Publications by authors named "Petr Hubacek"

33 Publications

Rhizoferrin Glycosylation in .

J Fungi (Basel) 2020 Jun 18;6(2). Epub 2020 Jun 18.

Institute of Microbiology of the Czech Academy of Sciences, Vídeňská 1083, 142 20 Prague, Czech Republic.

spp. are the most common etiological agents of mucormycosis, causing over 90% mortality in disseminated infections. The diagnosis relies on histopathology, culture, and/or polymerase chain reaction. For the first time, the glycosylation of rhizoferrin (RHF) was described in a clinical isolate by liquid chromatography and accurate tandem mass spectrometry. The fermentation broth lyophilizate contained 345.3 ± 13.5, 1.2 ± 0.03, and 0.03 ± 0.002 mg/g of RHF, imido-RHF, and bis-imido-RHF, respectively. Despite a considerable RHF secretion rate, we did not obtain conclusive RHF detection from a patient with disseminated mucormycosis caused by the same strain. We hypothesize that parallel antimycotic therapy, RHF biotransformation, and metabolism compromised the analysis. On the other hand, the full profile of posaconazole metabolites was retrieved by our in house software CycloBranch.
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http://dx.doi.org/10.3390/jof6020089DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7344610PMC
June 2020

The Power Gain Difference Method Analysis.

Sensors (Basel) 2020 May 26;20(11). Epub 2020 May 26.

Department of Communication Technologies, Electronic Warfare and Radars, University of Defence in Brno, 66210 Brno, Czech Republic.

In this paper, we propose a new approach to passively locate the 3D position of a signal source. This novel technique, called the power gain difference (PGD), is based only on measuring the received signal strength (RSS) with multiple sensors deployed in the area of interest, while the target transmit power or the equivalent isotropic radiated power (EIRP) is assumed to be unknown. Next, the signal source position is estimated using the knowledge of the ratios of RSS measured on different sensors. First, this article presents the geometric representation and the analytical solution of the model of the PGD technique. Second, the PGD dilution of precision was analyzed in order to gauge the accuracy of measuring the RSS. Finally, a numerical simulation of the performance of the proposed method was carried out and the results are discussed. It seems that the PGD technique has the potential to be a simple and effective solution of the 3D localization problem.
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http://dx.doi.org/10.3390/s20113018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7308829PMC
May 2020

[Comparing traditional and commercial molecular biology detection of gastrointestinal pathogens with AusDiagnostics panels in Motol University Hospital].

Klin Mikrobiol Infekc Lek 2019 Dec;25(4):132-139

Department of Medical Microbiology, 2nd Faculty of Medicine, Charles University in Prague and Motol University Hospital, Czech Republic, e-mail:

Background: The aim was to do an internal audit of gastrointestinal pathogen detection at the Department of Medical Microbiology, Motol University Hospital between the years 2014 and 2018 and to test two commercial multiplex molecular biology assays potentially improving the diagnostic process and reducing costs.

Material And Methods: Based on data from a laboratory information system (LIS), a total of 45,888 samples were identified which had been tested for the presence of gastrointestinal pathogens using culture, immunochromatographic, microscopic and molecular biology techniques between 2014-2018. Novel multiplex molecular biology detection was used to test 182 nucleic acid isolates obtained from stool samples with the Enteric Viruses (8-well) assay (Viral Panel, EVP) or Faecal Pathogens M (16-well) assay (Microbial Panel, FPM) manufactured by AusDiagnostics.

Results: The LIS data showed 6.2 % of positive pathogens causing diarrhea from all tested samples (detection rates: 4.5 % for bacterial agents, 21.6 % for viral agents and 0.4 % for parasitic agents). Valid samples (98.9 % of all tested samples) tested by the molecular biology technique yielded, in descending order: C. difficile toxin B (19 %), norovirus (9 %), astrovirus (8 %), Campylobacter (7 %), sapovirus (6 %), Yersinia enterocolitica (6 %), rotavirus (4 %), enterovirus (3 %), Aeromonas (3 %), adenovirus (2 %) and Salmonella (1 %). There was found at least 1 additional new positive detection in 27 % of stools tested by the Viral Panel and in 40 % of stools tested by the Microbial Panel in comparison with the traditional approach. Introducing the panels into routine diagnostic practice will not reduce the costs.

Conclusions: The introduction of novel multiplex molecular biology assays for detecting gastrointestinal pathogens will considerably increase pathogen detection rates even though the costs will be higher for the Department of Medical Microbiology.
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December 2019

Role of Epstein-Barr Virus in Pathogenesis and Racial Distribution of IgA Nephropathy.

Front Immunol 2020 28;11:267. Epub 2020 Feb 28.

Department of Medicine, University of Alabama at Birmingham, Birmingham, AL, United States.

