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.

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:

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 we’re 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.


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...."


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.