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Galaxy Diagnostics recently hosted a webinar where our CEO, Dr. Amanda Elam, and chief scientific officer, Dr. Ed Breitschwerdt, provided clinicians with an update on our Bartonella test offerings and discussed current recommendations for use based on the published research. Specific topics included:
- Bartonella pathogenesis and disease prevalence
- Bartonella ddPCR, Bartonella Digital ePCR™, and the recently launched 4-species IFA Serology Panel
- Three case studies that emphasized the importance of combining direct and indirect test methods to confirm diagnoses
We are happy to now make this resource accessible to healthcare providers, patients, researchers, and others who may be interested in learning about bartonellosis. A full transcript is also available below the video.
Full Transcript
AE: Dr. Amanda Elam, PhD, Galaxy Diagnostics cofounder & CEO
EB: Dr. Ed Breitschwerdt, DVM, Galaxy Diagnostics cofounder & chief scientific officer and NCSU – CVM Research Professor
AE:
First, I want to give you a quick overview about our company. So Galaxy Diagnostics is located in the Research Triangle Park in North Carolina. Our mission is to advance the detection of low abundance infections, flea, and tick-borne infections for better clinical outcomes.
We identify as a One Health company, so that little symbol up in the right-hand corner represents the One Health concept. One Health is a global movement looking at the intersection of health and disease, for humans, animals, and the environment. And this concept is really core to what we do and how we’ve approached diagnostic development as well as serving different communities.
We do testing for clinicians in human health. We do testing for clinicians in animal health. We also provide testing support for clinical researchers because this is an important area of emerging infectious disease. And we know that as we advance the diagnostics, those diagnostics are going to drive research.
So long story short, you know, we’re partnering with people in academia and industry to really drive these advances. And we are publishing along the way, and that’s really our DNA as a company. On our executive team. Here’s a photo of the three founders. I was teaching at the business school at NC State when I met Ed Breitschwerdt and Ricardo Maggi and heard about their work, the great science that they’re doing at North Carolina State University.
And I partnered with them to launch Galaxy Diagnostics back in 2009. We also have Dr. Robert Mozayeni who is our medical director at Galaxy. He is a clinician and founder of Translational Medicine Group, and past president of ILADS, and passionate about translational medicine. We have Dr. Susan Orton as our lab director. She works full time at North Carolina Public Health and has helped us guide the development of our quality program at Galaxy. Dr. Jennifer Miller is our director of research and development, and currently our assistant lab director. An excellent scientist like Ed and Ricardo are. And Ann Miller is our director of finance and operations.
So that’s our executive team. And then of course we have a wonderful team of lab technicians and administrators in the office. A lot of people ask us, what’s the relationship between Galaxy Diagnostics and NC State. When you look at this executive team, you get this idea that it all is kind of mixed up, but we’ve worked very hard to kind of keep the activities at NC State distinct from what we’re doing at Galaxy.
So Galaxy, we are commercializing the diagnostics developed at NC state, along with some other diagnostic technologies developed by research teams around the country. And so a way to think about that is that NC state is doing diagnostic development and clinical research and Galaxy is commercialization. So we’re doing some of our own prototyping at Galaxy but it is definitely directed by our collaboration with the NC State research team.
Just a quick overview of Bartonella for those of you who may be new to it. Bartonella is a stealth pathogen. I first came across this term reading a 2004 Nature article, by Stanley Falco and Robert Merrill.
And they had a brilliant article where they listed Bartonella along with H. pylori as the two most important microbes on the medical microbiology radar because of what these pathogens can do at the cellular level. So this is the key agent in cat scratch disease. There are estimated 30,000 cases of acute cat scratch disease in the US every year.
And about 10% of those are hospitalized. Now this is a number that. Doesn’t take into account a lot of what the NC State research has revealed, which is chronic bartonellosis and the role that these pathogens are playing in chronic disease.
