Viruses are evolving at an alarming rate, spreading and inconspicuously adapting to cutting-edge therapies. Therefore, the search for rapid, informative and reliable diagnostic methods is becoming urgent as ever. Conventional clinical tests (PCR, serology, etc.) are being continually optimized, yet provide very limited data. Could high throughput sequencing (HTS) become the future gold standard in molecular diagnostics of viral infections? Compared to conventional clinical tests, HTS is universal and more precise at profiling pathogens. Nevertheless, it has not yet been widely accepted as a diagnostic tool, owing primarily to its high cost and the complexity of sample preparation and data analysis. Those obstacles must be tackled to integrate HTS into daily clinical practice. For this, three objectives are to be achieved: (1) designing and assessing universal protocols for library preparation, (2) assembling purpose-specific pipelines, and (3) building computational infrastructure to suit the needs and financial abilities of modern healthcare centers. Data harvested with HTS could not only augment diagnostics and help to choose the correct therapy, but also facilitate research in epidemiology, genetics and virology. This information, in turn, could significantly aid clinicians in battling viral infections.

Background/Objectives: Carbapenem-resistant Enterobacterales (CRE) are a global health threat due to their high morbidity and mortality rates and limited treatment options. This study examines the plasmid-mediated transmission of virulence and antibiotic resistance determinants in carbapenem-resistant Klebsiella pneumoniae (Kpn) and Escherichia coli (E. coli) isolated from Russian hospitals. Methods: We performed short- and long-read whole-genome sequencing of 53 clinical isolates (48 Kpn and 5 E. coli) attributed to 15 genetic lineages and collected from 21 hospitals across nine Russian cities between 2016 and 2022. Results: The plasmid analysis identified 18 clusters that showed high concordance with replicon typing, with all clusters having a major replicon type. The majority of plasmids in the IncHI1B(pNDM-MAR)/IncFIB(pNDM-Mar)-like cluster (79.16%) carried both antibiotic resistance genes (e.g., bla_NDM-1 and bla_OXA-48) and virulence factors (VFs) such as siderophore genes. We hypothesized that hybrid plasmids could play a critical role in the dissemination of antibiotic resistance genes and VFs. Comparative analyses with global plasmid databases revealed high-risk lineages of hybrid plasmids that are predominantly spread throughout Russia at present. Conclusions: Our findings underscore the importance of monitoring plasmid backbones for clinical management, surveillance, and infection control activities.

Cystic fibrosis (CF) is one of the most common life-threatening genetic disorders. Around 2000 variants in the CFTR gene have been identified, with some proportion known to be pathogenic and 300 disease-causing mutations have been characterized in detail by CFTR2 database, which complicates its analysis with conventional methods. We conducted next-generation sequencing (NGS) in a cohort of 89 adult patients negative for p.Phe508del homozygosity. Complete clinical and demographic information were available for 84 patients. By combining MLPA with NGS, we identified disease-causing alleles in all the CF patients. Importantly, in 10% of cases, standard bioinformatics pipelines were inefficient in identifying causative mutations. Class IV-V mutations were observed in 38 (45%) cases, predominantly ones with pancreatic sufficient CF disease; rest of the patients had Class I-III mutations. Diabetes was seen only in patients homozygous for class I-III mutations. We found that 12% of the patients were heterozygous for more than two pathogenic CFTR mutations. Two patients were observed with p.[Arg1070Gln, Ser466*] complex allele which was associated with milder pulmonary obstructions (FVC 107 and 109% versus 67%, CI 95%: 63-72%; FEV 90 and 111% versus 47%, CI 95%: 37-48%). For the first time p.[Phe508del, Leu467Phe] complex allele was reported, observed in four patients (5%). NGS can be a more information-gaining technology compared to standard methods. Combined with its equivalent diagnostic performance, it can therefore be implemented in the clinical practice, although careful validation is still required.

Advances in the next generation sequencing (NGS) technologies have significantly increased our ability to detect new viral pathogens and systematically determine the spectrum of viruses prevalent in various biological samples. In addition, this approach has also helped in establishing the associations of viromes with many diseases. However, unlike the metagenomic studies using16SrRNA for the detection of bacteria, it is impossible to create universal oligonucleotides to target all known and novel viruses, owing to their genomic diversity and variability. On the other hand, sequencing the entire genome is still expensive and has relatively low sensitivity for such applications. The existing approaches for the design of oligonucleotides for targeted enrichment are usually involved in the development of primers for the PCR-based detection of particular viral species or genera, but not for families or higher taxonomic orders. In this study, we have developed a computational pipeline for designing the oligonucleotides capable of covering a significant number of known viruses within various taxonomic orders, as well as their novel variants. We have subsequently designed a genus-specific oligonucleotide panel for targeted enrichment of viral nucleic acids in biological material and demonstrated the possibility of its application for virus detection in bird samples. We have tested our panel using a number of collected samples and have observed superior efficiency in the detection and identification of viral pathogens. Since a reliable, bioinformatics-based analytical method for the rapid identification of the sequences was crucial, an NGS-based data analysis module was developed in this study, and its functionality in the detection of novel viruses and analysis of virome diversity was demonstrated.

Neuroinfections rank among the top ten leading causes of child mortality globally, even in high-income countries. The crucial determinants for successful treatment lie in the timing and swiftness of diagnosis. Although viruses constitute the majority of infectious neuropathologies, diagnosing and treating viral neuroinfections remains challenging. Despite technological advancements, the etiology of the disease remains undetermined in over half of cases. The identification of the pathogen becomes more difficult when the infection is caused by atypical pathogens or multiple pathogens simultaneously. Furthermore, the modern surge in global passenger traffic has led to an increase in cases of infections caused by pathogens not endemic to local areas. This review aims to systematize and summarize information on neuroinvasive viral pathogens, encompassing their geographic distribution and transmission routes. Emphasis is placed on rare pathogens and cases involving atypical pathogens, aiming to offer a comprehensive and structured catalog of viral agents with neurovirulence potential.