Combining deep learning and droplet microfluidics for rapid and label-free antimicrobial susceptibility testing of colistin.

Efficient tools for rapid antibiotic susceptibility testing (AST) are crucial for appropriate use of antibiotics, especially colistin, which is now often considered a last resort therapy with extremely drug resistant Gram-negative bacteria. Here, we developed a rapid, easy and miniaturized colistin susceptibility assay based on microfluidics, which allows for culture and high-throughput analysis of bacterial samples. Specifically, a simple microfluidic platform that can easily be operated was designed to encapsulate bacteria in nanoliter droplets and perform a fast and automated bacterial growth detection in 2 h, using standardized samples. Direct bright-field imaging of compartmentalized samples proved to be a faster and more accurate detection method as compared to fluorescence-based analysis. A deep learning powered approach was implemented for the sensitive detection of the growth of several strains in droplets. The DropDeepL AST method (Droplet and Deep learning-based method for AST) developed here allowed the determination of the colistin susceptibility profiles of 21 fast-growing Enterobacterales (E. coli and K. pneumoniae), including clinical isolates with different resistance mechanisms, showing 100 % categorical agreement with the reference broth microdilution (BMD) method performed simultaneously. Direct AST of bacteria in urine samples on chip also provided accurate results in 2 h, without the need of complex sample preparation procedures. This method can easily be implemented in clinical microbiology laboratories, and has the potential to be adapted to a variety of antibiotics, especially for last-line antibiotics to optimize treatment of patients infected with multi-drug resistant strains.

Metabolic activation of short-chain alkyl N-nitrosamines using Aroclor 1254 or phenobarbital/beta-naphthoflavone-induced rat or hamster S9 - A comparative analysis.

N-nitrosamines, a very heterogeneous class of chemicals, may enter humans in small amounts through various sources and are produced endogenously, too. Some are known to be mutagenic carcinogens and have recently been detected as impurities in several marketed pharmaceuticals. Despite their known mutagenic properties, the suitability of the bacterial reverse mutation (Ames) assay and in particular the use of induced rat liver S9 to detect their mutagenic potential, is often discussed. Recently, it could be demonstrated that induced rat liver S9 is capable of metabolizing small alkyl nitrosamines to exert their mutagenic potential (Bringezu & Simon, 2022). In this project, the mutagenic potential of nitrosamines in vitro under different S9 conditions applying the preincubation protocol and OECD 471-compliant standard Ames test recommendations was investigated. These conditions included various amounts of S9 fraction from hamster and rat, uninduced or induced with Aroclor 1254 or Phenobarbital/beta-Naphthoflavone (PB/NF). The findings indicated that in addition to induced S9, uninduced hamster S9 also demonstrated effectiveness. Moreover, both rat and hamster S9 fractions exhibited suitable responses in terms of mutation frequencies. Increasing the S9 content did not increase the sensitivity of the Ames test. However, above 20% S9, reduced mutation frequency was observed in the higher concentration range suggesting cytotoxicity to the bacteria. Thus, limiting the S9 content to 10% provides reliable results and relates to a lower number of animals required for S9 production which is in concordance with the 3R principles (reduce, refine, replace) for animal testing. In addition, results obtained show that uninduced and induced hamster S9 are similarly effective, doubting the requirement of pretreating animals with enzyme inducers. Further investigations to compare mutagenicity data and rat and hamster S9 proteome analyses are ongoing.

Revisiting the Landscape of Potential Small and Drug Substance Related Nitrosamines in Pharmaceuticals.

