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The article discusses a study aimed at determining if whole body calcium retention reflects skeletal uptake of calcium as well as evaluating the feasibility of using urinary calcium for measurement of bone resorption. Adult female Sprague-Dawley rats, injected with intra-pertineally, were used as subjects for the study. Results suggest that whole body calcium retention may be used as an accurate surrogate measure of calcium retention in the skeleton.

OXA-48-like enzymes represent the most frequently detected carbapenemases in Enterobacterales in Western Europe, North Africa and the Middle East. In contrast to other species, the presence of OXA-48-like in Proteus mirabilis leads to an unusually susceptible phenotype with low MICs for carbapenems and piperacillin-tazobactam, which is easily missed in the diagnostic laboratory. So far, there is little data available on the genetic environments of the corresponding genes, bla OXA-48 -like, in P. mirabilis. In this study susceptibility phenotypes and genomic data of 13 OXA-48-like-producing P. mirabilis were investigated (OXA-48, n  = 9; OXA-181, n  = 3; OXA-162, n  = 1). Ten isolates were susceptible to meropenem and ertapenem and three isolates were susceptible to piperacillin-tazobactam. The gene bla OXA-48 was chromosomally located in 7/9 isolates. Thereof, in three isolates bla OXA-48 was inserted into a P. mirabilis genomic island. Of the three isolates harbouring bla OXA-181 one was located on an IncX3 plasmid and two were located on a novel MOB F plasmid, pOXA-P12, within the new transposon Tn 7713 . In 5/6 isolates with plasmidic location of bla OXA-48- like, the plasmids could conjugate to E. coli recipients in vitro . Vice versa , bla OXA-48 -carrying plasmids could conjugate from other Enterobacterales into a P. mirabilis recipient. These data show a high diversity of bla OXA-48 -like genetic environments compared to other Enterobacterales, where genetic environments are quite homogenous. Given the difficult-to-detect phenotype of OXA-48-like-producing P. mirabilis and the location of bla OXA-48 -like on mobile genetic elements , it is likely that OXA-48-like-producing P. mirabilis can disseminate, escape most surveillance systems, and contribute to a hidden spread of OXA-48-like.

Carbapenemase-producing Klebsiella pneumoniae (CP-Kp) isolates are a public health concern as they can cause severe hospital-acquired infections that are difficult to treat. It has recently been shown that CP-Kp can take up virulence factors from hypervirulent K. pneumoniae lineages. In this study, 109 clinical CP-Kp isolates from the University Hospital Cologne were examined for the presence of acquired virulence factors using whole-genome sequencing and phenotypic tests, and results were linked to clinical data. The virulence factor iuc was present in 18/109 of the CP-Kp isolates. Other acquired virulence factors, such as ybt , cbt , iro , rmpA/rmpA2 , peg-344 , and hypervirulence-associated capsule types were detected in various combinations among these isolates. The iuc -positive isolates produced OXA-232 ( n = 7), OXA-48 ( n = 6), OXA-48+NDM ( n = 3), NDM, and KPC (each n = 1), and 7/18 isolates were resistant to ceftazidime-avibactam, colistin, and/or cefiderocol. Four isolates carried hybrid plasmids that harbored acquired virulence factors alongside the carbapenemase genes bla NDM-1/5 or bla OXA-48 . In 15/18 patients, iuc -positive CP-Kp were isolated from a clinically manifest infection site. Among these, four patients had osteomyelitis, and four patients died from pneumonia with OXA-232-producing ST231 isolates, three of them as part of an outbreak. In conclusion, acquired virulence factors are frequently detected in various combinations in carbapenemase-producing K. pneumoniae isolates in Germany, warranting continuous monitoring of infections caused by these strains., Competing Interests: The authors declare no conflict of interest.

BackgroundCarbapenemase-producing Enterobacterales are a public health threat worldwide and OXA-48 is the most prevalent carbapenemase in Germany and western Europe. However, the molecular epidemiology of OXA-48 in species other than Escherichia coli and Klebsiella pneumoniae remains poorly understood.AimTo analyse the molecular epidemiology of OXA-48 and OXA-48-like carbapenemases in Citrobacter species (spp.) in Germany between 2011 and 2022.MethodsData of 26,822 Enterobacterales isolates sent to the National Reference Centre (NRC) for Gram-negative bacteria were evaluated. Ninety-one Citrobacter isolates from 40 German hospitals harbouring bla OXA-48/OXA-48‑like were analysed by whole genome sequencing and conjugation experiments.ResultsThe frequency of OXA-48 in Citrobacter freundii (CF) has increased steadily since 2011 and is now the most prevalent carbapenemase in this species in Germany. Among 91 in-depth analysed Citrobacter spp. isolates, CF (n = 73) and C. koseri (n = 8) were the most common species and OXA-48 was the most common variant (n = 77), followed by OXA-162 (n = 11) and OXA‑181 (n = 3). Forty percent of the isolates belonged to only two sequence types (ST19 and ST22), while most other STs were singletons. The plasmids harbouring bla OXA‑48 and bla OXA-162 belonged to the plasmid types IncL (n = 85) or IncF (n = 3), and plasmids harbouring bla OXA‑181 to IncX3 (n = 3). Three IncL plasmid clusters (57/85 IncL plasmids) were identified, which were highly transferable in contrast to sporadic plasmids.ConclusionIn CF in Germany, OXA-48 is the predominant carbapenemase. Dissemination is likely due to distinct highly transmissible plasmids harbouring bla OXA‑48 or bla OXA-48-like and the spread of the high-risk clonal lineages ST19 and ST22.

