Valued Added by a point of care, rapid, precise diagnostic device for animal infectious disease control
- Point of care (field) testing ability
- The reliable detection of Bsal at low prevalence requires sampling a large number of individuals (DiGiacomo & Koepsell) and has therefore been expensive.
- Passive surveillance for infection in newts during their aquatic phase is unreliable, as hosts can carry infections asymptomatically (Stegen et al.), and dead newts quickly decompose in water.
- Temporal gap between sample collection and result significantly narrowed
- Data Management
- Power of full cycle involvement
- Use case varies widely and customizing our approach if need be or just simply being engaged with stakeholders lets us accomplish our goal at a greater scale.
- Current biosecurity methods are based on a PCR testing pathway that constitutes high costs and latent responses
- A newer diagnostic device would rewrite how we deal with infectious animal diseases
- Trying to retrofit the diagnostic device in existing protocols might not be beneficial
- Disease outbreaks that result from wildlife trade have severe impacts on native wildlife populations, ecosystems, livestock, and human health, and they are estimated to have caused hundreds of billions of dollars in economic loss (Daszak et al. 2000; Karesh et al. 2005; Fe`vre et al. 2006; Jones et al. 2008).
- The fungus, called Batrachochytrium salamandrivorans (Bsal in short), killed 99.9% of the Dutch population of fire salamanders in a short period of time. Bsal-infected salamanders were ever since discovered in three other European countries: Belgium, Germany, and the UK. If no measures are taken, our salamanders and newts may be extinct within 25 to 50 years.
- Despite small sample sizes, the experimental evidence to date further indicates that Bsal is associated with disease and death in 12 European and in 3 Asian salamander species, and is associated with high mortality rate outbreaks in kept salamanders. Experimental infection by Bsal was successful in individuals of at least one species pertaining to the families Salamandridae, Plethodontidae, Hynobiidae and Sirenidae. EFSA et al., 2017
Use cases and identification process
Mapping users and exploring the why behind diagnostic devices necessitated an understanding of where diagnosis falls in infectious disease control. The user mapping team worked to understand where a diagnostic device falls into ensuring animal biosecurity by identifying stakeholders. Based on Langwig et al., 2015's work, we divided our user mapping process using three stages based on differences in disease dynamics and appropriate management actions: Pre‐arrival, Invasion front, Epidemic/Established.
- "Identifying the invasion stage of the pathogen in a region requires data on host abundance and pathogen prevalence, making the initial collection of these data a critical part of disease management." Langwig et al., 2015
- The identified usage of a diagnostic device in animal biosecurity are the following:
- Active testing as part of risk assessment
- Pre-import testing
- Personal usage by pet owners, stores, and zoos
- Testing as part of prevention using host culling
- Disease prevalence test during invasion front
- Disease prevalence test during epidemic
The pre-arrival stage is one of caution where a given disease could potentially spread to a given area. In the case of the Bsal and Bd for instance, even though cases have not reached an alarming rate in most parts of Europe, models indicate areas are susceptible to spread pending physical spread and climatic conditions amongst other reasons. Import restrictions, monitoring of target species, and pubic engagement are actions that could be taken during this first stage. Langwig et al., 2015 In the pre-arrival stage, we identified that most of the usage is by researchers working to assess the risk of the disease by identifying prevalence spread. After collecting sample from a live animal, the amphibian is released at the point of capture. Tarrant et al., 2013 The addition of a field use device at this stage would thus not contribute to biosecurity, besides perhaps to convenience. Stakeholders will be queried to see if they would be keen on not releasing animals if the diagnostic method gave quick results. A potential method of advancement for a fully functional diagnostic device is through international membership organizations that have under their mandate reporting of diseases.
Active testing as part of risk assessment:
- Unless part of a mandated risk assessment in the wake of cases/symptoms, there doesn't seem to be any case of frequent testing (monitoring) to ensure a habitat/species has been compromised.