N-Nitrosamines are a class of indirect acting mutagens, as their metabolic degradation leads to the formation of the DNA-alkylating diazonium ion. Following up on the in-silico identification of thousands of nitrosamines that can potentially be derived from small molecule drugs and their known impurities described in a previous publication, we have now re-analyzed this dataset to apply EMA's Carcinogenic Potency Categorization Approach (CPCA) introduced with the 16th revision of their Q&A document for Marketing Authorization Holders. We find that the majority of potential nitrosamines from secondary amine precursors belongs to potency categories 4 and 5, corresponding to an acceptable daily intake of 1500 ng, whereas nitrosamines from tertiary amine precursors distribute more evenly among all categories, resulting in a substantial number of structures that are assigned the more challenging acceptable intakes of 18 ng/day and 100 ng/day for potency categories 1 and 2, respectively. However, the nitrosative dealkylation pathway for tertiary amine is generally far slower than the direct nitrosation on secondary amines, with a direct nitrosation mechanism suspected only for structures featuring electron-rich (hetero)aromatic substituents. This allows for greater focus towards those structures that require further review, and we demonstrate that their number is not substantial. In addition, we reflect on the nitrosamine risk posed by secondary amine API impurities and demonstrate that based on the ICH Q3A/B identification threshold unknown impurities may exist that could be transformed to relevant amounts of NA. We also demonstrate that the analytical sensitivity required for the quantification of high potency nitrosamines can be problematic especially for high dose APIs. In summary, the regulatory framework rolled out with the latest Q&A document represents a substantial improvement compared with the previous situation, but further refinement through interaction between manufacturers, regulators, not-for-profit and academic institutions will be required to ensure patient access to vital medicines without compromising safety.

Multititration: The New Method for Implementing Ultrasensitive and Quantitative Multiplexed In-Field Immunoassays Despite Cross-Reactivity?

The accurate in-field titration of multiple pathogens is essential to efficiently describe and monitor environmental or biological contamination, isolate, act, and treat adequately. This underscores the requirement of portable, fast, quantitative, and multiplexed detection technologies, which, however, have not been properly developed so far, notably because it has been hindered by the phenomenon of cross-reactivity. In this work, we proposed a new analytical method based on the imaging through a portable device of lanthanide-based nanoparticles (YVO4:Eu) for spatially multiplexed detection, relying on a multiparameter analysis, i.e., a simultaneous analysis of all of the luminescence signals through the comparison to a calibration surface built in the presence of multiple analytes of interest. We then demonstrated the possibility to simultaneously quantify by multiplexed lateral flow assay (xLFA) the three enterotoxins SEG, SEH, and SEI in unknown mixtures, over two concentration decades (from a dozen of pg·mL-1 to few ng·mL-1). Assays were performed in less than an hour (25 min of strip migration followed by 30 min of drying at room temperature), the time during which the presence of the operator was not required for more than 5 min, in order to dip the strip and have it imaged by the reader. The concepts of nominal concentration recovery, coefficient of variation (CV), limit of blank (LOB), and limit of detection (LOD) were discussed in detail in the context of multiplexed assays. With our new definitions, quantitative results demonstrated a high recovery of the nominal concentrations (115%), reliability (CV = 20%), and sensitivity (LOBs of 3, 27, and 6 pg·mL-1 for SEG, SEH, and SEI respectively, and LODs of 6, 48, and 11 pg·mL-1 for SEG, SEH, and SEI, respectively). Based on this method, we observed an increase in sensitivity of 100 compared to the other multiplexed LFA labeled with gold particles and we approached the sensitivity of the simplex enzyme-linked immunosorbent assay (ELISA) performed with the same capture and detection antibodies. To conclude, our results, which are applicable to virtually any kind of multiplexed test, pave the way to the next generation of in-field analytical immunoassays by providing fast, quantitative, and highly sensitive multiplexed detection of biomarkers or pathogens.

From Foodborne Disease Outbreak (FBDO) to Investigation: The Plant Toxin Trap, Brittany, France, 2018.

On 6 July 2018, the Center for Epidemiology and Public Health of the French Armed Forces was informed of an outbreak of acute gastroenteritis among customers of a dining facility at a military base in Brittany, France. A total of 200 patients were reported out of a population of 1700 (attack rate: 12%). The symptoms were mainly lower digestive tract disorders and occurred rapidly after lunch on 5 July (median incubation period: 3.3 h), suggesting a toxin-like pathogenic process. A case-control survey was carried out (92 cases and 113 controls). Statistical analysis pointed to the chili con carne served at lunch on 5 July as the very likely source of poisoning. Phytohaemagglutinin, a plant lectin, was found in the chili con carne at a concentration above the potentially toxic dose (400 HAU/gram). The raw kidney beans incorporated in the chili con carne presented a high haemagglutination activity (66,667 HAU/gram). They were undercooked, and the phytohaemagglutinin was not completely destroyed. FBDOs due to PHA are poorly documented. This study highlights the need to develop methods for routine testing of plant toxins in food matrices. Improved diagnostic capabilities would likely lead to better documentation, epidemiology, and prevention of food-borne illnesses caused by plant toxins.

