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.

A real-time PCR diagnostic method for detection of Naegleria fowleri.

Naegleria fowleri is a free-living amoeba that can cause primary amoebic meningoencephalitis (PAM). While, traditional methods for diagnosing PAM still rely on culture, more current laboratory diagnoses exist based on conventional PCR methods; however, only a few real-time PCR processes have been described as yet. Here, we describe a real-time PCR-based diagnostic method using hybridization fluorescent labelled probes, with a LightCycler instrument and accompanying software (Roche), targeting the Naegleria fowleriMp2Cl5 gene sequence. Using this method, no cross reactivity with other tested epidemiologically relevant prokaryotic and eukaryotic organisms was found. The reaction detection limit was 1 copy of the Mp2Cl5 DNA sequence. This assay could become useful in the rapid laboratory diagnostic assessment of the presence or absence of Naegleria fowleri.

[Monitoring the prevalence of Streptococcus pneumoniae in various groups]. / Monitoring prevalencie Streptococcus pneumoniae v rôznych kolektívoch.

BACKGROUND: The aim of the present study was to monitor the nasopharyngeal presence of Streptococcus pneumoniae in different age groups (especially children) in Banská Bystrica, Slovakia. The purpose of this screening was to determine the prevalence of different serotypes and to follow up the presence of pneumococcus in these children after the vaccination with heptavalent protein-conjugate vaccine. A contribution of molecular biology techniques was the detection of S. pneumoniae DNA by PCR and also the typisation and comparison of pneumococcal strains by pulsed-field gel electrophoresis. METHODS: S. pneumoniae in nasopharyngeal swabs was detected by cultivation on blood agar plates. Serotypisation was performed by standard Quellung reaction. The commercial diagnostic kit was used for PCR detection of S. pneumoniae DNA. Pulsed-field electrophoresis was performed by modified scheme according to literature. RESULTS: The incidence of pneumococcus is decreasing and less significant with the increasing age. Among youngest children is relatively high prevalence of pneumococci and the relatedness of isolated strains is high as well. After the vaccination, the less invasive serotypes were detected, although the overall incidence of S. pneumoniae was similar. CONCLUSIONS: The monitoring of S. pneumoniae in population is important according to variability of this bacteria with respect to possible changes in pneumococcal types as a consequence of vaccination.

Polymerase chain reaction (PCR)--an efficient tool for diagnosis of influenza and acute respiratory illnesses during influenza season.

Many severe diseases of the respiratory tract lead to hospitalisation. These diseases are often caused by viral infections and may cause increased mortality. The most common viral pathogens involved in these cases, which are also associated with significant morbidity and mortality during the influenza seasons are influenza viruses. Rapid differential diagnosis of influenza viruses is therefore of great importance. Classical diagnosis of these viruses involves virus cultures. Of the rapid diagnostic methodologies which have been developed are RT-PCR, multiplex PCR, real-time PCR. In the present study we have monitored clinical samples from patients of different age groups from selected regions in Slovakia and compared the effectiveness of the classical and molecular biological diagnostic methods. The molecular biological methods proved to be rapid, accurate and effective. Application of these techniques in diagnosis of the respiratory illnesses should help in the prevention, therapy and disease control.

Proliferation-linked expression of the novel murine gene m4mbt encoding a nuclear zinc finger protein with four mbt domains.

Here we describe the cloning and characterization of a novel murine gene named m4mbt that encodes a homolog of the lethal (3) malignant brain tumor (l(3)mbt) and Scm proteins. It is localized on mouse chromosome 15E2 and is organized into 17 exons. As demonstrated by Northern blot analysis, m4mbt mRNA is expressed in virtually all tested tissues and cell lines with the exception of stomach and muscle. The m4mbt transcript was most abundant in the testes. m4mbt expression was shown to initiate early during mouse embryonal development (before day 7) and continue until adulthood. The expression of m4mbt mRNA also appears to correlate with cellular proliferation, since we observed down-regulation of m4mbt expression during terminal monocytic differentiation and in contact-inhibited fibroblasts. Computer analysis of the amino acid (aa) sequence revealed that the M4mbt protein comprises an amino-terminally located atypical C2C2 zinc finger and four centrally located mbt repeats. Mbt repeats are also found in proteins of the Polycomb group (PcG) that associate with heterochromatin and function as long-term repressors of transcription. Using Western blot analysis and confocal fluorescent microscopy, we demonstrated that the M4mbt protein is localized in the nucleus. Since M4mbt has structural domains similar to chromatin-associated proteins, its expression is associated with proliferation, and it has a nuclear localization, it may have a regulatory role related to proliferation and/or differentiation.