IgA nephropathy (IgAN) is the dominant type of primary glomerulonephritis worldwide. However, IgAN rarely affects African Blacks and is uncommon in African Americans. Polymeric IgA1 with galactose-deficient hinge-region glycans is recognized as auto-antigen by glycan-specific antibodies, leading to formation of circulating immune complexes with nephritogenic consequences. Because human B cells infected with Epstein-Barr virus (EBV) secrete galactose-deficient IgA1, we examined peripheral blood B cells from adult IgAN patients, and relevant controls, for the presence of EBV and their phenotypic markers. We found that IgAN patients had more lymphoblasts/plasmablasts that were surface-positive for IgA, infected with EBV, and displayed increased expression of homing receptors for targeting the upper respiratory tract. Upon polyclonal stimulation, these cells produced more galactose-deficient IgA1 than did cells from healthy controls. Unexpectedly, in healthy African Americans, EBV was detected preferentially in surface IgM- and IgD-positive cells. Importantly, most African Blacks and African Americans acquire EBV within 2 years of birth. At that time, the IgA system is naturally deficient, manifested as low serum IgA levels and few IgA-producing cells. Consequently, EBV infects cells secreting immunoglobulins other than IgA. Our novel data implicate Epstein-Barr virus infected IgA cells as the source of galactose-deficient IgA1 and basis for expression of relevant homing receptors. Moreover, the temporal sequence of racial-specific differences in Epstein-Barr virus infection as related to the naturally delayed maturation of the IgA system explains the racial disparity in the prevalence of IgAN.
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http://dx.doi.org/10.3389/fimmu.2020.00267DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7058636PMC
March 2021

Heterologous Cytomegalovirus and Allo-Reactivity by Shared T Cell Receptor Repertoire in Kidney Transplantation.

Front Immunol 2019 31;10:2549. Epub 2019 Oct 31.

Transplant Laboratory, Institute for Clinical and Experimental Medicine, Prague, Czechia.

Cytomegalovirus (CMV) infection is associated with allograft rejection but the mechanisms behind are poorly defined yet. Although cross-reactivity of T cells to alloantigen and CMV has been hypothesized, direct evidence in patients is lacking. In this observational cohort study, we tested the pre-transplant effector/memory T cell response to CMV peptide pools and alloantigen in 78 living donor/recipient pairs using the interferon-gamma Enzyme-Linked ImmunoSpot (ELISPOT) assay. To prove the hypothesis of cross-reactivity, we analyzed by applying next-generation sequencing the T cell receptor ß (TCR- ß) repertoire of CMV- and alloantigen-reactive T cells enriched from peripheral pre-transplant blood of 11 CMV-seropositive and HLA class I mismatched patients. Moreover, the TCR-repertoire was also analyzed in the allograft biopsies of those patients. There was a significant association between the presence of pre-transplant CMV immediate-early protein 1 (IE-1)-specific effector/memory T cells and acute renal allograft rejection and function ( = 0.01). Most importantly, we revealed shared TCR-ß sequences between CMV-IE1 and donor alloantigen-reactive T cells in all pre-transplant peripheral blood samples analyzed in CMV-seropositive patients who received HLA class I mismatched grafts. Identical TCR sequences were also found in particular in post-transplant allograft biopsies of patients with concomitant CMV infection and rejection. Our data show the presence of functional, cross-reactive T cells and their clonotypes in peripheral blood and in kidney allograft tissue. It is therefore likely that CMV-donor cross-reactivity as well as CMV specific T cell elicited inflammation is involved in the processes that affect allograft outcomes.
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http://dx.doi.org/10.3389/fimmu.2019.02549DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6834532PMC
October 2020

Multiparametric flow cytometry analysis of peripheral blood B cell trafficking differences among Epstein-Barr virus infected and uninfected subpopulations.

Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub 2020 Sep 12;164(3):247-254. Epub 2019 Oct 12.

Department of Immunology, Faculty of Medicine and Dentistry, Palacky University Olomouc and University Hospital Olomouc, Czech Republic.

Aims: Epstein-Barr virus (EBV) targets predominantly B cells and these cells could acquire new phenotype characteristics. Here we analyzed whether EBV-infected and -uninfected B cells from healthy subjects differ in proportion of dominant phenotypes, maturation stage, and homing receptors expression.

Methods: EBV-infected and -uninfected cells were identified by flow cytometry using fluorophore-labeled EBV RNA-specific DNA probes combined with fluorophore-labeled antibody to surface lineage markers, integrins, chemokine receptors, and immunoglobulin isotypes, including intracellular ones.

Results: Our results show that the trafficking characteristics of EBER B cells are distinct from EBER B cells with most dominant differences detected for α4β1 and α4β7 and CCR5 and CCR7. EBV-positive cells are predominantly memory IgM B cells or plasmablasts/plasma cells (PB/PC) positive for IgA or less for IgM. In comparison to uninfected B cells, less EBV-positive B cells express α4β7 and almost no cells express α4β1. EBV-positive B cells contained significantly higher proportion of CCR5 and CCR7 cells in comparison to EBV-negative cells. In vitro exposure of blood mononuclear cells to pro-inflammatory cytokine IL-6 reduces population of EBV-positive B cell.

Conclusion: Although EBV-infected B cells represent only a minor subpopulation, their atypical functions could contribute in predisposed person to development abnormities such as some autoimmune diseases or tumors. Using multi-parameter flow cytometry we characterized differences in migration of EBV-positive and -negative B cells of various maturation stage and isotype of produced antibodies particularly different targeting to mucosal tissues of gastrointestinal and respiratory tracts.
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http://dx.doi.org/10.5507/bp.2019.052DOI Listing
September 2020

Guidelines from the 2017 European Conference on Infections in Leukaemia for management of HHV-6 infection in patients with hematologic malignancies and after hematopoietic stem cell transplantation.

Haematologica 2019 11 29;104(11):2155-2163. Epub 2019 Aug 29.

Department of Haematology, Henri Mondor Hospital, Assistance Publique-Hopitaux de Paris, Université Paris-Est Créteil, Créteil, France.

