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Rapid Diagnosis RT PCR - " a transforming moment"

"Fortunately, we were able to take the devices and test system into the field in Uruguay in November 2001, where they performed splendidly on farm in a remote area...." The statement made by Roger Breeze to the Royal Society Enquiry (pdf)

More emails from Dr Breeze

From "Making better use of technological advances to meet stakeholder needs" (page 14) http://www.oie.int/doc/ged/D3274.PDF:

The role of clinical inspection should be reassessed, as a result of the advances in portable, rapid pathogen detection devices, although clinical diagnosis will still be the basis of suspicion of disease in the first instance.
j) The role of portable, rapid diagnostic technology should be reassessed, to aid in disease control and management and reduce the burden on reference laboratories during an epidemic."

 

".....effort need to be concentrated on the development of simple, rapid, noninvasive tests that can be performed without expensive laboratory equipment.."

Review Article: A Brief Review on Diagnosis of Foot-and-Mouth Disease of Livestock: Conventional to Molecular Tools Veterinary Medicine International Volume 2011 (2011), Article ID 905768, 17 pages doi:10.4061/2011/905768 Neeta Longjam,¹ Rajib Deb,² A. K. Sarmah,¹ Tilling Tayo,³ V. B. Awachat,⁴ and V. K. Saxena⁵(open access article distributed under the Creative Commons Attribution License)
"...Effective control of this disease needs sensitive, specific, and quick diagnostic tools at each tier of control strategy....
....Automated real-time RT-PCR has been compared with VI and antigen-detection ELISA with result being more positive by real-time PCR indicating that the real-time PCR has higher sensitivity than VI for the detection of FMDV in epithelial samples [181].The development of multicolour real-time PCR cyclers and "ready-to-use" commercial multiplex real-time PCR kits has also made it possible to combine several assays within a single tube. Major advantages of multiplexing include a reduced sample requirement, which is especially important when sample material is scarce [182, 183], and the ability to combine assays with an internal control system. However, it is important to optimize these assays in order to limit competitive interactions that may significantly impact upon assay sensitivity. The combined properties of high sensitivity and specificity, low contamination risk, and speed have made real-time PCR technology a highly attractive alternative to tissue culture- or immunoassay-based methods for diagnosing many infectious diseases [184]. Clinical diagnostic applications and the use of real-time PCRs are growing exponentially, and real-time PCR is rapidly replacing conventional PCR and other established diagnostic methods such as antigen-ELISA and cell culture isolation." Full paper

June 21st 2011 ~ "A research collaboration in the UK has shown that a portable optical sensor can identify the presence of foot-and-mouth disease (FMD) much faster than existing, laboratory-based methods.

The prototype sensor unit, which is based around optical microchip technology developed by the University of Southampton spin-out Stratophase (see also below), could allow veterinarians, inspectors and even farmers to diagnose the disease much more quickly than is possible today – potentially making widespread culls of healthy animals a thing of the past." link (The word "potentially" is a dampener. One wonders why - apart from the probably obvious commercial considerations - the UK is so keen to reinvent the wheel and so slow to implement and adopt technology that is tried and tested, such as the Genestat device described below, backed by the FAO and IAEA, successfully evaluated, already available and in use.)
UPDATE - We hear that the Stratophase project, like the other home-grown diagnostic machine from Smiths Detection, the BioSeeq machine with which Pirbright collaborated, has been discontinued.

June 3rd 2011 ~ "...we're able to take our device, literally plug it in to the cigarette lighter of a car, and run molecular diagnostic tests out in the field..."

Interview with Gerrit Viljoen, Head of the Animal Section of the International Atomic Energy Agency on the subject of portable on-site diagnosis "...we want them to be used at the point where they do the diagnosis..In the animal field, in the veterinary field we really need them at the "point of care".
"... we're able to take our device, literally plug it in to the cigarette lighter of a car, and run molecular diagnostic tests out in the field.. the platform technology is there to do all the range of pathogens and viruses.... In your back pocket you will have let's say a library of five or six different cassettes having the building blocks of the different tests: Newcastle Disease, Avian Influenza,Swine Flu, Foot and Mouth disease..." (MP3)
The GeneStat portable diagnostic kit has been described (to an English interviewer) by Craig Mosman, Vice President, DxNA: "Our system processes the sample and gives a result within 45 minutes" If a central lab is used, there is delay while the samples are sent, then several hours of sample preparation and only then a result "in an hour or two" Extract - audio file - :
"... The system carries with it our remote reporting capability - you can have a test being done at a remote laboratory, "pen-side" as we say or in a remote village, and communicate those results back to a central laboratory or indeed back to any other spot on earth.....Because the sample is not transported back to a Central Laboratory, it's actually done on-site, we are able to save hours, or in some cases even days...we're able to take our device, literally plug it in to the cigarette lighter of a car, and run molecular diagnostic tests out in the field..."
Listen in full

June 3rd 2011 ~ "the collaboration with the FAO in the establishment of a portable and mobile diagnostic system that demonstrates high sensitivity and specificity characteristics, but is also affordable throughout the world."

Mass slaughter is not necessary with modern surveillance diagnostic and response capabilities. Eighteen months ago, the publication Virus Weekly reported that the DxNA GeneSTAT PCR System had successfully completed its evaluation and quoted DxNA Chief Executive Officer Phillip Grimm who acknowledged the importance of the collaboration with the FAO in the establishment of "a portable and mobile diagnostic system that demonstrates high sensitivity and specificity characteristics, but is also affordable throughout the world."
Pirbright collaborated with the Smiths Detection system (Bioseeq) and thousands of pounds were spent in research grants by DEFRA (see below). In 2007 the Smiths system looked promising but seems now to have dropped out of sight. Is that a reason for the UK not to use the DxNA system, already evaluated and proven in the field? We should be very grateful if anyone were able to explain to us via email why the UK continues to prevaricate when such an affordable portable diagnostic system, backed by the FAO and IAEA, successfully evaluated, already available and in use - is not even mentioned in the current UK Contingency Plan. The e-coli outbreak in Germany provides a stark reminder of how easy today it is for a pathogen to get a hold and spread. Our island is not immune to the viruses and bacteria that threaten both animal and human health - and they can arrive at any moment.

May 31st 2011 ~ Genestat machine is ideally suited to early detection of foot and mouth

An email received suggested that the rapid diagnosis machines mentioned below are "not yet 100% accurate - though under continual appraisal at Pirbright".
We find the lengths to which the UK has gone in order to prevaricate until it can produce its own commercial machine quite extraordinary. David King said ten years ago, when justifying that he chose not to use the USDA rapid detection machine offered to the UK for tests, that it was not "validated" - (the same machine was used in Uruguay's FMD crisis with excellent results.) David King omitted to mention on that Today Programme that the mathematical model that drove the mass culling policy, used instead of vaccination, was itself "unvalidated" and was highly flawed.
Hundreds of thousands of UK taxpayers' money have been poured into developing the Smiths Detection BioSeeq machine with which Pirbright collaborated. Yet this machine, although excellent, proved too expensive for commercial use. The DxNA machine, on the other hand, is already accessible and, as the website says,
"The advent of GeneStat PCR means that DNA and RNA analyses can now be accurately and rapidly done almost anywhere, indoors or outdoors, by virtually anybody."
This does, of course, mean the end of the monopoly enjoyed for so long by the laboratories which were alone allowed to handle samples in the past. It would be unusual were there not the extreme reluctance we are seeing from those involved. But that the GeneStat can be operated by "virtually anybody" I know at first hand. This was the machine I handled personally in Brussels at the end of the 'Towards a Durable Global Animal Health Policy' conference when I learned within five minutes how to use it. (Please note that I have no financial interest at all in anything mentioned on this website.)

May 30th 2011 ~ Nigel Gibbens:" these types of tests aren't currently practical for use on the ground during an outbreak" But DxNA Genestat would seem to prove the CVO mistaken

Available on-site rapid PCR diagnosis, validated and less expensive than others, is the DxNA system. www.dxna.com: "...transportation of samples to large, centralized laboratories creates issues with sample degradation, contamination, cost, and delays...Long-awaited, point-of-care technology..DxNA has developed a diagnostic platform technology allows individuals with approximately five minutes of training to conduct accurate real-time diagnostic testing almost anywhere." Highly infectious and contagious pathogens detected by the system include Foot and Mouth Disease as well as many others such as Anthrax, African Swine Fever, Exotic Newcastle Disease, Classic Swine Fever, Highly Pathogenic Avian Influenza (More)
The UK and DEFRA continue to claim (See the UK's Chief Vet, Nigel Gibbens, Guardian 5 May 2011)
".. these types of tests aren't currently practical for use on the ground during an outbreak...we are continuing to fund their development..."
How can DEFRA continue to assert that such technology hasn't been properly developed yet and is not as yet, as effective as laboratory testing, when DxNA dares to make such claims for the efficacy of GeneStat? When the new Woolhouse/Charleston research was published, DEFRA argued for
"... strong support for investment in the development of practical tools for preclinical diagnosis..."
Surely, the funding urgently needed now is not to spend vital taxpayers' money in reinventing a wheel that is already proven and effective - but to obtain the already proven Genestat technology. DEFRA's intention, apparent from the words of the CVO, to pour yet more thousands into research, is inexplicable. Six years of funding between 2003 and 2009 spent by DEFRA on (Projects SE1120, SE1123) have not produced anything that has found its way into DEFRA's Contingency Plan - yet the proven DxNA machine is already developed and apparently available for purchase.

May 30th 2011 ~ Is DEFRA aware of the following points from the DxNA website about the cheapest, most effective - and already available - on-site diagnosis system?

An effective, affordable (compared to the enormous financial and social costs involved in an outbreak) system - but is DEFRA aware of it? See the following points from the DxNA website?
Drawbacks of DxNA's competitors:
• Commercial systems purported to be portable are too bulky for convenient transport. Samples still require labor-intensive, manual preparation. In contrast, this is fully automated with DxNA technology.
• Considerable time is required to obtain reportable results
• Higher costs of devices and tests
• Higher power demands
Unique aspects of DxNA's technology:
• Speed: results in 60 minutes or less
• Ease of use: the need for highly-trained technicians is eliminated
• Portability: lightweight and can be powered through a wall outlet or car battery
• Lyophilzed PCR reagents: stable without refrigeration
• Accuracy: unique, closed system rendering valid, trusted data
• Economical: less costly than current real-time PCR technologies
• Multiplexed assays test for three targets at the same time.
As the ProMed moderator, Dr Martin Hugh Jones, said this month ".... tests must be run cow-side. There is no time for the delays inherent in sending off samples to a distant laboratory.... if you can get ahead of the disease, time is on your side...."

February 28th 2011 ~ The cost of using real-time diagnosis - and of not using it

We are very grateful to Tetracore and to our American friend, Gary, for giving us the maximum prices for just one example of the rapid RT-PCR technology that has been developed and that could be used on-site to discover, before clinical symptoms appear, whether animals have been infected with the FMD virus. Extract:
"...The VetAlert^TM Foot-and-Mouth Disease (FMD) Virus target specific reagents are a set of primers, fluorogenic probe and mastermix for the specific identification of FMD viral RNA by real-time reverse transcription polymerase chain reaction (RT-PCR)....The single tube method is easy to perform and results are obtained in less than 2 hours.
The assays are optimized for use on multiple platforms including the Cepheid SmartCycler, Roche LightCycler, ABI Prism, BioRad iCycler, Stratagene MX3005P, and Qiagen Rotor-Gene Q...."
See documentation (pdf) The latest version "TCor4" costs the equivalent (maximum price) of about £9.900 but comes with a mini centrifuge and case to carry everything in. The FMD assays is sold as units of 64rxns at £7.4 per rxn, so about £473 per unit.
The total cost for each culled farm in 2001, including compensation, cleaning up etc was at least £250,000 each. Gordon Brown estimated £2.6 billion for the cost of FMD2001 but a more comprehensive estimate (More on costs) puts the financial costs at £20 billion.

August 17th 2010 ~ Why is such "heavy lifting" needed to get rapid test technology into suspected premises - and into the Contingency Plan?

Suitcase sized systems for detecting foot and mouth disease have been effective and available since 2001. They have not yet been used in the UK. Indeed, the obstacles placed in the path of getting them into the Contingency Plan seem wholly out of proportion to their usefulness.
The UK-based company, Stratophase (thanks to the FMD news service for this link) is hoping to create something "smaller, more robust and easier to use" than the UK's current system that:
"...sends a sample to a lab but the time associated with this can have a massive impact. We hope to produce a system the size of a briefcase that can detect whether there is foot-and-mouth present in real time."
(Stratophase has no current plans for turning their own optical technology into products, speaking of the "heavy lifting" needed to get the product to the consumer.)

August 17th 2010 ~ Successful detection kits are manufactured and are already in use elsewhere for rapid lightweight and on-site diagnosis - Why are we not using them?

Technology that could have saved priceless breeding stock, farmers' livelihoods and a great deal of misery in the last two UK outbreaks in 2001 and 2007 - and in Japan this year - continues to be ignored. The reasons are unfathomable. See for example the very positive conclusions of "Real-Time Reverse Transcription PCR Detection of Foot-and-Mouth-Disease Virus Using the R.A.P.I.D. System" (pdf) (The RAPID "suitcase" can be used by field personnel with minimal training.)
The UK based Smiths Detection also has a portable, briefcase sized device for on-farm detection of foot and mouth disease . Their Bioseeq^TM gets a result from the sample in about 90 minutes on farm - yet although the study Diagnosis of FMD by RT-PCR: prospects for mobile and portable assays (pdf) funded by DEFRA concluded that such instruments
"provide realistic options for performing molecular assays for FMDV away from centralized laboratories"
the study then went on to say:
"validation of these and other mobile and portable instruments is required before RT-PCR methodology can reliably be used in the field or closer to suspect premises for FMD outbreak diagnosis and control."
Can anyone explain why "validation" takes so long when what needs to be validated has already been so successfully tried and tested in such studies?

