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

In 2019, Pantoea piersonii was initially isolated from the interior surfaces of the International Space Station. This microorganism is a species within the genus Pantoea in the family Erwiniaceae, belonging to the order Enterobacterales. Recent literature has documented four cases of its isolation. Despite initial predictions suggesting the non-pathogenicity of P. piersonii strains, evidence from observed cases indicates potential pathogenicity. According to documented evidence in the literature, this microorganism is capable of causing severe and life-threatening conditions, including sepsis. Traditional tests, as well as automated systems, may fail to provide complete differentiation due to these similarities. While MALDI-TOF MS is a valuable tool for identification in clinical diagnostic microbiology, sequencing may be necessary for precise identification. To determine the antibiotic susceptibility profile, various methods can be utilized, including minimum inhibitory concentration determination, disk diffusion testing (Kirby-Bauer test), genotypic resistance assays (PCR and sequencing), and automated systems. The literature reports a limited number of cases associating P. piersonii with human infection. This study contributes to this body of knowledge by reporting a novel case in which P. piersonii was isolated from a tissue sample for the first time. In this case report, the patient achieved recovery following the administration of appropriate antibiotic treatment based on the diagnosis. It underscores the need for precise identification and understanding of its pathogenicity.

• MeSH
• antibakteriální látky * farmakologie   terapeutické užití   MeSH
• enterobakteriální infekce * mikrobiologie   diagnóza   farmakoterapie   MeSH
• lidé MeSH
• mikrobiální testy citlivosti * MeSH
• Pantoea * izolace a purifikace   genetika   patogenita   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• kazuistiky 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

We analyzed mucormycosis data from the Zygomyco.net registry (2009-2022), encompassing cases from 16 countries. India, Russia and the Czech Republic provided the largest contributions. India reported the highest case number, consistent with its substantially higher incidence compared to that of high-income countries. Among the 382 patients with mucormycosis, 236 (61.8%) were male (male-to-female ratio 1.6). The median age was 48 years [interquartile range (IQR) 32-60]. There were 59 pediatric patients (median age ranging from < 1 month to 19 years). Diabetes mellitus type 2 was the most common underlying condition (39%), with significant geographic variation (> 70% of cases in India and Iran but only 6.9% in Europe). Hematologic malignancies (HM, 31.4%), the second most common underlying condition, were absent in India and Iran. The primary clinical presentations were rhino-orbito-cerebral mucormycosis (ROCM, 36.6%), pulmonary (33.2%) and cutaneous mucormycosis (17.5%). Patients with diabetes mellitus typically developed ROCM (55.9%), while pulmonary infections were more common in those with HM or hematopoietic cell transplantation (HCT) (47.5%, p < 0.001). Rhizopus was the leading fungal genus (58%), followed by Lichtheimia (13.7%) and Mucor (7%), with regional variations. Pulmonary infections in HM patients were linked to L. corymbifera and R. microsporus, while Apophysomyces spp. and Saksenaea spp. were more frequent in Indian healthcare-associated cutaneous cases. Concomitant infections were observed in 8.7% of patients with HM, complicating diagnosis and treatment. In most of them (57.1%), Aspergillus spp. was involved. Improved diagnostic practices, including direct microscopy and cultures, showed higher positivity rates, although PCR remained underutilized. Antifungal therapy, primarily with an amphotericin B formulation, combined with surgery, was the most common therapeutic approach. Overall mortality was high (47.8%), particularly in disseminated or advanced ROCM cases. Multivariable analysis identified older age, advanced ROCM, and HM/HCT as independent mortality risk factors (p < 0.05); whereas localized sinusitis and combined medical and surgical therapy were independently associated with improved outcomes (p < 0.006). This study underscores regional disparities in the mucormycosis epidemiology and species distribution. Improved early detection is needed, particularly in immunocompromised populations with HM. Enhanced surveillance and tailored public health strategies are crucial to address this ongoing global health threat.

• MeSH
• celosvětové zdraví MeSH
• dítě MeSH
• dospělí MeSH
• incidence MeSH
• kojenec MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• mukormykóza * epidemiologie   mikrobiologie   farmakoterapie   MeSH
• novorozenec MeSH
• předškolní dítě MeSH
• registrace * MeSH
• Check Tag
• dítě MeSH
• dospělí MeSH
• kojenec MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• novorozenec MeSH
• předškolní dítě MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Evropa 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

x

x

• MeSH
• leukocidiny * izolace a purifikace   MeSH
• lidé MeSH
• polymerázová řetězová reakce MeSH
• stafylokokové infekce diagnóza   patologie   MeSH
• Staphylococcus * izolace a purifikace   patogenita   MeSH
• Check Tag
• lidé MeSH
• Geografické názvy
• Česká republika MeSH

