Impact of the introduction of EUCAST's concept of "area of technical uncertainty".

On the first of January 2019, the European Committee on Antimicrobial Susceptibility Testing, EUCAST, introduced the concept of "area of technical uncertainty" (ATU). The aim was to report on the incidence of ATU test results in a selection of common bacterial species and the subsequent impact on antimicrobial resistance categorization and workload. A retrospective analysis of clinical samples collected from February 2019 until November 2019 was performed. Susceptibility to amoxicillin-clavulanic acid and piperacillin-tazobactam in Enterobacterales (Escherichia spp., Klebsiella spp., Proteus spp.), piperacillin-tazobactam in Pseudomonas aeruginosa, and amoxicillin-clavulanic acid and cefuroxime in Haemophilus influenzae was studied. Disk diffusion antibiotic susceptibility testing was read and interpreted by ADAGIO 93400 automated system (Bio-Rad, France). In case of an inhibition zone in the ATU, strains were retested using gradient minimal inhibitory concentration method (Etest, BioMérieux, France). Overall, 14,164 isolate-antibiotic combinations were tested in 7922 isolates, resulting in 1204 (8.5%) disk zone diameters in the ATU region. Retesting of ATUs with Etest resulted in a category change from S to R for amoxicillin-clavulanic acid in 63/498 (12.7%) of Escherichia spp., 2/58 (3.4%) of Klebsiella spp., 2/37 (5.4%) of Proteus spp., and 6/125 (4.8%) of Haemophilus influenzae. For piperacillin-tazobactam, a category change from S to R was found in 33/92 (35.9%) of Pseudomonas aeruginosa. We conclude that ATU testing has a substantial impact on the correct interpretation of antimicrobial resistance, at the expense of turn-around time and with the cost of additional workload.

Diagnosing COVID-19; towards a feasible COVID-19 rule-out protocol.

INTRODUCTION: We present the results of the COVID-19 rule-out protocol at Ghent University Hospital, a step-wise testing approach which included repeat NFS SARS-CoV-2 rRT-PCR, respiratory multiplex RT-PCR, low-dose chest CT and bronchoscopy with BAL to confirm or rule-out SARS-CoV-2 infection in patients admitted with symptoms suggestive of COVID-19. RESULTS: Between 19 March 2020 and 30 April 2020, 455 non-critically ill patients with symptoms suspect for COVID-19 were admitted. The initial NFS for SARS-CoV-2 rRT-PCR yielded 66.9%, the second NFS 25.4% and bronchoscopy with BAL 5.9% of total COVID-19 diagnoses. In the BAL fluid, other respiratory pathogens were detected in 65% (13/20) of the COVID-19 negative patients and only in 1/7 COVID-19 positive patients. Retrospective antibody testing at the time around BAL sampling showed a positive IgA or IgG in 42.9 % of the COVID-19 positive and 10.5% of the COVID-19 negative group. Follow-up serology showed 100% COVID-19 positivity in the COVID-19 positive group and 100% IgG negativity in the COVID-19 negative group. CONCLUSION: In our experience, bronchoscopy with BAL can have an added value to rule-in or rule-out COVID-19 in patients with clinical and radiographical high-likelihood of COVID-19 and repeated negative NFS testing. Furthermore, culture and respiratory multiplex PCR on BAL fluid can aid to identify alternative microbial etiological agents in this group. Retrospective analysis of antibody development in this selected group of patients suggests that the implementation of serological assays in the routine testing protocol will decrease the need for invasive procedures like bronchoscopy.

Toilet drain water as a potential source of hospital room-to-room transmission of carbapenemase-producing Klebsiella pneumoniae.

