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The article discusses the area of technical uncertainty (ATU) in ciprofloxacin susceptibility testing for Enterobacterales using automated systems like BD Phoenix™ and VITEK® 2. The study found that Enterobacterales with a ciprofloxacin MIC of 0.5 mg/L often appeared susceptible when tested with disc diffusion (DD) or gradient strip tests (GST), despite being classified as ATU/I by automated systems. Retesting these isolates using alternative methods frequently resulted in classification as susceptible or intermediate, rather than resistant. The authors recommend retesting ATU category isolates to avoid disqualifying effective treatment options, but caution that regional differences and antibiotic coverage changes may impact these findings. [Extracted from the article]

Background: The Netherlands is currently considered a low endemic country for carbapenem-resistant Enterobacterales (CRE) and carbapenemase-producing Enterobacterales (CPE), experiencing only sporadic hospital outbreaks. This study aims to describe susceptibility to carbapenems and the epidemiology of carbapenemase production in Enterobacterales in the Netherlands in 2017–2019. Methods: Three complementary nationwide surveillance systems are in place to monitor carbapenem susceptibility in the Netherlands. Routine antimicrobial susceptibility test results from medical microbiology laboratories were used to study phenotypic susceptibility of Escherichia coli and Klebsiella pneumoniae. Pathogen surveillance (of all Enterobacterales species) and mandatory notifications were used to describe the characteristics of CPE positive isolates and affected persons. Results: The prevalence of isolates with gradient strip test-confirmed elevated meropenem (> 0.25 mg/L) or imipenem (> 1 mg/L) minimum inhibitory concentration (MIC) in the Netherlands was very low in 2017–2019, with percentages of 0.06% in E. coli and 0.49% in K. pneumoniae, and carbapenem resistances of 0.02% and 0.18%, respectively. A total of 895 unique species/carbapenemase-encoding allele combinations of CPE from 764 persons were submitted between 2017 and 2019, with the annual number of submissions increasing slightly each year. Epidemiological data was available for 660 persons. Screening because of presumed colonisation risk was the reason for sampling in 70.0% (462/660) of persons. Hospitalization abroad was the most common risk factor, being identified in 45.9% of persons. Conclusions: Carbapenem resistance of E. coli and K. pneumoniae remains low in the Netherlands. The annual number of CPE isolates slightly increased during the period 2017–2019. Recent hospitalization abroad is the main risk factor for acquisition of CPE. [ABSTRACT FROM AUTHOR]

The Staphylococcus aureus immune evasion cluster (IEC), located on β-haemolysin-converting bacteriophages (βC-Φs), encodes the immune-modulating proteins chemotaxis inhibitory protein, staphylococcal complement inhibitor (SCIN), staphylococcal enterotoxin A and staphylokinase. Its precise role in S. aureus colonization is unclear. We studied the presence of the IEC-carrying bacteriophages in human and animal S. aureus isolates, using PCR for the gene encoding SCIN (scn). Human isolates were obtained by collecting serial nasal swabs from 21 persistent carriers. S. aureus strains from 19 (90%) persistent carriers contained an IEC that was present and indistinguishable in 95% of cases at all five sampling moments over a 3-month period. Of the 77 infectious animal strains included in the study, only 26 strains (34%) were IEC-positive. Integration of these IEC-positive strains into an amplified fragment length polymorphism genotype database showed that 24 of 53 (45%) strains were human-associated and only two of 24 (8%) were 'true' animal isolates (p <0.001). The high prevalence and stability of IEC-carrying βC-Φs in human strains suggested a role for these βC-Φs in human nasal colonization. To test this hypothesis, 23 volunteers were colonized artificially with S. aureus strain NCTC 8325-4 with or without the IEC type B-carrying βC-Φ13. Intranasal survival was monitored for 28 days after inoculation. The strain harbouring βC-Φ13 was eliminated significantly faster (median 4 days; range 1-14 days) than the strain without βC-Φ13 (median 14 days; range 2-28 days; p 0.011). In conclusion, although IEC-carrying βC-Φs are highly prevalent among human colonizing S. aureus strains, they are not essential in the first stages of S. aureus nasal colonization. [ABSTRACT FROM AUTHOR]