Rapid and reliable detection of carbapenemase-producing Enterobacterales (CPE) is crucial for prompt treatment and infection control. Most assays target the primary four enzymes (KPC, OXA-48-like, VIM, and NDM), often missing less common variants (e.g., GES, IMI, OXA-23, and OXA-58). Therefore, assays based on the hydrolysis of carbapenems are recommended in addition to differentiation tests such as PCR or immunochromatographic assays. The aim of this study was to compare the currently Clinical and Laboratory Standards Institute (CLSI)-recommended tests mCIM (modified carbapenem inactivation method) and Carba NP with new colorimetric tests (NitroSpeed-Carba NP) and novel variations of the carbapenem inactivation method (CIM) such as simplified CIM (sCIM) or modified zinc-supplemented CIM (mzCIM). The challenge collection included 205 clinical isolates, 139 CPE vs 66 non-CPE. Among all 205 isolates, the sensitivity/specificity of mCIM was 81.3%/98.5%, Carba NP 76.3%/100%, NitroSpeed-Carba NP 86.3%/78.8%, sCIM 100%/94%, and mzCIM 97.8%/98.5%. For rare carbapenemases ( n = 48), the sensitivity of mzCIM (98.3%) and sCIM (100%) was higher than that of mCIM (60.4%), Carba NP (50%), or NitroSpeed-Carba NP (70.2%). Most indeterminate results occurred for mCIM (14.4%), Carba NP (8.2%), and sCIM (6.3%). The detection of rare carbapenemases remains challenging with the currently recommended assays. The CIM-based tests demonstrated superior sensitivity, with sCIM and mzCIM outperforming the currently recommended mCIM and Carba NP, especially among isolates with weakly hydrolyzing carbapenemases (e.g., OXA-23 and OXA-58). Although colorimetric assays provide more rapid results, laboratories have to be aware of the low sensitivity for rare carbapenemases. Both sCIM and the new mzCIM performed well, are cost-effective, and can easily be implemented in any laboratory.IMPORTANCEDetection of so-called rare carbapenemases (e.g., GES, IMI, OXA-23, and OXA-58) in Enterobacterales is challenging, and data on the performance of currently available assays are scarce. This study systematically assessed the performance of currently recommended and novel hydrolysis-based assays on a set of molecularly characterized isolates. It demonstrates that the currently recommended assays mCIM and Carba NP perform well on isolates producing common carbapenemases such as KPC, VIM, NDM, and OXA-48, but have only a moderate sensitivity in the detection of rare carbapenemases. In contrast, the newer CIM-based variants, sCIM and mzCIM, are equally capable of detecting frequent and uncommon carbapenemases. These assays could potentially help to improve our knowledge on the epidemiology of these "rare" enzymes., Competing Interests: A.H. has received speaker honoraria from BD, bioMérieux, and Beckman Coulter; received research support from bioMérieux and Coris; and served on the advisory board of bioMérieux. S.G. has received speaker honoraria from bioMérieux, Beckman Coulter, and Thermo Fisher. All other authors declare no conflicts of interest.

Objectives: To analyse carbapenemases in Proteus mirabilis and assess the performance of carbapenemase detection assays., Methods: Eighty-one clinical P. mirabilis isolates with high-level resistance at least to ampicillin (>32 mg/L) or previous detection of carbapenemases were selected and investigated by three susceptibility testing methods (microdilution, automated susceptibility testing, and disk diffusion), six phenotypic carbapenemase assays (CARBA NP, modified carbapenemase inactivation method [CIM], modified zinc-supplemented CIM, simplified CIM, faropenem, and carbapenem-containing agar), two immunochromatographic assays, and whole-genome sequencing., Results: Carbapenemases were detected in 43 of 81 isolates (OXA-48-like [n = 13]; OXA-23 [n = 12]; OXA-58 [n = 12]; New Delhi metallo-β-lactamase (NDM) [n = 2]; Verona integron-encoded metallo-β-lactamase (VIM) [n = 2]; Imipenemase (IMP) [n = 1]; Klebsiella pneumoniae carbapenemase (KPC) [n = 1]). Carbapenemase-producing Proteus were frequently susceptible to ertapenem (26/43; 60%), meropenem (28/43; 65%), ceftazidime (33/43; 77%), and some even to piperacillin-tazobactam (9/43; 21%). Sensitivity/specificity of phenotypic tests were 30% (CI: 17-46%)/89% (CI: 75-97%) for CARBA NP, 74% (CI: 60-85%)/82% (CI: 67-91%) for faropenem, 91% (CI: 78-97%)/82% (CI: 66-92%) for simplified CIM, and 93% (CI: 81-99%)/100% (CI: 91-100%) for modified zinc-supplemented CIM. An algorithm for improved detection was developed, which demonstrated sensitivity/specificity of 100% (CI: 92-100%)/100% (CI: 91-100%) on the 81 isolates, and 100% (CI: 29-100%)/100% (CI: 96-100%) in a prospective analysis of additional 91 isolates. Interestingly, several OXA-23-producing isolates belonged to the same clonal lineage reported previously from France., Discussion: Current susceptibility testing methods and phenotypic tests frequently fail to detect carbapenemases in P. mirabilis, which could result in inadequate antibiotic treatment. In addition, the non-inclusion of bla OXA-23/OXA-58 in many molecular carbapenemase assays further impedes their detection. Therefore, the prevalence of carbapenemases in P. mirabilis is likely underestimated. With the herein proposed algorithm, carbapenemase-producing Proteus can be easily identified., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The emergence of extended-spectrum β-lactamase and carbapenemase-producing Enterobacterales (ESBL-E and CPE, respectively) is a threat to modern medicine, as infections become increasingly difficult to treat. These bacteria have been detected in aquatic environments, which raises concerns about the potential spread of antibiotic resistance through water. Therefore, we investigated the occurrence of ESBL-E and CPE in surface water in Lower Saxony, Germany, using phenotypic and genotypic methods. Water samples were collected from two rivers, five water canals near farms, and 18 swimming lakes. ESBL-E and CPE were isolated from these samples using filters and selective agars. All isolates were analyzed by whole genome sequencing. Multidrug-resistant Enterobacterales were detected in 4/25 (16%) water bodies, including 1/2 rivers, 2/5 water canals and 1/18 lakes. Among all samples, isolates belonging to five different species/species complexes were detected: Escherichia coli ( n  = 10), Enterobacter cloacae complex ( n  = 4), Citrobacter freundii ( n  = 3), Citrobacter braakii ( n  = 2), and Klebsiella pneumoniae (n = 2) . Of the 21 isolates, 13 (62%) were resistant at least to 3rd generation cephalosporins and eight (38%) additionally to carbapenems. CPE isolates harbored bla KPC-2 ( n  = 5), bla KPC-2 and bla VIM-1 ( n  = 2), or bla OXA-181 ( n  = 1); additionally, mcr-9 was detected in one isolate. Two out of eight CPE isolates were resistant to cefiderocol and two to colistin. Resistance to 3rd generation cephalosporins was mediated by ESBL ( n  = 10) or AmpC ( n  = 3). The presence of AmpC-producing Enterobacterales, ESBL-E and CPE in northern German surface water samples is alarming and highlights the importance of aquatic environments as a potential source of MDR bacteria., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Axel Hamprecht reports financial support was provided by Lower Saxony State Ministry of Science and Culture., (© 2023 The Authors.)

