No abstract

Late in December 2019, an outbreak of an unknown coronavirus, later identified as SARS-CoV- 2, emerged in the city of Wuhan, China. It causes a dangerous respiratory coronavirus disease in humans - COVID-19. Objective. To detect cases of the disease and prevent its spread across the Russian Federation it is necessary to create an effective diagnostic test system. Material and methods. Based on the analysis of the alignment of the SARS-CoV-2 nucleotide sequences, primers and a probe for RT-PCR were selected, and the analysis conditions were optimized. Results. The diagnostic system was developed and registered in the shortest possible time in real-time RT-PCR format for detecting SARS-CoV-2 coronavirus RNA in smears from the nasopharynx and oropharynx, sputum and feces. The high specificity of the system was verified on a representative set of viruses and microorganisms, the analytical sensitivity was 1x103 copies / ml in smears from the mucous membrane of the nasopharynx and oropharynx and sputum, 5x104 copies / ml in fecal samples. Diagnostic sensitivity and specificity established during clinical trials on samples from patients with confirmed COVID-19 infection, from patients with a different etiology of a disease and clinically healthy people were to 100% (range 94.2-100% with a confidence level of 95%).

The emergence of novel SARS-CoV-2 genetic variants with increased transmissivity and reduced antibody neutralization efficiency is a threat to global public health. Reverse transcription polymerase chain reaction (RT-PCR) with the use of fluorescent probes, which make it possible to detect the single nucleotide substitutions, is a technique suitable for screening the SARS-CoV-2 RNA-containing samples for the already known functionally significant mutations in the S-gene, identification of which allows to define and differentiate the most epidemiologically significant genetic variants. The study was aimed to develop an assay for the large-scale monitoring of the spread of the SARS-CoV-2 top-priority variants. Based on the whole-genome alignment of the SARS-CoV-2 sequences, deposited in the GISAID database, primers and LNA-modified probes were selected to detect mutations in the S gene, typical for the Alpha, Beta/Gamma and Delta variants of concern (VOC). The developed reagent kit for detection of the key mutations in the SARS-CoV-2 S gene by the real time RT-PCR has good analytical and diagnostic characteristics and was authorized as a medical device (reagent) for in vitro use. The results of detecting the VOC and the key mutations with the use of the developed reagent kit were consistent with the data of the whole genome sequencing of 1,500 SARS-CoV-2 RNA samples. The developed reagent kit and the subsequent SARS-CoV-2 RNA sequencing assay used to perform the epidemiological monitoring of SARS-CoV-2 variants made it possible to promptly report the emergence of the Delta genetic variant in Russia, and to trace the dynamic changes in the prevalence of Delta in Moscow Region in April–September 2021.

In April 2022, several cases of acute hepatitis of unknown origin were reported in children from 12 countries. In many cases, enteric adenovirus or SARS-CoV-2 and adenovirus coinfection were detected.

Background One of the main challenges for the mass introduction of molecular diagnostics of soil-transmitted helminths (STHs) into clinical practice is the lack of a generally recognized effective method for isolating parasitic DNA from fecal samples. In the present study, we assessed the effect of various pretreatment procedures on the efficiency of removing PCR inhibitors and extracting Toxocara canis DNA from feces. Methodology and main results In the first part of the work, we evaluated the effectiveness of four destructive methods (bead beating, the action of temperature-dependent enzymes, freeze-heat cycles, and incubation in a lysis buffer of a commercial kit) on the integrity of Toxocara eggs using microscopy and the efficiency of DNA extraction using PCR. Our results showed that Toxocara eggs were most effectively destroyed using the bead beating procedure, while the effect of enzymes and freeze- heat cycles did not lead to significant destruction of the eggs or the release of Toxocara DNA. In the second part of the work, we evaluated the effect of prewashes with 0.1% Tween-20 solution and the use of commercial concentrators on DNA extraction from fecal samples contaminated with T. canis eggs. We have shown that the use of commercial concentrators in combination with sample washing can significantly increase the DNA yield and reduce PCR inhibition. Conclusions A bead beating procedure for 30 minutes at a shaking frequency of 50 Hz was sufficient to completely destroy the Toxocara canis eggs. Helminth DNA isolation protocols that do not include a bead beating step are not preferred. The use of a commercial concentrator followed by washing with a 0.1% Tween-20 solution can significantly increase the yield of STHs DNA and reduce PCR inhibition.