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Portable automated test for fast detection and surveillance of influenza

Final Report Summary - PORTFASTFLU (Portable automated test for fast detection and surveillance of influenza)

Executive summary:

The project objective was to develop and validate a rapid diagnostic test for human influenza that would be used for surveillance and early detection of influenza and as a point-of-care tool in developed and developing countries. The diagnostic test had to enable the rapid detection of influenza infection in a fast and specific way (typing and sub-typing) using a monolithic disposable cartridge placed in a compact, portable analytical instrument.

Project context and objectives:

Infectious disease represents the greatest risk to global human health. This can range from classical infectious diseases, such as tuberculosis, cholera, dysentery, and typhoid or annual epidemics, such as norovirus, Influenza, and seasonal colds or emerging infectious diseases, such as avian influenza and haemorrhagic fevers, to global pandemics, such as Human immunodeficiency virus (HIV) and the current newly emerged H1N1v outbreak (commonly referred to as swine flu). Infectious diseases account for 10 % of all deaths recorded annually, and are responsible for 1 / 3 of all general practitioner consultations. The projected total cost for treatment of infectious diseases in the US alone is around USD 120 billion per annum.

With diagnosis of infectious disease firmly entrenched in classical culture techniques developed in the 19th and 20th century, there is a clear need for the development of fully automated, accurate and robust rapid diagnostic devices to alleviate the economic and health burden presented by pathogenic viruses and bacteria. In particular molecular assays which can distinguish pathogenic subtypes within species would allow fine detailed diagnosis of infections, as well as allow more rapid assessment of effective treatment measures and more rapid initiation of relevant control measures and epidemiological analysis.

The PORTFASTFLU project produced a novel diagnostic system that allows rapid automated detection and subtyping of influenza viruses in clinical and field samples. The approach is based on the integration of a lab-on-a-chip (LOC) consumable cartridge for automated extraction and amplification of the Ribonucleic acid (RNA) of the virus (carrying its genetic information), followed by hybridisation and real-time detection on a microarray, in a single portable and easy to use machine called the GeneSpress® platform.

Project results:

The initial objective of the PORTFASTFLU was to identify the influenza virus for the most usual types, A or B, and amongst the A types various subtypes H1, H2, H3, H5, H7, N1 and N2. However, while the project was well into its second year, the H1N1v new virus appeared worldwide (April 2009) up to a situation where a pandemic was declared, and now the H1N1v has become the 'standard' seasonal species of H1N1. The PORTFASTFLU team demonstrated quickly that the PORTFASTFLU system was capable of detecting the new influenza subtype and the final detection kit is able to detect the original target influenza subtypes, as well as the new one.

To ensure a reliable and verifiable operation of the PORTFASTFLU system, a positive control RNA molecule is added to the patient virus in order to verify that the diagnostic system has been operating well on the patient's sample. The PORTFASTFLU diagnostics system concept is demonstrated through the use of a disposable LOC cartridge, inserted in a machine which performs the various steps of the diagnostic protocol, detects the hybridised species and processes the signals and data.

In order to reach its objectives, the PORTFASTFLU project let to the assembling of a team with wide-reaching competences: the coordinator, Genewave (Paris, France) is a molecular diagnostic company with all competences from molecular diagnostics kit design to large scale fabrication of consumables and automated diagnostics systems; Biosensia (Dublin Ireland is a company devoted to point-of-care in vitro diagnostics; Ikerlan (Mondragon, Basque region, Spain) is a technology centre devoted to microtechnologies for in vitro diagnostics, Gaiker ( Bilbao, Basque region, Spain) is a technological centre with competences in molecular biology, microbiology, immunochemistry and enzymology to develop innovative biodetection systems; The molecular virology group at VIB (University of Gent, Belgium) is a scientific research institute for molecular biomedical research; Nottingham Trent University, School of Biomedical and Natural Sciences (Nottingham, UK), is a group devoted to research into emerging food-borne pathogens, molecular mechanisms of pathogenicity in bacteria and viruses; CIRAD (Montpellier, France) is a French public institute that makes research in agronomy for developing countries, strongly involved in research and development for the control of infectious diseases of cattle, small ruminants, swine and poultry. Whatman (part of GE healthcare) is a global leader in separations technology for the research and diagnostic community which has developed total sample preparation solutions; Foundation BIOEF and Hospital Donostia (San Sebastian, , Basque region, Spain) which form a joint research unit, in which Hospital Donostia is the Basque country reference laboratory for influenza virus.