- The European Food Safety Authority has found that enacting legislation that requires testing of the animals to demonstrate freedom from Bsal, before movement can take place, complemented with other measures such as quarantining, tracking and public awareness are possible risk mitigation measures to ensure sage international and EU trade of salamanders and their products. EFSA et al., 2017
The OIE has set principles and methods of validation of diagnostic assays for infectious diseases that need to be fulfilled for acceptance and usage in regulations. OIE, 2019
An assay that has completed the first three stages of the validation pathway (figure above), including performance characterization, can be designated as “validated for the original intended purpose(s) and become accepted by the OIE.
Currently, the OIE only has clinical signs and behavioral changes listed under field use methods.
Some of the mitigation measures proposed by Grant et l., 2017 and evaluated by EFSA et al., 2017 as ineffective have their roots in a lack of quick response which could potentially be enhanced with a diagnostic device.
[Quarantine] measures could be applied for both imports into the EU and intra-EU movements. The recommended duration of quarantine is 6–8 weeks during which the salamanders are to be sampled by skin swabs and tested by PCR assay at the beginning and the end of quarantine, although the test, at this point in time, has not been yet validated and the uncertainty related to its performance is very high. EFSA et al., 2017
When testing a conignment of amphibians during import or assessment, ensuring high confidence (95%) with worst-case DSe identified by EFSA et al., 2017 (0.5) needs large sample sizes from the consignment.
Our diagnostic device could thus improve the process in two ways: increase the sensitivity and specificity (requiring lower sample sizes) or have comparable detection metrics to current methods but output results at a much lower turnover time.
Discussion from Dr. Penner, our primary contact for field researchers, indicate that host culling is not actively sought for Bd and Bsal. However, some of that non-reliance of host culling as a containment major has to do with the unreliability of current diagnostic methods. Dr. Penner also confirmed that there would be an added advantage to a rapid field use device besides convenience since the researchers would then have the option of not releasing infected animals back into the habitat. Beyond managing the diagnostic techniques, Dr. Penner pointed out that additional skills are required for finding and identifying animals in the wild.
Active surveillance is carried out varyingly amongst nations and governing bodies. The government of Germany, for example, only takes an active role with diseases related to birds and mammals. Amphibian and reptile diseases are analyzed by researchers.
Given the other elements of this stage (testing, quarantine, and banning), Dr. Penner indicated that the cost of testing as a limitation for the biosecurity measures in trade is high.
... there are "waves" of higher rates of testing and lower efforts but not really continuous comparable standardized monitoring. Your machine could enable that.
The EPA (US Environmental Protection Agency) states high costs, some requiring sacrificing animals, others being invasive but nonlethal, while only a few being noninvasive to recommend laboratory investigations be considered on a higher-order or tier than routine bioassessments.
Why PCR? Several diagnostic methods for Bsal detection have been described including histology/histopathology, PCR, qPCR, and lateral flow technology. However, the limitations of some of these techniques include (i) the need for experienced operators or experts to perform these assays, (ii) the ability of the tests to only detect and not quantify the pathogen in samples, (iii) the inability to detect low numbers of the pathogen and (iv) poor diagnostic sensitivity and specificity. The Bsal real-time PCR adequately addresses these limitations and is one of the premier methods for Bsal diagnostics in use. Thomas et al, 2017
The team also looked into related infectious diseases where learnings from our project and the final product can be utilized.
- The outbreak of monkeypox in the US was traced to the sale of Gambian pouched rats (Cricetomys gambianus), restrictions were put in place that prevented trade of these animals from this region Bengis et al., 2014
White Nose Syndrome
- Screening travelers to identify those that have visited caves or mines could similarly reduce the accidental spread of the fungus causing white‐nose syndrome from areas where it is present. Langwig et al., 2015
Current Testing Pathway
- Animal Capture and Sample Collection
- Preferably, capture amphibians by hand. Wear gloves when swabbing animals and change gloves between animals. If you are using a dip net, be aware that B.dendrobatidis zoospores could be caught on the net and transferred between individuals, therefore, use different nets whenever possible, or disinfect the net as often as you can (there is no perfect solution to this problem).