OXA-48 is the most common carbapenemase in Enterobacterales in Germany and many other European countries. Depending on the genomic location of bla OXA-48 , OXA-48-producing isolates vary in phenotype and intra- and interspecies transferability of bla OXA-48 . In most bacterial isolates, bla OXA-48 is located on one of seven variants of Tn 1999 (Tn 1999.1 to Tn 1999.6 and invTn 1999.2 ). Here, a novel Tn 1999 variant, Tn 1999.7 , is described, which was identified in 11 clinical isolates from 2016 to 2020. Tn 1999.7 differs from Tn 1999.1 by the insertion of the 8,349-bp Tn 3 family transposon Tn 7442 between the lysR gene and bla OXA-48 open reading frame. Tn 7442 carries genes coding for a restriction endonuclease and a DNA methyltransferase as cargo, forming a type III restriction modification system. Tn 1999.7 was carried on an ~71-kb IncL plasmid in 9/11 isolates. In one isolate, Tn 1999.7 was situated on an ~76-kb plasmid, harboring an additional insertion sequence in the plasmid backbone. In one isolate, the plasmid size is only ~63 kb due to a deletion adjacent to Tn 7442 that extends into the plasmid backbone. Mean conjugation rates of the Tn 1999.7 -harboring plasmids in J53 ranged from 4.47 × 10 -5 to 2.03 × 10 -2 , similar to conjugation rates of other pOXA-48-type IncL plasmids. The stability of plasmids with Tn 1999.7 was significantly higher than that of a Tn 1999.2 -harboring plasmid in vitro . This increase in stability could be related to the insertion of a restriction-modification system, which can promote postsegregational killing. The increased plasmid stability associated with Tn 1999.7 could contribute to the further spread of OXA-48.

Bauhinia forficata Link is a plant rich in polyphenols that has been used mainly for its hypoglycemic activity, which is related to its antioxidant and anti-inflammatory potential. However, the beneficial effect of these bioactive compounds is directly dependent on their bioaccessibility and bioavailability, requiring processing techniques that can improve and preserve their biological activities. This work aimed to obtain nanocapsulated extracts from the infusion (ESIN) and decoction (ESDC) of B. forficata Link leaves, by spray drying. The encapsulating agents used were maltodextrin and colloidal silicon dioxide. The nanocapsules were characterized by HPLC-PDA-ESI-IT-MS n , evaluated the bioaccessibility of polyphenols after simulated digestion and their antioxidant activity. Additionally, an extensive physicochemical characterization of the nanocapsulated extracts was carried out and their stability and technological parameters were evaluated. The ESIN and ESDC extracts had yields of 57.3 % and 62.7 %, with average nanocapsules sizes of 0.202 μm and 0.179 μm, low humidity and water activity (<0.5), powder density and proper flow properties (Hausner ratio ≤ 1.25; Carr index 18-19 %). Scanning electron microscopy showed a spherical and amorphous morphology and low viscosity, which may have favored the solubility profile. The phenolic compounds of the nanocapsules degraded after 400 °C, showing high thermal stability. The infrared spectra identified the presence of maltodextrin and phenolic compounds and that there were no reactions between them. Chromatography confirmed the presence of phenolic compounds, mainly flavonols and their O-glycosylated derivatives, as well as carbohydrates, probably maltodextrin. Simulated in vitro digestion showed that polyphenols and flavonoids from ESIN and ESDC nanocapsules were bioaccessible after the gastric phase (49.38 % and 64.17 % of polyphenols and 64.08 % and 36.61 % of flavonoids) and duodenal (52.68 % and 79.06 % of polyphenols and 13.24 % and 139.03 % of flavoids), with a variation from 52.27 % to 70.55 % of the antioxidant activity maintained, by the ORAC method, after gastric digestion and still 25 %, after duodenal. Therefore, the nanoencapsulation of extracts of B. forficata is a viable option for the preservation of their bioactive compounds, making them bioaccessible and with antioxidant activity, which make them suitable for incorporation into various nutraceutical formulations, such as capsules, tablets and sachets., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Author(s).)

Neurofilament light chain (NfL), released during central nervous injury, has evolved as a powerful serum marker of disease severity in many neurological disorders, including infectious diseases. So far NfL has not been assessed in cerebral malaria in human or its rodent model experimental cerebral malaria (ECM), a disease that can lead to fatal brain edema or reversible brain edema. In this study we assessed if NfL serum levels can also grade disease severity in an ECM mouse model with reversible (n = 11) and irreversible edema (n = 10). Blood-brain-barrier disruption and brain volume were determined by magnetic resonance imaging. Neurofilament density volume as well as structural integrity were examined by electron microscopy in regions of most severe brain damage (olfactory bulb (OB), cortex and brainstem). NfL plasma levels in mice with irreversible edema (317.0 ± 45.01 pg/ml) or reversible edema (528.3 ± 125.4 pg/ml) were significantly increased compared to controls (103.4 ± 25.78 pg/ml) by three to five fold, but did not differ significantly in mice with reversible or irreversible edema. In both reversible and irreversible edema, the brain region most affected was the OB with highest level of blood-brain-barrier disruption and most pronounced decrease in neurofilament density volume, which correlated with NfL plasma levels (r = - 0.68, p = 0.045). In cortical and brainstem regions neurofilament density was only decreased in mice with irreversible edema and strongest in the brainstem. In reversible edema NfL plasma levels, MRI findings and neurofilament volume density normalized at 3 months' follow-up. In conclusion, NfL plasma levels are elevated during ECM confirming brain damage. However, NfL plasma levels fail short on reliably indicating on the final outcomes in the acute disease stage that could be either fatal or reversible. Increased levels of plasma NfL during the acute disease stage are thus likely driven by the anatomical location of brain damage, the olfactory bulb, a region that serves as cerebral draining pathway into the nasal lymphatics., (© 2022. The Author(s).)