They were discovered in HIV/AIDS patients in the us around 1990. Prior to that, we knew one species in South America, Bartonella bacilliformis, and we knew cat scratch disease was a thing, but we didn’t know what caused it until the HIV/AIDS epidemic. And this is really important because this is not an infection the HIV/AIDS patients picked up in the hospital that contributed to complications [such as] heart, liver, lesions on the skin, and neurologic issues. This is a pathogen they already had in their bodies when they became immune compromised by HIV. So I think this is a really important paradigm to understand when we’re looking at chronic infectious disease and the role that Bartonella is playing.
They are transmitted by fleas, ticks, lice, and biting flies. Also, zoonotically by animal bites and scratches. Ed likes to remind us that they’re carried by a large and diverse animal population, including pets. And this is what makes these bacteria quite unusual from a transmission standpoint. There are 40 plus species and counting globally. The research is at a very early stage and there are a lot of excellent scientists around the world driving discovery here. About half of those species have been implicated in animal and human disease.
They’ve been implicated in a range of disseminated diseases, affecting the heart, central nervous system, joints, eyes, liver, spleen, and even certain cancers. And that’s where a lot of the clinical research is focused is on, clarifying the clinical importance of these bacteria. They have demonstrated some level of antibiotic resistance as a complicated problem set, but we know treatment failure is common and they’re involved in polymicrobial infections and the tick-borne disease pathobiome. Again, this kind of hearkens back to the discovery of HIV/AIDS with Bartonella henselae and Bartonella quintana.
So in medicine, you know, you can be colonized by infections, by pathogens, but that doesn’t necessarily mean that they cause disease. So in science, we really look to the pathogenesis research to guide our understanding of why a particular pathogen might be so important clinically and Bartonella checks a lot of the really important boxes from a pathogenesis standpoint.
They’re slow growing, they’re intracellular, and they’re immune evasive. Bartonella cycle in and out of the blood over a five-to-seven-day period. So they’re in the blood, which presents an opportunity for blood-based testing. But they’re elusive, right? And they hide inside cells. In fact, they infect a broad range of cell types, including endothelial cells, red blood cells, CD-34 progenitor cells, microglial cells, and many others.
They are known to hide in bone marrow. They can hide in eyes. A number of ophthalmologists have told me that they often diagnose CSD through eye exams long before the other symptoms present. And we’ve also found them in joints, and they contribute to rheumatologic disease.
These bacteria can also cause vasoproliferative lesions and tumors. In fact, they override key cell functions, which make them oncogenesis candidates. So they’re really high on the medical microbiology radar. What are these pathogens potentially doing in their human and animal hosts?
So the real challenge diagnostically around these pathogens is that they’re low abundance. And to put this in context, this is a chart that I think helps a lot of people understand that most of the tests that are available out there for detection, those conventional limits of detection work great for infections like E. Coli where the pathogen grows very rapidly, where there’s a clear humoral immune response to the presence of the pathogen in the host.
But for stealth pathogens, it’s a completely different story. These pathogens are able to, you know, evade both direct and indirect detection. So from a diagnostic development strategy, we have to lower the limits of detection and that has been core to our mission at Galaxy. So we’re really working with three different technologies that increase the sensitivity of direct detection of low abundance pathogens.
So just very generally, we’re working with the Bartonella alpha-proteo growth medium. This is our proprietary enrichment medium that is only available commercially through Galaxy. And we use that to increase the bacterial load in the sample up to detectable levels for Bartonella.
We’re also working with droplet digital PCR, which is part of our presentation today. We’re very excited about this new technology. Instead of running one PCR on a small aliquot from the patient sample, effectively we generate 10 to 20,000 droplets out of that aliquot and we run a reaction in every droplet and this reduces the inhibition and increases the sensitivity of DNA detection. So we’re really excited about the potential of this technology for Bartonella and other low abundance pathogens.
And then we’re also working with the Nanotrap capture particles. These particles, right now, work for capturing proteins in a patient sample. So we can directly capture surface proteins and spin them down with magnetic beads and then test the concentrated proteins.
We’re excited about some of the advances behind this idea of capture. Some capture technologies that are going after RNA and DNA technologies. And that’s part of how we’re looking forward on sample enrichment, is how can we combine these different technologies to advance detection.