A large travel-associated outbreak of iatrogenic botulism in four European countries following intragastric botulinum neurotoxin injections for weight reduction, Türkiye, February to March 2023.

In March 2023, 34 associated cases of iatrogenic botulism were detected in Germany (30 cases), Switzerland (two cases), Austria (one case), and France (one case). An alert was rapidly disseminated via European Union networks and communication platforms (Food- and Waterborne Diseases and Zoonoses Network, EpiPulse, Early Warning and Response System) and the International Health Regulation mechanism; the outbreak was investigated in a European collaboration. We traced sources of the botulism outbreak to treatment of weight loss in Türkiye, involving intragastric injections of botulinum neurotoxin. Cases were traced using a list of patients who had received this treatment. Laboratory investigations of the first 12 German cases confirmed nine cases. The application of innovative and highly sensitive endopeptidase assays was necessary to detect minute traces of botulinum neurotoxin in patient sera. The botulism notification requirement for physicians was essential to detect this outbreak in Germany. The surveillance case definition of botulism should be revisited and inclusion of cases of iatrogenic botulism should be considered as these cases might lack standard laboratory confirmation yet warrant public health action. Any potential risks associated with the use of botulinum neurotoxins in medical procedures need to be carefully balanced with the expected benefits of the procedure.

Acceptable intakes (AIs) for 11 small molecule N-nitrosamines (NAs).

Low levels of N-nitrosamines (NAs) were detected in pharmaceuticals and, as a result, health authorities (HAs) have published acceptable intakes (AIs) in pharmaceuticals to limit potential carcinogenic risk. The rationales behind the AIs have not been provided to understand the process for selecting a TD50 or read-across analog. In this manuscript we evaluated the toxicity data for eleven common NAs in a comprehensive and transparent process consistent with ICH M7. This evaluation included substances which had datasets that were robust, limited but sufficient, and substances with insufficient experimental animal carcinogenicity data. In the case of robust or limited but sufficient carcinogenicity information, AIs were calculated based on published or derived TD50s from the most sensitive organ site. In the case of insufficient carcinogenicity information, available carcinogenicity data and structure activity relationships (SARs) were applied to categorical-based AIs of 1500 ng/day, 150 ng/day or 18 ng/day; however additional data (such as biological or additional computational modelling) could inform an alternative AI. This approach advances the methodology used to derive AIs for NAs.

Selective Wee1 Inhibitors Led to Antitumor Activity In Vitro and Correlated with Myelosuppression.

Wee1 is a tyrosine kinase that is highly expressed in several cancer types. Wee1 inhibition can lead to suppression of tumor cell proliferation and sensitization of cells to the effects of DNA-damaging agents. AZD1775 is a nonselective Wee1 inhibitor for which myelosuppression has been observed as a dose-limiting toxicity. We have applied structure-based drug design (SBDD) to rapidly generate highly selective Wee1 inhibitors that demonstrate better selectivity than AZD1775 against PLK1, which is known to cause myelosuppression (including thrombocytopenia) when inhibited. While selective Wee1 inhibitors described herein still achieved in vitro antitumor efficacy, thrombocytopenia was still observed in vitro.

Group trauma focused cognitive behavior therapy for parents of premature infants compared to individual therapy intervention.

BACKGROUND: The current study compares results of a group-based intervention developed to reduce symptoms of posttraumatic stress, depression, and anxiety in parents of premature infants with a prior study using an individual version of the treatment manual. METHODS: 26 mothers of preterm infants (25-34 weeks' gestational age; >600 g) received 6 sessions of trauma-focused cognitive behavior therapy (CBT). Outcomes were compared with those of a previously published RCT, which tested an individual therapy based on the same model in a group of 62 mothers. Results were also compared across in-person and telehealth treatment. RESULTS: From baseline to follow up, the individual intervention showed greater improvement in trauma symptoms assessed with the Davidson Trauma Scale (d = 0.48, p = 0.016), although both conditions showed clinically significant improvement. Similar patterns were found for maternal depression and anxiety. In-person treatment was found to be superior to telehealth treatment administered during the COVID-19 pandemic, although the difference was not significant. CONCLUSIONS: Group-based trauma focused CBT is an effective treatment modality for parents of premature infants with symptoms of psychological distress but not as effective as individual therapy using the same treatment model.

