Surface enhanced Raman spectroscopy and machine learning for identification of beta-lactam antibiotics resistance gene fragment in bacterial plasmid.

BACKGROUND: Antibiotic resistance stands as a critical medical concern, notably evident in commonly prescribed beta-lactam antibiotics. The imperative need for expeditious and precise early detection methods underscores their role in facilitating timely intervention, curbing the propagation of antibiotic resistance, and enhancing patient outcomes. RESULTS: This study introduces the utilization of surface-enhanced Raman spectroscopy (SERS) in tandem with machine learning (ML) for the sensitive detection of characteristic gene fragments responsible for antibiotic resistance appearance and spreading. To make the detection procedure close to the real case, we used bacterial plasmids as starting biological objects, containing or not the characteristic gene fragment (up to 1:10 ratio), encoding beta-lactam antibiotics resistance. The plasmids were subjected to enzymatic digestion and without preliminary purification or isolation the created fragments were captured by functional SERS substrates. Based on subsequent SERS measurements, a database was created for the training and validation of ML. Method validation was performed using separately measured spectra, which did not overlap with the database used for ML training. To check the efficiency of recognising the target fragment, control experiments involved bacterial plasmids containing different resistance genes, the use of inappropriate enzymes, or the absence of plasmid. SIGNIFICANCE: SERS-ML allowed express detection of bacterial plasmids containing a characteristic gene fragment up to the 10-7 concentration of the initial plasmid, despite the complex composition of the biological sample, including the presence of interfering plasmids. Our approach offers a promising alternative to existing methods for monitoring antibiotic-resistant bacteria, characterized by its simplicity, low detection limit, and the potential for rapid and straightforward analysis.

A retrospective cohort analysis of factors influencing continuous antibiotic therapy with ampicillin.

AIMS: There are limited data on ampicillin/sulbactam, both for continuous infusion and for use in critically ill patients. We aimed to identify factors that help predict ampicillin plasma levels during continuous antibiotic therapy in intensive care patients. MAIN METHODS: We retrospectively reviewed and retrieved a large dataset of patients who received continuous ampicillin infusion with therapeutic drug monitoring between 2015 and 2022. Patients initially received standard dosing (single shot of 2/1 g followed by continuous infusion of 6/3 g ampicillin/sulbactam per day), which was then adjusted based on the results of regular therapeutic drug monitoring and according to a target range of 30-60 mg/l (equivalent to four to eight times the minimum inhibitory concentration of ampicillin for Enterobacterales). MAIN RESULTS: 466 measurements from 225 patients (152 male, mean age 61 years) were analyzed. Initial measurements of ampicillin plasma levels were below the predefined optimal therapeutic range in 50 %, within the range in 30 % and above the range in 20 %. Target attainment increased to 70 % by the 4th measurement. There was a significant negative correlation between ampicillin plasma levels and estimated glomerular filtration rate (eGFR) (r = -0.74; p < 0.001) and, to a lesser extent, with height (r = -0.31; p < 0.001). Based on multiple linear regression, eGFR and body weight or height were the factors accounting for 63 % of the variability in the data. SIGNIFICANCE: To optimise target achievement, ampicillin dosing in critically ill patients requires a personalized approach based on renal function. Importantly, patients with normal or augmented eGFR require higher standard doses of ampicillin/sulbactam.

National Survey and consensus document on dosing strategies for beta-lactam antibiotics against multidrug-resistant gram-negative bacilli (MDR-GNB) in critically ill patients undergoing extracorporeal life support techniques: The DOSEBL study protocol. / Encuesta nacional y elaboración de un documento de consenso sobre estrategias de dosificación de antibióticos beta-lactámicos frente a bacterias gramnegativas multirresistentes (BGN-MDR) en pacientes críticos que son tratados con técnicas de soporte vital extracorpóreas: protocolo de estudio DOSEBL.