Background: With increasing resistance to common antibiotics the treatment of urinary tract infections has become challenging and alternative therapeutic options are needed. In the present study, we evaluate the activity of three older and less frequently used antibiotics against MDR Enterobacterales., Methods: Susceptibility of mecillinam, temocillin and nitroxoline was assessed in Enterobacterales isolated from urinary specimens with elevated MICs of third-generation cephalosporins. Susceptibility was determined by the recommended reference MIC methods and additionally by disc diffusion. All isolates were characterized for common β-lactamases by phenotypic and molecular assays., Results: In total 394 Enterobacterales were included. The most common resistance mechanisms were ESBLs ( n  = 273), AmpC ( n  = 132), carbapenemases [ n  = 12, including OXA-48-like ( n  = 8), VIM ( n  = 2), KPC ( n  = 1) and NDM ( n  = 1)] or others ( n  = 2). Resistance was observed in 59% of isolates to ceftazidime, in 41% to piperacillin/tazobactam and in 54% to ciprofloxacin. In comparison, resistance was less frequent against mecillinam (15%), temocillin (13%) or nitroxoline (2%). Mecillinam showed higher activity in Enterobacter spp., Escherichia coli and in OXA-48-like-producing isolates compared with temocillin, which was more active in Proteus mirabilis and in ESBL-producing isolates. Activity of nitroxoline was high against all isolates, including carbapenemase-producing isolates. Correlation between disc diffusion and MIC methods was good for mecillinam and moderate for temocillin and nitroxoline., Conclusions: Mecillinam, temocillin and nitroxoline show good to excellent in vitro activity in MDR Enterobacterales. The activity of mecillinam and temocillin was higher in certain species and restricted depending on β-lactamase production while nitroxoline showed universally high activity irrespective of species or β-lactamase present., (© The Author(s) 2022. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy.)

Human-induced changes in climate and habitats push populations to adapt to novel environments, including new sensory conditions, such as reduced visibility. We studied how colonizing newly formed glacial lakes with turbidity-induced low-visibility affects anti-predator behaviour in Icelandic threespine sticklebacks. We tested nearly 400 fish from 15 populations and four habitat types varying in visibility and colonization history in their reaction to two predator cues (mechano-visual versus olfactory) in high versus low-visibility light treatments. Fish reacted differently to the cues and were affected by lighting environment, confirming that cue modality and light levels are important for predator detection and evasion. Fish from spring-fed lakes, especially from the highlands (likely more diverged from marine fish than lowland fish), reacted fastest to mechano-visual cues and were generally most active. Highland glacial fish showed strong responses to olfactory cues and, counter to predictions from the flexible stem hypothesis, the greatest plasticity in response to light levels. This study, leveraging natural, repeated invasions of novel sensory habitats, (i) illustrates rapid changes in anti-predator behaviour that follow due to adaptation, early life experience, or both, and (ii) suggests an additional role for behavioural plasticity enabling population persistence in the face of frequent changes in environmental conditions.

Detection of carbapenemases in Enterobacterales is crucial for patient treatment and infection control. Among others, combination disc tests (CDTs) with different inhibitors (e.g., EDTA) and variations of the carbapenem inactivation method (CIM) are recommended by EUCAST or the CLSI and are used by many laboratories as they are relatively inexpensive. In this study, we compare three commercially available CDTs, faropenem disc testing (FAR), and the zinc-supplemented CIM (zCIM) test for the detection of carbapenemase-producing Enterobacterales (CPE). The Rosco KPC/MBL and OXA-48 Confirm kit (ROS-CDT), the Liofilchem KPC&MBL&OXA-48 disc kit (LIO-CDT), Mastdiscs Combi Carba plus (MAST-CDT), FAR, and zCIM were challenged with 106 molecularly characterized CPE and 47 non-CPE isolates. The sensitivities/specificities were 86% (confidence interval [CI], 78 to 92%)/98% (CI, 89 to 100%) for MAST-CDT and ROS-CDT, 96% (CI, 91 to 99%)/87% (CI, 74 to 95%) for LIO-CDT, and 99% (CI, 95 to 100%)/81% (CI, 67 to 91%) for FAR compared to 98% (CI, 93 to 100%)/100% (CI, 92 to 100%) for zCIM. The CDTs showed great performance differences depending on the carbapenemase class, with MAST-CDT and LIO-CDT best detecting class B, ROS-CDT best detecting class A, and LIO-CDT best detecting class D carbapenemases. The overall performance of commercially available CDTs was good but varied greatly for different carbapenemases and between manufacturers, compared with FAR and zCIM, which performed well for all carbapenemase types. For reliable carbapenemase detection, CDTs should preferably not be used as the sole test but can be part of a diagnostic strategy when combined with other assays (e.g., CIM-based, immunochromatographic, or molecular tests).

OXA-48-like carbapenemases are among the most frequent carbapenemases in Gram-negative Enterobacterales worldwide with the highest prevalence in the Middle East, North Africa and Europe. Here, we investigated the so far uncharacterized carbapenemase OXA-484 from a clinical E. coli isolate belonging to the high-risk clone ST410 regarding antibiotic resistance pattern, horizontal gene transfer (HGT) and genetic support. OXA-484 differs by the amino acid substitution 214G compared to the most closely related variants OXA-181 (214R) and OXA-232 (214S). The bla OXA - 484 was carried on a self-transmissible 51.5 kb IncX3 plasmid (pOXA-484) showing high sequence similarity with plasmids harboring bla OXA - 181 . Intraspecies and intergenus HGT of pOXA-484 to different recipients occurred at low frequencies of 1.4 × 10 -7 to 2.1 × 10 -6 . OXA-484 increased MICs of temocillin and carbapenems similar to OXA-232 and OXA-244, but lower compared with OXA-48 and OXA-181. Hence, OXA-484 combines properties of OXA-181-like plasmid support and transferability as well as β-lactamase activity of OXA-232., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Sommer, Gerbracht, Krause, Wild, Tietgen, Riedel-Christ, Sattler, Hamprecht, Kempf and Göttig.)

