Final Report Summary - POC4PETS (Point of Care Diagnostics for rapid and cheap pathogen detection of companion animals)
Executive Summary:
The necessity of finding innovative and improved diagnostics - to complement or substitute the available ones was considered strategic in the context of Animal Health Strategy (2007-2013). POCS4PETS consortium represented a newly-formed cluster covering the entire knowledge chain to guarantee a high efficiency in transforming scientific research into diagnostic innovation. POCS4PETS project has brought together three RTD performers working in the fields of animal health, veterinary, biotech and nanotechnology and three innovation-oriented SMEs working in the molecular devices, markers and detection systems. POC4PETS focused in filling the gaps of diagnostics availability and enhancing technology transfer to increase competitiveness of the Veterinary Diagnostic industry. The project plan was designed after identifying technology gaps for selected diseases through a preliminary analysis on the available tools carried out on manufacturers’ web sites. For Leishmania, current tools are mainly based on serology that document only the exposure to the agents, furthermore the presence of low antibody levels is not necessarily indicative of the disease and further work-up is necessary to confirm or exclude clinical leishmaniasis. Horse papillomavirus and zoonotic poxviruses are currently lacking proper diagnostic tools for routine testing. For canine and feline parvovirosis, the available diagnostic tools are represented by Rapid in-house test based on ELISA and immunochromatographic technology. These kits are characterized by a low sensitivity with consequently false negative results in the beginning and the end of diseases and in subclinical forms; they can also give false positive few days after vaccination. Laboratory tests for agent detection such as viral isolation or molecular methods (PCR, Real-Time) are expensive and time consuming. For Feline Herpesvirus and Feline Calicivirus Infections FHV/FCV pen-side tests for Ag detection are not available while serological tests based on ELISA and immunofluorescence antibody test (IFA) to detect IgG do exist, even if serology is not useful for the diagnosis and the control of feline upper respiratory tract diseases (URTD) infection due to widespread of vaccination. To fill the above gaps, the following goals have been reached: the POC4PETS consortium developed diagnostic technologies based on recent advances technologies (for the development of new rapid, sensitive and specific tests) for selected pet diseases which have significant incidence in Europe and at global scale that will enable the detection of nucleic acids of different pet animals’ pathogens. Such technological breakthrough achievement provides and will potentially enhance the prevention, early diagnosis and control of the selected animal and human diseases. The two years' project overall generated exploitable results and products for the benefit of the three SMEs involved in the consortium.
Project Context and Objectives:
Veterinary diagnostics is a key tool in the prevention and control of infectious diseases in animals. It is increasingly recognized that there is an urgent need for innovation in the animal infectious diseases testing field, bringing to market specific, rapid and efficiently validated diagnostic tests. Such development is commonly missing at European scale, as different constrains are currently tackling cost efficacy, the need to contribute to help interested SMEs working in the animal health field to bring to veterinary professionals i) early detection kits without need for externalization to specialized laboratories, ii) easy to apply and to transport (portable) equipment and detection systems iii) in field tests where the outbreak is taking place.
The current market of veterinary diagnostic tools is actually rather fragmented as it is requiring different step forwards in terms of different technical, technological and science - based constrains, which can be identify, among others: i) ability to perform serology and antigen detection effectively; ii) high sensitivity; iii) quantification ability; iv) ease of use by minimally trained or untrained users; v) low cost; vi) speed; vii) robustness and stability in the field makes them suitable for animal-side testing; viii) and ability to transport and store without the need for refrigeration.
Such challenges have been the base onto which POC4PETS project's multi-disciplinary approach is developed, as it tackled such innovations in order to contribute to deepening the knowledge of the selected animal diseases. Nucleic acid diagnostic techniques have become routine diagnostic tools in veterinary laboratories not only to make specific typing determinations but also to rapidly screen large numbers of samples during disease outbreaks. POC4PETS consortium included in its experimental work plan some of the most promising new technologies mainly based on the potential that biotechnology can improve the current diagnostics for veterinary pathogens of companion animal species.
The veterinary diagnostic industry is under continuous development and it is finding its own identity; furthermore, diagnostics is an area of veterinary medicine that has considerable growth potential and limited regulatory restrictions. In recent years, there has been a continuous and constant growth of the global veterinary devices and, so, of the diagnostics industry.
Currently, more than half of all diagnostic testing by veterinarians is still done at outside reference laboratories. However, as vets endeavor to provide better preventive healthcare in clinics increasingly devoted to small animal medicine, the uptake of in-clinic tests is likely to increase. Rapid immunoassays provide veterinarians with important information, which can be acted upon before the pet’s owner leaves the clinic. The POC4PETS project addressed such technological constrains through a strong collaboration among the SME participants and the complementary RTD performer skills.
The proposed work will enable the SME participants to improve their competitive position and to validate and fully exploit the research results provided by the RTD performers. The Key strengths of POC4PETS Work plan can be summarized as follows:
1) Adopting the concepts of low cost, simplicity, rapidity, stability for the new diagnostic tools. This was achieved by developing simple tests that allow field diagnosis as well as the discrimination of several diseases with the same sample and possibly the differential diagnosis in case of syndromes.
2) Improving the basic qualities required for the new tools in terms of specificity and sensitivity. This was achieved by probing detection systems ensuring high specificity. Sensitivity was mainly assured by methods based on the nucleic acid amplification and detection.
According to such challenges, the progress of POC4PETS project can be measured against:
1) Scientific objectives of the project:
a. Set up advanced, reliable and effective new diagnostic technologies suitable to be used at point of care to enable the detection of nucleic acids of pet animals’ pathogens.
b. Develop, standardize and disseminate novel testing procedures that can be used by “first line” diagnosticians.
c. Apply recent advances in technologies (nanotechnologies, fluorescence technologies and probing) for the development of new rapid, sensitive and specific pen-side and laboratory-based tests for selected pet diseases which have significant incidence in Europe and at global scale.
d. Make available diagnostic tests for pet animal diseases actually lacking specific diagnostic tools.
e. Implement veterinary diagnostic systems that confer robustness to the whole detection on the field without the needing of fully equipped laboratories.
In order to facilitate the achievement of such challenges, POC4PETS project created a manufacturing process which enabled the consortium to effectively perform field testing to evaluate the reliability and performance on real pathological samples under field conditions. This has been done in close collaboration with the SME industrial partners. As a consequence, a series of technical objectives has been foreseen as follows:
• To develop novel pen-side PCR assays for the detection of selected pathogens. This technology will be indeed implemented by further optimization of a portable real-time PCR instrumentation (DNA/RNA Finder) and luminescent labeling technology.
• To implement and apply nucleic acid detection technique without thermal cycling for Leishmania detection (with special regards to the isothermal amplification).
• To develop a mini-array and Lateral Flow kits for the rapid field diagnosis of zoonotic poxviruses. This will be achieved by the development of pen-side assays for rapid and simple detection of pathogens in the field.
• To develop in all the detection devices obtained, a specific and tailored sample preparation to improve, as a whole, the test sensitivity.
• To improve already available kits by means of at least new POC diagnostic tools.
• To provide new POC diagnostics for those tackled pet animal diseases that are currently lacking tools.
• To integrate, in a single system (kit) all the necessary reagents and tools allowing rapid on site diagnosis which wouldn’t require skilled personnel.
• To apply the so obtained new methods for the “on site” rapid field diagnosis for the robust confirmatory tests in laboratory.
Such POC4PETS evidence – based breakthrough was ensured by the combination with an economic business plan which lead to the identification of different main industry- driven strategic goals that, taking into account that the POC4PETS consortium as a whole, enabled the SME participants to gain a competitive advantage by the up taking of a innovative set of technologies.
Project Results:
The main S&T results (products) and company foreground obtained by the POC4PETS consortium can be summarized as follows:
WP 2 Rapid DNA extraction kit is based on magnetic beads (NBE's foreground)
WP 3 Fluorescent End Point PCR system (DNA/RNA Finder) (NBE's foreground)
WP 3 Mini Kit for the detection of Feline Calicivirus (NBE's foreground)
WP 3 Mini Kit for the detection of Feline Herpesvirus (NBE's foreground)
WP 3 Mini Kit for the detection of Bovine Papilloma Virus (NBE's foreground)
WP 4 Leishmania DNA amplification and electrochemical detection kit (VETGEN's foreground)
WP 4 Leishmania DNA amplification and lateral flow detection kit (VETGEN's foreground)
WP 5 Point of care diagnostics for the rapid detection of Parapoxviruses (ACOBIOM's foreground )
WP 5 Point of care diagnostics for the rapid detection of Orthopoxviruses (ACOBIOM's foreground)
For each POC4PETS technological innovation, a technical card (attached) has been produced and distributed during dissemination events and training (see attached POC4PETS training and dissemination video). Further details of generated POCPETS' foreground is given below.
Ennebi's foreground:
1. Rapid DNA extraction kit
The Rapid DNA extraction kit is based on magnetic beads. The super-paramagnetic particles are nominal 1 μm magnetic carboxylate-modified base particles (MG-CM) made by a core-shell process. They combine a fast magnetic response time with a large surface area and fast reaction kinetics. Typically, these particles are used in various molecular biology, nucleic acid isolation, research and clinical diagnostic immunoassay applications.
The innovative aspect of the product is the low, non-specific binding of serum proteins and other interfering substances, Non-leaching, encapsulated magnetite and high surface area per unit mass, high ligand binding capacity and slow settling rate in the absence of a magnetic field.
The Rapid DNA extraction kit has numerous advantages over other molecular diagnostic tools that make the kit suitable for a rapid and simple extraction of nucleic acid: easy to use, easy to interpret, little equipment required, rapidity, usable for technical personnel without specific training in molecular biology.
2. Fluorescent End Point PCR system
The developed system is a platform which enables end-point fluorescent PCR amplification of nucleic acids and their revelation by reading the initial and final value of fluorescence, the measured values have significance in the field of scientific research. The system is designed to detect emissions at defined wavelengths, through the excitation of a sample by means of a light source. The sample in the analysis is previously amplified through a heating unit that will perform the predetermined cycles in the process of set-up. The sections will be managed by a microcontroller and the user control via a touch screen TFT, size and weight of the system will be contained in such a way as to facilitate transport. The device is compact and portable all in one Polymeras Chain Reaction system for DNA and RNA detection integrating a thermocycler and a fluorometer.
The device incorporates a fully configurable thermocycler and a fluorescent detector targeted for point of care diagnostic applications. An all in one Fluorescent End Point PCR solution within a single low cost portable case allowing on site analysis without the costs of Real Time PCR detectors and with multiple usage scenarios such as isothermal and PCR based amplification optional dual wave length detector for multiplex PCR analyses.
3. Mini Kit for the detection of Feline Calicivirus
Although several different viruses and bacteria can cause respiratory disease in cats, calicivirus is one of the more common infectious agents isolated in cats with a respiratory infection.
Feline calicivirus (FCV) is a virus that is an important cause of upper respiratory infections and oral disease in cats. This virus infects cats throughout the world, and can cause disease in both domestic and exotic species of the cat family. There are different strains of FCV, and the virulence or severity of the disease caused by them may vary significantly.
The Mini kit method is based on detection that combines nucleic acid extraction and target nucleic acid amplification by end point PCR on a portable device. The Mini kit has numerous advantages over other molecular diagnostic tools that make the assay suitable for a rapid and simple detection of pathogens in the field: easy to use, easy to interpret, little equipment required, high sensitivity, high specificity, rapidity, usable for technical personnel without specific training in molecular biology and applicable to different biological matrices.
