A comparison of the new ROTEM® sigma with its predecessor, the ROTEMdelta.

Thromboelastometry point-of-care coagulation testing facilitates optimised management of bleeding. Previous thromboelastometry systems required the blood sample and liquid reagents to be pipetted in several manual steps by trained personnel. The ROTEMsigma coagulation analyser is a fully automated point-of-care device. We aimed to assess the reference ranges of the new device and to compare the results with those of the predecessor device, the ROTEMdelta. We took blood from healthy volunteers and from hyper- or hypocoagulable patients; blood samples from healthy volunteers served to determine reference ranges for the most important parameters for the ROTEMsigma: CTEXTEM 48-61 s; A5EXTEM 30-51 mm; MCFEXTEM 54-70 mm; CTINTEM 138-174 s; MCFINTEM 51-67 mm and MCFFIBTEM 5-24 mm. We then used blood samples from patients to compare the results obtained between the old and the new device. We found a strong correlation between the same tests performed on two ROTEMsigma devices and between the ROTEMsigma and the ROTEMdelta with respect to the determination of thromboelastometry parameters of hyper- and hypocoagulable patients (all p < 0.001 and R > 0.8). Performance evaluation for the ROTEMsigma device showed very high precision (R > 0.99, p < 0.001). Our reference ranges can serve as an important aid for other hospitals using this new device.

Retrograde Trafficking Inhibitor of Shiga Toxins Reduces Morbidity and Mortality of Mice Infected with Enterohemorrhagic Escherichia coli.

The most deadly outbreak of Escherichia coli O104:H4 occurred in Europe in 2011. Here, we evaluated the effects of the retrograde trafficking inhibitor Retro-2(cycl) in a murine model of E. coli O104:H4 infection. Systemic treatment with Retro-2(cycl) significantly reduced body weight loss and improved clinical scores and survival rates for O104:H4-infected mice. The present data established that Retro-2(cycl) contributes to the protection of mice against O104:H4 infection and may represent a novel approach to limit Shiga toxin-producing Escherichia coli (STEC)-induced toxicity.

Defining pathogenic verocytotoxin-producing Escherichia coli (VTEC) from cases of human infection in the European Union, 2007-2010.

During 2007-2010, 13 545 confirmed human verocytotoxin (VT)-producing Escherichia coli (VTEC) infections were reported in the European Union, including 777 haemolytic uraemic syndrome (HUS) cases. Clinical manifestations were reported for 53% of cases, 64% of which presented with diarrhoea alone and 10% with HUS. Isolates from 85% of cases were not fully serotyped and could not be classified on the basis of the Karmali seropathotype concept. There is no single or combination of phenotypic or genetic marker(s) that fully define 'pathogenic' VTEC. Isolates which contain the vtx2 (verocytotoxin 2) gene in combination with the eae (intimin-encoding) gene or aaiC (secreted protein of enteroaggregative E. coli) and aggR (plasmid-encoded regulator) genes have been associated with a higher risk of more severe illness. A molecular approach targeting genes encoding VT and other virulence determinants is thus proposed to allow an assessment of the potential severity of disease that may be associated with a given VTEC isolate.

LMA ProSeal(TM) vs. i-Gel(TM) in ventilated children: a randomised, crossover study using the size 2 mask.

BACKGROUND: The Laryngeal Mask Airway (LMA) ProSeal(TM) and the i-Gel(TM) are two extraglottic devices with either an inflatable cuff or a non-inflatable cuff. AIM: We test the hypothesis that oropharyngeal leak pressure and fiberoptic position of the airway tube differ between the size 2 LMA ProSeal(TM) and the i-Gel(TM) in non-paralysed ventilated children. METHODS: Fifty-one children aged 1.5-6 years weighing 10-25 kg were studied using a crossover design. Anaesthesia was with remifentanil/propofol mixture. The LMA ProSeal(TM) and the i-Gel(TM) were inserted into each patient in random order. RESULTS: Oropharyngeal leak pressure for the LMA ProSeal(TM) and the i-Gel(TM) was similar at 22 (5) and 21 (5) cm H(2) O, respectively. Fiberoptic position of the airway tube for the LMA ProSeal(TM) and the i-Gel(TM) was similar, with the vocal cords visible from the distal airway tube in 94% and 96%, respectively. CONCLUSION: We conclude that oropharyngeal leak pressure and fiberoptic position of the airway tube are similar for the size 2 LMA ProSeal(TM) and i-Gel(TM) in non-paralysed ventilated children.

