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.

Current Challenges of Methylation-Based Liquid Biopsies in Cancer Diagnostics.

In current clinical practice, effective cancer testing and screening paradigms are limited to specific types of cancer, exhibiting varying efficiency, acceptance, and adherence. Cell-free DNA (cfDNA) methylation profiling holds promise in providing information about the presence of malignity regardless of its type and location while leveraging blood-based liquid biopsies as a method to obtain analytical samples. However, technical difficulties, costs and challenges resulting from biological variations, tumor heterogeneity, and exogenous factors persist. This method exploits the mechanisms behind cfDNA release but faces issues like fragmentation, low concentrations, and high background noise. This review explores cfDNA methylation's origins, means of detection, and profiling for cancer diagnostics. The critical evaluation of currently available multi-cancer early detection methods (MCEDs) as well as tests targeting single genes, emphasizing their potential and limits to refine strategies for early cancer detection, are explained. The current methodology limitations, workflows, comparisons of clinically approved liquid biopsy-based methylation tests for cancer, their utilization in companion diagnostics as well as the biological limitations of the epigenetics approach are discussed, aiming to help healthcare providers as well as researchers to orient themselves in this increasingly complex and evolving field of diagnostics.

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.

Copy number variations in malignant melanoma: genomic regions, biomarkers, and therapeutic targets.

Malignant melanoma is a skin tumor arising from melanocytes, occurring mostly in predisposed individuals. Melanomas are frequently present with copy number variations (CNVs), i.e., gains or losses of specific DNA regions that have provided immense potential for disease diagnosis and classification. The methodology of CNV detection has revolutionized in past decades, and current high throughput technologies enable us to analyze the entire spectrum of CNV alterations at the whole genome scale. Thus, identifying novel CNV biomarkers and evaluating their applicability in biomedicine are becoming increasingly important. The aim of this review was to summarize copy number changes occurring in malignant melanomas. We made an overview of specific genes and chromosomal locations affected in sporadic and familial melanoma and also of known germline alterations in melanoma-prone families. We summarized genomic regions aberrant in malignant melanoma and highlighted those frequently discussed in the literature, suggesting 7q, 11q, 12q, 9p, and 1q, but also others, as the most affected ones.

Understanding genetic variability: exploring large-scale copy number variants through non-invasive prenatal testing in European populations.

Large-scale copy number variants (CNVs) are structural alterations in the genome that involve the duplication or deletion of DNA segments, contributing to genetic diversity and playing a crucial role in the evolution and development of various diseases and disorders, as they can lead to the dosage imbalance of one or more genes. Massively parallel sequencing (MPS) has revolutionized the field of genetic analysis and contributed significantly to routine clinical diagnosis and screening. It offers a precise method for detecting CNVs with exceptional accuracy. In this context, a non-invasive prenatal test (NIPT) based on the sequencing of cell-free DNA (cfDNA) from pregnant women's plasma using a low-coverage whole genome MPS (WGS) approach represents a valuable source for population studies. Here, we analyzed genomic data of 12,732 pregnant women from the Slovak (9,230), Czech (1,583), and Hungarian (1,919) populations. We identified 5,062 CNVs ranging from 200 kbp and described their basic characteristics and differences between the subject populations. Our results suggest that re-analysis of sequencing data from routine WGS assays has the potential to obtain large-scale CNV population frequencies, which are not well known and may provide valuable information to support the classification and interpretation of this type of genetic variation. Furthermore, this could contribute to expanding knowledge about the central European genome without investing in additional laboratory work, as NIPTs are a relatively widely used screening method.

Insights into non-informative results from non-invasive prenatal screening through gestational age, maternal BMI, and age analyses.

The discovery of cell-free fetal DNA fragments in the maternal plasma initiated a novel testing method in prenatal care, called non-invasive prenatal screening (NIPS). One of the limitations of NIPS is the necessity for a sufficient proportion of fetal fragments in the analyzed circulating DNA mixture (fetal fraction), otherwise, the sample is uninterpretable. We present the effect of gestational age, maternal body mass index (BMI), and maternal age on the fetal fraction (FF) of the sample. We retrospectively analyzed data from 5543 pregnant women with a single male fetus who underwent NIPS from which 189 samples received a repeat testing due to an insufficient FF. We showed the relationship between the failure rate of the samples after the repeated analysis, the FF, and the gestational age at the first sampling. Next, we found that different maternal BMI categories affect the FF and thus the chance of an informative redraw. A better understanding of the factors affecting the FF will reduce the number of non-informative calls from repeated analyzes. In this study, we provide helpful information to clinicians on how to approach non-informative analyses.

