warmwell.com

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 of USDA to the Royal Society Enquiry (pdf)

December 2007 ~ "......The issue is whether that test should be done on the farm or in a fixed lab,

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

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

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"

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

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"

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"

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

January 26-28 2007 ~ Silence on validation

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

See also background material

About rapid diagnosis tests

PCR for Foot and Mouth - click here



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.

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

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.

"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

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).

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.

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. . ..."

 

 

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

"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

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

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?

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."

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

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

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

"... 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

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

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

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

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

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.

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...."

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

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

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.

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

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.)

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)

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."

"... 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

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

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."

"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

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.

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...."

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.

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. >