The early identification of asymptomatic yet infectious cases is vital to curb the 2019 coronavirus (COVID-19) pandemic and to control the disease in the post-pandemic era. In this paper, we propose a fast, inexpensive and high-throughput approach using painless nasal-swab self-collection followed by direct RT-qPCR for the sensitive PCR detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This approach was validated in a large prospective cohort study of 1038 subjects, analysed simultaneously using (1) nasopharyngeal swabs obtained with the assistance of healthcare personnel and analysed by classic two-step RT-qPCR on RNA isolates and (2) nasal swabs obtained by self-collection and analysed with direct RT-qPCR. Of these subjects, 28.6% tested positive for SARS-CoV-2 using nasopharyngeal swab sampling. Our direct RT-qPCR approach for self-collected nasal swabs performed well with results similar to those of the two-step RT-qPCR on RNA isolates, achieving 0.99 positive and 0.98 negative predictive values (cycle threshold [Ct] < 37). Our research also reports on grey-zone viraemia, including samples with near-cut-off Ct values (Ct ≥ 37). In all investigated subjects (n = 20) with grey-zone viraemia, the ultra-small viral load disappeared within hours or days with no symptoms. Overall, this study underscores the importance of painless nasal-swab self-collection and direct RT-qPCR for mass testing during the SARS-CoV-2 pandemic and in the post-pandemic era.
- MeSH
- COVID-19 diagnóza prevence a kontrola MeSH
- diagnostické testy rutinní metody MeSH
- klinické laboratorní techniky metody MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- lidé MeSH
- odběr biologického vzorku metody MeSH
- plošný screening metody MeSH
- průzkumy a dotazníky MeSH
- samovyšetření metody MeSH
- SARS-CoV-2 genetika MeSH
- senzitivita a specificita MeSH
- testování na COVID-19 metody MeSH
- virová nálož metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- validační studie MeSH
BACKGROUND: PNPLA3 rs738409 minor allele c.444G represents a risk factor for liver steatosis and fibrosis progression also in chronic hepatitis C (HCV). We investigated its impact on the timing of liver transplantation (LT) in patients with genotype 1b HCV cirrhosis. METHODS: We genotyped and evaluated 172 LT candidates with liver cirrhosis owing to chronic HCV infection, genotype 1b. One hundred patients needed LT for chronic liver failure (CLF) and 72 for a small hepatocellular carcinoma (HCC) in the cirrhotic liver without CLF. Population controls (n = 647) were selected from the Czech cross-sectional study MONICA. RESULTS: The CLF patients were younger (53.5 ± 7.2 vs. 59.6 ± 6.6, P < 0.001) with more advanced liver disease than HCC patients (Child-Pugh's score 9.1 ± 1.8 vs. 7.1 ± 1.9, P < 0.001, MELD 14.1 ± 3.9 vs. 11.1 ± 3.7, P < 0.001). PNPLA3 G allele increased the risk of LT for CLF in both allelic and recessive models (CG + GG vs. CC: OR, 1.90; 95% CI, 1.017-3.472, P = 0.045 and GG vs. CC + CG: OR, 2.94; 95% CI, 1.032-7.513, P = 0.042). Multivariate analysis identified younger age (P < 0.001) and the G allele (P < 0.05) as risk factors for CLF. The genotype frequencies between the CLF group and MONICA study significantly differed in both, allelic and recessive model (P = 0.004, OR 1.87, 95% CI 1.222-2.875; P < 0.001, OR 3.33, 95% CI 1.824-6.084, respectively). The OR values almost doubled in the recessive model compared with the allelic model suggesting the additive effect of allele G. In contrast, genotype frequencies in the HCC group were similar to the MONICA study in both models. Pretransplant viral load was significantly lower in GG than in CC + CG genotypes (median, IQR; 162,500 (61,550-319,000) IU/ml vs. 570,000 (172,000-1,595,000) IU/ml, P < 0.0009). CONCLUSIONS: Our results suggest that PNPLA3 rs738409 G allele carriage may be associated with a faster progression of HCV cirrhosis to chronic liver failure.
