Characterization of bacteriophages infecting multidrug-resistant uropathogenic Escherichia coli strains.

Uropathogenic Escherichia coli (UPEC) is the most common causative agent of urinary tract infections, and strains that are resistant to antibiotics are a major problem in treating these infections. Phage therapy is a promising alternative approach that can be used to treat infections caused by polyresistant bacterial strains. In the present study, 16 bacteriophages isolated from sewage and surface water were investigated. Phage host specificity was tested on a collection of 77 UPEC strains. The phages infected 2-44 strains, and 80% of the strains were infected by at least one phage. The susceptible E. coli strains belonged predominantly to the B2 phylogenetic group, including strains of two clones, CC131 and CC73, that have a worldwide distribution. All of the phages belonged to class Caudoviricetes and were identified as members of the families Straboviridae, Autographiviridae, and Drexlerviridae and the genera Kagunavirus, Justusliebigvirus, and Murrayvirus. A phage cocktail composed of six phages - four members of the family Straboviridae and two members of the family Autographiviridae - was prepared, and its antibacterial activity was tested in liquid medium. Complete suppression of bacterial growth was observed after 5-22 hours of cultivation, followed by partial regrowth. At 24 hours postinfection, the cocktail suppressed bacterial growth to 43-92% of control values. Similar results were obtained when testing the activity of the phage cocktail in LB and in artificial urine medium. The results indicate that our phage cocktail has potential to inhibit bacterial growth during infection, and they will therefore be preserved in the national phage bank, serving as valuable resources for therapeutic applications.

Evaluation and limitations of different approaches among COVID-19 fatal cases using whole-exome sequencing data.

BACKGROUND: COVID-19 caused by the SARS-CoV-2 infection may result in various disease symptoms and severity, ranging from asymptomatic, through mildly symptomatic, up to very severe and even fatal cases. Although environmental, clinical, and social factors play important roles in both susceptibility to the SARS-CoV-2 infection and progress of COVID-19 disease, it is becoming evident that both pathogen and host genetic factors are important too. In this study, we report findings from whole-exome sequencing (WES) of 27 individuals who died due to COVID-19, especially focusing on frequencies of DNA variants in genes previously associated with the SARS-CoV-2 infection and the severity of COVID-19. RESULTS: We selected the risk DNA variants/alleles or target genes using four different approaches: 1) aggregated GWAS results from the GWAS Catalog; 2) selected publications from PubMed; 3) the aggregated results of the Host Genetics Initiative database; and 4) a commercial DNA variant annotation/interpretation tool providing its own knowledgebase. We divided these variants/genes into those reported to influence the susceptibility to the SARS-CoV-2 infection and those influencing the severity of COVID-19. Based on the above, we compared the frequencies of alleles found in the fatal COVID-19 cases to the frequencies identified in two population control datasets (non-Finnish European population from the gnomAD database and genomic frequencies specific for the Slovak population from our own database). When compared to both control population datasets, our analyses indicated a trend of higher frequencies of severe COVID-19 associated risk alleles among fatal COVID-19 cases. This trend reached statistical significance specifically when using the HGI-derived variant list. We also analysed other approaches to WES data evaluation, demonstrating its utility as well as limitations. CONCLUSIONS: Although our results proved the likely involvement of host genetic factors pointed out by previous studies looking into severity of COVID-19 disease, careful considerations of the molecular-testing strategies and the evaluated genomic positions may have a strong impact on the utility of genomic testing.

Repression of a large number of genes requires interplay between homologous recombination and HIRA.

