Úvod: Virové hemoragické horečky jsou způsobovány zástupci čtyř virových čeledí a patří mezi choroby s velmi vysokou smrtností. Pro jejich diagnostiku není dostupný žádný komerční kit. Výsledky: Vytvořili jsme real time RT-PCR panel pro detekci a identifikaci virů Ebola, Marburg, Lassa, Guanarito, Machupo, Sabiá, Seoul, Puumala, Hantaan, virus krymsko-konžské hemoragické horečky a horečky Rift Valley. Protokol funguje na principu jednotných reakčních podmínek a byl testován na různých PCR přístrojích jak kapilárních, tak i destičkových včetně polní verze termocykleru Ruggedized Advanced Pathogen Identification Device (R.A.P.I.D.) (Idaho Technology, Inc.). Závěr: V kombinaci s adaptovanými protokoly z již publikovaných prací představujeme jednoduchý detekční systém pro rychlou identifikaci původců virových hemoragických horeček z čeledí filoviru, arenavirů a bunyavirů s dostatečnou senzitivitou a specificitou pro použití laboratořích prvního kontaktu a pro diagnostiku v polních podmínkách.
Background: Viral hemorrhagic fevers are caused by viruses from four viral families and develop diseases with high fatality rates. However, no commercial diagnostic assay for these pathogens is available. Findings: We developed real-time RT-PCR assays for viruses Ebola, Marburg, Lassa, Guanarito, Machupo, Junin, Sabiá, Seoul, Puumala, Hantaan, Crimean-Congo hemorrhagic fever virus and Rift Valley fever virus. The assays were optimized for identical reaction conditions and can be performed using several types of real-time PCR instruments, both capillary and plate, including a portable Ruggedized Advanced Pathogen Identification Device (R.A.P.I.D.) (Idaho Technology, Inc.). Conclusions: In combination with primers and probes from previously published studies, we present a simple system for rapid identification of hemorrhagic filoviruses, arenaviruses and bunyaviruses with sufficient sensitivity for first contact laboratory and diagnosis under field conditions.
- MeSH
- Hemorrhagic Fever, Ebola diagnosis MeSH
- Hemorrhagic Fevers, Viral * diagnosis classification MeSH
- Lassa Fever diagnosis MeSH
- Arenaviridae Infections diagnosis MeSH
- Humans MeSH
- Marburg Virus Disease diagnosis MeSH
- Reverse Transcriptase Polymerase Chain Reaction * utilization MeSH
- RNA analysis MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
Úvod. Virová hepatitida C zůstává i přes zavedení a výrazné zdokonalení testů pro detekci anti-HCV u dárců krve a krevních složek významným rizikem potransfuzní hepatitidy. Cílem této práce bylo zhodnocení možnosti zavedení kombinovaného testu pro detekci HCV antigenu a anti-HCV v ČR. Soubor a metody. V lednu až dubnu 2005 jsme vyšetřili 1164 sér dárců krve/krevních složek (soubor 1) testem Monolisa HCV Ag-Ab Ultra (BioRad) a Monolisa anti-HCV Plus Version 2 (BioRad), dále testem Monolisa HCV Ag-Ab Ultra 106 HCV RNA pozitivních a anti-HCV pozitivních sér (soubor 2), séra 15 pacientů s prokázanou HBV infekcí (soubor 3), 41 sér reaktivních ve 3 EIA anti- -HCV testech nebo RIBA 3.0 HCV pozitivních (soubor 4), 109 sér reaktivních v alespoň jednom anti-HCV EIA testu a RIBA 3.0 nejasných nebo negativních (soubor 5), panel s nízkým titrem anti-HCV (soubor 6) a panel se smíšeným titrem anti-HCV (soubor 7) a 2 séra dárce HCV RNA pozitivní, anti-HCV negativní (soubor 8). Výsledky. Výsledky vyšetření souborů sér testem Monolisa HCV Ag-Ab Ultra, procento shodných nálezů uvedeno v závorce. Soubor 1: 1krát nespecifická reaktivita pouze v testu Monolisa HCV Ag-Ab Ultra (specificita 99,91 %); soubor 2: 106 sér pozitivních (100 %); soubor 3: 15 sér negativních (100 %); soubor 4: 38 reaktivních (92,7 %) a 3 negativní (7,3 %); soubor 5: 40 sér anti-HCV (BioRad) reaktivních a HCV Ag-Ab (BioRad) negativních (36,7 %), 23 sér anti-HCV (BioRad) negativních a HCV Ag-Ab (BioRad) reaktivních (21,1 %), 12 sér v obou testech reaktivních a 34 sér v obou testech negativních; soubor 6 a 7: všechny výsledky souhlasné s panelem; soubor 8: obě séra v testu HCV Ag-Ab (BioRad) negativní (S/CO 0,13 a 0,58). Diskuse a závěr. Naše pilotní studie ukazuje, že test Monolisa HCV Ag-Ab Ultra je vysoce specifický. Zkracuje diagnostické okno oproti protilátkovému testu o 17-49 dní (literární údaje), citlivost testu se zvyšuje rozšířením intervalu „šedé zóny“ od S/CO 0,50. Je vhodné uvažovat o zavedení kombinovaného testu HCV Ag-Ab do transfuzní praxe v ČR. Pro reálné hodnocení prospěchu testování HCV antigenu kombinovaným testem jsou potřebné další studie s rutinním testováním dárců krve a krevních složek.