BACKGROUND: Carbapenemase-producing Enterobacterales (CPE) have rapidly emerged in Europe, being responsible for nosocomial outbreaks. AIM: Following an outbreak in the burn unit of Ghent University Hospital, we investigated whether CPE can spread between toilets through drain water and therefrom be transmitted to patients. METHODS: In 2017, the burn centre of our hospital experienced an outbreak of OXA-48-producing Klebsiella pneumoniae that affected five patients staying in three different rooms. Environmental samples were collected from the sink, shower, shower stretcher, hand rail of the bed, nursing carts, toilets, and drain water to explore a common source. Whole-genome sequencing and phylogenetic analysis was performed on K. pneumoniae outbreak isolates and two random K. pneumoniae isolates. FINDINGS: OXA-48-producing K. pneumoniae was detected in toilet water in four out of six rooms and drain water between two rooms. The strain persisted in two out of six rooms after two months of daily disinfection with bleach. All outbreak isolates belonged to sequence type (ST) 15 and showed isogenicity (<15 allele differences). This suggests that the strain may have spread between rooms by drain water. Unexpectedly, one random isolate obtained from a patient who became colonized while residing at the geriatric ward clustered with the outbreak isolates, suggesting the outbreak to be larger than expected. Daily application of bleach tended to be superior to acetic acid to disinfect toilet water; however, disinfection did not completely prevent the presence of carbapenemase-producing K. pneumoniae in toilet water. CONCLUSION: Toilet drain water may be a potential source of hospital room-to-room transmission of carbapenemase-producing K. pneumoniae.

Methicillin-resistant staphylococcus aureus pneumonia in older people: a diagnostic and therapeutic challenge.

Pneumonia is one of the leading causes of death in older people, with high mortality rates (> 80%). One of the bacterial pathogens causing pneumonia is Staphylococcus aureus. The unique adaptive ability of S. aureus to a broad range of antibiotics has led to the emergence of methicillin-resistant S. aureus (MRSA) strain. MRSA pneumonia remains a relatively uncommon infection in older people, but it is associated with a very high mortality rate. We report two cases of MRSA pneumonia that highlight the severe clinical presentation and virulence of MRSA infections in geriatric population. MRSA pneumonia can present with mostly an uncontrollable clinical evolution and an infaust prognosis. Therefore, clinicians should be aware of MRSA pneumonia in patients with comorbidities, recent hospitalization with antibiotic treatment, previous MRSA infections and also in patients residing in nursing homes/revalidation centers. Low prevalence of MRSA combined with a lack of highly distinctive clinical features makes accurate targeting of empirical treatment with antibiotics very difficult. Currently, monotherapy with linezolid or vancomycin remain the first choice, in adult patients with proven MRSA infection. Despite the higher age related mortality rates, there are no specific treatment guidelines for older patients.

Clinical, cytogenetic and molecular characteristics of 14 T-ALL patients carrying the TCRbeta-HOXA rearrangement: a study of the Groupe Francophone de Cytogénétique Hématologique.

Recently, we and others described a new chromosomal rearrangement, that is, inv(7)(p15q34) and t(7;7)(p15;q34) involving the T-cell receptor beta (TCRbeta) (7q34) and the HOXA gene locus (7p15) in 5% of T-cell acute lymphoblastic leukemia (T-ALL) patients leading to transcriptional activation of especially HOXA10. To further address the clinical, immunophenotypical and molecular genetic findings of this chromosomal aberration, we studied 330 additional T-ALLs. This revealed TCRbeta-HOXA rearrangements in five additional patients, which brings the total to 14 cases in 424 patients (3.3%). Real-time quantitative PCR analysis for HOXA10 gene expression was performed in 170 T-ALL patients and detected HOXA10 overexpression in 25.2% of cases including all the cases with a TCRbeta-HOXA rearrangement (8.2%). In contrast, expression of the short HOXA10 transcript, HOXA10b, was almost exclusively found in the TCRbeta-HOXA rearranged cases, suggesting a specific role for the HOXA10b short transcript in TCRbeta-HOXA-mediated oncogenesis. Other molecular and/or cytogenetic aberrations frequently found in subtypes of T-ALL (SIL-TAL1, CALM-AF10, HOX11, HOX11L2) were not detected in the TCRbeta-HOXA rearranged cases except for deletion 9p21 and NOTCH1 activating mutations, which were present in 64 and 67%, respectively. In conclusion, this study defines TCRbeta-HOXA rearranged T-ALLs as a distinct cytogenetic subgroup by clinical, immunophenotypical and molecular genetic characteristics.

A new recurrent inversion, inv(7)(p15q34), leads to transcriptional activation of HOXA10 and HOXA11 in a subset of T-cell acute lymphoblastic leukemias.