March 18th 2010 ~ President Obama's National Strategy for Countering Biological Threats

Available here This is a very important document published in November 2009. The program Roger Breeze advises will be the principal actor in this for human and animal infections caused by US select agents (including AHS) and new and emerging infections. This is what his team will be implementing in several continents including East Africa. The importance of the document lies in its expression that the US cannot shelter from the world of dangerous diseases by stockpiling vaccines and diagnostics at home. "Rather we have to be out and about where these diseases arise and persist to reduce the threat to everyone at the source." This is exactly what Dr Breeze has been preaching for several years. So, for example, the place to limit AHS is in Africa- and that is what Dr Breeze's teams are starting to do. As he says,
".. it will take decades and there will be many setbacks. But the US seeks partnerships with all others to build on the base we establish. One of the innovations we will bring is rapid and accurate diagnostics for "point of care" - at bedside or in the village. These may be like a sheet of postage stamps where the diagnostic reagents are printed onto paper using an inkjet printer and a single test torn off. Or a simple device linked to a cell phone to send data."

March 12th 2010 ~".The matter of pathogen detection has been trivial for some time and the tough question has become how to best use the new tests and the information they generate."."

In continuing disappointment at Britain's apparent ignorance of significant technology advances in rapid diagnostic tests: ( examples are mass tag, FilmArray and the multi-pathogen chip) we once again quote from the 2006 OIE paper by Roger Breeze 'Technology, public policy and control of transboundary livestock diseases in our lifetimes'
"....We need to get these new generations of tests validated and out where they can be used within one year or less ..
There are repeated calls for cheap, rapid pen-side tests that can detect FMD and other pathogens.... Such tests have existed for some years. The FMD real-time PCR test (and others for CSF, etc.) was always intended to be the trigger for regulatory action and response when used to detect and report infection, in close to real time, from the index farm over the Internet. .... The differential test that discriminates animals vaccinated against FMD from those that were previously infected should also not be limited to rare use in developed countries after vaccination programmes..."
As the paper reminds us, the research that defined real-time PCR tests for FMD, CSF and other transboundary diseases "was disclosed and demonstrated to veterinary regulatory agencies in the USA and Great Britain and to members of the US Animal Health in 2001; the scientific paper was published in 2002 (3). In 2006, a joint effort by British and US FMD diagnostic scientists essentially confirmed what was known in 2001 (7). But the test has still not been recognised by the OIE for international use for the diagnosis of FMD, even though the 2006 study (7) rediscovered that it would detect the presence of dead virus that could not be grown in cell culture and was, therefore, the new state of the art, as it had been since 2001. .." (Read paper in full - pdf)

March 2010 ~ African Horse Disease - "a real-time RT-PCR method has been developed"

From Journal of Veterinary Diagnostic Investigation Vol. 20 Issue 3, 325-328 by the American Association of Veterinary Laboratory Diagnosticians Real-time fluorogenic reverse transcription polymerase chain reaction assay for detection of African horse sickness virus (Abstract) by Montserrat Agüero et al
"....Currently, only conventional (gel-based) reverse transcription polymerase chain reaction (RT-PCR) protocols are available for its detection; however, these methods are cumbersome and difficult to apply when large numbers of samples are to be tested, as in the case of epizootics. To overcome this problem, a real-time RT-PCR method has been developed, based on a 5'-Taq nuclease-3'-minor groove binder-DNA probe (TaqMan MGB) for detection of a wide range of AHSV serotypes and strains designed to the highly conserved region of the VP7 gene (segment 7). The method was able to detect all prototype strains from the 9 known serotypes of the virus, with a high analytical sensitivity; no cross-reactions were observed with other orbiviruses or with other viruses affecting horses.... This method, which can be performed in 96-well format, is suitable for large-scale surveillance of AHSV in areas where it can potentially spread."

November 5 2009 ~ Animal and Veterinary Molecular Testing - "increasingly important for modern veterinary and agricultural applications"

Qiagen, the company that has just announced an expansion of its strategic partnership with the VLA (the Veterinary Laboratories Agency), has an interesting website:
"Molecular testing of animals has become increasingly important for modern veterinary and agricultural applications. Diagnostic tests using PCR (e.g., for Johne's disease in cattle or herpesvirus in dogs and cats) are supplanting traditional ELISA or culture-based methods due to higher sensitivity and speed. Genotyping provides a valuable tool for selective breeding, from scrapie resistance in sheep ...This section of our Web site is designed to provide the technologies you need for molecular testing of animals."
The website has been newly updated. It does not seem very encouraging to read that the "listed testing kits, reagents, and methods are not approved by the USDA in the USA" but this is perhaps because the USDA and FDA cost a great deal to satisfy and that FDA / USDA approved testing is out of date (because most manufacturers cannot afford to go back again). "Cador BVDV real-time PCR kits are approved by the Friedrich-Loeffler-Institute in Germany for veterinary diagnostic use."
See also FAO news

September 9 2009 ~ Pirbright's lateral flow device: "a supportive method ...before diagnosing the disease in the laboratory.."

The abstract of the paper, "A simple and rapid lateral flow assay for the detection of foot and mouth disease virus" (PubMed) (September 2009) on Pirbright's lateral flow device, tested on samples from naive animals from Korea: "... Diagnostic sensitivity and specificity of the LFA for FMDV types O, A, C and Asia 1 was similar at approximately 87.3% compared to 87.7% obtained from the antigen ELISA. The diagnostic specificity of the LFA was 98.8% compared to 100% for the antigen ELISA. These results demonstrate that the LFA using the FMDV Mab 70-70 to FMDV is a supportive method for taking a rapid measurement at the site of a suspected FMD outbreak in Asia before diagnosing the diseases in the laboratory, thereby offering the possibility of implementing control procedures more rapidly."
The conclusions were only slightly different from that of Ferris et al at Pirbright (Jan 2009) whose abstract, Development and laboratory validation of a lateral flow device for the detection of foot-and-mouth disease virus in clinical samples, concluded: " These data illustrate the potential for the LFD to be used next to the animal in the pen-side diagnosis of FMD and for providing rapid and objective support to veterinarians in their clinical judgment of the disease."

September 8 2009 ~ Diagnosing bTB - a breakthrough? New technology can test for "any viral or bacterial disease using any body fluid"

"These devices are specifically designed to be field deployable and ultraportable allowing for very rapid detection of various viral and bacterial pathogens by relatively untrained personnel outside of the laboratory setting" (Source). The website of Biomagnetics Diagnostics Corporation describes the HTS-MTP - designed to detect, in a single test, the virus and count the viral load in a matter of minutes, not just simply screen for the presence of viral antibodies.
"No other screening, or confirmatory test, has the ability to measure viral load, a critical measurement .... It has the capability to screen several hundred percent more assays, per hour, at a fraction of the cost of current technology."
Bovine TB is now recognised as a growing problem throughout the world with an estimated 1.3 billion cattle at risk so the company cites bTB particularly - although it claims the technology can test for any viral or bacterial disease using any body fluid , "Field deployable integrated optical biosensor systems hold the promise to significantly speed the diagnostic testing process and to meaningfully lower costs..... Biomagnetics hopes to have products in use in China by late 2010/ early 2011, followed by US and European sales sometime in 2012, pending successful financing and approvals." (Animated diagram gives a demonstration of the machine here.)

August 26, 2009 ~ Emergency authorisation for the rapid PCR device, JBAIDS, to diagnose H1N1

It was a whole year ago that Dr Roger Breeze wrote: "It has been possible and practical to use the RAPID device to detect avian influenza virus on farm and in the field since 2000. The RAPID is sold in a small suitcase.." Today, we read at www.medpagetoday.com not only that the JBAIDS device, Idaho's successor to RAPID, is to be used by the US military to perform rapid diagnosis of H1N1 in the field - but also that "military doctors are going to request FDA approval to use the JBAIDS device for diagnosing H5N1 avian flu".
One continues to share Dr Breeze's wry astonishment that this technology - whose effective use against H5N1 by the Royal Thai Army and the US Defense Department in Thailand he described last year with such admiration - has not been in use for years and in all countries for the rapid on-site diagnosis of all dangerous animal and human pathogens for which a suitable assay has been developed. As he wrote in 2008, "I can only wonder how different things might have been had Thailand had a Chief Scientific Advisor to the Government of the stature demanded in UK, or a resource like Imperial College, or even the benefits of the research just funded by the EU"(see below)

19 July 2009 ~ "The aim of these new tools is to detect the presence of a pathogen agent before the onset of disease. "

From Advances in viral disease diagnostic and molecular epidemiological technologies Authors: Belák, Sándor1; Thorén, Peter; LeBlanc, Neil; Viljoen, Gerrit Source: Expert Review of Molecular Diagnostics, Volume 9, Number 4, May 2009 , pp. 367-381(15)".....The newer-generation molecular diagnostic technologies offer, hitherto, unparalleled detection and discrimination methodologies, which are vital for the positive detection and identification of pathogenic agents, as well as the effects of the pathogens on the production of antibodies. The development phase of the novel technologies entails a thorough understanding of accurate diagnosis and discrimination of present and emerging diseases. The development of novel technologies can only be successful if they are transferred and used in the field with a sustainable quality-assured application to allow for the optimal detection and effective control of diseases. The aim of these new tools is to detect the presence of a pathogen agent before the onset of disease. This manuscript focuses mainly on the experiences of two World Organisation for Animal Health collaborating centers in context to molecular diagnosis and molecular epidemiology of transboundary and endemic animal diseases of viral origin, food safety and zoonoses."

1 July 2009 ~ "Enigma FL provides a PCR result in less than one hour, a timescale needed to allow field-based & pen-side diagnosis"

Good news from Enigma Diagnostics, the private company at Porton Down that specialises in on-site diagnostics. The press release yesterday (pdf) was to publicise their Enigma FL (Field Laboratory) and its independent testing by the Veterinary Laboratories Agency. It seems that the VLA has successfully evaluated the use of the Enigma FL to diagnose bovine viral diarrhoea (BVD).
As for "exotic" diseases, with the best will in the world, and as we saw in 2001, sending samples of suspected disease to the international reference laboratory at Pirbright takes up vitally needed time. Even those animals who did get tested in recent outbreaks of foot and mouth (and many did not) were killed by officialdom choosing not to wait for a definitive answer. It was rare for farmers to be told that their killed animals had in fact returned a negative test - but tragically many healthy animals were summarily slaughtered and in 2001 and even 2007, many more were killed simply because they were within the "contiguous" area of a suspected 'Infected Premise' (often a misnomer)
The Enigma FL is, of course, just one of the new generation of machines to pinpoint the presence of pathogens in real time before clinical symptoms are apparent - but, given that commercial interests tend to be given a higher priority than animal health- the fact of its being British may make its adoption and use in the UK more likely.

June 3 2009 ~ "when there's a new outbreak of a serious virus in a remote area of Africa, it can take weeks for the information to reach the state veterinary services. Efforts to control the disease are hampered by this delay."

Telegraph
"... the combination of new types of mobile phones combined with increasing availability of broadband internet access in Africa may help.. Locally trained veterinary assistants on the ground will be able to log suspected cases of new illnesses as they happen.....clear, real-time mapping of the spread of livestock diseases, so that vaccines and treatments can be targeted much more effectively."
Meanwhile, in the UK, while similar rapid disease mapping for livestock is still not in use and the rapid portable RT-PCR technology is still not being spoken about, a new form of disease mapping for pets has just been launched. CICADA-Live was launched on Monday by Intervet/Schering-Plough Animal Health as an interactive register of serious viral diseases of small companion animals.

May 1 2009 ~ "The Department of Defense can fast track a reagent within hours for use in hand held satellite linked PCR device..."

On the subject of the inability of states to conduct on site testing for pandemic H1N1, Stephen M. Apatow, Founder, Director of Research and Development Pathobiologics International, has written a memo mentioning the rapid diagnostic kit by which the Department of Defense can fast track a reagent within hours.
"This molecular diagnostics technology has existed for years but has not yet reached the grassroots physician/veterinarian level."
Without validated tests, he says, there is a wait of 2-3 days because samples need to be shipped to the CDC lab in Atlanta. He calls this "a public health infrastructure disgrace" He recalls in the midst of a previous WHO pandemic level 5 scenario,
"..Roger Breeze did the PCR presentation at this meeting and my paper "Agricultural Security and Emergency Preparedness: Protecting One of America's Infrastructures" was a reference point for agricultural security. .."
Link to Humanitarian Resources Institute.

May 1 2009 ~ Rapid diagnostic test: "this identifies the current H1 and future changes in that target that have not yet evolved"

Meanwhile, Dr Breeze has given warmwell.com permission to quote from a letter whose subject concerns the ability of the TessArray® RPM-Flu assay to accurately detect the current H1N1 flu. Dr Breeze comments:
"This technology provides the actual sequence of up to 1500 bases of the nucelic acid target so if the H1 target mutates this test tells you the mutation directly. It is biosafety level 2 and does not require Biosafety level 3 containment because no virus is grown."
Extracts from the letter The language is highly technical - but we read that TessArae makes the following offer to recipients of the letter:
"If you want to analyze clinical specimens with suspected 2009 outbreak swine flu, we are available to support your use of the TessArray RPM-Flu 3.1 assay.
If you are willing and able to share your assay results with the community, then we are willing to replace a few of your TessArray RPM-Flu 3.1 assay kits at no charge."
The key point - as Roger Breeze makes clear- is that this rapid diagnosis kit "identifies the current H1 and future changes in that target that have not yet evolved."