Introduction. Besides implementation and considerable improvement of screening tests for anti-HCV detection in blood and blood components donors, hepatitis C virus remains the significant risk of post-transfusion hepatitis. The aim of our study was to evaluate the possibilities of introduction the assay for combined detection of HCV antigen and HCV antibodies in the Czech republic. Materials and methods. From January to April 2005 1164 serum samples of blood and blood components donors were tested simultaneously by Monolisa HCV Ag-Ab Ultra (BioRad) and Monolisa anti-HCV Plus Version 2 (BioRad) (group 1). Following samples were tested by Monolisa HCV Ag-Ab Ultra: 106 HCV RNA positive and anti-HCV positive samples (group 2); 15 samples from patients with proved HBV infection (group 3); 41 samples reactive in 3 EIA anti-HCV tests or RIBA 3.0 HCV positive (group 4); 109 samples reactive at least in one anti-HCV EIA test and RIBA 3.0 indeterminate or negative (group 5); panel of samples with low titre anti-HCV (group 6); panel of samples with mixed titre anti-HCV (group 7) and 2 samples from blood donor HCV RNA positive, anti-HCV negative (group 8). Results. Group 1: one unspecific reactivity only in Monolisa HCV Ag-Ab Ultra (specificity 99,91 %); group 2: 106 positive samples (100 %); group 3: 15 negative samples (100 %); group 4: 38 reactive samples (92,7 %) a 3 negative samples (7,3 %); group 5: 40 samples anti-HCV (BioRad) reactive and HCV Ag-Ab (BioRad) negative (36,7 %), 23 samples anti-HCV (BioRad) negative and HCV Ag-Ab (BioRad) reactive (21,1 %), 12 samples reactive in both tests and 34 samples negative in both tests; group 6 a 7: all results were accordant with panels; group 8: both samples were negative by HCV Ag-Ab (BioRad) (S/CO 0,13 a 0,58). Discussion. Our pilot study shows that Monolisa HCV Ag-Ab Ultra assay is highly specific. It shortens diagnostic window in comparison to antibody assay by 17-49 days (literature data), test sensitivity is increased by extension of the interval of „the grey zone“ from S/CO 0,50. It is suitable to consider the introduction of combined HCV antigen-antibody assay in transfusion service in the Czech republic. However, further studies with routine testing of blood and blood components donors are necessary for real evaluation of benefit of HCV antigen testing.
- MeSH
- Blood Donors MeSH
- Enzyme-Linked Immunosorbent Assay methods instrumentation utilization MeSH
- Hepatitis C Antigens diagnostic use immunology blood MeSH
- Hepatitis C Antibodies diagnostic use immunology blood MeSH
- Hepatitis C diagnosis prevention & control therapy MeSH
- Humans MeSH
- Serologic Tests methods utilization MeSH
- Check Tag
- Humans MeSH
Background The lack of effective biomarkers for the screening and early detection of ovarian cancer (OC) is one of the most pressing problems in oncogynecology. Because epigenetic alterations occur early in the cancer development, they provide great potential to serve as such biomarkers. In our study, we investigated a potential of a four-gene methylation panel (including CDH13, HNF1B, PCDH17 and GATA4 genes) for the early detection of high-grade serous ovarian carcinoma (HGSOC). Methods For methylation detection we used methylation sensitive high-resolution melting analysis and real-time methylation specific analysis. We also investigated the relation between gene hypermethylation and gene relative expression using the 2-ΔΔCt method. Results The sensitivity of the examined panel reached 88.5%. We were able to detect methylation in 85.7% (12/14) of early stage tumors and in 89.4% (42/47) of late stage tumors. The total efficiency of the panel was 94.4% and negative predictive value reached 90.0%. The specificity and positive predictive value achieved 100% rates. Our results showed lower gene expression in the tumor samples in comparison to control samples. The more pronounced downregulation was measured in the group of samples with detected methylation. Conclusions In our study we designed the four-gene panel for HGSOC detection in ovarian tissue with 100% specificity and sensitivity of 88.5%. The next challenge is translation of the findings to the less invasive source for biomarker examination, such as plasma. Our results indicate that combination of examined genes deserve consideration for further testing in clinical molecular diagnosis of HGSOC.