INTRODUCTION: Infections caused by multidrug-resistant gram-negative bacilli (MDR-GNB) in critically ill patients present a challenge for timely and appropriate antibiotic treatment. This is particularly important in patients undergoing extracorporeal life support techniques such as renal replacement therapy and extracorporeal membrane oxygenation. These techniques can introduce additional pharmacokinetic alterations, potentially leading to suboptimal exposure to antibiotics. This study aims to outline dosing strategies and therapeutic drug monitoring protocols for new ß-lactam antibiotics effective against MDR-GNB in critically ill patients undergoing extracorporeal life support techniques at a national level. Additionally, the study seeks to develop a consensus document, based on available evidence. METHODS: The project will comprise two main phases: I) A national survey, and II) the development of a consensus document. This consensus document, undertaken according to ACCORD guidelines, will encompass: a) establishment of a multidisciplinary panel of experts, b) prospective registration of the consensus, c) evidence synthesis, d) modified Delphi rounds. The antimicrobials to be included will be: meropenem, ceftazidime/avibactam, ceftolozane/tazobactam, cefiderocol, meropenem/vaborbactam, imipenem/relebactam, and aztreonam. Extracorporeal life support techniques will include continuous renal replacement therapy, conventional intermittent hemodialysis, and extracorporeal membrane oxygenation. DISCUSSION: The availability of extracorporeal life support techniques has expanded significantly in recent years, alongside a rise in the prevalence of infections caused by multidrug-resistant gram-negative bacilli (MDR-GNB). There is a need to develop evidence-based tools of high quality to standardize dosing and monitoring strategies for new ß-lactam antibiotics.

Penicillin binding proteins-based immunoassay for the selective and quantitative determination of beta-lactam antibiotics.

An immunoassay method based on penicillin-binding protein (PBP) was developed for the quantitative determination of 10 kinds of beta-lactam antibiotics (BLAs). First, two kinds of PBPs, which are named PBP1a and PBP2x, were expressed and purified, and they were characterized by SDS-PAGE and western blotting analysis. Then, the binding activity of PBP1a and PBP2x to template BLAs, cefquinome (CEFQ) and ampicillin (AMP), was determined. The effect of the buffer solution system, e.g., pH, ion concentration, and organic solvent, on the immune interaction efficiency between PBPs and BLAs was also evaluated. In the end, the PBP-based immunoassay method was developed and validated for the detection of 10 kinds of BLAs. Under optimal conditions, PBPs exhibited high binding affinity to BLAs. In addition, this method showed a high sensitivity for the detection of 10 kinds of BLAs with the limits of detection from 0.21 to 9.12 ng/mL, which are much lower than their corresponding maximum residual limit of European Union (4-100 ng/mL). Moreover, the developed PBP-immunoassay was employed for BLA detection from milk samples, and satisfactory recoveries (68.9-101.3 %) were obtained.

Unraveling the impact of therapeutic drug monitoring via machine learning for patients with sepsis.

Clinical studies investigating the benefits of beta-lactam therapeutic drug monitoring (TDM) among critically ill patients are hindered by small patient groups, variability between studies, patient heterogeneity, and inadequate use of TDM. Accordingly, definitive conclusions regarding the efficacy of TDM remain elusive. To address these challenges, we propose an innovative approach that leverages data-driven methods to unveil the concealed connections between therapy effectiveness and patient data, collected through a randomized controlled trial (DRKS00011159; 10th October 2016). Our findings reveal that machine learning algorithms can successfully identify informative features that distinguish between healthy and sick states. These hold promise as potential markers for disease classification and severity stratification, as well as offering a continuous and data-driven "multidimensional" Sequential Organ Failure Assessment (SOFA) score. The positive impact of TDM on patient recovery rates is demonstrated by unraveling the intricate connections between therapy effectiveness and clinically relevant data via machine learning.

Towards pharmacokinetic boosting of phenoxymethylpenicillin (penicillin-V) using probenecid for the treatment of bacterial infections.

In the face of increasing antimicrobial tolerance and resistance there is a global obligation to optimise oral antimicrobial dosing strategies including narrow spectrum penicillins, such as penicillin-V. We conducted a randomised, crossover study in healthy volunteers to characterise the influence of probenecid on penicillin-V pharmacokinetics and estimate the pharmacodynamics against Streptococcus pneumoniae. Twenty participants took six doses of penicillin-V (250 mg, 500 mg or 750 mg four times daily) with and without probenecid. Total and free concentrations of penicillin-V and probenecid were measured at two timepoints. A pharmacokinetic model was developed, and the probability of target attainment (PTA) calculated. The mean difference (95% CI) between penicillin-V alone and in combination with probenecid for serum total and free penicillin-V concentrations was significantly different at both timepoints (total: 45 min 4.32 (3.20-5.32) mg/L p < 0.001, 180 min 2.2 (1.58-3.25) mg/L p < 0.001; free: 45 min 1.15 (0.88-1.42) mg/L p < 0.001, 180 min 0.5 (0.35-0.76) mg/L p < 0.001). There was no difference between the timepoints in probenecid concentrations. PTA analysis shows probenecid allows a fourfold increase in MIC cover. Addition of probenecid was safe and well tolerated. The data support further research into improved dosing structures for complex outpatient therapy and might also be used to address penicillin supply shortages.