April 7/8 2009 ~ A test that can detect MRSA without culturing the bacteria or amplifying the DNA

Rapid diagnostics are making advances all the time in both human and veterinary medicine. A new test from Adnavance, for example, is built on top of a gold electrode that is covered with a proprietary marker that binds to the drug-resistant bacteria. As current flows through the electrode, it attracts negatively charged DNA, generating an easily detectable change in the surface charge of the electrode. The Adnavance website says its platform will "de-centralize this market and provide rapid turnaround of medically important tests." and they expect to launch the product as early as 2010 "targeting sales in the US, Canada and Europe"
Technology Review says,
"...In an effort to stop hospital outbreaks, a growing number of states require that hospitals test patients for MRSA before admitting them, or are considering legislation that would require them to do so. ... To identify whether a patient is infected with MRSA, hospitals either culture bacteria collected from nasal and other samples, which can take several days, or perform newer molecular tests, which are much more expensive. Only about 35 percent of hospitals in the United States are certified to perform the newer type of testing. The others must send samples out for analysis, which can also take time....Adnavance is developing a new test that can detect MRSA bacteria directly, without requiring that the DNA be amplified, as is done in existing molecular tests..."
Read in full

January 20 2009 ~ Spectrosens - an optical sensor chip developed by Southampton University spin-off Stratophase - will form the basis of a £1m project

www.eurekamagazine.co.uk
"...The new system should enable inspectors, and ultimately vets or farmers, to identify the infection on the spot. The collaborative project will bring together teams from Stratophase, the University of Cambridge, Bristol Industrial and Research Associates Limited and Chelsea Technologies Group. It should be more sensitive and accurate than existing field-deployable systems, and much faster than laboratory-based techniques. The system will collect pathogens from the air and put them into a liquid stream. The liquid will then be analysed 'in the field' using the Spectrosens detectors. When a specific pathogen sticks to the surface, the chip undergoes a tiny optical change.
Devaki Bhatta, project leader from Stratophase, said: "The ability to collect and identify airborne pathogens will remove the need for swabs and blood samples. This project is to develop a foot and mouth detection unit but could be used to identify TB and a range of other serious illnesses that affect livestock."
One year ago, a German university began a project to assess the chip's suitability for a range of applications.
The first complete sensing unit based on the technology was chosen by the University of Applied Sciences at Aschaffenburg for a three-year research project. Aschaffenburg believes the technology could have widespread commercial application in food, beverage and pharmaceutical manufacturing industries, drug discovery and process control. It will investigate how to combine the technology with microfluidic structures to create a robust, cost-effective biochemical sensor for commercial use. Stratophase's technology detects toxins, viruses and bacteria in real time. It has an optical silicon chip with an integral planar Bragg grating, using a fibre-optic cable to transmit light to the chip. When contaminants are carried in liquid across the surface, tiny changes in composition can be detected by precise, continuous monitoring of the wavelength of light reflected from the sensor. The surface can be pre-treated to make it sensitive to certain biological or chemical reactions.
The university's Dr Ralf Hellmann said: "As a purely optical process, it relies on changes in the refractive index on the surface of the sensor. This eliminates the usual and costly complication with other technologies of preparing the liquid specimen with fluorescent tags."
See also below

January 4 2009 ~ The biggest breakthrough for 50 years? Really?

Today's news about Pirbright's "lateral flow device" at the FMD newsportal at (UC Davis ) is baffling. Has anything actually changed since we last reported on this? It doesn't matter how quickly the penside lateral flow device is used at the lab or on the farm - our understanding is that the lateral flow device is designed to detect antigen and this can only be detected from lesions. The animals must already have developed lesions, hence the instruction in 2007 to look for lesions twice a day, before the penside test could be used at all. Informed comment would be very gratefully received.

January 4 2009 ~ But infection can be detected before it reaches the stage when a number of animals can be seen to be clinically infected.

Rapid on-site RT-PCR tests that can find disease in animals before they show any clinical signs at all are what will revolutionise diagnosis. IAH's 2007 statement 14 claimed that their lateral flow device could return a positive result within an hour "enabling DEFRA to take action" However, the "action" was always going to be slaughter - slamming the stable door long after the virus was on the move. Has the situation changed?
On December 10 2008 we reported that Biodetection expert Stratophase was working to develop a detector system using immunoassay diagnosis with teams from the University of Cambridge, Bristol Industrial and Research Associates Limited and Chelsea Technologies Group. (See www.cattlenetwork.com) and that the "Portable Direct Immunoassay Diagnosis Device for Animals and Humans (PDIDDAH)" will be "significantly more sensitive and accurate" than the lateral flow tests used at Pirbright. Its sensor system collects pathogens from the air and puts them into a liquid stream thus removing the need for swabs and blood samples.
The paper published this week by BMC Veterinary Research 2008 "Diagnosis of foot-and mouth disease by real time reverse transcription polymerase chain reaction under field conditions in Brazil" by Tatiane A Paixao et al.describes rapid and reliable detection of FMDV allowing identification of infected animals before development of clinical signs - under field conditions and in a fraction of the time required for combined virus isolation/ELISA.
In order to give retrospective legality to the mass slaughter of 2001 the 2002 Animal Health Act made provision for pre-emptive slaughter "on suspicion". What is now certain is that the development of rapid diagnosis for FMD and other pathogens is proceeding very fast. Any publicity that alerts farmers and vets to the fact that state of the art technology makes the slaughter of uninfected animals unnecessary must be good news.

Dec 31 2008 ~ ".... this approach allows identification of infected animals even before development of clinical signs..."

Published today by BMC Veterinary Research 2008 is "Diagnosis of foot-and mouth disease by real time reverse transcription polymerase chain reaction under field conditions in Brazil" by Tatiane A Paixao et al. Extract (BioMed Central provisional pdf):
"... several groups have been developing faster diagnostic methods for FMD based on amplification of specific sequences of the viral genome by reverse transcription polymerase chain reaction (RT-PCR) [8-21], which can be applied to different kinds of biological samples such as fluids and tissues, and in some cases, this approach allows identification of infected animals even before development of clinical signs or positive virus isolation as well as identification of positive cattle at the end of the course of infection when virus isolation may be negative [12, 22]. In addition, in a study in which results were compared between reference laboratories, RT-PCR results were more consistent among laboratories as compared to virus isolation and ELISA results..."
The rapid and reliable detection of FMDV allowing identification of infected animals even before development of clinical signs - under field conditions and in a fraction of the time required for combined virus isolation/ELISA - has once again been demonstrated by this team. Pre-emptive slaughter on suspicion - with all its waste of life and attendant miseries - can surely now be consigned to the dustbin of history.

December 10 2008 ~ Another rapid portable test being developed

Biodetection expert Stratophase is working to develop a detector system using immunoassay diagnosis. The collaborative development project is being co-funded by the UK government-sponsored Technology Strategy Board and brings together teams from Stratophase, the University of Cambridge, Bristol Industrial and Research Associates Limited and Chelsea Technologies Group. We read on www.cattlenetwork.com
"The sensor system will collect pathogens from the air and put them into a liquid stream. The liquid will then be analysed 'in the field' using Stratophase's optical sensor technology. The optical chips are coated with antibodies designed to attract specific antigens, such as those for TB or foot and mouth. If the targeted agent is present, it sticks to the surface and the chip undergoes a detectable change in optical spectrum, confirming the presence of the disease."
This Portable Direct Immunoassay Diagnosis Device for Animals and Humans (PDIDDAH) will be significantly more sensitive and accurate than the lateral flow tests used during the 2007 FMD crisis. Dr Devaki Bhatta, project leader from Stratophase, said, "One of the most important developments that will be realised in this system is the ability to collect and identify airborne pathogens. This will remove the need for swabs and blood samples. This project is to develop a foot and mouth detection unit, but using different antibodies the system could be used to identify TB and a range of other serious illnesses that affect livestock."

November 29 2008 ~ Smiths Detection is seeking partners to develop tests for its Bio-Seeq molecular diagnostics instrument

The UK company hopes that British hospitals will evaluate a multiplex assay for MRSA. The website www.genomeweb.com says:
".. ..the Bio-Seeq platform, is initially being marketed to government veterinarians doing outbreak containment and monitoring work for diseases such as foot and mouth disease and avian influenza..."
Genome Web adds that the director of business development for Smiths Detection, says he believes Smiths' assay will be easier to use than the Cepheid test. The company holds the exclusive rights to the Brandeis university's LATE (Linear After The Exponential Polymerase Chain Reaction) PCR technology which eliminates the need for real-time PCR and the associated licences. Czajka believes that LATE PCR's sensitivity is "often as good if not better than real-time PCR." While most real-time PCR multiplex tests run about four to 10 targets per assay LATE PCR assays will "be on the order of 20 to 50".
As we have been saying for seven years now, rapid diagnostic testing should be considered vital in the fight against diseases such as FMD and avian influenza since rapid detection of the virus before clinical signs are apparent should make pre-emptive slaughter a thing of the past. (More on rapid diagnosis)

November 10 2008 ~ Rapid test for C. difficile infection gets EU go-ahead. And for animal diseases...?

C. difficile is resistant to most antibiotics. There are roughly 58,000 detected C. difficile cases in the UK each year, and the number of deaths rose by around 55 percent to 8,500 in England and Wales in 2007. The virulent Type 027 strain of C.difficile has been reported in 16 European countries. C. difficile infections are estimated to cost the NHS up to £8,000 pounds per patient and more than £200 million each year. Now Cepheid has launched its molecular diagnostic test as a European CE IVD Mark product under the European Directive on In Vitro Diagnostic Medical Devices.
"European hospitals will have access to a reliable, rapid test for Clostridium difficile, which simultaneously detects and identifies the commonly occurring strains and specifically identifies the more virulent Type 027 strain. The new 50-minute test runs on Cepheid's GeneXpert(R) System.." Cepheid
The GeneXpert(R) System is "a closed, self-contained, fully-integrated and automated platform that represents a paradigm shift in the automation of molecular analysis, producing accurate results in a timely manner with minimal risk of contamination".
Such a system can also, with the appropriate reagents, detect such animal diseases as Foot and Mouth before the onset of clinical signs. The 2001USDA test, offered to the UK for field testing in 2001 detected the presence of FMD virus in experimentally infected cattle, sheep and pigs before clinical signs of disease were detectable. It took about 60 minutes. Sample preparation was minimal. The offer was turned down by Professor David King and his colleagues.
That was seven years ago.
Had such available kits been used in 2001 and 2007 to detect which animals really were infected there would have been no unnecessary culling with all the attendant trauma and financial loss. News that such systems are also validated and used in Europe for early pre-clinical-sign diagnosis of virulent animal disease is surely long overdue.

August 18 2008 ~ Can the EU really - possibly - not know that such a small portable machine has been in existence for years?

According to the BBC website, the EU is to fund a £2.3 million project to develop a diagnostic machine for H5N1. UK experts have called for a national surveillance programme to detect H5N1 cases in Indonesians and Dr Alan McNally, from Nottingham Trent University, "believes his technology could make a difference."
However, Dr Roger Breeze tells us that the Indonesian government and US Department of Defense Global Emerging Infections Surveillance System (GEISS) have already been monitoring, detecting and responding to avian influenza H5N1 in Indonesia for several years. GEISS uses PCR devices in labs and also the Idaho Technology RAPID for portable field detection.

August 18 2008 ~"... It has been possible and practical to use the RAPID device to detect avian influenza virus on farm and in the field since 2000."