Clinical onset of CNS demyelinating disease after COVID-19 vaccination: denovo disease?

BACKGROUND: Clinical onset of multiple sclerosis (MSpostvacc) and myelin-oligodendrocyte-glycoprotein-antibody-associated disease (MOGADpostvacc) has been reported in association with SARS-CoV-2-vaccination. There is uncertainty as to whether this is causality (denovo disease) or temporal coincidence (manifestation of a preexisting, subclinical neuroinflammation). OBJECTIVES: Comparing the clinical characteristics of MSpostvacc-patients versus patients with MS (PwMS) whose clinical onset occurred independently of vaccination (MSreference). METHODS: Consecutive patients with clinical onset ≤30 days after SARS-CoV-2-vaccination were included. Clinical data, cerebrospinal fluid (CSF) parameters and magnetic resonance imaging (MRI) as well as optical coherence tomography (OCT) data were compared to an age- and sex-matched MSreference-cohort. RESULTS: We identified 5 MSpostvacc and 1 MOGADpostvacc patients who developed their clinical onset ≤ 30 days after SARS-CoV-2-vaccination. Clinical characteristics, CSF, MRI and OCT parameters from MSpostvacc patients were comparable to the MSreference cohort and showed evidence of preexisting subclinical CNS disease. The single case with MOGADpostvacc clearly differed from PwMS in higher CSF cell counts, remission of MRI lesions during follow-up, and absence of oligoclonal bands. CONCLUSIONS: Our case series indicates that MSpostvacc patients showed a rather typical initial manifestation in temporal association with SARS-CoV-2-vaccination and harbored preexisting subclinical neuroinflammation. This argues against the denovo development of MS in this cohort.

Simplex and multiplex real-time PCR assays for the detection of flagellar (H-antigen) fliC alleles and intimin (eae) variants associated with enterohaemorrhagic Escherichia coli (EHEC) serotypes O26:H11, O103:H2, O111:H8, O145:H28 and O157:H7.

AIMS: To develop real-time PCR assays targeting genes encoding the flagellar antigens (fliC) and intimin subtypes (eae) associated with the five most clinically important serotypes of enterohaemorrhagic Escherichia coli (EHEC), i.e. O26:H11, O103:H2, O111:H8, O145:H28 and O157:H7. METHODS AND RESULTS: Primers and probes specific to fliC(H2) , fliC(H7) , fliC(H8) , fliC(H11) , fliC(H28) , eae-ß1, eae-γ1, eae-ε and eae-θ were combined in simplex and multiplex 5'-nuclease PCR assays. The specificity of the assays was assessed on 201 bacterial strains and the sensitivity determined on serially diluted EHEC genomes. The developed PCR assays were found to be highly specific and detected as few as five EHEC genome equivalents per reaction. Furthermore, it was possible to detect the five major EHEC serotypes in cheese samples inoculated at concentration levels of ≤5CFU per 25g after overnight enrichment using the PCR assays. CONCLUSIONS: The PCR assays developed here were found to be sensitive and specific for the reliable detection of genes encoding the flagellar antigens and intimin variants belonging to the five most clinically relevant EHEC serotypes. SIGNIFICANCE AND IMPACT OF THE STUDY: Application of real-time PCR assays should improve the identification of foods contaminated by EHEC and facilitate the molecular typing of these organisms.

Thromboelastometry (ROTEM) in children: age-related reference ranges and correlations with standard coagulation tests.