Of the two human herpesvirus 6 (HHV-6) species, human herpesvirus 6B (HHV-6B) encephalitis is an important cause of morbidity and mortality after allogeneic hematopoietic stem cell transplant. Guidelines for the management of HHV-6 infections in patients with hematologic malignancies or post-transplant were prepared a decade ago but there have been no other guidelines since then despite significant advances in the understanding of HHV-6 encephalitis, its therapy, and other aspects of HHV-6 disease in this patient population. Revised guidelines prepared at the 2017 European Conference on Infections in Leukaemia covering diagnosis, preventative strategies and management of HHV-6 disease are now presented.
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http://dx.doi.org/10.3324/haematol.2019.223073DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6821622PMC
November 2019

Guidelines for the management of cytomegalovirus infection in patients with haematological malignancies and after stem cell transplantation from the 2017 European Conference on Infections in Leukaemia (ECIL 7).

Lancet Infect Dis 2019 08 29;19(8):e260-e272. Epub 2019 May 29.

Division of Infection and Immunity, University College London, London, UK.

Cytomegalovirus is one of the most important infections to occur after allogeneic haematopoietic stem cell transplantation (HSCT), and an increasing number of reports indicate that cytomegalovirus is also a potentially important pathogen in patients treated with recently introduced drugs for hematological malignancies. Expert recommendations have been produced by the 2017 European Conference on Infections in Leukaemia (ECIL 7) after a review of the literature on the diagnosis and management of cytomegalovirus in patients after HSCT and in patients receiving other types of therapy for haematological malignancies. These recommendations cover diagnosis, preventive strategies such as prophylaxis and pre-emptive therapy, and management of cytomegalovirus disease. Antiviral drugs including maribavir and letermovir are in development and prospective clinical trials have recently been completed. However, management of patients with resistant or refractory cytomegalovirus infection or cytomegalovirus disease is a challenge. In this Review we summarise the reviewed literature and the recommendations of the ECIL 7 for management of cytomegalovirus in patients with haematological malignancies.
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http://dx.doi.org/10.1016/S1473-3099(19)30107-0DOI Listing
August 2019

Human Cytomegalovirus Genomes Sequenced Directly From Clinical Material: Variation, Multiple-Strain Infection, Recombination, and Gene Loss.

J Infect Dis 2019 07;220(5):781-791

Medical Research Council-University of Glasgow Centre for Virus Research, United Kingdom.

The genomic characteristics of human cytomegalovirus (HCMV) strains sequenced directly from clinical pathology samples were investigated, focusing on variation, multiple-strain infection, recombination, and gene loss. A total of 207 datasets generated in this and previous studies using target enrichment and high-throughput sequencing were analyzed, in the process enabling the determination of genome sequences for 91 strains. Key findings were that (i) it is important to monitor the quality of sequencing libraries in investigating variation; (ii) many recombinant strains have been transmitted during HCMV evolution, and some have apparently survived for thousands of years without further recombination; (iii) mutants with nonfunctional genes (pseudogenes) have been circulating and recombining for long periods and can cause congenital infection and resulting clinical sequelae; and (iv) intrahost variation in single-strain infections is much less than that in multiple-strain infections. Future population-based studies are likely to continue illuminating the evolution, epidemiology, and pathogenesis of HCMV.
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http://dx.doi.org/10.1093/infdis/jiz208DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6667795PMC
July 2019

Clinical and genotypic CMV drug resistance in HSCT recipients: a single center epidemiological and clinical data.

Bone Marrow Transplant 2019 01 27;54(1):146-149. Epub 2018 Jun 27.

4th Department of Internal Medicine, Haematology, University Hospital and Faculty of Medicine, Hradec Kralove, Czech Republic.

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http://dx.doi.org/10.1038/s41409-018-0257-7DOI Listing
January 2019

Proven Invasive Pulmonary Aspergillosis in Stem Cell Transplant Recipient Due to Aspergillus sublatus, a Cryptic Species of A. nidulans.

Mycopathologia 2018 Apr 11;183(2):423-429. Epub 2017 Nov 11.

Department of Medical Microbiology, 2nd Faculty of Medicine, Charles University and Motol University Hospital, V Uvalu 84, 15006, Prague 5, Czech Republic.

Invasive fungal disease represents one of the severe complications in haematopoietic stem cell transplant recipients. We describe a case of a patient treated for relapse of chronic lymphoblastic leukaemia 6 years after HSCT. The patient was treated for invasive pulmonary aspergillosis but died 3 months later from multiple organ failures consisting of haemorrhagic necrotizing fungal pneumonia, refractory chronic hepatic graft versus host disease and cytomegalovirus hepatitis. Autopsy samples revealed histopathological evidence of fungal hyphae and an unusual Aspergillus nidulans-like species was isolated in pure culture. More precise identification was achieved by using scanning electron microscopy of ascospores and sequencing of calmodulin gene, and the isolate was subsequently re-identified as A. sublatus (section Nidulantes) and showed good in vitro susceptibility against all classes of antifungals. Commonly used ITS rDNA region and β-tubulin gene fail to discriminate A. sublatus from related pathogenic species, especially A. quadrilineatus and A. nidulans. Although this is the first case of proven IPA attributed to A. sublatus, we demonstrated that at least some previously reported infections due to A. quadrilineatus were probably caused by this cryptic species.
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http://dx.doi.org/10.1007/s11046-017-0223-8DOI Listing
April 2018

Appearance of cytomegalovirus-specific T-cells predicts fast resolution of viremia post hematopoietic stem cell transplantation.