Evaluation of a new rapid immunochromatographic assay for the detection of GES-producing Gram-negative bacteria.

BACKGROUND: As carbapenemase-producing Enterobacterales are increasingly reported worldwide, their rapid detection is crucial to reduce their spread and prevent infections and outbreaks. Lateral flow immunoassays (LFIAs) have become major tools for the detection of carbapenemases. However, as for most commercially available assays, only the five main carbapenemases are targeted. OBJECTIVES: Here, we have developed and evaluated an LFIA prototype for the rapid and reliable detection of the increasingly identified GES-type ß-lactamases. METHODS: The GES LFIA was validated on 103 well-characterized Gram-negative isolates expressing various ß-lactamases grown on Mueller-Hinton (MH) agar, chromogenic, and chromogenic/selective media. RESULTS: The limit of detection of the assay was 106 cfu per test with bacteria grown on MH agar plates. GES LFIA accurately detected GES-type ß-lactamases irrespective of the culture media and the bacterial host. The GES LFIA was not able to distinguish between GES-ESBLs and GES-carbapenemases. Because GES enzymes are still rare, their detection as an ESBL or a carbapenemase remains important, especially because extensive use of carbapenems to treat ESBL infections may select for GES variants capable of hydrolysing carbapenems. CONCLUSIONS: The GES LFIA is efficient, rapid and easy to implement in the routine workflow of a clinical microbiology laboratory for the confirmation of GES-type ß-lactamases. Combining it with immunochromatographic assays targeting the five main carbapenemases (KPC, NDM, VIM, IMP and OXA-48) would improve the overall sensitivity for the most frequently encountered carbapenemases and ESBLs, especially in non-fermenters.

The Landscape of Potential Small and Drug Substance Related Nitrosamines in Pharmaceuticals.

This article reports the outcome of an in silico analysis of more than 12,000 small molecule drugs and drug impurities, identifying the nitrosatable structures, assessing their potential to form nitrosamines under relevant conditions and the challenges to determine compound-specific AIs based on data available or read-across approaches for these nitrosamines and their acceptance by health authorities. Our data indicate that the presence of nitrosamines in pharmaceuticals is likely more prevalent than originally expected. In total, 40.4 % of the analyzed APIs and 29.6 % of the API impurities are potential nitrosamine precursors. Most structures identified through our workflow could form complex API-related nitrosamines, so-called nitrosamine drug substance related impurities (NDSRIs), although we also found structures that could release the well-known small and potent nitrosamines NDMA, NDEA, and others. Due to common structural motifs including secondary or tertiary amine moieties, whole essential drug classes such as beta blockers and ACE inhibitors are at risk. To avoid the risk of drug shortages or even the complete loss of therapeutic options, it will be essential that the well-established ICH M7 principles remain applicable for nitrosamines and that that the industry and regulatory authorities keep an open communication not only about the science but also to make sure there is a good balance between risk and benefit to patients.

Rapid detection of CTX-M-type ESBLs and carbapenemases directly from biological samples using the BL-DetecTool.

BACKGROUND: Lateral flow immunoassays (LFIA) have shown their usefulness for detecting CTX-M- and carbapenemase-producing Enterobacterales (CPEs) in bacterial cultures. Here, we have developed and validated the BL-DetecTool to detect CTX-M enzymes and carbapenemases directly from clinical samples. METHODS: The BL-DetecTool is an LFIA that integrates an easy sample preparation device named SPID (Sampling, Processing, Incubation and Detection). It was evaluated in three University hospitals on urine, blood culture (BC) and rectal swab (RS) specimens either of clinical origin or on spiked samples. RS evaluation was done directly and after a 24 h enrichment step. RESULTS: The CTX-M BL-DetecTool was tested on 485 samples (154 BC, 150 urines, and 181 RS) and revealed a sensitivity and specificity of 97.04% (95% CI 92.59%-99.19%) and 99.43% (95% CI 97.95%-99.93%), respectively. Similarly, the Carba5 BL-DetecTool was tested on 382 samples (145 BC, 116 urines, and 121 RS) and revealed a sensitivity and specificity of 95.3% (95% CI 89.43%-98.47%) and 100% (95% CI 98.67%-100%), respectively. While with the Carba5 BL-DetecTool five false negatives were observed, mostly in RS samples, with the CTX-M BL-DetecTool, in addition to four false-negatives, two false-positives were also observed. Direct testing of RS samples revealed a sensitivity of 78% and 86% for CTX-M and carbapenemase detection, respectively. CONCLUSIONS: BL-DetecTool showed excellent biological performance, was easy-to-use, rapid, and could be implemented in any microbiology laboratory around the world, without additional equipment, no need for electricity, nor trained personnel. It offers an attractive alternative to costly molecular methods.

