Rozšíření borreliózy závisí na geografických, ekologických a klimatických faktorech, stejně jako napatogenezi vlastního agens skupiny Borrelia burgdorferi sensu lato. Zájem vzbuzuje nárůst případůtéto choroby a objevení nových příznaků. Vztah genodruhů k symptomům, jejich geografické rozšířenía možnost spolupůsobení dalších patogenů je předmětem naší studie.Do studie bylo zahrnuto 87 pacientů s borreliózou ze středních, východních Čech a z Moravy.Pacienti skupiny 1 (celkem 49) byli zařazeni podle pozitivní kliniky, pacienti skupiny 2 (celkem 21)se rekrutovali z pozitivních screeningovou metodou PCR a pacienti skupiny 3 (celkem 17) bylipozitivní kultivačně. Celkem 48 pacientů a 17 izolovaných kmenů bylo pozitivních v konvenčnínested PCR pro plazmidy a genom B. burgdorferi sensu lato. Genodruhy a subtypy borrelií bylyprokázány přímou sekvenací OspA a OspC produktů. Kvantitativní data byla determinována pomocíkřivky tání specifického produktu užitím PCR v reálném čase.Sekvenací fragmentů OspA genu jsme u pacientů ze středních Čech prokázali přítomnost B. garinii,subtypy 6, 5, 4, 3 v 51,8 % (14 z 27), B. burgdorferi s.s v 29,6 % (8 z 27) a B. afzelii v 18,5 % (5 z 27). Vevýchodní oblasti prevalovala B. garinii, subtyp 5 a v 7,6 % byla společná infekce s Anaplasmaphagocytophilum. Prevalence B. afzelii v 44 % (11 z 25) následovaná B. burgdorferi s.s. v 36 % (9 z 25)a v 20 % zbývající infekcí B. garinii byla u pacientů z Moravy. Sekvence dvou hypervariabilníchoblastí OspC a OspA genů a vzdálenosti sekvencí ve fylogenetických stromech ukázaly rozdíly nejenmezi genodruhy a subtypy ale i mezi „divokými“ kmeny, prokázanými přímou sekvencí ve vzorcíchpacientů a „arteficiálními“ kmeny, kultivovanými in vitro. Největší rozdíly byly u dlouhodoběnemocných borreliózou.

The spread of borreliosis depends on geographical, environmental and climatic factors as well ason the pathogenesis of the causative agent of the group of Borrelia burgdorferi sensu lato. The risein the incidence of the disease and emergence of new symptoms are of concern. Relationshipsbetween genospecies and symptoms, their geographical spread and possible interference of otherpathogens are the subject of the present study. Eighty-seven patients with borreliosis from Centraland Eastern Bohemia and Moravia were enrolled in the study. Forty-nine patients of group 1 showedclinical positivity, 21 patients of group 2 tested positive at PCR screening and 17 patients of group3 were culture positive. Forty-eight patients and 17 isolated strains showed positivity for plasmidsand the Borrelia burgdorferi sensu lato genome in conventional nested PCR. Borrelial genotypesand subtypes were detected by direct sequencing of OspA and OspC products. Quantitative datawere determined from specific product melting temperature curves for real time PCR.Based on sequencing of the OspA gene, B. garinii (subtypes 6, 5, 4 and 3), B. burgdorferi s.s. and B.afzelii were detected in 14 (51.8 %), 8 (29.6 %) and 5 (18.5 %) out of 27 Central Bohemian patients,respectively. Eastern Bohemian patients showed predominance of B. garinii subtype 5 and co-infectionwith Anaplasma phagocytophilum in 7.6 %. The predominant causative agent in 25 Moravianpatients was B. afzelii (11 patients, i.e. 44 %), followed by B. burgdorferi s.s. (9 patients, 36 %) and B.garinii 5 patients, i.e. 20 %). Sequences of two hypervariable regions of the OspA and OspC genesand distances in phylogenetic trees showed differences not only between genospecies and subtypesbut also between wild strains detected by direct sequencing from patient specimens and in vitrocultured strains. The greatest differences were found for patients with long-termborrelial infection.

