UNLABELLED: The aim of this study was to identify parameters influencing DNA extraction and PCR amplification efficiencies in an attempt to standardize Mucorales qPCR. The Fungal PCR Initiative Mucorales Laboratory Working Group distributed two panels of simulated samples to 26 laboratories: Panel A (six sera spiked with Mucorales DNA and one negative control serum) and Panel B (six Mucorales DNA extracts). Panel A underwent DNA extraction in each laboratory according to the local procedure and were sent to a central laboratory for testing using three different qPCR techniques: one in-house qPCR assay and two commercial assays (MucorGenius and Fungiplex). Panel B DNA extracts were PCR amplified in each laboratory using local procedures: nine in-house qPCR assays and two commercial kits (MucorGenius and MycoGENIE). All data were compiled and anonymously analyzed at the central laboratory. For Panel A, a total of six different automated platforms and five manual extraction methods were used. Positive rates were 64%, 70%, and 89%, for the MucorGenius, Fungiplex, and the in-house qPCR assay, respectively. Using a large volume of serum for DNA extraction provided the highest analytical sensitivity (82.5% for 1 mL compared with 62.7% for smaller volumes, P < 0.01). For Panel B, five in-house qPCR assays and two commercial kits had >78% positivity. Using larger PCR input volumes (≥7 μL) was associated with the highest sensitivity at 95.5% compared to 58.3% when lower input volumes were used (P < 0.01). Using larger sample volumes for nucleic acid extraction and DNA template volumes for PCR amplification significantly improves the performance of Mucorales qPCR when testing serum. IMPORTANCE: Mucormycosis is a life-threatening mold infection affecting immunosuppressed patients but also other patients with diabetes or trauma. Better survival is linked to shorter delays in diagnosis and treatment initiation. Detection of Mucorales-free DNA in serum or plasma using quantitative PCR allows a prompt diagnosis and earlier treatment. Several techniques and protocols of quantitative Mucorales PCR are used in Europe, and improving performance remains a common objective of laboratories participating in the fungal PCR Initiative Working Group. This study, which combined results from 26 laboratories in Europe, showed that the main parameters underpinning sensitivity are the preanalytical variables (volume of serum used for DNA extraction and DNA template volume), irrespective of the extraction platforms and qPCR assay/platform.
- MeSH
- diagnostické techniky molekulární normy metody MeSH
- DNA fungální * krev genetika MeSH
- kvantitativní polymerázová řetězová reakce * normy metody MeSH
- lidé MeSH
- Mucorales * genetika izolace a purifikace MeSH
- mukormykóza * diagnóza mikrobiologie krev MeSH
- senzitivita a specificita * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
The largest obstacle in the promotion of biopesticides is the existence of counterfeit products available in the market. Identification and quantification of antagonistic organisms in biopesticide products are the key to the reduction of spurious microbial pesticides. In this study, we have developed a simple, sensitive, isothermal-based colourimetric assay for specific detection of Bacillus subtilis from the biopesticide formulations and soil samples. A region specific to B. subtilis which codes for shikimate dehydrogenase was identified through in silico analysis. We employed conventional PCR, loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), and qPCR for specific detection of B. subtilis in soil samples and biopesticide formulations. Specificity tests showed that the PCR primers amplified an amplicon of 521 bp in four strains of B. subtilis only, and no amplification was found in negative control samples. Similarly, the LAMP assay showed sky blue colour in all four strains of B. subtilis and violet colour in negative control samples. Whereas in the RPA assay, upon the addition of SYBR Green dye, a bright green colour was seen in B. subtilis strains, while a brick-red colour was observed in negative control samples by visualizing under a UV transilluminator. The qPCR assay showed specific amplifications with a Ct value of 12 for B. subtilis strains and no amplification in negative control samples. In the sensitivity test, PCR could amplify DNA of B. subtilis up to 500 pg/μL. DNA concentration as low as 10 pg/μL was enough to show the colour change in the LAMP as well as the RPA assays, whereas the qPCR assay showed sensitivity till 100 pg/μL. All four diagnostic assays developed in the study have been validated in soil samples and B. subtilis-based biopesticides. Compared to conventional PCR, the qPCR assay has the advantage of quantification and visualizing the result in real-time, whereas LAMP and RPA assays have the benefits of being colourimetric and less time-consuming. The other advantages are that the results can be visualized with the naked eye, and these assays do not require a costly thermal cycler and gel documentation system. Hence, LAMP and RPA assays are highly suitable for developing point-of-need diagnostic kits and, in turn, help regulators assess the quality of biopesticides in the market.