4. Mini Kit for the detection of Feline Herpesvirus
Feline herpesvirus is a major cause of feline morbidity, following the exposure to the virus, virtually all cats become persistently infected and many of these will develop recrudescent disease on one or more occasions during their lifetime. Acute ocular herpetic disease manifests as conjunctivitis, corneal ulceration and keratitis, and can be severe and painful. Repeated bouts of recrudescent ocular disease can lead to progressive corneal pathology that can be ultimately blinding in affected cats. FHV has a worldwide distribution, with reported exposure rates in some cat populations of up to 97%. As such it is a significant cause of clinical disease in the global cat population. The Mini kit method is based on detection that combines nucleic acid extraction by DNAzol protocol and target nucleic acid amplification by end point PCR on a portable device. The Mini kit has numerous advantages over other molecular diagnostic tools that make the assay suitable for a rapid and simple detection of pathogens in the field: easy to use, easy to interpret, little equipment required, high sensitivity, high specificity, rapidity, usable for technical personnel without specific training in molecular biology and applicable to different biological matrices .
5. Mini Kit for the detection of Bovine Papilloma Virus
Equine sarcoids are common, non-metastasising, locally aggressive skin tumours that are induced by bovine papillomavirus types 1 and 2 (BPV-1, BPV-2). They are the most common neoplasm in horse, representing 67% of all equine tumours and for this reason they are of considerable veterinary importance. In contrast to BPV-induced bovine papillomas, which usually resolve spontaneously, equine sarcoids mostly persist and tend to recrudesce following treatment with considerable expense for horse owners. Sarcoids can become ulcerated and infected and, when occurring near the eyes or on the eyelids, can impair vision so that their progression to more aggressive types can compromise the use and welfare of affected animals. The presence of sarcoid is a major cause of “failure” at pre-purchase examination. In spite of the fact that the condition is restricted to the skin, many horses are destroyed every year as a result of fly worry, infection, blood loss and failure to thrive or for ‘social’ reasons. Some are destroyed because they are unsaleable even though they may perform well at their chosen discipline. The presence of BPV DNA in PBMCs suggests a possible contribution to virus spread. Virus may spread in utero from infected mares to their foals, as has been shown in cattle. Moreover, infected PBMCs may propagate disease within one individual, as they migrate to sites of inflammation where they may take up the virus and function as a carrier. There is no routine tests for the molecular detection of BPV even if it will be strongly recommended to relate this finding to the observed recurrence after surgery. A penside test is particularly required to differentiate proliferative lesions caused by BPV. Furthermore, a diagnostic tool able to evidence bovine papilloma virus (BPV) DNA in PBMCs and in apparently normal skin around equine sarcoids after surgical removal, will be vital to under take a proper therapeutic and prophilactic approach.
The Mini kit method, developed within POC4PETS project, is based on detection that combines nucleic acid extraction by DNAzol and target nucleic acid amplification by end point PCR on a portable device. The kit is standardized for BPV1 and BPV2 detection in horse samples. The Mini kit has numerous advantages over other molecular diagnostic tools that make the assay suitable for a rapid and simple detection of pathogens in the field: easy to use, easy to interpret, little equipment required, high sensitivity, high specificity, rapidity, usable for technical personnel without specific training in molecular biology and applicable to different biological matrices including skin biopsies, swabs and blood.
Comments:
Ennebi elettronica in collaboration with the University of Verona and Bologna optimised the Fluorescent End Point PCR system, named DNA/RNA Finder and developed associated diagnostic kits for pathogen detection.
To ensure an affordable analysis of device market on today’s scenario, ENNEBI has been written a new business plan. Input help to identify target and limit, analysis and output points show new evaluation of market. Commercially existing devices are either not portable or do not integrate a fluorometer for End Point PCR. There are some “portable” real time PCR systems but they are costly and somehow fragile. We did not find any integrated end time PCR system.
Device total power consumption is estimated to be lower than 90W. Similar devices with fully configurable fast thermal cycler are shown to have power requirements of about 200W or more. Limited power consumption allows this device to be powered from car dc socket thus allowing an increased flexibility where no wall power is available.
The previous business plan has been taken as refererence for the new one. A scalable and configurable device allows a cost reduction; Ennebi estimates an average price of 3200 euro for the Fluorescent End Point PCR system.
New multiplex kits are under development to allow multiple pathogens detection with a single reaction. These version of the kit will be targeted to the detection of animal (pets) pathogens but it can be later validated for IDV or alimentary purposes to extend market.
Ennebi Elettronica has a sales network for electromedical devices for dentistry, and is evaluating the use of the device as IVD or first analysis point for dentistry. Furthermore a part of same sales network will be used for veterinary market.
No patent will be registered. The technologies used are known and non-patentable. Enneby will register the trade name.
Vetgenomic's foreground
1. Leishmania DNA amplification and electrochemical detection kit
Diagnosis of zoonotic canine Leishmaniosis is complex due to the broad clinical spectrum. Moreover, it is important to differentiate Leishmania infection from clinical disease and to apply different diagnostic techniques for each state. The presence of low antibody levels is not necessarily indicative of the disease and further work‐up is necessary to confirm or exclude clinical leishmaniosis. Detection of Leishmania DNA in tissues by PCR allows sensitive and specific diagnosis of infection, but is of limited use for the need of skilled workers, the high cost and the fact that samples must be send to a reference lab. FP7 POC4PETS project developed a Kit for the rapid, accurate and cost‐effective detectionof Leishmania easily adapted for to be performed on clinics. It will combine the advantages of PCR (direct detection of the parasite) and serology (low cost and point of care device) to be easily adapted to the daily work of the clinician
The Leishmania DNA amplification and detection kit is a simple and easy way to detect Leishmania infantum DNA. The Leishmania kit is based on RPA isothermal amplification (Recombinase Polymerase Amplification, TwistDxTM) and involves a set of specific homemade designed primers that amplify the kinetoplast, a specific DNA region of the Leishmania genome. These specific primers are respectively labelled with Thiol and Biotin and then are conjugated with AuNps and Magnetic Beads. The kit combines the isothermal amplification (10’ at 37ºC) and the detection method based on electrochemical reading of the amplified product. To validate each assay an internal control is needed. For this purpose a region of the canine 18sRNA gene (endogenous control) is amplified to check for inhibitory effects or problems with the sample to validate negative results.
2. Leishmania DNA amplification and lateral flow detection kit
Isothermal amplification of DNA is rapid, cost effective, easy-to use and more tolerant to inhibitory components from a crude sample compared with PCR, showing at the same time equivalent or higher sensitivity and reliability in clinical diagnosis. The Leishmania DNA amplification and detection kit is a simple and easy way to detect Leishmania infantum DNA. The Leishmania kit is based on RPA isothermal amplification (Recombinase Polymerase Amplification, TwistDxTM) and involves a set of specific homemade designed primers labelled with FITC and Biotin, that amplify the kinetoplast, a specific DNA region of the Leishmania genome. The kit combines the isothermal amplification (10’ at 37ºC) and the detection method based on immunochromatographic detection method. To validate each assay an internal control is needed. For this purpose we amplify a region of the canine 18sRNA gene (endogenous control) to check for inhibitory effects or problems with the sample to validate negative results.
Comments:
Vetgenomics activity is focused in the veterinary genetic diagnostic fields, currently offered as an externalized service to the veterinary clinics. However, the strategy for the future growth of the company is focused on the development and commercialization of genetic diagnostic devices as final products with a greater added value, instead of services. As a proof of concept, Vetgenomics has been focused on the development of a biosensor to detect Leishmania DNA at the point of care, which takes advantage of their previous experience in Leishmania genetic diagnostic. POC4PETS has allowed Vetgenomics to successfully fulfill the main objectives proposed in the project, through the application of emergent technologies for the detection of pathogen DNA in a more rapid, accurate, and cost-effective way compared with traditional approaches: the prototype developed is based on isothermal amplification followed by lateral flow or electrochemical detection of the amplified product. At the technological level, the biosensor developed by Vetgenomics is intended to overpass the limitations of current techniques of diagnostic, providing clear advantages in terms of sensitivity and in detection of false positives (detecting the pathogen directly, no antibodies against it) or false negatives (detection of phase of acute infection prior to seroconversion).
The collaboration with technological partners as RTD performers allowed Vetgenomics to evaluate emergent and innovative technologies and the technology behind the biosensor prototype has been protected to establish the prior-art of the technology (EP14382266/P10398EP00).
Canine leishmaniasis is endemic in Southern Europe. It is estimated that there are 2.5 million infected dogs in southern Europe (4 CVBD Symposium Proceedings 2009), 80% of which are asymptomatic without clinical signs and act as sentinels of infection. In a reference veterinary hospital analysis for Leishmania infantum is requested in 5% of the visits (Veterinary Teaching Hospital, UAB, 2009), although the prevalence of infection varies in each country. Moreover, in recent years, the vectors that transmit the infection (sand-flies) are spreading to northern Europe and it is an emerging disease in some breeds from the USA (foxhounds). Moreover, movement of pets for holidays increases the need for monitoring the infection. The dog population at risk is estimated to be near 46 million dogs in Europe.
In human health Leishmaniasis threatens about 350 million men, women and children in 88 countries around the world. As many as 12 million people are believed to be currently infected, with about 1–2 million estimated new cases occurring every year. Moreover, the disease is an emerging thread as a coinfection in HIV patients and in people travelling to endemic areas.
Leishmania infantum is currently the only significant causative agent of visceral and cutaneous human leishmaniasis endemic in Europe. Its high prevalence in asymptomatic humans and in the widespread reservoir host (the domestic dog) means there is a high risk of emergence in parts of Europe further north. Risk of emergence or re-emergence of leishmaniasis in Europe are associated with different scenarios, such as the increasing worldwide travelling of people and domestic dogs, the re-emergence of disease in the Mediterranean region caused by an increase in the number of immunosuppressed people, and the natural spread of the endemic species of the pathogen from the Mediterranean region of Europe to neighbouring temperate areas (Temperate broadleaf forsts’ biome), where there are vectors without the disease. Additionally to the Mediterranean region, the vectors of Leishmania infantum are also abundant in northern Spain and central France, and small numbers occur as far north as Paris and the upper Rhine valley in Germany. The occurrence of “vectors without disease” poses a significant risk for the emergence of leishmaniasis in temperate regions of Europe. The domestic dog is the only reservoir host of major veterinary importance, and in Europe there is a large market for prophylactic drugs and treatment of canine leishmaniasis caused by L. Infantum. According to CVBD Forum (Bayer Healthcare Animal Healthcare) clinical diagnosis of canine leishmaniasis (including non-specific laboratory parameters) is unreliable for the following reasons:
- More than 50% of dogs with proven established infections are apparently healthy on clinical diagnosis (asymptomatic).
- When present, clinical signs can be variable and mimic those caused by other diseases.
- Atypical forms are reported in dogs.
For final diagnosis three types of tests are available:
- parasitological methods aiming at the detection of parasites (100% specificity, but are not very sensitive)
- serological methods with detection of anti-leishmanial antibodies in the blood. Seroprevalence, regarded as an intermediate measure between disease and the prevalence of infection, is influenced by the fact that on the one hand serology can reveal a high proportion of asymptomatic carriers which represent half of all seropositive animals; and on the other hand the limitation due to the long periods of serological latency. Antibodies are often detectable only several months after infection. Furthermore, a proportion of seropositive infected dogs spontaneously converts to negative, or does not develop specific antibodies at all. Thus a maximum estimated sensitivity of the most efficient technique (IFAT) hardly reaches 80% and only during a few months after exposure to Leishmania in the previous season.
- molecular biology techniques like the polymerase-chain reaction (PCR), and more recently quantitative PCR (qPCR), detecting the parasite DNA in host tissue after multiplication. PCR provides a highly sensitive and specific diagnostic for Leishmania, although limited to centralized reference laboratories due to the equipment and trained personnel required.
The Leishmania biosensor will be a product clearly differentiated from the actual portfolio of Vetgenomics and will represent a novelty with greater added value for the veterinary clinician. Its simplicity will allow implementing the assays to be used as pen-side devices in the veterinary hospitals, with two main advantages for the clinician: by two main reasons:
- By the change it will represent in the value chain compared to the need of reference labs
- By the added value of the analysis and the greater reimbursement for the professional due to the intrinsic characteristics of the device, such as integration, portability, ease of use, no need for trained personnel or specialized equipment and especially by the time to result, as it is shown in the value chart of the new product .
