Cíl studie: Identifikace a kvantifikace prediktivních microRNA (miRNA) markerů umožňujících včasnou diagnostiku COVID-19. Typ studie: prospektivní Název a sídlo pracoviště: Ústav laboratorní medicíny, Fakultní nemocnice Ostrava, 17. listopadu 1790/5, 708 52 Ostrava Poruba Materiál a metody: Do studie bylo zařazeno osm pacientů, kteří se nakazili virem SARS-CoV-2, a byli sledováni v čase. Odběr vzorků (krevní sérum a výtěr z nosohltanu) byl proveden na Klinice infekčního lékařství a Klinice anesteziologie, resuscitace a intenzivní medicíny Fakultní nemocnice Ostrava. Stanovení microRNA bylo provedeno metodou Two-Tailed qPCR (TT-qPCR), izolace microRNA probíhala na izolátoru iCatcher 12 (CatchGene Co.) a pro reverzní transkripci s následnou detekcí byl použit detekční systém CFX96TM Real-Time (Bio-Rad). Ke statistickému zpracování dat byl použit software MS Excel a MedCalc®. Výsledky: Do studie bylo zařazeno sedm mužů (87,5 %) a jedna žena (12,5 %). Bland-Altmanova analýza neprokázala statisticky významný rozdíl mezi vzorky krevního séra a výtěry z nosohltanu u všech studovaných miRNA. Nejvýznamnější změny v expresi microRNA během onemocnění COVID-19 byly zjištěny u hsa-miR-23a-3p, hsa-miR-146a-5p a hsa-miR-222-3p. Tyto microRNA byly označeny jako potenciální prognostické diagnostické markery na základě shody výsledků dvou statistických testů. Závěr: Využití TT-qPCR pro kvantifikaci exprese miRNA představuje inovativní a perspektivní strategii v diagnostice COVID-19. Identifikace miRNA biomarkerů s dynamickými změnami exprese v průběhu infekce může významně přispět k rozvoji moderních diagnostických nástrojů. Pro potvrzení těchto nálezů a jejich integraci do klinické praxe je však nezbytné provést další validace prostřednictvím rozsáhlých klinických studií.
Settings: Department of Laboratory Medicine, University Hospital Ostrava, 17. listopadu 1790/5, 708 52 Ostrava Poruba Material and Methods: Eight patients who were infected with SARS-CoV-2 virus a were tracked over time were included in the study. Sampling (blood serum and nasopharyngeal swab) was performed at the Department of Infectious Medicine and the Department of Anesthesiology, Resuscitation and Intensive Care Medicine of the University Hospital Ostrava. Determination of microRNA was performed by Two-Tailed qPCR (TT-qPCR), isolation of microRNA was performed on the iCatcher 12 isolator (CatchGene Co.) and the CFX96TM Real-Time detection system (Bio-Rad) was used for reverse transcription followed by detection. MS Excel and MedCalc® software were used for statistical data processing. Results: Seven men (87.5%) and one woman (12.5%) were included in the study. Bland-Altman analysis showed no statistically significant difference between blood serum samples and nasopharyngeal swabs for all studied miRNAs. The most significant changes in microRNA expression during COVID-19 disease were found in hsa-miR-23a-3p, hsa-miR-146a-5p and hsa-miR-222-3p. These microRNAs were identified as potential prognostic diagnostic markers based on the concordance of the results of two statistical tests. Conclusion: The use of TT-qPCR for quantification of miRNA expression represents an innovative and promising strategy in the diagnosis of COVID-19. Identification of miRNA biomarkers with dynamic changes in expression during infection may significantly contribute to the development of modern diagnostic tools. However, further validation through large-scale clinical studies is necessary to confirm these findings and integrate them into clinical practice.