BACKGROUND: The small sample volume needed and the prompt availability of results make viscoelastic methods like rotational thromboelastometry (ROTEM) attractive for monitoring coagulation in small children. However, data on reference ranges for ROTEM parameters in children are scarce. METHODS: Four hundred and seven children (ASA I and II) undergoing elective surgery were recruited for this prospective, two-centre, observational study. Subjects were grouped as follows: 0-3, 4-12, 13-24 months, 2-5, 6-10, and 11-16 yr. Study objectives were to establish age-dependent reference ranges for ROTEM assays, analyse age dependence of parameters, and compare ROTEM data with standard coagulation tests. RESULTS: Data from 359 subjects remained for final analysis. Except for extrinsically activated clot strength and lysis, parameters for ROTEM assays were significantly different among all age groups. The most striking finding was that subjects aged 0-3 months exhibited accelerated initiation (ExTEM coagulation time: median 48 s, Q1-Q3 38-65 s; P=0.001) and propagation of coagulation (α angle: median 78(o), Q1-Q3 69-84(o); P<0.001) and maximum clot firmness (median 62 mm, Q1-Q3 54-74 mm), although standard plasma coagulation test results were prolonged (prothrombin time: median 13.2 s, Q1-Q3 12.6-13.6 s; activated partial thromboplastin time: median 42 s, Q1-Q3 40-46 s). Lysis indices of <85% were observed in nearly one-third of all children without increased bleeding tendency. Platelet count and fibrinogen levels correlated significantly with clot strength, and fibrinogen levels correlated with fibrin polymerization. CONCLUSIONS: Reference ranges for ROTEM assays were determined for all paediatric age groups. These values will be helpful when monitoring paediatric patients and in studies of perioperative coagulation in children.

Reduced memory B cells in patients with hyper IgE syndrome.

Dominant-negative mutations in STAT-3 have recently been found in the majority of patients with sporadic or autosomal-dominant hyper IgE syndrome (HIES). Since STAT-3 plays a role in B cell development and differentiation, we analyzed memory B cells in 20 patients with HIES, 17 of which had STAT-3 mutations. All but four patients had reduced non-switched and/or class-switched memory B cells. No reduction in these B cell populations was found in 16 atopic dermatitis patients with IgE levels above 1000 KU/L. There was no correlation between the reduction of memory B cells and the ability to produce specific antibodies. Moreover, there was no correlation between the percentage of memory B cells and the infection history. Analysis of memory B cells can be useful in distinguishing patients with suspected HIES from patients with atopic disease, but probably fails to identify patients who are at high risk of infection.

Comparison of high- and low-dose intrathecal morphine for spinal fusion in children.

BACKGROUND: The purpose of this prospective study was to assess the blood-sparing effect, the quality of analgesia, and the incidence of side-effects of a low-dose regime of intrathecal opioids (ITO) when compared with those of a high-dose regime in scoliosis surgery in children. METHODS: Forty-six children were randomly included into one of the three groups to receive morphine 5 microg kg(-1) plus sufentanil 1 microg kg(-1) [low-dose intrathecal opioid (LITO)], morphine 15 microg kg(-1) plus sufentanil 1 microg kg(-1) [high-dose intrathecal opioid (HITO)] intrathecally, or no intrathecal opioid. Postoperative analgesia was provided by i.v. opioids. Intraoperative blood loss, postoperative quality of analgesia, opioid requirements, and the incidence of side-effects were recorded for 3 days. RESULTS: Intraoperative blood loss was significantly reduced by ITOs [LITO: 41.4 (sd 18.8) ml kg(-1); HITO: 37.5 (6.9) ml kg(-1); control: 76.9 (15.3) ml kg(-1), P<0.001], with no difference between the two intrathecal opioid groups. Mean pain scores on the day of surgery were lower in both intrathecal opioid groups (LITO: 2.2 and HITO: 2.1) when compared with the control group (4.1, P<0.03) and opioid consumption was significantly decreased [LITO: 304.3 (65.0) microg kg(-1); HITO: 224.1 (51.8) microg kg(-1); control: 667.7 (89.5) microg kg(-1), P<0.002]. Side-effects of intrathecally administered opioids were similarly frequent in all groups. CONCLUSIONS: Intrathecal administration of opioids significantly reduces blood loss and postoperative opioid demand, thereby showing side-effects comparable with the control group. These effects were already seen with the low-dose regimen and high dose did not further improve efficacy.