During homologous recombination, Dbl2 protein is required for localisation of Fbh1, an F-box helicase that efficiently dismantles Rad51-DNA filaments. RNA-seq analysis of dbl2Δ transcriptome showed that the dbl2 deletion results in upregulation of more than 500 loci in Schizosaccharomyces pombe. Compared with the loci with no change in expression, the misregulated loci in dbl2Δ are closer to long terminal and long tandem repeats. Furthermore, the misregulated loci overlap with antisense transcripts, retrotransposons, meiotic genes and genes located in subtelomeric regions. A comparison of the expression profiles revealed that Dbl2 represses the same type of genes as the HIRA histone chaperone complex. Although dbl2 deletion does not alleviate centromeric or telomeric silencing, it suppresses the silencing defect at the outer centromere caused by deletion of hip1 and slm9 genes encoding subunits of the HIRA complex. Moreover, our analyses revealed that cells lacking dbl2 show a slight increase of nucleosomes at transcription start sites and increased levels of methylated histone H3 (H3K9me2) at centromeres, subtelomeres, rDNA regions and long terminal repeats. Finally, we show that other proteins involved in homologous recombination, such as Fbh1, Rad51, Mus81 and Rad54, participate in the same gene repression pathway.

Uncovering Microbial Composition in Human Breast Cancer Primary Tumour Tissue Using Transcriptomic RNA-seq.

Recent research studies are showing breast tissues as a place where various species of microorganisms can thrive and cannot be considered sterile, as previously thought. We analysed the microbial composition of primary tumour tissue and normal breast tissue and found differences between them and between multiple breast cancer phenotypes. We sequenced the transcriptome of breast tumours and normal tissues (from cancer-free women) of 23 individuals from Slovakia and used bioinformatics tools to uncover differences in the microbial composition of tissues. To analyse our RNA-seq data (rRNA depleted), we used and tested Kraken2 and Metaphlan3 tools. Kraken2 has shown higher reliability for our data. Additionally, we analysed 91 samples obtained from SRA database, originated in China and submitted by Sichuan University. In breast tissue, the most enriched group were Proteobacteria, then Firmicutes and Actinobacteria for both datasets, in Slovak samples also Bacteroides, while in Chinese samples Cyanobacteria were more frequent. We have observed changes in the microbiome between cancerous and healthy tissues and also different phenotypes of diseases, based on the presence of circulating tumour cells and few other markers.

Increased Stromal Infiltrating Lymphocytes Are Associated with the Risk of Disease Progression in Mesenchymal Circulating Tumor Cell-Positive Primary Breast Cancer Patients.

Circulating tumor cells (CTCs) and the immune infiltration of tumors are closely related to clinical outcomes. This study aimed to verify the influence of stromal lymphocyte infiltration and the immune context of tumor microenvironment on the hematogenous spread and prognosis of 282 chemotherapy naïve primary BC patients. To detect the presence of mesenchymal CTCs, RNA extracted from CD45-depleted peripheral blood was interrogated for the expression of mesenchymal gene transcripts. Tumor-infiltrating lymphocytes (TILs) were detected in the stromal areas by immunohistochemistry, using CD3, CD8, and CD45RO antibodies. The concentrations of 51 plasma cytokines were measured by multiplex bead arrays. TILs infiltration in mesenchymal CTC-positive patients significantly decreased their progression-free survival (HR = 4.88, 95% CI 2.30-10.37, p < 0.001 for CD3high; HR = 6.17, 95% CI 2.75-13.80, p < 0.001 for CD8high; HR = 6.93, 95% CI 2.86-16.81, p < 0.001 for CD45ROhigh). Moreover, the combination of elevated plasma concentrations of transforming growth factor beta-3 (cut-off 662 pg/mL), decreased monocyte chemotactic protein-3 (cut-off 52.5 pg/mL) and interleukin-15 (cut-off 17.1 pg/mL) significantly increased the risk of disease recurrence (HR = 4.838, 95% CI 2.048-11.427, p < 0.001). Our results suggest a strong impact of the immune tumor microenvironment on BC progression, especially through influencing the dissemination and survival of more aggressive, mesenchymal CTC subtypes.

Monosomy 3 Influences Epithelial-Mesenchymal Transition Gene Expression in Uveal Melanoma Patients; Consequences for Liquid Biopsy.