Carbapenemase-producing Klebsiella pneumoniae emerged as a nosocomial pathogen causing morbidity and mortality in patients. For infection prevention it is important to track the spread of K. pneumoniae and its plasmids between patients. Therefore, the major aim was to recapitulate the contents and diversity of the plasmids of genetically related K. pneumoniae strains harboring the beta-lactamase gene blaKPC-2 or blaKPC-3 to determine their dissemination in the Netherlands and the former Dutch Caribbean islands from 2014 to 2019. Next-generation sequencing was combined with long-read third-generation sequencing to reconstruct 22 plasmids. wgMLST revealed five genetic clusters comprised of K. pneumoniae blaKPC-2 isolates and four clusters consisted of blaKPC-3 isolates. KpnCluster-019 blaKPC-2 isolates were found both in the Netherlands and the Caribbean islands, while blaKPC-3 cluster isolates only in the Netherlands. Each K. pneumoniae blaKPC-2 or blaKPC-3 cluster was characterized by a distinct resistome and plasmidome. However, the large and medium plasmids contained a variety of antibiotic resistance genes, conjugation machinery, cation transport systems, transposons, toxin/antitoxins, insertion sequences and prophage-related elements. The small plasmids carried genes implicated in virulence. Thus, implementing long-read plasmid sequencing analysis for K. pneumoniae surveillance provided important insights in the transmission of a KpnCluster-019 blaKPC-2 strain between the Netherlands and the Caribbean. [ABSTRACT FROM AUTHOR]

Objectives: The study institute was challenged with an outbreak of different vancomycin-resistant Enterococcus faecium (VREfm), including vanA- and/or vanB-containing isolates. Remarkably, screening overnight enriched specimens using a vanA and vanB real-time polymerase chain reaction (PCR) gave positive results for vanB with very low cycle threshold values, whereas VREfm-specific enrichment cultures remained negative. This paper describes the analysis of the diagnostic results leading to adaptation of the diagnostic algorithm., Methods: The results of vanA and vanB screening PCR and VREfm-specific culture (Brilliance VRE) were collected and combined with genotyping data of the identified VREfm isolates. During the outbreak, a second VREfm-specific culture medium (CHROMagar VRE) was introduced, and the results were compared with the results obtained with Brilliance VRE agar., Results: Thirty-five patients were identified as VREfm carriers, in which four different strains were identified: vanA (STnew-CT7088) and/or vanB (ST80-CT1065, ST117-CT7117 and ST117-CT7118). Complementing the PCR results, culture and genotyping revealed that culture with Brilliance VRE agar was inadequate for detection of the vanB ST117 isolates identified, irrespective of the minimum inhibitory concentration of vancomycin. In contrast, CHROMagar VRE was able to detect the vanB ST117 isolates and other tested isolates correctly., Conclusions: The vanB ST117 isolates were detected inadequately by the VREfm-specific culture media, possibly contributing to unnoticed spread of VREfm. For this reason, CHROMagar VRE was evaluated during the outbreak and subsequently implemented in routine diagnostics, replacing Brilliance VRE agar., Competing Interests: Conflict of interest statement None declared., (Copyright © 2024 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.)