- MeSH
- alely MeSH
- chronická hepatitida C genetika virologie MeSH
- genetická predispozice k nemoci genetika MeSH
- genotyp MeSH
- hepatocelulární karcinom genetika virologie MeSH
- jaterní cirhóza genetika virologie MeSH
- játra virologie MeSH
- jednonukleotidový polymorfismus genetika MeSH
- lidé středního věku MeSH
- lidé MeSH
- lipasa genetika MeSH
- membránové proteiny genetika MeSH
- nádory jater genetika virologie MeSH
- průřezové studie MeSH
- retrospektivní studie MeSH
- selhání jater genetika virologie MeSH
- studie případů a kontrol MeSH
- transplantace jater metody MeSH
- virová nálož metody MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Počet osob s infekcí způsobené lidským virem imunodeficience (Human Immunodeficiency Virus, HIV) v České republice stále stoupá. Narůstající počet HIV pozitivních gravidních žen není tolik výrazný a zásluhou profylaktického programu se riziko vertikálního přenosu výrazně snížilo. Profylaktický program je rozdělen do 4 částí (antiretrovirová léčba během gravidity, porod císařským řezem a nahrazení kojení mléčnou stravou, užívání antiretrovirové suspenze během prvních šesti týdnů života novorozence). Dispenzarizační program je do 18 měsíců věku dítěte, kdy je dítě po finálním negativním PCR testu (polymerázová řetězová reakce) vyřazeno z evidence.
A number of people with human immunodeficiency virus infection (Human Immunodeficiency Virus, HIV) are still rising in theCzech Republic. The increasing number of HIV-positive pregnant women is not so significant due to the prophylactic program,the risk of vertical transmission has significantly decreased. The prophylactic program is divided into 4 parts (antiretroviral treatmentduring pregnancy, birth by caesarean section and replacing breastfeeding with a milk diet, using antiretroviral suspensionduring the first six weeks of newborn life). The dispensation program is up to 18 months of child age when the child is removedfrom the evidence after the final negative PCR test (polymerase chain reaction).
- MeSH
- adherence k farmakoterapii MeSH
- antiretrovirové látky aplikace a dávkování MeSH
- chemoprofylaxe MeSH
- dítě MeSH
- HIV infekce diagnóza farmakoterapie MeSH
- infekční komplikace v těhotenství * diagnóza farmakoterapie ošetřování virologie MeSH
- lidé MeSH
- těhotenství MeSH
- vedení porodu MeSH
- vertikální přenos infekce prevence a kontrola statistika a číselné údaje MeSH
- virová nálož metody účinky léků MeSH
- zidovudin aplikace a dávkování MeSH
- Check Tag
- dítě MeSH
- lidé MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
After its computational inference from human stool metagenomes, the CrAssphage has proven to be the most prevalent phage in the human gut, with presumably very wide geographic distribution. The currently available molecular assays do not sufficiently reflect the CrAssphage sequence variability. Here, we report a novel real-time PCR assay whose primers and probes are derived from data of multiple CrAssphage strains obtained from gut viral metagenomes of European, Asian, and African subjects. This assay can be useful in analyses of putative bacterial host co-occurence, and in association studies of non-infectious diseases where the phage may modify the content of gut bacteriomes.
