Respiratory Syndrome Linked Pathogens Multiplex Detection and characterization – ReSynPlex

As soon as a respiratory syndrome is identified in a patient, clinician needs to rapidly characterise the ætiology of the syndrome to take appropriate decision. Following the sampling (bronchoalveolar lavage or bronchial aspirate), the total Nucleic Acids (NA) are extracted. A subsequent step of multiplexed PCR is performed in order to amplify the specific targets to be detected. It is foreseen that 50 probes will be implemented per well. 50 probes will allow detection of the most clinically relevant respiratory pathogens and for some of them their virulence factors and/or resistance traits to anti-infectious agents.

Following the multiplex PCR, all the amplicons of interest are labelled. The multiplex detection assay is then performed using an automated liquid handler using HIFI Technology. A custom ELISA plate format is used, composed of a translucent bottom arrayed with 50 oligo-probes in each single well. The hybridization will be revealed using an enzymatic label and a chromogenic substrate, which has been proven to be robust and reliable. Positives spots will appear on the bottom of each well, and the integrated densitometric imager (AXOware) will collect the data from each well/patient analysis. The software will automatically process the data, detecting the arrays and the different spots inside each well, validation PCR controls and spotting controls. The analytical results will be interpreted into an efficient and clear medical report that can be directly used by clinicians. The entire process will be built to be automated. The full test duration will be 4 hours, including only 30 minutes of hands-on time.

During the first phase, the proof of concept has been validated: the combination of PCRm and DNA chips permitted to identify the specific genetic characteristics of pathogens (bacteria and viruses at a time) and some resistance factors on known samples.
Including, the following points were developed and/or validated:
- The primary format (96-well plate ELISA)
- Testing the support (TKL adhesive based on patent held by AXO)
- Detection by colorimetry and automation of the test,
- Multiplexed detection, by the test of compounds pools of several samples of known pathogens,
- Possibility of detecting several types of characteristics: target genes from bacteria and viruses, resistance factors,
- Recent optimization of PCR conditions allowed to detect higher number of the targeted genes and to increase the sensitivity of other target genes.
- Development of the complete automated analytical protocol

The HIFI Resp product foreseen is designed to be adaptable to existing laboratory equipment: extraction, PCR amplification, automated liquid handling. The minimum required apparatus will be (i) a thermocycler and (ii) a microplate imager. For high-throughput needs, an automated liquid handler can run the full process (PCRm, hybridization assay on microarrays in plates, and reading). Based on the proof on concept and first results, performances of the test will be assessed (selectivity, sensibility).

A scientific article is being prepared on the basis of the first results in order to show the validation of the proof of concept.
A patent application is considered, based on coming results.

Respiratory syndromes associated to infections are threats to human health without geographical and societal spreading limitation. In France, as well as in similar countries across Europe, respiratory infections are the cause of 60 % of deaths in the category Communicable diseases, maternal and perinatal conditions and nutritional deficiencies (source: WHO "global burden disease 2008").
Respiratory syndromes are characterized by a large number of involved infectious agents (bacteria and viruses) that can become nosocomial (healthcare-associated pathogens) following their recurrent contact with antibiotics. As an example, pneumonia can be induced by at least 15 different pathogens, each one having different virulence and resistance factors. Typically, the clinician suspects a patient being infected during the post operatory wake up phase in the recovery room. Then a clear diagnostic is quickly needed in order to take appropriate decision for the health of the patient.
In this context, the attempt of the clinician to characterize the aetiology of a patient’s syndrome is a puzzle between the number of available tests (pathogen identification, resistance/virulence factors detection...) and the turnaround time of these tests that delays diagnosis and the allowed budget for each patient. At present, most pathogens are detected and identified individually either by culture or PCR-based techniques, and the multiplication of these tests are rarely time (from 48 hours to 7 days) and cost efficient (from 40 € for each bacterium, to 130 € for 13 PCR identifications). A real gap exists between the multiplicity of information required by the practitioner and the diagnostic tools available.
Today, the three key technologies of the integrated oligonucleotide diagnostic have evolved. Modern extraction system, PCR multiplexed and state of the art multiplex analysis platform exist independently. These systems need to be smartly combined, considering clinician requirements in order to create at the same time a powerful and flexible tool.
One can foresee a strong improvement of the analytical process by i) coupling multiplexed PCR with a sequence specific detection method (microarray like) and ii) identifying using the same multiplex PCR both the pathogens and their resistance/virulence factors. This is the concept and objectives of the ReSynPlex project: a cost efficient (less than 30 €) automated platform for the complete and rapid (4 hours for up to 96 samples) diagnosis of respiratory related syndromes.
In this concept, the clinician will have direct and fast access to the identity of involved pathogens and to the presence of resistance related genes. This new diagnostic tool will lead to more appropriate point-of-care patient management in the clinical setting and help reducing the spread of infections of global importance. The patients, the clinicians and the healthcare system will altogether benefit from the cost efficient technology platform created within ReSynPlex.
Over three years (2010 to 2013), AXO Science has been successfully leading a research program entitled HiFi-assays (ANR BiotecS 2010). The ReSynPlex project is aimed at developing an IVD product for pathogen advanced identification, taking advantage of the achievement completed within HiFi-assays. Especially, the technology developed and patented looks perfectly adapted to the application foreseen here. Thanks to HiFi-assays, a first product applied to extended blood group genotyping will be brought on the market. AXO Science and its historic partners (ICBMS and IAB) wish now to pursue the valorisation of the results by enlarging the field of application of the HIFI multiplexed technology. The expertise, know-how and IP recently achieved will be proficiently used in the ReSynPlex project.