Background: Quantifying morphologic changes is critical to our understanding of the pathophysiology of the lung. Mean linear intercept (MLI) measures are important in the assessment of clinically relevant pathology, such as emphysema. However, qualitative measures are prone to error and bias, while quantitative methods such as mean linear intercept (MLI) are manually time consuming. Furthermore, a fully automated, reliable method of assessment is nontrivial and resource-intensive., Methods: We propose a semi-automated method to quantify MLI that does not require specialized computer knowledge and uses a free, open-source image-processor (Fiji). We tested the method with a computer-generated, idealized dataset, derived an MLI usage guide, and successfully applied this method to a murine model of particulate matter (PM) exposure. Fields of randomly placed, uniform-radius circles were analyzed. Optimal numbers of chords to assess based on MLI were found via receiver-operator-characteristic (ROC)-area under the curve (AUC) analysis. Intraclass correlation coefficient (ICC) measured reliability., Results: We demonstrate high accuracy (AUC ROC  > 0.8 for MLI actual  > 63.83 pixels) and excellent reliability (ICC = 0.9998, p < 0.0001). We provide a guide to optimize the number of chords to sample based on MLI. Processing time was 0.03 s/image. We showed elevated MLI in PM-exposed mice compared to PBS-exposed controls. We have also provided the macros that were used and have made an ImageJ plugin available free for academic research use at https://med.nyu.edu/nolanlab., Conclusions: Our semi-automated method is reliable, equally fast as fully automated methods, and uses free, open-source software. Additionally, we quantified the optimal number of chords that should be measured per lung field.

Background: A substantial group of patients visit the emergency department (ED) with complaints of urinary tract infections (UTI). Treatment advice is based on national and local public health surveillance data. It is unclear whether this advice is adequate for hospitals with selected patient populations, such as university hospitals., Methods: We performed a retrospective study on patients visiting the ED of the Erasmus University Medical Center (Erasmus MC) in the Netherlands from January 1st, 2013 until December 31st, 2014 with a suspected complicated UTI (cUTI) and positive urinary cultures. Patient data, data concerning the ED visit and microbiological data were analysed., Results: 439 patients visited the ED, of whom 429 had a cUTI. Our results were compared with NethMap data. Distribution of uropathogens was comparable with the overall distribution in the Netherlands. Antibiotic susceptibility was comparable for intravenous antibiotics, but was lower for oral antibiotics. Susceptibility for empiric antibiotic therapy (i.e., cefuroxime and gentamyicin) was 96.2%. Pathogens differed from the index culture in 56.2% 104/185) of the urinary cultures available from the previous year. Using logistic regression, we found that a shorter time between last admission to the initiated antibiotic regimen was associated with lower susceptibility of cultured uropathogens., Conclusion: The distribution and antibiotic susceptibility of uropathogens for intravenous antibiotics in a Dutch university hospital is comparable with overall distribution in the Netherlands. Empiric antibiotic therapy in our local guideline appears to be an adequate antibiotic regimen for cUTI and we therefore recommend treating patients accordingly. Extension of the chosen regimen based on earlier cultured pathogens is advised, and narrowing of the antibiotic regimen strongly discouraged.

An odontogenic cause of a lung abscess is often overlooked. A 61-year-old man presented at an emergency department with a productive cough and dyspnoea. He was admitted to the pulmonary ward with a suspected odontogenic lung abscess. A thorax CT scan confirmed the diagnosis 'lung abscess', following which an oral-maxillofacial surgeon confirmed that the lung abscess probably had an odontogenic cause. The patient made a full recovery following a 6-week course of antibiotics, and his teeth were remediated by means of full extraction and the fabrication of immediate dentures. Poor oral hygiene can be a cause of a lung abscess. A patient with a lung abscess can be treated successfully with antibiotics. If, however, the odontogenic cause is not recognised the abscess can recur.

Background: An odontogenic cause of a lung abscess can easily be overlooked., Case Description: A 61-year-old man presented at the emergency department with a productive cough and dyspnoea. He was admitted to the pulmonary ward with a suspected odontogenic lung abscess. A thorax CT scan confirmed the diagnosis 'lung abscess', following which the dental surgeon confirmed that the lung abscess probably had an odontogenic cause. The patient made a full recovery following a 6-week course of antibiotics, and he received extensive dental treatment., Conclusion: Poor oral hygiene can be a cause of a lung abscess. A patient with a lung abscess can be treated successfully with a 6-week course of antibiotics; however, if the odontogenic cause is not recognised the abscess can recur.

