Beginning with the introduction to the problem to our current stage of product development, the team has engaged with a rigorous human centered design process that actively sought to map stakeholders and engage with. This page highlights the process the team went through and some key takeaways from stakeholder engagements that shaped the project. Late last year, after the group returned from the International Genetically Engineered Machine (iGEM) competition which we partake in annually, a conservation research scientist from the University of Freiburg, Dr. Johannes Penner, reached out to and proposed a problem for the group to address given our diagnostic history. After stating a multitude of use cases and problem-solving potential, the researcher indicated that a field use device similar to our past solutions, giving results within 20-30 minutes or so would be "a real game-changer" in monitoring amphibian populations and allowing conservationists, local agencies, etc. to act immediately.
Dr. Johannes Penner
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.
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
Following the identification of the three main aspects of animal biosecurity in this context (monitoring, research, and intervention), we broadly identified two major users: government institutions and researchers. Usage by private entities such as pet owners and stores, zoos etc is being researched more by the team to identify if there is a noticeable usage or need as the product development is shaped by the prior identified use cases.
To that end, the team has engaged with multiple stakeholders ranging from experts in fungal diseases, elements of point of care devices, to conservationist and field researchers that would be the end-users of our product.
Key takeaways from stakeholders
In the invasive front, host culling is a measure that can be taken. Is the culling contingent on the testing result?
- A key challenge when using culling to eradicate a pathogen (as opposed to reducing host densities to decrease transmission) is the identification and removal of all infected individuals, which is difficult even with effective diagnostic tools. Langwig et al., 2015 Does the difficulty have to do with the "effective" diagnostic device limitations?
(Dr. Penner) No, culling is not a strategy currently promoted for Bd or Bsal. This has several reasons. Partly because diagnostics are never 100% reliable. Partly because this will not stop the spread of the disease because it is also transmitted by other vectors (i.e. other species and other means (e.g. soil, water). Testing all vectors is impossible. For Bd and BSal other researchers test avenues to cure the infected individuals, which looks promising in the case of Bsal. So the current idea is to remove infected individuals, keep them in captivity until free of the pathogen, monitor the environment until pathogen free as well, then release the individuals back into the wild.
(Dr. Langwig) One issue with culling during pre-arrival (and culling in general) is that if the outbreak is much more widespread than your sampling, culling will be ineffective. However, if the spread is very localized, then knowing which animals are infected during pre-arrival would be helpful. Faster tests will always allow faster decisions.
On the roles of researchers: In pre-arrival, the protocols we have seen so far involve sample collection and release of animals. Would there be an added advantage to a field use device besides convenience in this case? If researchers were to get the results faster, would they not release? Would they be able to cull infected animals?
(Dr. Penner) Researchers would be faster and would not release infected animals. These would not be culled but cured and released at a later point in time. This is not possible with current methods. There one has to wait until infections have spread widely, then find living animals which will all be collected (not only infected individuals) and treated in captivity until release.
(Dr. Langwig) Most of the time, the complications [with field work in collecting sample] come from being to accurately identify the species and catch and hold the animal appropriately as opposed to collecting the sample (which in our case is just a swab).
Literature so far suggests that sample collection and management requires experience. Where does the necessity of proficiency stem from in the current procedures? Does it have to do with complexity in the collection method such as swabbing or perhaps later handling of sample in wet lab?
(Dr. Penner) In my opinion from two points: Finding and identifying the animals in the wild; second: later handling and analyses of the samples in the laboratory.
(Dr. Langwig) Sometimes researchers are leading the charge to collect samples, as has often been the case for chytridiomycosis. For other pathogens, sampling is more routine, usually conducted by government employees, often with more coordinated oversight by a central government.
Who carries out prevalence assessments in the invasion and epidemic stage? Is prevalence assessment taken out by governments seeking to contain spread or is there involvement by researchers in gathering sample, testing etc, and the government taking on a more managerial role?