Despite outstanding advances in diagnosis and the treatment of primary uveal melanoma (UM), nearly 50% of UM patients develop metastases via hematogenous dissemination, driven by the epithelial-mesenchymal transition (EMT). Despite the failure in UM to date, a liquid biopsy may offer a feasible non-invasive approach for monitoring metastatic disease progression and addressing protracted dormancy. To detect circulating tumor cells (CTCs) in UM patients, we evaluated the mRNA expression of EMT-associated transcription factors in CD45-depleted blood fraction, using qRT-PCR. ddPCR was employed to assess UM-specific GNA11, GNAQ, PLCß4, and CYSLTR2 mutations in plasma DNA. Moreover, microarray analysis was performed on total RNA isolated from tumor tissues to estimate the prognostic value of EMT-associated gene expression. In total, 42 primary UM and 11 metastatic patients were enrolled. All CD45-depleted samples were negative for CTC when compared to the peripheral blood fraction of 60 healthy controls. Tumor-specific mutations were detected in the plasma of 21.4% patients, merely, in 9.4% of primary UM, while 54.5% in metastatic patients. Unsupervised hierarchical clustering of differentially expressed EMT genes showed significant differences between monosomy 3 and disomy 3 tumors. Newly identified genes can serve as non-invasive prognostic biomarkers that can support therapeutic decisions.

A disintegrin and metalloprotease 23 hypermethylation predicts decreased disease-free survival in low-risk breast cancer patients.

A Disintegrin And Metalloprotease 23 (ADAM23), a member of the ADAM family, is involved in neuronal differentiation and cancer. ADAM23 is considered a possible tumor suppressor gene and is frequently downregulated in various types of malignancies. Its epigenetic silencing through promoter hypermethylation was observed in breast cancer (BC). In the present study, we evaluated the prognostic significance of ADAM23 promoter methylation for hematogenous spread and disease-free survival (DFS). Pyrosequencing was used to quantify ADAM23 methylation in tumors of 203 BC patients. Presence of circulating tumor cells (CTC) in their peripheral blood was detected by quantitative RT-PCR. Expression of epithelial (KRT19) or mesenchymal (epithelial-mesenchymal transition [EMT]-inducing transcription factors TWIST1, SNAI1, SLUG and ZEB1) mRNA transcripts was examined in CD45-depleted peripheral blood mononuclear cells. ADAM23 methylation was significantly lower in tumors of patients with the mesenchymal CTC (P = .006). It positively correlated with Ki-67 proliferation, especially in mesenchymal CTC-negative patients (P = .001). In low-risk patients, characterized by low Ki-67 and mesenchymal CTC absence, ADAM23 hypermethylation was an independent predictor of DFS (P = .006). Our results indicate that ADAM23 is likely involved in BC progression and dissemination of mesenchymal CTC. ADAM23 methylation has the potential to function as a novel prognostic marker and therapeutic target.

Decreased methylation in the SNAI2 and ADAM23 genes associated with de-differentiation and haematogenous dissemination in breast cancers.

BACKGROUND: In breast cancer (BC), deregulation of DNA methylation leads to aberrant expressions and functions of key regulatory genes. In our study, we investigated the relationship between the methylation profiles of genes associated with cancer invasivity and clinico-pathological parameters. In detail, we studied differences in the methylation levels between BC patients with haematogenous and lymphogenous cancer dissemination. METHODS: We analysed samples of primary tumours (PTs), lymph node metastases (LNMs) and peripheral blood cells (PBCs) from 59 patients with sporadic disseminated BC. Evaluation of the DNA methylation levels of six genes related to invasivity, ADAM23, uPA, CXCL12, TWIST1, SNAI1 and SNAI2, was performed by pyrosequencing. RESULTS: Among the cancer-specific methylated genes, we found lower methylation levels of the SNAI2 gene in histologic grade 3 tumours (OR = 0.61; 95% CI, 0.39-0.97; P = 0.038) than in fully or moderately differentiated cancers. We also evaluated the methylation profiles in patients with different cancer cell dissemination statuses (positivity for circulating tumour cells (CTCs) and/or LNMs). We detected the significant association between reduced DNA methylation of ADAM23 in PTs and presence of CTCs in the peripheral blood of patients (OR = 0.45; 95% CI, 0.23-0.90; P = 0.023). CONCLUSION: The relationships between the decreased methylation levels of the SNAI2 and ADAM23 genes and cancer de-differentiation and haematogenous dissemination, respectively, indicate novel functions of those genes in the invasive processes. After experimental validation of the association between the lower values of SNAI2 and ADAM23 methylation and clinical features of aggressive BCs, these methylation profiles could improve the management of metastatic disease.