Cíl: Těhotenství se nepovažuje za stav zvyšující náchylnost organismu k infekci SARS-CoV-2 (severe-acute-respiratory-syndrome-related coronavirus 2), avšak v případě nákazy v graviditě se zvyšuje riziko závažnějšího průběhu nemoci covid-19. Ve většině případů ovšem bývá průběh infekce v graviditě mírný nebo bezpříznakový. Hlavním cílem studie u těhotných žen s covidem-19 (coronavirové onemocnění) bylo dokázat, že způsob porodu se nemění a závažné komplikace jak porodnické, tak neonatologické se kvůli tomuto onemocnění nevyskytují častěji. Metody: Do retrospektivní, observační, multicentrické studie byly zařazeny pacientky s pozitivním testem na covid-19, které byly přijaty a následně porodily v období od 15. března 2020 do 15. března 2021. Data z porodnických oddělení pěti center v České republice během pandemie covidu-19 byla analyzována ve vztahu k metodě a době porodu, symptomům covidu-19 a potenciálním komplikacím s ohledem na demografii a komorbidity těhotných žen. Infekce koronavirem byla u pacientek potvrzena pomocí PCR (polymerázové řetězové reakce). Statistická analýza byla hodnocena pomocí programu Excel. Výsledky: Během sledovaného období bylo ve studijních centrech detekováno 236 těhotných žen s covidem-19. Většina pacientek byla asymptomatická (59,7 %). U symptomatických pacientek byly nejčastějšími příznaky kašel (52,6 %), nachlazení (43,2 %) a horečka (37,9 %) a covidová pneumonie byla diagnostikována u 8 pacientek. Porod byl proveden vaginálně u 52,5 % pacientek, těhotenství bylo ukončeno císařským řezem v 44,5 %, per VEX (vakuumextrakce) v 2,1 % a per forcipem v 0,8 % případů. Průměrný týden gravidity v době porodu byl 38 a předčasný porod byl proveden u 19,1 % pacientek. Výsledky tyto studie u těhotných žen s covidem-19 prokázaly, že způsob porodu se nezměnil a výskyt větších komplikací jak porodnických, tak neonatologických nebyl ve většině případů zaznamenán. Dva prezentované závažné průběhy covidu-19 u těhotných žen však vedly k předčasnému ukončení těhotenství. Jediným přidruženým rizikovým faktorem byla obezita pacientky. Závěry: Ačkoli je covid-19 onemocnění, které je u těhotných žen většinou asymptomatické nebo má pouze mírné příznaky podobné chřipce, je spojeno se zvýšenou nemocností a úmrtností ve srovnání s těhotnými ženami bez covidu-19. Výzvou do budoucna je možnost segregace pacientek do nízko a vysoce rizikových skupin na základě prokázaných rizikových faktorů a důsledné očkování těhotných žen nebo žen plánujících početí. V kritických případech je nutné správné načasování předčasného ukončení těhotenství a včasná indikace počátku zrání plic plodu.

Objective: Pregnancy is not considered a condition that increases the body‘s susceptibility to SARS-CoV-2 (severe-acute-respiratory-syndrome-related coronavirus-2) infection, but in the case of infection in pregnancy, there is an increased risk of a more severe course of COVID-19 (coronavirus disease-19). However, the course of infection in pregnancy is mild or asymptomatic in most cases. The main objective of the study in pregnant women with COVID-19 was to prove that the delivery method is not changed, and serious complications do not occur more frequently due to this disease. Methods: In a retrospective, observational, multicentric study, the pregnant women positively tested to COVID-19 were admitted and subsequently gave birth in the period from 15 March 2020 to 15 March 2021. Data from the delivery departments of five centers in the Czech Republic during COVID-19 pandemic were analyzed in relation to the delivery method and time, COVID-19 symptoms and potential complications with respect to demographics and comorbidities of pregnant women. COVID-19 positivity was confirmed with PCR (polymerase-chain reaction). The Excel program was used during statistical analysis. Results: During the observed study period, 236 pregnant women with COVID-19 were detected at study centers. Most of the patients were asymptomatic (59.7%). In symptomatic patients, most common symptoms were cough (52.6 %), cold (43.2%) and fever (37.9%), and COVID-19 pneumonia was diagnosed in 8 patients. The delivery was performed vaginally in 52.5% patients, the pregnancy was terminated by C-section (cesarean section) in 44.5%, per VEX (vacuum extractor) in 2.1% and per forcipem in 0.8% cases. The average week of pregnancy at the time of delivery was 38 (29–41) and preterm delivery was performed in 19.1% patients. The study results in pregnant women with COVID-19 demonstrated that the method of delivery was not changed and major delivery and neonatological complications did not develop in most cases. However, two presented serious courses of COVID-19 in pregnant women led to premature terminations of pregnancies. The only associated risk factor was the patient‘s obesity. Conclusions: Although COVID-19 is a disease that is mostly asymptomatic in pregnant women or with only mild flu-like symptoms, it is associated with increased morbidity and mortality compared to pregnant women without COVID-19. The challenge for the future is the possibility of segregating patients into lowand high-risk groups based on proven risk factors, and consistent vaccination of pregnant women or women planning conception. In critical cases, the correct timing of premature termination of pregnancy and early indication of the beginning of fetal lung maturation is necessary.