Dr Breeze recently visited the laboratory at Kamphaeng et, in northwest Thailand, operated by the Royal Thai Army and the US Defense Department and his email, complete with links, explains that when Indonesian patients are PCR positive (the hospital lab returns results within 2 hours), a mobile team then travels to the affected village to examine and test others with just such a portable RT-PCR kit as the EU now declares it "hopes" to develop.
"... I asked the lab director if the results of PCR tests performed in the village were regarded by public health authorities with the same validity as those conducted in the lab itself. She replied,"Of course. It is the same people performing the test in both places using the same controls, test reagents and equipment. Why would we not treat them identically?"
The EU, says the BBC, "hopes to come up with a version of the machine that can fit within a briefcase" Dr Breeze points out that the RAPID, in use now for several years, "is sold in a small suitcase".... Read in full

April 2008 ~ Svanova Biotech launches rapid test for FMDV

The press release
".. The test has been developed in co-operation with the OIE Community Reference Laboratory for FMD, Institute for Animal Health, Pirbright Laboratory, UK and Instituto Zooprofilattico Sperimentale della Lombardia e dell´Emilia Romagna (IZSLER), Italy, as part of the EU project Lab-On-Site, a project on new and emerging technologies for detection of important diseases in animals and animal products....The SVANODIP® FMDV-Ag is a simple direct test for the detection of all seven serotypes of the FMDV antigen in swab and tissue samples. It is a rapid test, which may be carried out on the field, next to the animal. It may be used for early detection of infection, as first line diagnostics in order to control the spreading of infection. The test procedure is rapid and simple, providing a result within 10 minutes."
(This is a test for antigen.)
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December 2007 ~ "......The issue is whether that test should be done on the farm or in a fixed lab,

See extract from email from Roger Breeze
"... If you detect FMD on farm within minutes - versus days in sending a sample to a lab - you will save enormous amounts of money in response costs downstream. If a farmer had the choice between paying 12 dollars for a test versus seeing his animals killed without testing because they were located within 3 miles of an FMD infected farm, all the farmers I know would be happy to pay for a daily test out of their own pockets. .... If there was an outbreak in the US, we could be testing herds. You would pool samples from several animals in a herd - not test each animal separately. Consider the cost of killing those herds that were not infected in Britain recently - was that a bargain compared to $12? I expect the cassette cost for the simple device will also be $12 to $15. There is an old saying that if you think education is expensive then try ignorance. ...."
Read in full

September 21 2007 ~ "We can now add DEFRA to the list of those who never miss an opportunity to miss an opportunity"

writes Roger Breeze in this article for warmwell today: (Dr Breeze's team has successfully set up rapid on-site testing in former Soviet Bloc countries.)
"..... In 2002 Callahan and colleagues (1) described a real time PCR test that could be performed within an hour on or close to premises where there were animals that might be infected... This test was more sensitive than traditional foot and mouth virus isolation and had the added advantage of detecting virus in non- optimal samples in which the virus was dead and not detectable at all by cell culture.
But Callahan and his colleagues also demonstrated something remarkable and previously unsuspected - that this PCR test could detect foot and mouth infected animals 2 or 3 days before they showed any outward signs of disease and before virus could be identified by any other means.
Had this rapid PCR test been performed on a few nasal swabs collected from each farm examined by animal health officers in Surrey in recent weeks there is no question but that infections would have been detected long ago even though lesions were not recognized.
The British veterinary authorities and the government's Chief Scientist, David King, have known about this technology since 2001 - ......
I also read that the Countess of Mar was told in the House of Lords that "tests have to be validated by the OIE"... This is not true....."
Read full article The conclusion that wilful ignorance has been displayed at the top of DEFRA seems inescapable.

August 10/13 ~ " the laboratory must move into the field and test animals quickly before irreversible actions are taken." ProMed

'ProMED' means 'Program for Monitoring Emerging Diseases' and is the Internet-based reporting programme of the International Society for Infectious Diseases (ISID). The moderators are international experts in their field who screen, review, and investigate reports before posting to the network. ProMED-mail is independent and free of political constraints. To read on ProMed that diagnostic testing should now be "out of the laboratory" is very cheering. On Saturday, a ProMED moderator, in the course of a five paragraph comment about the UK situation, (www.promedmail.org)wrote:
"....In the past -- that is, pre-1980 -- when we killed "contact" herds it was not questioned and laboratory techniques then could not have handled the volumes of samples. Today all that is different and thousands of samples are run each day. This brings home the point that the laboratory must move into the field and test animals quickly before irreversible actions are taken..."
(More) For six years warmwell and others have been asking that the analysis of samples should happen at the place where samples are actually taken rather than be taken by car, train or air to the reference laboratory. Results can now be obtained in the field within minutes rather than hours and days, can detect FMDv before the onset of clinical disease

August 2007 ~ " the case for a humane, civilised and scientifically sound policy has strengthened over the past few years to the point where it is beginning to look unassailable"

Magnus Linklater has kindly sent warmwell his article written for today's Times. He looks back shudderingly to six years ago when the "farming establishment closed ranks against any suggestion that there might be a more humane approach." On the question of vaccination, many readers will share our reaction to this gem:
" I remember asking the government's chief scientist, Sir David King, to explain to me why it was not being considered. "I would need five hours to explain the science to you," he said. "Unfortunately I don't have that time." ...."
But, " Let us not go back there, however. The fact is that there has been, since then, a sea change in attitudes within the Department ....the realisation that the science on which so many of those decisions in 2001 were based, was less sound than we were told..
..Again, there is no point in going back into that debate. What is important now is to record how far science has advanced in the meantime. There are accepted tests which can distinguish between infected and vaccinated animals....We know, too, that FMD "carriers" do not infect other animals... "pen-side" tests .. allow a vet to carry out on-the-spot checks to determine whether a herd of cattle or a flock of sheep have been infected, rather than having to send samples back to a laboratory. Rapid diagnosis of this kind means that biosecurity measures can be imposed immediately rather having to wait for the results of tests.
... vaccination can begin within that area as soon as it is available ...
.... I cannot, hand on heart, say that the battle for the vaccine has been won. There are still those in Defra and elsewhere, who will argue for slaughter as the only effective response to this disease. But the case for a humane, civilised and scientifically sound policy has strengthened over the past few years to the point where it is beginning to look unassailable..." Read in full (or on Times website)

August 7/8 2007 ~ A prototype on-site rapid diagnostic machine is being used in Surrey

A very impressed Bryn Wayt has sent this email to many contacts. "... a very nice and helpful lady vet and phoned and confirmed that, "a prototype RT-PCR unit had been used on the first IP, and the VO on the second site would be using it." His email is worth reading in full. We feel the manufacturer is irrelevant - as long as rapid test results can be obtained before the long wait for lab confirmation. It appears that Pirbright has been doing quite a bit behind the scenes with portable devices. (UPDATE Pirbright's portable machines are not performing RT-PCR it seems but the very good news is that the whole portable PCR field will be transformed with very cheap machines that are highly automated within the year, according to a reliable source. )
We find this news exciting - but once again, it has to be teased out and we are very grateful indeed both to those who ask the searching questions - and those who give the answers.

12/ 13 June 2007 ~Rapid diagnosis via automated multiplexing platform: "we have always known that the platform's flexibility confers benefit in other markets, such as veterinary diagnostics and the monitoring of bioterror threats such as foot and mouth"

A news release ( Thanks for news of this link to FMD News - a service provided by the FMD Surveillance and Modeling Laboratory, University of California at Davis ) from Nanogen, Inc reports on new funding and "collaborative agreement" with Canadian agencies which include the Canadian Food Inspection Agency (CFIA)
".... The purpose of the funding and collaborative agreement is to develop diagnostic tools for the detection of natural or potential bioterror threats to livestock, such as foot and mouth disease and avian flu, employing the company's NanoChip® platform ... The NanoChip® 400 is the company's second generation automated multiplexing platform....the system provides a simple, fast and cost effective means for performing molecular testing.."

March 20 2007 ~ "the main device under discussion at the meeting is a $1,000 mobile test system and reader the size of a small portable television"

Animal health experts from 15 nations, including the UK, are meeting today to discuss rapid diagnosis technology. The conference, The Early and Rapid Diagnosis of Transboundary Animal Diseases: Phase I- Avian Influenza is taking place in Vienna and is part of a $500,000 coordinated research project run jointly by the FAO and the International Atomic Energy Agency ( www-naweb.iaea.org/nafa). The project aims later to field-test devices, identify areas where the kits can be used first and explore sources of funding.
John Crowther of the Joint FAO/IAEA Programme's Animal Production and Health section points out:
"The genius here is that such mobile testers can be used by anyone, with the most basic training. Even farmers could do a test and the result could immediately be processed back to a central point, like a mobile phone message. Within two years, such tests could revolutionize disease diagnosis. Ultimately the tests would be done locally by people in their own countries, making schemes much more efficient in everything including speed, costs and local knowledge."
See more at www.un.org/apps/news/story
( It was in 2001 that Professor Fred Brown first argued for the UK to field trial such a device. As it was, with neither vaccination nor on-site diagnosis, carnage by computer, ruthlessly enforced, was the result of the UK's inability to pinpoint where the disease really was.)

February 25 2007 ~ Rapid Diagnosis for flu in birds: Multiplex test

From the latest report of the USDA/ARS Research Project: Development and Validation of Rapid Diagnostic Tests for Avian Influenza and Newcastle Disease
".....We have also developed an internal control to be run as a multiplex test with both the AIV and NDV tests to assure that the RRT-PCR reaction was performed correctly. The internal control should help to eliminate false-negatives. We have also developed hemagglutinin subtyping tests for most of the 16 described subtypes of AI. We remain active in the evaluation of primer sets to assure they work well with outbreak viruses from around the world. Finally, the development of dried down reagents to aid in the stability of the reagents, increased quality control, and ease of use of the test has already developed interest from a number of different diagnostic laboratories. The Southeast Poultry Research Laboratory (SEPRL) has been on the leading edge of use of real time PCR testing as a diagnostic test for viral pathogens of poultry with its work on avian influenza virus and Newcastle disease virus. Through collaborations with APHIS, the rapid diagnostic test for avian influenza and Newcastle Disease was validated and adopted by APHIS ...."

January 26-28 2007 ~ Silence on validation

Readers of warmwell may share our bafflement about how the grail of validation is to be achieved, especially for the rapid diagnosis on-site technology for foot and mouth. Pirbright's rapid diagnostic strip test device for FMD was discussed at the 2000 conference in Bulgaria, but, after 7 years, there are no signs that it is to be used in the field - or even "considered". As for the ARS machine, offered to the UK and used successfully in Uruguay in the same year as our disaster, the US GAO ( Government Accountability Office) paper, Homeland Security: Much Is Being Done to Protect Agriculture from a Terrorist Attack, but Important Challenges Remain GAO 2005: contained lines which were on the advice of the former Associate Administrator for Special Research Programs at USDA's Agricultural Research Service
".... Importantly, the tools can detect disease before the animal shows clinical signs of infection...... rapid diagnostic tools would not only allow for a rapid diagnosis but would also permit the monitoring of nearby herds before symptoms appeared so that only infected herds would have to be killed......rapid diagnostic tools would be helpful because FMD would be detected in less than an hour, informed control measures could be implemented, and herds in the area would be under regular surveillance."
But rapid diagnosis in the field seems as far away as ever. In 2005, when the Countess of Mar was told: "tests have to be validated by the OIE. We are waiting on that" her frustration was evident. " My Lords, it is coming up to five years since the foot and mouth disease outbreak. If that has not been done, can the Minister say why not? I remember the late Fred Brown coming over from America and telling us that they were using the tests in America. Why has that not been done in this country in the past five years?
It is now over six.

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A RAPID PCR machine (www.idahotech.com) costs about £40,000 pounds and the tests for exotic diseases like foot and mouth, classical swine fever, avian influenza, and Newcastle disease cost about £ 3 pounds each. Of course, with other test reagents, this same machine can detect all the common animal diseases too (with the exception of BSE and scrapie).

PCR assays were designed to be performed as real time assays outside BSL 3 containment either in a laboratory or on portable devices taken to the site of the problem.

For the latter purpose, there are three machines now available: the RAPID and RAZOR from Idaho Technology Inc. and the Smartcycler om Cepheid Inc (new window). In time, there will be other devices that are cheaper, faster and more robust. But all will share the same characteristics. Assays will be performed by persons of limited training (soldiers, technicians); assays will be performed using quality-controlled standardized reagents and protocols that are internationally consistent; results will be obtained in an hour or less; assays will be reviewed over the Internet as they take place by technical experts located at distant sites; and detection results will flow into an Internet-based Command and Control system

Extract from Dr Roger Breeze's letter to warmwell Jan 2006

"As you know from my papers, I believe the PCR offers a transforming moment for FMD control by which one can monitor all the herds in an area continuously and only slaughter those where there is infection (and I believe in vaccination immediately also).

... Many FMD viruses representative of all 7 serotypes were genetically sequenced by Dan Rock's team at Plum Island and the sequence information was transmitted electronically to Tetracore in Maryland. Rock and Tetracore worked together to compare the complete sequences of many different viruses simultaneously to find regions of the sequence that were identical between all the different viruses - these common regions would be targets for PCR tests. Tetracore have some proprietary software that eases this comparison.
Having identified likely targets, Tetracore made reagents to these targets and Rock tested these with real viruses at Plum Island. From this, the ARS Tetracore FMD PCR test was developed, and this did not require any materials that had ever been in contact with live viruses....
.... What happened with the FMD and other PCR tests is that "validation" became a smokescreen to preserve monopoly and jobs. And as was demonstrated by California, all the waffle from those insisting on "further validation" and federal not state responsibility disappears out the window when the fire starts in their own homes.
Currently, I understand that the ARS Tetracore FMD PCR test is very close to approval by USDA and OIE, a matter of months I hear." Read in full

March 2006 ~ On 13 March, a field laboratory with real-time polymerase chain reaction (RT-PCR) capacity to detect influenza A/H5 virus, was established by the US Navy Medical Research Unit 3 (NAMRU-3), Cairo (Egypt), at the Anti-Plague Station (APS) in Baku.

March 22 2006 ~ Cepheid's GeneXpert is the world's only system to combine sample preparation with real time PCR (polymerase chain reaction) amplification and detection

-......answers from unprocessed samples in minutes. ..... No specialized facilities or highly skilled laboratory training is required to operate the GeneXpert System "Cepheid (Nasdaq: CPHD), a broad-based molecular diagnostics company, announced today that it has received both certification and designation of the GeneXpert(R) technology as Qualified Anti-terrorism Technology (QATT). GeneXpert is now listed among products approved by the Department of Homeland Security and is granted coverage under the Support Anti-Terrorism by Fostering Effective Technology Act known as the SAFETY Act..."

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See also background material

About rapid diagnosis tests

PCR for Foot and Mouth - click here

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On-site use of tools such as these is critical to speeding diagnosis, containing the disease, and minimizing the number of animals that need to be slaughtered.