Chromosomal translocations with breakpoints in T-cell receptor (TCR) genes are recurrent in T-cell malignancies. These translocations involve the TCRalphadelta gene (14q11), the TCRbeta gene (7q34) and to a lesser extent the TCRgamma gene at chromosomal band 7p14 and juxtapose T-cell oncogenes next to TCR regulatory sequences leading to deregulated expression of those oncogenes. Here, we describe a new recurrent chromosomal inversion of chromosome 7, inv(7)(p15q34), in a subset of patients with T-cell acute lymphoblastic leukemia characterized by CD2 negative and CD4 positive, CD8 negative blasts. This rearrangement juxtaposes the distal part of the HOXA gene cluster on 7p15 to the TCRbeta locus on 7q34. Real time quantitative PCR analysis for all HOXA genes revealed high levels of HOXA10 and HOXA11 expression in all inv(7) positive cases. This is the first report of a recurrent chromosome rearrangement targeting the HOXA gene cluster in T-cell malignancies resulting in deregulated HOXA gene expression (particularly HOXA10 and HOXA11) and is in keeping with a previous report suggesting HOXA deregulation in MLL-rearranged T- and B cell lymphoblastic leukemia as the key factor in leukaemic transformation. Finally, our observation also supports the previous suggested role of HOXA10 and HOXA11 in normal thymocyte development.

Interleukin-1 is a motility factor for human breast carcinoma cells in vitro: additive effect with interleukin-6.

Interleukin-1 beta (Il-1 beta) and interleukin-1 alpha (Il-1 alpha) were shown to act as motility factors for the human breast carcinoma cell lines SK-BR-3 and ZR-75-1 in vitro. Both cytokines induced transition from the stationary to the motile phenotype (spreading). Il-1 beta stimulated translocation, shape change and random migration (chemokinesis) of SK-BR-3 cells as demonstrated by time-lapse video recordings and by a modified Boyden chamber assay. Interleukin-6 (Il-6) stimulated spreading of the SK-BR-3 cells; an additive effect with Il-1 beta on spreading and fast plasma membrane movements was evidenced. In the SK-BR-3 cell line, the signal transduction of Il-1 beta and Il-6 differed, since only the effect of Il-6 on spreading was sensitive to pertussis toxin. Both Il-1 beta and Il-6 required protein synthesis to stimulate spreading, since cycloheximide inhibited the effect of the cytokines. Induction of an autocrine loop of Il-6 in the SK-BR-3 cells by Il-1 beta was unlikely, since after stimulation with Il-1 beta, no induction of Il-6 activity was measured, nor was inhibition of stimulated spreading seen in the presence of an antiserum against Il-6. Addition of Il-8 or of an antiserum against Il-8 did not affect spreading. We concluded that Il-1 and Il-6 could act as motility factors for human breast carcinoma cells, in both an independent and an additive way.(ABSTRACT TRUNCATED AT 250 WORDS)

Arrest of MCF-7 cell migration by laminin in vitro: possible mechanisms.

Laminin, a major basement membrane component, arrested the migration of MCF-7/AZ human breast adenocarcinoma cells that were not invasive in vitro. Migration of invasive MCF-7/6 cells was not affected by laminin. Both cell types expressed the 67 kD laminin receptor, at both mRNA and protein level, but did not express the alpha 6 subunit of the VLA-6 integrin-type laminin receptor. The presence of YIGSR peptides (100 micrograms/ml), reported to block the interaction between laminin and its 67 kD receptor, did not change the migratory response of MCF-7/AZ or MCF-7/6 cells when meeting laminin lanes. In addition, the migration of these cell types was not affected by the presence of 17-beta-estradiol (10(-6) M) or all-trans retinoic acid (10(-6) M), which were both reported to increase the number of 67 kD receptors. We could therefore not assign an involvement of the 67 kD receptors in migration of MCF-7 cells on laminin, nor did we find evidence that conditioned medium of MCF-7/6 cells contains factors that are able to initiate migration of MCF-7/AZ cells on laminin.

Human T lymphopoiesis. In vitro and in vivo study models.