From an offering standpoint, at this point in time, we have direct detection options and indirect detection options. So when we’re looking for DNA detection for Bartonella, our latest offering is Bartonella Digital ePCR, where we’re combining the BAPGM media with droplet digital PCR. The data we have so far on this. Um, so we’re using genus level primers so we’re looking broadly for Bartonella species. But this technology does not, at this time, allow us to confirm positives by sequencing. And it really appears to be better for blood and fluid. That’s where we see the gains in the research studies.
We’re also offering Bartonella species ddPCR independent from BAPGM enrichment. Again, genus level primers, no sequence ID and best for blood and fluid. The premise behind droplet digital also includes sort of a flow cytometry approach for those droplets. They’re fed through and so tissue is a little harder to work with and doesn’t show the same gains that blood or fluid does. And then finally, we continue to offer qPCR or conventional PCR for Bartonella species. Again, we’re using DNA, we’re using genus level primers, and we are confirming by a sequence ID. And this approach is good for blood, fluid, tissue, a lot of different sample matrices.
And then on the indirect detection, we’re offering a new panel. So we have Bartonella IFA serology. We have expanded our initial panel for antibodies against Bartonella henselae and Bartonella quintana, and we’ve added Bartonella vinsonii berkhoffii and Bartonella koehlerae. The offering of this four X panel is very exciting for the company because it also indicates we’ve set up cultures in house. So we’re growing our own antigen. We have full control of quality program around the antigens that we grow and we’re just really excited about running our own cell culture and slide prep in house. This is going to serve as a foundation for expanding this kind of testing if we want to go forward.
The other important thing to know about IFA serology is that it’s the standard of diagnostic care for cat scratch disease and often other Bartonella infections. And so when we look at translating some of the novel assays that we’re developing, you know, it’s really important that we can compare that to the diagnostic standard of care. And also, you know, a way of working with our clinician population to understand which types of approaches are working best in their patient populations.
When it comes to test performance, I just want you all to know that we’re very systematic in the way we document our test performance. We have created performance characteristics and test interpretation documents, so test specification documents. They’re kind of like the product, they’re based on the idea of a product insert you would get in an IVD kit, and we have these available. I guess we don’t spend a lot of time leading with these in research because under experimental conditions, our assays work really well. Sensitivity, specificity, is really high.
We have a good solid biorepository where we’re able to test the performance of these assays in a high number of samples. 200 samples in terms of the clinical specificity for Bartonella Digital ePCR™ and, you know, highly accurate from a CFU per mil standpoint. However, this kind of validation data, while we’re happy to make it available to you, doesn’t necessarily answer the questions that most clinicians have, which we get all the time. What’s really the predictive probability of this assay finding or documenting a positive in my patient? And this is true for all our assays. So this information is available upon request, but I think it’s the clinical research that we’re publishing that is of more interest to our clinician population.
But here’s an example of some of the studies that we’ve done or NC state really. Ed I have to give you the credit on that, really driven a lot of the research. So in one study with 500 healthy blood donors with colleagues in Brazil using this back in the time, this was Bartonella ePCR. So it was BAPGM paired with PCR.
They found, the IgG antibody response was 16% and 32% in 500 healthy blood donors. And the DNA positive rate was about 3.2%. This is a really important percentage to understand when we’re looking at clinical populations is to be able to say, here’s a rate of what we expect to find potential asymptomatic infection, right?
And so we’ve done studies on veterinary workers where we found 28% of those individuals were actively infected. So evidence of DNA in blood and over half of them had some level of IgG antibody reactivity. And a large study with Bob Mozayeni’s patients, 296 rheumatologic patients. In this study, we found 41% of patients were DNA positive and 23% had a high IgG antibody positive rate.
Again, if you’re comparing this kind of data back to healthy blood donor rates, you know, you gain confidence that these pathogens are actually playing a role in disease. In a recent pilot study from NC State 17 schizophrenia patients. So newly diagnosed schizophrenia patients at UNC in North Carolina, 65% were DNA positive and this is using our BAPGM and ddPCR assay.