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Communications from and by Dr Roger Breeze, formerly of Plum Island

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Rapid PCR and Bovine TB Control in Great Britain

See the " Paper for Discussion " by the National Beef Association (page 10 of pdf file)

PCR (Polymerase Chain Reaction)

There are two forms of using this powerful technique by which an enzyme and a cycle of heating and cooling is used to generate billions of copies of segments of DNA (to make detection and spoligotyping easier). After multiplication, the system identifies TB, or any other bacteria, or virus or DNA material by comparison with a known sample, utilising the properties of florescent light to do so.

a. Laboratory-based conventional heating block thermocycler using agra gel electrophosesis; this has greatly facilitated research in the Badger Road Traffic Accident study.

b. A portable mini-lab which can give an on-the-spot diagnosis of infection within 30 minutes; this technique has been developed for detection of biological warfare agents on the battlefield in the US, and in this country by the Defence Science and Technology Laboratory. In the UK it is being "spun-out" by an offshoot of the MOD, Enigma Diagnostics, with investment led by Porton Capital, and including the Treasury and a private venture company, Partnerships UK, and was announced in the veterinary press in September. I

A variant of this system in the form of a machine called a Lightcycler, was recommended by Professor Fred Brown of the US Plum Island Animal Disease Research Center in 2001 to the UK Government to rapidly diagnose Foot and Mouth on site. One individual went as far as ordering one, at a cost of #20,000, but the Government intervened to prevent this without providing the industry or even the individual with an explanation.

See also Veterinary Times 27th Sept '04 "Battlefield technology deployed in fight against bovine TB" and BBC News 4th Oct '04

The obvious potential of a portable PCR cycler machine is to give a rapid identification of TB and the spoligotype of TB present in badgers. If one animal from a sett is found to have TB of a type causing infection in nearby cattle, then that sett could be treated with carbon monoxide with less nervousness by Ministers who would be able to give a better explanation to the general public.

There are 29 strains or spoligotypes of bovine TB, of which 17 are found very infrequently. In the UK the most common is type 9 with type 11 being more common in Devon, type 21 and 9 more common in Somerset and Dorset, and Cornwall being higher in types 9 and 15. The geographical distribution of spoligotypes of bovine TB in badgers has a high level of correlation with the distribution of spoligotypes in cattle. Spoligotype 35 has recently been identified in farmed deer near Ulverston, Cumbria, and linked to a spread to cattle there. The samples for multiplication in the PCR machine can be from any source and could merely be from a small amount of cattle blood or badger sputum or urine. Samples from several animals can be put in each of the glass testing tubes within the machine. A single case of infection in one animal would show up, allowing immediate rechecking of the animals in that batch.

The suitability of the portable PCR cycler machine for testing cattle for TB obviously depends on finding cattle that are shedding TB bacilli - either in milk, saliva, dung or urine - or which have bacilli in their blood.

The potential advantages of the PCR cycler over the gamma interferon test is that it should be able to differentiate between bovine TB and avian TB in blood and can be used on farm and give a result within 30 minutes. In the case of cattle this would save the wait of 3 days to read the skin test and the further wait of 6 to 12 weeks for confirmation of TB by culture test.

However the PCR cycle seems potentially to be of even more use in identifying bovine TB in badgers - which no other test can currently do satisfactorily. The sensitivity of the current (brock) ELISA blood test for badgers is only 40.7 per cent, and needs to be done 3 times at 28 to 42 day intervals, which entails keeping wild badgers in captivity for at least 84 days for a result. I

A further attraction of using this PCR technique is that it may be accurate enough to distinguish the TB status of individual badgers within a sett. If a half hour test can reveal this, then the targeted cull of badgers that we propose might be refined even further.

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"As we sit here now there are no validated tests and of course were all working hard towards that objective." Professor David King, Chief Scientific Advisor to the UK Government, on the Today programme, December 18 2002

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Professor King's careful use of the word "validated" notwithstanding, rapid diagnostic, portable PCR tests, invented in the US in the 1990s and offered to the UK at the start of FMD 2001, are being used around the world and have been for some years. In May 2005, a Canadian team deployed a battery-operated portable PCR machine in Angola to diagnose within four hours which patients were infected with the deadly Marburg virus. Otherwise blood samples would have had to be sent outside the country for testing. That would have taken days if not weeks. The same team used a geographic information mapping system to map the homes and contacts of infected people to direct and speed investigation of potentially-infected contacts. Details at (http://www.cmaj.ca/cgi/content/full/172/11/1430).

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These PCR assays were designed to be performed as real time tests outside specialized laboratories like Plum Island on portable PCR machines taken to the site of the problem. Most PCR machines are delicate and suited only to a controlled laboratory environment. But in the 1990s, Idaho Technology Inc. (www.idahotech.com) developed the RAPID, a ruggedized machine that could withstand the rigors of military field deployment to allow pathogen detection on over 30 samples to take place inside a vehicle. The latest version of this machine  the hand-portable, 9 lb RAZOR  is able to perform the analysis in about 30 minutes in a moving vehicle, since even 30 minutes of immobility makes for a vulnerable target on todays battlefield.

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About the RAZOR portable machine
"........allows non-traditional testers such as first responders and other non-laboratory personnel the ability to get valid results quickly. Reagents for the RAZOR are freeze-dried in plastic PathFinder(tm) pouches, which provide increased stability. Reagents can be stored at room temperature and are packaged for rugged shipment. Each sample pouch contains a sampling kit that includes all of the necessary items to perform an analysis of potential class A bio-threats. When preparing the test samples, the liquefied sample is automatically drawn from a syringe by the vacuum in the pouch, which eliminates the need for any measurement or pipetting by the user. This format can also be adjusted-one sample can be tested against multiple pathogens or multiple samples can be tested against a single target in the 12 lanes of the pouch. The freeze-dried reagents are the same as those used on the extensively deployed R.A.P.I.D. system. . ..."

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The PCR test identifies a small piece of the genetic material of a virus that is a fingerprint or signature for that virus by labeling this with a chemical that glows under a laser beam.

But viruses are exceedingly tiny and may be present in only small numbers in the sample taken for examination, making detection impossible. So the labeled virus piece is subjected to up to 40 cycles of heating and cooling over a period of about 45 minutes: after each cycle, the number of labeled pieces is doubled. This process involves an enzyme called a polymerase and the number of labeled pieces increases exponentially in a chain reaction: 1 piece becomes 2, 2 become 4, 4 become 8 and so on, so that by 20 cycles there are over 1 million daughters of the original labeled piece. As the number of labeled pieces increases, the glow under laser light becomes visible and is tracked on a computer screen cycle by cycle. Scientists can thus watch the analytical results as they are generated  in real time  and do not have to wait until the end of the test to learn that a sample is positive

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"U. S. public health and law enforcement officials .... have had to confront the problem of detecting high-consequence pathogens, outside the comfort of a reference laboratory and have again adopted portable real-time PCR devices as a way to provide fast, accurate answers wherever the pathogen might be present. ..." RAPID ON-SITE DETECTION 2002

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Speed is probably the most important advantage of the RRT-PCR technology as results may be obtained within three hours of sample collection, as opposed to virus isolation which takes a minimum of five days.

May 3 2005 "...According to experts, on-site use of these tools is critical to speeding diagnosis, containing the disease, and minimizing the number of animals that need to be slaughtered.

DOD uses rapid diagnostic tools to identify disease agents on the battlefield.." GAO report number GAO-05-214 entitled 'Homeland Security: Much Is Being Done to Protect Agriculture from a Terrorist Attack, but Important Challenges Remain' which was released on March 9, 2005. See http://www.warmwell.com/05may3gao.html

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the portable real-time PCR machine, which is now widely deployed

"....The term "real-time" refers to the ability to monitor all stages of the reaction and identification as it proceeds, rather than to have to wait until the end for a result. "Portable" means that the device is taken to the site of the problem and operated there by the military - it is not confined to a central laboratory staffed by trained microbiologists. Recently, as U. S. public health and law enforcement officials have come to realize that these same biological threat agents might be employed against civilian populations, they too have had to confront the problem of detecting high-consequence pathogens, outside the comfort of a reference laboratory and have again adopted portable real-time PCR devices as a way to provide fast, accurate answers wherever the pathogen might be present. ..." RAPID ON-SITE DETECTION OF FOOT AND MOUTH DISEASE VIRUS

"On-site detectors should transform disease surveillance and control"

PhD Roger Breeze, BVMS, PhD, MRCVS Centaur Science Group - U.S. Agricultural and Food Security: Who Will Provide the Leadership?

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Sept 12 - Sept 18 2004 ~ "It is fair to say that we have taken PCR out of the research lab and into the field where it is most needed."

The Telegraph (Wednesday) carries an article about what it calls the "first DNA-based test that can diagnose a range of diseases within 30 minutes" They refer to the "portable mini-lab" developed at the Defence Science and Technology Laboratory, Dstl, Porton Down which "could eventually cost less than #10 each." Trials are to take place at hospitals in Portsmouth and Liverpool and in-the-field testing for animal diseases, including foot and mouth and bovine TB.
Telegraph "The test could change the face of general practice and veterinary medicine. .... ..... Yesterday, speaking at a conference in Bournemouth, Dr Mullis said tests based on the method had huge potential because "you need to know what a disease is before you can do anything about it".
At first, Porton Down wanted to find a way to use PCR for fast battlefield detectors of biological warfare agents such as anthrax but its wider potential was realised ...... The team plans to provide two rapid, automated PCR machines for various uses...
...... There will also be in-the-field testing for animal diseases, including foot and mouth or tuberculosis in cattle within 30 minutes, rather than having to send samples to a lab. Tim Rubidge, Dstl head of technology transfer and investments group, said the idea of a tabletop DNA test laboratory was no longer a "a twinkle in the eye of a research scientist looking far out into the future". ..... The Porton team uses custom-built test tubes made from a novel, electrically-conducting polymer to heat and cool samples. This not only speeds up the process, but also creates a lighter, more portable instrument."
Can the UK Government continue now to ignore such rapid diagnostic tests in contingency planning for foot and mouth?

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March 2 2004 ~ RT-PCR diagnosis against FMD to be tested in Texas.

AgNews "Experimental technology to rapidly detect foot-and-mouth disease will be tested in Texas this spring as the result of an agreement between the U.S. Department of Agriculture and the Texas A&M University College of Veterinary Medicine. The agreement, which also will test classical swine fever, means the Texas A&M researchers will be responsible for testing cattle and hogs with new assays to determine the tests' accuracy in populations of disease-free animals. ....
Currently, foot-and-mouth testing may only be performed at the U.S. Plum Island Animal Disease Center in New York, a high-security biocontainment facility. The usual method of confirming foot-and-mouth includes virus isolation  a procedure that, while accurate, may take up to a week to obtain results plus the time required to ship samples to Plum Island. New, rapid tests that could be performed in the field would enable officials to quickly detect and stop massive spread in a disease outbreak, researchers said. ....
The new experimental testing procedures that will be evaluated use "real time" polymerase chain-reaction technology to identify genetic material specific for the viruses that cause foot-and-mouth and classical swine fever. No active foot-and-mouth virus will be used in Texas. "Such procedures can give results in less than one hour and could be modified to test livestock on location during outbreak situations," ...."
(The conference Pan American Health Organization (PAHO), gather in Houston, Texas to "give a final push towards the eradication of foot-and-mouth" tomorrow.)

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Feb 25 2004 ~ 80% of all state public health laboratories in the US now have and use SmartCyclers.

It is, as Fred Brown said, "a beautiful piece of kit, simple and not costly" - and making use of it three years ago, as it was used in Uruguay in the same year, would have avoided bloodshed and trauma. The RT-PCR rapid diagnosis kit now used by the Pennsylvania Veterinary Laboratory - ( "in a matter of hours, it can determine both the presence and strain of the disease") is the Smart Cycler . We have confirmation from the journalist who wrote last Sunday's article in the Philadelphia Inquirer that the "new machine" is indeed the same machine that was offered to the UK government by the Agricultural Research Service (ARS) of USDA in early March 2001 for field tests. It is, as we hear from the Cepheid installer, now in 80% of all US state public health laboratories.
As was noted in tactfully phrased evidence to the Royal Society Inquiry (pdf. new window)
"Unfortunately, yet understandably, Pirbright staff were too busy coping with the demands of epidemic control to explore new technology during the spring and summer of 2001 . As a result, my offer to provide the latest diagnostic technology was not taken up.
Fortunately, we were able to take the devices and test system into the field in Uruguay in November 2001, where they performed splendidly on farm in a remote area..."
The Philidephia article noted that even with the help of the older, slower Bio-rad machine: ".. the state was able to announce that it had confirmed avian influenza, that the particular strain was virulent in poultry, and that it was unrelated to the type implicated in Thailand and Vietnam. The public was reassured that there was no Asian connection. Agriculture officials knew how big a quarantine was needed, and scientists had crucial details needed to start tracing the source of the infection."
The Smart Cycler, with appropriate reagent, is considerably faster.

Even now, three years on, we can find no mention of RT-PCR technology in the141 pages of the latest DEFRA Foot and Mouth Contingency Plan (pdf - slow - new window)

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Feb 24 ~" a further indictment of the UK's refusal to recognise and grasp the same opportunity three years ago"

Comment from Alan Beat about the item on the Chinese tests posted on February 14, and which applies equally to the news of the RT-PCR test at the Pennsylvania Veterinary Laboratory :
"This technology is the very same that was offered to the UK authorities in the early stages of the 2001 FMD epidemic by their USA counterparts, and was bluntly refused on the grounds that it was "not validated" by the OIE (just before the contiguous cull, itself unvalidated, was introduced). By appropriate choice of reagent chemicals used, such tests can accurately identify and strain any viral infection, so the Chinese announcement is not in any sense a scientific advance, rather it is the practical application of well-established technology - and a further indictment of the UK's refusal to recognise and grasp the same opportunity three years ago."