Cytometry B Clin Cytom 2017 09 12;92(5):380-388. Epub 2016 Jan 12.

Department of Pediatric Hematology and Oncology, 2nd Faculty of Medicine, Charles University Prague and University Hospital Motol, V Uvalu 84, Prague, 150 06, Czech Republic.

Background: Cytomegalovirus (CMV) specific T-cells are known to provide long-term control of CMV reactivation, which is a frequent complication of hematopoietic stem cell transplantation. We have studied 58 pediatric patients after hematopoietic stem cell transplantation who suffered from CMV reactivation to reveal which functional T cell subset is best correlating with successful reactivation resolution and which protects from reactivation episode.

Methods: Detection of 30 combinatorial subsets of four types of response to ex vivo CMV stimulation (IFNγ secretion, IL-2 secretion, CD40L upregulation and degranulation) that were detectable on either CD8+ or CD4+ T cells through flow cytometry intracellular cytokine staining was used.

Results: We found that the presence of CD8+ dual positive (IFNγ+ and IL-2+) cells is the most accurate functional parameter that can predict fast resolution of CMV reactivation. Next, we show that the presence of CD8+ dual positive (IFNγ+ and IL-2+) and CD8+ IFNγ+ cells provides a protective effect (a hazard risk of 0.28 (confidence interval 0.18 - 0.43) and 0.45 (CI 0.27 - 0.75), respectively) and the presence of corticotherapy increases the risk of reactivation (HR 2.47 (CI 1.82-3.36)). Thus, a patient without corticotherapy and with both of the critical T cell subsets present has a cumulative 19.6 times lower risk of developing CMV reactivation than a patient on corticotherapy and without CD8+ dual positive (IFNγ+ and IL-2+) or CD8+ IFNγ+ cells.

Conclusions: We have established parameters of CMV specific functional response ex vivo that can be used in assisting clinical management of patients with CMV reactivation. © 2015 International Clinical Cytometry Society.
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http://dx.doi.org/10.1002/cyto.b.21348DOI Listing
September 2017

Seroprevalence of Epstein-Barr Virus, Cytomegalovirus, and Polyomaviruses in Children with Inflammatory Bowel Disease.

Dig Dis Sci 2015 Nov 20;60(11):3399-407. Epub 2015 Jun 20.

Department of Pediatrics, University Hospital Motol and Second Faculty of Medicine, Charles University in Prague, V Uvalu 84, Prague 5, 150 06, Czech Republic.

Background: Young age and thiopurine therapy are risk factors for lymphoproliferative disease among patients with inflammatory bowel disease (IBD).

Aims: The aims of this study were to evaluate the prevalence of seropositivity for the Epstein-Barr virus (EBV) and human cytomegalovirus (CMV) among children and adolescents with IBD, to assess the viral load of EBV, CMV, and BK and JC polyomaviruses (BKV, JCV) in these patients, and to assess the influence of different therapeutic regimens on seroprevalence and viral load.

Methods: Children who had been followed in our center were tested for EBV, CMV, BKV, and JCV in a cross-sectional study. One hundred and six children were included who had Crohn's disease (68%), ulcerative colitis (29%), and unclassified IBD (3%).

Results: We found that 64% of patients were EBV seropositive. The proportion of EBV seropositive patients increased during childhood. Azathioprine therapy (p = 0.003) was associated with EBV seropositivity in a multiple logistic regression model, after adjusting for gender, age, and disease activity at determination. We found a significant association between the number of polymerase chain reaction copies and infliximab dose (p = 0.023). We did not find any significant association between CMV serology and CMV, BKV, or JCV viral load, or any other therapeutic regimen or clinical characteristics.

Conclusions: Treatment with azathioprine appears to be a risk factor for early EBV seropositivity in children with IBD, and the infliximab dose was associated with a higher EBV viral load.
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http://dx.doi.org/10.1007/s10620-015-3764-zDOI Listing
November 2015

Diffuse parenchymal lung disease as first clinical manifestation of GATA-2 deficiency in childhood.

BMC Pulm Med 2015 Feb 10;15. Epub 2015 Feb 10.

Center for Chronic Immunodeficiency (CCI), University Medical Center and University of Freiburg, Freiburg im Breisgau, Germany.

Background: GATA-2 transcription factor deficiency has recently been described in patients with a propensity towards myeloid malignancy associated with other highly variable phenotypic features: chronic leukocytopenias (dendritic cell-, monocyto-, granulocyto-, lymphocytopenia), increased susceptibility to infections, lymphatic vasculature abnormalities, and sensorineural deafness. Patients often suffer from opportunistic respiratory infections; chronic pulmonary changes have been found in advanced disease.

Case Presentation: We present a case of a 17-year-old previously healthy Caucasian male who was admitted to the hospital with fever, malaise, headache, cough and dyspnea. A chest X-ray revealed bilateral interstitial infiltrates and pneumonia was diagnosed. Despite prompt clinical improvement under antibiotic therapy, interstitial changes remained stable. A high resolution computer tomography showed severe diffuse parenchymal lung disease, while the patient's pulmonary function tests were normal and he was asymptomatic. Lung tissue biopsy revealed chronic reparative and resorptive reaction with organizing vasculitis. At the time of the initial presentation to the hospital, serological signs of acute infection with Epstein-Barr virus (EBV) were present; EBV viremia with atypical serological response persisted during two-year follow up. No other infectious agents were found. Marked monocytopenia combined with B-cell lymphopenia led to a suspicion of GATA-2 deficiency. Diagnosis was confirmed by detection of the previously published heterozygous mutation in GATA2 (c.1081 C > T, p.R361C). The patient's brother and father were both carriers of the same genetic defect. The brother had no clinically relevant ailments despite leukocyte changes similar to the index patient. The father suffered from spondylarthritis, and apart from B-cell lymphopenia, no other changes within the leukocyte pool were seen.