(Dr. Penner) This differs from country to country. Currently the government in Germany only takes an active role with diseases related to birds and mammals. Amphibian and reptile diseases are analysed by researchers.
Are you aware of active frequent testing carried out as part of risk assessment (without any detected cases)?
(Dr. Penner) Yes, that used to be the case for Bd and Bsal.
One of the barriers to a rigorous measure during the pre-arrival stage is the significant cost and impact on trade. (Langwig et al., 2015) Given the other elements of this stage (testing, quarantine, and banning), how significant is the cost of testing as a limitation for the biosecurity measures?
(Dr. Penner) In my opinion costs are significant because research is conducted on the basis of money acquired by third party funds. Thus limited and only available for specific projects. Thus there are "waves" of higher rates of testing and lower efforts but not really continuos comparable standardised monitoring. Your machine could enable that.
(Dr. Langwig) Testing might be cheaper than implementing trade bans, depending on the product and specific risk. For example, the U.S. banned salamander imports, and while I don't know specific dollar amounts, I suspect the importation of salamanders to the U.S. is not generating a lot of value. (Dr. Karen Lips at U of Maryland was involved in this, and might be a good person to contact.) Therefore, banning salamander imports might be worth the cost tradeoff of testing every salamander. For something where profits were higher, I would think testing could be far more beneficial then costs associated with reductions in trade or sanitizing equipment.
How many amphibians need to be tested per trip to the field?
(Colleen Kamoroff) In my research with field based molecular techniques, I was able to detect Bd on every frog I swabbed, although Bb prevalence was known to be high. The swabbs were run in triplicate qPCR reactions (three technical replicates... three wells with DNA extract), and I did not detect Bd in every technical replicate. I cannot speak to how many frogs would need to be swabb in order to determine if Bd was present (especially if it was present at low densities), but I would suggest running the extracted DNA from the swabbs in triplicate qPCR reactions.
In your opinion, what is the most important factor to take into consideration when picking a point-of-care testing device for amphibian testing? Time, accuracy, cost, etc?
(Colleen Kamoroff) I just did a study where analyzed the effectiveness of using an in-situ DNA extraction method combined with a handheld mobile thermocycler for real-time qPCR analysis from Biomeme Inc. In this study, the limiting factor was accuracy. The probability of detecting Bd DNA in the technical replicates was lower for the Biomeme field protocol compared to samples extracted using the lab protocol, suggesting the field protocol has reduced sensitivity and may not detect low quantities of DNA. Our results suggest the field extraction protocol using a handheld qPCR platform is a promising tool for rapid detection of Bd in susceptible amphibian populations. The field protocol yielded accurate results in less than 60 minutes. However, the applied field protocol may be prone to false negatives when analyzing low-quantity DNA samples (i.e. eDNA).
Do the detection methods depend on the geographical location? If so, what are the factors that contribute to this dependence?
(Colleen Kamoroff) Aqueous eDNA detection is dependent on collection, extraction, and identification of target DNA particles. eDNA exists in heterogeneous clumps in the environment so collecting samples from multiple locations would increase your chances of collecting target eDNA. Also collecting eDNA samples near the target specimen (near frogs who are hosts of Bd) would increase chances of detection.
What kind of conditions will a point-of-care device for amphibians usually be used in? Are there any extreme conditions such as temperature that should be taken into account when developing a point-of-care device for amphibians?
(Colleen Kamoroff) This depends on the type of amphibian. I work with high elevation amphibian species... so we experience high UV conditions. DNA degrades more rapidly in high temp, pH, and UV conditions... so those are good conditions to take in account.
When testing for the presence of fungi on amphibians is the required result often qualitative or quantitative?