Preventing New Gram-negative Resistance Through Beta-lactam De-escalation in Hospitalized Patients With Sepsis: A Retrospective Cohort Study.

BACKGROUND: Whether antibiotic de-escalation reduces the risk of subsequent antibiotic resistance is uncertain. We sought to determine if beta-lactam (BL) antibiotic de-escalation is associated with decreased incidence of new Gram-negative resistance in hospitalized patients with sepsis. METHODS: In a retrospective cohort study, patients with sepsis who were treated with at least 3 consecutive days of BL antibiotics, the first 2 days of which were with a broad-spectrum BL agent defined as a spectrum score (SS) of ≥7 were enrolled. Patients were grouped into three categories: (1) de-escalation of beta-lactam spectrum score (BLSS), (2) no change in BLSS, or (3) escalation of BLSS. The primary outcome was the isolation of a new drug-resistant Gram-negative bacteria from a clinical culture within 60 days of cohort entry. Fine-Gray proportional hazards regression modeling while accounting for in-hospital death as a competing risk was performed. FINDINGS: Six hundred forty-four patients of 7742 (8.3%) patients developed new gram-negative resistance. The mean time to resistance was 23.7 days yielding an incidence rate of 1.85 (95% confidence interval [CI]: 1.71-2.00) per 1000 patient-days. The lowest incidence rate was observed in the de-escalated group 1.42 (95% CI: 1.16-1.68) per 1000 patient-days. Statistically significant reductions in the development of new gram-negative resistance were associated with BL de-escalation compared to no-change (hazards ratio (HR) 0.59 [95% CI: .48-.73]). CONCLUSIONS: De-escalation was associated with a decreased risk of new resistance development compared to no change. This represents the largest study to date showing the utility of de-escalation in the prevention of antimicrobial resistance.

Meropenem/Vaborbactam-A Mechanistic Review for Insight into Future Development of Combinational Therapies.

Beta-lactam antibiotics have been a major climacteric in medicine for being the first bactericidal compound available for clinical use. They have continually been prescribed since their development in the 1940s, and their application has saved an immeasurable number of lives. With such immense use, the rise in antibiotic resistance has truncated the clinical efficacy of these compounds. Nevertheless, the synergism of combinational antibiotic therapy has allowed these drugs to burgeon once again. Here, the development of meropenem with vaborbactam-a recently FDA-approved beta-lactam combinational therapy-is reviewed in terms of structure rationale, activity gamut, pharmacodynamic/pharmacokinetic properties, and toxicity to provide insight into the future development of analogous therapies.

[Penicillin allergy-Truth or duty?] / Penicillinallergie ­ Wahrheit oder Pflicht?

The beta-lactam antibiotics are some of the safest and best-tolerated antibiotic agents; however, many patients have reported allergies against penicillin. All beta-lactam antibiotics are only restrictively prescribed for these patients and alternative antibiotics are increasingly given, which carries the risk of negative clinical results and socioeconomic sequelae; however, over 95% of patients who reported an allergy to penicillin show a negative result in the allergy tests for penicillin and this antibiotic can safely be prescribed. The use of sensitive and specific instruments for identification of false penicillin allergies should be an important topic within the framework of antibiotic stewardship. Anesthesists can play a central role in the reduction of the enormous individual and public health burden associated with the classification of penicillin allergy by taking an appropriate medical history and a risk stratification for the identification of patients with a penicillin allergy. This overview article presents a possible delabelling algorithm within the framework of the clarification of a beta-lactam antibiotic allergy. The focus is on a structured allergy anamnesis using the penicillin allergy, five or fewer years ago, anaphylaxis/angioedema, severe cutaneous adverse reaction (SCAR) and treatment required for allergy episode (PEN-FAST) score.

