Differentiation of SARS-CoV-2 Variants Using RT-qPCRs by Targeting Recurrent Mutation Sites: A Diagnostic Laboratory Experience from Multi-Center Regional Study, August 2020-December 2021, Poland.

Rapid identification of SARS-CoV-2 variants is essential for epidemiological surveillance. RT-qPCR-based variant differentiation tests can be used to quickly screen large sets of samples for relevant variants of concern/interest; this study was conducted on specimens collected at 11 centers located in Poland during routine SARS-CoV-2 diagnostics between August 2020 and December 2021. A total of 1096 samples (with CT < 30) were screened for Alpha, Beta, Delta, Kappa and Omicron variants using commercial assays targeting repeat mutation sites. Variants were assigned to 434 (39.6%) specimens; the remaining 662 (60.4%) samples were not classified (no tested mutations detected). Alpha (n = 289; 66.59%), Delta (n = 115; 26.5%), Kappa (n = 30; 6.91%) and Omicron (n = 2; 0.46%) variants were identified and their distribution changed over time. The first Alpha variant appeared in October 2020, and it began to gradually increase its proportion of the virus population by June 2021. In July 2021, it was replaced by the Delta variant, which already dominated by the end of the year. The first Kappa was detected in October 2021, while Omicron was found in December 2021. The screening of samples allowed the determination of epidemiological trends over a time interval reflecting the national COVID-19 waves.

Viral Etiological Agent(s) of Respiratory Tract Infections in Symptomatic Individuals during the Second Wave of COVID-19 Pandemic: A Single Drive-Thru Mobile Collection Site Study.

One of the tools to contain the SARS-CoV-2 pandemic was to increase the number of performed tests and to improve the access to diagnostics. To this effect, mobile collection sites (MCSs) were established. This study was performed on samples collected at the MCS between November 2020 and March 2021. We aimed to confirm/exclude SARS-CoV-2, differentiate SARS-CoV-2 variants, and detect other respiratory pathogens. SARS-CoV-2 and other respiratory viruses were identified by RT-qPCRs. A total of 876 (46.35%) SARS-CoV-2 positive specimens in the diagnostic tests were identified. The wild-type variant was determined in 667 (76.14%) samples; the remaining 209 (23.86%) samples specimens were identified as Alpha variant. A total of 51 (5.6%) non-SARS-CoV-2 cases were detected in retrospective studies. These accounted for 33 cases of mono-infection including rhinovirus (RV), human adenovirus (HAdV), human metapneumovirus (HMPV), enterovirus (EV), and influenza virus, and 18 cases of co-infection (SARS-CoV-2 with RV or HAdV or HMPV, and RV with EV). Our research shows that the results obtained from the MCS have value in epidemiological studies, reflecting national trends on a micro scale. Although the spread of COVID-19 is a major public health concern, SARS-CoV-2 is not the only pathogen responsible for respiratory infections.

Impact of the Nucleic Acid Extraction Method and the RT-qPCR Assay on SARS-CoV-2 Detection in Low-Viral Samples.

COVID-19 was initially reported in China at the end of 2019 and soon thereafter, in March 2020, the WHO declared it a pandemic. Until October 2021, over 240 million COVID-19 cases were recorded, with 4.9 mln deaths. In order to stop the spread of this disease, it is crucial to monitor and detect any infected person. The etiologic agent of COVID-19 is a novel coronavirus called SARS-CoV-2. The gold standard for the detection of the virus is the RT-qPCR method. This study evaluated two RNA extraction methods and four commercial RT-qPCR assays routinely used in diagnostic laboratories for detecting SARS-CoV-2 in human specimens from the upper respiratory tract. We analyzed a panel of 70 clinical samples with varying RNA loads. Our study demonstrated the significant impact of the diagnostic methods selected by the laboratory on the SARS-CoV-2 detection in clinical specimens with low viral loads.

