by Galaxy Diagnostics recently hosted a webinar where our CEO, Dr. Amanda Elam, and our Director of Research and Development/Asst. Lab Director, Dr. Jennifer Miller, provided clinicians with an update on our Borrelia test offerings. Dr. Miller discussed current recommendations for use based on the published research. The webinar covered:
- Borrelia spp.infection prevalence and pathogenesis
- Utility and limitations of antibody testing
- Advantages of direct detection with Lyme Borrelia Nanotrap® Antigen Test
- Recommended testing strategies for acute and chronic Borrelia infection
We are happy to make this resource available to healthcare providers, patients, and researchers, and other who may be interested in learning about borreliosis. A full transcript of the video dialogue can be found below.
Full Transcript
AE: Dr. Amanda Elam, PhD, Galaxy Diagnostics cofounder & CEO
JM: Dr. Jennifer Miller, PhD, Galaxy Diagnostics Director of Research and Development | Assi. Lab Director
AE:
Alright well we’re going to welcome everyone to our webinar presentation today the topic is Borrelia diagnostic update. And I’d like to thank you for joining us today our speakers are me, Amanda, and Dr. Jennifer Miller, who’s our director of research and development, at Galaxy and assistant lab director. She, so, oversees all the day to day lab operations, at Galaxy.
A few notes for participants, this webinar is being recorded for future use everyone will be muted during the presentation, there will be a Q&A time at the end. And we invite you to drop any questions or comments in the chat we welcome to do that, during the presentation and during the Q&A period if there are fewer than 20 people on will promote everyone to panelists so we can have an open discussion and if you’re having any technical difficulties, please send a DM, a direct message, to Karen Kilburn she’s our tech support. And I’d also like to thank Karen and Victoria Quiett for their assistance with the webinar today.
So, to begin with, for those of you who are not familiar with Galaxy Diagnostics, we are a medical lab located in Research Triangle Park, North Carolina. Our mission is to advance molecular and immunologic testing for hard to detect flea and tick-borne diseases, notably, of course, Lyme disease, borreliosis or Borrelia species infection and also cat scratch disease, or bartonellosis and Bartonella species infection. These are emerging infectious diseases.
We’re a spin out from North Carolina State University. We have an ongoing collaboration with our co-founders and colleagues there, and at a number of other top academic institutions. We really see ourselves as a prototyping plat—and early commercialization platform for new technologies. Our goal is to identify tests that we can scale, either through you know transferring onto a high throughput platform and licensing out to big labs or creating IV kids that will run on high throughput systems.
We’re a one health company—a company, which is what signal that symbol is up in the top right hand corner. So we’re working at the nexus of human health, animal health and health of the environment. I think you’re—I think that flea and tick-borne diseases or vector borne diseases generally are really important one health topic and area of research and advancement.
We provide testing to providers in human health, but also to providers in veterinary sciences and medical practice, and we do a lot of clinical research support. So we provide testing for a number of different types of clinical research projects.
So that’s our company, and so when we come at this problem of advancing diagnos—diagnostic tools for providers and flea and tick-borne disease, I like to share this slide because people often ask us for examples [of] what’s your positive rate for your testing. And the answer is, you know, it’s not indicative. It’s really hard to tell you whether that’s a number, you should care about, right.
So, the reason we do research studies with tightly defined samples is to really effectively characterize the performance of the assay in targeted critical population. When we’re looking at what contributes to the test performance in our laboratory, we know that there are factors down here testing in the lab. We know that there are factors around how the samples are collected, sample integrity, chain of custody.
And we also know that some doctors are much more experienced and, and often skilled at figuring out what a which patients are likely to test positive and that really comes down to what, you know, prior exposure to vector borne the high-end high-risk groups and the associated symptoms and disease states and is, if you’re new to the area of flea and tick borne diseases, it’s important for you to know that this is a very early stage of research.
And so, our understandings of what clinically meaningful indicators are of potential positive patient are still evolving. So this is really important, and the reason why diagnostic technologies aren’t more advanced at this point is that there are some very clear technical challenges associated with advancing diagnostics, in the space.
Our focus, of course, is on slow growing low abundance infections and clinical samples. And these are, these are the types of infections, where conventional techniques often don’t really—are that accurate or reliable.