Management and Implications of Beta-Lactam Allergies.

Beta-lactam antibiotics are essential components in the current antimicrobial treatment strategy, playing a crucial role in ambulatory patients and hospitalized patients. Despite their prominent therapeutic index, the use of beta-lactam can lead to adverse effects, with allergic reactions being the most concerning because of their severity. Additionally, the phenomenon of cross-reactivity may occur among various beta-lactam families, with side chains significantly contributing to immunological recognition, making these structures often responsible for the cross-allergic reactivity of beta-lactams. Tools to assess beta-lactam allergy include taking a patient's medical history, performing skin tests, and conducting provocation tests. This research aims to analyze the relevant aspects related to the safe administration of beta-lactam antibiotics in hospitalized patients as well as provide knowledge on the proper management of patients with such hypersensitivity, by doing systemic research. This research was made using Google Scholar and keywords such as "Beta-lactam allergy," "Hypersensitivity," "Cross-reactivity," "Desensitization," and "Beta-lactam allergy management." In conclusion, substituting a beta-lactam antibiotic with an alternative antibiotic may not always be the best management option for these patients, as it may lead to more adverse effects, be less effective, and prolong hospitalization time. It may also result in higher rates of antibiotic-resistant infections and increased medical costs, as these alternatives are often more expensive. However, an alternative within the beta-lactam family can be sought by conducting the appropriate analyses. Although cross-reactivity does not always occur among all beta-lactams, potential cross-reactivity should always be considered.

The New Delhi metallo-ß-lactamase-1 biosensor rapidly and accurately detected antibiotic plasma concentrations in cefuroxime-treated patients.

OBJECTIVES: Therapeutic drug monitoring (TDM) of ß-lactam antibiotics in critically ill patients may benefit dose optimisation, thus improving therapeutic outcomes. However, rapidly and accurately detecting these antibiotics in blood remains a challenge. This research group recently developed a thermometric biosensor called the New Delhi metallo-ß-lactamase-1 (NDM-1) biosensor, which detects multiple classes of ß-lactam antibiotics in spiked plasma samples. METHODS: This study assessed the NDM-1 biosensor's effectiveness in detecting plasma concentrations of ß-lactam antibiotics in treated patients. Seven patients receiving cefuroxime were studied. Plasma samples collected pre- and post-antibiotic treatment were analysed using the NDM-1 biosensor and compared with liquid chromatography coupled with ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). RESULTS: The biosensor detected plasma samples without dilution, and a brief pre-treatment using a polyvinylidene fluoride filter significantly lowered matrix effects, reducing the running time to 5-8 minutes per sample. The assay's linear range for cefuroxime (6.25-200 mg/L) covered target concentrations during the trough phase of pharmacokinetics in critically ill patients. The pharmacokinetic properties of cefuroxime in treated patients determined by the NDM-1 biosensor and the UPLC-MS/MS were comparable, and the cefuroxime plasma concentrations measured by the two methods showed statistically good consistency. CONCLUSION: These data demonstrate that the NDM-1 biosensor assay is a fast, sensitive, and accurate method for detecting cefuroxime plasma concentrations in treated patients and highlights the NDM-1 biosensor as a promising tool for on-site TDM of ß-lactam antibiotics in critically ill patients.

The Rising Tide of Antibiotic Resistance: A Study on Extended-Spectrum Beta-Lactamase and Carbapenem-Resistant Escherichia coli and Klebsiella pneumoniae.