Determination of the Prevalence of Microsatellite Instability, BRAF and KRAS/NRAS Mutation Status in Patients with Colorectal Cancer in Slovakia.

Slovakia has one of the highest rates of colorectal cancer among the developed countries, ranking as the second highest in the incidence of this disease for men worldwide. Despite the significant burden on both quality of life and the healthcare system this disease imposes, data on molecular analysis of biomarkers in CRC-diagnosed patients is scarce. In our study, we analyzed confirmed CRC patients from the database of the National Cancer Institute (NCI) and evaluated the presence of 4 biomarkers in tumor tissues. Altogether, 83 FFPE tumor tissues from CRC patients listed in the NCI database were analyzed for microsatellite instability status, presence of BRAF and KRAS/NRAS mutations, and neoplastic cell percentage in tissue samples. We identified 4 MSI-high samples, 39 KRAS/NRAS mutations, and 5 BRAF p.V600E mutations, with one case of coexistence of all three markers in a single tumor sample. We also evaluated possible relationships between biomarkers, their coexistence, and the age and sex of the studied population.

Validated WGS and WES protocols proved saliva-derived gDNA as an equivalent to blood-derived gDNA for clinical and population genomic analyses.

BACKGROUND: Whole exome sequencing (WES) and whole genome sequencing (WGS) have become standard methods in human clinical diagnostics as well as in population genomics (POPGEN). Blood-derived genomic DNA (gDNA) is routinely used in the clinical environment. Conversely, many POPGEN studies and commercial tests benefit from easy saliva sampling. Here, we evaluated the quality of variant call sets and the level of genotype concordance of single nucleotide variants (SNVs) and small insertions and deletions (indels) for WES and WGS using paired blood- and saliva-derived gDNA isolates employing genomic reference-based validated protocols. METHODS: The genomic reference standard Coriell NA12878 was repeatedly analyzed using optimized WES and WGS protocols, and data calls were compared with the truth dataset published by the Genome in a Bottle Consortium. gDNA was extracted from the paired blood and saliva samples of 10 participants and processed using the same protocols. A comparison of paired blood-saliva call sets was performed in the context of WGS and WES genomic reference-based technical validation results. RESULTS: The quality pattern of called variants obtained from genomic-reference-based technical replicates correlates with data calls of paired blood-saliva-derived samples in all levels of tested examinations despite a higher rate of non-human contamination found in the saliva samples. The F1 score of 10 blood-to-saliva-derived comparisons ranged between 0.8030-0.9998 for SNVs and between 0.8883-0.9991 for small-indels in the case of the WGS protocol, and between 0.8643-0.999 for SNVs and between 0.7781-1.000 for small-indels in the case of the WES protocol. CONCLUSION: Saliva may be considered an equivalent material to blood for genetic analysis for both WGS and WES under strict protocol conditions. The accuracy of sequencing metrics and variant-detection accuracy is not affected by choosing saliva as the gDNA source instead of blood but much more significantly by the genomic context, variant types, and the sequencing technology used.

SnakeLines: integrated set of computational pipelines for sequencing reads.

With the rapid growth of massively parallel sequencing technologies, still more laboratories are utilising sequenced DNA fragments for genomic analyses. Interpretation of sequencing data is, however, strongly dependent on bioinformatics processing, which is often too demanding for clinicians and researchers without a computational background. Another problem represents the reproducibility of computational analyses across separated computational centres with inconsistent versions of installed libraries and bioinformatics tools. We propose an easily extensible set of computational pipelines, called SnakeLines, for processing sequencing reads; including mapping, assembly, variant calling, viral identification, transcriptomics, and metagenomics analysis. Individual steps of an analysis, along with methods and their parameters can be readily modified in a single configuration file. Provided pipelines are embedded in virtual environments that ensure isolation of required resources from the host operating system, rapid deployment, and reproducibility of analysis across different Unix-based platforms. SnakeLines is a powerful framework for the automation of bioinformatics analyses, with emphasis on a simple set-up, modifications, extensibility, and reproducibility. The framework is already routinely used in various research projects and their applications, especially in the Slovak national surveillance of SARS-CoV-2.

