Higher order multiplexing
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The digital polymerase chain reaction (dPCR) multiplexing method can simultaneously detect and quantify closely related deoxyribonucleic acid sequences in complex mixtures. The dPCR concept is continuously improved by the development of microfluidics and micro- and nanofabrication, and different complex techniques are introduced. In this review, we introduce dPCR techniques based on sample compartmentalization, droplet- and chip-based systems, and their combinations. We then discuss dPCR multiplexing methods in both laboratory research settings and advanced or routine clinical applications. We focus on their strengths and weaknesses with regard to the character of biological samples and to the required precision of such analysis, as well as showing recently published work based on those methods. Finally, we envisage possible future achievements in this field.
BACKGROUND: The aim was to do an internal audit of gastrointestinal pathogen detection at the Department of Medical Microbiology, Motol University Hospital between the years 2014 and 2018 and to test two commercial multiplex molecular biology assays potentially improving the diagnostic process and reducing costs. MATERIAL AND METHODS: Based on data from a laboratory information system (LIS), a total of 45,888 samples were identified which had been tested for the presence of gastrointestinal pathogens using culture, immunochromatographic, microscopic and molecular biology techniques between 2014-2018. Novel multiplex molecular biology detection was used to test 182 nucleic acid isolates obtained from stool samples with the Enteric Viruses (8-well) assay (Viral Panel, EVP) or Faecal Pathogens M (16-well) assay (Microbial Panel, FPM) manufactured by AusDiagnostics. RESULTS: The LIS data showed 6.2 % of positive pathogens causing diarrhea from all tested samples (detection rates: 4.5 % for bacterial agents, 21.6 % for viral agents and 0.4 % for parasitic agents). Valid samples (98.9 % of all tested samples) tested by the molecular biology technique yielded, in descending order: C. difficile toxin B (19 %), norovirus (9 %), astrovirus (8 %), Campylobacter (7 %), sapovirus (6 %), Yersinia enterocolitica (6 %), rotavirus (4 %), enterovirus (3 %), Aeromonas (3 %), adenovirus (2 %) and Salmonella (1 %). There was found at least 1 additional new positive detection in 27 % of stools tested by the Viral Panel and in 40 % of stools tested by the Microbial Panel in comparison with the traditional approach. Introducing the panels into routine diagnostic practice will not reduce the costs. CONCLUSIONS: The introduction of novel multiplex molecular biology assays for detecting gastrointestinal pathogens will considerably increase pathogen detection rates even though the costs will be higher for the Department of Medical Microbiology.
- MeSH
- diagnostické techniky molekulární * ekonomika normy MeSH
- feces mikrobiologie virologie MeSH
- gastrointestinální nemoci * mikrobiologie virologie MeSH
- lidé MeSH
- multiplexová polymerázová řetězová reakce MeSH
- nemocnice univerzitní MeSH
- průjem mikrobiologie virologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- srovnávací studie MeSH
- Geografické názvy
- Česká republika MeSH
PURPOSE OF THE STUDY: In the framework of a prospective study on transposition of the m. pectoralis major according to Clark in patients with type I arthrogryposis multiplex congenita, electromyography was carried out in order to determine pre-operative states of the elbow joint flexors and m. pectoralis major and then the post-operative electric activity of a transposed muscle and to correlate changes with clinical findings. Histological examination was performed to reveal changes in muscle morphology and to complete a comprehensive assessment of muscle transposition. MATERIAL AND METHODS: Electromyography was carried out on nine upper limbs of five pediatric patients aged 4.3 to 8.9 years. Using a needle electrode, activities of the elbow flexors (m. biceps and m. brachialis), m. pectoralis major, m. triceps brachii and m. deltoideus were examined. In the post-operative period, activity was repeatedly measured in both the transposed and non-transposed parts of the m. pectoralis major. In one patient, histological examination of muscle tissue was performed at 26 months after transposition; light