Relationship between circulating tumor cells, blood coagulation, and urokinase-plasminogen-activator system in early breast cancer patients.

Cancer is a risk factor for venous thromboembolism (VTE) and plasma d-dimer (DD) and tissue factor (TF) are established VTE associated markers. Circulating tumor cells (CTCs) are associated with the risk of VTE in metastatic breast cancer. This study aimed to correlate CTCs, blood coagulation and the urokinase plasminogen activator (uPA) system in primary breast cancer (PBC) patients. This prospective study included 116 PBC patients treated by primary surgery. CTCs were detected by quantitative RT-PCR assay for expression of epithelial (CK19) or epithelial-mesenchymal transition (EMT) genes (TWIST1, SNAIL1, SLUG, ZEB1, FOXC2). Plasma DD, TF, uPA system proteins were detected by enzyme-linked immunosorbent assays, while expressions of uPA system in surgical specimens were evaluated by immunohistochemistry. CTCs were detected in 27.6% patients. Patients with CTCs had a significantly higher mean plasma DD (ng/mL) than those of patients without CTCs (632.4 versus 365.4, p = 0.000004). There was no association between plasma TF and CTCs. Epithelial CTCs exhibit higher expression of uPA system genes compared to EMT_CTCs. Patients with CTCs had higher plasma uPA proteins than those of patients without CTCs; there was no correlation between tissue expression of uPA system, CTCs, DD or TF levels. In multivariate analysis CTCs and patients age were independent factors associated with plasma DD. We found association between plasma DD and CTCs indicating a potential role for activation of the coagulation cascade in the early metastatic process. CTCs could be directly involved in coagulation activation or increased CTCs could be marker of aggressive disease and increased VTE risk.

Effect of different DNA concentration methods on performance of non-invasive fetal y-chromosomal short tandem repeat profiling from maternal plasma.

BACKGROUND: The accuracy and reliability of detection of free fetal DNA in plasma of pregnant women can be significantly improved by increasing the overall DNA concentration following the isolation from maternal plasma. The aim of our study was to compare DNA concentration methods on samples with free fetal DNA. MATERIALS AND METHODS: DNA isolated from plasma samples of pregnant women carrying a male fetus were concentrated by 3 different methods: vacuum concentration, centrifugal filters and spin columns. Their performance was evaluated using PCR-based Y-chromosomal short tandem repeat (Y-STR) genotyping of the fetus. RESULTS: A statistically significant difference was found between the 3 tested methods (F = 15.57, p < 0.0001). Using vacuum concentration 85.3% of paternally inherited Y-STR alleles were correctly identified. A significantly smaller proportion of alleles was correctly identified in samples concentrated by centrifugal filters and spin columns - 75.9 and 66.5%, respectively. DISCUSSION: The highest proportion of paternally inherited Y-STR alleles was found in samples concentrated with the use of vacuum concentration. This concentration procedure does not require further handling of the sample either, which is an advantage because it avoids potential sample contamination. On the other hand, when automation is considered, vacuum concentration is less suitable because of an uneven and unpredictable sample evaporation rate. © 2014 S. Karger AG, Basel.

Matrix metalloproteinase 1 and circulating tumor cells in early breast cancer.