Identification of intimin alleles in pathogenic Escherichia coli by PCR-restriction fragment length polymorphism analysis.

A rapid two-step identification method based on PCR-RFLP analysis of the intimin gene was developed to differentiate specific alleles in pathogenic Escherichia coli. This technique, tested on isolates eae-positive, accurately detects eae and resolves alleles encoding the alpha1, alpha2, beta, gamma1, gamma2/theta, kappa, epsilon, zeta, and iota intimin variants.

Conjoined epigastric heteropagus twins: excision of a parasitic twin from the anterior abdominal wall of her sibling.

Epigastric heteropagus twins (EHT) are an exceedingly rare form of asymmetric conjoined twins in whom the dependent twin (parasite) is attached to the right or left upper abdomen of the dominant part (autosite). Such a case observed at our institution with 34 month follow-up is presented here and the surgical technique described. A magnetic resonance imaging (MRI)-supported surgical separation of the parasite with successful closure of the abdominal wall defect of the autosite was performed. Follow-up studies showed an autosite which was alive and in optimal health. A comprehensive review including data from English and non-English literature is presented.

Rapid Isolation of Staphylococcus aureus Pathogens from Infected Clinical Samples Using Magnetic Beads Coated with Fc-Mannose Binding Lectin.

Here we describe how Staphylococcus aureus bacteria can be rapidly isolated from clinical samples of articular fluid and synovial tissue using magnetic beads coated with the engineered chimeric human opsonin protein, Fc-mannose-binding lectin (FcMBL). The FcMBL-beads were used to capture and magnetically remove bacteria from purified cultures of 12 S. aureus strains, and from 8 articular fluid samples and 4 synovial tissue samples collected from patients with osteoarthritis or periprosthetic infections previously documented by positive S. aureus cultures. While the capture efficiency was high (85%) with purified S. aureus strains grown in vitro, direct FcMBL-bead capture from the clinical samples was initially disappointing (< 5% efficiency). Further analysis revealed that inhibition of FcMBL binding was due to coating of the bacteria by immunoglobulins and immune cells that masked FcMBL binding sites, and to the high viscosity of these complex biological samples. Importantly, capture of pathogens using the FcMBL-beads was increased to 76% efficiency by pretreating clinical specimens with hypotonic washes, hyaluronidase and a protease cocktail. Using this approach, S. aureus bacteria could be isolated from infected osteoarthritic tissues within 2 hours after sample collection. This FcMBL-enabled magnetic method for rapid capture and concentration of pathogens from clinical samples could be integrated upstream of current processes used in clinical microbiology laboratories to identify pathogens and perform antibiotic sensitivity testing when bacterial culture is not possible or before colonies can be detected.

The bacterial cytolethal distending toxin (CDT) triggers a G2 cell cycle checkpoint in mammalian cells without preliminary induction of DNA strand breaks.

The bacterial cytolethal distending toxin (CDT) was previously shown to arrest the tumor-derived HeLa cell line in the G2-phase of the cell cycle through inactivation of CDK1, a cyclin-dependent kinase whose state of activation determines entry into mitosis. We have analysed the effects induced in HeLa cells by CDT, in comparison to those induced by etoposide, a prototype anti-tumoral agent that triggers a G2 cell cycle checkpoint by inducing DNA damage. Both CDT and etoposide inhibit cell proliferation and induces the formation of enlarged mononucleated cells blocked in G2. In both cases, CDK1 from arrested cells could be reactivated both in vitro by dephosphorylation by recombinant Cdc25B phosphatase and in vivo by caffeine. However, the cell cycle arrest triggered by CDT, unlike etoposide, did not originate from DNA strand breaks as demonstrated in the single cell gel electrophoresis assay and by the absence of slowing down of S phase in synchronized cells. Together with additional observations on synchronized HeLa cells, our results suggest that CDT triggers a G2 cell cycle checkpoint that is initiated during DNA replication and that is independent of DNA damage.