And then there were high rates of antibody positivity as well. I believe the overall rate was about 71% to any Bartonella species, but specifically 65% to B. henselae and 41% to B. quintana. And then there are researchers around the world who are also using PCR and IFA to document rates of possible infection in different populations.
And one of the studies I found really to be important is one that looked at 106 culture-negative endocarditis patients in France where they found over half of the patients had high antibody positivity and 60% they found PCR positive. So not even with some of the advanced techniques we’ve been using, but that suggests that that Bartonella is probably playing a much larger role in endocarditis than is currently appreciated.
And then of course, with the comparative medicine focus of the NCSU research program on Bartonella, the Bartonella project there, we also have a lot of data on canine bartonellosis and one recent study I think is really important was a study looking at a hundred canine hemangiosarcoma tumors and the DNA positivity rate there was 73% looking in tissue and blood. And I believe the IFA positive rate was around 6%. So it was really low, I just didn’t have that number when I was preparing the slide. So Bartonella is playing a really important role.
One of the first findings we discovered when we, as a team kind of came at this, you know, antibody versus DNA evidence. I think is well illustrated in this recent study that was done on those canine hemangiosarcoma cases. This is table five from the first, the most recent publication, in Pathogens 2021, which followed another study on the same population published in 2020 from the NCSU team. Table five looked at, you know, the correlation, the correspondence between serology results and molecular results.
And this is where this idea that we need to combine methods and look for both antibodies and DNA evidence of infection in patients come from. And that was one of the first understandings established by the NCSU research and the early Galaxy commercialization efforts.
So this study showed that ddPCR in blood was about 36% sensitive. Actually, this is ddPCR and PCR, right Ed? So this is combined, molecular sensitivity was 36% in blood and tissue was 89% and that was primarily PCR. And then the IFA sensitivity was about 6% and blood tissue qPCR sensitivity was zero. So qPCR did not reveal many positives at all and ddPCR made a big difference.
We recently published some of the first kind of clinical population looking at establishing utility in patients with high vector and animal contact. So these are not known, infected cases. These are people who are high clinical suspicion. So like some of the cases that walk into your office, right? So they have symptoms considered consistent with what chronic bartonellosis literature says and these 38 patients had high risk factors. So high vector or animal contact and multiple samples were taken from these patients.
So we had a total of 112 samples. The blood samples were tested before and after BAPGM culture at 7, 14 and 21 days in the study and the experiment was performed twice. And with similar results for each run. I think it’s really important to look at, again, we’re looking at a correspondence table here and look at ddPCR versus qPCR.
The correlation is only 3%, but the ddPCR positivity rate was effectively 10 times what was achieved by qPCR. And this is why we’re so excited about this technology, and where we can move this forward to advance. Now for our clinician audience, I’ll tell you that the purchase of the droplet digital PCR equipment it came in around $150,000. Whereas we can pick up a PCR machine for, you know, 25 to $30,000. So this is really expensive. The test prices are high. We know that, but with innovation, we often start in this expensive place, and we try to find our way to a less expensive place.
From an enrichment standpoint these samples that are reflected here in the 38 patients we use BAPGM enrichment over time. And to give you an idea of the utility of BAPGM enrichment. In this scenario, we’re talking, this is out of the 38 patients, the first positive that showed up. What you find here is if you do seven days of enrichment, we capture five more cases than we would have if we had just done ddPCR in blood.
So that’s why Galaxy is continuing to offer the enrichment PCR, ePCR, approach is because you can pick up people you wouldn’t otherwise document. On a research basis, these samples are cultured out to 14 to 21 days and Ed will speak to this later, but this is the conundrum that drives him nuts as a clinician. He said over and over is that every single time he sees a sample picked up at 14 or 21 days.
Um, you know, some people don’t test positive until then. Some animals don’t test positive until the cultures run longer. But it’s not really a feasible test. This turns into a test turnaround time of about four weeks and that’s why Galaxy is not doing extended cultures outside of research studies. But we do do it for research studies so BAPGM can enhance detection I think in a really, really important way and pick up another 20 to 30% of patients who are positive.