- MeSH
- alkoholoxidoreduktasy * genetika MeSH
- Bacillus subtilis * genetika izolace a purifikace enzymologie MeSH
- bakteriální proteiny genetika MeSH
- diagnostické techniky molekulární * metody MeSH
- kolorimetrie * metody MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- půdní mikrobiologie MeSH
- senzitivita a specificita MeSH
- techniky amplifikace nukleových kyselin * metody MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND: Filoviruses, including Ebola and Marburg viruses, cause severe hemorrhagic fever in humans and primates. These viruses pose significant threats to public health, making rapid and sensitive detection critical for controlling outbreaks. We developed and validated a hemi-nested generic PanFilo assay to detect all Ebola virus species, Marburg viruses, and recently discovered bat filoviruses. This assay was deployed to 15 European laboratories and evaluated through testing of eight non-infectious samples. OBJECTIVES: Laboratories were asked to determine the detection limit of positive controls and test all samples using the assay provided. The deployed assay enables direct Nanopore sequencing of PCR products, by using tagged primers during the second round of PCR. Sequencing of the samples was carried out on a voluntary basis. RESULTS: Multicenter validation revealed a 95 % limit of detection of 5309 RNA copies/μL for Ebola, 10,273 copies/μL for Marburg, and 2145 copies/μL for Mengla virus. In an implementation quality assessment, 93.3 % (84/90) of samples containing filovirus RNA were correctly identified and 100 % (30/30) of filovirus-negative samples were correctly identified. Thirteen laboratories sequenced PCR products, with nine identifying all positive samples correctly. CONCLUSION: The assay enables rapid and reliable detection of filoviruses, with sequencing capabilities for identifying both known and novel variants. This assay might be used for detection during the initial phase of an emerging filovirus outbreak, before a specific assay has been developed. However, our distribution across 15 laboratories revealed variability challenges due to reagents, human performance, and sequencing capacity, emphasizing the need for more training and standardization.
- MeSH
- diagnostické techniky molekulární metody MeSH
- hemoragická horečka Ebola * diagnóza virologie MeSH
- lidé MeSH
- limita detekce MeSH
- marburgská horečka * diagnóza virologie MeSH
- polymerázová řetězová reakce * metody MeSH
- RNA virová genetika MeSH
- senzitivita a specificita MeSH
- virus Ebola * izolace a purifikace genetika MeSH
- virus Marburg * izolace a purifikace genetika MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- multicentrická studie MeSH
- validační studie MeSH
- Geografické názvy
- Evropa MeSH
INTRODUCTION: Central nervous system (CNS) involvement in diffuse large B-cell lymphoma (DLBCL) is a rare but serious condition requiring accurate diagnostics. Cerebrospinal fluid (CSF) analysis plays a crucial role, particularly in cases where biopsy is not feasible, and imaging is inconclusive. AREAS COVERED: Chemical markers have limitations, particularly in low-cellularity samples. Novel molecular techniques, including circulating tumor DNA (ctDNA) analysis and microRNAs (miRNAs), are gaining prominence for their ability to detect gene mutations at diagnosis and monitor minimal residual disease during follow-up. The sensitivity and specificity of genetic mutations, particularly MYD88 L265P, in combination with interleukin-10 (IL-10) levels, are discussed. The literature search methodology involved reviewing relevant studies and clinical data.This review examines both traditional and emerging methods for CSF analysis in diagnosing CNS involvement in DLBCL. Conventional approaches such as cytomorphology, flow cytometry, and biochemical markers have limitations, particularly in low-cellularity samples. Novel molecular techniques, including ctDNA analysis and miRNAs, are gaining prominence for their ability to detect gene mutations at diagnosis and monitor minimal residual disease during follow-up. The sensitivity and specificity of genetic mutations, particularly MYD88 L265P, in combination with interleukin-10 (IL-10) levels, are discussed. The literature search methodology involved reviewing relevant studies and clinical data. EXPERT OPINION: Advancements in CSF biomarker analysis are improving the diagnosis of CNS lymphoma, aiding early detection and personalized treatment approaches. However, further research and broader clinical validation are necessary for their routine implementation.