At the beginning of this project, a market survey was designed and several veterinary practices in Germany and Spain received a questionnaire regarding general occurrence of leishmaniasis, routinely used diagnostic methods and willingness to test a new diagnostic kit for the direct Leishmania detection. Results of this survey showed that incidence of dogs with leishmaniasis per year is 18.3% in Spain. The incidence is lower in Germany and was mostly associated to travel history of the dogs. More than 85% practices in Germany and 70% practices in Spain agreed that direct detection of the parasite instead of anti-Leishmania antibodies (serology) offers advantages in the diagnosis and PCR testing was evaluated with highest priority (75.0%) concerning its use as diagnostic procedure for canine leishmaniasis. Summarizing, 71% of practices in Germany and 85% of Spanish practices think it could be useful to have a new diagnostic kit to detect Leishmania DNA (instead of anti-leishmania antibodies) in house. In both countries veterinary practices are willing to test a new diagnostic kit (71% in Germany and 90% in Spain).
The potential market for the Leishmania biosensor is greater than the market for genetic diagnostic centralized in reference laboratories with sophisticated equipment and skilled personnel. Geographic barriers disappear when the analysis is done in the veterinary clinic, so local market becomes global and opens a potential market of more than 200,000 clinicians only in Europe. It is worthy to consider also at this point that the first vaccine for canine leishmaniasis has been recently approved in Europe (CaniLeish from Virbac; EMA/296955/2010; EMEA/V/C/002232) and is being first commercialized in Portugal, to be afterwards extended to Spain, France, Italy and Greece. CaniLeish is to be used only in ‘leishmania-negative’ dogs. The need of discarding Leishmania infection before vaccination increases the market for a POC biosensor to detect Leishmania DNA.
A comprehensive field study including interviews with a representative sample of veterinarians in Central and Southern Europe was subcontracted to Trifermed (as described in B2.4) to confirm three key aspects: (1) knowledge and sensitivity to the existence o the disease, (2) perception of incidence and (3) action protocol used for both diagnosis and treatment. The study has been focused on the German and Spanish markets, for their representativeness, size and validity for the conclusions sought for the commercialization of the device. It is worthy to note that the success in this project will not only consolidate the Leishmania biosensor as the first product for Vetgenomics and a new business unit in the company, but also place Vetgenomics as a partner for technology owners to jointly develop future applications and innovative products for the veterinary diagnostic market. The most important customers are veterinary medical clinicians, especially those placed in Mediterranean Europe, and from Central and North Europe for extending commercialization. The main advantages of the biosensor for veterinarians are rapid turnaround, greater control over the methods used, and potentially lower cost than analysis in centralized laboratories. Moreover, an integrated disease management solution that leverage the advantages of both animal-side and laboratory testing by offering both rapid diagnostic for use in the clinic and outside laboratory services would represent a market opportunity. Vetgenomics is offering Leishmania diagnostic by qPCR as a reference lab, which allows the company to assist the clinician with both the in-house (POC) and outsourced (qPCR) assays. Vetgenomics will offer the possibility of a qPCR confirmatory analysis in its own reference lab at a special rate for the biosensor customers.
At the commercial level, it is worth to consider the potential market, not only in veterinary but also even in human health. Worldwide, leishmaniasis is one of the most important vector-borne diseases (besides malaria, lymphatic filariosis and sleeping sickness) and is the third zoonotic disease (animal disease transmitted to man) in global importance, where the dog acts as the main reservoir of the causative agent. Commercialization strategy needs a technological partner for final product development, and a strategic distributer to reach the maximum market share. Vetgenomics is subcontracting these activities to Trifermed (www.trifermed.com) a business development company introduced in national and international markets, to better define the business development programme and the global expansion strategy.
Industrialization of the prototype will be outsourced. Vetgenomics intends to commercialize directly the biosensor in the Mediterranean European market, and will negotiate commercialization license with worldwide leaders in veterinary diagnostic for Central and North Europe and US. Vetgenomics will also evaluate the application to other CVBD pathogens or even to the human health market.
The estimated time to market is three years from now on, considering 3 phases:
- Initial exploitation (Year 1&2): for the industrialization and commercialization of the launching units
- European Expansion (Year 3&4): commercialization of the biosensor in Europe
- Global Expansion (Year 5): commercialization of the biosensor at a global scale.
While it is a growing market, it is not a market without competitors. There are at least 75 vendors worldwide that market rapid test kits for emerging infectious diseases in human health and they could enter easily the veterinary market, although the smaller global volume. The current market leaders are companies that have invested in sophisticated new technologies and those that have established relationships with governments and international aid agencies. Main competitors in the veterinary market for the Leishmania biosensor would be:
- Multinational companies devoted to veterinary diagnostic (ANTECH, IDDEX) opening new business units complementary to serology POC assays. They are currently offering DNA detection of the pathogens by means of PCR in central reference labs.
- Immunoassays for indirect detection of infection, such as IFAT (ImmunoFluorescence Antibody Test) or ELISA (Enzyme-Linked ImmunoSorbent Assay). Some of the assays are LEISH and IFI LEISH (ID VET), SNAP LEISHMANIA (IDEXX), SensPERT (MATERLAB) or Leishmania IFA Canine IgG Antibody Kit (FULLER Laboratories).
Competitors are actually offering POC devices based on serology. The main advantage over competitor products is that the biosensor will detect DNA of Leishmania instead of an antibody, which indicates the presence of the pathogen, and thus current infection and not a previous exposure to it, and distinguish between acute disease, asymptomatic infections, relapses and cured cases.
As in other market segments, large companies may prefer to use alliances with smaller companies when adding non-traditional products such as diagnostics or novel biological to their product portfolios, which would also represent a market opportunity for Vetgenomics.
Other competitors would be the current market leaders in molecular diagnostic, companies that have invested in sophisticated new technologies and those that have established relationships with governments and international aid agencies.
A dipstick format was developed for detection of PCR products by hybridization with a gold-conjugate probe, and a PCR oligochromatographic leishmania-specific test is being commercialized using this technology (Leishmania OligoC-test, Coris Bioconcep) (Srividya et al., 2012). In 2011, the US Food and Drug Administration (FDA) approved the Smart Leish PCR assay (Cepheid USA), a qualitative real-time PCR test for diagnosing individuals with cutaneous leishmaniasis. Both systems didn’t overcome the limitations of PCR for which sophisticate and expensive equipment are needed to perform the precise and repeated heating cycles required.
On the other hand, a new POC assay for Leishmania DNA detection has been recently developed by BIOGAL (Israel) based on isothermal amplification and lateral flow detection. This product could be considered a direct competitor, but the biosensor prototype developed by Vetgenomics in this project represents some technological advantages, such as temperature of amplification (37ºC over 65ºC), time to result (20 minutes for amplification and detection vs 1.5h) and the possibility of semiquantitative approach when electrochemical detection is applied. However, it could represent a market opportunity, as an advanced player opening the new market for these POC devices.
Main competitors are selling POC for Leishmania based on serology (detecting antibodies) at 10-15 € or quantitative PCR (detecting Leishmania DNA) in central labs at 30-35 €. BIOGAL price for the new developed DNA POC assay will be 35 €. The biosensor developed by Vetgenomics intends to be an innovative product in the veterinary market, detecting Leishmania DNA (as qPCR in central labs) in a POC device (as serology) at an average price of 17 €.
The biosensor for Leishmania should be sold at € 17/unit to be competitive in front serological POC devices that are already in the market, considering a production cost of € 4/unit plus a commercialization cost of € 2/unit. There is an estimated population of 18 million dogs in southern Europe (Spain, Italy, France and Portugal), 26 million in the rest of Europe and 70 million in the USA.
Given an infection rate of 10%, the volume of potential market is:
• 34 million € per year in endemic areas (Mediterranean Europe) (2 million tests at 17 € each)
• 200.6 million € considering the European and USA markets (11 million tests at 17 € each).
The following table shows an estimate of overall annual market volume for the Leishmania biosensor, considering the number of dogs in each region and a price of 17 € for the device.
With an objective of 2.5% share for the first year of commercialization rising up to 10% after 5 years, revenues for Vetgenomics are be as shown in the next figure and are estimated in different scenarios, depending on the geographic market: Mediterranean Europe (worst), Europe (normal), Europe and US (best scenario).
In this way, with a launching price of 17 € and a market quote of 2.5% the first year of commercialization of the biosensor, incomes for Vetgenomics are estimated in 850,000 € (50,000 units) in Southern Europe and 4.85 million € (11 million tests) when commercialization is extended to the European and North American markets in the best scenario.
Two people have been hired for the development of the prototype during the project, added to the SME owners: A Senior scientist with more than 10 years expertise in the field of Leishmania genetic diagnostic and canine genomics related to resistance to infection as the project manager and a young researcher responsible for the laboratory assays.
VETGEN is financing the first steps of the final product development with own resources, and intends to apply for Horizon 2020 dedicated SME Instrument - Phase 1 2014 and to raise external finance (bank loan and or Venture Capital) to develop the detection of DNA without amplification, which was the more risky proposal in this project. Results based on impedance were promising, but further work is needed to integrate it into a portable device.
Skuldtech's (now ACOBIOM) foreground
1. Point of care diagnostics for the rapid detection of Parapoxviruses
Zoonotic poxviruses (ZPs) fall into the category of neglected zoonoses. No universal diagnostic tests are in use; when available, is mainly performed at academic institutions or in public health reference laboratories. The majority of ZP are considered an occupational hazard even if an increasing number of cases in children exposed to infected animals in an occupational setting or during recreation. At present, there are no diagnostic tools available for the detection of zoonotic poxviruses. Rapid and reliable tools will be required at the point of patient care so that physicians can manage patients appropriately and avoid severe complications of infection in children and the immunocompromised; furthermore an early recognition of poxvirus-associated infections will allow to reduce the spread of virus between companion animals and their owners. FP7 POC4PETS developed a Lateral flow strip test for the rapid detection of Parapoxviruses (PPV) in animals allowing differential diagnosis from other life threatening infections.
The Parapoxvirus Lateral Flow Detection kit is designed for the detection of Pseudocowpox virus (PCPV), Bovine papular stomatitis virus (BPSV) and orf virus (ORFV) in bovine and ovine.
Parapoxvirus detection kit is based on the enzymatic amplification of a specific Parapoxvirus sequence by the PCR (Polymerase Chain Reaction) technique. It involves a set of specific primers that amplifies a specific DNA fragment of different Parapoxvirus species.
A positive control for the DNA sample preparation, the PCR reaction and the detection, called “internal control”, is present in each reaction. It is a conserved sequence of the gene encoding the cytochrome c oxidase subunit I (COX1).
The detection and the visualization of the amplified products are carried out by lateral flow immunochromatographic assay with dipsticks. The parapoxvirus infection is evaluated by the presence of a dark grey band on the dipstick.
The immunoassay-based lateral flow dipstick test developed is intended for the simple, sensitive and rapid field diagnosis of parapoxvirus in bovine and ovine. It is simple to use and provides results in a few minutes.
2. Point of care diagnostics for the rapid detection of Orthopoxviruses
At present, there are no diagnostic tools available for the detection of zoonotic poxviruses. The majority of ZP are considered an occupational hazard even if an increasing number of cases in children exposed to infected animals in an occupational setting or during recreation. In late 2008-early 2009 an outbreak of pet-associated Orthopoxvirus, CPXV occurred in the EU resulting in >30 human cases in France, Germany and the Netherlands (ECDC report 11/02/2009). Infections was due to either direct contact with infected rats, or contact with other animals (mangoose, cat) that had presumably became infected following contact with a rat or other small rodent harbouring CPXV.