BACKGROUND: The global spread of extended-spectrum beta-lactamase (ESBL)-producing and carbapenem-resistant Enterobacterales (CRE) poses a significant concern. Acquisition of antimicrobial resistance genes leads to resistance against several antibiotics, limiting treatment options. We aimed to study ESBL-producing and CRE transmission in clinical settings. METHODS: From clinical samples, 227 ESBL-producing and CRE isolates were obtained. The isolates were cultured on bacterial media and confirmed by VITEK 2. Antibiograms were tested against several antibiotics using VITEK 2. The acquired resistance genes were identified by PCR. RESULTS: Of the 227 clinical isolates, 145 (63.8%) were Klebsiella pneumoniae and 82 (36.1%) were Escherichia coli; 76 (33.4%) isolates were detected in urine, 57 (25.1%) in pus swabs, and 53 (23.3%) in blood samples. A total of 58 (70.7%) ESBL-producing E. coli were resistant to beta-lactams, except for carbapenems, and 17.2% were amikacin-resistant; 29.2% of E. coli isolates were resistant to carbapenems. A total of 106 (73.1%) ESBL-producing K. pneumoniae were resistant to all beta-lactams, except for carbapenems, and 66.9% to ciprofloxacin; 38 (26.2%) K. pneumoniae were resistant to carbapenems. Colistin emerged as the most effective antibiotic against both bacterial types. Twelve (20.6%) E. coli isolates were positive for blaCTX-M, 11 (18.9%) for blaTEM, and 8 (33.3%) for blaNDM. Forty-six (52.3%) K. pneumoniae isolates had blaCTX-M, 27 (18.6%) blaTEM, and 26 (68.4%) blaNDM. CONCLUSION: This study found a high prevalence of drug-resistant ESBL-producing and CRE, highlighting the need for targeted antibiotic use to combat resistance.

Potential involvement of beta-lactamase homologous proteins in resistance to beta-lactam antibiotics in gram-negative bacteria of the ESKAPEE group.

BACKGROUND: Enzymatic degradation mediated by beta-lactamases constitutes one of the primary mechanisms of resistance to beta-lactam antibiotics in gram-negative bacteria. This enzyme family comprises four molecular classes, categorized into serine beta-lactamases (Classes A, C, and D) and zinc-dependent metallo-beta-lactamases (Class B). Gram-negative bacteria producing beta-lactamase are of significant concern, particularly due to their prevalence in nosocomial infections. A comprehensive understanding of the evolution and dissemination of this enzyme family is essential for effective control of these pathogens. In this study, we conducted the prospecting, phylogenetic analysis, and in silico analysis of beta-lactamases and homologous proteins identified in 1827 bacterial genomes with phenotypic data on beta-lactam resistance. These genomes were distributed among Klebsiella pneumoniae (45%), Acinetobacter baumannii (31%), Pseudomonas aeruginosa (14%), Escherichia coli (6%), and Enterobacter spp. (4%). Using an HMM profile and searching for conserved domains, we mined 2514, 8733, 5424, and 2957 proteins for molecular classes A, B, C, and D, respectively. This set of proteins encompasses canonical subfamilies of beta-lactamases as well as hypothetical proteins and other functional groups. Canonical beta-lactamases were found to be phylogenetically distant from hypothetical proteins, which, in turn, are closer to other representatives of the penicillin-binding-protein (PBP-like) and metallo-beta-lactamase (MBL) families. The catalytic amino acid residues characteristic of beta-lactamases were identified from the sequence alignment and revealed that motifs are less conserved in homologous groups than in beta-lactamases. After comparing the frequency of protein groups in genomes of resistant strains with those of sensitive ones applying Fisher's exact test and relative risk, it was observed that some groups of homologous proteins to classes B and C are more common in the genomes of resistant strains, particularly to carbapenems. We identified the beta-lactamase-like domain widely distributed in gram-negative species of the ESKAPEE group, which highlights its importance in the context of beta-lactam resistance. Some hypothetical homologous proteins have been shown to potentially possess promiscuous activity against beta-lactam antibiotics, however, they do not appear to expressly determine the resistance phenotype. The selective pressure due to the widespread use of antibiotics may favor the optimization of these functions for specialized resistance enzymes.

Quantification of cefuroxime and flucloxacillin in synovial tissue and bone using ultra-performance convergence chromatography-tandem mass spectrometry.

