Diagnostic tools and their role in the global control of foot and mouth disease

W. Vosloo

Commonwealth Scientific and Industrial Research Organisation (CSIRO) – Australian Animal Health Laboratory, Private Bag 24, Geelong, Australia Correspondence: Wilna.Vosloo@csiro.au

Summary

Global control of an infectious disease such as foot and mouth disease (FMD) is not possible without the knowledge generated by diagnostic assays. For both endemic countries that have embarked on a control plan and free countries suffering outbreaks, information is needed that could inform control plans and strategies. Data regarding the prevalence of infection in various species, the serotypes and topotypes involved in outbreaks and, where vaccination is used, the immune profile of the animals, as well as potential sub-clinical infection and carrier status, are essential when controlling FMD. This information is also vital for countries embarking on the Progressive Control Pathway to determine baseline epidemiological information and to act as an incentive to progress along the pathway towards improved disease control and, ultimately, eradication. The specific need for diagnostic assays will change as countries move along the pathway. FMD is currently endemic mostly in resource-poor countries, which implies a need for cost-effective, but accurate, assays. Although maintaining laboratories and trained staff is expensive, their role is essential in ensuring diagnostic test results are delivered in an accredited and reliable manner. The cost related to performing tests (both the consumables and the overheads) is probably the determining factor in how widespread the use of diagnostic assays will be in assisting with FMD control. However, even inexpensive assays will be of less value if the expenditure for sampling and other control issues, such as movement control and vaccination, cannot be covered.

Keywords

Diagnostic assays – Diagnostic laboratories – Fit-for-purpose – Foot and mouth disease – OIE – Pen-side assays – Progressive control – Quality control – Validation – World Organisation for Animal Health.

Introduction

Foot and mouth disease (FMD) incursions can potentially cause severe economic losses to non-endemic countries and it is therefore important to have an accurate diagnosis to ensure that clinical signs are not due to another vesicular disease. In endemic countries it is equally important to know whether the virus is present and determine the serotype, especially when the country is embarking on a plan to control or eradicate the disease. Not only is it important to ensure that the virus is present, but data on the prevalence and incidence are needed which can be obtained accurately only with diagnostic assays. It is also essential to determine what serotypes and topotypes are prevalent as this information feeds into decisions regarding choice of vaccine strains and vaccination. In addition, diagnostic support is needed to measure immune profiles, especially after vaccination, to ensure that the vaccines are effective and the campaigns are successful, and also to prove freedom of infection post outbreaks. Sensitive assays are essential to detect sub-clinical infection and carriers. All these data provide epidemiological information vital for countries embarking on the Progressive Control Pathway (PCP) and are indispensable in designing control strategies. They provide a measure of success for countries involved with control and can act as an incentive to improve control and, ultimately, eradication of the disease. However, the specific need for diagnostic assays will change as countries move along the pathway. In Stage 1, where it is necessary to improve the understanding of the epidemiology of FMD, a country or region should perform serological assays to determine the prevalence of infection in various husbandry systems and collect samples for further characterisation. If this serotyping and genetic characterisation cannot occur in-country due

to a lack of specialised laboratories, the assistance of a reference laboratory can be requested. The information should be recent; therefore, these tests have to be performed on at least an annual basis. Stage 2 requires the implementation of a risk-based control programme to reduce the impact of FMD, which implies the ongoing monitoring of circulating FMD strains and, in this case, targeted serological surveys to determine prevalence. It is also necessary to assess vaccination coverage where vaccines are used and to provide evidence that the appropriate vaccine strains are used against the circulating viruses. Success in the previous stages can lead to Stage 3 with a reduction in outbreak incidence and elimination of FMD circulation in at least a zone of the country. This requires rapid detection of FMD outbreaks and detailed characterisation of the viruses with improved monitoring of vaccination and population immunity. In Stage 4 the focus is to maintain successes and aim to reach a status of free from FMD with vaccination. The laboratory will be responsible for testing to ensure that the FMD virus is not circulating and to assist with the detailed investigations of any incursions.