Conclusion: We conclude that a diagnosis of GATA-2 deficiency should be considered in all patients with diffuse parenchymal lung disease presenting together with leukocytopenia, namely monocyto-, dendritic cell- and B-lymphopenia, irrespective of severity of the clinical phenotype. Genetic counseling and screening for GATA2 mutations within the patient's family should be provided as the phenotype is highly variable and carriers without apparent immunodeficiency are still in danger of developing myeloid malignancy. A prompt recognition of this rare condition helps to direct clinical treatment strategies and follow-up procedures.
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http://dx.doi.org/10.1186/s12890-015-0006-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4340788PMC
February 2015

[Guidelines for antifungal prophylaxis, diagnosis and therapy in pediatric hematology and oncology - a review of literature and expert recommendations].

Klin Mikrobiol Infekc Lek 2014 Sep;20(3):85-91

Department of Pediatric Hematology and Oncology, Teaching Hospital Motol, 2nd Medical School, Charles University Prague, Czech Republic, e-mail:

Objectives: Problems with importing non-registered medicines for treating rare life-threatening infectious diseases led to establishment of the Emergency Anti-Infective Drug Reserve (EAIDR) for the Czech Republic.

Methods: Thirteen anti-infective drugs are included in the project: antisera against rabies virus, varicella-zoster virus, and botulinum toxin; antituberculosis drugs (intravenous rifampicin and isoniazid; capreomycin, cycloserine, and clofazimine); antiparasitics (intravenous quinine, primaquine, meglumine antimoniate, and praziquantel); and pentamidine. These drugs are imported according to the Czech drug legislation (specific drug availability programs). Realization: The project, approved by the Czech Ministry of Health in September 2013, was started in January 2014. The anti-infective drugs sufficient for 2-4 patients are permanently available in the Toxicological Information Center (TIC) in Prague. The medicines can be applied in any hospital throughout the Czech Republic within several hours.

Conclusions: All but three drugs are available at present; the remaining ones will be imported after new batches of these drugs are released.
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September 2014

Polyclonal, newly derived T cells with low expression of inhibitory molecule PD-1 in tonsils define the phenotype of lymphocytes in children with Periodic Fever, Aphtous Stomatitis, Pharyngitis and Adenitis (PFAPA) syndrome.

Mol Immunol 2015 May 3;65(1):139-47. Epub 2015 Feb 3.

CLIP, Department of Paediatric Haematology/Oncology, 2nd Faculty of Medicine, Charles University in Prague and University Hospital Motol, Czech Republic. Electronic address:

Purpose: PFAPA syndrome is a benign, recurrent inflammatory disease of childhood. Tonsillectomy is one of the therapeutic options with a yet unexplained biological mechanism. We tested whether specific lymphocyte subsets recruited from blood to human tonsils participate in PFAPA pathogenesis.

Methods: Paired tonsils/peripheral blood (PB) samples were investigated (a) from children with PFAPA that successfully resolved after tonsillectomy (n=10) (b) from children with obstructive sleep apnoea syndrome as controls (n=10). The lymphocyte profiles were analysed using 8-colour flow cytometry, immunoglobulin (IGH) and T-cell receptor (TCR) gene rearrangements via PCR and next generation sequencing; a TREC/KREC analysis was performed using qPCR.

Results: The PFAPA tonsils in the asymptomatic phase had a lower percentage of B-lymphocytes than controls; T-lymphocyte counts were significantly higher in PB. The percentages of cytotoxic CD8pos T-lymphocytes were approximately 2-fold higher in PFAPA tonsils; the transitional B cells and naïve stages of both the CD4pos and CD8pos T-lymphocytes with a low expression of PD-1 molecule and high numbers of TREC were also increased. With the exception of elevated plasmablasts, no other differences were significant in PB. The expression levels of CXCL10, CXCL9 and CCL19 genes were significantly higher in PFAPA tonsils. The IGH/TCR pattern showed no clonal/oligoclonal expansion. DNA from the Epstein-Barr virus, Human Herpervirus-6 or adenovirus was detected in 7 of 10 PFAPA tonsils but also in 7 of 9 controls.

Conclusions: Our findings suggest that the uninhibited, polyclonal response of newly derived lymphocytes participate in the pathogenesis of PFAPA. Because most of the observed changes were restricted to tonsils and were not present in PB, they partly explain the therapeutic success of tonsillectomy in PFAPA syndrome.
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http://dx.doi.org/10.1016/j.molimm.2015.01.004DOI Listing
May 2015

Possible Tyromyces fissilis (Basidiomycota, Polyporales) co-infection in a lung transplant recipient.

Folia Microbiol (Praha) 2015 Jan 27;60(1):33-5. Epub 2014 Jul 27.