Case Report of COVID-19 after Full Vaccination: Viral Loads and Anti-SARS-CoV-2 Antibodies.

The introduction of effective vaccines against SARS-CoV-2 is expected to prevent COVID-19. However, sporadic cases of infection in vaccinated persons have been reported. We describe a case of a double-dose vaccinated woman with COVID-19. All stages of infection were observed, from no identification of virus, then the start of the infection, a high viral load, coming out of viraemia, and finally no detection of the virus. Despite the high viral load, the woman demonstrated mild COVID-19 symptoms, manifested only by a sore throat. The antibody results showed that she produced both post-infectious and post-vaccination immune responses. Phylogenetic analysis of the obtained viral genome sequence indicated that the virus belonged to the UK SARS-CoV-2 lineage B.1.1.7 (GR 501Y.V1; 20I/S:501Y.V1; Alpha variant).

Neutralization of cholera toxin by Rosaceae family plant extracts.

BACKGROUND: Cholera is one of the most deadly diarrheal diseases that require new treatments. We investigated the neutralization of cholera toxin by five plant extracts obtained from the Rosaceae family that have been traditionally used in Poland to treat diarrhea (of unknown origin). METHODS: Hot water extracts were prepared from the dried plant materials and lyophilized before phytochemical analysis and assessment of antimicrobial activity using microdilution assays. The ability of the plant extracts to neutralize cholera toxin was analyzed by measurement of cAMP levels in cell cultures, enzyme-linked immunosorbent assay and electrophoresis, as well as flow cytometry and fluorescence microscopy studies of fluorescent-labeled cholera toxins with cultured human fibroblasts. RESULTS: The antimicrobial assays displayed modest bacteriostatic potentials. We found that the plant extracts modulate the effects of cholera toxin on intracellular cAMP levels. Three plant extracts (Agrimonia eupatoria L., Rubus fruticosus L., Fragaria vesca L.) suppressed the binding of subunit B of cholera toxin to the cell surface and immobilized ganglioside GM1 while two others (Rubus idaeus L., Rosa.canina L.) interfered with the toxin internalization process. CONCLUSIONS: The traditional application of the Rosaceae plant infusions for diarrhea appears relevant to cholera, slowing the growth of pathogenic bacteria and either inhibiting the binding of cholera toxin to receptors or blocking toxin internalization. The analyzed plant extracts are potential complements to standard antibiotic treatment and Oral Rehydration Therapy for the treatment of cholera.

Activation of mammalian terget of rapamycin kinase and glycogen synthase kinase-3ß accompanies abnormal accumulation of cholesterol in fibroblasts from Niemann-Pick type C patients.

BACKGROUND: Niemann Pick type C (NPC) lysosomal disorder is linked to the disruption of cholesterol transport. Recent data suggest that the molecular background of this disease is more complex. It was found that accumulation of cholesterol and glycolipids in the late endosomal/lysosomal compartment of NPC1 cells may affect mitochondrial functions. MATERIALS AND METHODS: In this study, primary skin fibroblasts derived from skin biopsies of two anonymous patients with NPC-carrying mutations in the NPC1 gene, characterized by a high total cholesterol content, as well as two healthy donors were used. The presence of signaling proteins in the whole cell lysates and mitochondrial fractions were examined by Western blotting assay. RESULTS: In this report, we provide experimental evidence that in NPC1 cells, dysfunction of mitochondria and cellular metabolism, as reported by Wos et al in 2016, coexist with alterations in signal transduction pathways, such as the mammalian target of rapamycin, AKT, phosphoinositide-dependent protein kinase-1, glycogen synthase kinase-3 ß, and Jun amino-terminal kinase, leading to abnormal cholesterol accumulation and distribution. CONCLUSION: Differences in signal transduction between control and NPC1 cells may suggest that the latter cells experienced significant alterations in the complex molecular mechanisms that control cellular energy metabolism and vesicular transport.