So at this point, I’m going to invite Ed to take over and share his perspective on the test modalities that are available and some examples of what he sees in specific cases and what next steps are from his perspective.
EB:
Thanks, Amanda. And I want to start by thanking everyone for being with us today. As a clinician and internist and a researcher, I realize that all of us have extremely full lives, professionally and hopefully some time left outside our professions to spend with family and friends.
So one of the important aspects of Bartonella that is becoming very clear. And Dr. Elam emphasized the fact that this organism, one infects many cells and two, it persists for a very long time as we’ve come to appreciate primarily from work out of our laboratory and now more work from other laboratories. And so I think the challenge, and we’ll see this in the three cases that I present, is although a patient might present for seizures in a detailed history, you’ll find that they often have other systemic manifestations, headaches, periodic joint, myalgias, that type thing.
So clearly a multi-system disease, but we as clinicians often like to put our diseases into a specific system. Any time I see a patient with multi-system disease, I’m often thinking of a vector transmitted organism.
So in regard to test recommendations that actually Bob Mozayeni and I went back and forth over for a period of time that represent the current recommendations from Galaxy Diagnostics. Ideally, we would all like to be testing patients before they are treated with antibiotics or multiple antibiotics, which certainly complicates both serologic and culture and molecular diagnostic testing.
So for pre-treatment, we would certainly say the most sensitive modality would be the triple draw ePCR. Once treatment is started then, following serology, you can actually, as I’ll show you in the examples, be quite helpful in some instances. And again, combining that with the ePCR, would allow us to say that the patient went from a positive status hopefully to a negative status.
That antibody titers are decreasing over time. And the patient’s responding clinically to the antibiotic regimen that’s being used. And then finally because we’ve seen enough recrudescent disease, which we believe is treatment failures, following up with IFA serology after what is perceived to be successful treatment along with a triple draw, particularly if the patient re-presents with symptoms and again, prior to antibiotics would be beneficial.
First, I’m just going to have one slide on serology and one slide on molecular diagnostic testing, because what I really want to share with you is our experience in regard to specific patients and the specific use of these tests and their advantages and disadvantages and limitations.
And again, in, in lecturing, primarily to veterinarians about diagnostic testing modalities, I generally start with the fact that every diagnostic test we do has limitations and that although it’s difficult for the clinicians out there in the real world seeing patients to understand those limitations in the detail that the laboratory has to understand them.
It’s important for all of us to realize that there are limitations and do the best we can to understand and overcome them. Which is what we’ve tried to do on a research basis at NC State and on a commercial diagnostic basis at Galaxy Diagnostics.
So Bartonella serology advantage is it’s widely available and widely used. We all know that antibody detection can only indicate exposure and clearly in humans, there’s questions about specificity which I’ll again address a little bit when we get to the cases. In regard to disadvantages, one, it’s hard to standardize across laboratories two, it’s difficult, particularly in regard to stealth pathogens to establish meaningful cutoff values.
That’s a lot easier to do in an acute infection or a severe infection like Rocky Mountain Spotted Fever where antibody titers go sky high in a short period of time. I am suspicious that there’s less cross-reactivity based on what we’ve done, experimentally and clinically, in dogs than has been reported in human literature, but Coxiella, Toxoplasma and Chlamydia have all been implicated as cross-reacting genera to the genus Bartonella.
And then there’s variable epitope expression due to genetic rearrangement. And this is problematic because I think the longer an individual is infected, the less predictable the antibody response is going to be and certainly the less specific it will be to the infecting Bartonella species. And then in regard to the assay it’s dependent on the species and strain. Both Galaxy and our research group at NC state uses the Bartonella henselae San Antonio-II strain.
That is the most frequent strain that we will find by PCR amplification and DNA sequencing in a cat, adult human, or a horse in the United States of America. And then CDC back in the early nineties demonstrated that using cell culture is important and what cells you’re using and whether you’re not using a cell cultured base assay for IFA will clearly indicate the titers that are generated.