- MeSH
- cirkulující nádorová DNA mozkomíšní mok genetika MeSH
- diagnostické techniky molekulární metody MeSH
- difúzní velkobuněčný B-lymfom * diagnóza mozkomíšní mok genetika patologie MeSH
- interleukin-10 genetika mozkomíšní mok MeSH
- lidé MeSH
- meningeální nádory * diagnóza mozkomíšní mok genetika MeSH
- mikro RNA genetika mozkomíšní mok MeSH
- mutace MeSH
- myeloidní diferenciační faktor 88 genetika MeSH
- nádorové biomarkery * mozkomíšní mok genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
OBJECTIVES: The aim of the study was to evaluate the efficiency of molecular diagnostics of tick-borne encephalitis (TBE) and to correlate viral RNA (vRNA) detection with the clinical and laboratory data. METHODS: Clinical samples from 1125 patients from South Bohemia, Czech Republic, a highly endemic TBE region, were screened for TBE virus (TBEV) RNA by RT-qPCR. Samples included blood, serum, cerebrospinal fluid (CSF), and urine. RESULTS: TBEV RNA was detected in 14 patients with clinically proven TBE. TBEV RNA was most frequently detected in sera during early infection (11/37 patients tested, 29.7%) but decreased with rising IgG antibody response (3/228, 1.3%). Detection in CSF and urine was infrequent (1/30, 3.3% and 1/52, 1.9%, respectively). Additionally, five patients initially not diagnosed with TBE were retrospectively found to have TBEV RNA in serum, indicating possible underdiagnosis, particularly in mild or atypical presentations. The study also highlighted the diagnostic challenge of an immunocompromised patient whose delayed antibody response hindered timely diagnosis. In such cases, RT-qPCR could significantly shorten the diagnostic timeline. CONCLUSIONS: These findings underscore the value of early RNA detection in improving the diagnosis of TBE and may in the future facilitate the early administration of potential treatment, thereby improving patient outcomes.
- MeSH
- diagnostické techniky molekulární metody MeSH
- dítě MeSH
- dospělí MeSH
- imunoglobulin G krev MeSH
- klíšťová encefalitida * diagnóza virologie MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladiství MeSH
- mladý dospělý MeSH
- předškolní dítě MeSH
- protilátky virové krev MeSH
- RNA virová * krev mozkomíšní mok izolace a purifikace MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- viry klíšťové encefalitidy * izolace a purifikace genetika MeSH
- Check Tag
- dítě MeSH
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladiství MeSH
- mladý dospělý MeSH
- mužské pohlaví MeSH
- předškolní dítě MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Česká republika MeSH
Epilepsia je komplexné neurologické ochorenie, ktoré postihuje 40 - 60 miliónov ľudí na celom svete. V patogenéze epilepsie zohrávajú významnú úlohu viaceré genetické faktory, čo vedie k rastúcemu významu genetiky v oblasti epileptológie. S rozvojom metodík využívajúcich masívne paralelné sekvenovanie boli identifikované mnohé DNA varianty spôsobujúce epilepsiu, čím sa zlepšuje naše chápanie molekulárnych mechanizmov súvisiacich s klinickými prejavmi geneticky podmienených epilepsií. V tejto práci ponúkame prehľad súčasných, ale aj budúcich možností genetickej diagnostiky epilepsie, ktorá prostredníctvom určenia génových variantov u pacientov s monogénovou aj polygénovou epilepsiou môže otvoriť cestu k cielenej personalizovanej diagnostike a liečbe.
Epilepsy is a complex neurological disease that affects 40-60 million people worldwide. Multiple genetic factors play a significant role in the pathogenesis of epilepsy, leading to the growing importance of genetics in the field of epileptology. With the development of methodologies using massively parallel sequencing, many DNA variants causing epilepsy have been identified, improving our understanding of the molecular mechanisms involved in the clinical manifestations of genetically determined epilepsies. In this paper, we offer an overview of current but also future possibilities for genetic diagnostics of epilepsy, which, by identifying gene variants in patients with both monogenic and polygenic epilepsy, may open the way to targeted personalized diagnosis and treatment.