Successive steps in T lymphocyte differentiation and T potential of human stem cells (HSC) can be tested in the following models: (a) the infusion of cells in NOD-SCID mice, (b) the injection of cells in renconstituted SCID/hu mice, (c) the differentiation of cells in fetal thymus organ culture (FTOC), and (d) on thymic stromal layers. Using mixed human-murine FTOC, we showed (a) TCR alpha beta, TCR gamma delta lymphocytes, NK cells, and dendritic cells complete their differentiation, (b) IL-7R alpha signaling and IL-7 are essential, (c) a detailed phenotypic and functional analysis of discrete successive steps of positively selected thymocytes, (d) an efficient transduction of genes in HSC with persistent gene expression throughout the T-lymphocyte differentiation, and (e) adaptation to submerging high oxygen culture increases the test sensitivity to a clonal assay. Other approaches are the in vivo SCID/hu reconstitution model. With this method small fragments of human fetal liver and thymus are implanted under the kidney capsule of an adult SCID mouse with result in an impressive human thymus organ, six months after transplantation. We use this model to study thymus T-cell developmental kinetics, development of gene-marked precursor cells and thymic homing of precursor cells.

Efficient gene transfer in CLL by mRNA electroporation.

Chronic lymphocytic leukemia (CLL) consists of at least two major prognostic subgroups, characterized by different cellular and molecular markers. This observation sparked studies on the function and clinical importance of these markers. In order to address their function adequately, an efficient and reliable method for gene transfer is needed. In this study, we compared efficiency and utility of different gene transfer techniques in CLL. Lenti-, retro- and adenoviral transduction did not yield appreciable numbers of marker gene enhanced green fluorescent protein (EGFP) positive CLL cells, despite various prestimulation protocols. Efficient transgene expression was observed after nucleofection of CLL cells with plasmid DNA, at the expense of low survival rates. After optimization, electroporation of in vitro transcribed mRNA yielded up to 90% EGFP+CLL cells without affecting survival. Transgene expression remained detectable for at least 2 weeks after electroporation. Furthermore, we could demonstrate overexpression of ZAP70 and of a ZAP70-EGFP fusion protein after electroporation with ZAP70 or ZAP70-EGFP mRNA. We conclude that mRNA electroporation is a novel and straightforward method for highly efficient gene transfer in CLL. The application of this technique should facilitate functional studies on CLL cells, as well as clinical research.

Generation of embryonic stem cell lines from mouse blastocysts developed in vivo and in vitro: relation to Oct-4 expression.

Embryonic stem (ES) cells are the source of all embryonic germ layer tissues. Oct-4 is essential for their pluripotency. Since in vitro culture may influence Oct-4 expression, we investigated to what extent blastocysts cultured in vitro from the zygote stage are capable of expressing Oct-4 and generating ES cell lines. We compared in vivo with in vitro derived blastocysts from B6D2 mice with regard to Oct-4 expression in inner cell mass (ICM) outgrowths and blastocysts. ES cells were characterized by immunostaining for alkaline phosphatase (ALP), stage-specific embryonic antigen-1 (SSEA-1) and Oct-4. Embryoid bodies were made to evaluate the ES cells' differentiation potential. ICM outgrowths were immunostained for Oct-4 after 6 days in culture. A quantitative real-time PCR assay was performed on individual blastocysts. Of the in vitro derived blastocysts, 17% gave rise to ES cells vs 38% of the in vivo blastocysts. Six-day old outgrowths from in vivo developed blastocysts expressed Oct-4 in 55% of the cases vs 31% of the in vitro derived blastocysts. The amount of Oct-4 mRNA was significantly higher for freshly collected in vivo blastocysts compared to in vitro cultured blastocysts. In vitro cultured mouse blastocysts retain the capacity to express Oct-4 and to generate ES cells, be it to a lower level than in vivo blastocysts.

Nasal polyps in patients with and without cystic fibrosis: a differentiation by innate markers and inflammatory mediators.