And then finally, the longer you passage, these Bartonella, the less likely they are to be expressing proteins on their surface that would be detected by antibodies in your patients. So using bacteria that are low passage number is absolutely critical when generating these reagents for testing.
So in regard to DNA amplification, whether you’re talking about conventional quantitative PCR or droplet digital PCR, the good part is we can go in regard to qPCR from two copies of DNA to literally 3.5 times 10 to the 13 copies of DNA. Regardless of the modality, it works for most types of samples, blood tissues, CSF, effusions, pathological effusions in the abdomen or in the chest. It’s highly specific and sensitive under ideal conditions. It’s easily adapted and customized to various laboratories.
Despite those very good points, there are some bad points. One, it cannot detect what is not there. And I just basically say to clinicians, a negative PCR has never ruled out an infectious agent in any patient, unless you actually extracted all the DNA from that patient and did PCR, and you still would not have truly eliminated the possibility of infection in that patient due to technical reasons.
It’s prone to be variable under reagent and design quality and enzyme and format and protocol. So it’s kind of like making a cake. Everybody can make a cake, everybody can make a chocolate cake, but that cake can taste very different depending on the ingredients that are being used.
And that is very true in regards to PCR its sensitivity and specificity. And then what you’ll see on the right side, what you’ll recognize are frequently in patient samples, are inhibitors, known inhibitors of PCR reactions. And this is where quantitative PCR, qPCR, differs from ddPCR because the advantage of ddPCR is because we’re doing literally 20,000 PCR reactions rather than starting with a single PCR reaction, inhibitors are not as much of a problem.
And then I really think the ugly part, particularly in vector-borne diseases, unlike many other infectious diseases, where there would be standardization across laboratories. There’s no standardization that I’m aware of across any laboratory for either qPCR or ddPCR testing.
So now we’re just going to discuss three cases and then we’ll bring it back to Dr. Elam for concluding comments on what’s occurring with Galaxy. So it’s really rare in the context of the research we’ve done to know when an individual, particularly a veterinarian who’s constantly exposed to cats, dogs, and oftentimes fleas and ticks, becomes infected.
And in this instance, what you’ll see is one of the veterinarians that actually worked here at the college of veterinary medicine was obtaining a diagnostic sample from a fractious dog and, after aspirating this cutaneous lesion, managed to stick the needle in her hand. The hospital administrator contacted me because six months earlier, the dog had a history of ehrlichiosis.
It was treated with doxycycline as would be appropriate. And the initial question to me is could this veterinarian become infected with Ehrlichia? What I knew that the veterinarian and the hospital administrator didn’t know on the basis of prior publications from our laboratory, is that 33% of dogs in the Southeastern United States that have antibodies to Ehrlichia canis have antibodies to Bartonella vinsonii berkhoffii. So this is a very, very high percentage where we generally see in our blood donors, less than 3% actually having antibodies by IFA to any of our Bartonella antigens.
So we’re pretty sure when this veterinarian got infected, we’re pretty sure how the veterinarian got infected and I’m just going to call your attention to a couple things on this slide in regard to this particular case. So initially this veterinarian was negative serologically and negative by PCR. On day 34, she had started to develop headaches and kind of space sensory neuropathy and you’ll see that by direct extraction, we managed to amplify and sequence Bartonella vinsonii berkhoffii genotype I.
But our enrichment culture, unfortunately, did not grow the organism. Again, you need to have enough organisms that are viable in the sample that you’re dealing with to get enrichment culture to work and even when there are viable organisms there, it doesn’t always grow as easy as E. coli would grow on a blood agar plate. By day 81, we have a 1: 32 antibody titer, which again, we would report that as negative from our laboratory, but by the time we get to day 97, we have now developed an antibody titer of 1:64. And by day 144, an antibody titer of 1:128.
What’s important is this veterinarian clearly seroconverted to the organism that we found in her blood. And you’ll see that the second time she was tested, well actually the third time she was tested, we did not amplify Bartonella vinsonii berkhoffii directly from her blood, but we did amplify it from her enrichment culture. And regrettably, which is something that we have worked on for over a decade, we continue to work on, is trying to get this organism to come out of enrichment culture on the blood agar plates and be able to actually have isolates for more research purposes.