(Colleen Kamoroff) Binary detection (i.e. presence/absence) of Bd DNA is an important metric for understanding disease dynamics and host risk. However, DNA quantification of both eDNA and swab samples is critical to the ecological interpretation of the results. Determining when Bd levels and infection intensities rapid/ exponential grown before lethal threshold levels is critical for management to implement conservation strategies. Such determination can only be accomplished through accurate quantification of Bd load on skin swabs and potentially eDNA samples.
From your experience ,on average, what is the cost of a lab PCR?
(Colleen Kamoroff) Typical costs for lab extraction and analysis of swabs and eDNA samples are ~$10-35 and ~$50-150 respectively depending on type of lab, extraction method, and number of samples processed. Typical qPCR machines used in lab analysis have a 96 well capacity and can multiplex up to five targets per well resulting in a high-volume throughput per run.
What species of amphibians is your research team testing on for Bd and Bsal diagnosis?
(Dr. Penner) My team and I would like to conduct research with such a device on Grass frogs (Rana temporaria) to test for Bd and on Fire salamanders (Salamandra salamandra) to test for Bsal. However, in general all 3 orders of amphibians (Anura, Gymnophiona and Urodela) have Bd and Bsal has been found on several species of Anura and Urodela. So for my research tests would involve all amphibians encountered in suitable habitats of Rana temporaria and Salamandra salamandra
During your research with the fungus Bd, is it preferred for the point-of-care device to give out quantitative or qualitative results?
(Dr.Penner) The main aim would be quantitative results for Bd.
What exactly do they calculate, how, and what do they infer from those quantitative measurements? what does low accuracy in quantification mean, how exactly do we determine the accuracy?
(Research and Colleen Kamoroff) The quantitative measurements allow for the researchers to know the Bd/ Bsal load present in the amphibian this is essential as only once the fungal load reaches a certain level does it become lethal and at another fungal load it becomes contagious. Low accuracy means that when there is a low fungal load the testing results for presence of the fungi will come back negative when in fact there is just a low trace of the fungus. (Steffanie) Accuracy is basically at what quantity of fungi present will the test start to give negative results due to low traces of the fungi.
Why do the researchers use qPCR?
Answer: (Colleen Kamoroff) qPCR is used due to the cost and time efficiency, three test can be carried out at once. Result are also quantitative.
Map of Bd for finding out climate conditions.
Prevalence of Bd in an area decreases with increasing temperature, the exact statistic is "decreased by 8.8% with each degree increase in temperature range at a site" although the prevalence begins to decrease once temperatures exceed 28 degrees as Bd does not grow well at temperatures≥28°C. Research found increased infection prevalence and mortality under cooler conditions. The presence of Bd also increases with proximity to a water source. As seen in the maps most sites that are pinned as positive sites are prone to be damp areas. Barely any positive sites are in landlocked areas. Montane, grasslands and shrublands have the highest occurrence of Bd.
As can be seen in the section above, stakeholder input was taken using questions and interviews which has been extremely important to the development of the device as these inputs provide the requirements/constraints/criterion for the device to follow. For example, using input from Amphibian researchers such as Dr. Penner and Colleen Kamoroff, we were able to base requirements on the device time and ability to multiplex, determine that we needed quantitative detection rather than qualitative, and focus on accuracy as one of the most important factors.
The team also utilized the expertise available in the university itself by contacting laboratories, professors, and researchers to provide opinions and insights regarding the project components. For example, we reached out to NYUAD Professor Mohammed Qasaimeh whose lab works with point of care technologies. Prof. Qasaimeh provided the team with his opinions working with multiple technologies such as microfluidics and electrochemical sensing, and his feedback was valuable for us in determining which options to proceed with. Similarly, researchers at NYUAD Boissinot lab provided the team with further insights regarding the sample collection method such as number of amphibians sampled per trip, time of collection, etc. which have been instrumental in finding the best device strategies.
The PIs of the iGEM project also provided regular feedback on the project progress, helping the team make decisions on which options to proceed with and helped answer the team's technical questions about specific fields they are working with.