After a revision surgery, approximately 1-2 % of patients will develop a periprosthetic joint infection (PJI). During the revision surgery, the infected prosthesis is removed, a debridement is performed and a new or temporary spacer is placed. Additionally, patients are treated with antibiotics during and after the surgery. Adequate exposure of the administered antibiotic to the pathogen is of crucial importance during the treatment of any infection. Inadequately low concentrations are associated with an increase in antibiotic resistance, antibiotic related side effects, treatment failures and prolonged infections. While high concentrations may lead to serious adverse events and potential lasting damage. Despite the importance of optimal dosing, there is a lack of knowledge with respect to the correlation between the plasma concentrations and target site concentrations of the antibiotics. Two of the commonly administered antimicrobial agents during the arthroplasty exchange are cefuroxime and flucloxacillin. Therefore, an accurate, specific, and sensitive quantification method is required in order to assess pharmacokinetics of cefuroxime and flucloxacillin in synovial tissue and bone. The aim of this study is to develop and validate a quantification method for the measurement of cefuroxime and flucloxacillin in human synovial tissue and bone using the UPC2-MS/MS conform Food and Drug Administration guidelines. The method was found linear for both compounds in both matrices (r2 > 0.990) from 1 µg/g to 20 µg/g, except for cefuroxime in bone, which was validated from 1 µg/g to 15 µg/g. We developed and validated a quantification method for cefuroxime and flucloxacillin in synovial tissue and bone using a simple sample preparation and a short analysis run time of 5.0 min, which has been already successfully applied in a clinical study. To our knowledge, no methods have been described earlier for the simultaneous quantification of cefuroxime and flucloxacillin in synovial tissue and bone.

Comparing prolonged infusion to intermittent infusion strategies for beta-lactam antibiotics in patients with gram-negative bacterial infections: a systematic review and meta-analysis.

INTRODUCTION: Our objective is to determine whether prolonged infusion (PI) of beta-lactam antibiotics yields superior outcomes compared to intermittent infusion (II) in patients with Gram-Negative Bacterial (GNB) infections. METHODS: We systematically searched papers from PubMed, the Cochrane Library, Embase, and Clinicaltrials.gov, targeting mortality as the primary outcome and looking at the clinical cure rate, hospital and intensive care unit (ICU) stay lengths, antibiotic treatment duration, and mechanical ventilation (MV) duration as secondary outcomes. RESULTS: Our meta-analysis of 18 studies, including 5 randomized control trials and 13 observational studies, with a total of 3,035 patients-1,510 in the PI group and 1,525 in the II group, revealed significant findings. PI was associated with reduced mortality (RR, 0.67; 95% CI, 0.55-0.81; p = 0.001; I2 = 4.52%) and a shorter MV duration (SMD, -0.76; 95% CI, -1.37 to -0.16; p = 0.01; I2 = 87.81%) compared to II. However, no differences were found in clinical cure rates, antibiotic treatment duration, length of hospital stay, or length of ICU stay. CONCLUSIONS: The PI approach for administering beta-lactam antibiotics in patients with suspected or confirmed GNB infections may be advantageous in reducing mortality rates and the duration of MV when compared to the II strategy.

A Rare Case of Gemella haemolysans Endocarditis: A Challenging Diagnosis.

Gemella haemolysans bacterium is an opportunistic pathogen that can cause localized or systemic infections. Here we describe a rare case of infective endocarditis secondary to Gemella haemolysans infection. In our case, although the bacteremia was cleared with antibiotics, the mitral valve vegetations continued to enlarge and the course was complicated by septic brain emboli.

In silico prediction and a systematic toxicology-based in vivo investigation uncovering the mechanism of aquatic toxicity caused by beta-lactam antibiotics.

Recently, beta-lactam antibiotics have gained attention as significant contributors to public health and environmental issues due to their potential toxicity. Our study employed machine learning to develop a model for assessing the aquatic toxicity of beta-lactam antibiotics on zebrafish. Notably, aztreonam (AZT), a synthetic monobactam and a subclass of beta-lactam antibiotics, demonstrated developmental effects in zebrafish embryos comparable to cephalosporins, indicating a potential for toxicity. Using a systems toxicology-based approach, we identified apoptosis and metabolic disorders as the primary pathways affected by AZT and its impurity F exposure. During the administration of monobactams, we noted that ctsbb, nos2a, and dgat2, genes associated with apoptosis and the metabolic pathway, exhibited significant differential expression. Molecular docking studies were conducted to ascertain the binding affinity between monobactam compounds and their potential targets-Ctsbb, Nos2a, and Dgat2. Furthermore, our research revealed that monobactams influence pre-mRNA alternative splicing, resulting in disruptions in the expression of genes involved in hair cells, brain, spinal cord, and fin regeneration (e.g., krt4, krt5, krt17, cyt1). Notably, we observed a correlation between the levels of rpl3 and rps7 genes, both important ribosomal proteins, and the detected alternative splicing events. Overall, this study enhances our understanding of the toxicity of beta-lactam antibiotics in zebrafish by demonstrating the developmental effects of monobactams and uncovering the underlying mechanisms at the molecular level. It also identifies potential targets for further investigation into the mechanisms of toxicity and provides valuable insights for early assessment of biological toxicity associated with antibiotic pollutants.