I’d also emphasize that she never seroconverted to Ehrlichia and she remained PCR negative to Ehrlichia at all those data points that you see there. And importantly, she never seroconverted whatsoever to genotype two, and she did develop titers to genotype three, which is actually what we were able to PCR out of the dog. So my suspicion is she was infected with both genotype one and genotype three. We were able to prove one in the dog and one in her, which always ruins a good case presentation
And so on this particular slide, this is a young woman that lived in the Bali area whose mother was on the internet and following our Bartonella research and reached out to me by email. She had a four-year history of peripheral visual loss, sensory neuropathy and hallucinations. You’ll see, this was published in 2011 and the story actually started with us in 2009.
And she also, as I mentioned, had other symptoms in addition to the neuropathy and hallucinations that included joint pain, back pain, mood swings, and dizziness. As you would expect, she had been evaluated by numerous physicians, including psychiatrists and neuro ophthalmologists.
So I think if we just look at the very top line for 1/13/09. And what you’ll see is initially by our testing, is she was negative to all the antigens that we were using at that time, which included Bartonella quintana that I didn’t put on this particular slide in the interest of space or table. But after we detected Bartonella koehlerae in cats and we started seeing Bartonella koehlerae by PCR amplification and sequencing in a small series of veterinary workers.
We developed an IFA test specifically to a cat isolate of Bartonella koehlerae, and then we went back and tested some of the people that we, retrospectively had documented as having Bartonella species DNA in their blood. So you’ll see on day 14 in culture, we were able to get an amplicon, but we were not able to sequence it.
And ultimately by developing a Bartonella specific IFA and a Bartonella specific intergenic spacer target for Bartonella koehlerae, we were able to document that on the very first sample we ever got from this young lady that she was truly seroreactive to B. koehlerae and and she was infected with B. koehlerae. But if we had only tested her to henselae and quintana, she would have been negative by our testing.
And again, I think this gets to the specificity of Bartonella across species. And certainly as Dr. Elam mentioned now with over 40 species and half of them being implicated in pathology in animals and humans, this genus is not going to get any easier as we march forward in our understanding. But you’ll see that after treatment by Chris Woods over at Duke, we had a very hard time in eliminating the infection and I’ll show you another example in the next slide. Initially, that was attributed to the fact that despite the fact that she was dispensed doxycycline and rifampin, she was non-compliant and didn’t take a lot of her medications.
So after we proved subsequently that she was still infected with Bartonella koehlerae, and we did manage to document in enrichment culture several times that she was infected and the organism was viable, she was treated for over a year and ultimately went negative and remained negative.
The last case is another veterinarian and this I think is more support that Bartonella, as a stealth pathogen. is playing a role in regards to rheumatologic symptoms. And this again is a veterinarian, in her early thirties, who actually was initially infected with Bartonella koehlerae, but did not have antibodies to Bartonella koehlerae. And, unfortunately, the sample that we tested did not grow in enrichment culture.
And at that point, we were only doing our enrichment cultures 7 days and 14 days and we ultimately moved it out to 21 days with the idea that some people would not be positive until a 21-day culture. Again, reflecting the fact that we’re starting with a very, very low bacterial load and requiring a long period of time for those organisms to increase to a level of detection.
What you can see here is that she was treated starting in late June of 2010 by Dr. Mozayeni and Bob was treating her with doxycycline and rifampin and clarithromycin at that point in time. And she initially on the 9/17, very specifically seroconverted to the Bartonella that we had found in her blood and we were very happy.
Just after that, she managed to go on vacation down in Georgia, stay in a hotel that was animal friendly because all veterinarians take their pets with them when they go to vacation, and she and the pet got fleas. And so, what we think happened is that she actually became super infected by a second Bartonella exposure.
And after that, we were able to identify multiple Bartonella strains over time, and she was very refractory to treatment. She became pregnant in 2012 ultimately had a baby and in the manuscript, you’ll see, we follow this individual till 2018. And as Paul Harvey would say, there’s more to that story, but you’ll have to stay tuned to future publications coming out of our research laboratory.