• MeSH
• Borrelia burgdorferi genetika   izolace a purifikace   klasifikace   MeSH
• lymeská nemoc diagnóza   epidemiologie   MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• prevalence MeSH
• sekvenční analýza metody   přístrojové vybavení   MeSH
• senzitivita a specificita MeSH
• zdroje nemoci MeSH
• Publikační typ
• přehledy MeSH
• srovnávací studie MeSH

Cíl práce: 1. zavedení, optimalizace a validace polymerázové řetězové reakce v realném čase pro detekci DNA borélií včetně kontroly přítomnosti inhibitorů DNA polymerázy; 2. shrnutí dosavadních výsledků získaných pomocí zavedené metody. Materiál a metody: Izolace DNA byla prováděna pomocí komerčního kitu MagNA Pure Isolatin Kit III (Bacteria, Fungi) na automatickém izolačním přístroji MagNA Pure LC fi rmy Roche. Metoda byla koncipována jako multiplex real-time PCR s amplifi kací DNA borélií a koamplifi kací inhibiční kontroly v jedné zkumavce. Výsledky: Zavedená metoda byla plně optimalizována, validována a následně použita pro vyšetření 1822 materiálů, z toho 1151 likvorů, 609 krví a 62 jiných materiálů. Pouze 17 vzorků bylo pozitivních a 4 vzorky vykazovaly přítomnost inhibitorů DNA polymerázy. Závěr: Námi zavedená a validovaná metoda je velice rychlá, citlivá a specifi cká pro detekci DNA borélií. Přesto v diagnostice lymeské boreliózy není vždy stoprocentně spolehlivá a klinická korelace, stejně jako u jiných metod, není uspokojivá.

Objective: The aims of our work were to introduction, optimization and validation of real-time PCR for detection Borrelia DNA including control of DNA polymerase inhibitors presence and summary of the results obtained by the new method. Material and methods: DNA was isolated by commercial kit MagNA Pure Isolatin Kit III (Bacteria, Fungi) on automatic isolation instrument MagNA Pure LC (Roche). The method is created as multiplex real-time PCR with Borrelia DNA amplifi cation and co-amplifi cation of inhibitor control in one tube. Results: Established method was optimised, validated and then used for analysis of 1,822 biological materials, of it 1,151 cerebrospinal fl uids, 609 blood samples and 62 other biological samples. Only 17 samples were positive and 4 samples contained inhibitors of DNA polymerase. Conclusion: The established and validated method is very fast, sensitive and specifi c for detection of Borrelia DNA. In spite of, Lyme borreliosis diagnostics is not always unimpeachable and clinical correlation is not satisfactory as well as other methods.

• MeSH
• Borrelia burgdorferi genetika   izolace a purifikace   MeSH
• in situ značení DNA s primerem MeSH
• lidé MeSH
• lymeská nemoc diagnóza   MeSH
• odběr biologického vzorku MeSH
• polymerázová řetězová reakce metody   MeSH
• senzitivita a specificita MeSH
• Check Tag
• lidé MeSH

Cíl studie: Rozlišit genotypy a subtypy Borrelia burgdorferi sensu lato a Ehrlichia spp. V kulturách, ve vzorcích mozkomíšního moku a krve pacientů a ve tkáních zvířat. Studie byla zaměřena na střední a východní Čechy a Moravu. Materiál: Zkoumáno bylo 16 kmenů borelií, izolovaných z krve a moku pacientů, dále 49 vzorků mozkomíšního moku a krve pacientů s boreliózou, tkáně 80 kusů lovné zvěře a krev 55 krav. Metody: Diferenciaci a kvantifikaci obou patogenů umožnilo užití LightCycler PCR v reálném čase s příměry 16S rRNA, OspA, recA genů a přímá PCR-sekvenační analýza produktů. Výsledky: Prokázali jsme 11 kmenů Borrelia garinii, OspA-typy 5,6,4 a pět kmenů B. afélii. B. burgdorferi sensu stricto nebyla v kulturách prokázána. Sekvenační analýzou PCR produktů ze 49 vzorků mozkomíšního moku jsme prokázali B. burgdorferi s.s. v 20,4 %, B. afzeJii v 12,2 %, B. garinii, subtypy 6, 5, 4 a 3 v 61,3 %, z nichž 8,2 % vzorků obsahovalo kombinace blízkých OspA-typů. Koinfekce s EhrJichia phagocytophiia byla zjištěna v 6,1 % vzorků. E. phagocytophiia subtyp Frankonia I a II jsme prokázali u 12,5 % lovné zvěře a u 9 % skotu. B. burgdorferi sensu strigo a B. garinii byla zjištěna u 17,5 % lovné zvěře. Light Cycler PCR v reálném čase byla méně sensitivní v tekutinách pacientů než v kulturách a v tkáních zvířat. Závěr: Prokázali jsme, že neuroboreliózu mohou působit všechny tři genospecies B. burgdorferi sensu lato. Průběh infekce a symptomy mohou ovlivnit různé OspA- subtypy.