We’re also designing tests that focused on the path of biology of infection which can vary considerably across, you know, different general species of infection. And of course, you know we tend to be a little US focused as US labs tend to be, but a lot of these infections have variable geographic distribution not only across the United States but around the world. And people travel. So as clinicians you want to know—you want access to tools that help you diagnose a patient, no matter where they’ve—they’ve traveled in the world.
And then, how we’re going after sample enrichment is really the term of ours, that we use to describe how we’re approaching, you know, diagnostic improvements for these low abundance infections. We’re working with three different technologies.
When Galaxy launched, we launched using this enrichment methods so Bartonella alpha Proteobacteria Growth Medium for increasing the sensitivity of DNA detection for Bartonella species infection. This is a medium where we can increase the bacterial load so that we’re more likely to aliquot the small bit we take out for DNA testing. Aliquot out, you know, something that actually has the organisms in it.
Then we started working with ddPCR, drop– digital droplet PCR or digital PCR, which effectively does manipulate the sample. So instead of running one PCR and an aliquot, we’re—you’re—we’re running—we’re taking that aliquot and breaking it into 10 to 20,000 droplets and running a reaction in each droplet. This is a technology we’re applying Borrelia, Bartonella, Babesia, and other tick-borne diseases. And we have our first offer our first offerings out right now for about Bartonella.
And then the third technology we’re working with is Nanotrap capture and our first application here is Borrelia, which is an important focus of our presentation here today. And Nanotrap tech– capture we’re using protein capture right now. And so using magnetic fields, we can effectively, you know, capture any of the proteins that are in the test sample. Sort of like a magnet pulling it in, spinning it down and then test the concentrate. And these are really powerful technologies we’re going after different applications on.
And so, at this point I’m going to invite Jen, who is a Borrelia expert, well published, and has been working in this disease area for a long time, to walk you through an introduction to, to the disease and talk about some of the diagnostic advances we’re working on Jen.
JM:
Thanks, thanks Amanda so I just wanted to start, start by talking about a few things about Lyme disease and Lyme Borrelia disease associations. So, what you’re looking at in the bar graph below it to the left of your screen are annual cases of Lyme disease in the US. And very quickly what you’ll see is that the number of cases reported to the CDC and estimated total cases has been steadily up ticking throughout the years.
This particular bar graph goes back to 1988 and what you see is a very steady uptick both as the incidence of Lyme disease United States grows and as our awareness of the disease grows. And so right now we’re looking at an estimated 467,000 cases per year in the United States. And, of course, this is a very large number Lyme disease is the number one vector borne disease and United States.
So Lyme disease was, was, as we’ll get into in a minute, first described many years ago. And so, there’s a couple different definitions of patients that are tracked patients that exhibit persistent symptoms following antibiotic treatment.
Lyme disease dates back to studies done in the 80s, and so the term post treatment Lyme disease syndrome is, is really a disease definition that was designated so that specific research studies could be conducted. It’s not really meant to exclude or diminish those with chronic Lyme disease, of which post treatment line is a form of chronic Lyme disease, but certainly there are other forms of chronic Lyme disease as well that that are utilized by clinicians and certainly by patients that are exhibiting [a] myriad of symptoms.
The first case definition for post treatment Lyme disease or PTLDS was first described in 2003 and then again in 2006. And like I said, this is very important as a research tool, but in no way covers the full spectrum of, of chronic disease and that’s because as, as many of you know what is depicted here on the right, is there’s multiple different signs and symptoms of Lyme disease that can emerge days to months after tick bite including neurological symptoms like: facial palsy, short term memory loss, headache, respiratory episodes, dizziness, shortness of breath, heart issues, heart palpitations, irregular heartbeat. The case of severe Lyme carditis, heart block. Other neuroborreliosis symptoms, you can get inflammation of the brain, spinal cord, nerve or radiculopathies that can radiate down your arm. Intermittent pain, so various myalgias. Of course a symptom that many people are familiar with is, is arthritis usually affecting a large joint. You can get severe joint pain and swelling.
And then the other thing that is documented in the literature is, of course, people know that some people start out with a Lyme disease rash, but in some instances, you can get what’s known as a disseminated EM rash where the rash will show up on other parts of your body, sometimes quite distinct from the area where it first originated.
Next slide.
