The genome of the Lyme disease-causing Borrelia burgdorferi is one of the most complex of any bacterium—but could genetic sequencing prove key to next-generation diagnostics? Having studied Lyme disease and other tick-borne pathogens over the course of their careers, Dr. John Branda and Dr. Jacob Lemieux separately concluded that genetic sequencing has the potential to improve testing. Now, their team is one of 10 Phase 1 winners that have advanced to Phase 2 of the LymeX Diagnostics Prize, a prize competition to accelerate the development of Lyme disease diagnostics.

Through September 2023, the Phase 2 cohort is participating in a virtual accelerator designed to help them refine their concepts for detecting active Lyme disease infections in people. The goal of the multiphase LymeX Innovation Accelerator (LymeX) competition is to nurture the development of diagnostics toward Food and Drug Administration (FDA) review.

We spoke with Massachusetts General Hospital’s Associate Pathologist Dr. John Branda and Assistant in Medicine and Physician-Investigator Dr. Jacob Lemieux to understand how the team is applying their expertise in genetic sequencing, considering applications of different testing methodologies, and looking ahead to future needs for patients.

Your proposed test looks for genetic material in plasma samples. Why did you decide to utilize genetic sequencing in your diagnostic, and what are the benefits of that approach?
Lemieux: “A year ago, John did a study that showed that if you did whole genome sequencing on plasma samples collected from patients, you could detect sequence fragments of B. burgdorferi in a surprisingly high number of [samples]. I had also done a related study of babesiosis showing that if you performed a target enrichment technique prior to whole genome sequencing, you could pick up lots of fragments of the microbe in the patient’s blood, and pick up less interfering human material.

Since the LymeX Diagnostics Prize started, we’ve been able to start testing our ideas in earnest. We’ve been able to try out different enrichment and isolation methods, and have seen a pretty substantial boost in the signal. There’s some good preliminary data that we can actually do better than standard next-generation sequencing by having a way to fish out the sequences that we’re interested in. If we can also look at DNA and RNA at the same time—RNA is something that is produced by actively replicating organisms—that may give us some clue into whether or not detected sequences are coming from live bacteria that require antimicrobial therapy.

A related issue is that the tick that transmits Lyme disease also transmits other pathogens, such as the agents of anaplasmosis or babesiosis. Ideally, we would have one or a smaller number of diagnostic tests that could detect multiple organisms. The approach that John and I are working on is very promising in that regard because sequencing is unbiased; it can detect nucleic acid from any microbe. We can purify the sequences that are of particular interest to us for diagnostic purposes.”

Both of you have extensive knowledge of Lyme disease, tick-borne diseases, and diagnostics. How have you applied your expertise to your work in the LymeX Diagnostics Prize?
Branda: “Jacob and I have complimentary training and skills. He’s a clinical infectious disease expert; he sees patients in the clinic, and then he has this great special expertise that he developed in his postdoc training relating to sequencing methods and all the things that would need to be done for the project that we’re doing. As a pathologist specializing in medical microbiology,  have some expertise in diagnostic testing development and evaluation, and have worked in Lyme disease for long enough that I have a good sample repository that I can bring to bear. As he said, I have also worked in a preliminary way on the kinds of things we’re trying to accomplish in this project.

We reside in an area where there’s a lot of Lyme disease and we have access to patients and samples at least on some scale. And I think we’re really well poised to take this next step, which is this project that’s combining the technique of unbiased or shotgun metagenomic sequencing of plasma samples with something Jacob has really been on the forefront of, which is enriching samples like plasma prior to doing the sequencing to improve signal detection.”

Lemieux: “The COVID-19 pandemic really shut everything down. But the flip side is that it actually accelerated a lot of what we want to do in a number of really exciting ways. For one thing, John and I and others had this preexisting relationship around tick-borne disease. We were able to work together to do a lot of COVID-19 stuff that we wouldn’t otherwise have been able to do. We collaborated to develop one of the first laboratory-developed tests to diagnose COVID-19, which got emergency-use approval from the FDA, and then we were involved in various ways in bringing forward different diagnostic or genetic sequencing methodologies. The field of infectious disease diagnostics is much more open now to genetic sequencing as a diagnostic tool than it was just a few years ago. So in some ways there’s been an interruption in the work, but in other ways the pandemic has set the stage forsomething like this. It’s given us experience that we wouldn’t otherwise have had with some of the practical and regulatory aspects of this kind of approach.”

Traditionally, Lyme disease diagnostics have relied on detecting the presence of IgM and IgG antibodies through ELISA and western blot tests. How will your concept improve on current diagnostics?
Branda: “Right now, the diagnostic methodology is a two-tiered approach where the first test would be a screening ELISA, and if that is positive, then you go on to do a secondary antibody test. There are a number of different ways of doing secondary tests these days. And I would say that method is pretty accurate in later stages of Lyme disease. 