Pen-side test for ZP will be important in both veterinary and human diagnostics, in cases when differential diagnosis from other skin tropic infections, especially vesicular diseases and those other life-threatening infections which are all mandatory for risk assessment. Rapid and reliable tools will be required at the point of patient care so that physicians can manage patients appropriately and avoid severe complications of infection in children and the immunocompromised; furthermore an early recognition of poxvirus-associated infections will allow to reduce the spread of virus between companion animals and their owners. FP7 POC4PETS developed a Lateral flow strip test for the rapid detection of Orthopoxviruses (OPV) in animals allowing differential diagnosis from other life threatening infections. The Orthopoxvirus Lateral Flow Detection kit is designed for the detection of Vaccinia virus (VACV), Cowpox virus (CPXV), monkeypox virus (MPXV) and Ectromelia virus (ECTV) in bovine, feline and rodents. Orthopoxvirus detection kit is based on the enzymatic amplification of a specific orthopoxvirus sequence by the PCR (Polymerase Chain Reaction) technique. It involves a set of specific primers that amplifies a specific DNA fragment of different orthopoxvirus species. A positive control for the DNA sample preparation, the PCR reaction and the detection, called “internal control”, is present in each reaction. It is a conserved sequence of the gene encoding the cytochrome c oxidase subunit I (COX1). The detection and the visualization of the amplified products are carried out by lateral flow immunochromatographic assay with dipsticks. The orthopoxvirus infection is evaluated by the presence of a dark grey band on the dipstick. The immunoassay-based lateral flow dipstick test developed is intended for the simple, sensitive and rapid field diagnosis of orthopoxvirus in bovine, feline and rodents. It is simple to use and provides results in a few minutes.
Comments:
POC4PETS outcomes allowed Acobiom to benchmark a new diagnostic approach on the basis of Acobiom technologies for quick cost effective and easy to handle kit able to identify the aforementioned infectious agents. It will therefore increase Acobiom pipeline and provide the opportunity to reach markets which are long to penetrate. The business model developed by Acobiom for dealing the new diagnostic kits is to include Acobiom manufacturing and distribution licensing.
Through POC4PETS, Acobiom was able to validate its genomics technologies and expertise to launch on the market new innovative diagnostic in the area of infectious disease in veterinary field. Three kits for zoonotic poxviruses detection have been developed, evaluated by beta testers and are now marketable: one for orthopoxvirus detection, a second one for parapoxvirus detection and a third one for specific orthopoxvirus detection in humans. IPR exploitable measures for the above mentioned kits are intended to be undertaken in the near future.
Acobiom will manufacture in-house the new diagnostic kits. A specific manufacturing line will be set-up. This manufacturing will be fulfilled in France for EU and US and will be assembled in local countries for South America and Asia. Acobiom has already some partnerships and business contact in EU, USA and Brazil to develop distribution licensing for such kind of innovative diagnostic. However POC4PETS project will also help to set-up EU commercial partnership increasing Acobiom EU network through POC4PETS partners. First diagnostics kit launch on the market is forecasted for 2015 including health authorities’ registration and approval as well as marketing and outcomes dissemination.
Acobiom will use already existing distribution channel through partnerships with companies or laboratories involved in veterinary diagnostic testing as Royal Canin (Mars group, US), Vetoquinol (France), CEVA (France). Acobiom is currently in contact with these companies.
Pricing is very low to be competitive and to reach the aforementioned markets which are depending on 3 lines: low cost, quick and specific, easy to handle and do not required specific equipment.
New diagnostic kits developed through POC4PETS project are innovative diagnostics able to reply to unmet needs especially within the field of quick, specific, easy to handle and low cost diagnostics kits.
There are no available kits on the market (no competitors), diagnosis is only made in research laboratories at an average price of 50 €. The kits, subjected to field validation, are new and innovative products, and are able to detect Zoonotic poxvirus DNA using POC devices at an average price of 8€.
Acobiom estimated its revenues on the basis of the new outcomes of POC4PETS and for 5 years of sales. Considering the European market only, expected revenues are 1,2M€ and 4,2 M€ in respectively 1 and 5 years. Considering the global market, including USA, Japan and other extra EU markets, expected revenues are 3M€ and 13 M€ in respectively 1 and 5 years.
For Europe and for the global market, assuming an average scenario and different market penetration rates, the penetration rates have been estimated at low rates because of the size of the markets and the barriers to entry of the market.
Through POC4PETS project, Acobiom directly hired one person, a sale manager (Laurent PASCAL, the 1st March 2014). At long term, POC4PETS project will allow Acobiom to hire approximately 6 additional employees dedicated to health regulation and marketing.
Concerning the zoonotic poxvirus, the market has been appraised on the basis of the population of cats which account for 130M and with a growing population of exotic pet animals and fancy rats. In humans, poxvirus infections are neglected zoonoses and are undoubtedly under-reported, so the true impact of these diseases remains unknown.
To meet the expectations of veterinarians working with farm animals and consequently to widen Acobiom market over companion animals, the kits would need to be further improved in terms of simplicity, rapidity and reduced costs. Indeed, an additional important step in the development of POC diagnostic devices for the detection of poxviruses is the replacement of the PCR step by a LAMP amplification step which will be combined to the LFD assay. Nevertheless, a first commercial approach has been undertaken to the french veterinary companies.
Potential Impact:
POCS4PETS consortium recognized the importance to facilitate and accelerate the development of effective tools to counteract companion animal diseases. It recognized the importance of the potential infectious disease hazards linked to close interactions with pets, especially for immune-comprised individuals. Others at higher risk for severe zoonotic infection include those with particularly prolonged animal contact or occupational exposure, such as pet care-takers, veterinary professionals, animal handlers and laboratory researchers.
In the EU there are more than 200 million pets in total (204,947,400) and 72 million homes have at least one pet animal. According to the European Pet Food Association (FEDIAF), there are: 66 million cats, 60 million dogs, 9 million ornamental fish (number of aquaria), 39 million cage birds, 21 million small mammals (Fig 1 - Source: FEDIAF Facts and Figures 2012).
There is an important socio-economic value of companion animals. This can be in part calculated directly as reduced number of medical consultations and more rapid recovery after surgery for pet owners, and indirectly by improved quality of life and thus better function in society. Scientific studies have recognized that pets provide social support and positive effects are generated by human-animal interactions. One of the most important positive aspects of companion animals is a direct effect on the owner’s health, both socially as well as directly related to health issues and economy.
It has been proposed three mechanisms to explain the association between companion animal ownership and health benefits for humans:
1. There is no real association between the two, but cofactors such as personal traits, economy and desire to own a pet
2. Pets may enhance social interactions with other people, thus providing an indirect effect on wellbeing
3. Pet ownership may exert a direct effect on human health and wellbeing through the nature of the relationship
The pets play a key role in every stage of human development: child, adult, elderly.
- Child: for the child, a pet animal encourages a sense of responsibility, caring and communication. The relationship instils confidence and friendship - qualities, which can endure and grow as the child moves on through life. Children’s exposure to companion animals may also ease anxiety. For example, one study measured blood pressure, heart rate, and behavioral distress in healthy children aged 3 to 6 at two different doctor visits for routine physicals. At one visit a dog (unrelated to the child) was present in the room and at the other visit the dog was absent. When the dog was present, children had lower blood pressure measures, lower heart rates, and less behavioral distress
- Adults: health benefits of pets can, although these factors overlap to some extent, be divided into therapeutic, psychological, physiological and psychosocial. For adults, the pet providing companionship for those living alone; giving stimulation to make contact with others as in, for instance, an owner walking his dog. Compared with people without a pet, people who keep a pet have been reported to require fewer visits to the doctor with direct economic impact and a consequent benefit to public health services.: in a country like Norway, with a population of 4 mill inhabitants, this means a net saving of € 88 million annually for the ten largest hospitals (average cost/day estimated low: € 625); for Belgium with 10,5 mill inhabitants, this equals € 231 million annually. A large German study collected pet information (dog, cat, horse, fish, bird or other pet ownership) from over 9,000 people at two different times (1996 and 2001). The survey included a number of health, economic, and labor issues, so that respondents would not realize the researchers’ interest in a link between pets and health. Researchers found that people who said they had a pet in both 1996 and 2001 had the fewest doctor visits, followed by people who had acquired a pet by 2001; the group of people who did not have a pet at either time had the highest number of doctor visits.
Pet owners adapt more quickly to stress associated with bereavement and other adverse events; have stronger emotional stability and maintain a generally sounder state of health. Bonding with a companion animal was credited with leading to beneficial effects on the human cardiovascular system. In patients with hypertension who were under stress, patting and talking to a pet dog or cat may have prevented increased blood pressure. In a study investigating risk factors for cardiovascular disease, it was found that dog owners had lower systolic blood pressure and lower levels of serum triglycerides than non-dog owners. Dog owners are physically more active, have lower blood pressure and cholesterol levels which helps ease the strain on healthcare resources. Companion animals may improve heart health by lowering blood pressure and regulating the heart rate during stressful situations. In a 2002 study, researchers measured changes in heart rate and blood pressure among people who had a dog or cat, compared to those who did not, when participants were under stress (performing a timed math task). People with a dog or cat had lower resting heart rates and blood pressure measures at the beginning of the experiment than non-pet owners. People with a dog or cat were also less likely to have spikes in heart rates and blood pressure while performing the math task, and their heart rates and blood pressure returned to normal more quickly. They also made fewer errors in their math when their pet was present in the room. All these findings indicated that having a dog or cat lowered the risk of heart disease, as well as lowering stress so that performance improved.
- Elderly: The direct health effects are similar to what is described for adults. Elderly people with dogs visit physicians 21% less often than do those without a dog. Elderly people constitute a segment of the population at heightened risk for a variety of physical and emotional problems. In one study, elderly individuals that had a dog or cat were better able to perform certain physical activities deemed “activities of daily living,” such as the ability to climb stairs; bend, kneel, or stoop; take medication; prepare meals; and bathe and dress oneself. The use of specially trained companion animals in institutions are increasingly being used, and the positive effect of this intervention has been documented.
In addition, there is increased awareness of the value of companion animals in animal assisted therapy for different groups of people and may mitigate the stresses of urban living and counteract what has been called “nature-deficit disorder.”
The social support provided by a pet might also encourage more social interactions with people, reducing feelings of isolation or loneliness. For example, walking with a dog has been found to increase social interaction, especially with strangers, compared to walking without a dog.
Throughout Europe, an estimated 550.000 direct and indirect jobs are generated by pet ownership such as veterinarians or breeders or connected supply industries. Indirect employment is an estimation based on an approximate 200.000 veterinarians in Europe, an estimated 60.000 pet specialist stores, medication/vaccination, suppliers to the pet food industry, pet accessories industry, direct employment trade shows, pet press, breeders, pet food industry: 50.000 animal welfare organizations, transport. The pet food industry and related supply and services represent a combined annual turnover of over € 24 billion.
The global dog and cat animal health market was valued at approximately $ 5 000 million in 2005 and accounted for nearly 40 % of the global animal health market. Dog and cat sales in Europe were approximately 35 % = $ 1 750. As for the future the companion animal health market is expected to account for almost 60 % of the growth in the animal health market. The dog and cat market is expected to grow most strongly in the geriatric and juvenile areas because pets live longer and their owners are more willing to spend on them.
The etiology and prevalence of many diseases of companion animals are still unknown since these pathologies are often difficult to detect for their long period of latency or for their asymptomaticity. For these reasons it is necessary to screen large numbers of animals and so new tests are continuously needed and developed. The independence from the major centers of analysis also allows the diagnostic kits’ market not to be constrained by geographical boundaries. Today there are more than 35 000 vets in Europe who work with companion animals. Most of them are general practitioners, many of whom have additional qualifications like national specialization, etc. At present, 50% of the veterinarians who graduate will work in companion animal practice. (Source: The economic importance of companion animals FECAVA - February 2007).
While clinical practice for companion animals is the core of the industry, providing treatment and prevention services to pet-owning households is a matter still not properly considered, with implications for both human and animal health. It can easily be assumed that the growing awareness on animal health and benefits of early detection and preventive medicines will drive the demand for veterinary diagnostic kits over the next few years.
Such technological breakthrough achievement will provide and consequently enhance the prevention, early diagnosis (and thereof a reduction of their economic impact) and control of a wide range of animal diseases so that it will in turns contribute to better preventing the human transmission, in case of zoonoses. Thereof, the public health impact will be benefiting in 2 directions, both in terms of i) global animal health as well as of ii) human disease prevention.