The PORTFASTFLU consortium partners had well defined roles:
- Genewave acted as the architect of the diagnostics system and as the integrator of the various technologies.
- Biosensia worked on microfluidic designs.
- Ikerlan developed the LOC cartridge and associated hardware and electronics transfer and adapted the sample preparation and polymerase chain reactions (PCR) from the tube to the chip. Ikerlan developed fabrication techniques for large-scale production of diagnostics kits.
- Gaiker developed and validated biochemical protocols for influenza diagnostics for LOC operation.
- VIB University of Gent developed, produced and purified large preparations of influenza A and B viruses, and an internal reference RNA used as an internal control for real-time PCR (rt-PCR). VIB provided biological material containing known and unknown amounts of IVA, IVB and RSV virus for validation testing of the new device, and tested the PORTFASTFLU system.
- Nottingham University provided recent influenza isolates. They acted as experts for the definition of the PORTFASTFLU products.
- CIRAD designed and validated primers and probes, worked on amplification techniques, tested the sensitivity, specificity and reproducibility of the PORTFASTFLU test.
- Whatman provided FTA filter paper technology for the processing of samples to yield RNA for amplification.
- Hospital Donostia provided human samples infected with other respiratory viruses (VRS, parainfluenza, adenovirus, metapneumovirus, coronavirus, bocavirus, and rhinovirus) as well as samples infected with influenza A H1N1, H3N2, influenza B, and influenza C. They compared extraction methods of RNA / Deoxyribonucleic acid (DNA). They evaluated sensitivity of the PORTFASTFLU system with clinical samples.

The PORTFASFLU system comprises three items:

- the consumable LOC cartridge, which performs sample preparation, RNA transcription into DNA, DNA amplification, microarray hybridisation;
- the portable automated system houses the fluidics, the pneumatic actuation system, the electronics and the interface with the control computer;
- the measurement and analysis system performs the readout of the hybridisation result, the signal and data automated analysis.

The PORTFASTLU consortium developed a portable microfluidic cartridge for viral RNA isolation, amplification and hybridisation taking into account the LOC concept. The cartridge detects in a specific and rapid way human influenza viruses from clinical samples (nasopharyngeal and throat swabs). The samples used for this work consisted of viral cultures and nasopharyngeal samples from human patients prepared and supplied by Hospital Donostia and VIB.

The packaged microfluidic chip performs viral RNA extraction, amplification by rt-PCR reaction and hybridisation.

The microdevice can extract viral RNA from real samples, generate cDNA and amplify influenza molecular markers by PCR inside one-single-chamber chip. The reduction of the biochemical steps has allowed us to simplify the LOC concept avoiding reaction yield losses. Two materials (SU-8 and Cyclic olefin copolymer or COC) have been successfully assayed for the microdevice manufacturing, but the COC cartridges prove to be amenable to large scale, low cost production through injection moulding.

A COC cartridge integrating the single chamber chip of above plus a hybridisation chamber has been developed. The fabrication process is based on the bonding between an injected COC piece with the desired channels and chambers already patterned and a thin (100 micrometre) COC film.

The complexity of a multi chamber cartridge is quite high as it involves elements / structures that allow the proper handling of the various liquids required to perform the various steps of the process leading from the viral sample to the readout of a hybridised microarray. Thus 'out-line' and 'in-line' microvalves have been developed and fabricated able to open or close the liquid flow at different places than the inlet and outlet ports.