- MeSH
- Early Detection of Cancer MeSH
- Humans MeSH
- DNA Methylation * MeSH
- Biomarkers, Tumor blood MeSH
- Ovarian Neoplasms blood diagnosis genetics MeSH
- Sensitivity and Specificity MeSH
- Check Tag
- Humans MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
Východisko. Hemofilie A je jednou z nejčastějších vrozených krvácivých poruch. Znalost molekulární příčiny onemocnění je využívána při genetickém poradenství a pomáhá odhadnout riziko závažné komplikace standardní léčby (inhibitor faktoru VIII). Deficit faktoru VIII je způsoben širokým spektrem mutací. Pouze inverze s místy zlomu v intronech 22, respektive 1 se vyskytují častěji. Ostatní mutace je nutno hledat v celém rozsahu 26 exonů kódujících 9 kb mRNA a v přilehlých nekódujících sekvencích. Cílem práce bylo metodicky zajistit urychlení genotypizace hemofilických rodin v České republice využitím analýzy amplikonů jednotlivých exonů genu faktoru VIII pomocí denaturační vysokotlaké kapalinové chromatografie. Metody a výsledky. Citlivost metody jsme ověřovali na panelu vzorků DNA se 49 dříve charakterizovanými různými sekvenčními variantami genu faktoru VIII, celkem ve 21 exonech. Všechny testované sekvenční změny byly snadno detekovány. Testování příbuzných ukázalo velmi dobrou reprodukovatelnost jednotlivých elučních profilů. Navíc se podařilo detekovat kauzální mutace u 4 z 5 zjevně nepříbuzných hemofilických rodin, u kterých nebyla předchozí analýza metodou multiplexní CSGE úspěšná. Závěry. Zavedená metoda významně urychlí genotypizaci rodin s hemofilií A v České republice.
Background. Haemophilia A is one of the most prevalent inherited bleeding disorders. Causal mutations in the factor VIII gene are detected to facilitate the genetic counselling and to estimate the risk of serious complication associated with standard treatment (factor VIII inhibitor). Wide range of mutations located across the entire length of the factor VIII gene underlies the factor VIII deficiency of variable severity. The only two common recurrent mutations in the factor VIII gene are intron 22 and intron 1 inversions. In the remaining cases it is necessary to screen all 26 exons encoding 9kb mRNA together with adjacent nonncoding sequences. In order to speed up genotyping in haemophilia A families in the Czech Republic we evaluated DHPLC-based screening technique. Methods and Results.We tested sensitivity of the analysis on a panel of DNA samples containing 49 different sequence variations distributed over 21 exons. All the genetic alterations were readily detected. Analysis of family members has shown good reproducibility of the respective elution patterns. DHPLC analysis detected mutations in 4 out of 5 samples from apparently unrelated haemophilia patients, where previously applied multiplex CSGE was not successful. Conclusions. Establishing of DHPLC analysis will substantially speed up the genotyping of haemophilia A families in the Czech Republic
studie byla zaměřena na stanovení exprese membránových cytokinových receptorů na kryostatových tkáňových řezech s použitím vysoce citlivého systému s biotin-streptavidinem. Byly použity monoklonální protilátky proti cytokinovým receptorům vybrané ze 6. mezinárodního workshopu o lidských leukocytámích diferenciačních antigenech: CD25 (IL.2Ra), CD95 (FAS antigen), CD116 (GM-CSFR), CD117 (SCFR), CD120a (TNFR I), CD120b (TNFR H), CD121a (IL-IR I), CDwl23 (IL3R), CD124 (IL.4R), CD126 (DL-GR), CD127 (IL-TR), CDwl28 (IL-8R), CD130 (gp 130), CD131 (IL-3Rβ), CD132 (IL.2Ry), CD134 (OX-40), CD135 (FLT3/FLK2). Patologické tkáně (uzliny a sleziny) byly odebrány 12 pacientům s diagnózami: folikulárního non-Hodgkinova lymfomu, periferního T non Hodgkinova lymfomu, B-lymfomu, myelomu, Hodgkinovy choroby, dva případy B-lymfomu bohatého na T lymfocyty, autoimunitní hemolytické anémie, rudimentární granulomatózní lymfadenitidy, dva případy idiopatické trombocytopenické purpury a lymfedému. Z výsledků vyplývá, že imunohistologická metoda využívající při průkazu cytokinových receptorů nativních zmrazených tkání, monoklonálních protilátek a vizualizačního systému s biotin-streptavidinem je mimořádně vhodnou detekční metodou pro přesnější lokalizaci cytokinových receptorů ve tkáních.