List of Websites:
http://www.poc4pets-fp7.eu
The necessity of finding innovative and improved diagnostics - to complement or substitute the available ones was considered strategic in the context of Animal Health Strategy (2007-2013). POCS4PETS consortium represented a newly-formed cluster covering the entire knowledge chain to guarantee a high efficiency in transforming scientific research into diagnostic innovation. POCS4PETS project has brought together three RTD performers working in the fields of animal health, veterinary, biotech and nanotechnology and three innovation-oriented SMEs working in the molecular devices, markers and detection systems. POC4PETS focused in filling the gaps of diagnostics availability and enhancing technology transfer to increase competitiveness of the Veterinary Diagnostic industry. The project plan was designed after identifying technology gaps for selected diseases through a preliminary analysis on the available tools carried out on manufacturers’ web sites. For Leishmania, current tools are mainly based on serology that document only the exposure to the agents, furthermore the presence of low antibody levels is not necessarily indicative of the disease and further work-up is necessary to confirm or exclude clinical leishmaniasis. Horse papillomavirus and zoonotic poxviruses are currently lacking proper diagnostic tools for routine testing. For canine and feline parvovirosis, the available diagnostic tools are represented by Rapid in-house test based on ELISA and immunochromatographic technology. These kits are characterized by a low sensitivity with consequently false negative results in the beginning and the end of diseases and in subclinical forms; they can also give false positive few days after vaccination. Laboratory tests for agent detection such as viral isolation or molecular methods (PCR, Real-Time) are expensive and time consuming. For Feline Herpesvirus and Feline Calicivirus Infections FHV/FCV pen-side tests for Ag detection are not available while serological tests based on ELISA and immunofluorescence antibody test (IFA) to detect IgG do exist, even if serology is not useful for the diagnosis and the control of feline upper respiratory tract diseases (URTD) infection due to widespread of vaccination. To fill the above gaps, the following goals have been reached: the POC4PETS consortium developed diagnostic technologies based on recent advances technologies (for the development of new rapid, sensitive and specific tests) for selected pet diseases which have significant incidence in Europe and at global scale that will enable the detection of nucleic acids of different pet animals’ pathogens. Such technological breakthrough achievement provides and will potentially enhance the prevention, early diagnosis and control of the selected animal and human diseases. The two years' project overall generated exploitable results and products for the benefit of the three SMEs involved in the consortium.
Project Context and Objectives:
Veterinary diagnostics is a key tool in the prevention and control of infectious diseases in animals. It is increasingly recognized that there is an urgent need for innovation in the animal infectious diseases testing field, bringing to market specific, rapid and efficiently validated diagnostic tests. Such development is commonly missing at European scale, as different constrains are currently tackling cost efficacy, the need to contribute to help interested SMEs working in the animal health field to bring to veterinary professionals i) early detection kits without need for externalization to specialized laboratories, ii) easy to apply and to transport (portable) equipment and detection systems iii) in field tests where the outbreak is taking place.
The current market of veterinary diagnostic tools is actually rather fragmented as it is requiring different step forwards in terms of different technical, technological and science - based constrains, which can be identify, among others: i) ability to perform serology and antigen detection effectively; ii) high sensitivity; iii) quantification ability; iv) ease of use by minimally trained or untrained users; v) low cost; vi) speed; vii) robustness and stability in the field makes them suitable for animal-side testing; viii) and ability to transport and store without the need for refrigeration.
Such challenges have been the base onto which POC4PETS project's multi-disciplinary approach is developed, as it tackled such innovations in order to contribute to deepening the knowledge of the selected animal diseases. Nucleic acid diagnostic techniques have become routine diagnostic tools in veterinary laboratories not only to make specific typing determinations but also to rapidly screen large numbers of samples during disease outbreaks. POC4PETS consortium included in its experimental work plan some of the most promising new technologies mainly based on the potential that biotechnology can improve the current diagnostics for veterinary pathogens of companion animal species.
The veterinary diagnostic industry is under continuous development and it is finding its own identity; furthermore, diagnostics is an area of veterinary medicine that has considerable growth potential and limited regulatory restrictions. In recent years, there has been a continuous and constant growth of the global veterinary devices and, so, of the diagnostics industry.
Currently, more than half of all diagnostic testing by veterinarians is still done at outside reference laboratories. However, as vets endeavor to provide better preventive healthcare in clinics increasingly devoted to small animal medicine, the uptake of in-clinic tests is likely to increase. Rapid immunoassays provide veterinarians with important information, which can be acted upon before the pet’s owner leaves the clinic. The POC4PETS project addressed such technological constrains through a strong collaboration among the SME participants and the complementary RTD performer skills.
The proposed work will enable the SME participants to improve their competitive position and to validate and fully exploit the research results provided by the RTD performers. The Key strengths of POC4PETS Work plan can be summarized as follows:
1) Adopting the concepts of low cost, simplicity, rapidity, stability for the new diagnostic tools. This was achieved by developing simple tests that allow field diagnosis as well as the discrimination of several diseases with the same sample and possibly the differential diagnosis in case of syndromes.
2) Improving the basic qualities required for the new tools in terms of specificity and sensitivity. This was achieved by probing detection systems ensuring high specificity. Sensitivity was mainly assured by methods based on the nucleic acid amplification and detection.
According to such challenges, the progress of POC4PETS project can be measured against:
1) Scientific objectives of the project:
a. Set up advanced, reliable and effective new diagnostic technologies suitable to be used at point of care to enable the detection of nucleic acids of pet animals’ pathogens.
b. Develop, standardize and disseminate novel testing procedures that can be used by “first line” diagnosticians.
c. Apply recent advances in technologies (nanotechnologies, fluorescence technologies and probing) for the development of new rapid, sensitive and specific pen-side and laboratory-based tests for selected pet diseases which have significant incidence in Europe and at global scale.
d. Make available diagnostic tests for pet animal diseases actually lacking specific diagnostic tools.
e. Implement veterinary diagnostic systems that confer robustness to the whole detection on the field without the needing of fully equipped laboratories.
In order to facilitate the achievement of such challenges, POC4PETS project created a manufacturing process which enabled the consortium to effectively perform field testing to evaluate the reliability and performance on real pathological samples under field conditions. This has been done in close collaboration with the SME industrial partners. As a consequence, a series of technical objectives has been foreseen as follows:
• To develop novel pen-side PCR assays for the detection of selected pathogens. This technology will be indeed implemented by further optimization of a portable real-time PCR instrumentation (DNA/RNA Finder) and luminescent labeling technology.
• To implement and apply nucleic acid detection technique without thermal cycling for Leishmania detection (with special regards to the isothermal amplification).
• To develop a mini-array and Lateral Flow kits for the rapid field diagnosis of zoonotic poxviruses. This will be achieved by the development of pen-side assays for rapid and simple detection of pathogens in the field.
• To develop in all the detection devices obtained, a specific and tailored sample preparation to improve, as a whole, the test sensitivity.
• To improve already available kits by means of at least new POC diagnostic tools.
• To provide new POC diagnostics for those tackled pet animal diseases that are currently lacking tools.
• To integrate, in a single system (kit) all the necessary reagents and tools allowing rapid on site diagnosis which wouldn’t require skilled personnel.
• To apply the so obtained new methods for the “on site” rapid field diagnosis for the robust confirmatory tests in laboratory.
Such POC4PETS evidence – based breakthrough was ensured by the combination with an economic business plan which lead to the identification of different main industry- driven strategic goals that, taking into account that the POC4PETS consortium as a whole, enabled the SME participants to gain a competitive advantage by the up taking of a innovative set of technologies.
Project Results:
The main S&T results (products) and company foreground obtained by the POC4PETS consortium can be summarized as follows:
WP 2 Rapid DNA extraction kit is based on magnetic beads (NBE's foreground)
WP 3 Fluorescent End Point PCR system (DNA/RNA Finder) (NBE's foreground)
WP 3 Mini Kit for the detection of Feline Calicivirus (NBE's foreground)
WP 3 Mini Kit for the detection of Feline Herpesvirus (NBE's foreground)
WP 3 Mini Kit for the detection of Bovine Papilloma Virus (NBE's foreground)
WP 4 Leishmania DNA amplification and electrochemical detection kit (VETGEN's foreground)
WP 4 Leishmania DNA amplification and lateral flow detection kit (VETGEN's foreground)
WP 5 Point of care diagnostics for the rapid detection of Parapoxviruses (ACOBIOM's foreground )
WP 5 Point of care diagnostics for the rapid detection of Orthopoxviruses (ACOBIOM's foreground)
For each POC4PETS technological innovation, a technical card (attached) has been produced and distributed during dissemination events and training (see attached POC4PETS training and dissemination video). Further details of generated POCPETS' foreground is given below.
Ennebi's foreground:
1. Rapid DNA extraction kit
The Rapid DNA extraction kit is based on magnetic beads. The super-paramagnetic particles are nominal 1 μm magnetic carboxylate-modified base particles (MG-CM) made by a core-shell process. They combine a fast magnetic response time with a large surface area and fast reaction kinetics. Typically, these particles are used in various molecular biology, nucleic acid isolation, research and clinical diagnostic immunoassay applications.
The innovative aspect of the product is the low, non-specific binding of serum proteins and other interfering substances, Non-leaching, encapsulated magnetite and high surface area per unit mass, high ligand binding capacity and slow settling rate in the absence of a magnetic field.
The Rapid DNA extraction kit has numerous advantages over other molecular diagnostic tools that make the kit suitable for a rapid and simple extraction of nucleic acid: easy to use, easy to interpret, little equipment required, rapidity, usable for technical personnel without specific training in molecular biology.
2. Fluorescent End Point PCR system
The developed system is a platform which enables end-point fluorescent PCR amplification of nucleic acids and their revelation by reading the initial and final value of fluorescence, the measured values have significance in the field of scientific research. The system is designed to detect emissions at defined wavelengths, through the excitation of a sample by means of a light source. The sample in the analysis is previously amplified through a heating unit that will perform the predetermined cycles in the process of set-up. The sections will be managed by a microcontroller and the user control via a touch screen TFT, size and weight of the system will be contained in such a way as to facilitate transport. The device is compact and portable all in one Polymeras Chain Reaction system for DNA and RNA detection integrating a thermocycler and a fluorometer.
The device incorporates a fully configurable thermocycler and a fluorescent detector targeted for point of care diagnostic applications. An all in one Fluorescent End Point PCR solution within a single low cost portable case allowing on site analysis without the costs of Real Time PCR detectors and with multiple usage scenarios such as isothermal and PCR based amplification optional dual wave length detector for multiplex PCR analyses.
3. Mini Kit for the detection of Feline Calicivirus
Although several different viruses and bacteria can cause respiratory disease in cats, calicivirus is one of the more common infectious agents isolated in cats with a respiratory infection.
Feline calicivirus (FCV) is a virus that is an important cause of upper respiratory infections and oral disease in cats. This virus infects cats throughout the world, and can cause disease in both domestic and exotic species of the cat family. There are different strains of FCV, and the virulence or severity of the disease caused by them may vary significantly.
The Mini kit method is based on detection that combines nucleic acid extraction and target nucleic acid amplification by end point PCR on a portable device. The Mini kit has numerous advantages over other molecular diagnostic tools that make the assay suitable for a rapid and simple detection of pathogens in the field: easy to use, easy to interpret, little equipment required, high sensitivity, high specificity, rapidity, usable for technical personnel without specific training in molecular biology and applicable to different biological matrices.
4. Mini Kit for the detection of Feline Herpesvirus
Feline herpesvirus is a major cause of feline morbidity, following the exposure to the virus, virtually all cats become persistently infected and many of these will develop recrudescent disease on one or more occasions during their lifetime. Acute ocular herpetic disease manifests as conjunctivitis, corneal ulceration and keratitis, and can be severe and painful. Repeated bouts of recrudescent ocular disease can lead to progressive corneal pathology that can be ultimately blinding in affected cats. FHV has a worldwide distribution, with reported exposure rates in some cat populations of up to 97%. As such it is a significant cause of clinical disease in the global cat population. The Mini kit method is based on detection that combines nucleic acid extraction by DNAzol protocol and target nucleic acid amplification by end point PCR on a portable device. The Mini kit has numerous advantages over other molecular diagnostic tools that make the assay suitable for a rapid and simple detection of pathogens in the field: easy to use, easy to interpret, little equipment required, high sensitivity, high specificity, rapidity, usable for technical personnel without specific training in molecular biology and applicable to different biological matrices .