Purpose of the study: To differentiate the genotypes and subtypes of Borreiia burgdorferi sensu lato and Ehrlichia spp. in cultures, in samples of patients' cerebrospinal fluid and blood and in animal tissues. The study focuses on southern and eastern Bohemia and on Moravia. Material: The investigation involved 16 borrelia strains isolated from the blood and CSP of patients and further 49 samples of CSF and blood of borreliosis patients, tissues from 80 pieces of game animals and blood from 55 cows. Methods: The differentiation and quantification of the two pathogenes was done using real-time LightCycler PCR with the primers loS rRNA, OpA, recA genes and direct PCR sequential analysis of the products. Results: We demonstrated 11 strains of Borreiia garinii, OspA types 5, 6, 4 and 5 strains of B. afzelii. B. burgdorferi sensu stricto was not demonstrated in the cultures. The sequential analysis PCR of the products from 49 CSF samples demonstrated B, burgdorferi s.s. in 20.4 %, B. afzelii in 12.2 %, B. garnii subtypes 6, 5, 4 and 3 in 61.3 % - out of these 8.2 % of the samples contained combinations similar to OspA types. Co-infection with Ehrlichia phagocytophila was found in 6.1 % of samples. E. phagocytophila subtype Frankonia I and II was demonstrated in 12.5 % of game animals and in 9 % of cattle. B. burgdorferi sensu stricto and B. garinii were identified in 17.5 % of game. Real-time LightCycler PCR was less sensitive in patients' fluids than in cultures and animal tissues. Conclusions: We demonstrated that all three genotypes of B. burgdorferi sensu lato are capable of causing neuroborreliosis. The course of the infection and its symptoms may be affected by various OspA subtypes.

• MeSH
• Borrelia genetika   klasifikace   patogenita   MeSH
• Ehrlichia genetika   patogenita   MeSH
• ehrlichióza diagnóza   mikrobiologie   MeSH
• finanční podpora výzkumu jako téma MeSH
• lidé MeSH
• lymeská nemoc diagnóza   mikrobiologie   MeSH
• polymerázová řetězová reakce s reverzní transkripcí metody   MeSH
• příznaky a symptomy diagnóza   MeSH
• sekvenční analýza metody   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• přehledy MeSH
• srovnávací studie MeSH

The development and use of a fast method employing a direct analysis in real time (DART) ion source coupled to high-resolution time-of-flight mass spectrometry (TOFMS) for the quantitative analysis of caffeine in various coffee samples has been demonstrated in this study. A simple sample extraction procedure employing hot water was followed by direct, high-throughput (<1 min per run) examination of the extracts spread on a glass rod under optimized conditions of ambient mass spectrometry, without any prior chromatographic separation. For quantification of caffeine using DART-TOFMS, an external calibration was used. Isotopically labeled caffeine was used to compensate for the variations of the ion intensities of caffeine signal. Recoveries of the DART-TOFMS method were 97% for instant coffee at the spiking levels of 20 and 60 mg/g, respectively, while for roasted ground coffee, the obtained values were 106% and 107% at the spiking levels of 10 and 30 mg/g, respectively. The repeatability of the whole analytical procedure (expressed as relative standard deviation, RSD, %) was <5% for all tested spiking levels and matrices. Since the linearity range of the method was relatively narrow (two orders of magnitude), an optimization of sample dilution prior the DART-TOFMS measurement to avoid saturation of the detector was needed.

• MeSH
• analýza potravin metody   MeSH
• časové faktory MeSH
• hmotnostní spektrometrie MeSH
• káva chemie   MeSH
• kofein analýza   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

A direct analysis in real-time (DART) ion source coupled to a high-resolution orbitrap mass spectrometer was used for the quantitative analysis of isoflavones isolated from soybeans. For the isolation of genistein, daidzein, glycitein, and their respective acetyl, malonyl, and glucoside forms, an extraction employing 80% aqueous MeOH enhanced by sonication was used. As far as the total isoflavones (expressed as aglycones) were to be determined, an acid hydrolysis with 80% aqueous EtOH and refluxing had to be employed, while in the latter case a good agreement of the results with the data generated by the UHPLC-orbitrap MS method was achieved, in the case of the analysis of non-hydrolyzed extracts, some overestimation of the results as compared with those generated by UHPLC-orbitrap MS was observed. A careful investigation of this phenomenon showed that the free aglycones originated from the conjugated forms of isoflavones in the DART ion source, thus contributing significantly to the "free" genistein/daidzein/glycitein signals during the DART analysis. Good recoveries (95-102%) and repeatabilities (RSD: 7-15%) were obtained at the spiking levels of 0.5, 1, and 0.05 g/kg, for daidzein, genistein, and glycitein, respectively. The limits of detection estimated for the respective analytes were 5 mg/kg.