We have studied tissue expression of the cytokine receptors using a high sensitivitity biotin-streptayidm system on cryostat sections. We used a panel of monoclonal antibodies from the 6th International Workshop on Human Leukocyte Differentiation Antigens, namely CD25 (IL-2Rα), CD95 (FAS antigen), CD116 (GM CSFR), CD117 (SCFR), CD120a (TNFR I), CD120b (TNFR II), CD121a (IL-IR I), CDwl23 (IL.3R), CD124 (n.-4R), CD126 (IL-6R), CD127 (IL-7R), CDwl28 (IL-8R), CD130 (gpll30), CD131 (IL-SR), CD132 (IL-2Ry), CD134 (OC-40), CD135 (FLT3/FLK2). Examined tissues (lymph nodes and spleens) were obtained from 12 patients with folicular non-Hodgkin's lymphoma, periferal T non-Hodgkin's lymphoma, B lymphoma, myeloma, Hodgkin's disease, two cases of T cell rich B-lymphoma, autoimmune haemolytic anemia and two cases of rudimentary trombocytopenic purpura. Our results indicate that immunohistological technology using native tissues on cryostat sections, monoclonal antibodies and the visualisation with biotin-streptavidin is a particularly suitable supplementary staining procedure for detection of the cytokine receptors in tissues.
1st ed. 87 s.
- MeSH
- Cholesterol MeSH
- Hypercholesterolemia MeSH
- Publication type
- News MeSH
- Conspectus
- Lékařské vědy. Lékařství
- NML Fields
- vnitřní lékařství
In the microbiological diagnosis of bloodstream infections (BSI), blood culture (BC) is considered the gold standard test despite its limitations such as low sensitivity and slow turnaround time. A new FDA-cleared and CE-marked platform utilizing magnetic resonance to detect amplified DNA of the six most common and/or problematic BSI pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Escherichia coli; referred to as ESKAPEc) is available and may shorten the time to diagnosis and potentially improve antimicrobial utilization. Whole blood samples from hospitalized patients with clinical signs of sepsis were analyzed using the T2Bacteria Panel (T2Biosystems) and compared to simultaneously collected BC. Discrepant results were evaluated based on clinical infection criteria, combining supporting culture results and the opinion of treating physicians. A total of 55 samples from 53 patients were evaluated. The sensitivity and specificity of the T2Bacteria panel was 94% (16 out of 17 detections of T2Bacteria-targeted organisms) and 100%, respectively, with 36.4% (8 of 22) causes of BSI detected only by this method. The T2Bacteria Panel detected pathogens on average 55 hours faster than standard BC. In our study, 9 of 15 patients with positive T2Bacteria Panel results received early-targeted antibiotic therapy and/or modification of antimicrobial treatment based on T2Bacteria Panel findings. Given the high reliability, faster time to detection, and easy workflow, the technique qualifies as a point-of-care testing approach.