Carbapenem-Resistant Klebsiella pneumoniae in COVID-19 Era-Challenges and Solutions.

The COVID-19 era brought about new medical challenges, which, together with nosocomial bacterial infections, resulted in an enormous burden for the healthcare system. One of the most alarming nosocomial threats was carbapenem-resistant Klebsiella pneumoniae (CRKP). Monitoring CRKP incidence and antimicrobial resistance globally and locally is vitally important. In a retrospective study, the incidence of CRKP in the pre-COVID-19 period (2017-2019) and the COVID-19 pandemic (2020-2022) was investigated in the Central Military Hospital in Ruzomberok, Slovak Republic. The relative incidence of CRKP significantly increased during the COVID-19 period-by 4.8 times, from 0.18 to 0.76%. At the same time, 47% of CRKP-positive patients also had COVID-19. Twenty-six KPC and sixty-nine NDM-producing isolates were identified. CRKPs isolated in the year 2022 were submitted to whole genome sequencing, and their susceptibility was tested to cefiderocol, ceftazidime-avibactam, imipenem-relebactam and meropenem-vaborbactam, with excellent results. KPC-producing isolates were also highly susceptible to colistin (92%). The NDM isolates revealed lower susceptibility rates, including only 57% colistin susceptibility. ST-307 prevailed in KPC and ST-11 in NDM isolates. Despite the excellent activity of new antimicrobials, rational antibiotic policy must be thoroughly followed, supported by complementary treatments and strict anti-epidemic precautions.

Cross-Kingdom Interaction of miRNAs and Gut Microbiota with Non-Invasive Diagnostic and Therapeutic Implications in Colorectal Cancer.

Colorectal cancer (CRC) has one of the highest incidences among all types of malignant diseases, affecting millions of people worldwide. It shows slow progression, making it preventable. However, this is not the case due to shortcomings in its diagnostic and management procedure and a lack of effective non-invasive biomarkers for screening. Here, we discuss CRC-associated microRNAs (miRNAs) and gut microbial species with potential as CRC diagnostic and therapy biomarkers. We provide rich evidence of cross-kingdom miRNA-mediated interactions between the host and gut microbiome. miRNAs have emerged with the ability to shape the composition and dynamics of gut microbiota. Intestinal microbes can uptake miRNAs, which in turn influence microbial growth and provide the ability to regulate the abundance of various microbial species. In the context of CRC, targeting miRNAs could aid in manipulating the balance of the microbiota. Our findings suggest the need for correlation analysis between the composition of the gut microbiome and the miRNA expression profile.

New Possible Ways to Use Exosomes in Diagnostics and Therapy via JAK/STAT Pathways.

Exosomes have the potential to be the future of personalized diagnostics and therapy. They are nano-sized particles between 30 and 100 nm flowing in the extracellular milieu, where they mediate cell-cell communication and participate in immune system regulation. Tumor-derived exosomes (TDEs) secreted from different types of cancer cells are the key regulators of the tumor microenvironment. With their immune suppressive cargo, TDEs prevent the antitumor immune response, leading to reduced effectiveness of cancer treatment by promoting a pro-tumorigenic microenvironment. Involved signaling pathways take part in the regulation of tumor proliferation, differentiation, apoptosis, and angiogenesis. Signal transducers and activators of transcription factors (STATs) and Janus kinase (JAK) signaling pathways are crucial in malignancies and autoimmune diseases alike, and their potential to be manipulated is currently the focus of interest. In this review, we aim to discuss exosomes, TDEs, and the JAK/STAT pathways, along with mediators like interleukins, tripartite motif proteins, and interferons.

PHERI-Phage Host ExploRation Pipeline.