Metabolism of naturally occurring propenylbenzene derivatives. III. Allylbenzene, propenyl benzene, and related metabolic products.

The acidic and neutral urinary metabolites of allylbenzene, propenylbenzene, 1'-hydroxyallylbenzene, and cynnamyl alcohol were identified and related through a common metabolic scheme. The rat metabolizes allylbenzene to 1'-hydroxyallylbenzene which can rearrange to yield cinnamyl alcohol whick is further metabolized. This mechanism is proposed to account for the appearance of "propenyl type" metabolites from allylbenzene compounds. Propenylbenzene is oxidized to cynnamyl alcohol. Both 1'-hydroxyallylbenzene and cunnamyl alcohol are excreted unchanged in the neutral extract when given to rats. Allylbenzene and 1'-hydroxyallylbenzene yield basic ninhydrin-positive metabolites. Allylbenzene is first oxidized on the benzylic carbon to form 1'-hydroxyallylbenzene, which is further oxidized to form phenyl vinyl ketone, which condenses with the secondary amines piperidine and dimethylamine to form tertiary aminopropiophenones (Mannich bases). Analogous compounds, cinnamyl alcohol and propenylbenzene, do not yield Mannich base metabolites. This proposed metabolic scheme is consistent with the chemical mechanism operative in the synthesis of Mannich base from allylbenzene via chromic acid oxidation followed by amine addition.

Colibactin biosynthesis and biological activity depend on the rare aminomalonyl polyketide precursor.

The as-yet unidentified E. coli metabolite colibactin induces DNA damage in eukaryotic cells and promotes tumorigenesis. Its wide distribution in pathogenic and probiotic strains has raised great interest in its structure and biosynthesis. Here we show that colibactin formation involves a rare aminomalonyl unit used as a building block.

Cytolethal distending toxin (CDT): a bacterial weapon to control host cell proliferation?

Cytolethal distending toxins (CDT) constitute a family of genetically related bacterial protein toxins able to stop the proliferation of numerous cell lines. This effect is due to their ability to trigger in target cells a signaling pathway that normally prevents the transition between the G2 and the M phase of the cell cycle. Produced by several unrelated Gram-negative mucosa-associated bacterial species, CDTs are determined by a cluster of three adjacent genes (cdtA, cdtB, cdtC) encoding proteins whose respective role is not yet fully elucidated. The CDT-B protein presents sequence homology to several mammalian and bacterial phosphodiesterases, such as DNase I. The putative nuclease activity of CDT-B, together with the activation by CDT of a G2 cell cycle checkpoint, strongly suggests that CDT induces an as yet uncharacterized DNA alteration. However, the effective entry of CDT into cells and subsequent translocation into the nucleus have not yet been demonstrated by direct methods. The relationship between the potential DNA-damaging properties of this original family of toxins and their role as putative virulence factors is discussed.

A single protein of 110 kDa is associated with the multinucleating and necrotizing activity coded by the Vir plasmid of Escherichia coli.

A lethal and necrotic factor which causes cell multinucleation in HeLa cell cultures has previously been shown to be coded by the Vir plasmid of Escherichia coli. Using an absorbed rabbit antiserum which neutralized the Vir toxic properties, we have compared the SDS-PAGE immunoblots from laboratory and field strains which either produce or do not produce Vir toxicity. A single band of 110 kDa was found to be specifically associated with vir toxicity in E. coli strains. This antiserum also recognized the 115 kDa protein band which was previously identified as the cytotoxic necretozing factor (CNF) of certain E. coli strains. These results suggest that the toxin coded by the Vir plasmid is a protein of 110 kDa distinct from, but immunologically related to CNF.