- MeSH
- Acinetobacter baumannii drug effects genetics isolation & purification MeSH
- Anti-Bacterial Agents pharmacology MeSH
- Antimicrobial Stewardship methods MeSH
- Bacteremia blood drug therapy microbiology MeSH
- Enterococcus faecium drug effects genetics isolation & purification MeSH
- Escherichia coli drug effects genetics isolation & purification MeSH
- Klebsiella pneumoniae drug effects genetics isolation & purification MeSH
- Blood microbiology MeSH
- Blood Culture MeSH
- Humans MeSH
- Prospective Studies MeSH
- Pseudomonas aeruginosa drug effects genetics isolation & purification MeSH
- Staphylococcus aureus drug effects genetics isolation & purification MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
OBJECTIVE: Developmental and epileptic encephalopathies (DEEs) are a group of severe, early-onset epilepsies characterised by refractory seizures, developmental delay, or regression and generally poor prognosis. DEE are now known to have an identifiable molecular genetic basis and are usually examined using a gene panel. However, for many patients, the genetic cause has still not been identified. The aims of this study were to identify causal variants for DEE in patients for whom the previous examination with a gene panel did not determine their genetic diagnosis. It also aims for a detailed description and broadening of the phenotypic spectrum of several rare DEEs. METHODS: In the last five years (2015-2020), 141 patients from all over the Czech Republic were referred to our department for genetic testing in association with their diagnosis of epilepsy. All patients underwent custom-designed gene panel testing prior to enrolment into the study, and their results were inconclusive. We opted for whole exome sequencing (WES) to identify the cause of their disorder. If a causal or potentially causal variant was identified, we performed a detailed clinical evaluation and phenotype-genotype correlation study to better describe the specific rare subtypes. RESULTS: Explanatory causative variants were detected in 20 patients (14%), likely pathogenic variants that explain the epilepsy in 5 patients (3.5%) and likely pathogenic variants that do not fully explain the epilepsy in 11 patients (7.5%), and variants in candidate genes in 4 patients (3%). Variants were mostly de novo 29/40 (72.5%). SIGNIFICANCE: WES enables us to identify the cause of the disease in additional patients, even after gene panel testing. It is very important to perform a WES in DEE patients as soon as possible, since it will spare the patients and their families many years of a diagnostic odyssey. In particular, patients with rare epilepsies might significantly benefit from this approach, and we propose using WES as a new standard in the diagnosis of DEE instead of targeted gene panel testing.
- MeSH
- Epilepsy, Generalized * genetics MeSH
- Epilepsy * diagnosis genetics MeSH
- Phenotype MeSH
- Genetic Association Studies MeSH
- Genetic Testing MeSH
- Humans MeSH
- Exome Sequencing MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
Currently, the influenza virus infects millions of individuals every year. Since the influenza virus represents one of the greatest threats, it is necessary to develop a diagnostic technique that can quickly, inexpensively, and accurately detect the virus to effectively treat and control seasonal and pandemic strains. This study presents an alternative to current detection methods. The flow-injection analysis-based biosensor, which can rapidly and economically analyze a wide panel of influenza virus strains by using paramagnetic particles modified with glycan, can selectively bind to specific viral A/H5N1/Vietnam/1203/2004 protein-labeled quantum dots. Optimized detection of cadmium sulfide quantum dots (CdS QDs)-protein complexes connected to paramagnetic microbeads was performed using differential pulse voltammetry on the surface of a hanging mercury drop electrode (HMDE) and/or glassy carbon electrode (GCE). Detection limit (3 S/N) estimations based on cadmium(II) ions quantification were 0.1 μg/mL or 10 μg/mL viral protein at HMDE or GCE, respectively. Viral protein detection was directly determined using differential pulse voltammetry Brdicka reaction. The limit detection (3 S/N) of viral protein was estimated as 0.1 μg/mL. Streptavidin-modified paramagnetic particles were mixed with biotinylated selective glycan to modify their surfaces. Under optimized conditions (250 μg/mL of glycan, 30-min long interaction with viral protein, 25°C and 400 rpm), the viral protein labeled with quantum dots was selectively isolated and its cadmium(II) content was determined. Cadmium was present in detectable amounts of 10 ng per mg of protein. Using this method, submicrogram concentrations of viral proteins can be identified.
- MeSH
- Biosensing Techniques instrumentation methods MeSH
- Biotin chemistry metabolism MeSH
- Electrochemical Techniques instrumentation methods MeSH
- Electrodes MeSH
- Hemagglutinin Glycoproteins, Influenza Virus analysis metabolism MeSH
- Cadmium analysis MeSH
- Quantum Dots MeSH
- Limit of Detection MeSH
- Linear Models MeSH
- Magnetite Nanoparticles chemistry MeSH
- Flow Injection Analysis instrumentation methods MeSH
- Mercury chemistry MeSH
- Cadmium Compounds chemistry MeSH
- Streptavidin chemistry metabolism MeSH
- Sulfides chemistry MeSH
- Carbon chemistry MeSH
- Influenza A Virus, H5N1 Subtype chemistry isolation & purification MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