microscopy of paraffin-mounted sections stained with hematoxylin-eosin was used. RESULTS: Out of seven arms examined by electromyography before muscle transfer, six showed complete and one incomplete atrophy of the m. biceps brachii and m. brachialis. The m. pectoralis major had a five- to four-degree electric activity, which provided enough strength for transposition. Post-operative examination revealed changes leading to re-innervation of the transposed muscle, which corresponded to a partial denervation of the muscle followed by repair of innervation. None of the muscles was markedly atrophic due to denervation. In muscles with a higher electric activity, clinical outcomes were better, although electric activity always slightly exceeded clinical activity. In terms of electric activity, the transposed muscle was stabilized a year after surgery. Non-transposed parts of the muscle were not damaged by the surgical procedure, as shown by electromyography. Histological examination showed the muscle at a state of partial atrophy but with signs of ongoing regeneration of muscle fibers. DISCUSSION: No data on examination of the electric activity of the m. pectoralis major following its transposition in patients with arthrogryposis multiplex congenita have been reported in the literature. Electromyography in this study proved useful for providing information on the electric activity of a muscle before transposition and on contractility of the muscle after surgery; it also allowed us to distinguish between a mechanical failure of transfer and muscle atrophy due to neurogenic or vascular causes. All transposed muscles that were examined revealed changes indicating a minimum denervation followed by re-innervation. This finding was confirmed by the results of histological examination. CONCLUSIONS: Electromyography showed that the electric activity of a transposed muscle corresponded to the clinical presentation of this muscle and thus became an indispensable part of both pre- and post-operative examination. Both electromyographic and histological examination confirmed the applicability of the treatment described here.
- MeSH
- artrogrypóza patofyziologie chirurgie MeSH
- dítě MeSH
- elektromyografie MeSH
- lidé MeSH
- loketní kloub patofyziologie chirurgie MeSH
- předškolní dítě MeSH
- prospektivní studie MeSH
- prsní svaly patologie patofyziologie transplantace MeSH
- Check Tag
- dítě MeSH
- lidé MeSH
- předškolní dítě MeSH
- Publikační typ
- anglický abstrakt MeSH
- časopisecké články MeSH
- práce podpořená grantem MeSH
Methicillin-resistant Staphylococcus aureus (MRSA) presenting spa type t899 is commonly associated with sequence type 9 (ST9) but is also increasingly linked to ST398. This study provides genomic insight into the diversity of t899 isolates using core genome multilocus sequence typing (cgMLST), single nucleotide polymorphism (SNP)-based phylogeny, and the description of selected antimicrobial resistance and virulence markers. The SNP-based phylogenic tree showed that isolates sharing the same spa type (t899) but different STs highly diverged in their core and accessory genomes, revealing discriminant antimicrobial resistance (AMR) and virulence markers. Our results highlighted the idea that in a surveillance context where only spa typing is used, an additional multiplex PCR for the detection of the tet(M), sak, and seg genes would be valuable in helping distinguish ST9 from ST398 isolates on a routine basis.IMPORTANCE This study showed the genetic diversity and population structure of S. aureus presenting the same spa type, t899, but belonging to different STs. Our findings revealed that these isolates vary deeply in their core and accessory genomes, contrary to what is regularly inferred from studies using spa typing only. Given that identical spa types can be associated with different STs and that spa typing only is not appropriate for S. aureus isolates that have undergone major recombination events which include the passage of the spa gene (such as in t899-positive MRSA), the combination of both MLST and spa typing methods is recommended. However, spa typing alone is still largely used in surveillance studies and basic characterization. Our data suggest that additional markers, such as tet(M), sak, and seg genes, could be implemented in an easy and inexpensive manner in order to identify S. aureus lineages with a higher accuracy.