So, Borrelia are stealth pathogens and as we just mentioned United States there’s more than 76,000 live diagnosed Lyme disease cases where people are treated. And there’s greater than 200,000 cases per year just in Western Europe alone. Of course, Lyme disease also affects other parts of the world, and including parts of Asia. Many of you know that the type member of the genus Borrelia burgdorferi was originally discovered in Lyme Connecticut, hence the name Lyme disease. Alan Steer discovered it as an agent that was causing juvenile arthritis and in a group of children back in 1977. The spirochete was first grown and visualized and isolated by Willy Burgdorfer in the early 80s, hence the name burgdorferi, named for him.
Quite revolutionary the first studies, where this organism was able to be grown in a very specialized and complex media. So, Borrelia, all members of genus Borrelia, are—or most Members—are transmitted by ticks carried by rodents, deer, and birds.
There are 52 Borrelia species that fall into two different phylogenetic groups. You have Lyme disease agents and the relapsing fever group. The reference I made the most being carried by, by ticks is, of course, louse, louse born relapsing fever, which is obviously transmitted by the lice.
So Lyme Borrelia has been implicated in a whole range of disseminated diseases as we just discussed affecting the heart central nervous system in joints and Borrelia are spirochetes. Spirochetes are sort of an unusual group of bacteria. They’re not technically gram negative or gram positive. If you were to attempt to gram-stain a spirochete it would vary weekly stain gram negative, this is due to their unique membrane architecture.
And because spirochetes are unique, as we’ll discuss in a few minutes, they have a unique way of burrowing into tissues in hiding, and for this reason, as well as many others have treatment failure with Lyme disease is unfortunately common. The other thing I want to point out is that one of the other vexing features of Borrelia burgdorferi and other Lyme spirochete infections, is that the acquired immune responses is complicated and delayed.
And so, typically with an antibody response you get an early antibody response, where you get a peaking of IgM antibodies and for Borrelia this is delayed. You often don’t see complete conversion to an IgM response for four to six weeks. And by this time, you know your patients that have erythema migrans, which is certainly not everybody, and flu like illnesses may very well have gone on to develop other symptoms before they’ve even produced detectable antibodies.
And then, of course, you have early disseminated Lyme disease, where you can have neurological symptoms of inflammatory symptoms of the joints or even heart symptoms. And in a group of these patients, they may have class switched from IgM to IgG. And again, this take, this is delayed with Borrelia can occurs after six to eight weeks of infection. But it’s been well documented in the literature that certain patients don’t flash which effectively or, in some cases at all, which can make tracking of Borrelia infection by antibodies a bit problematic.
And then, of course, in late phases of Lyme disease, you can continue to have neurological symptoms and you can have chronic arthritis symptoms as well. And then, of course, not on this chart are other forms of chronic or post treatment Lyme disease, where you can have these symptoms or other symptoms. So continued memory loss, pain, intermittent fatigue, other symptoms as well.
Next slide please.
And so, the other final thing I’ll point out is that these Borrelia, Lyme Borrelia, and also relapsing fever earlier, to some extent are involved in polymicrobial infections this idea that tick borne diseases form part of the pathobiome or entire panel of infections that can be picked off sometimes simultaneously from a vector.
For example, we know that ticks can transmit a wide variety of pathogens sometimes all at once, not only Borrelia burgdorferi, but other viral pathogens, the tick-borne pathogens like Anaplasma, Ehrlichia, Babesia parasites, you know and we’re now beginning to understand that this pathobiome is, is really complicated it can affect pathogenesis of, of multiple infections, depending upon which was present first and also the immune response because you’re getting assaulted on all fronts.
And the various forms of tissue or other damage that can occur as a result of, of infections. And so studying the pathobiome as a whole, becomes very important.
Next slide please.
So this is just a brief pictorial slide, as we said there’s two different classes of Borrelia. There’s Lyme disease and relapsing fever. This looks like a color wheel to the right here, is a phylogenetic image and what you’ll notice is that the various, various species of infection are highlighted.
So, the Lyme Borrelia species are highlighted in blue primarily but, as you move sort of counterclockwise around the circle, the blue runs into the purple. The purple is the recently discovered and described Borrelia mayonii species, which as you’ll see shares features with Lyme Borrelia, but it also shares some features relapsing fever Borrelia, which are sort of at the top of the circle. And you have various colors yellow, magenta, red. The red is the Borrelia miyamotoi, which was recently discovered. This relapsing fever species and with the miyamotoi, you know, it shares a lot of features with Lyme Borrelia, but one of the relapsing fever features that it shares, is it replicates to higher levels in the blood then Lyme Borrelia do. And so, this is kind of a phylogenetic highlighting of the different species within the genus.