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"... the real-time PCR assay for foot and mouth disease virus detects all seven FMD virus serotypes

and differentiates this virus from other RNA viruses of animals and man and specifically from three viruses that cause almost identical diseases in livestock, namely swine vesicular disease, vesicular exanthema and vesicular stomatitis viruses. The assay detects as few as 10 virus particles, well below the number required to establish an infection and is more sensitive than cell culture. Significantly, this has been found to be a preclinical test: in experimentally infected cattle, sheep and pigs, foot and mouth virus can be detected 24 to 48 hours before the onset of clinical signs of disease. A single assay costs about £5. ..." See full article

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Feb 22 2004 ~ New DNA-based test speeds diagnosis of avian influenza

Philadelphia Inquirer Sunday 22Feb
" ...a new DNA-based test, capable of confirming the disease within hours, has arrived in labs around the region at a fortuitous time. ....About a year ago, the state bought DNA-testing equipment that can identify a virus within 24 hours of collecting the sample. A newer model, with a turnaround of several hours, arrived less than two weeks ago at the Pennsylvania Veterinary Laboratory in Harrisburg.
The staff have yet to be fully trained and validated on the new equipment, but they lost no time running samples from the Lancaster farm on the new machine, known as real-time RT-PCR (reverse transcriptase-polymerase chain reaction). Their findings, confirmed by the National Veterinary Services Laboratories in Ames, Iowa, identified an H2 strain of avian flu - a concern, but not nearly as bad as the type found in Delaware.
A quarantine of the farm and screening of 16 flocks within five miles appear to have contained the infection, said state veterinarian John Enck...."
See also MasterAmp Real-Time RT-PCR Kit (pdf file), an example of the sort of kit now available. We assume that DEFRA has been looking into this new technology fo many months now. We mentioned the Chinese PCR kit on February 14th2004

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Jan 9 2003 ~ Rapid Diagnosis PCR tests: a peer-reviewed publication, lab validation, and successful field tests in South America.

Roger Breeze's ProMed posting of May 2001. ...getting on for TWO years ago. Much has moved on since then, including a peer-reviewed publication, lab validation, and successful field tests in South America.
Extract: "These devices offer rapid real-time detection and identification by polymerase chain reaction (PCR) assays, are designed for use on farm at the site of the problem as hand-held or portable units, and communicate real-time data via the Internet to those who need to know in order that immediate action can be taken. ..... The system is specifically intended to support immediate detection on-site by operators with limited training, not just by highly-trained personnel geographically restricted to centralized laboratories. ........ The FMD assay requires minimal sample preparation and results are available in less than 2 hours after collection. The assay detects all 7 FMD virus serotypes and differentiates the virus from near relatives and from swine vesicular disease, vesicular exanthema and vesicular stomatitis viruses. In experimentally-infected animals, FMD virus can be detected well before the onset of clinical signs of disease. ...... instead of taking the sample to the expert in a central laboratory, the system takes the analytical data from the farm to the expert, so that any comment can be immediately returned to the person performing the analysis on the site. The system thus offers a time saving of at least 24 to 48 hours in definitive detection of virus. If time is gained, multiple alternate courses of action become possible for those charged with controlling the disease outbreak. This is the true significance of the system."

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"we did attempt to validate Fred Brown's test and it didn't pass the validation"

said Professor David King on the Today Programme (Dec 18 2002) (See transcript below)
But here is an extract of the letter sent as evidence to the Royal Society Enquiry
It was also sent to the Lessons Learned Inquiry and given by hand to Lord Whitty

...Our real time PCR assay for foot and mouth disease (W) has been validated in the laboratory: it has proven to be a pre-clinical test for infection in cattle, swine and sheep, it detects all 7 serotypes of FMD virus and differentiates this infection from other viral diseases that cause similar clinical signs. The test is more sensitive than viral culture and will detect as few as 10 virus particles....
Some eight months after we had disclosed the existence of our x;h/lD test to Dr. Donaldson, we read in the Veterinary Record that the Pirbright Laboratory had subsequently established a relationship with Cepheid and conducted some experiments with FpvfB reagents supplied by that company (data published by Alex Donaldson and others in the Veterinary Record, 2001).
I have no idea what those reagents were because the paper does not describe them. But I can be sure that these reagents were not those developed by USDA-ARS and Tetracore because Cepheid does not have this proprietary information.
I hope there has been no confusion in Britain between the Cepheid mystery test and the real time PCR test developed at Plum Island...."
The research paper with these results appeared in the Journal of the American Veterinary Medical Association.
For Prof King to continue to suggest that "we did attempt to validate Fred Brown's test" is absurd. There was indeed "confusion in Britain between the Cepheid mystery test and the real time PCR test". In the Veterinary Record on 6 October 2001, "Evaluation of a portable, 'real-time' PCR machine for FMD diagnosis", Alex Donaldson and his team reported poor results, stating that:"The reagents used in the assay were recommended by the manufacturer of the instrument" - but of course they were not recommended by the manufacturer, only by Cepheid - who, of course, didn't know and were guessing. Had the real time PCR test been properly trialled with the correct reagents - in other words, if the US offer had been courteously accepted - the story of FMD in 2001 would be very different. But Professor King told the EFRA Committee that "there are very serious questions to be asked about the use of that machine in the field, in particular the problem of cross-contamination". If one studies the letter and compares it with what Professor King and Dr Donaldson were saying in March 2001 at that EFRA Committee meeting one is struck by a feeling of great regret at what may well have been a genuine but tragic mistake.
This transcript was sent to Alan Beat's smallholders.org newsletter - and we reproduce it here with gratitude

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On the 9th of March 2001, an offer of help came from the USDA collaborating with Tetracore to provide a sensitive real time PCR farmgate test

and, if required, an experienced team to carry out the work. It had been successfully laboratory tested by the USDA and required validation in the field. Its convenient size, speed and simplicity of use was even demonstrated here on BBC television by Tetracore. But Pirbright turned down the offer on the grounds of lack of time.
Seven months later Pirbright took the very same machine and started their own laboratory trials Failing in the first instance to get good results, they went to press (The Veterinary Record 6 Oct 2001)* where they falsely claimed that Cepheid, the manufacturer of the PCR machine, had recommended and provided the wrong materials. Later in the same letter they triumphantly claim success by changing to those they would normally use - Cepheid do not provide or give advice on test materials.

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lack of cooperation may have been due to an economic conflict of interest

Evidence submitted to the Royal Society Inquiry of Edinburgh by the Director Patent and Licensing Affairs United Biomedical Inc. "... Clearly, safety and MAFF regulations were not the controlling factors here, but rather a desire to exploit their privileged position as the World Reference Centre to restrict competition. IAH-Pirbright is now involved with a competitor of UBI for the commercialization of their own NS test so it appears that their lack of cooperation may have been due to an economic conflict of interest. To put a kinder light on it, perhaps they simply decided to retain an intellectual exclusivity to FMD immunoassays. Either motivation is equally unethical and retarded development of FMD immunoassays...."

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Rapid on-site PCR

"....the real-time PCR assay for foot and mouth disease virus detects all seven FMD virus serotypes and differentiates this virus from other RNA viruses of animals and man and specifically from three viruses that cause almost identical diseases in livestock, namely swine vesicular disease, vesicular exanthema and vesicular stomatitis viruses. The assay detects as few as 10 virus particles, well below the number required to establish an infection and is more sensitive than cell culture. Significantly, this has been found to be a preclinical test: in experimentally infected cattle, sheep and pigs, foot and mouth virus can be detected 24 to 48 hours before the onset of clinical signs of disease." (read in full)

http://www.nuengr.unl.edu/cet/Research/LabNotes/nworthy1.html

Professors Develop Advanced DNA Replication Technology

by Roxane Gay

Imagine being able to diagnose a viral strain at the site of an outbreak or identifying a deadly pathogen on the battlefield.  Professors Hendrik Viljoen (chemical engineering) and George Gogos (mechanical engineering), both from the University of NebraskaLincoln, have been awarded a $1.44 million grant over five years from the National Institutes of Health (NIH) to further their development of faster Polymerase Chain Reaction (PCR) technology.  PCR is a technique for amplifying DNA for diagnostic purposes wherein a sample of target DNA is replicated many times over to enable gene sequencing and identification. The key to our technology is that were able to amplify DNA in five to ten minutes, Viljoen said. Our device is also rugged, transportable and reliable. The rapid PCR uniquely positions us to pursue two other technologies. We can measure the PCR kinetics and the results are included in intelligent software, which will be part of the next generation of PCR thermocyclers. We have also started a program in assembly PCR. Short pieces of DNA are assembled, under conditions to minimize mutations, into larger structures.  

To enable this rapid amplification, Viljoen and Gogos have integrated a novel device to produce the heating and cooling gases necessary for the thermocycling of the DNA. This device makes our technology amenable to field usage, Viljoen said. And it can handle volumes from 5 microliters to 40 microliters with outstanding sensitivity, while producing a high yield. The grant will be used to add optical detection, establish protocols for reverse transcription and the quantification of PCR, as well as to collect kinetic data of various polymerase enzymes to create mathematical models. The researchers have partnered with Michael Nelson, president of local biotechnology firm Megabase Research Products. The firm provides the required biochemistry expertise. This is an interdisciplinary project. Putting together a group of chemical engineers, mechanical engineers and biochemists has been very critical to the success of the project, Gogos said. We have built a device with vast medical and non-medical applications which at the same time is a scientific instrument for basic studies.

The implications for this innovative technology are many. We hope to form cross-disciplinary teams of researchers to look at parallel applications of this technology, said Dipanjan Nag, a technology development associate with the UNL Office of Technology Development, which provided the researchers with an additional $75,000 grant for further development of the technology. This NIH grant demonstrates that we are at the cutting edge of research, Nag said. And it has tremendous potential in biosecurity and bioterrorism related research.
See also: http://www.dailynebraskan.com/vnews/display.v/ART/2004/07/19/40fc058f93db5


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http://www.medinews.com/GMEDTS32olcgi/ts.cgi?tsurl=0.59.13813.0.0

30-Minute PCR Technology

By medinews.com staff writers
Posted on 21 December 2004
A portable polymerase chain reaction (PCR) system automates all the steps, including sample preparation, allowing operation by nonscientist users in the field and providing results in only 30 minutes.

All the instruments used in the process are protected by a portfolio of 30 patents and incorporate a direct heating system that uses a plastic to rapidly heat and cool the samples. When combined with the detection and sample preparation chemistries, direct heating allows several test assays to be run in a single rapid reaction.

The process was originally developed for fast, accurate battlefield detection of biologic warfare agents such as anthrax, but has wider potential applications in human care. Two systems close to market launch are the NPT (near-patient testing) Gold for detection of chlamydia in hospitals and clinics and the PCR-Light, for field applications such as detection of foot-and-mouth disease and outbreaks of food poisoning. The systems are being marketed by Enigma Diagnostics (Porton Down, UK).

My task is now to devise and implement a licensing and commercial strategy that will enable us to successfully exploit the many potential applications areas both in the UK and internationally, noted John Thornback, new CEO of Enigma Diagnostics.

pdf file of Real-Time Reverse Transcription PCR Detection of Foot-and-Mouth-Disease Virus Using the R.A.P.I.D.® System by Katy M. Andrews, Michael D. Powers, Gordon B. Ward, Thomas McKenna, Deepika de Silva Idaho Technology Inc., Salt Lake City, UT, 2APHIS USDA, Plum Island Animal Disease Ctr., Plum Island, NY.

"This study evaluates the ability of two real-time Reverse Transcription Polymerase Chain Reaction (RT-PCR) assays to independently detect the presence of Foot-and-Mouth-Disease Virus (FMDV) RNA. Assays to amplify the internal ribosomal entry site (IRES) and RNA polymerase regions of the FMDV genome recognize all seven existing serotypes. Both assays can effectively evaluate the presence of FMDV in various bovine samples, including blood, serum, saliva, nasal swabs, and epithelial tissue..... Real-time RT-PCR using the Ruggedized Advanced Pathogen Identification Device (R.A.P.I.D.) System is an ideal platform for fast, sensitive, and specific identification of FMDV...."

USDA's Dr Roger Breeze on the Rapid Diagnosis technology (Royal Society report)

 

FMD Test from Joint FAO/IAEA

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From the submission to the Royal Inquiry by the FMDF

RAPID ON-SITE DETECTION OF FOOT AND MOUTH DISEASE VIRUS

Those puzzled and frustrated by the seeming lack of technological breakthroughs in control of foot and mouth and other foreign animal diseases should not be discouraged by recent lacklustre results reported for foot and mouth virus detection by real-time polymerase chain reaction (PCR) on a portable device (Donaldson et al Vet Rec 149 6 Oct 2001).

This technology is now so well developed for high-consequence pathogen detection in defence, public health, law enforcement and agriculture in the United States (U.S.) that it is clear that those indifferent results can be explained entirely by the inappropriate choice of reagents and assay conditions.

Furthermore, the caveats raised about potential limitations on applicability of the technology have not been found valid in experienced hands.

To understand the current frontiers of high-consequence pathogen detection elsewhere in the world, British government officials, livestock owners, practitioners and other interested parties should know the pedigree of this technology.

It was essentially driven by the needs of the U. S. Defense Department, to detect biological threat agents quickly, in the field and with such a high level of confidence that there would be time to take protective measures (Higgins et al., 1999).

To solve these problems, the Defense Department initiated revolutionary approaches to pathogen detection.

One of these is the portable real-time PCR machine, which is now widely deployed.