As you all know, I’m a veterinarian and I got into this business because I want to protect the health of pets and the people that own them and Bartonella has become very disconcerting to me in that regard. The reason that Galaxy tests for Bartonella henselae, Bartonella vinsonii berkhoffii, and Bartonella koehlerae is those are the organisms that we find in pets that increasingly, because of the importance of the companion animal bond, people have not only brought into their house, but they brought them into their bedroom, which is great. But it may not be so great if these animals are reservoirs for these specific Bartonellas.
It’s also the reason that we do not specifically test for Bartonella elizabethae, which is a rat associated Bartonella. Exposure to rats is probably far less likely in most of the patients that you see than exposure to a cat and a dog.
So the disconcerting part about Bartonella is that there is a large and diverse pet and wildlife reservoir population. There are more vectors that transmit Bartonella than any other vector-borne organism I’m aware of. We’ve mentioned the increasing number of Bartonella species. The fact that these bacteria are definitely capable of inducing persistent intradermal and intravascular infections in both animals and humans.
They’re difficult to detect and isolate diagnostically. And they’re difficult to eliminate with the combination of antibiotics, which is the reason that the Cohen Foundation has recently funded NC State university, Duke university, and Tulane university to develop a Bartonella specific drug. Research that is in progress.
So I think at this point I’ll ask Amanda to unmute and turn it back to her. I hope some of what I said will be helpful to you when you’re examining your next patient.
AE:
Thank you so much, Ed, really appreciate your expertise on this topic. I’m going to come back to the company for a minute.
And that’s just to say that again, we’re this early commercialization platform, we’re working with partners on prototyping different diagnostics. But we are thinking, you know, a lot about the patients and we’re thinking a lot about the clinicians, and we understand that while our focus has been largely on quality, that there are concerns around price and there are concerns around convenience.
And so I just want everyone to know what that insurance reimbursements for the novel test panels that we are coming up with are a priority. We’ve received codes for Bartonella ddPCR, Bartonella Digital ePCR, and for our Lyme Nanotrap kit. It’s a two-year process, but we’re on track and fingers crossed that we get good pricing from CMS and we can move this forward.
We are looking to launch ddPCR assays for all the other flea and tick borne pathogens on our list. And we think that it’s that this approach, that advancing direct detection, not only will facilitate documentation of these low abundance pathogens in patient samples. We think it will also facilitate earlier diagnosis, even for faster growing pathogens, has been demonstrated with some of the technologies we’re using.
We’re looking to expand the standard of care serology test options as we go after other targets, and we’ll continue to clarify the clinical importance of these infections through research. We all have this goal of doing prospective research, but the research funding is really challenging, but we’ll continue to work on that with partners.
And we’ll continue to advance the sample enrichment techniques that we’re using to increase sensitivity. Optimization is always a goal. I think from a cost standpoint we’re really interested in developing multiplex assays. So NC state and Galaxy recently published the first multiplex, sort of feasibility data for, multiplexing using ddPCR.
We’re going to continue working on that development pathway. You know, if we can look for multiple pathogen targets, whether we’re looking at multiple species in one genus or we’re looking for multiple genera, then we can actually reduce the cost of the overall test to the patient.
So multiplexing is very appealing on a number of levels. But the regulatory is not well-defined around that the same way it’s not well-defined around any novel technology that is in development. So that’s , part of the challenge that we’re facing is as we’re advancing these diagnostics. And you know, again, we’re partnering to advance the solutions and ultimately what we’re hoping is to find the right prototype, where it makes sense to take it through FDA approval to productize.
And work with some of our partners to get IVD kits out into every lab running some of these technologies. But you may all know that 80% of clinical testing in the US is lab developed tests for better or for worse. And so some tests never graduate right to the product phase. But we do recognize that in the world of flea and tick-borne disease, that this is really critical. That these may appear to be rare diseases, but until we are testing for them, until clinicians are documenting infections in patients, we really don’t know how prevalent they really are.