Some of you may be aware that there was a fairly recent proposal to reclassify Borrelia as a genus into the genus Borreliella. And in the Borreliella designation the Borreliella would apply to the Lyme Borrelia and the designation Borrelia would be reserved for relapsing fever. There’s still a lot of debate about this, so we chose not to use that terminology here.
Another thing to think about with various Borrelia spirochetes is, you have also have designations based upon conserved versus variable protein. So, for example with Lyme Borrelia the outer surface protein A, which many of you are familiar with as OspA, is actually conserved fairly well across all Lyme Borrelia. You don’t really have a major sequence derivation, and this was part of the reason that it was thought to originally have been a good vaccine candidate and why it’s often a diagnostic target of interest.
In contrast OspC, or outer surface protein C, also a major variant to determined Borrelia burgdorferi. It varies widely across various streams of Borrelia. In other words, the B31 OspC looks different from an N40 from a ZOsp7, and so there’s lots of different stereotypes of OspC. And that’s something to keep in mind when anytime you’re talking about serology is that the ability of antibody to recognize a target can differ if the sequence of the target is buried.
And then relapsing fever, some of you may be familiar with the glove Q protein, which is a diagnostic marker for relapsing fever spirochetes. There’s also different clinical implications for the spirochetal infections.
Relapsing fever Borrelia, as the name implies are characterized by a high cyclical relapsing, resolving bacteremia in the blood or the bacterial replicates the high numbers in the blood. The immune system starts to catch up right around the time you get antibody mediated kill off the bacteria changes surface proteins and evade the antibody response, so you get this up and down patter.
Whereas Lyme Borrelia are more stealth, they don’t replicate at high volumes in the blood, you know they get into the blood they get out and they really like to hide out and do their damage within tissue cells and systems of the body they can really borough in and do their thing.
So, because you have all of these different basically spectrum of Borrelia infections it’s always important to ask patients about travel history and vector contact and occupational exposure. Basically, so that we can cover the basis on how to detect and treat these very complicated pathogens.
Next slide please.
So pathogenesis of Lyme Borrelia is, is worth spending a couple minutes on. Borrelia are very slow growing you’ve got a doubling time of 12 hours. While they’re extra cellular pathogens, they can invade tissues and cells. And while they’re inside those cells, you know, while they’re in a tug of war with the cellular machinery, they can—they can cause quite a bit of damage. Because like I said they’re spirochetes, they’re very corkscrew in their motion so imagine a drill burrowing into tissues and cells and doing damage to underlying tissues.
So they cloak themselves with tissue components at the bind to various surface proteins they put on their surface fibronectin decorin, just to name a few. They will, they will form biofilms sometimes within the tissues. All this makes it a lot harder for the immune system to detect them and eliminate them. They do trigger an innate immune response you know they’re, they’re delivered via tick bite, so you have you know, the first line of defense are, of course, the cells within the skin.
And they do respond to Borrelia, but Borrelia also has a way of getting around that and can hijacking so that they can disseminate spread the distant tissue sites and cause inflammation, flu like symptoms. Some of this is, is through changing of the surface proteins that it displays on its surface. To buy itself time, to disseminate from the skin into the blood and then to tissue systems. The other thing to point out is that only about a quarter of all patients develop in EM rash.
So for a lot of the surveillance case definitions, with the serological practices that are recommended by the CDC and others, the initial criteria is the visualization of EM rash, but only a quarter the patient’s develop the and rash. And of course, it’s not always easy to see and EM rash on, on the skin of ethnicities that are, that are darker. It can be very hard to visualize.
As I said before, the Lyme agency in the blood generally within two weeks, and they, they do hide in biofilms. And they also form around body forums or persisters. And the whole reason for this is unknown. One hypothesis is in the presence of factories static antibiotic slicked oxycycline that work by slowing down the replication of the bacteria. The bacteria tried, it may try to get around the mode of action of the antibiotic by slowing down the replication, which causes them to change into a form where they’re less metabolically active and therefore the antibiotic either doesn’t penetrate or have as much of a mode of action.
Next slide please.