BACKGROUND: Matrix metalloproteinases (MMPs) are involved in cancer invasion and metastasis. Circulating tumor cells (CTCs) play role in tumor dissemination and are an independent survival predictor in breast cancer (BC) patients. The aim of this study was to assess correlation between CTCs and tumor MMP1 in BC. METHODS: Study included 149 primary BC patients treated by surgery from March 2012 to March 2013. Peripheral blood mononuclear cells (PBMC) were depleted of hematopoietic cells using RossetteSep(TM) selection kit. RNA extracted from CD45-depleted PBMC was interrogated for expression of EMT (TWIST1, SNAIL1, SLUG, ZEB1) and epithelial (CK19) gene transcripts by qRT-PCR. Patient samples with higher epithelial and/or mesenchymal gene transcripts than those of healthy donors (n = 60) were considered as CTC positive. Expression of MMP1 in surgical specimens was evaluated by immunohistochemistry. RESULTS: CTCs were detected in 24.2% patients. CTCs exhibiting only epithelial markers were present in 8.7% patients, whereas CTCs with epithelial-mesenchymal transition (EMT) markers (CTC_EMT) were observed in 13.4% of patients and CTCs co-expressing both markers were detected in 2.0% patients. Patients with CTC_EMT in peripheral blood had significantly increased expression of MMP1 in tumor cells (p = 0.02) and tumor associated stroma (p = 0.05) than those of patients without CTC_EMT. In multivariate analysis, CTC_EMT and tumor grade were independently associated with MMP1 expression in cancer cells, while CTC_EMT and Ki67 were independently associated with MMP1 expression in cancer associated stroma. CONCLUSION: Our data suggest link between MMP1 and CTCs with EMT phenotype and support role of MMPs and EMT in tumor dissemination.

Selection of an optimal method for co-isolation of circulating DNA and miRNA from the plasma of pregnant women.

BACKGROUND: Circulating nucleic acids acquired non-invasively have been confirmed as useful biomarkers in cancer and prenatal medicine. The most important molecules in the field of circulating nucleic acids research are circulating DNA and miRNA. In this study, the possibility of co-isolation of total circulating DNA, cell-free fetal DNA and miRNA from the plasma of pregnant women was tested, and the yields of co-isolated circulating nucleic acids using two commercial kits and three protocols were compared. METHODS: Cell-free fetal DNA and miRNA from the plasma of pregnant women carrying male fetuses were co-isolated with the miRCURY RNA Isolation Kit according to the original protocol and the QIAamp Circulating Nucleic Acid Kit (CNA kit) according to the manufacturer's protocol for DNA isolation and miRNA isolation. For comparison of DNA isolation, the AR and DYS14 gene-based assays were used for the detection and quantification of total circulating and cell-free fetal DNA. For miRNA detection and quantification, the miR-16 and miR-451 assays were used. RESULTS: Two different protocols for isolation using the CNA kit did not significantly differ in the yields of isolated tcDNA and cffDNA; however, the amount of isolated cffDNA using the miRCURY RNA Isolation Kit was significantly less (p<0.05, F=4.776). There was a statistically significant difference for miRNA isolation (p<0.0001, F=859 for miR-16 and p<0.0001, F=854.4 for miR-451), with the highest amount of isolated miRNA obtained using the miRCURY RNA Isolation Kit. CONCLUSIONS: All three methods used in our study were successful in the co-isolation of tcDNA, cffDNA and miRNA from the same sample. The best combined results were obtained with the miRCURY RNA Isolation Kit.

Evaluation of wastewater surveillance results for SARS-CoV-2 at the national scale in the Slovak Republic.

As the COVID-19 transits to endemicity, the frequency of clinical testing and its utility for determining lineage prevalence has declined. This situation is not unique to Slovakia but reflects a global trend, as attention shifts from COVID-19 to other post-pandemic issues and emerging global health challenges. Nevertheless, the pandemic itself has spurred advancements in monitoring the epidemiological situation. At the beginning of the pandemic, genomic surveillance was carried out through sequencing of individual COVID-19 cases. Subsequently, many countries implemented wastewater surveillance to monitor the prevalence of SARS-CoV-2 variants in the community. In the present study, we collected and analysed 1715 virus-positive samples from 64 wastewater treatment plants across Slovakia, serving 69 % of the population connected to the wastewater treatment pipelines. Here, we show that wastewater sequencing is effective in detecting the emergence of new virus lineages. Additionally, we can assume that wastewater surveillance provides results that are approximately consistent when compared with clinical testing at both national and city levels, concurrently providing information on variant lineages which have not been detected in clinical cases due to reduced clinical testing. Our study demonstrates and concludes the value of wastewater-based surveillance strategies in the Slovakia, establishing it as an important and supportive tool for monitoring public health and serving as an early warning system in times when clinical testing is either declining or unavailable.