• MeSH
• časové faktory MeSH
• Glycine max chemie   MeSH
• hmotnostní spektrometrie s elektrosprejovou ionizací MeSH
• isoflavony analýza   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

A combination of direct analysis in real time (DART) ionization coupled to time-of-flight mass spectrometry (TOFMS) and chemometrics was used for animal fat (lard and beef tallow) authentication. This novel instrumentation was employed for rapid profiling of triacylglycerols (TAGs) and polar compounds present in fat samples and their mixtures. Additionally, fat isolated from pork, beef, and pork/beef admixtures was analyzed. Mass spectral records were processed by principal component analysis (PCA) and stepwise linear discriminant analysis (LDA). DART-TOFMS profiles of TAGs were found to be more suitable for the purpose of discrimination among the examined fat types as compared to profiles of polar compounds. The LDA model developed using TAG data enabled not only reliable classification of samples representing neat fats but also detection of admixed lard and tallow at adulteration levels of 5 and 10% (w/w), respectively. The presented approach was also successfully applied to minced meat prepared from pork and beef with comparable fat content. Using the DART-TOFMS TAG profiles of fat isolated from meat mixtures, detection of 10% pork added to beef and vice versa was possible.

• MeSH
• diskriminační analýza MeSH
• hmotnostní spektrometrie přístrojové vybavení   metody   MeSH
• maso analýza   MeSH
• prasata MeSH
• řízení kvality MeSH
• skot MeSH
• triglyceridy analýza   MeSH
• tuky chemie   MeSH
• zvířata MeSH
• Check Tag
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH

The use of fast semi-automated method employing direct analysis in real time (DART) ion source coupled to time-of-flight mass spectrometry (TOFMS) for analysis of melamine (MEL) and cyanuric acid (CYA) in milk powder and milk based products has been demonstrated in this study. Simple sample extraction procedure employing methanol-5% aqueous formic acid mixture, which enabled disruption of melamine-cyanurate complex, was followed by direct, high-throughput (30s per run) examination of sample extracts spread on a glass rod by mass spectrometry under ambient conditions, without any prior chromatographic separation. After optimization of instrument parameter settings, limits of detection (LODs) 170 and 450microgkg(-1) were achieved for MEL and CYA, respectively. In the final phase of study, the possibility of minimizing spectral interference, thus improving method performance characteristics through the use of ultrahigh resolving power offered by Orbitrap based mass analyzer is demonstrated.

• MeSH
• hmotnostní spektrometrie metody   MeSH
• kontaminace potravin analýza   MeSH
• limita detekce MeSH
• mléko chemie   MeSH
• prášky, zásypy, pudry analýza   MeSH
• skot MeSH
• triaziny analýza   MeSH
• zvířata MeSH
• Check Tag
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH

RATIONALE: Direct analysis in real time (DART) is a novel ionization technique that has been demonstrated in numerous applications as a useful tool for fast and convenient mass spectrometry (MS)-based analysis of complex samples. In this study, the feasibility of DART ionization coupled to a high-resolution mass spectrometer utilizing an orbitrap mass analyzer (orbitrap MS) for high-throughput analysis of antiparasitic veterinary drugs was explored. METHODS: To obtain the best DART-orbitrap MS performance, stepwise optimization of instrumental parameter settings, such as ionization gas temperature and mass resolving power, was performed. The optimized method was applied to feed and bovine milk samples previously extracted following a QuEChERS-like strategy. RESULTS: Most antiparasitic drugs could be analyzed following the described method. Positive DART ionization provided the protonated molecules [M+H](+); in negative DART ion mode, deprotonated molecules [M-H](-) were observed. As an exception, polyether ionophores could be observed as the sodiated adducts [M+Na](+). Samples of milk and feed were extracted using a modified QuEChERS method for the determination of benzimidazoles and coccidiostats respectively and quantification was carried out by matrix-matched calibration curves. CONCLUSIONS: The combination of an analysis time of less than 1 min per sample and the possibility to acquire accurate masses under high mass resolving power (HR) makes the DART-HRMS technique an effective tool for rapid qualitative screening of antiparasitic veterinary drugs. Additionally, the results obtained in this study demonstrated the feasibility of this approach to quantify target analytes at levels down to 1 µg kg(-1) for benzimidazolic compounds in milk and 0.25 mg kg(-1) for coccidiostats in chicken feed.