Analysis of Minerals Produced by hFOB 1.19 and Saos-2 Cells Using Transmission Electron Microscopy with Energy Dispersive X-ray Microanalysis.

This video presents the use of transmission electron microscopy with energy dispersive X-ray microanalysis (TEM-EDX) to compare the state of minerals in vesicles released by two human bone cell lines: hFOB 1.19 and Saos-2. These cell lines, after treatment with ascorbic acid (AA) and ß-glycerophosphate (ß-GP), undergo complete osteogenic transdifferentiation from proliferation to mineralization and produce matrix vesicles (MVs) that trigger apatite nucleation in the extracellular matrix (ECM). Based on Alizarin Red-S (AR-S) staining and analysis of the composition of minerals in cell lysates using ultraviolet (UV) light or in vesicles using TEM imaging followed by EDX quantitation and ion mapping, we can infer that osteosarcoma Saos-2 and osteoblastic hFOB 1.19 cells reveal distinct mineralization profiles. Saos-2 cells mineralize more efficiently than hFOB 1.19 cells and produce larger mineral deposits that are not visible under UV light but are similar to hydroxyapatite (HA) in that they have more Ca and F substitutions. The results obtained using these techniques allow us to conclude that the process of mineralization differs depending on the cell type. We propose that, at the cellular level, the origin and properties of vesicles predetermine the type of minerals.

Vesicles of the intracellular and extracellular transport - key structures in the process of tissue differentiation towards bone and cartilage.

Differentiation of cells of the skeletal tissue, such as osteoblasts and chondrocytes, into mineralization-competent cells is a necessary step of the physiological process of bone and cartilage mineralization. Vascular cell calcification accompanies a pathological process of atherosclerotic plaque formation, which occurs due to trans-differentiation of vascular smooth muscle cells into cells resembling bone mineralization-competent cells. The activity of tissue-nonspecific alkaline phosphatase (TNAP), an enzyme necessary for physiological mineralization, is also induced in vascular cells in response to inflammation. TNAP acquires its mineralizing function when anchored to the plasma membrane (PM) of mineralizing cells and to the surface of vesicles derived from these cells. Numerous important reports indicate that various types of vesicles play a crucial role in initiating cell differentiation. In this review, we would like to highlight various functions of different types of vesicular structures of the cellular transport machinery such as intracellular vesicles (IVs), extracellular vesicles (EVs) or matrix vesicles (MVs) at distinct stages of both physiological and pathological processes of tissue differentiation.

Bacterial type AB5 enterotoxins--structure, function and mechanism of action.

One of the main factors causing bacterial diarrhea are AB5 enterotoxins. This group is divided into four families: pertussis toxin, cholera toxin, shiga toxin and subtilase cytotoxin. In this review we will describe the activity, structure and function of the cholera and shiga toxin families. The AB5 enterotoxins contain a catalytic subunit A and pentameric subunit B, which binds to the cell surface within lipid rafts. The cholera toxin family cause the constitutive activation of Gsa protein, which results in cAMP production, an opening of the chloride channels and releases chloride ions into the lumen of the small intestine. In contrast, the shiga toxin family has a cytotoxic effect on epithelial cells. It can inhibit protein synthesis leading to cell death. Although AB5 has a toxic activity, the B5 subunits have a significant potential as a transporter for proteins with anticancer activity and as a tool for the visualization of lipid rafts and cancer cells.

Use of plant extracts to control and treat AB5 enterotoxin-related diarrhea.

Plants contain a broad spectrum of small molecules with potential antimicrobial properties. Here, we review the antimicrobial activities of plant extracts against enterotoxic bacteria encoding AB5 toxins, including Vibrio cholerae, Shigella dysenteriae and enterotoxic Escherichia coli strains. Several plant extracts have strong antimicrobial effects and the potential to boost Oral Rehydration Therapy, which is the first line of treatment for acute diarrhea.