Candida auris is an emerging pathogen with resistance to many commonly used antifungal agents. Infections with C. auris require rapid and reliable detection methods to initiate successful medical treatment and contain hospital outbreaks. Conventional identification methods are prone to errors and can lead to misidentifications. PCR-based assays, in turn, can provide reliable results with low turnaround times. However, only limited data are available on the performance of commercially available assays for C. auris detection. In the present study, the two commercially available PCR assays Auris ID (OLM, Newcastle Upon Tyne, UK) and Fungiplex Candida Auris RUO Real-Time PCR (Bruker, Bremen, Germany) were challenged with 29 C. auris isolates from all five clades and eight other Candida species as controls. Auris ID reliably detected C. auris with a limit of detection (LoD) of 1 genome copies/reaction. However, false positive results were obtained with high DNA amounts of the closely related species C. haemulonii, C. duobushaemulonii and C. pseudohaemulonii . The Fungiplex Candida Auris RUO Real-Time PCR kit detected C. auris with an LoD of 9 copies/reaction. No false positive results were obtained with this assay. In addition, C. auris could also be detected in human blood samples spiked with pure fungal cultures by both kits. In summary, both kits could detect C. auris -DNA at low DNA concentrations but differed slightly in their limits of detection and specificity.

Introduction. Carbapenemase-producing Enterobacterales (CPE) are an increasing threat to global health. Fast detection is crucial for patient management and outbreak control. Hypothesis/Gap statement. Recently, a new commercial colorimetric test, CARBA PAcE, was released that has not yet been scientifically evaluated. Aim. Our goals were to evaluate the performance of CARBA PAcE using a large variety of different CPE. Methodology. CARBA PAcE was challenged with 107 molecularly characterized CPE and 53 non-CPE controls. Isolates were grown on Mueller-Hinton agar (MHA); in the case of a false-negative result, isolates were additionally inoculated on Columbia blood agar (CBA) and CARBA PAcE was repeated. The test was performed according to the manufacturer's protocol. Results. CARBA PAcE showed an overall sensitivity and specificity of 72 % [confidence interval (CI) 62-80 %] and 91 % (CI 79-97 %), respectively, when isolates were grown on MHA. With growth on CBA, detection improved (especially of metallo-β-lactamases), resulting in an extrapolated sensitivity of 89 % (CI 81-94 %) for all carbapenemases and 96 % (CI 89-99 %) for the four major carbapenemases (NDM, OXA-48-like, KPC, VIM). Conclusion. CARBA PAcE is a simple and very rapid test for the detection of CPE which performs well for the major carbapenemases when isolates are grown on CBA. Laboratories should be aware of the limitations of this assay, such as moderate sensitivity when isolates are grown on more challenging agars such as MHA and the poor detection of some rare carbapenemases (e.g. IMI, OXA-58).

Background and Aims: Transoral incisionless fundoplication (TIF) is an effective endoscopic treatment for refractory GERD with small or absent hiatal hernia (< 2 cm in length and width). The single-session laparoscopic hernia repair followed by transoral incisionless fundoplication (HH + TIF) aims to repair mechanical defects in the lower esophageal sphincter that leads to GERD in patients with hiatal hernias ≥ 2 cm. The procedure effectively treats GERD without causing added post-surgical dysphagia and gas bloating commonly associated with partial laparoscopic fundoplication. We aimed to assess patient satisfaction, symptom resolution, safety, and proton pump inhibitor use following the HH + TIF procedure., Methods: Thirty-three patients underwent single-session laparoscopic hernia repair with intraoperative TIF using the EsophyX Z device (EndoGastric Solutions, Inc.) between June 2015 and June 2018. The presence of GERD and normal esophageal motility were confirmed with pH testing and manometry prior to the procedure. Data were collected on pre- and post-procedure symptoms, patient satisfaction, PPI use, and complications. Median post-procedure follow-up with symptom surveys was 9 months (11-29 months)., Results: Patients reported significant decreases in common GERD symptoms including heartburn, regurgitation, cough, and hoarse voice. Eighty-one percent (27/33) of patients were off daily PPIs. Ninety-four percent (31/33) of patients reported 75% or greater satisfaction with the procedure and outcomes. One patient had a superficial mucosal laceration after the procedure, likely due to vomiting, which was treated conservatively., Conclusions: The majority of patients reported 75% or greater satisfaction with the procedure and had an improvement in GERD symptoms as well as decreased PPI use. There were no serious adverse events.

Substrate-dependent gliding motility is key to malaria transmission. It mediates host cell traversal, invasion and infection by Plasmodium and related apicomplexan parasites. The 110 amino acid-long cell surface protein LIMP is essential for P. berghei sporozoites where it is required for the invasion of the mosquito's salivary glands and the liver cells of the rodent host. Here we define an additional role for LIMP during mosquito invasion by the ookinete. limp mRNA is provided as a translationally repressed mRNP (messenger ribonucleoprotein) by the female gametocyte and the protein translated in the ookinete. Parasites depleted of limp (Δlimp) develop ookinetes with apparent normal morphology and no defect during in vitro gliding motility, and yet display a pronounced reduction in oocyst numbers; compared to wildtype 82 % more Δlimp ookinetes remain within the mosquito blood meal explaining the decrease in oocysts. As in the sporozoite, LIMP exerts a profound role on ookinete infection of the mosquito., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Inserted (I) domains function as ligand-binding domains in adhesins that support cell adhesion and migration in many eukaryotic phyla. These adhesins include integrin αβ heterodimers in metazoans and single subunit transmembrane proteins in apicomplexans such as TRAP in Plasmodium and MIC2 in Toxoplasma . Here we show that the I domain of TRAP is essential for sporozoite gliding motility, mosquito salivary gland invasion and mouse infection. Its replacement with the I domain from Toxoplasma MIC2 fully restores tissue invasion and parasite transmission, while replacement with the aX I domain from human integrins still partially restores liver infection. Mutations around the ligand binding site allowed salivary gland invasion but led to inefficient transmission to the rodent host. These results suggest that apicomplexan parasites appropriated polyspecific I domains in part for their ability to engage with multiple ligands and to provide traction for emigration into diverse organs in distant phyla., Competing Interests: DK, SG, JK, JS, LS, MS, CL, TS, FF No competing interests declared, (© 2020, Klug et al.)