Melarsoprol is the only currently available drug for treatment of the late stage of African trypanosomiasis (sleeping sickness). Unfortunately, the arsenic-containing drug causes serious side effects, for which the mechanisms have not been elucidated so far. This investigation describes the study of the melarsoprol biotransformation processes by electrochemical (EC) techniques. Based on EC, potential oxidation reactions of melarsoprol are examined. Moreover, the reactivity of melarsoprol, its metabolite melarsen oxide, and their oxidation products toward the tripeptide glutathione and the proteins hemoglobin and human serum albumin is evaluated. The combination of different analytical techniques allows the identification as well as the quantification of the biotransformation products. The hyphenation of liquid chromatography (LC) and electrospray ionization mass spectrometry (ESI-MS) is applied for identification and structure elucidation, which implies the determination of exact masses and fragmentation patterns. For the selective detection of arsenic containing metabolites, LC coupled to inductively coupled plasma mass spectrometry is utilized. Based on the obtained data, the oxidative biotransformation of melarsoprol can be predicted, revealing novel species which have been suspected, but not been identified up to now. The results of the protein studies prove that melarsen oxide, the active derivative of melarsoprol, strongly binds to human hemoglobin and forms different adducts via the free cysteinyl groups of the hemoglobin α- and β-chain.

Typing of methicillin-resistant Staphylococcus aureus (MRSA) remains necessary in order to assess whether transmission of MRSA occurred and to what extent infection prevention measures need to be taken. Raman spectroscopy (SpectraCellRA [SCRA]; RiverD International, Rotterdam, The Netherlands) is a recently developed tool for bacterial typing. In this study, the performance (typeability, discriminatory power, reproducibility, workflow, and costs) of the SCRA system was evaluated for typing of MRSA strains isolated from patients and patients' household members who were infected with or colonized by MRSA. We analyzed a well-documented collection of 113 MRSA strains collected from 54 households. The epidemiological relationship between the MRSA strains within one household was used as the gold standard. Pulsed-field gel electrophoresis (PFGE) was used for discrepancy analysis. The results of SCRA analysis on the strain level corresponded with epidemiological data for 108 of 113 strains, a concordance of 95.6%. When analyzed at the household level, the results of SCRA were correct for 49 out of 54 households, a concordance of 90.7%. Concordance on the strain level with epidemiological data for PFGE was 93.6% (103/110 isolates typed). Concordance on the household level with epidemiological data for PFGE was 93.5% (49/53 households analyzed). With PFGE regarded as the reference standard, the conclusions reached with Raman spectroscopy were identical to those reached with PFGE in 100 of 105 cases (95.2%). The reproducibility of SCRA was found to be 100%. We conclude that the SpectraCellRA system is a fast, easy-to-use, and highly reproducible typing platform for outbreak analysis that can compete with the currently used typing techniques.

Skin sensitization is initiated by the modification of proteins located in the skin. After oxidative activation, eugenol and isoeugenol have the potential to modify skin proteins and therefore cause sensitization processes. Despite their known skin sensitizing properties, they are of common use in cosmetic products. According to the European Commission regulation No. 1223/2009, animal tests have to be banned for substances intended for cosmetic use. Therefore, alternative methods of investigation need to be developed for the approval of future substances. For this reason, eugenol and isoeugenol were selected as model substances to be investigated in a purely instrumental approach comprising electrochemistry, liquid chromatography and mass spectrometry. In the present work, reactive oxidation products of eugenol and isoeugenol were electrochemically generated. Reactive quinones and quinone methides were formed. Surprisingly, eugenol and isoeugenol differ significantly in their oxidation behaviour. Isoeugenol exhibits the formation of quinones and quinone methides of an alkylated and dealkylated species, respectively, whereas eugenol shows the formation of quinoid species only after dealkylation. Reactive quinoid species could be trapped with glutathione and the protein β-lactoglobulin A. The results are comparable to the ones with conventional animal studies in literature, which attribute the adverse effects of eugenol and isoeugenol to the formation of reactive quinones or quinone methides, which are reactive intermediates, able to react with proteins. Such species were successfully generated and investigated by the use of electrochemistry coupled to mass spectrometry. Above all, the investigation of adduct formation by the additional use of liquid chromatography allowed the assessment of the mechanism of oxidation, as it might happen in the skin. Both substances were proven to be trapped by the protein β-lactoglobulin A after electrochemical oxidation. However, isoeugenol formed the larger variety of adducts compared to eugenol., (Copyright © 2012 Elsevier B.V. All rights reserved.)