Department of Medical Microbiology 2nd Faculty of Medicine, Charles University in Prague and Motol University Hospital, V Uvalu 84, 15006, Prague 5, Czech Republic,

Invasive fungal diseases are severe complication of the lung transplant patients' follow-up as they are increasing the risk of rejection. We report a patient who developed possible Tyromyces fissilis co-infection during graft rejection episode 2 years after bilateral lung transplantation for cystic fibrosis. The fungus was detected using conventional culture methods as a filamentous basidiomycete and further placed to T. fissilis species based on internal transcribed spacer (ITS) rDNA sequences. The patient was treated according to the susceptibility testing results by voriconazole in combination with the anti-rejection therapy and recovered completely within few weeks. This is, to our knowledge, the first published case report of T. fissilis as a possible causative agent of an infection/rejection episode in a lung transplant recipient.
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http://dx.doi.org/10.1007/s12223-014-0336-0DOI Listing
January 2015

Response to rituximab-based therapy and risk factor analysis in Epstein Barr Virus-related lymphoproliferative disorder after hematopoietic stem cell transplant in children and adults: a study from the Infectious Diseases Working Party of the European Group for Blood and Marrow Transplantation.

Clin Infect Dis 2013 Sep 13;57(6):794-802. Epub 2013 Jun 13.

Department of Pediatric Hematology and Oncology, Collegium Medicum, Nicolaus Copernicus University, Bydgoszcz, Poland.

Background:  The objective of this analysis was to investigate prognostic factors that influence the outcome of Epstein-Barr virus (EBV)-related posttransplant lymphoproliferative disorder (PTLD) after a rituximab-based treatment in the allogeneic hematopoietic stem cell transplant (HSCT) setting.

Methods:  A total of 4466 allogeneic HSCTs performed between 1999 and 2011 in 19 European Group for Blood and Marrow Transplantation centers were retrospectively analyzed for PTLD, either biopsy-proven or probable disease.

Results:  One hundred forty-four cases of PTLD were identified, indicating an overall EBV-related PTLD frequency of 3.22%, ranging from 1.16% for matched-family donor, 2.86% for mismatched family donor, 3.97% in matched unrelated donors, and 11.24% in mismatched unrelated donor recipients. In total, 69.4% patients survived PTLD. Multivariable analysis showed that a poor response of PTLD to rituximab was associated with an age ≥30 years, involvement of extralymphoid tissue, acute GVHD, and a lack of reduction of immunosuppression upon PTLD diagnosis. In the prognostic model, the PTLD mortality increased with the increasing number of factors: 0-1, 2, or 3 factors being associated with mortality of 7%, 37%, and 72%, respectively (P < .0001). Immunosuppression tapering was associated with a lower PTLD mortality (16% vs 39%), and a decrease of EBV DNAemia in peripheral blood during therapy was predictive of better survival.

Conclusions:  More than two-thirds of patients with EBV-related PTLD survived after rituximab-based treatment. Reduction of immunosuppression was associated with improved outcome, whereas older age, extranodal disease, and acute graft-vs-host disease predicted poor outcome.
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http://dx.doi.org/10.1093/cid/cit391DOI Listing
September 2013

[Chromosomal integration of the sixth human herpes virus (HHV-6)].

Epidemiol Mikrobiol Imunol 2012 Sep;61(3):58-66

Ustav lékarské mikrobiologie 2. lékarské fakulty UK a Fakultní nemocnice v Motole.

Two closely related and commonly found human herpesviruses HHV-6 A and HHV-6 B are classified into the sixth human herpes virus complex (HHV-6). Primary infection with HHV-6 often takes place in early childhood and it can be either asymptomatic or manifests itself as sixth disease (caused by HHV-6 B). HHV-6 remains present in a latent form in the body with the potential for virus reactivation. The article points out the phenomenon of chromosomal integration of HHV-6 (Ci-HHV-6) which is found in about 1% of the population and, unlike the commonly spread HHV-6 infection, has become hereditary, with its pathological potential in Ci-HHV-6 DNA carriers remaining unknown. Therefore, the focus on clinical consequences of Ci-HHV-6 is of high relevance to the therapeutic strategy for patients with high HHV-6 positivity in molecular biological tests.
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September 2012

[Invasive mucormycosis in pediatric hematology patients--single-center experience from 2005-2010].

Klin Mikrobiol Infekc Lek 2012 Aug;18(4):102-8

Institute of Medical Microbiology, Charles University, 2nd Medical Faculty Prague.

Background: Mucormycosis is an invasive fungal disease severely complicating treatment of patients with hematologic diseases. Effective therapy is represented by the combination of surgery and amphotericin B administration and early initiation of the therapy is necessary for favorable outcome. The first clinical symptoms are usually non-specific and this can lead to late therapy onset. The objective of this retrospective work was to determine the frequency, risk factors and outcome of invasive mucormycosis in pediatric hematology patients.

Material And Methods: The study cohort comprised 399 patients diagnosed with hematologic diseases in the Department of Pediatric Hematology and Oncology (DPHO), University Hospital Motol, Prague between 2005 and 2010. Risk factors for the development of mucormycosis, clinical symptoms and radiology and laboratory results were retrospectively evaluated. So were the therapy used and outcomes. The findings were analyzed using Fisher's exact test.

Results: During the selected period, mucormycosis was detected in 8 patients diagnosed with hematologic disease. The incidence of mucormycosis was 1.75 %. These conditions accounted for 20.6 % of all mycoses. In five patients, it was found as isolated infection; three cases were associated with other mycoses (one with candidiasis, two with aspergillosis). The most frequent underlying disease was acute leukemia; the most common risk factor was severe prolonged neutropenia (median duration 21.5 days). Three of eight patients survived mucormycosis, a mortality rate of 62.5 %. The effective therapy was amphotericin B administration in three patients (p = 0.02); in two of them, it was combined with radical surgery.

