumor * genetics blood MeSH
- Prostatic Neoplasms, Castration-Resistant genetics blood diagnostic imaging MeSH
- Prostatic Neoplasms * genetics blood diagnostic imaging MeSH
- Positron Emission Tomography Computed Tomography * methods MeSH
- Prognosis MeSH
- Prostate-Specific Antigen * blood genetics MeSH
- Cross-Sectional Studies MeSH
- Gallium Radioisotopes * MeSH
- Aged, 80 and over MeSH
- Aged MeSH
- Check Tag
- Middle Aged MeSH
- Humans MeSH
- Male MeSH
- Aged, 80 and over MeSH
- Aged MeSH
- Publication type
- Journal Article MeSH
- Comparative Study MeSH