During the development of new materials demonstrating biological activity, prediction and identification of reactive intermediates generated in the course of drug metabolism in the human liver is of great importance. We present a rapid and purely instrumental method for the structure elucidation of possible phase I metabolites. With electrochemical (EC) conversion adopting the oxidative function of liver-inherent enzymes and nuclear magnetic resonance (NMR) spectroscopy enabling structure elucidation, comprehensive knowledge on potential metabolites can be gained. Paracetamol (APAP) has been known to induce hepatotoxicity when exceeding therapeutic doses and was therefore selected as the test compound. The reactive metabolite N-acetyl-p-benzoquinone imine has long been proven to be responsible for the toxic side effects of APAP and can easily be generated by EC. EC coupled online to NMR is a straightforward technique for structure elucidation of reactive drug intermediates at an early stage in drug discovery.

Oxidative and potentially metabolic pathways of the five most frequently used contrast agents for magnetic resonance imaging (MRI) based on gadolinium (Gd) are examined. The oxidation of gadopentetate (Gd-DTPA) was studied with a focus on electrochemical oxidation coupled to analytical separation methods and mass spectrometric detection. Mass voltammograms generated with online electrochemistry/electrospray ionization mass spectrometry (EC/ESI-MS) gave a first overview of oxidation products. Two potential metabolites could be detected, with the major metabolite originating from an N-dealkylation (M1). Four other Gd complexes used as MRI contrast agents showed similar reactions in the EC/ESI-MS set-up. To obtain more information about the properties and the quantity of the generated products, a wide range of separation and detection techniques was applied in further experiments. Gd-DTPA and its N-dealkylation product were successfully separated by capillary electrophoresis (CE) and detected by ESI-MS and inductively coupled plasma (ICP)-MS, respectively. CE experiments indicated that the second oxidation product (M2) detected in the mass voltammogram is unstable and decomposes to M1. Employing EC/CE/ICP-MS, the quantification of the metabolites could be achieved. Under the employed conditions, 8.8% of Gd-DTPA was oxidized. Online experiments with high performance liquid chromatography (HPLC) coupled to ESI-MS confirmed the decomposition of M2. Time-resolved measurements showed a decrease of M2 and a simultaneous increase in M1 within only a few minutes, confirming the conclusion that M2 degrades to M1, while EC/LC/ICP-MS measurements provided quantitative evidence as well. The EC/MS simulation shows that a metabolic transformation should not be disregarded in further research regarding the trigger of nephrogenic systemic fibrosis (NSF), a disease exclusively observed for several hundred dialysis patients after delivery of Gd-based MRI contrast agents with linear structure. Furthermore, the used methods may allow the prediction of options for the oxidative removal of these contrast agents from wastewaters., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Historically, skin sensitization tests are typically based on in vivo animal tests. However, for substances used in cosmetic products, these tests have to be replaced according to the European Commission regulation no. 1223/2009. Modification of skin proteins by electrophilic chemicals is a key process associated with the induction of skin sensitization. The present study investigates the capabilities of a purely instrumental setup to determine the potential of commonly used non-electrophilic chemicals to cause skin sensitization by the generation of electrophilic species from the parent compound. In this work, the electrophiles were generated by the electrochemical oxidation of aniline, a basic industrial chemical which may also be released from azo dyes in cosmetics. The compound is a known sensitizer and was oxidized in an electrochemical thin-layer cell which was coupled online to electrospray ionization-mass spectrometry. The electrochemical oxidation was performed on a boron-doped diamond working electrode, which is able to generate hydroxyl radicals in aqueous solutions at high potentials. Without any pretreatment, the oxidation products were identified by electrospray ionization/time-of-flight mass spectrometry (ESI-ToF-MS) using their exact masses. A mass voltammogram was generated by plotting the obtained mass spectra against the applied potential. Oligomerization states with up to six monomeric units in different redox states of aniline were observed using this setup. This approach was extended to generate adducts between the oxidation products of aniline and the tripeptide glutathione. Two adducts were identified with this trapping experiment. Protein modification was carried out subsequently: Aniline was oxidized at a constant potential and was allowed to react with β-lactoglobulin A (β-LGA) or human serum albumin (HSA), respectively. The generated adducts were analyzed by liquid chromatography coupled to ESI-ToF-MS. For both β-LGA and HSA, aniline adducts were successfully generated and identified.