- Swab the underside or ventrum of adult/metamorphs 30 times. Remember you are in effect scraping small amounts of tissue from the skin. Some pressure must be applied, but this does not mean that you must squash the animal. Areas to target are the drink patch, thighs and webbing between the toes.
- Air dry the swab for approximately 5 minutes, avoid direct sunlight if possible (if conditions are too humid to air dry then store in 95% EtOH).
- Break swab ~3cm from tip and drop into empty screw cap tube. The swab stick should not touch or bump against the top of the vial. Screw the cap on the vial and store in the shade.
- Samples can be kept a room temperature for a week or maybe longer, but it is best to keep the samples cool and placed as soon as possible in a 4° C freezer (the kind you have at home is fine). Avoid extreme high temperature and direct sunlight. Samples may be stored in a freezer for many months without problems.
DOD Amphibian Swapping Video
How to Collect a chytrid swab from a frog
How to collect a chytrid swab from a salamander
How to swab a preserved amphibian
Important to note that current procedures (including labor) are based on diagnostic methods with low turnover rate, time, and expert sample collection in the field.
- Comparison of methods for detection of chytrid fungus:
KADEKARU, UNE , 2017
- "...we compared Bd detection rates in swab-scraped and resected mouthpart samples, using nested polymerase chain reaction (PCR). The resulting detection rates for swab-scraped and resected specimens were 67 and 65%, respectively, with no significant difference. Furthermore, we performed a histopathological examination for Bd distribution in the mouthparts; we found that Bd infection occurred in the tip and basement of the jaw sheaths and tooth rows. We recommend using swab-scraped samples for Bd detection. Moreover, careful attention should be paid to scraping the tip and basement of the jaw sheaths and the entire oral cavity to reduce the rates of false-negative results on nested PCR of the mouthparts of bullfrog tadpoles."
- Swabs are originally placed in a sterile 2.0 microcentrifuge tube and stored on ice until they could be stored at 4°C in the laboratory.
- The temporal gap between collecting the samples and analyzing could range from hours to years depending on the need for testing
- For example, in D. IWANOWICZ et al, 2017 samples were extracted within four months of collection and analyzed for Bd by qPCR in 2012. All extracted DNA from these 2012 archived samples have been stored for three years at -20°C prior to Bsal analyses for this study in 2015.
- Postmortem testing: directly after death of an animal, it was transferred to a passively aerated plastic container with moist soil and leaf litter to mimic conditions under which dead Bsal-infected salamanders were found in the field. The containers were placed in a climate-controlled room set at 15°C. Directly after death, and after 1, 2, 3, 4, 5, 7, and 10 days, samples (swabs and tissue samples) for the detection of Bsal were taken. Each sampling occasion consisted of collecting swabs of the ventral and dorsal skin by placing a swab on the skin and rotating it in place for 20 full rotations, collecting full-thickness single skin samples of the ventral and dorsal skin with the use of a 3-mm biopsy punch (Stiefel, Germany) and collecting a tail clip of the animal (tissue samples were weighed to allow correction for variability in tissue size). All samples were taken from new, unsampled areas of skin to avoid interference in the detection of Bsal. Swab samples and tail tissues were stored at 20°C and analyzed for the presence of Bsal with use of a Bsal-specific simplex real-time PCR (Blooi et al., 2013). Skin biopsy samples were stored in formaldehyde and processed for histological examination (haematoxylin and eosin [HE] stain) for the detection of Bsal. Thomas et al, 2017
- Costs associated with equipment (also represents equipment personnel take with them to the field)
- Nitrile gloves
- Vials: $214.00 - $305.00 for 500 tubes
- Sterile cotton swabs: $392.50 for 1250 swabs
- Plastic bags
- Costs and time associated with
labor in field (not quantified):
- Wet lab work:
- Result based action