- MeSH
- diagnostické techniky molekulární metody MeSH
- epilepsie * diagnóza genetika klasifikace MeSH
- genetické testování * metody MeSH
- karyotypizace metody MeSH
- lidé MeSH
- sekvenování celého genomu metody MeSH
- sekvenování exomu metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
- přehledy MeSH
Cíl: Zjistit výskyt potenciálně patogenních druhů babesií pro člověka v klíšťatech a v krvi psů a jelenů ve vybraných regionech České republiky. Prevalenci Babesia spp. v klíšťatech porovnat s výskytem jiných patogenů přenášených klíšťaty jako Borrelia spp., Anaplasma spp., Rickettsia spp. Materiál a metody: Vzorky klíšťat byly jednotlivě homogenizovány, ze vzorků klíšťat a krve živočichů provedena izolace DNA. Detekce Babesia spp. byla stanovena metodou PCR 18S rRNA genu a sekvenační analýzou PCR produktů určeny jednotlivé druhy babesií. Výsledky: V letech 2014–2016 byla analyzována klíšťata a krev psů a jelenů na různých místech České republiky. Ze souboru 675 klíšťat Ixodes ricinus dosahovala pozitivita na přítomnost Babesia spp. hodnot od 0,0 do 3,3 %. Sekvenační analýzou byly v klíšťatech identifikovány druhy Babesia venatorum, Babesia microti (patogenní druhy pro člověka) a druh Babesia capreoli. Prevalence Babesia spp. v klíšťatech byla v porovnání s výskytem jiných patogenů jako Borrelia burgdorferi s. l. (29,3 %), Anaplasma phagocytophilum (4,9 %) nižší a srovnatelná s Rickettsia spp. (1,6 %). U třetiny pozitivních klíšťat na babesie byla zjištěna koinfekce s Borrelia burgdorferi s. l. (B. venatorum – Borrelia garinii, Borrelia afzelii a B. microti – B. afzelii). Ze 109 vzorků krve psů bylo 3,7 % pozitivních na Babesia spp. s výskytem druhů Babesia gibsoni a Babesia vulpes. Z 50 vzorků krve jelenů z přírodního ekosystému dosahovala pozitivita 4,0 %. Identifikován byl druh Babesia divergens, nejvíce patogenní druh Babesia spp. pro člověka. Z 80 vzorků krve jelenů chovaných na farmách bylo pozitivních 5,0 % s výskytem druhu Babesia odocoilei. Nukleotidové sekvence babesií způsobujících humánní babesiózu byly zaslány do genové banky a přijaty pod čísly ON892053 (B. venatorum), ON892061 (B. microti), ON892067 (B. divergens). Závěr: Metodou PCR 18S rRNA genu a sekvenací amplikonů byly na území České republiky detekovány tři druhy babesií patogenních pro člověka: B. divergens, B. venatorum, B. microti. Výskyt těchto druhů babesií znamená potenciální riziko onemocnění babesiózou, zejména pro asplenické a imunokompromitované pacienty. Zjištěné koinfekce s Borrelia burgdorferi s. l. mohou být příčinou komplikovaného průběhu onemocnění.
Aim: To determine the occurrence of species of Babesia potentially pathogenic for humans in ticks and in the blood of dogs and deer in selected regions of the Czech Republic. To compare the prevalence of Babesia spp. in ticks with that of other tick-borne pathogens, such as Borrelia spp., Anaplasma spp., and Rickettsia spp. Material and Methods: Tick samples were individually homogenized. DNA was isolated from tick samples and animal blood. The detection of Babesia spp. was based on PCR of the 18S rRNA gene, and the identification to the species level was done by sequencing analysis of the PCR products. Results: In 2014–2016, ticks and blood of dogs and deer collected in various areas of the Czech Republic were analyzed. In a set of 675 Ixodes ricinus ticks, the positivity rate for Babesia spp. varied from 0.0 to 3.3 %. The species Babesia venatorum, Babesia microti (both pathogenic for humans), and Babesia capreoli were identified in ticks by sequencing analysis. The prevalence of Babesia spp. in ticks compared to that of other pathogens such as Borrelia burgdorferi s. l. (29.3 %) or Anaplasma phagocytophilum (4.9 %) was lower and comparable to that of Rickettsia spp. (1.6 %). Co-infection with Borrelia burgdorferi s.l (B. venatorum – Borrelia garinii, Borrelia afzelii, and B. microti – B. afzelii) was found in a third of Babesia spp. positive ticks. Out of 109 dog blood samples, 3.7 % were positive for Babesia spp., specifically Babesia gibsoni and Babesia vulpes. Of 50 blood samples of wild deer from the natural ecosystem, the positivity rate reached 4.0 %. The species Babesia divergens, a major human pathogen, was identified. Out of 80 blood samples from farmed deer, 5.0 % were positive for the species Babesia odocoilei. Nucleotide sequences of the agents causing human babesiosis were deposited in the gene bank under accession numbers ON892053 (B. venatorum), ON892061 (B. microti), and ON892067 (B. divergens). Conclusions: Using PCR of the 18S rRNA gene and amplicon sequencing, three species of Babesia causing human babesiosis were detected in the Czech Republic: B. divergens, B. venatorum, and B. microti. Babesia spp. pathogenic for humans pose a potential risk especially in asplenic and immunocompromised patients. The detected co-infections with Borrelia spp. can be the cause of a complicated course of the disease.