Antibiotic resistance is becoming a common problem in medicine, food, and industry, with multidrug-resistant bacterial strains occurring in all regions. One of the possible future solutions is the use of bacteriophages. Phages are the most abundant form of life in the biosphere, so we can highly likely purify a specific phage against each target bacterium. The identification and consistent characterization of individual phages was a common form of phage work and included determining bacteriophages' host-specificity. With the advent of new modern sequencing methods, there was a problem with the detailed characterization of phages in the environment identified by metagenome analysis. The solution to this problem may be to use a bioinformatic approach in the form of prediction software capable of determining a bacterial host based on the phage whole-genome sequence. The result of our research is the machine learning algorithm-based tool called PHERI. PHERI predicts the suitable bacterial host genus for the purification of individual viruses from different samples. In addition, it can identify and highlight protein sequences that are important for host selection.

Combination of expert guidelines-based and machine learning-based approaches leads to superior accuracy of automated prediction of clinical effect of copy number variations.

Clinical interpretation of copy number variants (CNVs) is a complex process that requires skilled clinical professionals. General recommendations have been recently released to guide the CNV interpretation based on predefined criteria to uniform the decision process. Several semiautomatic computational methods have been proposed to recommend appropriate choices, relieving clinicians of tedious searching in vast genomic databases. We have developed and evaluated such a tool called MarCNV and tested it on CNV records collected from the ClinVar database. Alternatively, the emerging machine learning-based tools, such as the recently published ISV (Interpretation of Structural Variants), showed promising ways of even fully automated predictions using broader characterization of affected genomic elements. Such tools utilize features additional to ACMG criteria, thus providing supporting evidence and the potential to improve CNV classification. Since both approaches contribute to evaluation of CNVs clinical impact, we propose a combined solution in the form of a decision support tool based on automated ACMG guidelines (MarCNV) supplemented by a machine learning-based pathogenicity prediction (ISV) for the classification of CNVs. We provide evidence that such a combined approach is able to reduce the number of uncertain classifications and reveal potentially incorrect classifications using automated guidelines. CNV interpretation using MarCNV, ISV, and combined approach is available for non-commercial use at https://predict.genovisio.com/ .

Complete Genome Sequence of Cronobacter dublinensis Bacteriophage vB_Cdu_VP8.

Cronobacter dublinensis is a Gram-negative pathogen that is capable of causing infection in humans. In this announcement, we describe the characterization of bacteriophage vB_Cdu_VP8, which is able to lyse a Cronobacter dublinensis strain. Related to phages belonging to the genus Muldoonvirus, such as Muldoon and SP1, vB_Cdu_VP8 contains 264 predicted protein-coding genes and 3 tRNAs.

Telomere Length Changes in Cancer: Insights on Carcinogenesis and Potential for Non-Invasive Diagnostic Strategies.

Telomere dynamics play a crucial role in the maintenance of chromosome integrity; changes in telomere length may thus contribute to the development of various diseases including cancer. Understanding the role of telomeric DNA in carcinogenesis and detecting the presence of cell-free telomeric DNA (cf-telDNA) in body fluids offer a potential biomarker for novel cancer screening and diagnostic strategies. Liquid biopsy is becoming increasingly popular due to its undeniable benefits over conventional invasive methods. However, the organization and function of cf-telDNA in the extracellular milieu are understudied. This paper provides a review based on 3,398,017 cancer patients, patients with other conditions, and control individuals with the aim to shed more light on the inconsistent nature of telomere lengthening/shortening in oncological contexts. To gain a better understanding of biological factors (e.g., telomerase activation, alternative lengthening of telomeres) affecting telomere homeostasis across different types of cancer, we summarize mechanisms responsible for telomere length maintenance. In conclusion, we compare tissue- and liquid biopsy-based approaches in cancer assessment and provide a brief outlook on the methodology used for telomere length evaluation, highlighting the advances of state-of-the-art approaches in the field.

Complete Genome Sequence of New Cronobacter-Specific Bacteriophage Dev_CS701.

Cronobacter is a ubiquitous Gram-negative bacterium linked with serious infections. In this report, we describe the characterization of Cronobacter phage Dev_CS701, which was isolated from wastewater. Related to phages belonging to the Straboviridae family and Pseudotevenvirus genus, such as vB_CsaM_IeB, Dev_CS701 contains 257 predicted protein-coding genes and a tRNA gene.

APOC3 and ABCA1 variants in unusual combined hypolipidaemia showing premature peripheral vascular disease.

