Fast and accurate detection of causative agents of bloodstream infections remains a challenge of today's microbiology. We compared the performance of cutting-edge technology based on polymerase chain reaction coupled with electrospray ionization-mass spectrometry (PCR/ESI-MS) with that of conventional broad-range 16S rRNA PCR and blood culture to address the current diagnostic possibilities for bloodstream infections. Of 160 blood samples tested, PCR/ESI-MS revealed clinically meaningful microbiological agents in 47 samples that were missed by conventional diagnostic approaches (29.4% of all analyzed samples). Notably, PCR/ESI-MS shortened the time to positivity of the blood culture-positive samples by an average of 34 hr. PCR/ESI-MS technology substantially improved current diagnostic tools and represented an opportunity to make bloodstream infections diagnostics sensitive, accurate, and timely with a broad spectrum of microorganisms covered.

• MeSH
• Bacteremia * diagnosis   MeSH
• Spectrometry, Mass, Electrospray Ionization methods   MeSH
• Humans MeSH
• Microbiological Techniques methods   MeSH
• Polymerase Chain Reaction methods   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Clinical Study MeSH
• Research Support, Non-U.S. Gov't MeSH

Fast and accurate detection of causative agents of bloodstream infections remains a challenge of today's microbiology. We compared the performance of cutting-edge technology based on polymerase chain reaction coupled with electrospray ionization-mass spectrometry (PCR/ESI-MS) with that of conventional broad-range 16S rRNA PCR and blood culture to address the current diagnostic possibilities for bloodstream infections. Of 160 blood samples tested, PCR/ESI-MS revealed clinically meaningful microbiological agents in 47 samples that were missed by conventional diagnostic approaches (29.4% of all analyzed samples). Notably, PCR/ESI-MS shortened the time to positivity of the blood culture-positive samples by an average of 34 hr. PCR/ESI-MS technology substantially improved current diagnostic tools and represented an opportunity to make bloodstream infections diagnostics sensitive, accurate, and timely with a broad spectrum of microorganisms covered.

• MeSH
• Bacteremia diagnosis   microbiology   MeSH
• Molecular Diagnostic Techniques methods   MeSH
• Adult MeSH
• Spectrometry, Mass, Electrospray Ionization methods   MeSH
• Middle Aged MeSH
• Humans MeSH
• Microbiological Techniques MeSH
• Young Adult MeSH
• Polymerase Chain Reaction methods   MeSH
• Reproducibility of Results MeSH
• RNA, Ribosomal, 16S MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Sepsis diagnosis   microbiology   MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Young Adult MeSH
• Male MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Požadavek časné identifikace infekčních agens zůstává v mikrobiologické diagnostice výzvou zvláště u pacientů, jež infekce ohrožuje na životě. Standardní postupy založené na kultivaci jsou zatíženy časovou prodlevou, navíc mohou selhat u obtížně kultivovatelných agens či u pacienta na antibiotické léčbě. Cílem projektu je zhodnotit úspěšnost nejnovějších molekulárně genetických metod, u nichž se předpokládá, že řeší nedostatky kultivace v klinicky vypjatých situacích. Zaměříme se na diagnostiku infekcí krevního řečiště u vysoce rizikových hematoonkologických pacientů a na identifikaci původců infekční endokarditidy, kultivačně často negativní. K tomu využijeme nejnovějšího postupu kombinace hmotnostní spektrometrie a ionizace produktů PCR elektrosprejem (ESI-MS). Identifikaci mikroorganismu cestou patogen-specifického PCR zvolíme pro další klinicky významné situace v pediatrii, kde je kultivační diagnostika suboptimální: pneumokoková pneumonie, herpesvirová encefalitis a znovu se objevující pertuse. Detekce bude probíhat v uzavřeném systému s extrakcí DNA a PCR v reálném čase.; Early identification of infectious agents is still an unmet need in microbiological diagnostics especially for patients with life threatening infections. Downside of standard culture based methods is their inherent time delay; in addition they may fail in fastidious organisms or in a patient on antibiotic therapy. The aim of the project is to evaluate usefulness of the latest molecular genetic methods where one expects they solve the drawbacks of cultivation in emergency situations. We will focus on diagnostics of bloodstream infections in high risk haematooncological patients and on identification of causes of infective endocarditis, frequently culture negative. We will utilize a cutting edge technology of electrospray ionization-mass spectrometry (ESI-MS). Microorganism identification via pathogen-specific PCR will be applied to other clinically serious situations in paediatrics where culture diagnostics is suboptimal: pneumococcal pneumonia, herpesviral encephalitis and re-emerging pertussis. Detection will be carried out in a closed system with DNA extraction and real time PCR.

• MeSH
• Bacterial Infections diagnosis   MeSH
• Early Diagnosis MeSH
• Endocarditis MeSH
• Hematologic Neoplasms MeSH
• Encephalitis, Herpes Simplex diagnosis   MeSH
• Spectrometry, Mass, Electrospray Ionization MeSH
• Critical Illness MeSH
• Vascular Diseases diagnosis   MeSH
• Whooping Cough diagnosis   MeSH
• Pneumonia, Pneumococcal MeSH

• Conspectus
• Patologie. Klinická medicína
• NML Fields
• infekční lékařství
• mikrobiologie, lékařská mikrobiologie
• NML Publication type
• závěrečné zprávy o řešení grantu AZV MZ ČR