Background: Although pain treatment is an important objective in prehospital emergency medicine the incidence of oligoanalgesia is still high in prehospital patients. Given that prehospital emergency medicine in Germany is open for physicians of any speciality, the prehospital pain treatment may differ depending on the primary medical education. Aim of this study was to explore the difference in pain treatment between surgeons and anaesthesiologists in a physician staffed emergency medical service., Methods: Retrospective single centre cohort analysis in a physician staffed ground based emergency medical service from January 2014 until December 2016. A total of 8882 consecutive emergency missions were screened. Primary outcome measure was the difference in application frequency of prehospital analgesics by anaesthesiologist or surgeon. Univariate and multivariate logistic regression analysis was used for statistical analysis including subgroup analysis for trauma and acute coronary syndrome., Results: A total of 8238 patients were included in the analysis. There was a significant difference in the application frequency of analgesics between surgeons and anaesthesiologists especially for opioids (p < 0.001, OR 0.68 [0.56-0.82]). Fentanyl was the most common administered analgesic in the trauma subgroup, but significantly less common used by surgeons (p = 0.005, OR 0.63 [0.46-0.87]). In acute coronary syndrome cases there was no significant difference in morphine administration between anaesthesiologists and surgeons (p = 0.49, OR 0.88 [0.61-1.27])., Conclusions: Increased training for prehospital pain treatment should be implemented, since opioids were administered notably less frequent by surgeons than by anaesthesiologists.

Patients with chronic immune-mediated arthritis exhibit abnormal hypothalamo-pituitary-adrenal (HPA) axis activity. The basis for this abnormality is not known. Immune-mediated arthritis is associated with increased extra-adrenal synthesis of active glucocorticoids by the 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) enzyme. 11β-HSD1 is expressed in the central nervous system, including regions involved in HPA axis regulation. We examined whether altered 11β-HSD1 expression within these regions contributes to HPA axis dysregulation during arthritis. The expression of 11β-HSD1, and other components of glucocorticoid signaling, were examined in various brain regions and the pituitary gland of mice with experimentally induced arthritis. Two arthritis protocols were employed: The K/BxN spontaneous arthritis model for chronic arthritis and the K/BxN serum transfer arthritis model for acute arthritis. 11β-HSD1 mRNA ( Hsd11b1 ) was expressed in the hippocampus, hypothalamus, cortex, cerebellum and pituitary gland. Hypothalamic Hsd11b1 expression did not change in response to arthritis in either model. Pituitary Hsd11b1 expression was however significantly increased in both chronic and acute arthritis models. Hippocampal Hsd11b1 was decreased in acute but not chronic arthritis. Chronic, but not acute, arthritis was associated with a reduction in hypothalamic corticotropin-releasing hormone and arginine vasopressin expression. In both models, serum adrenocorticotropic hormone and corticosterone levels were no different from non-inflammatory controls. These findings demonstrate inflammation-dependent regulation of Hsd11b1 expression in the pituitary gland and hippocampus. The upregulation of 11β-HSD1 expression in the pituitary during both chronic and acute arthritis, and thus, an increase in glucocorticoid negative feedback, could contribute to the abnormalities in HPA axis activity seen in immune-mediated arthritis., (© 2018 The authors.)

Background: Neurocysticercosis (NCC), the central nervous system infection by Taenia solium larvae, is a preventable and treatable cause of epilepsy. In Sub-Saharan Africa, the role of NCC in epilepsy differs geographically and, overall, is poorly defined. We aimed at contributing specific, first data for Rwanda, assessing factors associated with NCC, and evaluating a real-time PCR assay to diagnose NCC in cerebrospinal fluid (CSF)., Methodology/principal Findings: At three healthcare facilities in southern Rwanda, 215 people with epilepsy (PWE) and 51 controls were clinically examined, interviewed, and tested by immunoblot for cysticerci-specific serum antibodies. Additionally, CSF samples from PWE were tested for anticysticercal antibodies by ELISA and for parasite DNA by PCR. Cranial computer tomography (CT) scans were available for 12.1% of PWE with additional symptoms suggestive of NCC. The Del Brutto criteria were applied for NCC diagnosis. Cysticerci-specific serum antibodies were found in 21.8% of PWE and 4% of controls (odds ratio (OR), 6.69; 95% confidence interval (95%CI), 1.6-58.7). Seropositivity was associated with age and lack of safe drinking water. Fifty (23.3%) PWE were considered NCC cases (definitive, based on CT scans, 7.4%; probable, mainly based on positive immunoblots, 15.8%). In CSF samples from NCC cases, anticysticercal antibodies were detected in 10% (definitive cases, 25%) and parasite DNA in 16% (definitive cases, 44%). Immunoblot-positive PWE were older (medians, 30 vs. 22 years), more frequently had late-onset epilepsy (at age >25 years; 43.5% vs. 8.5%; OR, 8.30; 95%CI, 3.5-20.0), and suffered from significantly fewer episodes of seizures in the preceding six months than immunoblot-negative PWE., Conclusions/significance: NCC is present and contributes to epilepsy in southern Rwanda. Systematic investigations into porcine and human cysticercosis as well as health education and hygiene measures for T. solium control are needed. PCR might provide an additional, highly specific tool in NCC diagnosis.

The combination of an oxidation and a reduction in a cascade allows performing transformations in a very economic and efficient fashion. The challenge is how to combine an oxidation with a reduction in one pot, either by running the two reactions simultaneously or in a stepwise fashion without isolation of intermediates. The broader availability of various redox enzymes nowadays has triggered the recent investigation of various oxidation-reduction cascades., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Chronic disease management requires a clear vision, collaboration, and continuing program development to be successful. The patient benefits from an approach that is patient-centered and goal directed. Our challenge in this environment of declining healthcare reimbursement is to stay the course and continue measurement of our outcomes to determine our success in meeting our goals.