Fitness for purpose

The various requirements outlined for the different PCP stages all necessitate different laboratory and field-based assays where applicable. The tests also differ in their complexity, costs and need for specialised laboratories, equipment and trained operators. For example, in Stage 1 it is necessary to ascertain the level of virus circulation. In this instance, serological assays that measure virus circulation by assessing antibodies to the non-structural proteins (NSPs) can be used (2). These are mostly enzyme-linked immunosorbent assays (ELISAs) that do not require expensive equipment and can be performed in the majority of laboratories. Stage 1 also requires the identification of the serotypes of circulating virus. Although ELISA-based techniques are available that can distinguish between the seven serotypes, they require serotype-specific reagents that are often expensive to obtain and should be suitable for the specific virus serotypes and genotypes that occur in the region (see below). The later PCP stages require not only improved control measures and plans, but also improved laboratories, training and investment in equipment and reagents. In these stages, it is necessary to determine titres during vaccination campaigns, hence additional work over and above simply finding a sero-positive animal. It will also become necessary to perform polymerase chain reaction (PCR) to detect virus presence and to determine the serotype(s) responsible for the outbreaks. This requires specialised equipment, good workflow and quality assurance to prevent cross-contamination in laboratories. Where viruses are isolated on cell cultures, improved biosecurity will be required, especially when the incidence of FMD decreases and there is a risk of accidental introduction of a virus from the laboratory to the field. In addition, the higher PCP stages require further characterisation of the circulating viruses that may involve nucleotide sequencing and other specialised techniques, such as vaccine matching and antigenic cartography.

Importance of appropriate reagents

Globally, the circulating FMD viruses are divided into pools where defined geographical regions are affected by similar serotypes and genotypes (15). The aim is to customise the vaccine needs for each pool to fit the specific circulating strains in regions and thereby ensure an improved regional approach to control. The emphasis has always been on the need for appropriate vaccine strains, but the same is true for the reagents used in diagnostics, both for serology and, to a lesser extent, for molecular-based assays. The reagents should closely match the viruses circulating in a specific region. In serology, it is well established that heterologous reactions, where there are antigenic differences between the reagents in the assays and the virus circulating in the field, give lower titres than homologous reactions, i.e. where the reagents are the same as the virus tested (14). This could lead to an incorrect interpretation of vaccine reactions, mostly resulting in lower titres than are actually the case, or the incorrect serotype determination when using an ELISA. Cross-reactions between the various serotypes make it difficult to determine the serotype using sera from infected and/or vaccinated animals. Sera may also react non-specifically to several FMD serotypes, which is more problematic when animals have been exposed to more than one serotype or have been vaccinated with multivalent vaccines.

Point-of-care devices

There is some debate over the use of penside or point-of-care (POC) diagnostic devices. They have been publicised for their ease of use, speed and relative high sensitivity and specificity. Several devices are commercially available, of which a number are lateral flow devices that can either diagnose multiple serotypes of FMD (8) or are serotype specific (7, 10) and use antigens as the diagnostic input. However, most of these are expensive, and therefore not accessible to developing countries. There are a number of field-based assays available that amplify the genomic material of the virus, such as PCR (12) and loop-mediated amplification (1). Although these assays could be applied in the field, there is currently no obvious commercial interest and great care will be needed to prevent contamination. Most of these assays have also not been fully validated in the field.