Conclusion: In the cohort, the proportion of mucormycosis cases was surprisingly high when compared with other fungal diseases. Continuous surveillance of mucormycosis in the DPHO is needed. There was no significant influence of the combination of radical surgery and amphotericin B administration as compared to administration of amphotericin B alone. Nevertheless, according to the published data, we consider this approach as an optimal strategy for the management of mucormycosis at the present time.
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August 2012

Impact of novel mutations of herpes simplex virus 1 and 2 thymidine kinases on acyclovir phosphorylation activity.

Antiviral Res 2012 Dec 3;96(3):386-90. Epub 2012 Oct 3.

UPMC Univ Paris 06, ER1 DETIV, Paris, France.

The acyclic analogue of guanosine acyclovir (ACV) constitutes the first-line drug for the treatment of herpes simplex virus (HSV) infections. ACV activation requires primophosphorylation by virus-encoded HSV thymidine kinase (TK). In 95% of cases, HSV resistance to ACV is associated with mutations located in TK. The aim of this work was to address the question of the potential involvement of novel HSV-1 and HSV-2 TK mutations in reduced susceptibility to ACV using a novel nonradioactive method, based on luminescent quantitation of ADP, for the evaluation of in vitro phosphorylation activity of TK. All recombinant TKs tested exhibited significantly lower ACV phosphorylation activities in comparison with those of reference KOS or gHSV-2 TKs (p<0.015), therefore indicating that amino acid changes Y53D, L170P, R176W, A207P (HSV-1) and S66P, A72S, I101S, M183I (HSV-2) were likely to be involved in HSV resistance to ACV.
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http://dx.doi.org/10.1016/j.antiviral.2012.09.016DOI Listing
December 2012

Prevalence of chromosomally integrated HHV-6 in patients with malignant disease and healthy donors in the Czech Republic.

Folia Microbiol (Praha) 2013 Jan 15;58(1):87-90. Epub 2012 Jul 15.

Department of Paediatric Haematology and Oncology, 2nd Faculty of Medicine of Charles University and Motol University Hospital, V Uvalu 84, Prague 5, CZ 150 06, Czech Republic.

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http://dx.doi.org/10.1007/s12223-012-0180-zDOI Listing
January 2013

No association between the TP53 codon 72 polymorphism and risk or prognosis of Hodgkin and non-Hodgkin lymphoma.

Leuk Res 2011 Aug 5;35(8):1117-9. Epub 2011 May 5.

1st Department of Medicine, 1st Faculty of Medicine, Charles University in Prague and General University Hospital in Prague, Prague, Czech Republic.

The role of the TP53 gene's R72P polymorphism in non-Hodgkin lymphoma (NHL) has been analyzed in several studies but it has not been studied in Hodgkin lymphoma (HL). We have evaluated the role of R72P in 340 NHL and 298 HL patients. There was no difference in the R72P frequency between analyzed lymphoma cases and 749 controls. We found no association of R72P with the risk of NHL and HL development [OR(ArgPro/ProPro)=0.9 (95% CI 0.7-1.2) and 1.2 (95% CI 0.9-1.5), respectively] or with survival. Our results support the evidence that R72P is not a prognostic factor in Caucasian NHL patients, and they indicate its irrelevance for HL development or prognosis.
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http://dx.doi.org/10.1016/j.leukres.2011.04.001DOI Listing
August 2011

Plasma EBV-DNA monitoring in Epstein-Barr virus-positive Hodgkin lymphoma patients.

APMIS 2011 Jan 25;119(1):10-6. Epub 2010 Oct 25.

Department of Clinical Hematology, University Hospital Kralovske Vinohrady, Charles University, Prague, Czech Republic.

Epstein-Barr virus (EBV) is associated with approximately one-third of Hodgkin lymphoma (HL) cases. EBV-DNA is often present in the plasma and whole blood of EBV-associated HL patients. However, the significance of EBV-DNA monitoring is debated. In a cohort of 165 adult HL patients, EBV-DNA viral load was prospectively monitored both in the plasma and whole blood. Diagnostic tissue samples of all patients were histologically reviewed; in 72% nodular sclerosis was detected, 24% presented with mixed cellularity (MC), and 5% had other type of HL. Tissues from 150 patients were also analyzed for the presence of latent EBV infection using in situ hybridization for EBV-encoded RNA (EBER) and immunohistochemistry for latent membrane protein (LMP1). Using these methods, 29 (19%) patients were classified as EBV positive. Using real-time quantitative PCR, 22 (76%) of EBV-positive HL patients had detectable EBV-DNA in the plasma and 19 (66%) patients in whole blood prior to therapy. In the group of EBV-negative HL cases, three (2%) patients had detectable plasma EBV-DNA and 30 (25%) patients whole blood EBV-DNA before treatment. EBV-positive HL was significantly associated with EBV-DNA positivity both in the plasma and whole blood in pretreatment samples, increasing age and MC subtype. Serial analysis of plasma EBV-DNA showed that response to therapy was associated with decline in viral load. Moreover, significantly increased plasma EBV-DNA level recurred before disease relapse in one patient. Our results further suggest that the assessment of plasma EBV-DNA viral load might be of value for estimation of prognosis and follow-up of patients with EBV-positive HL.
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http://dx.doi.org/10.1111/j.1600-0463.2010.02685.xDOI Listing
January 2011

HHV-6 DNA throughout the tissues of two stem cell transplant patients with chromosomally integrated HHV-6 and fatal CMV pneumonitis.