Lipoprotein(a) [Lp(a)] consists of LDL and the glycoprotein apolipoprotein(a) [apo(a)], which are covalently linked via a single disulfide bridge. The formation of Lp(a) occurs extracellularly, but an intracellular assembly in human liver cells has also been claimed. The human apo(a) gene locus is highly polymorphic due to a variable number of tandemly arranged kringle IV repeats. The size of apo(a) isoforms correlates inversely with Lp(a) plasma concentrations, which is believed to reflect different synthesis rates. To examine this association at the cellular level, we analyzed the subcellular localization and fate of apo(a) in stably transfected HepG2 cells. Our results demonstrate that apo(a) is synthesized as a precursor with a lower molecular mass which is processed into the mature, secreted form. The retention times of the precursor in the ER positively correlated with the sizes of apo(a) isoforms. The mature form was observed intracellularly at low levels and only in the Golgi apparatus. No apo(a) was found to be associated with the plasma membrane. Under temperature-blocking conditions, we did not detect any apo(a)/apoB-100 complexes within cells. This finding was confirmed in HepG2 cells transiently expressing KDEL-tagged apo(a). The precursor and the mature forms of apo(a) were found in the ER and Golgi fractions, respectively, also in human liver tissue. From our data, we conclude that in HepG2 cells the apo(a) precursor, dependent on the apo(a) isoform, is retained in the ER for a prolonged period of time, possibly due to an extensive maturation process of this large protein. The assembly of Lp(a) takes place exclusively extracellularly following the separate secretion of apo(a) and apoB.

Histamine assays in gastroduodenal tissues and body fluids are not an absolute objective of scientific interest but are related to the role of histamine in health and disease. Hence, the reliability of histamine assays has to be assessed in relation to this aim. Sensitivity and specificity of the chemical histamine assays are similar in tissues and body fluids. The modern developments in a fluorometric-fluoroenzymatic assay guarantee the highest sensitivity and specificity, especially by tests that monitor specificity in each single run of histamine determinations. Precision and accuracy of histamine measurement were especially investigated for the fluorometric assay. They included tests on the coefficient of variation over the whole concentration range, long-term precision with double-sample standard control charts, comparison of several methods for histamine assay including bioassay, and long-term accuracy with the use of Cusum charts. Finally, appropriate sample preparation, sample-taking, relevant body fluids and tissues, and the right time for sample-taking were evaluated in extended methodologic studies. Histamine assays are not just methods for a normal routine laboratory. Extended knowledge about histamine release and metabolism will be necessary to analyse data in this particular field with reasonable validity.

Seventy-five women who had undergone augmentation mammaplasty responded to a questionnaire that obtained their perceptions of five areas of their personal and relationship functioning: (1) body and self-image, (2) attractiveness, (3) sensual sensitivity of breasts, (4) sexual life, and (5) relationship with partner. The women's perceptions of these areas before and after surgery were obtained retrospectively (between 3 months and 3 years after surgery). The 54 women in consistent relationships reported positive effects of the surgery on their relationship, although not to the extent that they had anticipated. However, regardless of this finding, these women perceived surgery to have had significant positive effects on their attractiveness, as well as on their body and self-image. Postoperatively, the partners of these women were viewed as having a significantly greater interest in sexual activity, as perceiving the women to be significantly more attractive, and as believing that the sexual relationship was significantly enhanced. The 21 women who were not in a consistent relationship also reported positive postoperative changes, although these were not statistically significant. Neither the women's age, length of time since surgery, nor the duration of the woman's relationship had any effect on the positive changes reported. The quantitative and qualitative data underscored the highly positive benefits of breast augmentation for the respondents.

The gangliosides GLac1, GGtri1 and GGtet1 in aqueous medium sediment in the ultracentrifuge as oligomeric aggregates independent of concentration at 10(-3)M to 10(-9)M with the sedimentation constants S20,W of 5.3, 7.5 and 10.0, respectively. If gangliosides dissolved in dimethylsulfoxide were diluted with water, this resulted in aggregates with lower sedimentation coefficients. In the presence of 1M tetramethylurea no sedimentation of gangliosides could be observed in the ultracentrifuge. A ganglioside analogue (gangliosidoide) with two hydrocarbon chains instead of the ceramide moiety shows a similar sedimentation behaviour as the native compound GGtet1. However, a ganglioside analogue with only one aliphatic hydrocarbon chain forms sedimenting aggregates only at concentrations exceeding 10(-3)M.

Binding parameters for the interaction of cholera toxin and choleragenoid with monosialo-gangliotetraose, the oligosaccharide moiety of ganglioside II3 NeuAc-GgOse4-Cer, have been measured by equilibrium displacement dialysis. The experimental data were evaluated by a curve fitting computer program. The binding curves obtained at 6 degrees C reflected positive cooperativity with average Hill coefficients in the range of 1.16 to 1.25. The cholera toxin as well as its ganglioside-binding protomer B-protein bind 4 mol of monosialogangliotetraitol per mol of protein. The presence of disulfide-cleaving agents like 2-mercaptoethanol did not eliminate the cooperativity of the binding characteristics, but increased the Hill coefficient.

Psychologists estimated "true IQs" or "effective intelligence" from WISC profiles that varied for ethnicity (black, Mexican-American, or white), social class (lower or middle), profile (three scatter patterns), and direction of Verbal-Performance Scale discrepancy. Psychologists gave higher IQ estimates to black and Mexican-American children's profiles than to the same profiles of white children. Social class was not a significant factor. Profiles with much scatter received higher IQs than profiles with limited scatter. The pattern of subtest scores also affected estimates, while the direction of the Verbal-Performance discrepancy was not significant. Finally, the WISC was judged to be more valid for white than for black and Mexican-American children. Explanations of the findings were discussed.

The two choleragen protein constituents were isolated and characterized. Protein I has a molecular weight of approximately 54000. It consists of subunits of approximate molecular weight 10000. Protein II with molecular weight of approximately 32000 is cleaved by 2-mercaptoethanol into two fragments, protein II1 (N-terminal Asx, Mr = 25000) and protein II2 (N-terminal Ser, Mr = 7000). Proteins II1 and II2 could be recombined by oxidation to yield protein II. Upon treatment of choleragem with 2-mercaptoethanol protein II1 precipitates quantitatively. The remaining protein consisting of proteins I and II2, was quantitatively precipitated by ganglioside GGtet1. Of the separated choleragen subunit proteins, only protein I and not protein II complexed specifically with ganglioside GGtet1. The isolated proteins I and II were considerably less toxic in the skin test but almost full toxicity was recovered after mixing the two proteins I and II. Antisera against protein I and protein II revealed no immuno-cross reactivity between the two proteins. Both antisera inhibited the biological effects of choleragen in the skin and ileal loop tests. A molecular model for the constitution of choleragen is proposed.