BACKGROUND: Familial combined hypolipidaemia is a condition characterised by very low concentrations of circulating very-low-density lipoprotein (VLDL), low-density lipoprotein cholesterol (LDL), and high-density lipoprotein cholesterol (HDL). It is thought that low LDL/combined hypolipidaemia can protect from cardiovascular disease (CVD), but this is not what we found in a case we present. OBJECTIVE: We report on a 57-years-old male patient with combined hypolipidaemia who presented with premature peripheral vascular disease. We investigated also his two sons, 32- and 27-years-old, who manifested a tendency to low lipid levels. METHODS AND RESULTS: We used Illumina exome analysis in all three individuals and in all of them we could exclude the major effect of the variants within the genes most frequently mutated in hypolipidaemia, including recently reported LIPC gene variant. Instead, in all three individuals we identified a novel ABCA1 variant, possibly responsible for the decreased HDL levels. The proband and one of his sons also share the splicing APOC3 variant rs138326449, known to be associated with decreased TG levels. CONCLUSION: The heterogeneous nature and the risk of atherosclerosis in combined hypolipidaemia seems to be variable, based on an interplay between low HDL and LDL levels, and it depends on the combination of variants that cause it (Tab. 2, Ref. 38).

Microsatellite instability assessment is instrumental for Predictive, Preventive and Personalised Medicine: status quo and outlook.

A form of genomic alteration called microsatellite instability (MSI) occurs in a class of tandem repeats (TRs) called microsatellites (MSs) or short tandem repeats (STRs) due to the failure of a post-replicative DNA mismatch repair (MMR) system. Traditionally, the strategies for determining MSI events have been low-throughput procedures that typically require assessment of tumours as well as healthy samples. On the other hand, recent large-scale pan-tumour studies have consistently highlighted the potential of massively parallel sequencing (MPS) on the MSI scale. As a result of recent innovations, minimally invasive methods show a high potential to be integrated into the clinical routine and delivery of adapted medical care to all patients. Along with advances in sequencing technologies and their ever-increasing cost-effectiveness, they may bring about a new era of Predictive, Preventive and Personalised Medicine (3PM). In this paper, we offered a comprehensive analysis of high-throughput strategies and computational tools for the calling and assessment of MSI events, including whole-genome, whole-exome and targeted sequencing approaches. We also discussed in detail the detection of MSI status by current MPS blood-based methods and we hypothesised how they may contribute to the shift from conventional medicine to predictive diagnosis, targeted prevention and personalised medical services. Increasing the efficacy of patient stratification based on MSI status is crucial for tailored decision-making. Contextually, this paper highlights drawbacks both at the technical level and those embedded deeper in cellular/molecular processes and future applications in routine clinical testing.

Extracellular vesicles derived from dental mesenchymal stem/stromal cells with gemcitabine as a cargo have an inhibitory effect on the growth of pancreatic carcinoma cell lines in vitro.

Extracellular vesicles (EVs) are nowadays a target of interest in cancer therapy as a successful drug delivering tool. Based on their many beneficial biocompatible properties are designed to transport nucleic acids, proteins, various nanomaterials or chemotherapeutics. Extracellular vesicles derived from mesenchymal stem/stromal cells (MSCs) possess their tumor-homing abilities. This inspired us to engineer the MSC's EVs to be packed with chemotherapeutic agents and deliver it as a Trojan horse directly into tumor cells. In our study, human dental pulp MSCs (DP-MSCs) were cultivated with gemcitabine (GCB), which led to its absorption by the cells and subsequent secretion of the drug out into conditioned media in EVs. Concentrated conditioned media containing small EVs (potentially exosomes) significantly inhibited the cell growth of pancreatic carcinoma cell lines in vitro. DP-MSCs were simultaneously engineered to express a suicide gene fused yeast cytosinedeaminase:uracilphosphoribosyltransferase (yCD::UPRT). The product of the suicide gene converts non-toxic prodrug 5-fluorocytosine (5-FC) to highly cytotoxic chemotherapeutic drug 5-fluorouracil (5-FU) in the recipient cancer cells. Conversion of 5-FC to 5-FU had an additional effect on cancer cell's growth inhibition. Our results showed a therapeutic potential for DP-MSC-EVs to be designed for successful delivering of chemotherapeutic drugs, together with prodrug suicide gene therapy system.