Fragmentation of DNA affects the accuracy of the DNA quantitation by the commonly used methods.

BACKGROUND: Specific applications and modern technologies, like non-invasive prenatal testing, non-invasive cancer diagnostic and next generation sequencing, are currently in the focus of researchers worldwide. These have common characteristics in use of highly fragmented DNA molecules for analysis. Hence, for the performance of molecular methods, DNA concentration is a crucial parameter; we compared the influence of different levels of DNA fragmentation on the accuracy of DNA concentration measurements. RESULTS: In our comparison, the performance of the currently most commonly used methods for DNA concentration measurement (spectrophotometric, fluorometric and qPCR based) were tested on artificially fragmented DNA samples. In our comparison, unfragmented and three specifically fragmented DNA samples were used.According to our results, the level of fragmentation did not influence the accuracy of spectrophotometric measurements of DNA concentration, while other methods, fluorometric as well as qPCR-based, were significantly influenced and a decrease in measured concentration was observed with more intensive DNA fragmentation. CONCLUSIONS: Our study has confirmed that the level of fragmentation of DNA has significant impact on accuracy of DNA concentration measurement with two of three mostly used methods (PicoGreen and qPCR). Only spectrophotometric measurement was not influenced by the level of fragmentation, but sensitivity of this method was lowest among the three tested. Therefore if it is possible the DNA quantification should be performed with use of equally fragmented control DNA.

Selection of the optimal manual method of cell free fetal DNA isolation from maternal plasma.

BACKGROUND: The cell free fetal DNA (cffDNA) present in plasma of pregnant women represents an important alternative source of DNA for non-invasive prenatal diagnosis. Due to the low quantity and increased fragmentation of cffDNA, the choice of DNA extraction method is a crucial step for downstream analyses. METHODS: In our study, the three spin column-based kits for isolation of cffDNA [DNA Blood Mini Kit (DBM), DSP Virus Kit (DSP) and Circulating Nucleic Acid (CNA) Kit] were compared. Original and optimized protocol were used in comparison and applied in the two phases of the study. RESULTS: A statistically significant difference in performance of the kits was determined based on the comparison of genomic equivalents per mL (GEq/mL) values (p<0.0001). The GEq/mL of isolated DNA was significantly higher using CNA and DSP Kits than DBM Kit. The CNA Kit and DSP Kit did not significantly differ in the GEq/mL values, although all tested samples isolated with CNA Kit showed higher values. CONCLUSIONS: According to our results the commonly used DBM Kit could be successfully replaced with CNA or DSP Kits. The replacement could be beneficial in qualitative as well quantitative tests (e.g., gender determination, aneuploidy detection) when the isolation yield limits subsequent analyses. However, there is an important decision to be made when switching DBM Kit for DSP or CNA Kits. The price of DBM Kit is two and six times lower than DSP and CNA Kits, respectively.

Vitamin D and circulating tumor cells in primary breast cancer.