NeuroTorp, a lateral flow test based on toxin-receptor affinity for in-situ early detection of cyclic imine toxins.

The emergent cyclic imine toxins produced by marine dinoflagellates are potent antagonists of nicotinic acetylcholine receptors. Shellfish accumulate cyclic imine toxins following filter-feeding on toxic dinoflagellates vectoring them to humans. Herein is presented a lateral flow test for the detection of cyclic imine toxins based on three new concepts for test strips: i) the immobilization of lipoprotein vesicles in the test-line, ii) the high affinity of neurotoxins for their receptor targets and iii) the use of high porosity glass fiber filter membranes as support for the fabrication of the lateral flow test NeuroTorp (WO2017108115). Purified electrocyte membrane vesicles from Torpedo marmorata were used as a source of receptor and were immobilized in the test-line. Biotin-α-bungarotoxin was used as toxin tracer for the NeuroTorp LFT given its high affinity for nicotinic acetylcholine receptors while neutravidin nanogold particle conjugates enable its visual detection. Herein is reported for the first time the use of GF/C glass fiber membranes as the stationary phase for a lateral flow test. The GF/C filter ensures both: the immobilization of a complex lipoprotein in the test-line and the capillary migration of the mobile phase. Scanning electron microscopy studies shed light into the mechanism by which Torpedo-electrocyte membranes vesicles are immobilized in the GF/C glass microfiber. The electrocyte membrane vesicles anchor in neighboring microfibers randomly disposed in the same plane of the GF/C filter forming stable microfilm structures ensuring the functionality of nicotinic acetylcholine receptors. NeuroTorp is a ready-to-use low-cost early warning device for rapid detection of cyclic imine toxins in shellfish by end-users.

The Revolution of Lateral Flow Assay in the Field of AMR Detection.

The global spread of antimicrobial resistant (AMR) bacteria represents a considerable public health concern, yet their detection and identification of their resistance mechanisms remain challenging. Optimal diagnostic tests should provide rapid results at low cost to enable implementation in any microbiology laboratory. Lateral flow assays (LFA) meet these requirements and have become essential tools to combat AMR. This review presents the versatility of LFA developed for the AMR detection field, with particular attention to those directly triggering ß-lactamases, their performances, and specific limitations. It considers how LFA can be modified by detecting not only the enzyme, but also its ß-lactamase activity for a broader clinical sensitivity. Moreover, although LFA allow a short time-to-result, they are generally only implemented after fastidious and time-consuming techniques. We present a sample processing device that shortens and simplifies the handling of clinical samples before the use of LFA. Finally, the capacity of LFA to detect amplified genetic determinants of AMR by isothermal PCR will be discussed. LFA are inexpensive, rapid, and efficient tools that are easy to implement in the routine workflow of laboratories as new first-line tests against AMR with bacterial colonies, and in the near future directly with biological media.

Proof of concept of a two-stage GMR sensor-based lab-on-a-chip for early diagnostic tests.

The development of rapid, sensitive, portable and inexpensive early diagnostic techniques is a real challenge in the fields of health, defense and in the environment. The current global pandemic has also shown the need for such tests. The World Health Organization has defined ASSURED criteria (affordable, sensitive, specific, user-friendly, rapid and robust, equipment-free and deliverable to end-users) that field diagnostic tests must fulfill, which proves the real need in terms of public health. Giant magnetoresistance (GMR) sensors, which have flourished in a wide variety of spintronic applications (automobile industry, Information Technology, etc.), also have real potential in the field of health, particularly for the development of early diagnostic point-of-care devices. This work presents a new type of innovative biochip, consisting of GMR sensors arranged on both sides of a microfluidic channel which allow on the one hand to count magnetic objects one by one but also to better distinguish false positives (aggregates of beads, etc.) from labelled biological targets of interest by determining their magnetic moment. We present the operating principle of this new tool and its great potential as a versatile diagnostic test.