5. Mini Kit for the detection of Bovine Papilloma Virus
Equine sarcoids are common, non-metastasising, locally aggressive skin tumours that are induced by bovine papillomavirus types 1 and 2 (BPV-1, BPV-2). They are the most common neoplasm in horse, representing 67% of all equine tumours and for this reason they are of considerable veterinary importance. In contrast to BPV-induced bovine papillomas, which usually resolve spontaneously, equine sarcoids mostly persist and tend to recrudesce following treatment with considerable expense for horse owners. Sarcoids can become ulcerated and infected and, when occurring near the eyes or on the eyelids, can impair vision so that their progression to more aggressive types can compromise the use and welfare of affected animals. The presence of sarcoid is a major cause of “failure” at pre-purchase examination. In spite of the fact that the condition is restricted to the skin, many horses are destroyed every year as a result of fly worry, infection, blood loss and failure to thrive or for ‘social’ reasons. Some are destroyed because they are unsaleable even though they may perform well at their chosen discipline. The presence of BPV DNA in PBMCs suggests a possible contribution to virus spread. Virus may spread in utero from infected mares to their foals, as has been shown in cattle. Moreover, infected PBMCs may propagate disease within one individual, as they migrate to sites of inflammation where they may take up the virus and function as a carrier. There is no routine tests for the molecular detection of BPV even if it will be strongly recommended to relate this finding to the observed recurrence after surgery. A penside test is particularly required to differentiate proliferative lesions caused by BPV. Furthermore, a diagnostic tool able to evidence bovine papilloma virus (BPV) DNA in PBMCs and in apparently normal skin around equine sarcoids after surgical removal, will be vital to under take a proper therapeutic and prophilactic approach.
The Mini kit method, developed within POC4PETS project, is based on detection that combines nucleic acid extraction by DNAzol and target nucleic acid amplification by end point PCR on a portable device. The kit is standardized for BPV1 and BPV2 detection in horse samples. The Mini kit has numerous advantages over other molecular diagnostic tools that make the assay suitable for a rapid and simple detection of pathogens in the field: easy to use, easy to interpret, little equipment required, high sensitivity, high specificity, rapidity, usable for technical personnel without specific training in molecular biology and applicable to different biological matrices including skin biopsies, swabs and blood.
Comments:
Ennebi elettronica in collaboration with the University of Verona and Bologna optimised the Fluorescent End Point PCR system, named DNA/RNA Finder and developed associated diagnostic kits for pathogen detection.
To ensure an affordable analysis of device market on today’s scenario, ENNEBI has been written a new business plan. Input help to identify target and limit, analysis and output points show new evaluation of market. Commercially existing devices are either not portable or do not integrate a fluorometer for End Point PCR. There are some “portable” real time PCR systems but they are costly and somehow fragile. We did not find any integrated end time PCR system.
Device total power consumption is estimated to be lower than 90W. Similar devices with fully configurable fast thermal cycler are shown to have power requirements of about 200W or more. Limited power consumption allows this device to be powered from car dc socket thus allowing an increased flexibility where no wall power is available.
The previous business plan has been taken as refererence for the new one. A scalable and configurable device allows a cost reduction; Ennebi estimates an average price of 3200 euro for the Fluorescent End Point PCR system.
New multiplex kits are under development to allow multiple pathogens detection with a single reaction. These version of the kit will be targeted to the detection of animal (pets) pathogens but it can be later validated for IDV or alimentary purposes to extend market.
Ennebi Elettronica has a sales network for electromedical devices for dentistry, and is evaluating the use of the device as IVD or first analysis point for dentistry. Furthermore a part of same sales network will be used for veterinary market.
No patent will be registered. The technologies used are known and non-patentable. Enneby will register the trade name.
Vetgenomic's foreground
1. Leishmania DNA amplification and electrochemical detection kit
Diagnosis of zoonotic canine Leishmaniosis is complex due to the broad clinical spectrum. Moreover, it is important to differentiate Leishmania infection from clinical disease and to apply different diagnostic techniques for each state. The presence of low antibody levels is not necessarily indicative of the disease and further work‐up is necessary to confirm or exclude clinical leishmaniosis. Detection of Leishmania DNA in tissues by PCR allows sensitive and specific diagnosis of infection, but is of limited use for the need of skilled workers, the high cost and the fact that samples must be send to a reference lab. FP7 POC4PETS project developed a Kit for the rapid, accurate and cost‐effective detectionof Leishmania easily adapted for to be performed on clinics. It will combine the advantages of PCR (direct detection of the parasite) and serology (low cost and point of care device) to be easily adapted to the daily work of the clinician
The Leishmania DNA amplification and detection kit is a simple and easy way to detect Leishmania infantum DNA. The Leishmania kit is based on RPA isothermal amplification (Recombinase Polymerase Amplification, TwistDxTM) and involves a set of specific homemade designed primers that amplify the kinetoplast, a specific DNA region of the Leishmania genome. These specific primers are respectively labelled with Thiol and Biotin and then are conjugated with AuNps and Magnetic Beads. The kit combines the isothermal amplification (10’ at 37ºC) and the detection method based on electrochemical reading of the amplified product. To validate each assay an internal control is needed. For this purpose a region of the canine 18sRNA gene (endogenous control) is amplified to check for inhibitory effects or problems with the sample to validate negative results.
2. Leishmania DNA amplification and lateral flow detection kit
Isothermal amplification of DNA is rapid, cost effective, easy-to use and more tolerant to inhibitory components from a crude sample compared with PCR, showing at the same time equivalent or higher sensitivity and reliability in clinical diagnosis. The Leishmania DNA amplification and detection kit is a simple and easy way to detect Leishmania infantum DNA. The Leishmania kit is based on RPA isothermal amplification (Recombinase Polymerase Amplification, TwistDxTM) and involves a set of specific homemade designed primers labelled with FITC and Biotin, that amplify the kinetoplast, a specific DNA region of the Leishmania genome. The kit combines the isothermal amplification (10’ at 37ºC) and the detection method based on immunochromatographic detection method. To validate each assay an internal control is needed. For this purpose we amplify a region of the canine 18sRNA gene (endogenous control) to check for inhibitory effects or problems with the sample to validate negative results.
Comments:
Vetgenomics activity is focused in the veterinary genetic diagnostic fields, currently offered as an externalized service to the veterinary clinics. However, the strategy for the future growth of the company is focused on the development and commercialization of genetic diagnostic devices as final products with a greater added value, instead of services. As a proof of concept, Vetgenomics has been focused on the development of a biosensor to detect Leishmania DNA at the point of care, which takes advantage of their previous experience in Leishmania genetic diagnostic. POC4PETS has allowed Vetgenomics to successfully fulfill the main objectives proposed in the project, through the application of emergent technologies for the detection of pathogen DNA in a more rapid, accurate, and cost-effective way compared with traditional approaches: the prototype developed is based on isothermal amplification followed by lateral flow or electrochemical detection of the amplified product. At the technological level, the biosensor developed by Vetgenomics is intended to overpass the limitations of current techniques of diagnostic, providing clear advantages in terms of sensitivity and in detection of false positives (detecting the pathogen directly, no antibodies against it) or false negatives (detection of phase of acute infection prior to seroconversion).
The collaboration with technological partners as RTD performers allowed Vetgenomics to evaluate emergent and innovative technologies and the technology behind the biosensor prototype has been protected to establish the prior-art of the technology (EP14382266/P10398EP00).
Canine leishmaniasis is endemic in Southern Europe. It is estimated that there are 2.5 million infected dogs in southern Europe (4 CVBD Symposium Proceedings 2009), 80% of which are asymptomatic without clinical signs and act as sentinels of infection. In a reference veterinary hospital analysis for Leishmania infantum is requested in 5% of the visits (Veterinary Teaching Hospital, UAB, 2009), although the prevalence of infection varies in each country. Moreover, in recent years, the vectors that transmit the infection (sand-flies) are spreading to northern Europe and it is an emerging disease in some breeds from the USA (foxhounds). Moreover, movement of pets for holidays increases the need for monitoring the infection. The dog population at risk is estimated to be near 46 million dogs in Europe.
In human health Leishmaniasis threatens about 350 million men, women and children in 88 countries around the world. As many as 12 million people are believed to be currently infected, with about 1–2 million estimated new cases occurring every year. Moreover, the disease is an emerging thread as a coinfection in HIV patients and in people travelling to endemic areas.
Leishmania infantum is currently the only significant causative agent of visceral and cutaneous human leishmaniasis endemic in Europe. Its high prevalence in asymptomatic humans and in the widespread reservoir host (the domestic dog) means there is a high risk of emergence in parts of Europe further north. Risk of emergence or re-emergence of leishmaniasis in Europe are associated with different scenarios, such as the increasing worldwide travelling of people and domestic dogs, the re-emergence of disease in the Mediterranean region caused by an increase in the number of immunosuppressed people, and the natural spread of the endemic species of the pathogen from the Mediterranean region of Europe to neighbouring temperate areas (Temperate broadleaf forsts’ biome), where there are vectors without the disease. Additionally to the Mediterranean region, the vectors of Leishmania infantum are also abundant in northern Spain and central France, and small numbers occur as far north as Paris and the upper Rhine valley in Germany. The occurrence of “vectors without disease” poses a significant risk for the emergence of leishmaniasis in temperate regions of Europe. The domestic dog is the only reservoir host of major veterinary importance, and in Europe there is a large market for prophylactic drugs and treatment of canine leishmaniasis caused by L. Infantum. According to CVBD Forum (Bayer Healthcare Animal Healthcare) clinical diagnosis of canine leishmaniasis (including non-specific laboratory parameters) is unreliable for the following reasons:
- More than 50% of dogs with proven established infections are apparently healthy on clinical diagnosis (asymptomatic).
- When present, clinical signs can be variable and mimic those caused by other diseases.
- Atypical forms are reported in dogs.
For final diagnosis three types of tests are available:
- parasitological methods aiming at the detection of parasites (100% specificity, but are not very sensitive)
- serological methods with detection of anti-leishmanial antibodies in the blood. Seroprevalence, regarded as an intermediate measure between disease and the prevalence of infection, is influenced by the fact that on the one hand serology can reveal a high proportion of asymptomatic carriers which represent half of all seropositive animals; and on the other hand the limitation due to the long periods of serological latency. Antibodies are often detectable only several months after infection. Furthermore, a proportion of seropositive infected dogs spontaneously converts to negative, or does not develop specific antibodies at all. Thus a maximum estimated sensitivity of the most efficient technique (IFAT) hardly reaches 80% and only during a few months after exposure to Leishmania in the previous season.
- molecular biology techniques like the polymerase-chain reaction (PCR), and more recently quantitative PCR (qPCR), detecting the parasite DNA in host tissue after multiplication. PCR provides a highly sensitive and specific diagnostic for Leishmania, although limited to centralized reference laboratories due to the equipment and trained personnel required.
The Leishmania biosensor will be a product clearly differentiated from the actual portfolio of Vetgenomics and will represent a novelty with greater added value for the veterinary clinician. Its simplicity will allow implementing the assays to be used as pen-side devices in the veterinary hospitals, with two main advantages for the clinician: by two main reasons:
- By the change it will represent in the value chain compared to the need of reference labs
- By the added value of the analysis and the greater reimbursement for the professional due to the intrinsic characteristics of the device, such as integration, portability, ease of use, no need for trained personnel or specialized equipment and especially by the time to result, as it is shown in the value chart of the new product .