The term "real-time" refers to the ability to monitor all stages of the reaction and identification as it proceeds, rather than to have to wait until the end for a result.

"Portable" means that the device is taken to the site of the problem and operated there by the military - it is not confined to a central laboratory staffed by trained microbiologists.

Recently, as U. S. public health and law enforcement officials have come to realize that these same biological threat agents might be employed against civilian populations, they too have had to confront the problem of detecting high-consequence pathogens, outside the comfort of a reference laboratory and have again adopted portable real-time PCR devices as a way to provide fast, accurate answers wherever the pathogen might be present.

Veterinary medicine is also deeply involved in high-consequence pathogen detection - for control of infectious diseases moving swiftly through international boundaries and perhaps also, deliberately introduced diseases for the purpose of overwhelming conventional defences.

Hence, the Agricultural Research Service (ARS) of the U.S. Department of Agriculture (USDA) has begun to develop a uniform system of animal, plant and zoonotic pathogen detection, identification and epidemic response.

There are two components:

1. Rapid on-site tests that detect and identify important animal and plant pathogens.
2. An Internet-based information technology programme that incorporates these assays into an integrated management scheme to focus assets for rapid outbreak control in real time. This system is fully compatible with that used by U. S. military, public health and law enforcement agencies, which obviously maximises its utility. This is not a new test system that is an incremental improvement over the past - it is a fundamentally new way to control highly-infectious disease epidemics.

The rapid detection and identification tests are performed with standardized reagents designed to work on a common device platform for all assays. Several devices are already commercially available and others are close to production. The most popular devices are made by Idaho Technology Inc. (www.idahotech.com) and Cepheid Inc. (www.cepheid.com).

These devices both work in the same way.

Future models from these and other manufacturers are expected to become smaller, quicker and cheaper to purchase and operate - hand held machines are already available. The devices cost between £15,000 and £30,000.

Real-time PCR assays are performed on a small, portable computer controlled device operating on mains or car battery power. The device is specifically designed to be taken to or near the site of the problem and used there by a person with limited training.

An integral global positioning system can identify the exact location of the device. A wireless Internet connection provides world-wide communication, so that distant experts can "look over the shoulder" of the person conducting the assay, to offer advice, expert analysis and validation of assay performance as it proceeds.

Instead of taking the sample to the expert, at a distant site for analysis, the sample is analysed on the spot and the data sent electronically to the expert and back to the operator with whatever advice is needed.

This immediately saves one or two or more days. If time is gained, multiple alternate courses of action become possible for those charged with taking action to control the disease outbreak. This is the true significance of the technology (Breeze, 2001).

Sample preparation is minimal and can be automated. The process of preparation inactivates infectious agents, including foot and mouth disease virus

. All necessary reagents are contained in a single-assay sealed tube in a freeze-dried form stable under a wide range of environmental conditions for over a year. Reagents are produced to ISO 9000 standards and weekly updates of quality control and quality assurance data for each batch are available over the Internet. Sample collection, preparation and the assay itself can be completed in 90 minutes after arriving on site, but positive results can be obtained much earlier.

Real-time PCR assays for foot and mouth disease and classical swine fever virus detection and identification were described and demonstrated by scientists from the USDA at the 105th Annual Meeting of the U.S. Animal Health Association/44th Annual meeting of the American Association of Veterinary Laboratory Diagnosticians held in November 2001 in Hershey, Pennsylvania.

Members of the FMD Forum were in the audience and had the opportunity to see the research results and watch assays being conducted.

Briefly, data were presented to show that the real-time PCR assay for foot and mouth disease virus detects all seven FMD virus serotypes and differentiates this virus from other RNA viruses of animals and man and specifically from three viruses that cause almost identical diseases in livestock, namely swine vesicular disease, vesicular exanthema and vesicular stomatitis viruses.

The assay detects as few as 10 virus particles, well below the number required to establish an infection and is more sensitive than cell culture.

Significantly, this has been found to be a preclinical test: in experimentally infected cattle, sheep and pigs, foot and mouth virus can be detected 24 to 48 hours before the onset of clinical signs of disease.

A single assay costs about £5.

The classical swine fever real time PCR assay detects all the strains of this virus and differentiates these from similar viruses, such as border disease and bovine viral diarrhoea viruses. This test is also more sensitive than cell culture and has again been found to be preclinical - detecting infected animals several days before the onset of clinical signs.

Once a positive identification is made on-site, the information technology part of the system allows those responsible to take immediate action in cooperation with other parties who must become involved.

Since the device location is known by global positioning, officials can immediately see electronically a map of the area around the infection, predict where infection may have been spread by recent wind, map this spread according to geography and topography, identify quarantine zones, set up control measures (such as road blocks) and identify farms at risk where animals should be tested immediately to detect any infection.

The system is designed to coordinate Government officials, academia and private industry, cooperatively, to focus all available resources on immediately stamping out such an introduction through quick, targeted and science-based interventions.

Consequentially, in future, the British people - once aware that such amazing and proven science is available from our friends across the water - will not tolerate the little Englander attitude and medieval approach, adopted for the control of this outbreak

References 1. Breeze, R.G. Foot and mouth disease preparedness -USA. Promed-mail, 20010520.0981, May 20, 2001. 2. Donaldson, A. L., Hearps, A., and Alexandersen, S. Evaluation of a portable, "real-time" PCR machine for FMD diagnosis, Veterinary Record,149, 430, 2001. 3. Higgins, J.A., Ibrahim, M.S., Knauert, F.K., Ludwig, G.V., Kijek, T.M., Ezzell, J.W., Courtney, B.C., and Henchal, E.A.. Sensitive and rapid identification of biological threat agents. In "Food and Agricultural Security: Guarding against natural threats and terrorist attacks affecting health, national food supplies and agricultural economics". Editors Frazier, T.W. and Richardson, D.C. Annals of the New York Academy of Sciences, 894, 130-148, 1999.

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About rapid diagnosis tests

"As we sit here now there are no validated tests and of course were all working hard towards that objective." Professor David King, Chief Scientific Advisor to the UK Government, on the Today programme, December 18 2002

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March 2 2004 ~ RT-PCR diagnosis against FMD to be tested in Texas.

AgNews "Experimental technology to rapidly detect foot-and-mouth disease will be tested in Texas this spring as the result of an agreement between the U.S. Department of Agriculture and the Texas A&M University College of Veterinary Medicine. The agreement, which also will test classical swine fever, means the Texas A&M researchers will be responsible for testing cattle and hogs with new assays to determine the tests' accuracy in populations of disease-free animals. ....
Currently, foot-and-mouth testing may only be performed at the U.S. Plum Island Animal Disease Center in New York, a high-security biocontainment facility. The usual method of confirming foot-and-mouth includes virus isolation  a procedure that, while accurate, may take up to a week to obtain results plus the time required to ship samples to Plum Island. New, rapid tests that could be performed in the field would enable officials to quickly detect and stop massive spread in a disease outbreak, researchers said. ....
The new experimental testing procedures that will be evaluated use "real time" polymerase chain-reaction technology to identify genetic material specific for the viruses that cause foot-and-mouth and classical swine fever. No active foot-and-mouth virus will be used in Texas. "Such procedures can give results in less than one hour and could be modified to test livestock on location during outbreak situations," ...."
(The conference Pan American Health Organization (PAHO), gather in Houston, Texas to "give a final push towards the eradication of foot-and-mouth" tomorrow.)

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Feb 25 2004 ~ 80% of all state public health laboratories in the US now have and use SmartCyclers.

It is, as Fred Brown said, "a beautiful piece of kit, simple and not costly" - and making use of it three years ago, as it was used in Uruguay in the same year, would have avoided bloodshed and trauma. The RT-PCR rapid diagnosis kit now used by the Pennsylvania Veterinary Laboratory - ( "in a matter of hours, it can determine both the presence and strain of the disease") is the Smart Cycler . We have confirmation from the journalist who wrote last Sunday's article in the Philadelphia Inquirer that the "new machine" is indeed the same machine that was offered to the UK government by the Agricultural Research Service (ARS) of USDA in early March 2001 for field tests. It is, as we hear from the Cepheid installer, now in 80% of all US state public health laboratories.
As was noted in tactfully phrased evidence to the Royal Society Inquiry (pdf. new window)
"Unfortunately, yet understandably, Pirbright staff were too busy coping with the demands of epidemic control to explore new technology during the spring and summer of 2001 . As a result, my offer to provide the latest diagnostic technology was not taken up.
Fortunately, we were able to take the devices and test system into the field in Uruguay in November 2001, where they performed splendidly on farm in a remote area..."
The Philidephia article noted that even with the help of the older, slower Bio-rad machine: ".. the state was able to announce that it had confirmed avian influenza, that the particular strain was virulent in poultry, and that it was unrelated to the type implicated in Thailand and Vietnam. The public was reassured that there was no Asian connection. Agriculture officials knew how big a quarantine was needed, and scientists had crucial details needed to start tracing the source of the infection."
The Smart Cycler, with appropriate reagent, is considerably faster.

Even now, three years on, we can find no mention of RT-PCR technology in the141 pages of the latest DEFRA Foot and Mouth Contingency Plan (pdf - slow - new window)

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Feb 24 ~" a further indictment of the UK's refusal to recognise and grasp the same opportunity three years ago"

Comment from Alan Beat about the item on the Chinese tests posted on February 14, and which applies equally to the news of the RT-PCR test at the Pennsylvania Veterinary Laboratory :
"This technology is the very same that was offered to the UK authorities in the early stages of the 2001 FMD epidemic by their USA counterparts, and was bluntly refused on the grounds that it was "not validated" by the OIE (just before the contiguous cull, itself unvalidated, was introduced). By appropriate choice of reagent chemicals used, such tests can accurately identify and strain any viral infection, so the Chinese announcement is not in any sense a scientific advance, rather it is the practical application of well-established technology - and a further indictment of the UK's refusal to recognise and grasp the same opportunity three years ago."

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"... the real-time PCR assay for foot and mouth disease virus detects all seven FMD virus serotypes

and differentiates this virus from other RNA viruses of animals and man and specifically from three viruses that cause almost identical diseases in livestock, namely swine vesicular disease, vesicular exanthema and vesicular stomatitis viruses. The assay detects as few as 10 virus particles, well below the number required to establish an infection and is more sensitive than cell culture. Significantly, this has been found to be a preclinical test: in experimentally infected cattle, sheep and pigs, foot and mouth virus can be detected 24 to 48 hours before the onset of clinical signs of disease. A single assay costs about £5. ..." See full article

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Feb 22 2004 ~ New DNA-based test speeds diagnosis of avian influenza

Philadelphia Inquirer Sunday 22Feb
" ...a new DNA-based test, capable of confirming the disease within hours, has arrived in labs around the region at a fortuitous time. ....About a year ago, the state bought DNA-testing equipment that can identify a virus within 24 hours of collecting the sample. A newer model, with a turnaround of several hours, arrived less than two weeks ago at the Pennsylvania Veterinary Laboratory in Harrisburg.
The staff have yet to be fully trained and validated on the new equipment, but they lost no time running samples from the Lancaster farm on the new machine, known as real-time RT-PCR (reverse transcriptase-polymerase chain reaction). Their findings, confirmed by the National Veterinary Services Laboratories in Ames, Iowa, identified an H2 strain of avian flu - a concern, but not nearly as bad as the type found in Delaware.
A quarantine of the farm and screening of 16 flocks within five miles appear to have contained the infection, said state veterinarian John Enck...."
See also MasterAmp Real-Time RT-PCR Kit (pdf file), an example of the sort of kit now available. We assume that DEFRA has been looking into this new technology fo many months now. We mentioned the Chinese PCR kit on February 14th2004

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Jan 9 2003 ~ Rapid Diagnosis PCR tests: a peer-reviewed publication, lab validation, and successful field tests in South America.

Roger Breeze's ProMed posting of May 2001. ...getting on for TWO years ago. Much has moved on since then, including a peer-reviewed publication, lab validation, and successful field tests in South America.
Extract: "These devices offer rapid real-time detection and identification by polymerase chain reaction (PCR) assays, are designed for use on farm at the site of the problem as hand-held or portable units, and communicate real-time data via the Internet to those who need to know in order that immediate action can be taken. ..... The system is specifically intended to support immediate detection on-site by operators with limited training, not just by highly-trained personnel geographically restricted to centralized laboratories. ........ The FMD assay requires minimal sample preparation and results are available in less than 2 hours after collection. The assay detects all 7 FMD virus serotypes and differentiates the virus from near relatives and from swine vesicular disease, vesicular exanthema and vesicular stomatitis viruses. In experimentally-infected animals, FMD virus can be detected well before the onset of clinical signs of disease. ...... instead of taking the sample to the expert in a central laboratory, the system takes the analytical data from the farm to the expert, so that any comment can be immediately returned to the person performing the analysis on the site. The system thus offers a time saving of at least 24 to 48 hours in definitive detection of virus. If time is gained, multiple alternate courses of action become possible for those charged with controlling the disease outbreak. This is the true significance of the system."

