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Teaser, summary, work performed and final results



Beta-lactams (BLCs) are the most frequently prescribed antibiotics worldwide. As consequence of their intake, hypersensitivity reactions can happen. Current figures show over 2.5 million (0.5%) people in Europe and more than 5.4 million (1.8%) north-Americans suffer from...


Beta-lactams (BLCs) are the most frequently prescribed antibiotics worldwide. As consequence of their intake, hypersensitivity reactions can happen. Current figures show over 2.5 million (0.5%) people in Europe and more than 5.4 million (1.8%) north-Americans suffer from hypersensitivity to BLCs. These figures are increasing due to the misuse and consumption of these antibiotics. Moreover, allergy to BLCs is the most frequent cause (47%) of drug anaphylaxis, becoming an important public health problem with estimated additional hospitalization costs of 1750-4500 €/patient.
The correct diagnosis of BLCs allergic patients as well as the proper identification of patients who erroneously carry a label of BLCs allergy, leads to the improvement in the use of antibiotics, contributing to reduce the spread of multiple drug-resistant bacteria.
The diagnosis of drug hypersensitivity is difficult and underdiagnosed, there is a lack of standardization of the in vivo and in vitro test procedures to detect drug hypersensitivity reactions.
The most common methods for drug hypersensitivity allergic diagnosis are: clinical history, invasive skin test, in-vivo basophil test, in-vitro tests (quantification of IgEs), and drug provocation test (DPT). Among these methods, in-vitro test would be the preferred option as they are not invasive, but only a few in vitro diagnostic (IVD) methods are available in the market for the tertiary health services. These tests lack of sensitivity and selectivity (resulting in an inaccurate diagnosis), analyse few drugs allergens, are time consuming, and expensive.
COBIOPHAD project results provide an innovative IVD device for IgE-mediated drug allergies, we can highlight the following achievements:
• Different electronic and photonic modules, integrated in an optical reader for performing the assay and providing the readout of the results.
• Reagents and protein- determinant conjugates developed for nine BLCs.
• Detection platform: a microfluidic disc where to develop simultaneously six immunoassays able to determine up to nine different specific IgEs.
The results achieved in serum samples show high analytical sensitivity (<0.1 kUA/L), good clinical performances (sensitivity and selectivity 55% and 85%, respectively) and multiplexing capabilities (9 BLCs). This assay is developed in a fluidic disc capable to process simultaneously 6 different samples. On each sample the nine BLCs targeted are addressed and quantitative data about the content of specific IgE can be obtained. The assay time takes 60 minutes. The software processes the analytical results in 15 minutes storing them in the cloud and providing the report for supporting the diagnostics.
The cost of the disc reader and the consumables are very competitive, considering the components existing in the market.

Work performed

Since the project launch, the work of the partners led us to explore different approaches on each step of development. The research and design of the sensing platform (the microfluidic multiplexed disc), the processing and reading unit (the reader) and the materials to allow performing the assay, required important efforts in the coordination of all the developments, to allow the full compatibility of technologies and the implementation of a single integrated solution. The resulting system passed through an intensive period of iteration and testing, using serum samples collected during the project.
The main results are:
• A reader designed, manufactured, debugged and validated, a quick and robust tool for biosensing (drug allergy testing), adaptable to other type of bioassays.
• A microfluidic disc has been designed, prototyped, tested and manufactured. The disc fulfils the requirements given by the chemical assay and the manufacturing restrictions.
• A pool of BLCs determinants synthetized, becoming promising reagents to reach good analytical performances for new diagnosis devices and clinical tests. Strategies for anchoring protein-conjugated probes.
• ARTHUS (artificial human serum) reagents, obtained for several BLCs targets, used as chimeric IgEs for the selection of the determinants.
• An analytical immunoassay to determine levels of specific IgEs in serum of allergic patients at very low level (0.1 IU/mL) working in a microfluidic-multiplexed disc (6 samples, 9 BLCs).
There are 5 outcomes identified as potentially patentable from the main results arising from the project.

Final results

The state of the art for drug allergy diagnosis includes in vitro and ex-vivo methods: immunoassays and the basophil activation test. Both have controversial performances, including the limited number of drugs.
The systems currently available in the market are devoted to determine specific IgE to a short menu of drug allergens. The current IVD tests are not well validated, providing different specific IgE concentrations and poor sensitivity (38-54%), probably because of the inadequate low detection limit (> 0.35 kU/L). The estimated cost is 29.6 €/drug (6-7 € in reagents). They are prone to use in well-equipped laboratories and hospitals facilities. Besides drawbacks, they do not determine specific IgEs for other commonly BLCs such as cephalosporins, monobactams and oxopenams as alternative drugs.
COBIOPHAD overcomes these limitations by the development of a new IVD device for the determination of specific IgEs for a large panel of BLCs with high sensitivity.
The progress achieved is:
• A new reading system. Currently there are 3 operative units: portable, user friendly, robust, high throughput capability and low cost, attributes demonstrated by the analysis of representative serum samples of patients and controls.
• New BLCs determinants synthetized and conjugated as haptenized determinants for improving the detection capabilities of current tests. The new synthetized determinants and proteins are patentable.
• An analytical platform fully compatible with mass production: microfluidic structures needed for multiplexing samples and performing the assay including 9 BLCs.
The COBIOPHAD system is a reliable, quick and affordable system, attractive to the companies working in the allergies market and useful to the Health Systems. The next steps will pass through industrialization (TRLs 8 and 9) becoming a fully tested system for IVD of human allergies to BLCs, being an important tool for clinicians. The obtained results can be transferred to other environmental or food allergies, widening the scope of applications.
As a final summary:
• There is a clear need for improved diagnosis of BLCs antibiotic allergies.
• The COBIOPHAD system provides improved analytical sensitivity, good clinical performances and multi-drug allergy detection.
• The system provides results in 75 minutes, the cost is very competitive.
After industrialization stages, the COBIOPHAD approach implemented in national health systems would improve the cost-effectiveness of consulting an allergist. Also, providing relevant information about allergy profiles, reducing the time for patient evaluation and the potential risks inherent to in vivo tests, decreasing misinterpretation of the results. All this will contribute to a reduction in the overall costs associated with allergy consultations and interventions.

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