So, as we’ve mentioned one really are low abundance infection. And so unlike with E. coli, where the bacteria again they replicate you know to very high levels and so conventional limited detection is, is a sufficient sensitivity to detect what’s there. When you have stealth pathogens that are present it much, much lower limits the conventional limited detection isn’t going to suffice, because it isn’t going to pick up a stealth pathogens. And so stealth pathogen numbers, you know, may or may not progress over time in the blood, but your symptoms progress and, certainly, you can have persistent microorganisms within tissues working in body sites away from the immune system, so we really need more sensitive detection methods for detecting these pathogens.
And as Amanda alluded, you know, this is our sample enrichment can become so very important, so that we can boost those lower limits to something that is more easily detectable using tools that we have at our disposal.
Next slide please.
So I want to introduce our newest test offering the Lyme Borrelia Nanotrap test, and this is utilizing the Nanotrap particle technology pioneered by Ceres biosciences and scientists at George Mason University. And so the, the Nanotrap is, is a direct detection method that confirms presence of–confirms actual presence of an organism.
And the way that it works is the beads target and concentrate the Borrelia burgdorferi OspA protein. And this is a urine test. And so the benefits of urine is that it’s easy to collect sample, especially for children it’s, it’s less invasive there’s a lot less anxiety involved.
And so studies have shown that that using the Nanotrap particles to, to concentrate and pull off a out of urine can improve detection by greater than 10%. And so, when you look at Nanotrap enrichment coupled with perhaps with the more conventional Western blot, you can increase the sensitivity engine detection and urine.
And so the George mason team is published on these studies extensively. The first publication was in 2015 this one is referenced here. And what we see is that the Nanotrap assay allows us to pick up cases that are not detected with standard two-tier testing.
So in this graphic, this two by– this two by two table that you see over to the left, 23% of samples were detected by Nanotrap, but were two-tier negative. And then, when you combine that with the samples that were detected by most methods, you were– they were able to detect have Lyme disease and 96% of the samples that they looked at. And this included 26 acute cases of Lyme disease, both with and without the clinically defined definition of EM rash.
And so the way this works is a pair of urine and serum were collected. The initial visit serum was tested by two tier testing for IgG, and IgM and the urine was tested with the Nanotrap assay. And then we conducted an internal analysis of the data published in this paper. And when we crunch, the numbers what we saw was that, based upon their designated patient cohort, it would appear that the essay had a 40% sensitivity and chronically ill patients, but this data requires more analysis and research to further define clinical utility in this and other relevant populations.
Next slide please.
So, I alluded to the history of Nanotrap, but this is giving a little more in-depth information for those that are interested, as well as relevant references for those that want to go and dig a little bit deeper. As I said, the technology was originally developed by scientists at George Mason University and licensed to Ceres nanosciences.
And you can see that they’ve published extensively on this technology. Antigen testing in urine has had a troubled history, due in part to low concentration of antigens but also stability of the antigens within urine. The Nanotrap particles that were pioneered by this group of really did a great job of, of changing that perception in dramatically increased the sensitivity, through the antigen capture. And how they did this, as is well laid out in the 2015 paper if it’s referenced here to the right.
And this technology is well published for Lyme disease, and it’s also been published for other tick-borne diseases if you’d like to reference the 2020 science report paper here. They investigate other tick-borne illnesses there. It’s also been used for tuberculosis, other parasites, including Toxoplasma gondii, Trypanosoma cruzi, and malaria parasites, Plasmodium falciparum. And then studies have been done with HIV and Zika viruses. And Nanotrap capture is currently used for wastewater surveillance testing for COVID-19. This has been combined with influenza. It’s being utilized on college campuses, United States coast guard installations, military vessels and certain towns and cities. And so this technology is really very powerful it’s got Y application to a host of pathogenically important microbes.
Next slide please.
So, I just want to briefly mention that we take quality very seriously Galaxy diagnostics and what you’re looking at are the test performance data for the Nanotrap urine antigen tests that we have validated and launched as license from Ceres. And so we did an analytical validation. We calculated analytical sensitivity and specificity.
We did a clinical validation. we utilized blinded de identified paired serum and urine obtained from Canadian patients presenting with Lyme borreliosis, look like illnesses, or healthy controls. This is, in collaboration with the Magnotta Research laboratory at the University of Wealth in Ontario—Ontario. Nice if I was able to talk. And we looked at serum reactivity using standard two-tier testing using the CDC criteria.
And we also did the Lyme Borrelia Nanotrap antigen test on the urine. And what you can see, is summarized below. We weren’t just able to detect more positives overall utilizing the Nanotrap antigen test, but what was interesting was it seems like there was a stratification, in the sense that Nanotrap picked up positives that two-tier doesn’t, and vice versa.