• MeSH
• antiparazitární látky analýza   chemie   MeSH
• benzimidazoly analýza   chemie   MeSH
• hmotnostní spektrometrie metody   MeSH
• krmivo pro zvířata analýza   MeSH
• mléko chemie   MeSH
• rychlé screeningové testy metody   MeSH
• skot MeSH
• veterinární léky analýza   chemie   MeSH
• zvířata MeSH
• Check Tag
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

In this study, direct analysis in real time-mass spectrometry (DART-MS) was assessed for the analysis of various pharmaceutical formulations with intention to summarize possible applications for the routine pharmaceutical development. As DART is an ambient ionization technique, it allows direct analysis of pharmaceutical samples in solid or liquid form without complex sample preparation, which is often the most time-consuming part of the analytical method. This makes the technique suitable for many application fields, including pharmaceutical drug development. DART mass spectra of more than twenty selected tablets and other common pharmaceutical formulations, i.e. injection solutions, ointments and suppositories developed in the pharmaceutical industry during several recent years are presented. Moreover, as thin-layer chromatography (TLC) is still very popular for the monitoring of the reactions in the synthetic chemistry, several substances were analyzed directly from the TLC plates to demonstrate the simplicity of the technique. Pure substance solutions were spotted onto a TLC plate and then analyzed with DART without separation. This was the first DART-MS study of pharmaceutical dosage forms using DART-Orbitrap combination. The duration of sample analysis by the DART-MS technique lasted several seconds, allowing enough time to collect sufficient number of data points for compound identification. The experimental setup provided excellent mass accuracy and high resolution of the mass spectra which allowed unambiguous identification of the compounds of interest. Finally, DART mass spectrometry was also used for the monitoring of the selected impurity distribution in the atorvastatin tablets. These measurements demonstrated DART to be robust ionization technique, which provided easy-to-interpret mass spectra for the broad range of compounds. DART has high-throughput potential for various types of pharmaceutical analyses and therefore eliminates the time for sample cleanup and chromatographic separation.

• MeSH
• chromatografie na tenké vrstvě metody   MeSH
• hmotnostní spektrometrie metody   MeSH
• léčivé přípravky analýza   chemie   MeSH
• objevování léků * MeSH
• pomocné látky chemie   MeSH
• tablety chemie   MeSH
• vysokoúčinná kapalinová chromatografie metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Ambient mass spectrometry employing a direct analysis in real time (DART) ion source coupled to a medium high-resolution/accurate mass time-of-flight mass spectrometer (TOFMS) was used as a rapid tool for metabolomic fingerprinting to study the effects of supplemental feeding with cereals (triticale) on the composition of muscle metabolites of common carp (Cyprinus carpio L.). First, the sample extraction and DART-TOFMS instrumental conditions were optimized to obtain the broadest possible representation of ionizable compounds occurring in the extracts obtained from common carp muscle. To this end, a simultaneous (all-in-one) extraction procedure was developed employing water and cyclohexane mixture as the extraction solvents. Under these conditions both polar as well as non-polar metabolites were isolated within a single extraction step. Next, the metabolomic fingerprints (mass spectra) of a large set of common carp muscle extracts were acquired. Finally, the experimental data were statistically evaluated using principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA). Using this approach, differentiation of common carp muscle in response to dietary supplementation (feeding with and without cereals) was feasible. Correct classification was obtained based on the assessment of polar and as well as non-polar extracts fingerprints. The current study showed that DART-TOFMS metabolomic fingerprinting represents a rapid and powerful analytical strategy enabling differentiation of common carp muscles according to feeding history by recording metabolomic fingerprints of ionizable components under the conditions of ambient MS.

• MeSH
• analýza hlavních komponent MeSH
• cyklohexany MeSH
• dieta MeSH
• extrakce kapalina-kapalina MeSH
• jedlá semena * MeSH
• kapři růst a vývoj   metabolismus   MeSH
• kosterní svaly chemie   metabolismus   MeSH
• metabolom * MeSH
• metoda nejmenších čtverců MeSH
• potravní doplňky * MeSH
• rozpouštědla MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice metody   MeSH
• voda MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH