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- MeSH
- kontrola potravin MeSH
- lidé MeSH
- potravní doplňky MeSH
- probiotika * MeSH
- Check Tag
- lidé MeSH
With the onset of the COVID-19 pandemic, a problem arose with classic body donation programmes for obtaining cadavers for anatomical dissections, science and research. The question has emerged whether bodies of individuals who died of COVID-19 or were infected by SARS-CoV-2 could be admitted to Departments of Anatomy. To determine the risk of SARS-CoV-2 transmission to employees or students, the presence and stability of SARS-CoV-2 RNA in cadavers after fixation agents' application and subsequent post-fixation baths over time were examined. The presence of viral RNA in swabs from selected tissues was assessed by the standardized routine RNA isolation protocol and subsequent real-time PCR analysis. To support the results obtained from the tissue swabs, samples of RNA were exposed in vitro to short and long-term exposure to the components of the injection and fixation solutions used for the bodies' conservation. Substantial removal of SARS-CoV-2 RNA was observed in post-mortem tissue following perfusion with 3.5% phenol, 2.2% formaldehyde, 11.8% glycerol and 55% ethanol, and subsequent post-fixation in an ethanol bath. In vitro experiments showed significant effects of formaldehyde on SARS-CoV-2 RNA, while phenol and ethanol showed only negligible effects. We conclude that cadavers subjected to fixation protocols as described here should not pose a considerable risk of SARS-CoV-2 infection while being handled by students and staff and are, therefore, suitable for routine anatomical dissections and teaching.
- MeSH
- COVID-19 * MeSH
- ethanol MeSH
- fenoly MeSH
- formaldehyd MeSH
- lidé MeSH
- mrtvola MeSH
- pandemie MeSH
- RNA virová MeSH
- SARS-CoV-2 * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
The risk of Alzheimer's disease (AD) has a strong genetic component, also in the case of late-onset AD (LOAD). Attempts to sequence whole genome in large populations of subjects have identified only a few mutations common to most of the patients with AD. Targeting smaller well-characterized groups of subjects where specific genetic variations in selected genes could be related to precisely defined psychological traits typical of dementia is needed to better understand the heritability of AD. More than one thousand participants, categorized according to cognitive deficits, were assessed using 14 psychometric tests evaluating performance in five cognitive domains (attention/working memory, memory, language, executive functions, visuospatial functions). CD36 was selected as a gene previously shown to be implicated in the etiology of AD. A total of 174 polymorphisms were tested for associations with cognition-related traits and other AD-relevant data using the next generation sequencing. Several associations between single nucleotide polymorphisms (SNP's) and the cognitive deficits have been found (rs12667404 with language performance, rs3211827 and rs41272372 with executive functions, rs137984792 with visuospatial performance). The most prominent association was found between a group of genotypes in six genetically linked and the age at which the AD patients presented with, or developed, a full-blown dementia. The identified alleles appear to be associated with a delay in the onset of LOAD. In silico studies suggested that the SNP's alter the expression of CD36 thus potentially affecting CD36-related neuroinflammation and other molecular and cellular mechanisms known to be involved in the neuronal loss leading to AD. The main outcome of the study is an identification of a set of six new mutations apparently conferring a distinct protection against AD and delaying the onset by about 8 years. Additional mutations in CD36 associated with certain traits characteristic of the cognitive decline in AD have also been found.
Introduction. Cystic fibrosis (CF) is a serious disease with multisystemic clinical signs that is easily and frequently complicated by bacterial infection. Recently, the prevalence of nontuberculous mycobacteria as secondary contaminants of CF has increased, with the Mycobacterium avium complex (MAC) and Mycobacterium abscessus complex (MABSC) being the most frequently identified. The MABSC includes subspecies of significant clinical importance, mainly due to their resistance to antibiotics.Gap statement. Sensitive method for early detection and differentiation of MABSC members and MAC complex for use in routine clinical laboratories is lacking. A method based on direct DNA isolation from sputum, using standard equipment in clinical laboratories and allowing uncovering of possible sample inhibition (false negative results) would be required. The availability of such a method would allow accurate and accelerated time detection of MABSC members and their timely and targeted treatment.Aim. To develop a real time multiplex assay for rapid and sensitive identification and discrimination of MABSC members and MAC complex.Methodology. The method of DNA isolation directly from the sputum of patients followed by quadruplex real-time quantitative PCR (qPCR) detection was developed and optimised. The sensitivity and limit of detection (LOD) of the qPCR was determined using human sputum samples artificially spiked with a known amount of M. abscessus subsp. massiliense (MAM).Results. The method can distinguish between MAC and MABSC members and, at the same time, to differentiate between M. abscessus subsp. abscessus/subsp. bolletii (MAAb/MAB) and MAM. The system was verified using 61 culture isolates and sputum samples from CF and non-CF patients showing 29.5 % MAAb/MAB, 14.7 % MAM and 26.2 % MAC. The LOD was determined to be 1 490 MAM cells in the sputum sample with the efficiency of DNA isolation being 95.4 %. Verification of the qPCR results with sequencing showed 100 % homology.Conclusions. The developed quadruplex qPCR assay, which is preceded by DNA extraction directly from patients' sputum without the need for culturing, significantly improves and speeds up the entire process of diagnosing CF patients and is therefore particularly suitable for use in routine laboratories.
- MeSH
- atypické mykobakteriální infekce * diagnóza farmakoterapie MeSH
- cystická fibróza * komplikace mikrobiologie MeSH
- DNA terapeutické užití MeSH
- intracelulární infekce bakterií Mycobacterium avium * epidemiologie MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- lidé MeSH
- Mycobacterium abscessus * genetika MeSH
- Mycobacterium avium komplex genetika MeSH
- netuberkulózní mykobakterie MeSH
- sputum mikrobiologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články 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
- azidy farmakologie MeSH
- biotest MeSH
- kvantitativní polymerázová řetězová reakce metody MeSH
- mikrobiální viabilita MeSH
- Mycobacterium avium subsp. paratuberculosis * genetika MeSH
- palladium farmakologie MeSH
- paratuberkulóza * mikrobiologie MeSH
- propidium farmakologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Mycobacterium avium subsp. paratuberculosis (MAP) represents a slow-growing bacterium causing paratuberculosis, especially in domestic and wild ruminants. Until recently, the assessment of MAP viability relied mainly on cultivation, which is very time consuming and is unable to detect viable but non-culturable cells. Subsequently, viability PCR, a method combining sample treatment with the DNA-modifying agent ethidium monoazide (EMA) or propidium monoazide (PMA) and quantitative PCR (qPCR), was developed, enabling the selective detection of MAP cells with an intact cell membrane. However, this technology requires a laborious procedure involving the need to work in the dark and on ice. In our study, a method based on a combination of platinum compound treatment and qPCR, which does not require such a demanding procedure, was investigated to determine mycobacterial cell viability. The conditions of platinum compound treatment were optimized for the fast-growing mycobacterium M. smegmatis using live and heat-killed cells. The optimal conditions consisting of a single treatment with 100 μM cis-dichlorodiammine platinum(II) for 60 min at 5°C resulted in a difference in quantification cycle (Cq) values between live and dead membrane-compromised mycobacterial cells of about 6 Cq corresponding to about 2 log10 units. This optimized viability assay was eventually applied to MAP cells and demonstrated a better ability to distinguish between live and heat-killed mycobacteria as compared to PMA. The viability assay combining the Pt treatment with qPCR thereby proved to be a promising method for the enumeration of viable MAP cells in foodstuffs, environmental, and clinical samples which could replace the time-consuming cultivation or laborious procedures required when using PMA.
- Publikační typ
- časopisecké články MeSH
At the time of sampling (2020/2021), the number of new cases of SARS-CoV-2-positive individuals in the Czech Republic significantly exceeded the numbers in neighboring countries and in the EU. In terms of the number of deaths, the country ranked near the top of the list. Legislative orders required wearing masks indoors, disinfecting surfaces in public places, and limiting the number of people per sales area in commercial spaces. Due to an situation, most schools and shops were closed. The entire country anticipated a total lockdown. To assess the risk to public health regarding SARS-CoV-2 transmission, air and surfaces were sampled in two public places: a post office and a shopping center. Samples were also collected at the COVID-19 unit at the local hospital. Neither air nor surface samples were positive for SARS-CoV-2 virus particles in the post office or shopping center. Positive results were found in the hospital ward, with floors being the most and highest contaminated surface. Based on our results, we believe that public places do not pose a risk in relation to SARS-CoV-2 transmission, especially when epidemiological measures to reduce transmission are followed, such as wearing masks, using disinfectant or limiting the number of customers per retail establishment.