And so, what this illustrates to us is that a combined approach utilizing both direct and indirect methods is going to be the best bet for maximizing diagnostic sensitivity. We have not been unblinded so I can’t speak to the clinical utility in depth, yet of the patient population that we analyzed because we, we have not been blinded. So stay tuned for more information there.
And then, I’ll just briefly mention that, even though this is not a quantitative essay, we assessed accuracy of—by comparing expected versus observed test results utilizing a twofold solution series of spiked urine samples. And we tested a wide range of, of Borrelia burgdorferi lysates spiked into the samples. And you can see that the accuracy is, is quite achievable over abroad dynamic range.
Next slide please.
So now, I want to get to what I think is, is a really important area of emerging research and really starting to speak to the clinical utility of this assay for clinicians because that’s where the rubber meets the road. And so I’d like to present three of our representative standout cases.
So case number one we analyzed urine and serum through various assays from a 57-year-old female from Singapore. We did not know anything about the clinical symptoms or signs that this particular patient. But the urine did test positive with the Borrelia Nanotrap antigen assay. While the ELISA was negative, this patient did have corresponding IgM Western blot serology. So that that’s an interesting case. Like I said we don’t know anything about the clinical symptoms and signs, but it was interesting to us that we got a positive Nanotrap antigen test and that lined up with a IgM Western blot positive serum response.
In our second case we tested an 18-year-old female from Hawaii. This particular patient, was assigned the icd-10 codes of Lyme disease joint pain, interestingly juvenile rheumatoid arthritis, and anxiety. While this patient did not order Borrelia serology from us, they were also Nanotrap managing positive and urine. This may harken back to some of the very early reports and literature in the late 80s early 90s. So, where there was an association made between expression of Borrelia burgdorferi OspA and arthritis. So this warrants more investigation, but I think it’s worth pointing out.
Case number three was a 42-year-old male from California. This particular patient had—was assigned neurological icd-10 codes for neurological disorders, nervous system disorders, also muscle spasms, and other myalgias. This patient—we also did not run serology on this patient, but this patient was also Nanotrap antigen positive in the urine. So this is intriguing to us because it would be nice to do further studies to kind of investigate utility of the Nanotrap antigen urine assay for Neuroborreliosis.
And the other reason I bring up these case studies is, is, we would like to invite interested clinicians to partner with us. So that we can do clinical utility studies utilizing paired urine and serum samples. So that we can better define clinical utility of the Nanotrap urine antigen assay for various disease stages of Lyme disease.
Next slide please.
So currently Galaxy offers both direct and indirect test offerings. Our direct detection methods, we’ve talked at length about the Nanotrap antigen assay, and this is for urine samples of 40 mL or above. We also do a Borrelia species PCR on non-blood fluid. These are genus level primers. Any positive results would be confirmed by sequence analysis. The idea being that if you get a sequence, you would then know the genus and species, these are research use only. And the reason we say their best for non-blood fluid is again Borrelia are very hard to detect in the blood. Their presence at very, very low numbers.
Similarly, we also offer a corresponding tissue PCR based assay. This works best on fresh or frozen tissue. This is because formalin and other cross linking DNA treating agents cross linked the DNA to a point where it really damages the DNA and it’s harder to detect. So indirect detection we offer the standard two-tier ELISA and Western blot assays for you know, detection of antibodies to Borrelia burgdorferi proteins.
So—that’s okay, you can go the next slide.
So, antibody testing is tricky. So originally two-tier serology was, was devised as a surveillance tool. It was really meant to go out into the field and, and surveil prevalence. And so, they needed something that was quick and amenable for surveillance, you know, responding to a wide range of antigens.
The ELISA, which is the first tier, is—it was always meant as a screening assay, okay. It’s widely used to indicate exposure. There is a tradeoff between sensitivity and specificity with serology. Whether you’re talking about the ELISA, or the second-tier Western blot. Disadvantages: it’s hard to standardize across labs. Different labs run different assays with different cutoff values. They utilize different sources of antigen.
They have different criteria for interpretation of those results, different antibodies you know, on and on and on different ways of imaging. There is potential cross reactivity with IgM. So as a molecule IgM is a pentagram, which means it has five arms, whereas IgG only has two arms. And so because of it, it’s more sticky and more things stick to it.