- Klíčová slova
- MLST, MRSA, cgMLST, spa type, t899, whole-genome sequencing,
- MeSH
- bakteriální geny MeSH
- bakteriální léková rezistence genetika MeSH
- faktory virulence genetika MeSH
- fylogeneze MeSH
- genom bakteriální MeSH
- genomika MeSH
- jednonukleotidový polymorfismus MeSH
- methicilin rezistentní Staphylococcus aureus genetika izolace a purifikace MeSH
- multilokusová sekvenční typizace MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- faktory virulence MeSH
Neonatal systemic inflammatory response and multiple organ dysfunction syndrome are the main postnatal insults influencing mortality and morbidity. Critically ill newborns with high predicted mortality are supported by extracorporeal membrane oxygenation (ECMO). Biomarkers of inflammatory response and endothelial injury can be used for early diagnosis and treatment of critical neonatal situations. The aim of our study was to explore plasma proteins and endothelial microvesicles as markers of inflammation and endothelial activation in newborns on ECMO and to compare them with healthy neonates. Thirteen newborns on ECMO and 13 healthy newborns were included in the study. Plasma soluble biomarkers were measured using multiplex immunoassay based on Luminex® xMAP multianalyte profiling platform. The total microvesicle count and plasma level of surface antigen-specific microvesicles were determined by flow cytometry. The plasma concentration of cell-derived microvesicles was measured using annexin-V labeling, and the endothelial origin of microvesicles was determined using lineage-specific antigen labeling of endothelial cell/microvesicle markers (endoglin/CD105, PECAM1/CD31, VEGFR2/CD309, and MadCAM1). Inflammatory markers (procalcitonin, IL-1β, IL-6, and IL-22) were increased in the ECMO group (P < 0.01). The assessment of endothelial markers showed higher concentrations of endocan and angiopoietin-2 (P < 0.01) in the ECMO group while VEGF in the ECMO group was significantly lower (P < 0.01). In the ECMO group, the concentration of annexin-V-positive microvesicles (total microvesicles) and endothelial microvesicles expressing mucosal vascular addressin cell adhesion molecule 1 (MadCAM1) was increased (P = 0.05). In summary, we found increased concentrations of soluble inflammatory and endothelial markers in the plasma of critically ill newborns with multiple organ dysfunction. Increased plasma concentrations of microvesicles may reflect the activation or damage of blood cells and vasculature including endothelial cells. The measurement of cell membrane-derived microvesicles may be added to the panel of established inflammatory markers in order to increase the sensitivity and specificity of the diagnostic process in critically ill newborns.
- MeSH
- biologické markery krev MeSH
- buněčná membrána metabolismus MeSH
- endotel metabolismus MeSH
- endoteliální buňky MeSH
- kritický stav MeSH
- lidé MeSH
- mikropartikule metabolismus MeSH
- mimotělní membránová oxygenace * MeSH
- novorozenec MeSH
- průtoková cytometrie MeSH
- zánět krev MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- novorozenec MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- biologické markery MeSH
OBJECTIVES: Normal pressure hydrocephalus (NPH) is a treatable neurological syndrome developing in the elderly. It is characterized by balance impairment, urinary incontinence and dementia development caused by disorders in the cerebrospinal fluid (CSF) circulation. The diagnosis can be easily mistaken for other neurodegenerative diseases, which are often accompanied by inflammation and the production of cytokines. The aim of our study was to determine and compare selected CSF and plasma cytokines with respect to their informative value for laboratory diagnostics of NPH. METHODS: The levels of IL-1β, IL-4, IL-6, IL-10, IL-17A, IL-17F, IL-21, IL-22, IL-23, IL-25, IL-31, IL-33, INF-γ, sCD40L and TNF-α were measured in the CSF and plasma in age-matched subjects with NPH (n=20) and controls (n=20) by multiplex assay. RESULTS: CSF IL-1β, IL-6 and IL-10 were significantly increased on the 1st day of lumbar drainage in NPH (p<0.01). No significant changes were observed in the plasma. The CSF cytokines were one to three orders of magnitude higher compared to the plasma. CONCLUSION: CSF can better show the neurodegenerative changes in the brain. The cytokines IL-1β, IL-6 and IL-10 may be helpful in NPH diagnostics.
- MeSH
- biologické markery mozkomíšní mok MeSH
- cytokiny mozkomíšní mok imunologie MeSH
- demence mozkomíšní mok diagnóza imunologie MeSH
- inkontinence moči mozkomíšní mok diagnóza imunologie MeSH
- lidé MeSH
- normotenzní hydrocefalus mozkomíšní mok diagnóza imunologie MeSH
- posturální rovnováha MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- stárnutí imunologie MeSH
- Check Tag
- lidé MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- biologické markery MeSH
- cytokiny MeSH