Dual Blockade of Misfolded Alpha-Sarcoglycan Degradation by Bortezomib and Givinostat Combination.

Limb-girdle muscular dystrophy type R3 (LGMD R3) is a rare genetic disorder characterized by a progressive proximal muscle weakness and caused by mutations in the SGCA gene encoding alpha-sarcoglycan (α-SG). Here, we report the results of a mechanistic screening ascertaining the molecular mechanisms involved in the degradation of the most prevalent misfolded R77C-α-SG protein. We performed a combinatorial study to identify drugs potentializing the effect of a low dose of the proteasome inhibitor bortezomib on the R77C-α-SG degradation inhibition. Analysis of the screening associated to artificial intelligence-based predictive ADMET characterization of the hits led to identification of the HDAC inhibitor givinostat as potential therapeutical candidate. Functional characterization revealed that givinostat effect was related to autophagic pathway inhibition, unveiling new theories concerning degradation pathways of misfolded SG proteins. Beyond the identification of a new therapeutic option for LGMD R3 patients, our results shed light on the potential repurposing of givinostat for the treatment of other genetic diseases sharing similar protein degradation defects such as LGMD R5 and cystic fibrosis.

Introduction to the Toxins Special Issue: "Antibodies for Toxins: From Detection to Therapeutics".

This Special Issue aims to provide an up-to-date investigation and reviews linked to antibody-based technologies for medical countermeasures and detection/diagnosis tools for toxins [...].

Deep mutational engineering of broadly-neutralizing nanobodies accommodating SARS-CoV-1 and 2 antigenic drift.

Here, we report the molecular engineering of nanobodies that bind with picomolar affinity to both SARS-CoV-1 and SARS-CoV-2 receptor-binding domains (RBD) and are highly neutralizing. We applied deep mutational engineering to VHH72, a nanobody initially specific for SARS-CoV-1 RBD with little cross-reactivity to SARS-CoV-2 antigen. We first identified all the individual VHH substitutions that increase binding to SARS-CoV-2 RBD and then screened highly focused combinatorial libraries to isolate engineered nanobodies with improved properties. The corresponding VHH-Fc molecules show high affinities for SARS-CoV-2 antigens from various emerging variants and SARS-CoV-1, block the interaction between ACE2 and RBD, and neutralize the virus with high efficiency. Its rare specificity across sarbecovirus relies on its peculiar epitope outside the immunodominant regions. The engineered nanobodies share a common motif of three amino acids, which contribute to the broad specificity of recognition. Our results show that deep mutational engineering is a very powerful method, especially to rapidly adapt existing antibodies to new variants of pathogens.

Multiplex Detection of 24 Staphylococcal Enterotoxins in Culture Supernatant Using Liquid Chromatography Coupled to High-Resolution Mass Spectrometry.

Staphylococcal food poisoning outbreaks are caused by the ingestion of food contaminated with staphylococcal enterotoxins (SEs). Among the 27 SEs described in the literature to date, only a few can be detected using immuno-enzymatic-based methods that are strongly dependent on the availability of antibodies. Liquid chromatography, coupled to high-resolution mass spectrometry (LC-HRMS), has, therefore, been put forward as a relevant complementary method, but only for the detection of a limited number of enterotoxins. In this work, LC-HRMS was developed for the detection and quantification of 24 SEs. A database of 93 specific signature peptides and LC-HRMS parameters was optimized using sequences from 24 SEs, including their 162 variants. A label-free quantification protocol was established to overcome the absence of calibration standards. The LC-HRMS method showed high performance in terms of specificity, sensitivity, and accuracy when applied to 49 enterotoxin-producing strains. SE concentrations measured depended on both SE type and the coagulase-positive staphylococci (CPS) strain. This study indicates that LC-MS is a relevant alternative and complementary tool to ELISA methods. The advantages of LC-MS clearly lie in both the multiplex analysis of a large number of SEs, and the automated analysis of a high number of samples.