EAACI/ENDA position paper on drug provocation testing.

In drug hypersensitivity, drug provocation testing (DPT), also called drug challenge, is the gold standard for investigation. In recent years, risk stratification has become an important tool for adjusting the diagnostic strategy to the perceived risk, whilst still maintaining a high level of safety for the patient. Skin tests are recommended before DPT but may be omitted in low-risk patients. The task force suggests a strict definition of such low-risk patients in children and adults. Based on experience and evidence from studies of allergy to beta-lactam antibiotics, an algorithm on how to adjust DPT to the risk, and when to omit skin tests before DPT, is presented. For other antibiotics, non-steroidal anti-inflammatory drugs and other drugs, skin tests are poorly validated and DPT is frequently necessary. We recommend performing DPT with chemotherapeutics and biologicals to avoid unnecessary desensitization procedures and DPT with skin tests negative contrast media. We suggest DPT with anesthetics only in highly specialized centers. Specifics of DPT to proton pump inhibitors, anticonvulsants and corticosteroids are discussed. This position paper provides general recommendations and guidance on optimizing use of DPT, whilst balancing benefits with patient safety and optimizing the use of the limited available resources.

Patient Adherence to Written Instructions following Complete Allergological Evaluation for Suspected Beta-Lactam Allergy: A Tertiary Hospital Study in Greece.

BACKGROUND: Beta-lactam (BL) antibiotics are among the most prescribed groups of drugs worldwide and have been implicated in a variety of allergic reactions. There is a paucity of literature regarding patient adherence to prescribed instructions following comprehensive allergy assessments. OBJECTIVE: The objective was to follow up the clinical course of BL allergy in patients who underwent thorough allergological investigation for suspected BL allergy at a tertiary hospital and ascertain patients' compliance with the provided written instructions. MATERIALS: An observational study in patients referred for suspected BL allergy who underwent a comprehensive allergy workup (in vivo ± in vitro tests, DPT in culprit and/or alternative BL) and who subsequently received written instructions was conducted. Data on the nature of the reported drug hypersensitivity reaction, the culprit BL drug, the allergological workup, and the detailed instructions provided in a written drug allergy report were collected retrospectively. Patients' compliance with the instructions was recorded by a telephone survey using a pre-defined questionnaire. RESULTS: Among the 212 patients meeting the inclusion criteria, 87 patients (72.4% women; mean age 50.1 years; age range 6-84 years) responded to the telephone survey and were included in this study. Surprisingly, 45 out of 87 (51.7%) patients did not adhere to the written instructions. The primary factor contributing to non-compliance was the fear of re-occurrence of a drug-induced allergic reaction (personal and/or triggered by their treating physician reluctance), accounting for 77.7% of cases. The analysis demonstrated that the initial reaction's severity and type, as well as the outcomes of skin testing, did not correlate with compliance to instructions (p > 0.05). Surprisingly enough, a drug provocation test (DPT), irrespectively of the result, emerged as a negative predictor for adherence, with only 40.6% of DPT patients complying compared to 77.8% of those who did not undergo DPT (p = 0.005; odds ratio = 0.195; 95% confidence interval: 0.058-0.655). Variables such as performing DPT with alternative or incriminated drugs or the result of the DPT (positive-negative) were not associated with patient compliance. Conversely, the type of instructions provided exhibited a noteworthy correlation with compliance. Patients who were explicitly instructed to entirely avoid all BL antibiotics demonstrated markedly higher adherence rates (83.3%) compared to those who were advised to have a partial or complete release of BLs (31.8% and 58.1%, respectively; p < 0.05). Notably, among compliant patients who received either the original culprit drug or the alternative (32 out of 87, 36.7%), no allergic reactions were reported. In contrast, among the 12 patients with written avoidance of all BLs, subsequent BL intake led to immediate reactions (Grade I and IV) in 2 patients (16.6%). CONCLUSIONS: A notable disparity in patient adherence to written instructions prohibiting or releasing beta-lactams was demonstrated. Less than half of the patients ultimately complied with the provided instructions, underscoring the need for tailored patients' education and strategies to improve adherence in the management of suspected BL allergy.