However, this approach is not directly detecting the agent of infection, and instead is detecting a host human antibody response to the agent of infection. This leads to two important limitations. One limitation with serologic testing is the difficulty of telling whether there’s an active ongoing infection or whether you’re seeing an antibody response relating to a more remote infection. And equally, it hasn’t worked very well to use serologic testing to try to monitor therapeutic success. So those are the things that usually could be addressed by direct detection methods, meaning trying to develop assays to detect the actual agent of infection.

In many infectious illnesses—COVID would be a good example—we usually could use a simple molecular test like PCR to detect the pathogen. Another very routine direct detection method would be culture. Lyme disease has been more challenging than that: PCR and cultures have not worked well at any stage of the illness. In a pilot study that Jacob and I did, we applied routine PCR to 28 blood samples from patients with known early-stage Lyme disease and the sensitivity was 7%, meaning that it was falsely negative in 93% [of samples]. 

But when we tried unbiased or shotgun metagenomic sequencing in plasma, we were able to detect sequences that map back to the agent of infection in 64% of the samples. Current serologic testing was somewhere in between. So we think that we’ve come to a method of direct detection that is vastly superior to a standard PCR that could be used on readily obtainable samples like blood or cerebrospinal fluid.”

Lemieux: “Right now, we rely on the patient’s immune system to develop a response to the bacteria and we then detect that response. If the patient’s treated right away, then they may have a small response. And so you have to define this threshold: You had Lyme disease or you didn’t have Lyme disease, and make it black and white. For patients who don’t have immune systems that are fully intact—they may be on medications that weaken the immune system or have a medical condition that limits the quality of the immune response that they can produce— the diagnostic assays are less useful and less able to be interpreted.”

The LymeX Diagnostics Prize uses an open innovation model to speed development of the next generation of Lyme disease diagnostics while sparking sustained innovation, participation, and capital investment. What has been the most useful resource for your team?
Lemieux: “The money is the most helpful. Sequencing is expensive; it uses expensive reagents and expensive equipment. So having financial support to generate some preliminary data—this concept has been tough to make progress with, because we haven’t had the funding to actually take it forward. We don’t work in a commercial environment. We work in an academic nonprofit environment where we need to raise money from grantmaking organizations.

It’s hard to get NIH funding for diagnostic assays, because the NIH funds basic research about how pathogens work. Most diagnostic development is done by big companies. There’s been some activation energy needed, some catalyst to move things forward. So we’re hugely grateful for the money. We would love to scale this up and really take this to the next step and study this in patients and show that it works.”

Branda: “The LymeX Diagnostics Prize is filling an important gap: The usual funding mechanisms for medical research have not really focused on this problem. There isn’t a lot of federal funding for a Lyme disease diagnostic or Lyme disease research, period. But when there is, it’s usually oriented toward what we think of as fundamental science, not translational or clinical projects. Industry often will help with funding for projects that they see a commercial value in. But it’s hard to get preliminary work done to a point where you’ll get a major investment from a for-profit or from the government. So it’s filling this gap where we’re trying to study a problem that there isn’t really a good funding mechanism for, but it’s a really important problem.”

Improving diagnostics is only the first step in improving outcomes for patients. If your test is successfully validated, what does your team want to turn to next?
Lemieux: “Lyme disease is such a challenging problem—diagnostically, the care of patients with Lyme disease, and the manifestations of Lyme disease, including medical issues, infection issues, rheumatologic issues, and neurological issues. Some patients who develop Lyme disease are treated and don’t return to health. John and I are both very interested in tackling these many challenges in Lyme disease. Our hope is to develop a multidisciplinary program for the diagnosis, treatment, and investigation of tick-borne pathogens, including Lyme disease.”

Branda: “A future direction would definitely include studies of patients with later manifestations of Lyme disease and potentially patients who have persistent symptoms after treatment of Lyme disease. We would try to work on diagnostics in those areas, but also on some spinoff questions. Why does one person get a more aggressive infection than another person, for example? Some of those things are not going to directly be answered in this project, but we’re laying the groundwork to get there.”

Looking ahead: Expert judging panel to convene in October 2023

Following the accelerator, the cohort will submit concept papers that detail solution refinement, clinical and patient input, and a roadmap from lab to market. The competition judging panel—composed of experts across biology, clinical and technology translation, patient experience and advocacy, diagnostic science and technology, exponential innovation, and ethics—will evaluate eligible submissions according to official Phase 2 evaluation criteria. Based on the judges’ evaluations, the panel will recommend up to five Phase 2 winners of the LymeX Diagnostics Prize.

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