Many laboratories worldwide have developed LOC systems for genetic or cell analysis. These however remain laboratory objects as they require a large range of devices and equipment surrounding them to operate. To reach its goals of portability and automation, the PORTFASTLU consortium developed a diagnostics system which acts as a docking station for the LOC consumable cartridge. It is highly integrated as it incorporates all the control electronics, the optical detection system of the microarray fluorescence, the pneumatic elements to control the fluids in the cartridge, the various fluids needed to perform the various biochemical steps of the molecular recognition protocol. The signal and data analysis is automatically done without supervision by a computer linked to the PORTFASTLU machine through a USB link. To keep the cartridge and system as simple as possible, only validated concepts of LOCs have been implemented, and versatility is obtained by having all reagents injected as required from an ensemble of 14 reagent bottles placed in the machine, two of them refrigerated for temperature-sensitive reagents.

The integration of the full protocol for the analyses of nasopharyngeal samples in the multichamber cartridges is carried out without supervision using the PORTFASTLU machine GeneSpress®. For that, the three different steps (extraction / purification, amplification, hybridisation) have been optimised separately on breadboard systems, and then adjustments has been made to link these steps together along with specific adaptation of the protocol to the GeneSpress® system and cartridges, specifically in regard to the fluid handling. We have demonstrated a full protocol implementation (figure 5). The fluorescence image resulting from the analysis of an H1N1v sample is shown, next to the display of the automated signal and data analysis.

Potential impact:

Purpose

The foreground developed by Genewave during the PORTFASTFLU project is very broad and encompasses all aspects of molecular diagnostics system, as the end product is a full diagnostics system, including the kit development and consumables, in addition to the diagnostic system itself.

Therefore, Genewave developed foreground or further developed background in the following areas:

- molecular diagnostic kit for influenza virus identification: primer and probe design;
- testing tools for molecular diagnostic kits based on its HybLive tool;
- protocol for the diagnostic chain of steps: RNA extraction, purification, reverse transcription, PCR amplification, hybridisation, revelation;
- design and fabrication techniques for the LOC and microarray cartridge (in strong cooperation with Ikerlan);
- docking system making the interface between the cartridge and the measuring system: it provides the following functionalities: mechanical placement, pneumatic (for cartridge valve operation), fluidic connections, heating (for PCR), optical coupling of the microarray to the image sensor;
- control software of the GeneSpress diagnostic system;
- image analysis and automated spot recognition and quantification;
- data analysis for automated display of the diagnostic results.

Thus, PORTFASTFLU provided at the end a full automated system for influenza diagnostic.

Foreseen foreground exploitation plan

In the course of PORTFASTFLU, Genewave explored the possibility of commercialising an influenza diagnostics kit. There are several reasons which make such a market unreliable:

- There is no clear cut demand for a systematic diagnosis of influenza. While such a system would be very useful in epidemiology for an early warning of an epidemic scenario or to detect and monitor a new mutation, in the case of a pandemic the dominant species is so prevalent that subspecies identification is not demanded.
- While there seems to be a demand for civil authorities such as airports or ports of entry, the access to the market for these entities is not yet identifiable.
- In emergency rooms of hospitals where patients with severe symptoms will be treated, advanced single-plex molecular diagnostics tools exist to determine if the patient is subject to the prevalent influenza subtype at the time.
- The PORTFASTFLU consortium does not possess any key marker IP, nor is it clear that such protecting IP would exist.
- The need clearly expressed by hospital practitioners is for a multiplex diagnostics of a panel of respiratory infectious diseases, of which influenza is just one among several other critical ones. Such applications of the GeneSpress® platform are planned for 2014.

Present plans for exploitation by Genewave

For the time being, Genewave plans to market molecular diagnostics kits and systems aimed at Hospital acquired infections (HAIs). The first product should be targeting Ventilatory acquired pneumonia (VAP) in 2012 and screening patients carrying Multi drug resistance (MDR) bacteria in 2013.

This exploitation will involve beneficiary Ikerlan for the manufacture of the LOC cartridge.