BACKGROUND: The dysfunction of the mucosal interface of the upper respiratory tract in cystic fibrosis (CF) patients is clinically visible by the development of nasal polyps (NP) at a young age. Innate defence markers and inflammatory mediators in NP from patients with CF were compared with non-cystic fibrosis nasal polyps (non-CF-NP) to determine a possible different immunological background in macroscopically similar tissue. METHODS: Surgical samples were obtained from patients with non-CF-NP, cystic fibrosis patients with nasal polyps (CF-NP) and control patients (CO). With real time PCR, the mRNA expression of human beta defensins (HBD) 2 and 3, toll-like receptors (TLR) 2 and 4 and the macrophage mannose receptor (MMR) were measured. On homogenates of the surgical samples eotaxin, myeloperoxidase (MPO), IL-5 and IL-8 protein content was measured using commercial ELISA kits; IgE and eosinophilic cationic protein (ECP) were measured by the Unicap system. RESULTS: In CF-NP we found a statistically significant higher mRNA expression of HBD 2 compared with non-CF-NP and CO and of TLR 2 compared with non-CF-NP. In the non-CF-NP group, MMR mRNA expression was significantly elevated compared with CO and CF-NP. For TLR 4 mRNA expression no statistically significant differences were found between groups. IL-5 was below detection level in all CO and CF-NP, but was measurable in 80% of the non-CF-NP. MPO and IL-8 concentrations were significantly higher in CF-NP compared with CO and non-CF-NP, whereas ECP, eotaxin and IgE were significantly higher in the non-CF-NP group. CONCLUSIONS: We here demonstrate that CF-NP and non-CF-NP not only differ in terms of inflammatory mediator profile, but also in terms of innate markers.

Efficiency of transgenic T cell generation from gene-marked cultured human CD34+ cord blood cells is determined by their maturity and the cytokines present in the culture medium.

Success of gene therapy for diseases affecting the T cell lineage depends on the thymic repopulation by genetically engineered hematopoietic progenitor cells (HPC). Although it has been shown that retrovirally transduced HPC can repopulate the thymus, little information is available on the effect of the culture protocol. Moreover, for expansion of the number of HPC, cytokine supplemented culture is needed. Here, we transduced purified human umbilical cord blood (CB) CD34+ cells in cultures supplemented with various combinations of the cytokines thrombopoietin (TPO), stem cell factor (SCF), flt3/flk-2 ligand (FL), interleukin-3 (IL-3) and IL-6, and investigated thymus-repopulating ability of gene-marked HPC in vitro. Irrespective of the cytokine cocktail used, transduced CD34+CD38- CB cells, expressing the marker green fluorescent protein (GFP) encoded by the MFG-GFP retrovirus, have both superior proliferative and thymus-repopulating potential compared with transduced CD34+CD38+ CB cells. Effectively transduced GFP+CD34+CD38- HPC, cultured for 3 or 17 days, more readily generated T cells than GFP- HPC from the same culture. The reverse was true in the case of CD34+CD38+ HPC cultures. Finally, our results indicate that the number of GFP+ T cell progenitors actually increased during culture of CD34+CD38- HPC, in a magnitude that is determined by the cytokine cocktail used during culture.

In vitro culture of chick down feather bulbi: a tool to obtain proliferating and differentiating keratinocytes in an organotypic structure.

Chick down feather bulbi can be cultured in different culture systems. Morphological analysis and 3H-thymidine incorporation measurements prove that the majority of cells are viable epithelial cells.

Effects of serotonin and ketanserin on the functional morphology of chick down feather bulbi in vitro.

Chick feather bulbi cultured in vitro showed an increased DNA synthesis and a delayed keratinization after treatment with ketanserin, a serotonin2 antagonist with wound-healing properties. In contrast, serotonin stimulates keratinization of the keratinocytes in the bulbus.

Case of recurrent Flavimonas oryzihabitans bacteremia associated with an implanted central venous catheter (Port-A-Cath): assessment of clonality by arbitrarily primed PCR.

Flavimonas oryzihabitans bacteremias, which occurred immediately after the flushing or use of an implanted central venous catheter (Port-A-Cath) in two patients at the same pediatric ward, were studied by arbitrarily primed PCR. We conclude that the colonization of the Port-A-Cath with F. oryzihabitans described here lasted for several months.

Differentiation to T helper cells in the thymus. Gradual acquisition of T helper cell function by CD3+CD4+ cells.