- MeSH
- COVID-19 * MeSH
- kontrola infekčních nemocí MeSH
- lidé MeSH
- masky MeSH
- nemocnice MeSH
- SARS-CoV-2 * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Quantitative PCR (qPCR) has become a frequently employed direct method for the detection and quantification of Mycobacterium avium subsp. paratuberculosis (MAP). The quantity of MAP determined by qPCR, however, may be affected by the type of qPCR quantification standard used (PCR product, plasmid, genomic DNA) and the way in which standard DNA quantity is determined (absorbance, fluorescence). In practice, this can be reflected in the inability to properly compare quantitative data from the same qPCR assays in different laboratories. Thus, the aim of this study was to prepare a prototype of an international MAP reference standard, which could be used to calibrate routinely used qPCR quantification standards in various laboratories to promote clinical data comparability. Considering stability, storage and shipment issues, a lyophilised fecal suspension artificially contaminated with a MAP reference strain was chosen as the most suitable form of the standard. The effect of five types of lyophilisation matrices on standard stability was monitored on 2-weeks interval basis for 4 months by F57 qPCR. The lyophilisation matrix with 10% skimmed milk provided the best recovery and stability in time and was thus selected for subsequent comparative testing of the standard involving six diagnostic and research laboratories, where DNA isolation and qPCR assay procedures were performed with the parallel use of the identical supplied genomic DNA solution. Furthermore, the effect of storage conditions on the standard stability was tested for at least 6 months. The storage at room temperature in the dark and under light, at + 4 °C, - 20 °C and - 80 °C showed no significant changes in the stability, and also no substantial changes in MAP viability were found using phage amplification assay. The prepared MAP quantification standard provided homogeneous and reproducible results demonstrating its suitability for utilisation as an international reference qPCR standard.
- MeSH
- DNA bakterií klasifikace genetika MeSH
- feces chemie mikrobiologie MeSH
- kvantitativní polymerázová řetězová reakce normy MeSH
- lyofilizace MeSH
- Mycobacterium avium subsp. paratuberculosis klasifikace genetika izolace a purifikace MeSH
- nemoci skotu diagnóza MeSH
- paratuberkulóza diagnóza mikrobiologie MeSH
- referenční standardy MeSH
- senzitivita a specificita MeSH
- skot MeSH
- zvířata MeSH
- Check Tag
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Viruses are common causes of food- and waterborne diseases worldwide. Conventional identification of these agents is based on cultivation, antigen detection, electron microscopy, or real-time PCR. Because recent technological advancements in detection methods are focused on fast and robust analysis, a rapid multiplexing technology, which can detect a broad spectrum of pathogenic viruses connected to food or water contamination, was utilized. A new semiquantitative magnetic bead-based multiplex system has been designed for simultaneous detection of several targets in one reaction. The system includes adenoviruses 40/41 (AdV), rotavirus A (RVA), norovirus (NoV), hepatitis E virus (HEV), hepatitis A virus (HAV), and a target for external control of the system. To evaluate the detection system, interlaboratory ring tests were performed in four independent laboratories. Analytical specificity of the tool was tested on a cohort of pathogenic agents and biological samples with quantitative PCR as a reference method. Limit of detection (analytical sensitivity) of 5 × 100 (AdV, HEV, and RVA) and 5 × 101 (HAV and NoV) genome equivalents per reaction was reached. This robust, senstivie, and rapid multiplexing technology may be used to routinely monitor and manage viruses in food and water to prevent food and waterborne diseases.
Animal or human protothecosis belongs to rather rare, endemic, pro-inflammatory infections. It is caused by achlorophyllous algae of the genus Prototheca. Especially, P. bovis (formerly P. zopfii genotype 2) is often inflected as a non-bacterial causative agent of dairy cattle mastitis. In this study, we present a multiplex real-time PCR (qPCR) system for rapid and exact Prototheca spp. detection and quantification. Limit of detection, diagnostic sensitivity, and specificity were determined. For the first time, specific sequences of AccD (encoding acetyl CoA reductase) for P. bovis, cox1 (encoding cytochrome C oxidase subunit 1) for P. wickerhamii, cytB (encoding cytochrome B) for P. blashkeae and atp6 (encoding transporting ATPase F0 subunit 6) for P. ciferrii (formerly P. zopfii genotype 1) were used for species identification and quantification together with 28S rRNA sequence detecting genus Prototheca. The developed qPCR assay was applied to 55 individual cow milk samples from a herd suspected of protothecosis, 41 bulk milk samples from different Czech farms, 16 boxed milk samples purchased in supermarkets and 21 environmental samples originating from a farm suspected of protothecosis. Our work thus offers the possibility to diagnose protothecosis in the samples, where bacterial mastitis is the most commonly presumed and thereby assisting adequate corrective measures to be taken.
- MeSH
- DNA chemie izolace a purifikace MeSH
- farmy MeSH
- klonování DNA MeSH
- kvantitativní polymerázová řetězová reakce metody MeSH
- limita detekce MeSH
- mikrobiologie životního prostředí MeSH
- mlékárenství MeSH
- mléko mikrobiologie MeSH
- multiplexová polymerázová řetězová reakce metody MeSH
- plazmidy genetika MeSH
- Prototheca genetika růst a vývoj izolace a purifikace MeSH
- senzitivita a specificita MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Česká republika MeSH