At the beginning of this project, a market survey was designed and several veterinary practices in Germany and Spain received a questionnaire regarding general occurrence of leishmaniasis, routinely used diagnostic methods and willingness to test a new diagnostic kit for the direct Leishmania detection. Results of this survey showed that incidence of dogs with leishmaniasis per year is 18.3% in Spain. The incidence is lower in Germany and was mostly associated to travel history of the dogs. More than 85% practices in Germany and 70% practices in Spain agreed that direct detection of the parasite instead of anti-Leishmania antibodies (serology) offers advantages in the diagnosis and PCR testing was evaluated with highest priority (75.0%) concerning its use as diagnostic procedure for canine leishmaniasis. Summarizing, 71% of practices in Germany and 85% of Spanish practices think it could be useful to have a new diagnostic kit to detect Leishmania DNA (instead of anti-leishmania antibodies) in house. In both countries veterinary practices are willing to test a new diagnostic kit (71% in Germany and 90% in Spain).
The potential market for the Leishmania biosensor is greater than the market for genetic diagnostic centralized in reference laboratories with sophisticated equipment and skilled personnel. Geographic barriers disappear when the analysis is done in the veterinary clinic, so local market becomes global and opens a potential market of more than 200,000 clinicians only in Europe. It is worthy to consider also at this point that the first vaccine for canine leishmaniasis has been recently approved in Europe (CaniLeish from Virbac; EMA/296955/2010; EMEA/V/C/002232) and is being first commercialized in Portugal, to be afterwards extended to Spain, France, Italy and Greece. CaniLeish is to be used only in ‘leishmania-negative’ dogs. The need of discarding Leishmania infection before vaccination increases the market for a POC biosensor to detect Leishmania DNA.
A comprehensive field study including interviews with a representative sample of veterinarians in Central and Southern Europe was subcontracted to Trifermed (as described in B2.4) to confirm three key aspects: (1) knowledge and sensitivity to the existence o the disease, (2) perception of incidence and (3) action protocol used for both diagnosis and treatment. The study has been focused on the German and Spanish markets, for their representativeness, size and validity for the conclusions sought for the commercialization of the device. It is worthy to note that the success in this project will not only consolidate the Leishmania biosensor as the first product for Vetgenomics and a new business unit in the company, but also place Vetgenomics as a partner for technology owners to jointly develop future applications and innovative products for the veterinary diagnostic market. The most important customers are veterinary medical clinicians, especially those placed in Mediterranean Europe, and from Central and North Europe for extending commercialization. The main advantages of the biosensor for veterinarians are rapid turnaround, greater control over the methods used, and potentially lower cost than analysis in centralized laboratories. Moreover, an integrated disease management solution that leverage the advantages of both animal-side and laboratory testing by offering both rapid diagnostic for use in the clinic and outside laboratory services would represent a market opportunity. Vetgenomics is offering Leishmania diagnostic by qPCR as a reference lab, which allows the company to assist the clinician with both the in-house (POC) and outsourced (qPCR) assays. Vetgenomics will offer the possibility of a qPCR confirmatory analysis in its own reference lab at a special rate for the biosensor customers.
At the commercial level, it is worth to consider the potential market, not only in veterinary but also even in human health. Worldwide, leishmaniasis is one of the most important vector-borne diseases (besides malaria, lymphatic filariosis and sleeping sickness) and is the third zoonotic disease (animal disease transmitted to man) in global importance, where the dog acts as the main reservoir of the causative agent. Commercialization strategy needs a technological partner for final product development, and a strategic distributer to reach the maximum market share. Vetgenomics is subcontracting these activities to Trifermed (www.trifermed.com) a business development company introduced in national and international markets, to better define the business development programme and the global expansion strategy.
Industrialization of the prototype will be outsourced. Vetgenomics intends to commercialize directly the biosensor in the Mediterranean European market, and will negotiate commercialization license with worldwide leaders in veterinary diagnostic for Central and North Europe and US. Vetgenomics will also evaluate the application to other CVBD pathogens or even to the human health market.
The estimated time to market is three years from now on, considering 3 phases:
- Initial exploitation (Year 1&2): for the industrialization and commercialization of the launching units
- European Expansion (Year 3&4): commercialization of the biosensor in Europe
- Global Expansion (Year 5): commercialization of the biosensor at a global scale.
While it is a growing market, it is not a market without competitors. There are at least 75 vendors worldwide that market rapid test kits for emerging infectious diseases in human health and they could enter easily the veterinary market, although the smaller global volume. The current market leaders are companies that have invested in sophisticated new technologies and those that have established relationships with governments and international aid agencies. Main competitors in the veterinary market for the Leishmania biosensor would be:
- Multinational companies devoted to veterinary diagnostic (ANTECH, IDDEX) opening new business units complementary to serology POC assays. They are currently offering DNA detection of the pathogens by means of PCR in central reference labs.
- Immunoassays for indirect detection of infection, such as IFAT (ImmunoFluorescence Antibody Test) or ELISA (Enzyme-Linked ImmunoSorbent Assay). Some of the assays are LEISH and IFI LEISH (ID VET), SNAP LEISHMANIA (IDEXX), SensPERT (MATERLAB) or Leishmania IFA Canine IgG Antibody Kit (FULLER Laboratories).
Competitors are actually offering POC devices based on serology. The main advantage over competitor products is that the biosensor will detect DNA of Leishmania instead of an antibody, which indicates the presence of the pathogen, and thus current infection and not a previous exposure to it, and distinguish between acute disease, asymptomatic infections, relapses and cured cases.
As in other market segments, large companies may prefer to use alliances with smaller companies when adding non-traditional products such as diagnostics or novel biological to their product portfolios, which would also represent a market opportunity for Vetgenomics.
Other competitors would be the current market leaders in molecular diagnostic, companies that have invested in sophisticated new technologies and those that have established relationships with governments and international aid agencies.
A dipstick format was developed for detection of PCR products by hybridization with a gold-conjugate probe, and a PCR oligochromatographic leishmania-specific test is being commercialized using this technology (Leishmania OligoC-test, Coris Bioconcep) (Srividya et al., 2012). In 2011, the US Food and Drug Administration (FDA) approved the Smart Leish PCR assay (Cepheid USA), a qualitative real-time PCR test for diagnosing individuals with cutaneous leishmaniasis. Both systems didn’t overcome the limitations of PCR for which sophisticate and expensive equipment are needed to perform the precise and repeated heating cycles required.
On the other hand, a new POC assay for Leishmania DNA detection has been recently developed by BIOGAL (Israel) based on isothermal amplification and lateral flow detection. This product could be considered a direct competitor, but the biosensor prototype developed by Vetgenomics in this project represents some technological advantages, such as temperature of amplification (37ºC over 65ºC), time to result (20 minutes for amplification and detection vs 1.5h) and the possibility of semiquantitative approach when electrochemical detection is applied. However, it could represent a market opportunity, as an advanced player opening the new market for these POC devices.
Main competitors are selling POC for Leishmania based on serology (detecting antibodies) at 10-15 € or quantitative PCR (detecting Leishmania DNA) in central labs at 30-35 €. BIOGAL price for the new developed DNA POC assay will be 35 €. The biosensor developed by Vetgenomics intends to be an innovative product in the veterinary market, detecting Leishmania DNA (as qPCR in central labs) in a POC device (as serology) at an average price of 17 €.
The biosensor for Leishmania should be sold at € 17/unit to be competitive in front serological POC devices that are already in the market, considering a production cost of € 4/unit plus a commercialization cost of € 2/unit. There is an estimated population of 18 million dogs in southern Europe (Spain, Italy, France and Portugal), 26 million in the rest of Europe and 70 million in the USA.
Given an infection rate of 10%, the volume of potential market is:
• 34 million € per year in endemic areas (Mediterranean Europe) (2 million tests at 17 € each)
• 200.6 million € considering the European and USA markets (11 million tests at 17 € each).
The following table shows an estimate of overall annual market volume for the Leishmania biosensor, considering the number of dogs in each region and a price of 17 € for the device.
With an objective of 2.5% share for the first year of commercialization rising up to 10% after 5 years, revenues for Vetgenomics are be as shown in the next figure and are estimated in different scenarios, depending on the geographic market: Mediterranean Europe (worst), Europe (normal), Europe and US (best scenario).
In this way, with a launching price of 17 € and a market quote of 2.5% the first year of commercialization of the biosensor, incomes for Vetgenomics are estimated in 850,000 € (50,000 units) in Southern Europe and 4.85 million € (11 million tests) when commercialization is extended to the European and North American markets in the best scenario.
Two people have been hired for the development of the prototype during the project, added to the SME owners: A Senior scientist with more than 10 years expertise in the field of Leishmania genetic diagnostic and canine genomics related to resistance to infection as the project manager and a young researcher responsible for the laboratory assays.
VETGEN is financing the first steps of the final product development with own resources, and intends to apply for Horizon 2020 dedicated SME Instrument - Phase 1 2014 and to raise external finance (bank loan and or Venture Capital) to develop the detection of DNA without amplification, which was the more risky proposal in this project. Results based on impedance were promising, but further work is needed to integrate it into a portable device.
Skuldtech's (now ACOBIOM) foreground
1. Point of care diagnostics for the rapid detection of Parapoxviruses
Zoonotic poxviruses (ZPs) fall into the category of neglected zoonoses. No universal diagnostic tests are in use; when available, is mainly performed at academic institutions or in public health reference laboratories. The majority of ZP are considered an occupational hazard even if an increasing number of cases in children exposed to infected animals in an occupational setting or during recreation. At present, there are no diagnostic tools available for the detection of zoonotic poxviruses. Rapid and reliable tools will be required at the point of patient care so that physicians can manage patients appropriately and avoid severe complications of infection in children and the immunocompromised; furthermore an early recognition of poxvirus-associated infections will allow to reduce the spread of virus between companion animals and their owners. FP7 POC4PETS developed a Lateral flow strip test for the rapid detection of Parapoxviruses (PPV) in animals allowing differential diagnosis from other life threatening infections.
The Parapoxvirus Lateral Flow Detection kit is designed for the detection of Pseudocowpox virus (PCPV), Bovine papular stomatitis virus (BPSV) and orf virus (ORFV) in bovine and ovine.
Parapoxvirus detection kit is based on the enzymatic amplification of a specific Parapoxvirus sequence by the PCR (Polymerase Chain Reaction) technique. It involves a set of specific primers that amplifies a specific DNA fragment of different Parapoxvirus species.
A positive control for the DNA sample preparation, the PCR reaction and the detection, called “internal control”, is present in each reaction. It is a conserved sequence of the gene encoding the cytochrome c oxidase subunit I (COX1).
The detection and the visualization of the amplified products are carried out by lateral flow immunochromatographic assay with dipsticks. The parapoxvirus infection is evaluated by the presence of a dark grey band on the dipstick.
The immunoassay-based lateral flow dipstick test developed is intended for the simple, sensitive and rapid field diagnosis of parapoxvirus in bovine and ovine. It is simple to use and provides results in a few minutes.
2. Point of care diagnostics for the rapid detection of Orthopoxviruses
At present, there are no diagnostic tools available for the detection of zoonotic poxviruses. The majority of ZP are considered an occupational hazard even if an increasing number of cases in children exposed to infected animals in an occupational setting or during recreation. In late 2008-early 2009 an outbreak of pet-associated Orthopoxvirus, CPXV occurred in the EU resulting in >30 human cases in France, Germany and the Netherlands (ECDC report 11/02/2009). Infections was due to either direct contact with infected rats, or contact with other animals (mangoose, cat) that had presumably became infected following contact with a rat or other small rodent harbouring CPXV.