Br J Haematol 2009 May 17;145(3):394-8. Epub 2009 Feb 17.

Department of Paediatric Haematology and Oncology, 2nd Faculty of Medicine and Motol University Hospital, Charles University, Prague, The Czech Republic.

Two patients with the characteristic high human herpesvirus 6 (HHV-6) DNA loads in peripheral blood caused by chromosomally integrated (CI) virus received a haematopoietic stem cell transplant (HSCT) from a donor without CI HHV-6. Both patients died in consequence of cytomegalovirus (CMV) pneumonitis. At autopsy, high amounts of CMV DNA were detected in lungs but at much lower levels in other organs. In contrast HHV-6 DNA was detected at high levels throughout the organs with the exception of donor-derived haematopoietic tissue. In individuals with chromosomal integration, HHV-6 DNA is found in every tissue of recipient origin indicating inheritance through the germ line.
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http://dx.doi.org/10.1111/j.1365-2141.2009.07622.xDOI Listing
May 2009

Prevalence of HHV-6 integrated chromosomally among children treated for acute lymphoblastic or myeloid leukemia in the Czech Republic.

J Med Virol 2009 Feb;81(2):258-63

Department of Paediatric Haematology and Oncology, Motol University Hospital, Prague, Czech Republic.

Chromosomal integration of human herpesvirus 6 (HHV-6) is a novel situation found in a small percentage of individuals. While active HHV-6 infection is treatable using antivirals, the abnormally high level of HHV-6 DNA found in chromosomal integration of HHV-6 (CI-HHV-6) is not affected by such drugs. Stored DNA samples taken originally for detection of fusion genes and minimal residual disease from 339 pediatric patients treated for leukemia in the Czech Republic between the years 1995-2007 were tested retrospectively. Using real-time quantitative PCR technology, the quantity of HHV-6 DNA detected was normalized to 100,000 human genome equivalents as assessed by quantitation of the albumin gene. HHV-6 DNA was detected in 107 samples from 91 patients (26.8%). In the majority of samples (99) only a minute level of normalized viral copies (NVCs) (median 1.84 NVCs) was detected. A high viral load of approximately 100,000 NVCs was detected in 5 patients (1.5%; median 140,150 NVCs), in all of whom were confirmed subsequently CI-HHV-6 by a detection of HHV-6 DNA in hair follicles or in the nails. In all but one patient with HHV-6 variant B, variant A of the virus was detected. None of the patients with CI-HHV-6 had complications attributable to HHV-6 infection. The prevalence of CI-HHV-6 in childhood leukemia does not differ from that published for other patients or healthy populations. Where high levels of HHV-6 DNA are present, CI-HHV-6 should be confirmed as soon as possible so that potentially toxic but ineffective antiviral treatment can be stopped.
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http://dx.doi.org/10.1002/jmv.21371DOI Listing
February 2009

Cytomegalovirus encephalitis/retinitis in allogeneic haematopoietic stem cell transplant recipient treated successfully with combination of cidofovir and foscarnet.

Pediatr Transplant 2009 Nov 1;13(7):919-22. Epub 2008 Nov 1.

Motol University Hospital, Prague, Czech Republic.

We report an 18-yr-old female patient with repeated CMV reactivations after HSCT treated by several pre-emptive courses of virostatic therapy. Seven months after HSCT, she developed CMV encephalitis/retinitis. Initial therapy with GCV and hyperimmune globulin failed, and later on GCV-resistant strain was detected. Continual increase of CMV DNA in peripheral blood led us to combined therapy with CDV and FCV, which was successful and free of severe renal toxicity. To our best knowledge, this is the first reported case of successful CMV treatment with a combination of CDV and FCV.
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http://dx.doi.org/10.1111/j.1399-3046.2008.01070.xDOI Listing
November 2009

Allogeneic stem cell transplantation in children with leukemia using human leukocyte antigen-mismatched unrelated donors.

Pediatr Transplant 2008 Feb;12(1):24-31

Department of Pediatric Hematology and Oncology, University Hospital Motol, 2nd Medical School, Charles University, Prague, Czech Republic.

Allogeneic HSCT is a curative treatment, when chemotherapy fails, for certain malignant diseases. In Europe, only 15% of the indicated children have an HLA-matched sibling available; in 65-70% of others, HLA allele-matched (9-10/10) UDs can be identified. For the rest, it is necessary to identify other alternative donors (HLA-mismatched family or unrelated cord blood). We present our data of HSCT using HLA partially allele-mismatched (7-8/10) UDs in 24 children with leukemia. Uniform GvHD prophylaxis was used (rATG, CsA and MTX). Acute GvHD grade II was diagnosed in 70.8% of the patients and grade III-IV in 12.5%. Overall incidence of chronic GvHD was 38.7% (extensive in 30%). The probability of EFS was 60.3% (95% CI 35.5-78.1) and OS was 74.9 (95% CI 49.1-88.9). No difference in survival between PBSC and BM recipients was observed. TRM at day + 100 was 4%, and overall was 12.5%. We conclude that used combination of drugs for GvHD prophylaxis is efficient even for patients transplanted with grafts from a HLA-mismatched UDs. It enables stable engraftment, good control of GvHD, full reconstitution of immunity, and is not connected with unacceptable transplant-related mortality.
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http://dx.doi.org/10.1111/j.1399-3046.2007.00762.xDOI Listing
February 2008