The future of penside point-of-care devices

Policies are needed for notifiable diseases when these devices are used. It is essential for control that disease is not concealed by farmers or operators who fear control measures. There should be control over sales and distribution and, preferably, there should not be sales without governmental approval. The devices should be used only by competent persons who have been trained not only in using the device, but also in reporting and further actions needed if there is a positive result. Training should focus on fitness for purpose, thereby ensuring that the correct device is used for the sample available. For example, when using a device that requires a virus antigen, such as is present in epithelium, a serum sample may not be suitable and could lead to a false-negative result and delayed actions to control an outbreak. Regulations are needed on the notification of positive and negative results, and protocols should be in place when a result is negative. This will in turn rely on the sensitivity of the assay and the impact a potential false-negative result could have on the overall economy or disease status of a country. As it is important to have virus available for further characterisation, regulations on submission of samples to laboratories should be clarified. It is not sufficient to base all diagnostic results on devices in the field without submitting clinical material to laboratories for confirmation and further characterisation. Finally, record keeping will be as important when using these devices as when taking samples for laboratory diagnosis. Information on species, age, epidemiological factors, etc. should be available to accompany the result. Most assays suffer from a lack of sufficient field validation, and it is also important that tests be validated in different regions using samples from local breeds and farming systems.

Role of laboratories

From the availability of POC devices, it could easily be extrapolated that investment in laboratories is not needed. However, the role of the laboratory cannot be underestimated. It is of utmost importance that an index case be confirmed in a quality-assured environment, especially in countries where the disease is mostly controlled. Once an outbreak has been confirmed, some reliance could be placed on diagnostics using field-based assays. As POC devices become more prevalent, laboratories will be needed to confirm positive/negative/inconclusive results. They should also take the responsibility for developing or validating devices and making recommendations on their use. During an outbreak where vaccination is used, post-outbreak sero-monitoring will invariably lead to the need for high-volume throughput of samples to be tested, which can be dealt with only within a laboratory. High-throughput testing has specific requirements for samples and data tracing that will be difficult to achieve in the field using local devices.

Laboratories should also take responsibility for keeping stockpiles of reagents to be used when needed, and ensure that these are maintained in a quality-assured manner. In addition, local laboratories should participate in proficiency testing rounds to ensure the accuracy of results and take responsibility for ensuring smaller or provincial laboratories also reach the required standards. In addition, laboratories have to ensure that tests are validated and uncertainty of measurement and precision in testing are determined. During the later stages of the PCP, information is needed about the outbreak isolates that will involve techniques such as sequencing and phylogenetic analysis to determine the potential origin of the incursion (11). Whole genome sequencing could be used to trace the epidemiological path of the outbreaks (4, 5), while vaccine matching using

r-values, epitope mapping and antigenic cartography could provide essential information on the use of vaccines, and therefore assist in control strategies (16). It is also an important responsibility of the laboratories to develop improved and novel vaccines and participate in their assessment and registration, such as the adenovirus vectored vaccines (13). This would be an important contribution to FMD control and eradication and especially to distinguish between vaccinated and infected animals. The use of antivirals to protect animals prior to the development of neutralising antibodies is an option in countries where rapid control is needed (3, 6, 9).

Quality control and validation

The accurate diagnosis of FMD is important, regardless of the stage a country is at in the PCP, and validation of tests is an important aspect that contributes to accuracy. The World Organisation for Animal Health (OIE) Manual of Diagnostic Tests and Vaccines for Terrestrial Animals (2012) explains in detail what is needed to validate tests (www. oie.int/fileadmin/Home/eng/Health_standards/tahm/1.01.05_VALIDATION.pdf). The cost of validated commercially available assays often compels countries to develop their own assays, not always realising the complexities and difficulties to fully validate these tests. This, in turn, could lead to inaccurate diagnosis with severe impact.

Conclusions

Diagnostic requirements will change as countries move through the PCP, and laboratories will remain essential to assist in reaching the overarching goals of the control plans. To facilitate this, several aspects will be increasingly important, such as access to region-specific reagents. This is even more important as most commercial assays are too expensive for widespread use in resource-poor countries, which often leads to the development of in-house assays. Although the perceived cost may be less, those tests need to be validated in a process that is expensive and often difficult, due to a lack of sufficient control material. In this regard, collaboration between different laboratories in a region would be beneficial. Finally, good laboratory diagnostics will be meaningful only when there are sufficient resources available not only to support the laboratory, but also to ensure that material can be collected in the field, submitted in a timely manner to the laboratory and acted upon if necessary.

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