Pen-side test for ZP will be important in both veterinary and human diagnostics, in cases when differential diagnosis from other skin tropic infections, especially vesicular diseases and those other life-threatening infections which are all mandatory for risk assessment. Rapid and reliable tools will be required at the point of patient care so that physicians can manage patients appropriately and avoid severe complications of infection in children and the immunocompromised; furthermore an early recognition of poxvirus-associated infections will allow to reduce the spread of virus between companion animals and their owners. FP7 POC4PETS developed a Lateral flow strip test for the rapid detection of Orthopoxviruses (OPV) in animals allowing differential diagnosis from other life threatening infections. The Orthopoxvirus Lateral Flow Detection kit is designed for the detection of Vaccinia virus (VACV), Cowpox virus (CPXV), monkeypox virus (MPXV) and Ectromelia virus (ECTV) in bovine, feline and rodents. Orthopoxvirus detection kit is based on the enzymatic amplification of a specific orthopoxvirus sequence by the PCR (Polymerase Chain Reaction) technique. It involves a set of specific primers that amplifies a specific DNA fragment of different orthopoxvirus species. A positive control for the DNA sample preparation, the PCR reaction and the detection, called “internal control”, is present in each reaction. It is a conserved sequence of the gene encoding the cytochrome c oxidase subunit I (COX1). The detection and the visualization of the amplified products are carried out by lateral flow immunochromatographic assay with dipsticks. The orthopoxvirus infection is evaluated by the presence of a dark grey band on the dipstick. The immunoassay-based lateral flow dipstick test developed is intended for the simple, sensitive and rapid field diagnosis of orthopoxvirus in bovine, feline and rodents. It is simple to use and provides results in a few minutes.
Comments:
POC4PETS outcomes allowed Acobiom to benchmark a new diagnostic approach on the basis of Acobiom technologies for quick cost effective and easy to handle kit able to identify the aforementioned infectious agents. It will therefore increase Acobiom pipeline and provide the opportunity to reach markets which are long to penetrate. The business model developed by Acobiom for dealing the new diagnostic kits is to include Acobiom manufacturing and distribution licensing.
Through POC4PETS, Acobiom was able to validate its genomics technologies and expertise to launch on the market new innovative diagnostic in the area of infectious disease in veterinary field. Three kits for zoonotic poxviruses detection have been developed, evaluated by beta testers and are now marketable: one for orthopoxvirus detection, a second one for parapoxvirus detection and a third one for specific orthopoxvirus detection in humans. IPR exploitable measures for the above mentioned kits are intended to be undertaken in the near future.
Acobiom will manufacture in-house the new diagnostic kits. A specific manufacturing line will be set-up. This manufacturing will be fulfilled in France for EU and US and will be assembled in local countries for South America and Asia. Acobiom has already some partnerships and business contact in EU, USA and Brazil to develop distribution licensing for such kind of innovative diagnostic. However POC4PETS project will also help to set-up EU commercial partnership increasing Acobiom EU network through POC4PETS partners. First diagnostics kit launch on the market is forecasted for 2015 including health authorities’ registration and approval as well as marketing and outcomes dissemination.
Acobiom will use already existing distribution channel through partnerships with companies or laboratories involved in veterinary diagnostic testing as Royal Canin (Mars group, US), Vetoquinol (France), CEVA (France). Acobiom is currently in contact with these companies.
Pricing is very low to be competitive and to reach the aforementioned markets which are depending on 3 lines: low cost, quick and specific, easy to handle and do not required specific equipment.
New diagnostic kits developed through POC4PETS project are innovative diagnostics able to reply to unmet needs especially within the field of quick, specific, easy to handle and low cost diagnostics kits.
There are no available kits on the market (no competitors), diagnosis is only made in research laboratories at an average price of 50 €. The kits, subjected to field validation, are new and innovative products, and are able to detect Zoonotic poxvirus DNA using POC devices at an average price of 8€.
Acobiom estimated its revenues on the basis of the new outcomes of POC4PETS and for 5 years of sales. Considering the European market only, expected revenues are 1,2M€ and 4,2 M€ in respectively 1 and 5 years. Considering the global market, including USA, Japan and other extra EU markets, expected revenues are 3M€ and 13 M€ in respectively 1 and 5 years.
For Europe and for the global market, assuming an average scenario and different market penetration rates, the penetration rates have been estimated at low rates because of the size of the markets and the barriers to entry of the market.
Through POC4PETS project, Acobiom directly hired one person, a sale manager (Laurent PASCAL, the 1st March 2014). At long term, POC4PETS project will allow Acobiom to hire approximately 6 additional employees dedicated to health regulation and marketing.
Concerning the zoonotic poxvirus, the market has been appraised on the basis of the population of cats which account for 130M and with a growing population of exotic pet animals and fancy rats. In humans, poxvirus infections are neglected zoonoses and are undoubtedly under-reported, so the true impact of these diseases remains unknown.
To meet the expectations of veterinarians working with farm animals and consequently to widen Acobiom market over companion animals, the kits would need to be further improved in terms of simplicity, rapidity and reduced costs. Indeed, an additional important step in the development of POC diagnostic devices for the detection of poxviruses is the replacement of the PCR step by a LAMP amplification step which will be combined to the LFD assay. Nevertheless, a first commercial approach has been undertaken to the french veterinary companies.
Potential Impact:
POCS4PETS consortium recognized the importance to facilitate and accelerate the development of effective tools to counteract companion animal diseases. It recognized the importance of the potential infectious disease hazards linked to close interactions with pets, especially for immune-comprised individuals. Others at higher risk for severe zoonotic infection include those with particularly prolonged animal contact or occupational exposure, such as pet care-takers, veterinary professionals, animal handlers and laboratory researchers.
In the EU there are more than 200 million pets in total (204,947,400) and 72 million homes have at least one pet animal. According to the European Pet Food Association (FEDIAF), there are: 66 million cats, 60 million dogs, 9 million ornamental fish (number of aquaria), 39 million cage birds, 21 million small mammals (Fig 1 - Source: FEDIAF Facts and Figures 2012).
There is an important socio-economic value of companion animals. This can be in part calculated directly as reduced number of medical consultations and more rapid recovery after surgery for pet owners, and indirectly by improved quality of life and thus better function in society. Scientific studies have recognized that pets provide social support and positive effects are generated by human-animal interactions. One of the most important positive aspects of companion animals is a direct effect on the owner’s health, both socially as well as directly related to health issues and economy.
It has been proposed three mechanisms to explain the association between companion animal ownership and health benefits for humans:
1. There is no real association between the two, but cofactors such as personal traits, economy and desire to own a pet
2. Pets may enhance social interactions with other people, thus providing an indirect effect on wellbeing
3. Pet ownership may exert a direct effect on human health and wellbeing through the nature of the relationship
The pets play a key role in every stage of human development: child, adult, elderly.
- Child: for the child, a pet animal encourages a sense of responsibility, caring and communication. The relationship instils confidence and friendship - qualities, which can endure and grow as the child moves on through life. Children’s exposure to companion animals may also ease anxiety. For example, one study measured blood pressure, heart rate, and behavioral distress in healthy children aged 3 to 6 at two different doctor visits for routine physicals. At one visit a dog (unrelated to the child) was present in the room and at the other visit the dog was absent. When the dog was present, children had lower blood pressure measures, lower heart rates, and less behavioral distress
- Adults: health benefits of pets can, although these factors overlap to some extent, be divided into therapeutic, psychological, physiological and psychosocial. For adults, the pet providing companionship for those living alone; giving stimulation to make contact with others as in, for instance, an owner walking his dog. Compared with people without a pet, people who keep a pet have been reported to require fewer visits to the doctor with direct economic impact and a consequent benefit to public health services.: in a country like Norway, with a population of 4 mill inhabitants, this means a net saving of € 88 million annually for the ten largest hospitals (average cost/day estimated low: € 625); for Belgium with 10,5 mill inhabitants, this equals € 231 million annually. A large German study collected pet information (dog, cat, horse, fish, bird or other pet ownership) from over 9,000 people at two different times (1996 and 2001). The survey included a number of health, economic, and labor issues, so that respondents would not realize the researchers’ interest in a link between pets and health. Researchers found that people who said they had a pet in both 1996 and 2001 had the fewest doctor visits, followed by people who had acquired a pet by 2001; the group of people who did not have a pet at either time had the highest number of doctor visits.
Pet owners adapt more quickly to stress associated with bereavement and other adverse events; have stronger emotional stability and maintain a generally sounder state of health. Bonding with a companion animal was credited with leading to beneficial effects on the human cardiovascular system. In patients with hypertension who were under stress, patting and talking to a pet dog or cat may have prevented increased blood pressure. In a study investigating risk factors for cardiovascular disease, it was found that dog owners had lower systolic blood pressure and lower levels of serum triglycerides than non-dog owners. Dog owners are physically more active, have lower blood pressure and cholesterol levels which helps ease the strain on healthcare resources. Companion animals may improve heart health by lowering blood pressure and regulating the heart rate during stressful situations. In a 2002 study, researchers measured changes in heart rate and blood pressure among people who had a dog or cat, compared to those who did not, when participants were under stress (performing a timed math task). People with a dog or cat had lower resting heart rates and blood pressure measures at the beginning of the experiment than non-pet owners. People with a dog or cat were also less likely to have spikes in heart rates and blood pressure while performing the math task, and their heart rates and blood pressure returned to normal more quickly. They also made fewer errors in their math when their pet was present in the room. All these findings indicated that having a dog or cat lowered the risk of heart disease, as well as lowering stress so that performance improved.
- Elderly: The direct health effects are similar to what is described for adults. Elderly people with dogs visit physicians 21% less often than do those without a dog. Elderly people constitute a segment of the population at heightened risk for a variety of physical and emotional problems. In one study, elderly individuals that had a dog or cat were better able to perform certain physical activities deemed “activities of daily living,” such as the ability to climb stairs; bend, kneel, or stoop; take medication; prepare meals; and bathe and dress oneself. The use of specially trained companion animals in institutions are increasingly being used, and the positive effect of this intervention has been documented.
In addition, there is increased awareness of the value of companion animals in animal assisted therapy for different groups of people and may mitigate the stresses of urban living and counteract what has been called “nature-deficit disorder.”
The social support provided by a pet might also encourage more social interactions with people, reducing feelings of isolation or loneliness. For example, walking with a dog has been found to increase social interaction, especially with strangers, compared to walking without a dog.
Throughout Europe, an estimated 550.000 direct and indirect jobs are generated by pet ownership such as veterinarians or breeders or connected supply industries. Indirect employment is an estimation based on an approximate 200.000 veterinarians in Europe, an estimated 60.000 pet specialist stores, medication/vaccination, suppliers to the pet food industry, pet accessories industry, direct employment trade shows, pet press, breeders, pet food industry: 50.000 animal welfare organizations, transport. The pet food industry and related supply and services represent a combined annual turnover of over € 24 billion.
The global dog and cat animal health market was valued at approximately $ 5 000 million in 2005 and accounted for nearly 40 % of the global animal health market. Dog and cat sales in Europe were approximately 35 % = $ 1 750. As for the future the companion animal health market is expected to account for almost 60 % of the growth in the animal health market. The dog and cat market is expected to grow most strongly in the geriatric and juvenile areas because pets live longer and their owners are more willing to spend on them.
The etiology and prevalence of many diseases of companion animals are still unknown since these pathologies are often difficult to detect for their long period of latency or for their asymptomaticity. For these reasons it is necessary to screen large numbers of animals and so new tests are continuously needed and developed. The independence from the major centers of analysis also allows the diagnostic kits’ market not to be constrained by geographical boundaries. Today there are more than 35 000 vets in Europe who work with companion animals. Most of them are general practitioners, many of whom have additional qualifications like national specialization, etc. At present, 50% of the veterinarians who graduate will work in companion animal practice. (Source: The economic importance of companion animals FECAVA - February 2007).
While clinical practice for companion animals is the core of the industry, providing treatment and prevention services to pet-owning households is a matter still not properly considered, with implications for both human and animal health. It can easily be assumed that the growing awareness on animal health and benefits of early detection and preventive medicines will drive the demand for veterinary diagnostic kits over the next few years.
Such technological breakthrough achievement will provide and consequently enhance the prevention, early diagnosis (and thereof a reduction of their economic impact) and control of a wide range of animal diseases so that it will in turns contribute to better preventing the human transmission, in case of zoonoses. Thereof, the public health impact will be benefiting in 2 directions, both in terms of i) global animal health as well as of ii) human disease prevention.
List of Websites:
http://www.poc4pets-fp7.eu