We investigated at which point during thymocyte differentiation functions were acquired that are characteristic for mature Th cells. Differentiation from CD3+CD69-, CD4+CD8+ double-positive (DP) cells to terminally differentiated CD3+, CD4+CD8- single-positive (SP) cells was broken down into six discrete stages that were purified by four-color sorting: CD69-CD3+DP (stage 0), CD69+CD27-DP (stage 1), CD69+CD27-CD4+SP (stage 2), CD27+CD1+CD4+SP (stage 3), CD1-CD45RO+CD4+SP (stage 4), and CD1-CD45RO-CD4+SP cells (stage 5). Phenotypically, these stages seem to describe consecutive steps in differentiation from immature stage 0 to the terminally matured stage 5. Functionally, the capacity to proliferate on IL-2 after stimulation was absent in CD69- stage 0 cells, but was acquired gradually during stages 1 to 4. Clonal expandability and the capacity to respond to stimulation with the production of cytokines were acquired later and rather abruptly by CD1- stage 4 and 5 cells. Activation markers such as CD69 expression and in vivo IL-2 gene transcription came up simultaneously at the DP stage and peaked at stage 3 to 4. These data suggest that functional maturation of Th cells occurs over an extended period in differentiation, stages 1 to 4, and coincides with a gradual increase in activation markers. After completion of functional differentiation, at stage 5, in vivo IL-2 mRNA transcription and CD69 expression are down-regulated, and the cells become functionally resting naive T cells expressing CD45RA+.

Interleukin-7 is a critical growth factor in early human T-cell development.

Highly purified human CD34+ fetal liver stem cells differentiate to mature T cells when seeded in vitro into isolated fetal thymic lobes of severe combined immunodeficient (SCID) mice followed by fetal thymus organ culture (FTOC). Here, this chimeric human-mouse FTOC was used to address the role of interleukin-7 (IL-7) and of the alpha chain of the IL-7 receptor (IL-7R alpha) in early human T-cell development. We report that addition of either the monoclonal antibody (MoAb) M25, which neutralizes both human and mouse IL-7, or the MoAb M21, which recognizes and blocks exclusively the human high-affinity alpha-chain of the IL-7R, results in a profound reduction in human thymic cellularity. Analysis of lymphoid subpopulations indicates that a highly reduced number of cells undergo maturation from CD34+ precursor cells toward CD4+CD3-CD1+ progenitor cells and subsequently toward CD4+CD8+ thymocytes. Our results reveal a critical role for IL-7 during early human thymocyte development, and may explain the absence or highly reduced levels of T cells in patients with X-linked SCID. The molecular defect in these patients has been shown to be a mutation in the gamma chain of the IL-2R. Although this gamma chain is not only present in the IL-2R, but also forms an essential part of other cytokine receptors, including IL-4, IL-7, IL-9, IL-13, and IL-15, the T-cell defect in these patients can be explained by the fact that IL-7 is not able to transduce its signal by the molecular defect of the common gamma (gamma c) chain and that IL-7 is indispensable for T-cell development.

Retrovirally transduced CD34++ human cord blood cells generate T cells expressing high levels of the retroviral encoded green fluorescent protein marker in vitro.

Human umbilical cord blood (UCB) hematopoietic stem cells (HSC) receive increased attention as a possible target for gene-transfer in gene therapy trials. Diseases affecting the lymphoid lineage, as adenosine deaminase (ADA) deficiency and acquired immunodeficiency syndrome (AIDS) could be cured by gene therapy. However, the T-cell progenitor potential of these HSC after gene-transfer is largely unknown and was up to now not testable in vitro. We show here that highly purified CD34++ Lineage marker-negative (CD34++Lin-) UCB cells generate T, natural killer (NK), and dendritic cells in a severe combined immunodeficient mouse fetal thymus organ culture (FTOC). CD34++Lin- and CD34++CD38-Lin- UCB cells express the retroviral encoded marker gene Green Fluorescent Protein (GFP) after in vitro transduction with MFG-GFP retroviral supernatant. Transduced cells were still capable of generating T, NK, and dendritic cells in the FTOC, all expressing high levels of GFP under control of the Moloney murine leukemia virus (MoMuLV) long terminal repeat promotor. We thus present an in vitro assay for thymic T-cell development out of transduced UCB HSC, using GFP as a marker gene.