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"we did attempt to validate Fred Brown's test and it didn't pass the validation"

said Professor David King on the Today Programme (Dec 18 2002) (See transcript below)
But here is an extract of the letter sent as evidence to the Royal Society Enquiry
It was also sent to the Lessons Learned Inquiry and given by hand to Lord Whitty

...Our real time PCR assay for foot and mouth disease (W) has been validated in the laboratory: it has proven to be a pre-clinical test for infection in cattle, swine and sheep, it detects all 7 serotypes of FMD virus and differentiates this infection from other viral diseases that cause similar clinical signs. The test is more sensitive than viral culture and will detect as few as 10 virus particles....
Some eight months after we had disclosed the existence of our x;h/lD test to Dr. Donaldson, we read in the Veterinary Record that the Pirbright Laboratory had subsequently established a relationship with Cepheid and conducted some experiments with FpvfB reagents supplied by that company (data published by Alex Donaldson and others in the Veterinary Record, 2001).
I have no idea what those reagents were because the paper does not describe them. But I can be sure that these reagents were not those developed by USDA-ARS and Tetracore because Cepheid does not have this proprietary information.
I hope there has been no confusion in Britain between the Cepheid mystery test and the real time PCR test developed at Plum Island...."
The research paper with these results appeared in the Journal of the American Veterinary Medical Association.
For Prof King to continue to suggest that "we did attempt to validate Fred Brown's test" is absurd. There was indeed "confusion in Britain between the Cepheid mystery test and the real time PCR test". In the Veterinary Record on 6 October 2001, "Evaluation of a portable, 'real-time' PCR machine for FMD diagnosis", Alex Donaldson and his team reported poor results, stating that:"The reagents used in the assay were recommended by the manufacturer of the instrument" - but of course they were not recommended by the manufacturer, only by Cepheid - who, of course, didn't know and were guessing. Had the real time PCR test been properly trialled with the correct reagents - in other words, if the US offer had been courteously accepted - the story of FMD in 2001 would be very different. But Professor King told the EFRA Committee that "there are very serious questions to be asked about the use of that machine in the field, in particular the problem of cross-contamination". If one studies the letter and compares it with what Professor King and Dr Donaldson were saying in March 2001 at that EFRA Committee meeting one is struck by a feeling of great regret at what may well have been a genuine but tragic mistake.

This transcript was sent to Alan Beat's smallholders.org newsletter - and we reproduce it here with gratitude

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On the 9th of March 2001, an offer of help came from the USDA collaborating with Tetracore to provide a sensitive real time PCR farmgate test

and, if required, an experienced team to carry out the work. It had been successfully laboratory tested by the USDA and required validation in the field. Its convenient size, speed and simplicity of use was even demonstrated here on BBC television by Tetracore. But Pirbright turned down the offer on the grounds of lack of time.
Seven months later Pirbright took the very same machine and started their own laboratory trials Failing in the first instance to get good results, they went to press (The Veterinary Record 6 Oct 2001)* where they falsely claimed that Cepheid, the manufacturer of the PCR machine, had recommended and provided the wrong materials. Later in the same letter they triumphantly claim success by changing to those they would normally use - Cepheid do not provide or give advice on test materials.

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lack of cooperation may have been due to an economic conflict of interest

Evidence submitted to the Royal Society Inquiry of Edinburgh by the Director Patent and Licensing Affairs United Biomedical Inc. "... Clearly, safety and MAFF regulations were not the controlling factors here, but rather a desire to exploit their privileged position as the World Reference Centre to restrict competition. IAH-Pirbright is now involved with a competitor of UBI for the commercialization of their own NS test so it appears that their lack of cooperation may have been due to an economic conflict of interest. To put a kinder light on it, perhaps they simply decided to retain an intellectual exclusivity to FMD immunoassays. Either motivation is equally unethical and retarded development of FMD immunoassays...."

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Jan 10 ~"Clearly, safety and MAFF regulations were not the controlling factors here, but rather a desire to exploit their privileged position as the World Reference Centre to restrict competition."

From the letter submitted as evidence to the Royal Society (Edinburgh) Inquiry by the Director Patent and Licensing Affairs United Biomedical Inc. about the reluctance of Pirbright to let them have access to sera "..... IAH-Pirbright is now involved with a competitor of UBI for the commercialization of their own NS test so it appears that their lack of cooperation may have been due to an economic conflict of interest. To put a kinder light on it, perhaps they simply decided to retain an intellectual exclusivity to FMD immunoassays.
Either motivation is equally unethical and retarded development of FMD immunoassays. Without access to the Pirbright World Reference Centre collection, we were unable to fully standardize the UBI tests at that time..We believe it is perfectly OK for them to profit from their own tests, but as the publicly funded OIE-designated World Reference Center, it is improper for them to reserve their resources to themselves or to use an arbitrary standard to exclude companies from the ranks of qualified FMD researchers. (Unfortunately, an anti-company attitude is more pervasive among FMD researchers than it is in most fields of the life sciences and in this age of biotechnology, this attitude needs to be seriously questioned.) ..Dr. Brown and others at the USDA also have been working on a device to detect FMDV RNA by PCR, in real-time, in collaboration with Tetracore, Inc., another U.S. biotechnology company. This project is also worth your looking into, independently of IAH-Pirbright. .UBI has not been involved with that project. UBI also has an FMD vaccine program. IAH-Pirbright has provided advice and has offered access to their facilities. The conflict of interest seems to be limited to diagnostics." More

Jan 10 ~ "it is a breach of duty that this has been allowed to pass"

Extract from Dr Watkins' Submission to the Royal Society of Edinburgh FMD Enquiry ".... During the early stages of the FMD epidemic Dr Noel Mowat offered to help educate MAFF officials and vets on FMD virology. Dr Mowat used to work at Pirbright and ran courses on FMD infection at Pirbright. His offer was refused.
On the 9th of March 2001, an offer of help came from the USDA collaborating with Tetracore to provide a sensitive real time PCR farmgate test and if required an experienced team to carry out the work. It had been successfully laboratory tested by the USDA and required validation in the field. Its convenient size, speed and simplicity of use was even demonstrated here on BBC television by Tetracore. But Pirbright turned down the offer on the grounds of lack of time.
Seven months later Pirbright took the very same machine and started their own laboratory trials Failing in the first instance to get good results, they went to press (The Veterinary Record 6 Oct 2001)* where they falsely claimed that Cepheid, the manufacturer of the PCR machine, had recommended and provided the wrong materials. Later in the same letter they triumphantly claim success by changing to those they would 'normally' use - Cepheid do not provide or give advice on test materials. ......They (Pirbright) have also insisted that they could not use an anti-NSP test, as it also was not validated. Antibody to non-structural virus proteins (NSP) enables vaccinated herds or flocks to be distinguished from infected ones. Again they use their own in-house test rather than validate any commercial anti-NSP tests, also offered them during this epidemic from UBI for example. What is going on at Pirbright? Pirbright has confined itself to in-house tests, producing the materials and developing its own protocols. It has refused to undertake validation of commercial FMD tests such as those produced by Michael Walker at Genesis. There is no other laboratory in Britain that is allowed or could undertake to validate FMD tests - it is a breach of duty that this has been allowed to pass..." (more)

Jan 9 ~ Rapid Diagnosis PCR tests: a peer-reviewed publication, lab validation, and successful field tests in South America.

Roger Breeze's ProMed posting of May 2001. Much has moved on since then, including a peer-reviewed publication, lab validation, and successful field tests in South America.
Extract: "These devices offer rapid real-time detection and identification by polymerase chain reaction (PCR) assays, are designed for use on farm at the site of the problem as hand-held or portable units, and communicate real-time data via the Internet to those who need to know in order that immediate action can be taken. ..... The system is specifically intended to support immediate detection on-site by operators with limited training, not just by highly-trained personnel geographically restricted to centralized laboratories. ........ The FMD assay requires minimal sample preparation and results are available in less than 2 hours after collection. The assay detects all 7 FMD virus serotypes and differentiates the virus from near relatives and from swine vesicular disease, vesicular exanthema and vesicular stomatitis viruses. In experimentally-infected animals, FMD virus can be detected well before the onset of clinical signs of disease. ...... instead of taking the sample to the expert in a central laboratory, the system takes the analytical data from the farm to the expert, so that any comment can be immediately returned to the person performing the analysis on the site. The system thus offers a time saving of at least 24 to 48 hours in definitive detection of virus. If time is gained, multiple alternate courses of action become possible for those charged with controlling the disease outbreak. This is the true significance of the system."

Jan 9 ~ FMD Contingency Plan: no reference to new technologies - rapid field diagnosis and linked GIS systems. On the contrary:

DEFRA's Contingency Plan (external link) "8.2 Transport of samples
8.2.1 DVMs will ensure they have access to local couriers to transport blood samples during an animal disease outbreak as per SVS standard instructions."
So is there really no plan to use the already available and excellent real time PCR tests? Defra is continuing to maintain its option of slaughter on contiguous premises and of "firebreak" culls:
"7. If FMD is Confirmed (through Clinical Examination or Laboratory Test) ..... Further action will depend on the circumstances of a particular outbreak and depending on the scientific and veterinary advice. Additional options and strategies which are potentially available include:
• emergency vaccination (either to live or to kill, within an area or in a ring around an area);
• culling of other livestock exposed to the disease (e.g. premises under virus plumes, contiguous premises); and
• (subject to the Government's Animal Health Bill becoming law) pre-emptive or 'firebreak' culling of animals not on infected premises not dangerous contacts or not necessarily exposed to the disease, in order to prevent the wider spread of the disease outwith an area."
  No stress on the importance of targeted vaccination, which requires better diagnostics/detection technology and better data management than indicated in this paper.
But an "open consultation" is certainly a welcome development - if it means what it says. It is hoped that interested parties will contact DEFRA about this. Email: contingency.comments@defra.gsi.gov.uk

Jan 1-6 ~ "we did attempt to validate Fred Brown's test and it didn't pass the validation"

said Professor David King on the Today Programme (Dec 18)
We will remind readers again of what actually happened since Professor King doesn't seem to remember. Here is an extract of the letter sent as evidence to the Royal Society Enquiry
It was also sent to the Lessons Learned Inquiry and given by hand to Lord Whitty

...Our real time PCR assay for foot and mouth disease (W) has been validated in the laboratory: it has proven to be a pre-clinical test for infection in cattle, swine and sheep, it detects all 7 serotypes of FMD virus and differentiates this infection from other viral diseases that cause similar clinical signs. The test is more sensitive than viral culture and will detect as few as 10 virus particles....
Some eight months after we had disclosed the existence of our x;h/lD test to Dr. Donaldson, we read in the Veterinary Record that the Pirbright Laboratory had subsequently established a relationship with Cepheid and conducted some experiments with FpvfB reagents supplied by that company (data published by Alex Donaldson and others in the Veterinary Record, 2001).
I have no idea what those reagents were because the paper does not describe them. But I can be sure that these reagents were not those developed by USDA-ARS and Tetracore because Cepheid does not have this proprietary information.
I hope there has been no confusion in Britain between the Cepheid mystery test and the real time PCR test developed at Plum Island...."
The research paper with these results appeared in the Journal of the American Veterinary Medical Association.
For Prof King to continue to suggest that "we did attempt to validate Fred Brown's test" is absurd. There was indeed "confusion in Britain between the Cepheid mystery test and the real time PCR test". In the Veterinary Record on 6 October 2001, "Evaluation of a portable, 'real-time' PCR machine for FMD diagnosis", Alex Donaldson and his team reported poor results, stating that:"The reagents used in the assay were recommended by the manufacturer of the instrument" - but of course they were not recommended by the manufacturer, only by Cepheid - who, of course, didn't know and were guessing. Had the real time PCR test been properly trialled with the correct reagents - in other words, if the US offer had been courteously accepted - the story of FMD in 2001 would be very different. But Professor King told the EFRA Committee that "there are very serious questions to be asked about the use of that machine in the field, in particular the problem of cross-contamination". If one studies the letter and compares it with what Professor King and Dr Donaldson were saying in March 2001 at that EFRA Committee meeting one is struck by a feeling of great regret at what may well have been a genuine but tragic mistake.

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Real-time PCR

Real-time PCR differs from standard PCR in that the amplified PCR products are detected directly during the amplification cycle using hybridisation probes, which enhance assay specificity. Various real-time methods, such as TaqMan, Scorpions, FRET, or Molecular Beacons assays, have become popular tools for detection of infectious agents. Real-time PCR has been used for the detection of bacteria, viruses or parasites from a range of animal species (2, 6, 8). These new assays have several advantages over the 'classical' conventional or nested PCR methods. Only one primer pair is used, providing sensitivity often close or equal to traditional nested PCR but with a much lower risk of contamination. Fluorescence, indicating the presence of the amplified product, is measured through the lid or side of the reaction vessel thus there is no need for post-PCR handling of the amplified DNA. These procedures are considerably less time-consuming compared with traditional post-amplification PCR product detection in agarose gels followed by ethidium bromide staining and again, the risk of contamination is reduced. The use of a 96-well microtitre plate format, without the need for nested PCR, allows the procedure to be automated and suitable for large-scale testing (5). Diagnosis can be further automated by using robots for DNA/RNA extractions and pipetting. Compared with classical amplification methods, a further advantage of the real-time PCR is that it is possible to perform quantitative assays (6).

Multiplex PCR

PCR reactions using multiple primers directed at different targets in a single assay are referred to as multiplex PCR assays. In multiplex PCR, various infectious agents can be detected and differentiated in a single reaction vessel at the same time. The different PCR targets amplified in a standard PCR assay are identified based on PCR product size. The use of 'classical' nested PCR methods for the construction of a multiplex assay is complicated by the need for targets of different sizes, as well as primers that may 'compete' with each other in the same reaction mix, both of which can negatively impact PCR efficiency. In contrast, the concept of real-time PCR (single primer pairs) provides excellent possibilities for the construction of highly sensitive multiplex systems (2, 4) based on more uniform target size, uniform amplification conditions, and differential detection of targets using specific hybridisation probes labelled with different fluorophores. >