Diclofenac is a frequently prescribed drug for rheumatic diseases and muscle pain. In rare cases, it may be associated with a severe hepatotoxicity. In literature, it is discussed whether this toxicity is related to the oxidative phase I metabolism, resulting in electrophilic quinone imines, which can subsequently react with nucleophiles present in the liver in form of glutathione or proteins. In this work, electrochemistry coupled to mass spectrometry is used as a tool for the simulation of the oxidative pathway of diclofenac. Using this purely instrumental approach, diclofenac was oxidized in a thin layer cell equipped with a boron doped diamond working electrode. Sum formulae of generated oxidation products were calculated based on accurate mass measurements with deviations below 2 ppm. Quinone imines from diclofenac were detected using this approach. It could be shown for the first time that these quinone imines do not react with glutathione exclusively but also with larger molecules such as the model protein β-lactoglobulin A. A tryptic digest of the generated drug-protein adduct confirms that the protein is modified at the only free thiol-containing peptide. This simple and purely instrumental set-up offers the possibility of generating reactive metabolites of diclofenac and to assess their reactivity rapidly and easily.

Electrochemistry/liquid chromatography/mass spectrometry is a powerful complementary tool for the simulation of the oxidative metabolism of drugs and other xenobiotics.

Unlabelled: EPIDEMIC: The annual number of new MRSA isolates in the Netherlands tripled in 2002 compared with previous years. This increase was in part due to a MRSA outbreak in the Rijnmond region. The outbreak occurred in two merged hospitals and is the largest ever to occur in the Netherlands. From November 2001 till June 2003 MRSA was isolated from 381 patients and 113 hospital employees. The worst affected departments were Surgery and Internal Medicine. One MRSA strain (pulsed-field gel electroforesis (PFGE) type 16) remained initially unrecognised and was therefore able to spread unnoticed. Soon two additional epidemic MRSA strains (types 37 and 38) were discovered., Discussion: Multiple factors played a role in the extent and duration of the outbreak. Because of the delayed detection and rapid spread of MRSA type 16, the outbreak grew too large once recognised to be resolved within the available infrastructure. Investments were needed at various fields, including the infection-control service and the microbiology laboratory. Employees had to be informed and motivated, and a separate MRSA ward and OPD were provided. New MRSA outbreaks occurred, despite extensive MRSA (contact) screening among patients and employees. The numbers of isolates began falling as from the beginning of 2003.

Patients with functional or anatomic asplenia are at a significantly increased risk of overwhelming infection, particularly involving the encapsulated bacteria Streptococcus pneumoniae and Haemophilus influenzae. The risk is highest in infants and young children, but adults also have an increased risk of infection. Preventive strategies are very important and fall into three major categories: immunoprophylaxis, antibiotic prophylaxis and education. Studies have shown that many asplenic patients are unaware of their increased risk for serious infection and the appropriate health precautions that should be undertaken. In this article we emphasise the need for preventive measures in hyposplenic and asplenic patients. We discuss the value of newly developed conjugate vaccines and the need for revaccination. Finally we draw up a recommendation for the preventive management in functional and anatomical asplenic patients.