Periodic Reporting for period 1 - ASSERT (Development of a nanotechnology-based innovative assay for the diagnosis of allergy to Betalactams)
Okres sprawozdawczy: 2021-09-10 do 2024-09-09
Allergy to betalactam antibiotics such as amoxicillin, is a worldwide problem affecting more than 10% people, partly due to high consumption. It is a burden for patients that must take less effective and more expensive second-line antibiotics and must stay longer at the hospital. Moreover, more than 90% of self-reported allergic individuals are not true allergic, carrying the label throughout life. The diagnosis of BL allergy is complex and requires provocation, which is not risk-free, and in vitro like the basophil activation test (BAT) tests show very little sensitivity. The main goal of ASSERT project was to optimize the BAT through (i)understanding basophil activation mechanisms, (ii) better antigen presentation by nanoengineering, and (iii) assay validation to fit in vitro diagnostic standards. The project has been successfully completed, achieving all its goals. We developed nanoparticles of different size and carrying different numbers of amoxicillin molecules. We found that larger nanoparticles with more amoxicillin molecules performed 20 times better than free amoxicillin, suggesting their potential use in allergy testing. The BAT was also improved by adding a bacterial-derived component to the reaction, improving sensitivity from 43% to 67%. Moreover, to identify new biomarkers of basophil activation we developed a basophil isolation method directly from blood, with reduced preparation time while maintaining high cell viability, making it easier to study allergic responses. Importantly, a quality management plan was implemented to fit diagnostic standards. The results of our work have been published in scientific journals and shared at international conferences, social media, and newspapers, to reach a broader audience. Additionally, we participated in public engagement activities, such as The European Researchers Night, where people could meet researchers and learn about our work. Overall, this project has made significant strides in improving allergy detection methods, for a better diagnosis and treatment and a reduction of the costs for the Health Systems. Our efforts not only advance scientific knowledge, but also aim to make allergy testing more accessible and effective for patients in the future. By engaging with the public, we hope to raise awareness of a conscious antibiotic usage and promote interest in scientific research among younger audiences.
Near 100 aged-matched, gender-balanced amoxicillin allergic and tolerant subjects, were recruited for this project, after agreeing to participate and signing the informed consent, as required by the national legislation. A series of nanoparticles of different sizes (20nm, 30nm or 50nm), containing different amount of amoxicillin (80, 100, 300 and 600 nM AX) were developed and evaluated for their capacity to better activate sample basophils than free amoxicillin. Basophil activation was measured by detection of CD63 and CD203c basophil activation markers. Parallel testing was done with free amoxicillin and nanoparticle-associated amoxicillin. We found that only bigger nanoparticles displaying the highest amount of amoxicillin (NP-50/600AX) were able to activate human basophils in the blood samples. Importantly, only allergic patient-derived basophils were activated by the nanoparticle, meaning that they retained specificity. In fact, NP-50/600AX was near 20 times more potent than free amoxicillin, yet conserving specificity to activate only basophils from allergic subjects and not from healthy controls. However, to achieve enough sensitivity very high amounts of NP-50/600AX were needed, affecting their stability and impeding a correct flow-cytometry measurements. In parallel, other optimization strategies were tested, like the addition of bacterial components to the sample with the aim to "prepare" the basophils before incubation with amoxicillin to obtain a higher degree of activation. All known bacterial derived molecules were tested as potential "co-factors" for the basophil activation test. Only bacterial lippopolisaccharyde (LPS) showed a significant increase in basophil activation upon incubation with amoxicillin in the basophil activation test, which was evident only in alergic subjects. The level of improvement in sensitivity raised from 46% to 68%, while specificity was kept at 95%, meaning that this can be an improvement that can be transferred to the clinic after a proper validation. In this line, a prospective study is being conducted to estimate the power of this optimization as well as the suitability to implement it into the clinical practice. In this sense, we developed a series of assays to standardize the basophil activation test, according to the European regulation for in vitro devices. Flow cytometers are the core instruments of the BAT. A risk-based approach was carried out to determine the best comparators for correlation studies among instruments. We found that %CD63 and %CD203c were the best comparators, to calculate analytical correlation, and clinical agreement between instruments. This qualification studies are part of the verifications required by the current european IVD regulation and are key to conduct multicentric studies, often important to measure reproducibility of the technique. In addition, we aim to study basophil interaction with the nanoparticles and with LPS, and for this we set up a rapid, medium throughput basophil isolation method, which gave highly pure, viable and functional basophils.The results of the nanoparticles, LPS, standardization were published in high impact journals, and disseminated through international meetings and activities to reach the general public such as the European researchers night or press articles and social media. Importantly, new nano structures displaying amoxicillin and LPS are planned to be developed soon, thanks to a national grant for the next three years.
The expected results beyond the state of the art have a potential socio-economic impact, by making innovative diagnostics that are affordable by the health systems. Allergy has already become a public health problem. Concerning allergy to antibiotics its increase is due to the increasing antibiotic consumption, the appearance of superressistant bacteria, or the great number of falsely-labelled individuals as allergic only based on history or isolated allergy-like episodes during infancy that are taken as allergy diagnosis, leading to a certain antiobiotic avoidance through life. The technology proposed by the development of the project is cost-effective and will allow to give a precise diagnosis of allergy, reducing the number of false allergy-labelled individuals, which will increase their quality of life by enabling the access to the right antibiotics when needed, and in turn will reduce the economic burden of the heath system linked to the management of the allergic patients.The proposed optimization of the diagnostic tests will have a significant impact on the quality of life of patients, and their socio-economic burden, since allergic patients need to take second-line antibiotics which are more expensive, and sometimes, less effective. The development of the optimized diagnostics assays that are proposed in this project will enable a precise diagnosis which a concomitant reduction of cost for the patients, the health care system and increase of the patient's quality of life.