So you also, with Borrelia, as we alluded to earlier, you have variation in gene expression and surface proteins worth both during times of infection and between strains, and that can make interpretation of serology a bit tricky. And then, of course, immunoassay sensitivity is always dependent upon the species or strain of, so there’s multiple, even with Borrelia burgdorferi right there’s multiple strains. And there can be differences and in migration patterns of the antigens of interest just, just as one example.
Other differences are whether you’re using cell cultivated or non-cell cultivated antigen. Whether that be a lie say whether you’re using recombinant proteins, what species, they come from, and then also the passage number of the bacteria.
I didn’t get into this, but Borrelia lose infectivity if their passage too many times in culture. And the reason for that is, they contain numerous extra chromosomal plasmids that are required for pathogenesis and they kick them out pretty quickly if their cultured too many times.
Next slide.
So what we’re recommending currently is in, and I alluded to this before is, is that clinicians utilize both direct and indirect testing methods to maximize their potential for detection of Borrelia species. And so obviously the indirect would be the two-tier testing, ELISA and Western blot. And direct methodology would be the Lyme borreliosis Nanotrap urine test. And again because our internal data suggests that there’s little overlap between patients the test positive with two tier testing versus those that test positive, with Nanotrap. If—you’re better off maximizing your chances by utilizing both since we suspect that we may be capturing different populations of patients.
Next slide please.
So what’s next, so Nanotrap is novel technology. There’s currently no insurance code for reimbursement. We have gone through—this is a, this is a complicated process—but I’m proud to say that we are most of the way through this process. We’ve been assigned a code, and very soon, we will be able to file insurance and Medicare reimbursements for the Nanotrap assay, which will, will be a great tool and relief to both providers and patients.
We are also looking at other applications of Nanotrap both to other protein-based methods for Borrelia and other pathogens, but also there may be other ways of utilizing the Nanotrap pop—cap—Nanotrap particles for nucleic acids. We are working on launching our droplet digital PCR technology for other pathogens, including Borrelia, Babesia, and other tick-borne pathogens. We are very interested in development of multiplex assays that are both serology and perhaps more detect—more direct detection, so that we can further reduce the cost for our patients. We’d like to expand the standard of care for serology for other pathogens such as Anaplasma, Rickettsia, Babesia.
And we really would like to clarify the clinical importance of all of our assets through perspective research with interested parties, pursue government funding through NIH, private foundation funding, because we really understand that we need to be able to define the clinical utility of each of these essays for clinicians.
And finally, as Amanda alluded to in the beginning of our presentation, we’d like to partner. We are a prototyping platform and we want to partner to advance solutions and develop FDA approved products and kits were clickable and, and necessary so that we can make more widely available all of these tools, not only for other pathogens, but for potentially other labs to utilize.
Next slide.
AE:
Right so Jen here—we’re here at the end, and so I’m going to sound in. And note that, you know, thank you so much for the overview. And, and the detail and testing and a lot of the reasoning for why we do things the way we do. I think there’s a number of teams working hard out there to advance the sensitivity and accuracy or liability of testing tools.
I think that, speaking for our team, we don’t think there’s going—that there’s going to be one tool. We think that, because of the, you know, broad range of presentations for Lyme borreliosis and the different requirements for confirming acute, you know, disseminated or chronic stage disease, we think that there’ll be a variety of tools.
And so, this is this part of what drives our partnering strategy, right. Where we’re interested in working with researchers who have promising technologies. We are working, obviously with research teams who have technologies that we are really excited about. Because we just want to advance detection. We really want to help doctors do a better job at providing excellent patient care.
And we also understand that this is emerging infectious disease, right. And so, the clinical knowledge is emerging one step at a time. And so we’re working with partners to drive that the clinical understandings, right. To clarify the clinical importance of these infections and medicine and—but we also have our business hat on too. And our focus is on quality, and that is our number one focus right now is to have the absolute best test.
And we recognize that new technologies are often more expensive, so the pricing can be challenging, but we want to reassure everyone that we’re going after the reimbursements and, and we are doing this the right way. We also recognize that there’s a convenience factor, right. So we do have a results portal for, for physicians and we understand that there, and you know, we have the sample collection kits so—we understand that there are other things that we can do in order to make ordering easier and, and we’re working on those things. But we’re, again, we’re a small tenacious dedicated team and working with partners in academia and industry to just get this problem solved right and get better tools out there.