The stimulation by cholera toxin of adenylate cyclase in Chinese hamster ovarian cells could be inhibited by various ligands. The latter have been shown to contain the structural oligosaccharide entities required for binding to cholera toxin, established as Galbeta1 leads to 3GalNAcbeta1 leads to 4Gal3 comes from 2alphaNeuAc. The different inhibitory potency of the ligands thereby correlates with the size of the aggregates formed with the toxin, which in turn depends on the valency of the ligands. The conclusion is drawn from a comparison of the interaction of cholera toxin and its B-protomer with ganglioside II3NeuAc-GgOse4-Cer, the newly synthesized bis-(monosialo-gangliotetraityl)amine and monosialogangliotetraose. In a double diffusion test cholera toxin B-protomer precipitated with the ganglioside II3 NeuAcGgOSE4-Cer and the divalent ligand bis(monosialo-gangliotetraityl)amine, suggesting the formation of high molecular weight aggregates, whereas no precipitation was observed with the monovalent monosialo-gangliotetraose. By ultracentrifugation analysis, aggregate formation of the cholera toxin B-protomer could be demonstrated with the ganglioside II3 NeuAc-GgOse4-Cer and bis(monosialo-gangliotetraityl)amine at a concentration at which the ganglioside was assumed to be monodisperse. Ganglioside/cholera toxin B-protomer complexes sediment faster than those of the toxin and bis(monosialo-gangliotetraityl)amine, suggesting higher aggregation of cholera toxin B-protomer with the former. On the other hand, no sedimentation with monosialo-gangliotetraose was observed. By equilibrium displacement dialysis, however, a comparable high affinity of binding to cholera toxin B-protomer of both the mono- and divalent oligosaccharides was demonstrated. Furthermore, values for the maximal concentration of the bound ligand from these binding experiments with cholera toxin B-protomer established molar ratios of ligand to protein of 4 to 1 and 2 to 1 for monosialo-gangliotetraose and bis(monosialo-gangliotetraityl)amine, respectively. From the results it is concluded that the lipophilic moiety of the ganglioside is not directly involved in the binding process to the toxin protein but leads to an oligovalency of this ligand, due to formation of micellar or submicellar structures.

Ceftriaxone and cefotaxime are third-generation cephalosporins with similar in vitro potencies and spectra. However, previous studies have shown that ceftriaxone had superior in vivo activity (mouse PD50 values greater than or equal to 2-fold lower) compared to cefotaxime in 23 of 46 tested enterobacteriaceae. This superior activity was thought to be due to ceftriaxone's 5- to 8-fold longer half-life. The relationship between half-life (ceftriax-one 6 h, cefotaxime 1 h) and potency was examined by following bacterial kill curves in a single chamber, open-ended perfusion model over an 8-hour period. Both antibiotics were compared for efficacy at both half-lives against four gram-negative bacteria. For two of the bacterial strains antibiotic potency differences in the perfusion model were determined largely by pharmacokinetics. For the other two strains intrinsic bacterial and antibiotic properties were of prime importance.

The binding between cholera toxin or its B-protein subunit and various ganglioside-related oligosaccharides was studied by equilibrium displacement dialysis. At low concentrations of ligand, the binding of monosialo-gangliotetraitol exceeded that of the parent ganglioside II3NeuAcGgOse4-Cer, the possible cell surface receptor for the toxin. The terminal galactose residue and an intact carboxyl group of the sialic acid moiety of monosialo-gangliotetraose were found to be necessary for strong binding to the toxin.

In 766 patients, 1413 polyps of the colon and the rectum were resected endoscopically. 79% of the polyps were located in the rectum and sigmoid colon. The histological examination revealed adenomatous polyps in 60%. 3.9% of the polyps showed a severe dysplasia in accordance with the WHO guidelines, and a further 3.1% already contained parts of an invasively growing carcinoma with involvement of the submucosa. Polyps in excess of 2 cm diameter had already undergone malignant transformation in 12%. However, the smallest malignant polyp was only 8 mm in size. Complications after endoscopic polypectomy occurred in 14 patients (1%); they could be controlled conservatively in 13 cases. Surgery was necessary in one case. No patient died.

Hardy, Welcher, Mellitis, and Kagan altered standard WISC administrative and scoring procedures and, from the resulting higher subtest scores, concluded that IQs based on standardized tests are inappropriate measures for inner-city children. Careful examination of their study reveals many methodological inadequacies and problematic interpretations. Three of these are as follows: (a) failure to use any external criterion to evaluate the validity of their testing-of-limits procedures; (b) the possibility of examiner and investigator bias; and (c) lack of any comparison group that might demonstrate that poor children would be helped more than others by the probes recommended. Their report creates misleading doubts about existing intelligence tests and does a disservice to inner-city children who need the benefits of the judicious use of diagnostic procedures, which include standardized intelligence tests. Consequently, their assertion concerning the inappropriateness of standardized test results for inner-city children is not only premature and misleading, but it is unwarranted as well.

The rat enteral and rabbit rectal models were utilized to study the effect of Capmul (medium chain glycerides) on the absorption of a selection of beta-lactam and aminoglycoside antibiotics. All tested non-orally available beta-lactam antibiotics (cefamandole, cefotaxime, moxalactam, cefoxitin, mezlocillin, carumonam, penicillin G and amdinocillin) showed increased absorption enterally in rats and rectally in rabbits when formulated with Capmul. The orally available beta-lactam antibiotics, cephalexin and cephradine, were not enhanced in their enteral or rectal absorption by Capmul in the two model systems. Ampicillin absorption was enhanced rectally and enterally by Capmul. Rectal absorption of the aminoglycoside antibiotics, tobramycin and gentamycin, was enhanced by Capmul while enteral absorption was not.