Cytotoxic effect of multinucleation in HeLa cell cultures associated with the presence of Vir plasmid in Escherichia coli strains.

Escherichia coli strain S5 possesses a virulent plasmid termed Vir, which codes for a lethal toxin and a surface antigen. This strain and two trans-conjugant strains, which have received the Vir plasmid from S5, produced a specific and thermolabile cytopathic effect of multinucleation in an HeLa cell cultures assay, whereas isogenic Vir- strains did not. Moreover sonicates of two epidemiologically unrelated Vir+ strains, exerted the same type of cytotoxicity. This effect, together with lethality for chicken, was specifically neutralized by a rabbit antiserum prepared against Vir+ sonicates. The Vir cytopathic effect appeared morphologically distinct from the one caused by the cytotoxic necrotizing factor of E. coli, which was partially related immunologically. We therefore propose to call this type of toxin 'Vir cytotoxin'.

Expression of P, S, and F1C adhesins by cytotoxic necrotizing factor 1-producing Escherichia coli from septicemic and diarrheic pigs.

Nineteen papC-positive cytotoxic necrotizing factor 1 (CNF1)-producing Escherichia coli isolates from pigs with septicemia or diarrhea were tested for the presence of pap-, sfa-, and afa-related sequences encoding P/Prs, S/F1C, and Dr/AFA adhesins respectively. Production of adhesins by isolates was tested by mannose-resistant hemagglutination (MRHA), sialidase treatment of erythrocytes and particle agglutination tests. Production of P, S, and F1C fimbriae by isolates was also examined by immunofluorescence. All isolates were pap+ by PCR. Eighteen isolates (95%) were MRHA for ovine and human A erythrocytes and exhibited GalNac-GalNac receptor specificity associated with class III P(Prs) adhesins. Fifteen (79%) of the 19 isolates reacted with antisera specific for one or more different P fimbrial serotypes on immunofluorescence. Three of these isolates also demonstrated Gal-Gal receptor specificity associated with class I or II P fimbrial adhesins. Fifteen (79%) of the isolates were sfa+ by PCR. Seven of these isolates exhibited sialidase-sensitive MRHA of bovine and human O erythrocytes and reacted with serum specific for S fimbriae on immunofluorescence. Seven of the 8 sfa+ isolates which were MRHA-negative for bovine erythrocytes reacted with serum specific for F1C fimbriae on immunofluorescence. All isolates produced type 1 fimbriae as determined by mannose-sensitive agglutination of yeast cells. None of the isolates were afa+ by PCR or colony hybridization. Results suggest that most pap+ porcine CNF1-producing E. coli isolates express P fimbriae bearing class III (Prs) type adhesins. In addition, most of these isolates also produce S or F1C fimbriae.

Specific DNA probes to detect Escherichia coli strains producing cytotoxic necrotising factor type 1 or type 2.

Cytotoxic necrotising factors type 1 (CNF1) and type 2 (CNF2) are produced by many Escherichia coli strains isolated from man and animals with intestinal or extra-intestinal colibacillosis. In most laboratories, CNF-producing strains are detected by a cell cytotoxicity assay and confirmed with a neutralisation assay or a mouse footpad assay. In this study, we sought to determine whether DNA probes could detect clinical isolates of E. coli producing CNF2 or CNF1, or both, without the need for cell cultures or animal assays. Two internal fragments of the gene encoding CNF2 were used as DNA probes: a 875-bp XhoI-PstI DNA fragment and an adjacent 335-bp PstI-ClaI fragment. A positive response with both DNA probes was associated with CNF2-producing strains, whereas a positive response with only the 335-bp probe was associated with CNF1-producing strains. Results of colony hybridisation experiments with 185 clinical isolates of E. coli demonstrated that these DNA probes detected CNF2-producing strains with a sensitivity and specificity of 100% and CNF1-producing strains with a sensitivity and specificity of 99%. These two DNA probes should greatly facilitate epidemiological studies to assess the importance of CNF-producing strains as agents of diarrhoea and septicaemia.