- MeSH
- Babesia mikrobiologie MeSH
- babezióza * epidemiologie krev přenos MeSH
- Borrelia burgdorferi MeSH
- diagnostické techniky molekulární metody MeSH
- klíšťata * mikrobiologie MeSH
- koinfekce diagnóza přenos MeSH
- krev mikrobiologie MeSH
- lidé MeSH
- nemoci přenášené klíšťaty epidemiologie přenos prevence a kontrola MeSH
- polymerázová řetězová reakce metody MeSH
- psi * mikrobiologie MeSH
- vysoká zvěř * krev mikrobiologie MeSH
- Check Tag
- lidé MeSH
- psi * mikrobiologie MeSH
- Geografické názvy
- Česká republika MeSH
Standardy molekulárně-patologického testování se s pátým vydáním WHO klasifikace nádorů centrálního nervového systému razantně mění. Významnou změnou je zapojení molekulárně genetického testování a využívání komplexního přístupu v diagnostice. Některé typy nádorů pro své správné zařazení vyžadují vyšetření molekulárního profilu. Nejčastěji jde o vyšetření jednonukleotidových mutací, delecí a kodelecí, fúzí, případně metylací. Molekulární metody rozšiřují diagnostické spektrum založené na histopatologickém a imunohistochemickém zhodnocení. Využívají se molekulární metody, jako je PCR, Sangerovo sekvenování, I‐FISH, MLPA a/nebo NGS. Zcela zásadní je pro diagnostiku úzká spolupráce vyšetřujícího neuropatologa, molekulárního genetika a neuroonkologa, bez níž může dojít nesprávně užitými postupy k významnému poškození pacienta.
The standards of molecular-pathological testing are transforming with the fifth edition of the WHO classification of central nervous system tumors. A significant change involves incorporating molecular genetic testing and adopting a comprehensive diagnostic approach. Certain tumors require examining the molecular profile for accurate classification, typically analyzing single nucleotide variants, deletions, codeletions, fusions, or methylation. Molecular methods extend the diagnostic spectrum beyond histopathological and immunohistochemical assessments, using techniques like PCR, Sanger sequencing, I-FISH, MLPA, and/or NGS. Collaboration among neuropathologists, molecular geneticists, and neurooncologists is crucial for accurate diagnosis and preventing potential harm to patients.
- MeSH
- diagnostické techniky molekulární metody MeSH
- hybridizace in situ fluorescenční MeSH
- imunohistochemie MeSH
- lidé MeSH
- metylace DNA MeSH
- multiplexová polymerázová řetězová reakce MeSH
- mutace genetika MeSH
- nádory centrálního nervového systému * diagnóza genetika patologie MeSH
- polymerázová řetězová reakce MeSH
- sekvenční analýza DNA MeSH
- vysoce účinné nukleotidové sekvenování MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- přehledy MeSH
- MeSH
- diagnostické techniky molekulární * metody MeSH
- lidé MeSH
- molekulární patologie * metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- úvodní články MeSH
- úvodníky MeSH
- Klíčová slova
- ivosidenib, azacitidine,
- MeSH
- akutní myeloidní leukemie * diagnóza farmakoterapie genetika MeSH
- diagnostické techniky molekulární metody MeSH
- genetické testování MeSH
- inhibitory enzymů farmakologie terapeutické užití MeSH
- lidé MeSH
- protokoly protinádorové kombinované chemoterapie MeSH
- randomizované kontrolované studie jako téma MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