Background: Circulating tumor cells (CTCs) contribute to the metastatic cascade and represent an independent survival predictor in breast cancer (BC) patients. Vitamin D has pleiotropic effects, and its low concentrations are associated with breast cancer and metastasis. The aim of this study was to assess plasma vitamin D in primary BC patients in relation to CTCs. Methods: This study included 91 non-metastatic BC patients (stage I-III) and 24 healthy donors. Blood samples for the analyses were drawn at the time of surgery. CTCs were assessed using a quantitative RT-PCR assay for expression of epithelial (CK19) or epithelial-to-mesenchymal transition (EMT) genes (TWIST1, SNAIL1, SLUG, and ZEB1). Total 25-OH vitamin D was measured in plasma using ELISA. Plasma cytokines and angiogenic factors were measured by enzyme-linked immunoassay. Results: CTCs were detected in 30 (33%) patients. Patients with detectable CTCs in peripheral blood had significantly lower vitamin D concentrations in comparison to patients without detectable CTCs ((mean ± SD) 8.50 ± 3.89 µg/L for CTC-positive vs 9.69 ± 3.49 µg/L for CTC-negative patients, p = 0.03). The mean ( ± SD) vitamin D plasma level was 9.3 ± 3.65 µg/L for breast cancer patients compared to 18.6 ± 6.8 for healthy donors (p < 0.000001). There was no association between plasma vitamin D and other patient/tumor characteristics. Plasma vitamin D levels are inversely correlated with plasma TGF-ß1, TGF-ß2, IL ß, IL-5, and eotaxin (all p < 0.05). Patients with vitamin D above the median had a better overall survival (hazard ratio (HR) = 0.36, 95% CI 0.16-0.80, p = 0.017), and combined analysis showed the best survival for CTC-negative patients with vitamin D levels above the median as compared to patients with opposite characteristics (HR = 0.18, 95% CI 0.05-0.63, p = 0.004). Conclusions: Low vitamin D could be a consequence and hence a biomarker of a more invasive disease. Alternatively, vitamin D could be associated with survival because of its role in tumor dissemination. Whether its supplementation affects the metastatic cascade should be tested in animal experiments and interventional studies.

Sequential development of several RT-qPCR tests using LNA nucleotides and dual probe technology to differentiate SARS-CoV-2 from influenza A and B.

Sensitive and accurate RT-qPCR tests are the primary diagnostic tools to identify SARS-CoV-2-infected patients. While many SARS-CoV-2 RT-qPCR tests are available, there are significant differences in test sensitivity, workflow (e.g. hands-on-time), gene targets and other functionalities that users must consider. Several publicly available protocols shared by reference labs and public health authorities provide useful tools for SARS-CoV-2 diagnosis, but many have shortcomings related to sensitivity and laborious workflows. Here, we describe a series of SARS-CoV-2 RT-qPCR tests that are originally based on the protocol targeting regions of the RNA-dependent RNA polymerase (RdRp) and envelope (E) coding genes developed by the Charité Berlin. We redesigned the primers/probes, utilized locked nucleic acid nucleotides, incorporated dual probe technology and conducted extensive optimizations of reaction conditions to enhance the sensitivity and specificity of these tests. By incorporating an RNase P internal control and developing multiplexed assays for distinguishing SARS-CoV-2 and influenza A and B, we streamlined the workflow to provide quicker results and reduced consumable costs. Some of these tests use modified enzymes enabling the formulation of a room temperature-stable master mix and lyophilized positive control, thus increasing the functionality of the test and eliminating cold chain shipping and storage. Moreover, a rapid, RNA extraction-free version enables high sensitivity detection of SARS-CoV-2 in about an hour using minimally invasive, self-collected gargle samples. These RT-qPCR assays can easily be implemented in any diagnostic laboratory and can provide a powerful tool to detect SARS-CoV-2 and the most common seasonal influenzas during the vaccination phase of the pandemic.

Systematic Genomic Surveillance of SARS-CoV-2 Virus on Illumina Sequencing Platforms in the Slovak Republic-One Year Experience.

To explore a genomic pool of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during the pandemic, the Ministry of Health of the Slovak Republic formed a genomics surveillance workgroup, and the Public Health Authority of the Slovak Republic launched a systematic national epidemiological surveillance using whole-genome sequencing (WGS). Six out of seven genomic centers implementing Illumina sequencing technology were involved in the national SARS-CoV-2 virus sequencing program. Here we analyze a total of 33,024 SARS-CoV-2 isolates collected from the Slovak population from 1 March 2021, to 31 March 2022, that were sequenced and analyzed in a consistent manner. Overall, 28,005 out of 30,793 successfully sequenced samples met the criteria to be deposited in the global GISAID database. During this period, we identified four variants of concern (VOC)-Alpha (B.1.1.7), Beta (B.1.351), Delta (B.1.617.2) and Omicron (B.1.1.529). In detail, we observed 165 lineages in our dataset, with dominating Alpha, Delta and Omicron in three major consecutive incidence waves. This study aims to describe the results of a routine but high-level SARS-CoV-2 genomic surveillance program. Our study of SARS-CoV-2 genomes in collaboration with the Public Health Authority of the Slovak Republic also helped to inform the public about the epidemiological situation during the pandemic.