IPR exploitable measures taken or intended

Genewave has applied for one patent on the cartridge architecture and is writing another one on the docking station.

Potential / expected impact

Under the PORTFASTFLU project, Genewave has successfully developed critical building blocks that are essential for the development of a point-of-care device for the rapid diagnostic of VAP and rapid detection of MDR bacteria.

Clinical impact

The clinical impact of rapid identification of pathogens and resistance may be summarised as follows:

- A novel diagnostic tool permitting rapid identification of pathogens and resistance genes in respiratory samples could positively affect the patient's outcome. Many previous studies have demonstrated a clear relationship between appropriate antibiotic use and survival in Intensive care units (ICU). Putative etiologic agents and their antibiotic susceptibility patterns are suggested by local epidemiologic studies, prior duration of hospitalisation / mechanical ventilation before the onset of VAP and prior exposure to antibiotics. This strategy is inaccurate and can lead to inappropriate antibiotic therapy, increasing mortality and morbidity. A targeted antibiotic would improve the outcome for the patient, constituting a strong benefit for the individual, as well as decreasing the length-of-stay in ICU, which is an economic benefit for the hospital.
- Several previous studies suggest that even a short duration of broad-spectrum antibiotics such as imipenem may modify digestive flora. This short duration corresponds to duration of empirical antibiotic therapy. By identifying resistance genes (or their absence), a new strategy, avoiding as much as possible the use of broad-spectrum molecules, would reduce the so called 'selective pressure', which is a long-term ecological benefit for the community.

Socio-economic impact

It has been estimated that VAP increases the risk of hospital death, the duration of ICU stay (approximately 6 days) and the cost (25 000 - 35 000?per episode). The socioeconomic impact of a novel diagnostic tool permitting very rapid identification of pathogens and resistance genes may be as follows:

- Improvement in outcome due to a more appropriate choice in antibiotic therapy could translate in in the number of lives saved. Because physicians now use very-broad spectrum antibiotics, the percentage of VAP patients with non-appropriate antibiotics is generally between 20 and 30 %. This figure is still high. Rapid detection of resistance could decrease this percentage to near zero. Moreover, an initial non appropriate antibiotic treatment is associated with rapid growing of lung bacterial inoculum. As a consequence, VAP may be more difficult to treat and this could result in a longer ICU stay.
- Less acquisition costs and side effects: several broad spectrum antibiotics such as new carbapenems and oxazolidinones are expensive. A strategy permitting to use these molecules parsimoniously will probably be cost-effective. In addition, some of these molecules may have severe side effects, which increases morbidity.
- Breaking the vicious circle of the administration of carbapenems to many patients because of the fear of extended-spectrum betalactamases enterobacteriaecae, resulting in an increase in carbapenem-resistant strains.
- Developing point of care tools enabling a fast diagnosis of bacteria and resistance assay may prove to be cost-effective in an era of resource-limited settings (decentralised biology lab) or out of hours.

Dissemination

All along the project, we have communicated a lot to relevant end users, including industry, authorities, regulatory bodies, citizens groups, etc. These contacts highlighted the scientific achievements of the project and its future commercial use.

Many public media were used, such as newspapers, radio or television broadcasts. This has been helped by the outbreak of the new pandemic species H1N1v which raised broad public interest about the means to control new infectious diseases by rapid diagnostics methods.

We have also been in contact with authorities, mainly through discussions with the national reference laboratories. Among the many activities, we can single out the exhibit by Genewave of the PORTFASTFLU prototype at a meeting of the Haut Comité Français pour la Défense Civile.

The publication output has been below our early expectations. As the technologies developed within the project evolved strongly (in particular the switch from SU8 to COC cartridges), biological results of interest came out only slowly. The final clinical validations are still in the making.

The results on the use of bio microtechnology for different processes require also more experiments. It should be however kept in mind the confidentiality of technology itself, and of some of the results.

On novel technologies developed within PORTFASTFLU, patents will be applied for whenever possible.

List of websites: www.portfastflu.com

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