• MeSH
• COVID-19 * komplikace   mortalita   MeSH
• diagnostické zobrazování metody   MeSH
• komplikace porodu * mikrobiologie   MeSH
• komplikace těhotenství * mikrobiologie   MeSH
• lidé MeSH
• retrospektivní studie MeSH
• rizikové faktory MeSH
• Check Tag
• lidé MeSH
• Geografické názvy
• Česká republika 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

Androgen deprivation therapy has long been the first-line treatment for hormone-sensitive prostate cancer (HSPC). After progression to castration-resistant prostate cancer (CRPC), androgen receptor pathway inhibitors (ARPIs) are commonly used. Recently, combined therapy with androgen deprivation and an ARPI has been recommended for metastatic HSPC patients. Novel markers are urgently needed for monitoring this disease and for making therapeutic decisions. Plasma samples were collected from 140 patients with either metastatic HSPC (n = 72) or CRPC (n = 68) before the start of ARPI therapy. Digital PCR was used to assess AR gene amplification, while the expression levels of miR-375 were measured by quantitative PCR. Sixteen other clinical markers were also evaluated, including prostate-specific antigen (PSA), chromogranin A (CGA), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), C-reactive protein (CRP), lymphocyte-to-monocyte ratio, and platelet count. A multivariate analysis, adjusted for age and metastatic dissemination, identified miR-375 expression and lymphocyte-to-monocyte ratio to be the independent negative predictors of ARPI therapy failure in CRPC patients. Regarding the HSPC patients, this article reports the primary finding of the independent negative predictive value of platelet count, CRP, and CGA for the failure of combined androgen deprivation therapy and ARPI.

• MeSH
• androgenní receptory genetika   MeSH
• C-reaktivní protein * metabolismus   MeSH
• chromogranin A * krev   MeSH
• lidé středního věku MeSH
• lidé MeSH
• metastázy nádorů MeSH
• mikro RNA * genetika   krev   MeSH
• nádorové biomarkery krev   MeSH
• nádory prostaty rezistentní na kastraci * krev   genetika   patologie   farmakoterapie   diagnóza   MeSH
• neúspěšná terapie MeSH
• prognóza MeSH
• prostatický specifický antigen * krev   MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• trombocyty * metabolismus   MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• Publikační typ
• časopisecké články MeSH

Autosomal dominant craniometaphyseal dysplasia (AD-CMD) is a rare condition defined by the occurrence of progressive diffuse hyperostosis of cranial bones and abnormal metaphyseal widening of the tubular bones. ANKH is known to be the only gene associated with AD-CMD. We present a case of a toddler boy with macrodolichocephaly, asymmetry of the skull, wide bulging forehead, gingival hypertrophy and irregular teeth. Physical examination, X-ray and DNA analysis were performed. All exons and flanking intron regions of ANKH were amplified by PCR and directly sequenced using the Sanger method. X-ray images showed diffuse osteosclerosis in the area of facial skeleton and skull base. Limbs exhibited club-shaped enlargement of the distal metaphysis of the femur and the proximal metaphysis of the tibia were described. The DNA analysis showed that the patient is a heterozygous carrier of the known pathogenic in-frame deletion (rs121908406; ANKH:c.1122-4delCTC, p.Ser375del), which has already been described in patients with AD-CMD.

• MeSH
• hyperostóza MeSH
• hypertelorismus MeSH
• kojenec MeSH
• kraniofaciální abnormality genetika   diagnostické zobrazování   MeSH
• lidé MeSH
• proteiny přenášející fosfát * genetika   MeSH
• radiografie MeSH
• vývojové onemocnění kostí genetika   diagnostické zobrazování   diagnóza   MeSH
• Check Tag
• kojenec MeSH
• lidé MeSH
• mužské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• kazuistiky MeSH