Ixodes ricinus ticks are vectors of numerous human and animal pathogens. They are host generalists able to feed on more than 300 vertebrate species. The prevalence of tick-borne pathogens is influenced by host-vector-pathogen interactions that results in spatial distribution of infection risk. Broad-range polymerase chain reaction electrospray ionization mass spectrometry (PCR/ESI-MS) was used to analyze 435 I. ricinus nymphs from four localities in the south of the Czech Republic for the species identification of tick-borne pathogens. Borrelia burgdorferi sensu lato spirochetes were the most common pathogen detected in the ticks; 21% of ticks were positive for a single genospecies and 2% were co-infected with two genospecies. Other tick-borne pathogens detected included Rickettsia helvetica (3.9%), R. monacensis (0.2%), Anaplasma phagocytophilum (2.8%), Babesia venatorum (0.9%), and Ba. microti (0.5%). The vertebrate host of the ticks was determined using PCR followed by reverse line blot hybridization from the tick's blood-meal remnants. The host was identified for 61% of ticks. DNA of two hosts was detected in 16% of samples with successful host identification. The majority of ticks had fed on artiodactyls (50.7%) followed by rodents (28.6%) and birds (7.8%). Other host species were wild boar, deer, squirrels, field mice and voles.

• MeSH
• Anaplasma phagocytophilum genetics   isolation & purification   MeSH
• Artiodactyla MeSH
• Arvicolinae MeSH
• Babesia classification   genetics   isolation & purification   MeSH
• Borrelia burgdorferi genetics   isolation & purification   MeSH
• Tick Infestations * MeSH
• Ixodes microbiology   parasitology   MeSH
• Humans MeSH
• Mice MeSH
• Surveys and Questionnaires MeSH
• Birds MeSH
• Rickettsia classification   genetics   isolation & purification   MeSH
• Sciuridae MeSH
• Sus scrofa MeSH
• Deer MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Geographicals
• Czech Republic MeSH

Skripta objasňují některé důležité termíny a fakta podstatná k pochopení významu potravinářské mikrobiologie pro bezpečnost potravin. Dále jsou zde popsány vybrané metody používané pro mikrobiologické zkoumání potravin k detekci (kvalitativní stanovení) a kvantifikaci patogenních mikroorganismů přítomných ve vzorcích potravin. Součástí uvedených metod jsou principy a postupy stanovení, včetně základní charakteristiky zkoumaných mikroorganismů.

• MeSH
• Food Analysis MeSH
• Laboratories MeSH
• Microbiology MeSH
• Food Microbiology MeSH

• Conspectus
• Mikrobiologie
• Učební osnovy. Vyučovací předměty. Učebnice
• NML Fields
• mikrobiologie, lékařská mikrobiologie
• NML Publication type
• učebnice vysokých škol

Genes for mannitol-metabolizing enzymes, mannitol phosphate dehydrogenase (MPDH) and mannitol dehydrogenase (MDH), have been recently identified in the genome of Acanthamoeba castellanii and their potential role in stress tolerance was proposed. Using qRT-PCR, comparison has been made of mRNA levels of the enzymes for mannitol metabolism at various time intervals during the stress defence reactions of encystation and pseudocyst formation. Gradual decrease of both enzymes during encystation and slight increases at the beginning of pseudocyst formation were observed. Detailed analysis of mRNA sequences of the two genes revealed similarities with various alcohol dehydrogenases rather than mannitol dehydrogenases. Our results indicate there is probably no protective role for mannitol in Acanthamoeba as no mannitol was detected using HILIC ESI MS, in any Acanthamoeba life cycle stage. Possible misinterpretation of previously published sequences as encoding enzymes of the mannitol metabolic pathway is discussed.

• MeSH
• Acanthamoeba castellanii enzymology   metabolism   physiology   MeSH
• Molecular Sequence Annotation MeSH
• Stress, Physiological MeSH
• Transcription, Genetic MeSH
• Conserved Sequence MeSH
• Real-Time Polymerase Chain Reaction MeSH
• Mannitol metabolism   MeSH
• Mannitol Dehydrogenases genetics   metabolism   MeSH
• Carbohydrate Metabolism MeSH
• Molecular Sequence Data MeSH
• Protozoan Proteins genetics   metabolism   MeSH
• Gene Expression Regulation, Enzymologic MeSH
• Amino Acid Sequence MeSH
• Spores, Protozoan enzymology   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

We have separated 2b myosin heavy chain (MyHC) isoform from the rat extensor digitorum longus muscle by SDSPAGE and analyzed it by two subsequent mass spectrometry techniques. After tryptic digestion, the obtained peptides were identified by Matrix-Assisted Laser Desorption/Ionisation reflectron Time of Flight mass spectrometry (MALDITOF MS) and sequenced by Liquid chromatography tandem mass spectrometry (ESI LC/MS/MS). The analyzed peptides proportionally covered 30 % of the 2b MyHC isoform sequence. The results suggest that the primary structure is identical with the highest probability to a NCBI database record ref|NP_062198.1|, representing the last updated record of rat 2b isoform. Nonetheless, four peptides carrying amino acid substitution(s) in comparison with the NCBI database record were identified.

• MeSH
• Financing, Organized utilization   MeSH
• Spectrometry, Mass, Electrospray Ionization methods   utilization   MeSH
• Data Interpretation, Statistical MeSH
• Reverse Transcriptase Polymerase Chain Reaction methods   utilization   MeSH
• Rats, Inbred Lew physiology   MeSH
• Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization methods   utilization   MeSH
• Myosin Heavy Chains physiology   isolation & purification   MeSH