BACKGROUND: The anti-PD-1 monoclonal antibody pembrolizumab has been shown to be associated with a good response in patients with metastatic gastric cancer. Excellent therapeutic results of pembrolizumab have been shown in patients with tumours showing a high microsatellite instability (MSI) and Epstein–Barr virus (EBV) positivity. GOAL: This is a retrospective study of 40 bioptic specimens from the patients, who underwent gastrectomy for gastric carcinoma. The goal of the study was to identify biomarkers (EBV, MLH-1, PDL-1 expression) that are potentially relevant for selecting the patients, who may benefit from PD-1 inhibition therapy. METHODS: Immunohistochemical (IHC) expression of PDL-1 and MSI, cytogenetic FISH amplification of the HER-2/neu gene and polymerase chain reaction of EBV RNA, including charge quantification, were performed in selected patients with metastatic or advanced gastric cancer. RESULTS: EBV-encoded RNA was detected in nine patients. None of them exhibited Her-2 overexpression or CMV infection. PD-L1 was detected in twelve patients. Ten patients were MLH1 positive. All nine cases of EBV infection showed a high expression of PD-L1 and MLH-1 (Tab. 1, Ref. 14).
- MeSH
- B7-H1 Antigen analysis immunology MeSH
- Biomarkers * analysis MeSH
- Gene Expression MeSH
- Epstein-Barr Virus Infections diagnosis immunology MeSH
- Clinical Laboratory Techniques methods MeSH
- Middle Aged MeSH
- Humans MeSH
- Microsatellite Instability MeSH
- MutL Protein Homolog 1 analysis immunology MeSH
- Stomach Neoplasms * immunology physiopathology MeSH
- Specimen Handling MeSH
- Receptor, ErbB-2 analysis immunology MeSH
- Retrospective Studies MeSH
- Aged MeSH
- Patient Selection MeSH
- Check Tag
- Middle Aged MeSH
- Humans MeSH
- Aged MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
OBJECTIVES: RA patients who fail to respond to MTX can receive biologic dMARDs (bDMARDs). The Torque Teno Virus (TTV) is a potential novel candidate for monitoring of immunosuppression. We explore TTV in these patients and its association with clinical response to bDMARDs. METHODS: The BioBio Study is a multicentre randomized open-label trial, including RA patients with insufficient response to MTX. Patients were randomized to either TNFi (infliximab, INF), anti-IL-6 (tocilizumab, TCZ), CTLA4-Ig (abatacept, ABA) or anti-CD20 (rituximab, RTX) in addition to MTX. PCR was used to quantify TTV in the peripheral blood. RESULTS: TTV was measured in 95 patients (INF, n = 23; TCZ, n = 22; ABA, n = 27; RTX; n = 23). TTV increased by a median of 4.5 × 104 copies/ml [c/ml; interquartile range (IQR) 0-7.5 × 105] after 3 months. TTV levels at month 3 were associated with the Simplified Disease Activity Index (SDAI) (P = 0.03) and the Clinical Disease Activity Index (CDAI) response (P = 0.026) at month 6. A TTV cut-off level of 1.2 × 106 c/ml at month 3 had a positive likelihood ratio of 2.7 for prediction of an 85% reduction in SDAI at month 6. CONCLUSION: Our data suggest that TTV levels increase upon TNF, CD20 and costimulation blockade and are associated with the clinical response to bDMARDs in RA patients. TRIAL REGISTRATION: ClinicalTrials.gov; https://clinicaltrials.gov; NCT01638715.
- MeSH
- Abatacept therapeutic use MeSH
- Antirheumatic Agents * therapeutic use MeSH
- Biological Products * therapeutic use MeSH
- Immunomodulation MeSH
- Humans MeSH
- Arthritis, Rheumatoid * drug therapy MeSH
- Torque teno virus * MeSH
- Treatment Outcome MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Multicenter Study MeSH
- Research Support, Non-U.S. Gov't MeSH
- Randomized Controlled Trial MeSH
The COVID-19 outbreak has raised questions about how vulnerable groups experience the pandemic. Research that focuses on the view of individuals with pre-existing mental health conditions is still limited, and so are cross-country comparative surveys. We gathered our sample of qualitative data during the first lockdown after governmental measures against the spread of the SARS-CoV-2 virus came into force in Austria, Czechia, Germany, and Slovakia. A total of n = 1690 psychotherapists from four middle European countries answered the question of how the COVID-19 pandemic was addressed in sessions by their patients during the early stage of unprecedented public health conditions. We employed a descriptive qualitative methodology to determine themes following levels of the social-ecological model (SEM) regarding how the COVID-19 pandemic affected patients. At the public policy level, stressful environmental conditions concerned the governmental mitigation efforts. At the level of community/society, reported key themes were employment, restricted access to educational and health facilities, socioeconomic consequences, and the pandemic itself. Key themes at the interpersonal level regarded forced proximity, the possibility of infection of loved ones, childcare, and homeschooling. Key themes at the individual level were the possibility of contracting COVID-19, having to stay at home/isolation, and a changing environment. Within the SEM framework, adaptive and maladaptive responses to these stressors were reported, with more similarities than differences between the countries. A quantification of word stems showed that the maladaptive reactions predominated.
- MeSH
- COVID-19 * epidemiology MeSH
- Communicable Disease Control MeSH
- Humans MeSH
- Pandemics MeSH
- Psychotherapists MeSH
- SARS-CoV-2 MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
Mycobacterium avium subsp. paratuberculosis (MAP) is a pathogenic bacterium causing the paratuberculosis, chronic and infectious disease common particularly in wild and domestic ruminants. Currently, culture techniques to detect viable MAP are still used most commonly, although these require a long incubation period. Consequently, a faster molecular method for assessing MAP cell viability based on cell membrane integrity was introduced consisting of sample treatment with the intercalation dye propidium monoazide (PMA) followed by quantitative PCR (qPCR). However, the PMA-qPCR assay is complicated by demanding procedures involving work in a darkroom and on ice. In this study, we therefore optimized a viability assay combining sample treatment with palladium (Pd) compounds as an alternative viability marker to PMA, which does not require such laborious procedures, with subsequent qPCR. The optimized Pd-qPCR conditions consisting of 90 min exposure to 30 μM bis(benzonitrile)dichloropalladium(II) or 30 μM palladium(II)acetate at 5 °C and using ultrapure water as a resuspension medium resulted in differences in quantification cycle (Cq) values between treated live and dead MAP cells of 8.5 and 7.9, respectively, corresponding to approximately 2.5 log units. In addition, Pd-qPCR proved to be superior to PMA-qPCR in distinguishing between live and dead MAP cells. The Pd-qPCR viability assay thus has the potential to replace time-consuming culture methods and demanding PMA-qPCR in the detection and quantification of viable MAP cells with possible application in food, feed, clinical and environmental samples.
- MeSH
- Azides pharmacology MeSH
- Biological Assay MeSH
- Real-Time Polymerase Chain Reaction methods MeSH
- Microbial Viability MeSH
- Mycobacterium avium subsp. paratuberculosis * genetics MeSH
- Palladium pharmacology MeSH
- Paratuberculosis * microbiology MeSH
- Propidium pharmacology MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
COVID-19 is caused by the SARS-CoV-2 virus and has spread globally in 2020. Cellular immunity may serve as an important functional marker of the disease, especially in the asymptomatic cases. Blood samples were collected from 46 convalescent donors with a history of COVID-19 and 38 control donors. Quantification of the T-cell response upon contact with SARS-CoV-2 proteins in vitro was based on IFN-γ. Significantly higher numbers of activated cells were measured in patients who underwent COVID-19. Anti-SARS-CoV-2 T cells were detected weeks after the active virus disappeared from the organism. Repeated sample collection after five months proved that the T-cell activation was weaker in time in 79 % of the patients. In the majority of cases, the CD4+ helper T-cell subpopulation was responsible for the immune reaction. Moreover, different viral proteins triggered activation in CD4+ helper and in CD8+ cytotoxic T cells. Together, these findings suggest that the T-cell activation level identifies the individuals who underwent COVID-19 and may become a diagnostic tool for the disease.
- MeSH
- Lymphocyte Activation MeSH
- COVID-19 * MeSH
- Humans MeSH
- Antibodies, Viral MeSH
- SARS-CoV-2 MeSH
- T-Lymphocytes MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
Proper assembly and disassembly of both immature and mature HIV-1 hexameric lattices are critical for successful viral replication. These processes are facilitated by several host-cell factors, one of which is myo-inositol hexaphosphate (IP6). IP6 participates in the proper assembly of Gag into immature hexameric lattices and is incorporated into HIV-1 particles. Following maturation, IP6 is also likely to participate in stabilizing capsid protein-mediated mature hexameric lattices. Although a structural-functional analysis of the importance of IP6 in the HIV-1 life cycle has been reported, the effect of IP6 has not yet been quantified. Using two in vitro methods, we quantified the effect of IP6 on the assembly of immature-like HIV-1 particles, as well as its stabilizing effect during disassembly of mature-like particles connected with uncoating. We analyzed a broad range of molar ratios of protein hexamers to IP6 molecules during assembly and disassembly. The specificity of the IP6-facilitated effect on HIV-1 particle assembly and stability was verified by K290A, K359A, and R18A mutants. In addition to IP6, we also tested other polyanions as potential assembly cofactors or stabilizers of viral particles.IMPORTANCE Various host cell factors facilitate critical steps in the HIV-1 replication cycle. One of these factors is myo-inositol hexaphosphate (IP6), which contributes to assembly of HIV-1 immature particles and helps maintain the well-balanced metastability of the core in the mature infectious virus. Using a combination of two in vitro methods to monitor assembly of immature HIV-1 particles and disassembly of the mature core-like structure, we quantified the contribution of IP6 and other small polyanion molecules to these essential steps in the viral life cycle. Our data showed that IP6 contributes substantially to increasing the assembly of HIV-1 immature particles. Additionally, our analysis confirmed the important role of two HIV-1 capsid lysine residues involved in interactions with IP6. We found that myo-inositol hexasulphate also stabilized the HIV-1 mature particles in a concentration-dependent manner, indicating that targeting this group of small molecules may have therapeutic potential.
- MeSH
- gag Gene Products, Human Immunodeficiency Virus chemistry genetics metabolism MeSH
- HIV-1 chemistry genetics MeSH
- Mutation, Missense MeSH
- Polymers chemistry MeSH
- Virus Assembly * MeSH
- Amino Acid Substitution MeSH
- Structure-Activity Relationship MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Mathematical models of in vitro viral kinetics help us understand and quantify the main determinants underlying the virus-host cell interactions. We aimed to provide a numerical characterization of the Zika virus (ZIKV) in vitro infection kinetics, an arthropod-borne emerging virus that has gained public recognition due to its association with microcephaly in newborns. The mathematical model of in vitro viral infection typically assumes that degradation of extracellular infectious virus proceeds in an exponential manner, that is, each viral particle has the same probability of losing infectivity at any given time. We incubated ZIKV stock in the cell culture media and sampled with high frequency for quantification over the course of 96 h. The data showed a delay in the virus degradation in the first 24 h followed by a decline, which could not be captured by the model with exponentially distributed decay time of infectious virus. Thus, we proposed a model, in which inactivation of infectious ZIKV is gamma distributed and fit the model to the temporal measurements of infectious virus remaining in the media. The model was able to reproduce the data well and yielded the decay time of infectious ZIKV to be 40 h. We studied the in vitro ZIKV infection kinetics by conducting cell infection at two distinct multiplicity of infection and measuring viral loads over time. We fit the mathematical model of in vitro viral infection with gamma distributed degradation time of infectious virus to the viral growth data and identified the timespans and rates involved within the ZIKV-host cell interplay. Our mathematical analysis combined with the data provides a well-described example of non-exponential viral decay dynamics and presents numerical characterization of in vitro infection with ZIKV.