Intrauterine Fetal Demise After Uncomplicated COVID-19: What Can We Learn from the Case?

BACKGROUND: SARS-CoV-2 infection in pregnant women can lead to placental damage and transplacental infection transfer, and intrauterine fetal demise is an unpredictable event. CASE STUDY: A 32-year-old patient in her 38th week of pregnancy reported loss of fetal movements. She overcame mild COVID-19 with positive PCR test 22 days before. A histology of the placenta showed deposition of intervillous fibrinoid, lympho-histiocytic infiltration, scant neutrophils, clumping of villi, and extant infarctions. Immunohistochemistry identified focal SARS-CoV-2 nucleocapsid and spike protein in the syncytiotrophoblast and isolated in situ hybridization of the virus' RNA. Low ACE2 and TMPRSS2 contrasted with strong basigin/CD147 and PDL-1 positivity in the trophoblast. An autopsy of the fetus showed no morphological abnormalities except for lung interstitial infiltrate, with prevalent CD8-positive T-lymphocytes and B-lymphocytes. Immunohistochemistry and in situ hybridization proved the presence of countless dispersed SARS-CoV-2-infected epithelial and endothelial cells in the lung tissue. The potential virus-receptor protein ACE2, TMPRSS2, and CD147 expression was too low to be detected. CONCLUSION: Over three weeks' persistence of trophoblast viral infection lead to extensive intervillous fibrinoid depositions and placental infarctions. High CD147 expression might serve as the dominant receptor for the virus, and PDL-1 could limit maternal immunity in placental tissue virus clearance. The presented case indicates that the SARS-CoV-2 infection-induced changes in the placenta lead to ischemia and consecutive demise of the fetus. The infection of the fetus was without significant impact on its death. This rare complication of pregnancy can appear independently to the severity of COVID-19's clinical course in the pregnant mother.

Prognostic role of matrix metalloproteinase 9 in early breast cancer.

MMP9 is involved in extracellular matrix degradation during various physiological and pathological conditions, including tumorigenesis. The present study aimed to assess the prognostic role of intratumoral MMP9 and to determine its association with circulating tumor cells (CTCs) in patients with early breast cancer. A total of 318 patients with primary breast cancer (PBC) were enrolled into the present study. Specimens were subjected to immunohistochemistry analysis, using the MMP9 monoclonal antibody. MMP9 expression was scored using a weighted histoscore (WH). The results demonstrated that the mean WH ± SEM for MMP9 expression was significantly higher in breast tumor cells compared with tumor associated stromas (132.0±5.2 vs. 50.8±3.7; P<0.00001). Furthermore, a positive association was observed between MMP9 expression, the hormone positive status and proliferation index of analysed breast cancer tumour cells. Notably, the prognostic role of MMP9 was not observed in tumor cells [hazard ratio (HR) =0.96; 95% confidence interval (CI), 0.58-1.59; P=0.864] or tumor associated stroma (HR=1.29; 95% CI, 0.60-2.78; P=0.547). Subgroup analysis demonstrated that patients that were HR negative or triple negative, with low MMP9 expression in tumor cells and stroma had a significantly improved disease-free survival than patients with high MMP9 expression. Taken together, the results of the present study demonstrated that high MMP9 expression in PBC was associated with favorable tumor characteristics. However, the prognostic value of MMP9 was limited to only the HR negative and CTC epithelial-to-mesenchymal transition positive subgroups. Thus, analyzing MMP9 tumor expression may help identify patients with increased risk of disease recurrence in these subgroups.