- MeSH
- bakteriofágy genetika izolace a purifikace MeSH
- DNA primery genetika MeSH
- kvantitativní polymerázová řetězová reakce metody MeSH
- lidé MeSH
- mikrobiota * MeSH
- oligonukleotidové sondy genetika MeSH
- střevní mikroflóra * MeSH
- virová nálož metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Afrika MeSH
- Asie MeSH
- Evropa MeSH
Mouse polyomavirus (MPyV) is a member of the Polyomaviridae family, which comprises non-enveloped tumorigenic viruses infecting various vertebrates including humans and causing different pathogenic responses in the infected organisms. Despite the variations in host tropism and pathogenicity, the structure of the virions of these viruses is similar. The capsid, with icosahedral symmetry (ø, 45 nm, T = 7d), is composed of a shell of 72 capsomeres of structural proteins, arranged around the nucleocore containing approximately 5-kbp-long circular dsDNA in complex with cellular histones. MPyV has been one of the most studied polyomaviruses and serves as a model virus for studies of the mechanisms of cell transformation and virus trafficking, and for use in nanotechnology. It can be propagated in primary mouse cells (e.g., in whole mouse embryo cells) or in mouse epithelial or fibroblast cell lines. In this unit, propagation, purification, quantification, and storage of MPyV virions are presented.
Quantitation of viruses is practised widely in both basic and applied virology. Infectious titration in cell cultures, the most common approach to it, is quite labour-intensive and alternative protocols are therefore sought. One of the alternatives is transmission electron microscope (TEM) quantitation using latex particles at a known concentration as a reference for counting virus particles. If virus TCID₅₀ is determined in parallel, the ratio of infectious to non-infectious virus particles may be established. This study employs such an approach to compute the number of virus particles and TCID₅₀, and establish their correlation for three viruses: Canine adenovirus 1 (CAdV-1), Feline calicivirus (FCV) and Bovine herpesvirus 1 (BoHV-1). Each of the viruses was grown in five replicates until complete cytopathology was recorded (time 0), then frozen. They were thawed, filter-sterilised and left for additional periods of 16, 32 and 48 h at 37°C. At each time point, the infectious ability of the virus was characterised by TCID50 and the number of virions quantified by TEM, in order to evaluate the influence of timing on virus harvest. The virus particle count determined by TEM did not change for any of the viruses throughout the experiment. The relationship between virus particle counts with TCID₅₀ at time 0 showed good linearity response; their ratio was almost constant. The virus particle-to-TCID₅₀ ratio varied between 146 and 426 (mean±SD: 282±103) for CAdV-1, between 36 and 79 (57±18) for FCV and between 110 and 249 (167±53) for BoHV-1. The proportion of non-infectious particles did not change throughout the experiment for either CAdV-1 or BoHV-1. However, a decrease in virus infectious ability disclosed by TCID₅₀ indicated that the fraction of non-infectious particles in FCV increased 300,000 times when time 0 and 48 h were compared. The quantitation of viruses with TEM is a simple and rapid protocol for virus quantitation but account must be taken of the type of virus and harvesting time as virus counts need not necessarily correlate with virus infectious ability.
- MeSH
- bovinní herpesvirus 1 izolace a purifikace fyziologie MeSH
- buněčné linie MeSH
- časové faktory MeSH
- kočičí kalicivirus izolace a purifikace fyziologie MeSH
- kultivace virů MeSH
- mikrobiální viabilita * MeSH
- psí adenoviry izolace a purifikace fyziologie MeSH
- transmisní elektronová mikroskopie metody MeSH
- virová nálož metody MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
A rapid method for detection, discrimination and quantification of wheat and barley strains of wheat dwarf virus (WDV) was successfully developed. The sensitivity of quantification of the wheat and barley strains of WDV ranged from an average of 1.2 × 10(7)-1.2 × 10(2) and from an average of 1.4 × 10(7)-1.4 × 10(4) copies of viral genome, respectively. These standard serial dilutions were applied to plant and vector tissues for virus titer calculations. Both strains of WDV were clearly discriminated by specific probes and melting curve analysis. Both TaqMan(®) and SYBR(®) Green technologies provided accurate and reliable methods for monitoring, detection, discrimination, and quantification of WDV.
- MeSH
- Geminiviridae klasifikace genetika izolace a purifikace MeSH
- ječmen (rod) virologie MeSH
- kvantitativní polymerázová řetězová reakce metody MeSH
- nemoci rostlin virologie MeSH
- pšenice virologie MeSH
- senzitivita a specificita MeSH
- virová nálož metody MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