- MeSH
- Chlorocebus aethiops MeSH
- Zika Virus Infection virology MeSH
- Kinetics MeSH
- Virus Replication * MeSH
- Reproducibility of Results MeSH
- Models, Theoretical * MeSH
- Vero Cells MeSH
- Viral Load MeSH
- Zika Virus growth & development physiology MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
Some viroids-single-stranded, non-coding, circular RNA parasites of plants-are not transmissible through pollen to seeds and to next generation. We analyzed the cause for the elimination of apple fruit crinkle viroid (AFCVd) and citrus bark cracking viroid (CBCVd) from male gametophyte cells of Nicotiana tabacum by RNA deep sequencing and molecular methods using infected and transformed tobacco pollen tissues at different developmental stages. AFCVd was not transferable from pollen to seeds in reciprocal pollinations, due to a complete viroid eradication during the last steps of pollen development and fertilization. In pollen, the viroid replication pathway proceeds with detectable replication intermediates, but is dramatically depressed in comparison to leaves. Specific and unspecific viroid degradation with some preference for (-) chains occurred in pollen, as detected by analysis of viroid-derived small RNAs, by quantification of viroid levels and by detection of viroid degradation products forming "comets" on Northern blots. The decrease of viroid levels during pollen development correlated with mRNA accumulation of several RNA-degrading factors, such as AGO5 nuclease, DICER-like and TUDOR S-like nuclease. In addition, the functional status of pollen, as a tissue with high ribosome content, could play a role during suppression of AFCVd replication involving transcription factors IIIA and ribosomal protein L5.
Cíl: Porovnat rozsah postižení plic u pacientů hospitalizovaných na standardním oddělení a na JIP na základě analýzy dat pomocí umělé inteligence. Metodika: Retrospektivně jsme zhodnotili soubor 50 pacientů, kteří měli provedené CT hrudníku indikované z důvodu podezření z nákazy virem SARS-CoV-2 s následně potvrzeným pozitivním RT-PCR testem. Nemocní byli vyšetřeni v době první vlny epidemie v březnu a dubnu 2020. Soubor pacientů byl rozdělen do dvou skupin podle hospitalizace na jednotce intenzivní péče (skupina A - 19) či na standardním oddělení (skupina B - 31). U těchto pacientů jsme provedli analýzu dat pomocí prototypu softwaru Siemens Healthineers´ interactive CT Pneumonia Analysis. Výsledky: Průměrný procentuální podíl opacit vůči celkovému objemu plic u souboru A (pacienti hospitalizovaní na JIP) byl 31,56 %, u souboru B (pacienti hospitalizovaní na standardním oddělení) byl 11,25 %. Procentuální podíl konsolidací vůči celkovému objemu plic byl u souboru A 5,25 % a u souboru B 2,09 %. Průměrné Infection Score je u souboru A 8,32 a u souboru B 3,35. Predispozice dolních laloků s nejvíce postiženým pravým dolním lalokem (39,17 % u souboru A a 18,65 % u souboru B). Závěr: Automatické hodnocení postižení plic s možností kvantifikace postižení plicní tkáně dovoluje rychlý staging plicního postižení, stanovuje podíl postižené plicní tkáně. Skóre rozsahu postižení dovoluje využít jako jeden z objektivizujících parametrů klinického stavu nemocného, který lze zvažovat při odhadnutí nutnosti intenzivní péče.
Aim: To compare the extent of the lung involvement hospitalized at the non-intensive care unit and intensive care unit using the analysis assisted by the artificial intelligence. Method: The retrospective analysis of the sample of 50 patients, who underwent chest CT indicated due to the suspected SARS-CoV-2 infection and followed by the confirmation of the diagnosis by the PCR test. The patients were investigated during spring wave of the epidemic between March and April 2020. The sample was split into two groups hospitalized at the non-ICU (31 pt.) and ICU (19 pt.). We analyzed the data in these patients using software prototype Interactive CT Pneumonia Analysis provided by Siemens Heathineers. The percentage of the consolidation and ground glass opacities were quantified in lobes and lungs. Results: The mean percentage of the opacities related to the whole volume of lungs in patients hospitalized in ICU was 31.56%, 11.25 in patients in non-ICU respectively; the consolidation was affected 5.2% in ICU group, 2.09% in non-ICU group. Domination of right lower lobe was in 39.17% in ICU patients, 18.65% in group of non-ICU patients. Conclusion: The assessment of the lung pa-renchyma load using lung infection AS assisted analysis enabling the quantification of the affected volume, makes possible to score the patients and making the parametric staging, it allows to estimate the need of intensive care.
